bims-proteo Biomed News
on Proteostasis
Issue of 2026–08–02
seventy papers selected by
Eric Chevet, INSERM
  1. ER-localized translational control as a nexus for proteostasis.
  2. DNAJB12/14 redox switching directs chaperone- and Bax/Bak-dependent ER protein reflux.
  3. Spatiotemporal control of Atg2 association with the ER during autophagosome formation.
  4. Two Mechanisms One Molecule: Developing a 'Truly' Bifunctional Degrader Targeting Rpn13 and CRBN.
  5. Vimentin intermediate filaments support autophagosome biogenesis at ER-endosome contact sites in response to starvation.
  6. ATL2 Recruits TRAK1 to Promote Mitochondrial Transport at ER-Mitochondria Contact Sites.
  7. Identifying endogenous substrates of the 26S proteasome through site-specific photocrosslinking.
  8. A Golgi-bypass secretion mechanism for proTGFα involving TMED9 and GRASP65.
  9. APOE4 disrupts the central dogma by arresting neuronal proteome dynamics.
  10. The Drosophila CEBPG homolog Irbp18 partners with crc (ATF4) to mediate the integrated stress response in degenerative disease models.
  11. Hexosamine Pathway Disruption by GFPT1 Loss Drives Coordinated Defects in Glycosylation, Autophagy, and Trafficking.
  12. Live-cell co-translational folding tracking reveals bidirectional coupling between translation and folding.
  13. Ribosome-associated chaperones integrate co-translational mitochondrial protein import, stress signaling and proteostasis in Saccharomyces cerevisiae.
  14. Cellular stress triggers degradation and global redistribution of RNAPII.
  15. Functional and transcriptomic analyses in Neurospora crassa reveal a crucial role for the N-glycoprotein deglycosylation process in fungal homeostasis.
  16. Quantitative profiling of basal and stress-induced ribosome collisions.
  17. EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression.
  18. Direct single-molecule visualization of Hsp90-mediated relief of an Hsp70-folding block.
  19. Prolines in linkers of multi-domain proteins: Intrinsic regulators of misfolding and aggregation.
  20. Autophagy prevents ER stress-induced Tight Junction barrier disruption via claudin-2 homeostasis.
  21. Retrograde trafficking inhibitors allosterically trap Get3 to block tail-anchored protein biogenesis.
  22. Trr1 (thioredoxin reductase 1) modulates ERphagy in aging via liquid-liquid phase separation.
  23. Linkerology in PROTACs: learnings for proximity-inducing therapeutics.
  24. Multidomain Relaxation Dispersion NMR Resolves an Intermediate-Gated Binding Pathway for Selective Recognition of Linear Diubiquitin by HOIL-1L.
  25. Mechanical forces stimulate Golgi export.
  26. Golgi CATCHR complexes function as organizing hubs for vesicle tethering and fusion.
  27. PARP1 hyperactivity stalls translation and triggers stress granule formation following DNA damage.
  28. Molecular basis of ubiquitin-independent recognition and degradation of ODC/Antizyme by the 26S proteasome.
  29. HERC4 synergizes with USP16 to promote lung adenocarcinoma growth by augmenting protein translation via stabilizing RPS15.
  30. Translational Recoding in Tumors.
  31. A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells.
  32. K63-linked ubiquitin chains mark inactive Smoothened for packaging into ciliary extracellular vesicles.
  33. Nutrient stress activates RAB5B-mediated autophagy to remodel the synaptic proteome.
  34. Spatiotemporal regulation of DNA repair proteins between Golgi and nucleus maintains genome stability.
  35. Substrate biasing in UCHL5 proteoforms.
  36. DNA-Alkyne-Chain Probes Enable N-Glycosite-Resolved GlycoBarcoding of Cell-Surface Proteins.
  37. A Structure-guided Active Site Affinity Ligand Unmasks a Stress-Sensitizing Role for BLVRB in the Endoplasmic Reticulum.
  38. Computational mapping of productive POI-E3 ligase conformations to guide de novo degrader design: application to WEE1 and PKMYT1 PROTACs.
  39. Profiling Multiplexed Protein-Specific Sialylation in Cancer Drug Resistance Using Proximity Ligation Sequencing.
  40. Overlapping upstream ORFs repress translation and expand proteome diversity in Arabidopsis.
  41. CRISPR activation screen uncovers MARCKSL1 as a gauge for extracellular vesicle secretion.
  42. The glycolytic enzyme PGAM1 functions as a metabolic-autophagy checkpoint to coordinate growth and stress tolerance.
  43. Discovery of Noninhibitory Macrocyclic Ligands for Protein Tyrosine Phosphatase 1B Using a Function-Based, Iterative Screening Strategy.
  44. Inhibition of mitochondrial protein translation sensitizes tumor cells to death receptor 5 agonistic antibody.
  45. Non-Canonical Activation of HSF1 Stimulates mTORC1-Mediated Translation in HCMV-Infected Monocytes.
  46. E3 Ubiquitin Ligase Mindbomb1 Is a Novel Negative Regulator of Tumor-Suppressive BMP Signaling.
  47. A Stress-Adaptive Lipid Kinase Axis Defines Metabolic Vulnerabilities in Neuroendocrine Prostate Cancer.
  48. Leveraging Supramolecular Polymers to Induce the Targeted Protein Degradation of α-Synuclein.
  49. Evolution of a core ribosomal innovation in octopus.
  50. Proteostatic defect drives biophysical remodeling that triggers cell competition.
  51. Live Visualization of Endoplasmic Reticulum Redox Potential in Zebrafish Embryos Reveals Region-Specific Heterogeneity.
  52. A domain-swapped proPC1/3 structure reveals a conformational checkpoint for ER export.
  53. Faf2 is required for neural differentiation in embryonic neural progenitor cells.
  54. A de novo designed enzyme for photo-proximity labeling of E3 ligase neighborhoods in live cells.
  55. Selective and Potent First-in-Class CRBN-Dependent Molecular Glue Degraders of WW Domain-Binding Protein 4.
  56. COG7 links Golgi integrity to stress signaling and senescence.
  57. Toxoplasma TgATG2 works with TgATG9 and TgProp1 to drive autophagic flux for parasite extracellular survival and persistence.
  58. ER shaping proteins guide spindle elongation and division during rapid cleavage mitoses.
  59. Protein thiol alterations drive pathologic liquid-liquid phase separation in the aging brain.
  60. Structural basis for substrate recognition and inhibition of human glucose-6-phosphate transporter SLC37A4.
  61. The LRRK2 G2019S variant rewires Rab GTPase phosphorylation after lysosomal damage and promotes cell death in human macrophages.
  62. A machine learning model predicts protein stability of annotated and alternate protein isoforms.
  63. Altered neuronal start codon stringency favors cap-independent repeat-associated non-AUG translation.
  64. Intermolecular disulfide bond formation promotes Hsp42 higher-order assembly and shapes client selection in yeast.
  65. OTUD6A promotes de novo pyrimidine synthesis by deubiquitinating and stabilizing CAD in cervical cancer.
  66. Human GBP4 promotes TRIM21-BIP-dependent autophagy to restrict M. tuberculosis infection by preventing SORT1-mediated progranulin degradation.
  67. Inference of secreted protein signaling activities in intercellular communication.
  68. LRMDA promotes intestinal mucosal barrier integrity and limits colitis and colon cancer.
  69. An AI-enabled structural atlas decodes kinase specificity across the human proteome.
  70. GABPA recruits the Integrator endonuclease complex to promote transcription elongation.
  1. FEBS J. 2026 Jul 27.
    ER-localized translational control as a nexus for proteostasis.
    Hideki Nishitoh, Hisae Kadowaki.
      Maintenance of proteostasis is essential for cellular and organismal homeostasis, and disruption of protein quality control (QC) networks underlies numerous human diseases. The endoplasmic reticulum (ER) functions as a central organelle for the synthesis, folding, maturation, and trafficking of secretory and membrane proteins, and serves as a central hub of intracellular proteostasis. Recent studies have established that the ER membrane serves not only as a site of protein translocation but also as a dynamic platform integrating translational regulation, RNA surveillance, and multiple QC pathways. During ER-associated translation, cells continuously monitor ribosome dynamics, mRNA integrity, nascent-chain folding, and transmembrane protein insertion processes to prevent the accumulation of aberrant proteins. These surveillance systems include the PKR-like ER kinase (PERK)-mediated integrated stress response (ISR), regulated IRE1-dependent decay (RIDD), nonsense-mediated mRNA decay (NMD), RNA silencing, ribosome-associated QC (RQC), ubiquitin-fold modifier 1 conjugation (UFMylation), ER-phagy, and ER stress-induced pre-emptive QC (ERpQC). Although these pathways were originally characterized independently, increasing evidence indicates that they function cooperatively on or near the ER membrane to coordinate translational attenuation, mRNA degradation, ribosome recycling, nascent-chain elimination, and organelle remodeling. In particular, UFMylation has emerged as a central mechanism linking ER-associated RQC, translocation-associated QC (TAQC), and ER-phagy. Dysfunction of these ER-localized translational QC pathways contributes to neurodegeneration, inflammation, fibrosis, cancer, and aging-related disorders. In this review, we summarize recent advances in ER-localized translational control and discuss how integrated QC networks on the ER membrane maintain proteostasis and influence disease pathogenesis.
    Keywords:  ER stress; ER stress‐induced pre‐emptive quality control; UFMylation; endoplasmic reticulum; proteostasis; ribosome‐associated quality control; translational control
    DOI:  https://doi.org/10.1111/febs.70665
  2. Redox Biol. 2026 Jul 25. pii: S2213-2317(26)00323-X. [Epub ahead of print]96 104324
    DNAJB12/14 redox switching directs chaperone- and Bax/Bak-dependent ER protein reflux.
    Laila Abu Madegam, Noa Gavriel, Raifu Tolulope Adebisi, Aeid Igbaria.
      Maintenance of endoplasmic reticulum (ER) proteostasis is essential for cellular homeostasis and survival during stress. Beyond canonical quality control pathways, ER-to-cytosol signaling (ERCYS) enables the reflux of ER-resident proteins into the cytosol, where they can acquire noncanonical functions that promote cell survival. However, the mechanisms governing ERCYS and its relationship to ER stress remain poorly understood. Here, we show that ER protein reflux is restricted to a defined stress window and is governed by the ER redox environment. Mild ER stress maximizes protein reflux, whereas severe or reductive stress markedly suppresses this process. Mechanistically, we identify the ER-resident cochaperones DNAJB12 and DNAJB14 as redox-sensitive regulators of ERCYS. Under mild stress, intramolecular disulfide bonds stabilize DNAJB12 and DNAJB14, thereby supporting efficient protein reflux. In contrast, severe or reductive stress increases intracellular glutathione, reducing these disulfide bonds and promoting degradation of DNAJB12 and DNAJB14, resulting in the loss of chaperone-mediated reflux. We further show that protein reflux requires cysteine-dependent interactions between refluxed substrates and the cytosolic cochaperone SGTA, revealing a previously unrecognized redox-sensitive step in the ERCYS pathway. When ERCYS is impaired during severe ER stress, cells instead engage an alternative apoptosis-associated pathway mediated by BAX/BAK-dependent ER membrane permeabilization. This transition is driven by enhanced recruitment of BAX and BAK to the ER by the BH3-only protein BIK, amplifying apoptotic signaling. Together, these findings establish redox regulation as a molecular switch that determines whether cells mount an adaptive ER protein reflux response or commit to BAX/BAK-dependent ER membrane permeabilization and apoptosis.
    DOI:  https://doi.org/10.1016/j.redox.2026.104324
  3. Proc Natl Acad Sci U S A. 2026 Aug 04. 123(31): e2606606123
    Spatiotemporal control of Atg2 association with the ER during autophagosome formation.
    Tetsuya Kotani, Haruki Tanabe, Shinri Kitta, Momoko Higashi, Tatsuya Niwa, Tatsuya Kaminishi, Chika Kakuta, Junko Shimasaki, Hidetaka Kosako, Tamotsu Yoshimori, Akiko Kuma, Hitoshi Nakatogawa.
      Autophagy degrades various intracellular components by sequestering them within membrane vesicles called autophagosomes and delivering them to lysosomes or vacuoles. Previous studies have revealed that the conserved, bridge-like lipid transfer protein Atg2 tethers autophagosome precursors to the endoplasmic reticulum (ER) and mediates lipid supply from the ER to drive their expansion into autophagosomes. However, how Atg2 docks onto the ER has remained unclear. Here, we show in Saccharomyces cerevisiae that Atg2 interacts with the ER-resident VAP family protein Scs2. This interaction is mediated by a phospho-FFAT motif in Atg2 and the major sperm protein domain of Scs2 and enhanced by phosphorylation of the motif by the autophagy-initiating kinase Atg1, which is activated at the autophagosome formation site upon autophagy induction. This interaction cooperates with the N-terminal region of Atg2, which contains a weakly amphipathic helix, to mediate Atg2 association with the ER. Thus, the Atg2-Scs2 interaction functions as a spatiotemporal switch that controls Atg2-ER association. We also show that mammalian ATG2 interacts with the VAP-like proteins MOSPD1 and MOSPD3 to promote autophagosome formation. Collectively, this study reveals a conserved mechanism that initiates lipid transfer during autophagosome formation.
    Keywords:  Atg2; VAP protein; autophagy; lipid transfer protein; phospho-FFAT motif
    DOI:  https://doi.org/10.1073/pnas.2606606123
  4. J Am Chem Soc. 2026 Jul 29. 148(29): 30850-30860
    Two Mechanisms One Molecule: Developing a 'Truly' Bifunctional Degrader Targeting Rpn13 and CRBN.
    Cody A Loy, Shawn E Vinogradsky, Darci J Trader.
      Targeted protein degradation (TPD) has emerged as a powerful strategy to eliminate disease-relevant proteins, yet current approaches remain largely constrained to hijacking ubiquitin ligases. We previously introduced ByeTACs, bifunctional molecules that directly recruit proteins to the proteasome for E-ligase independent degradation. Here, we report "Truly" degraders, a new class of dual-mechanism molecules that combine a ligand for the proteasomal receptor Rpn13 with a ligand for cereblon (CRBN) to simultaneously engage both ubiquitin-independent and ubiquitin-dependent degradation pathways. Structure-guided design identified an optimal linker length that supports efficient substrate processing, with the PEG4 derivative (Truly-4) inducing robust depletion of both Rpn13 and CRBN in several cancer cell types. Remarkably, Truly-4 is the first noncovalent small molecule shown to degrade full-length Rpn13, a target previously approached using covalent or domain-restricted strategies. Mechanistic studies confirmed that degradation of Rpn13 proceeds via CRBN-dependent E3 ligase activity, whereas CRBN degradation occurs through an E-ligase independent process, consistent with a ByeTAC mechanism. Importantly, Truly-4 induces selective cytotoxicity in hematologic and solid cancer cell lines but not in healthy cells, despite comparable Rpn13 depletion, indicating that dual degradation can uncouple target engagement from toxicity. These findings establish a generalizable framework for engineering bifunctional degraders that program the proteasome to execute parallel degradation mechanisms and highlight proteasome receptors as druggable nodes for selective destruction of disease-relevant proteins.
    DOI:  https://doi.org/10.1021/jacs.6c02914
  5. J Cell Sci. 2026 Jul 15. pii: jcs264635. [Epub ahead of print]139(14):
    Vimentin intermediate filaments support autophagosome biogenesis at ER-endosome contact sites in response to starvation.
    Quentin Frenger, Dobrawa Amiri Czajkowski, Carla Alemany, Damiana Lecoeuche, Cédric Delevoye, Etienne Morel.
      Macroautophagy (autophagy) is a fundamental catabolic process requiring the biogenesis of the autophagosome to support cell survival during stress. Although the roles of F-actin and microtubule cytoskeleton in autophagy are well established, the contribution of intermediate filaments (IFs) remains poorly understood. Here, we investigated the role of the type III IF vimentin in supporting the early steps of starvation-induced autophagy. We demonstrate that starvation triggers a rapid, perinuclear compaction of vimentin IFs, which showed enhanced overlap with the endoplasmic reticulum (ER) and correlated with transient vimentin phosphorylation at serine 56. We reveal that autophagic proteins accumulate at the vimentin-ER interface, physically connecting the autophagosome biogenesis machinery to the vimentin IF network. Knockout or pharmacological perturbation of vimentin-IFs dynamics using withaferin-A significantly impairs starvation-induced autophagic flux. Mechanistically, we reveal that vimentin IFs are essential coordinators for the mobilization of endosome-ER membrane contact sites (EERCS), a crucial hub for autophagosome nucleation. Together, our findings uncover a novel role for vimentin IFs as a dynamic cytoskeletal coordinator that spatially organizes membrane contact sites to promote the efficient initiation of autophagosome biogenesis in response to nutrient stress.
    Keywords:  Autophagosome; Autophagy; Endoplasmic reticulum; Intermediate filaments; Membrane contact sites; Vimentin
    DOI:  https://doi.org/10.1242/jcs.264635
  6. Adv Sci (Weinh). 2026 Jul 30. e76972
    ATL2 Recruits TRAK1 to Promote Mitochondrial Transport at ER-Mitochondria Contact Sites.
    Yiru Cheng, Peiyuan Chai, Xiayuhe Pei, Yiwen Chen, Xiaoshuai Huang, Bei Liu, Yiqian Wu, Junlin Teng, Pengli Zheng, Jianguo Chen.
      Mitochondrial transport and distribution are crucial for cellular homeostasis, yet whether and how they are regulated by endoplasmic reticulum (ER)-mitochondria contact sites remains unclear. Here, we demonstrate that the ER protein atlastin-2 (ATL2) orchestrates mitochondrial transport and distribution by promoting assembly of the transport machinery at ER-mitochondria contact sites. Mechanistically, ATL2 recruits the adaptor trafficking kinesin-binding protein 1 (TRAK1) to the ER membrane, strengthening the interaction of TRAK1 with the mitochondrial transport adaptor MIRO1 to promote anterograde mitochondrial transport. Loss of ATL2 disrupts this process, leading to perinuclear mitochondrial clustering. We further find that ATL2 stabilizes ER-mitochondria contact sites by interacting with MFN2, providing a platform for mitochondrial transport complex assembly. Moreover, in hypoxia, ATL2 is ubiquitinated at lysine 567 by the E3 ligase SYVN1, leading to its degradation and a resulting defect in mitochondrial distribution. Our findings elucidate a novel ER-mediated mechanism for mitochondrial transport.
    Keywords:  ATL2; ER–mitochondria contact sites; TRAK1; hypoxia; mitochondrial transport
    DOI:  https://doi.org/10.1002/advs.76972
  7. bioRxiv. 2026 Jul 29. pii: 2026.07.19.739412. [Epub ahead of print]
    Identifying endogenous substrates of the 26S proteasome through site-specific photocrosslinking.
    Santiago Yori Restrepo, Andreas Martin.
      The 26S proteasome is the hub for regulated protein turnover in eukaryotic cells. Degradation of proteins by the Ubiquitin-Proteasome System plays critical roles in every aspect of cell biology, such as the regulation of gene transcription, the quality control of translation and protein folding, and protein transport across membranes. While mRNA levels and protein abundances can be readily measured with a robust set of established tools, only a few methodologies exist to identify proteins that are degraded by the proteasome rather than the lysosome as the second major pathway for turnover. Here, we sought to address this by using genetic code expansion to introduce a photo-crosslinkable unnatural amino acid into the yeast 26S proteasome and capture cellular protein substrates as they translocate through the proteasomal ATPase motor. In vitro biochemical experiments confirmed that these modified proteasomes are functional, which allowed us to introduce them into live yeast cells for in vivo crosslinking and the identification of enriched ATP-dependent substrates by mass spectrometry. These experiments revealed a very diverse pool of proteasomal substrates that markedly changed upon cell exposure to endoplasmic reticulum stress. Together, our results represent an exciting avenue for probing the landscape of proteasomal substrates and its changes in response to various cellular conditions and stresses.
    DOI:  https://doi.org/10.64898/2026.07.19.739412
  8. J Cell Sci. 2026 Jul 27. pii: jcs.264719. [Epub ahead of print]
    A Golgi-bypass secretion mechanism for proTGFα involving TMED9 and GRASP65.
    Susanne S Steigleder, Isabel Döring, Marina Tauber, Julian Nüchel, Marius K Lemberg.
      We recently demonstrated that the endoplasmic reticulum export of proTGFα is tightly gated and that proteostasis factors, including the intramembrane protease RHBDL4, can promote its release by an unknown mechanism. While investigating a potential link between RHBDL4-regulated p24/TMED family proteins and proTGFα trafficking, we uncovered an RHBDL4-independent pathway in which the cargo receptor TMED9 induces unconventional protein secretion (UcPS) of ectopically expressed proTGFα. This pathway selectively requires GRASP65, but not the closely related GRASP55, and depends on components of the autophagic machinery and the ESCRT-associated protein ALIX, consistent with a Golgi-bypass secretion route. TMED9- and GRASP65-induced proTGFα secretion is insensitive to Brefeldin A treatment and involves trafficking through pre-Golgi compartments. Although largely based on overexpression systems, our findings identify a previously unrecognized UcPS mechanism and provide insight into alternative protein trafficking pathways that may be exploited under conditions of proteostasis stress and in pathological contexts such as cancer and inflammation. At present, our data establish the existence of a TMED9/GRASP65-dependent Golgi-bypass trafficking route under conditions of ectopic cargo expression, whereas its endogenous physiological relevance remains to be established.
    Keywords:  GRASP65; Golgi-bypass trafficking; Proteostasis; Secretory autophagy; TMED9; Unconventional protein secretion (UcPS)
    DOI:  https://doi.org/10.1242/jcs.264719
  9. bioRxiv. 2026 Jul 22. pii: 2026.07.15.738801. [Epub ahead of print]
    APOE4 disrupts the central dogma by arresting neuronal proteome dynamics.
    Einar K Krogsaeter, Justin McKetney, Lishi Li, Isabelle Liu, Alicia L Richards, Vishvak Subramanyam, Keyi Yin, Martin Gordon, Pedro Belio Mairal, Erica J Stevenson, Houzhen Qian, Yadong Huang, Hani Goodarzi, Nevan J Krogan, Danielle L Swaney.
      Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease and promotes neuronal dysfunction through incompletely understood mechanisms. Here, we integrated transcriptomic, translatomic, and proteomic profiling of isogenic APOE3 and APOE4 human iPSC-derived neurons and found that APOE4 fundamentally impairs neuronal proteome renewal. Although transcriptional changes were modest, APOE4 disrupted ribosome occupancy, altered translational dynamics, and uncoupled protein abundance from transcript levels. Proteome-wide turnover measurements revealed a global extension of protein half-lives and widespread accumulation of long-lived proteins. Functional proteomic analyses demonstrated concurrent lysosomal and proteasomal impairments associated with reduced proteasome activity and increased association of APOE with neuronal proteasomes. Longitudinal proteomics further showed that protein accumulation emerges during neuronal maturation and precedes a senescence-like cellular stress state. Together, these findings identify impaired proteome renewal as a central mechanism underlying neuronal vulnerability to APOE4 and establish defective proteostasis as an early pathogenic event in Alzheimer's disease.
    DOI:  https://doi.org/10.64898/2026.07.15.738801
  10. Cell Rep. 2026 Jul 27. pii: S2211-1247(26)00804-1. [Epub ahead of print]45(8): 117726
    The Drosophila CEBPG homolog Irbp18 partners with crc (ATF4) to mediate the integrated stress response in degenerative disease models.
    Sahana Mitra, Huai-Wei Huang, Rhihab Faruk, Arissa Low, Adam I Yeo, Hyung Don Ryoo.
      The integrated stress response (ISR) coordinates cellular adaptation to diverse stress conditions. In Drosophila, two bZIP transcription factors, Xrp1 and crc (ATF4 homolog), are induced during ISR. Crc protein can dimerize with two CEBP factors in vitro, but the in vivo relevance of those interactions remained unknown. Here, we report that the CEBPG homolog, Irbp18, is an essential partner of crc during ISR. Specifically, Irbp18 is broadly required for the transcriptional induction of ISR target genes in the photoreceptors of ninaEG69D, a Drosophila model of retinitis pigmentosa. Moreover, CUT&RUN analysis indicates that Irbp18 loss reduces or abolishes crc binding to target DNAs in photoreceptors and impairs crc's ability to induce target transcripts upon overexpression. Functionally, Irbp18 loss causes retinal degeneration and suppresses ISR signaling in parkin mutants, a model of Parkinson's disease. Together, these findings identify Irbp18 as a cofactor for crc, impacting pathological outcomes in Drosophila models of degeneration.
    Keywords:  ATF4; CEBP; CP: molecular biology; CP: neuroscience; ISR; Irbp18; bZIP; dimerization; integrated stress response; parkin; retinal degeneration; transcription factor
    DOI:  https://doi.org/10.1016/j.celrep.2026.117726
  11. Biomolecules. 2026 Jun 30. pii: 966. [Epub ahead of print]16(7):
    Hexosamine Pathway Disruption by GFPT1 Loss Drives Coordinated Defects in Glycosylation, Autophagy, and Trafficking.
    Stephen H Holland, Ricardo Carmona-Martinez, Andreas Hentschel, Alexa Derksen, Kaela O'Connor, Daniel O'Neil, Kelly Ho, Stephen D Baird, Andreas Roos, Sally Spendiff, Hanns Lochmüller.
      Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT1), the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP), provides the UDP-N-acetylglucosamine (UDP-GlcNAc) required for protein glycosylation. Biallelic mutations in GFPT1 cause congenital myasthenic syndromes (GFPT1-CMS), yet the molecular mechanisms linking impaired glycosylation to skeletal muscle dysfunction remain incompletely understood. Here, we combine cellular models of inducible Gfpt1 knockdown and a skeletal muscle-specific Gfpt1 knockout mouse (Gfpt1Tm1d/Tm1d) with whole-cell proteomics, immunoblot studies and secretomics to define glycosylation-dependent defects in intracellular trafficking, ER stress signaling and autophagy. Global proteomic profiling of Gfpt1-deficient myoblasts revealed marked downregulation of protein trafficking pathways and impaired secretion of key muscle cargo proteins, including serglycin (Srgn). Loss of GFPT1 reduced both high-molecular-weight glycosylated serglycin and its core protein, accompanied by intracellular retention and decreased secretion. These trafficking defects coincide with robust activation of the unfolded protein response (UPR), evidenced by increased Xbp1 expression and accumulation of spliced Xbp1s across pharmacologic, cellular, and mouse models of GFPT1 deficiency. Converging evidence from proteomics, immunoblotting, and immunofluorescence demonstrated impaired autophagy, including increased LC3-II accumulation, elevated p62/Sqstm1 levels, and enhanced p62-positive puncta in both Gfpt1-deficient C2C12 myoblasts and skeletal muscle. Soluble/insoluble fractionation further confirmed p62 accumulation, indicating defective autophagic flux and buildup of aggregated cargo. Together, these findings identify a glycosylation-dependent failure in protein trafficking that triggers ER stress, UPR activation, and autophagy impairment in Gfpt1-deficient skeletal muscle. This mechanistic cascade provides a unifying explanation for muscle pathology in GFPT1-CMS and suggests that restoring glycosylation or improving proteostasis may represent viable therapeutic approaches.
    Keywords:  GFPT1; autophagy; congenital myasthenic syndrome; glycosylation; trafficking
    DOI:  https://doi.org/10.3390/biom16070966
  12. bioRxiv. 2026 Jul 14. pii: 2026.07.12.738044. [Epub ahead of print]
    Live-cell co-translational folding tracking reveals bidirectional coupling between translation and folding.
    Rhiannon M Sears, Luis U Aguilera, Tristan M Bunting, Ning Zhao.
      Translation elongation and protein folding have long been proposed to coordinate during co-translational folding, yet the lack of technologies capable of simultaneously tracking both processes in live cells has hindered mechanistic understanding of this relationship. Here, we developed co-translational folding tracking (coTFT), a live-cell imaging platform that directly and simultaneously tracks translation and folding from individual mRNAs. Using reporters with distinct folding kinetics, we found that differences in folding kinetics were accompanied by corresponding changes in translation elongation rates. Conversely, altering translation elongation markedly affected protein folding outcomes. Combining coTFT with mathematical modeling enabled estimation of reporter folding times on translating ribosomes in live cells, confirming their distinct folding kinetics. Together, our results reveal that translation elongation and folding are bidirectionally coupled during co-translational folding.
    DOI:  https://doi.org/10.64898/2026.07.12.738044
  13. Genetics. 2026 Aug 01. pii: iyag200. [Epub ahead of print]
    Ribosome-associated chaperones integrate co-translational mitochondrial protein import, stress signaling and proteostasis in Saccharomyces cerevisiae.
    J Ernesto Bravo-Arévalo, Ariann E Mendoza-Martínez, Teresa Ballado, Ericka Moreno-Méndez, Alexander DeLuna, Soledad Funes.
      Mitochondrial biogenesis requires the coordinated synthesis, targeting, and import of nuclear-encoded mitochondrial precursor proteins. Although ribosome-associated chaperones support co-translational protein folding, their genetic contributions to mitochondrial protein import and cellular homeostasis remain incompletely defined. Here, we investigate the roles of the nascent polypeptide-associated complex (NAC) and the ribosome-associated Hsp70 system Ssb1/2 in Saccharomyces cerevisiae. We show that NAC and Ssb1/2 have distinct yet partially overlapping functions in the handling of mitochondrial precursor proteins. Loss of NAC activates the mitochondrial retrograde pathway and enhances growth on ethanol as a non-fermentable carbon source without compromising respiratory competence, indicating metabolic adaptation rather than overt mitochondrial dysfunction. In contrast, Ssb1/2 deficiency disrupts cytosolic proteostasis, sensitizes cells to TORC1 inhibition, and impairs autophagy and mitophagy. Using a TEV protease-based import reporter, we show that Ssb1/2 promotes efficient co-translational distribution of precursor proteins, whereas NAC limits the accumulation of misfolded proteins at the mitochondrial surface. Biochemical analyses further reveal that Ssb1/2 supports the association of translating cytosolic ribosomes with the mitochondrial outer membrane, while NAC loss partially restores this interaction in the absence of Ssb1/2. Together, these findings establish NAC and Ssb1/2 as key components of an integrated network linking co-translational targeting, mitochondrial signaling, and cellular homeostasis.
    Keywords:   Saccharomyces cerevisiae ; Ribosome-associated chaperones; TORC1 signaling; co-translational targeting; mitochondrial protein import; proteostasis; retrograde signaling
    DOI:  https://doi.org/10.1093/genetics/iyag200
  14. Sci Adv. 2026 Jul 31. 12(31): eaec6649
    Cellular stress triggers degradation and global redistribution of RNAPII.
    Hansong Qi, Kangning Chen, Ningyawen Liu, Mengqiu Yin, Yongfang Chen, Xiaoli Ren, Ke Yang, Guijun Chen, Fan Lai, Xinglou Yang, Jumin Zhou.
      Transcription is essential for cellular stress response. However, how RNAPII respond to and are regulated during stress are poorly understood. We show that RNAPII is degraded during many types of cellular stresses. In osmotic stressed cells, the TNFα-p38 pathway was activated and promoted the neddylation of the CUL1 E3 ligase complex, which interacted with RPB1 through FBXO11 to ubiquitylate and degrade RNAPII. This caused genome wide RNAPII binding reduction, but prevented RNAPII binding loss from genes with low promoter GC content. This redistribution protected the RNAPII loss from stress response genes in the cell adhesion, MAPK and GPCR pathways. RNAPII redistribution is vital for cell survival, as degradation blockage resulted in the loss of RNAPII from low GC promoters and compromised stress response from disrupted cell adhesion to increased apoptosis. Thus, rapid RNAPII degradation and RNAPII redistribution are components of the cellular stress response to benefit cell survival.
    DOI:  https://doi.org/10.1126/sciadv.aec6649
  15. FEBS J. 2026 Jul 29.
    Functional and transcriptomic analyses in Neurospora crassa reveal a crucial role for the N-glycoprotein deglycosylation process in fungal homeostasis.
    Anastasios Samaras, Tanim Jabid Hossain, Magnus Karlsson, Georgios Tzelepis.
      N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, efficient endoplasmic reticulum (ER) quality control, such as that provided by the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-β-n-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the cytosolic ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-β-n-acetylhexosaminidase under ER stress. These findings identify the cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.
    Keywords:  ER stress; GH18; N‐glycans; de‐n‐glycosylation; endo‐β‐n‐acetyloglucosaminidases
    DOI:  https://doi.org/10.1111/febs.70666
  16. bioRxiv. 2026 Jul 22. pii: 2026.07.21.739814. [Epub ahead of print]
    Quantitative profiling of basal and stress-induced ribosome collisions.
    James Marks, Pedro H Ayres-Galhardo, Bavavarshini Tamilselvam, Alexis Roberson, Ruby Pitts, Nicholas Guydosh, Sezen Meydan.
      Ribosomes stall when they encounter problematic codons or cellular stress that perturbs translation. Stalled ribosomes can lead to the formation of ribosome collisions, also known as disomes, that engage cellular surveillance and stress signaling pathways. How many disomes form during basal conditions and how disome levels change under stress remain poorly understood. Here, we used spike-in normalized Ribo-seq and Disome-seq to quantify transcriptome-wide disome levels. Applying this approach in yeast and human cells, we found that disomes comprise approximately 2-10% of translating ribosomes under basal conditions. A high-resolution Disome-seq experiment in human cells identified reproducible disome-forming sites that contribute to the basal level of disome formation in the cell. Exposure of yeast cells to methyl methanesulfonate and human cells to anisomycin increased disome abundance up to four-fold and changed the distribution of collisions in a stress-specific and context-dependent manner. Overall, these data provide a quantitative, transcriptome-wide framework for measuring disome levels and reveal how translational stress reshapes the landscape of ribosome collisions in cells.
    DOI:  https://doi.org/10.64898/2026.07.21.739814
  17. Res Sq. 2026 Jul 22. pii: rs.3.rs-8117355. [Epub ahead of print]
    EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression.
    Guan-Yu Xiao, Kevin Fulp, Oluwafunminiyi Obaleye, Shike Wang, Xin Liu, Jiang Yu, Jonathan M Kurie, Jun Xu, William Russell, Xiaochao Tan.
      Membrane trafficking governs the transport of molecules to both intracellular and extracellular locations, thereby maintaining cell homeostasis. During cancer progression, alterations in membrane trafficking are frequently observed. However, the mechanisms underlying the dysregulation of membrane trafficking in cancer progression remain largely unresolved. Recent evidence has demonstrated that epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma (LUAD) employs a membrane trafficking program to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME). To further dissect the pro-tumorigenic membrane trafficking program, here we conducted a CRISPR interference (CRISPRi) in vivo screen for membrane trafficking regulators in a syngeneic mouse model with a complete immune system. This screen identified REEP2, an endoplasmic reticulum (ER) shaping protein, as a novel regulator of the EMT-dependent membrane trafficking program, which is associated with a poor prognosis in LUAD patients and is required for LUAD metastasis in a syngeneic orthotopic LUAD mouse model. Mechanistically, the EMT activator ZEB1 upregulates REEP2 expression through miR-183- and miR-193a-mediated regulation that promotes the transportation of secretory cargoes from the ER exit site (ERES) to the Golgi, thereby augmenting the secretion of pro-tumorigenic factors. The REEP2-driven secretion promotes cancer cell proliferation, migration, and the infiltration of myeloid-derived suppressor cells (MDSCs) in the TME. These findings identify REEP2 as a critical mediator of the EMT-driven pro-metastatic membrane trafficking program, revealing a specific vulnerability in mesenchymal LUAD.
    DOI:  https://doi.org/10.21203/rs.3.rs-8117355/v1
  18. Sci Adv. 2026 Jul 31. 12(31): eaeg5464
    Direct single-molecule visualization of Hsp90-mediated relief of an Hsp70-folding block.
    Nicholas R Marzano, Bailey Skewes, Shannon McMahon, Lauren Rice, Dezerae Cox, Antoine M van Oijen, Heath Ecroyd.
      Heat shock protein 70 (Hsp70) and Hsp90 are essential molecular chaperones that cooperate to fold diverse client proteins, yet how their activities are coordinated to remodel clients remains unclear. To address this, we used a combination of single-molecule fluorescence resonance energy transfer and total internal reflection fluorescence microscopy to observe individual firefly luciferase proteins during sequential engagement with Escherichia coli Hsp70 (DnaK) and Hsp90 (HtpG). We show that HtpG reduces rebinding of DnaK to folding intermediates while still allowing engagement with misfolded clients, enabling productive refolding in the presence of typically inhibitory concentrations of DnaK. HtpG couples adenosine 5'-triphosphate binding and hydrolysis to promote progressive folding through localized compaction across multiple regions of the client, reducing misfolding and establishing native interdomain contacts. Kinetic simulations support a model whereby heterogeneous DnaK binding generates region-specific folding kinetics and conformational dynamics. This enables efficient subdomain folding by DnaK/HtpG and suggests that the number and position of DnaK binding sites on clients provide a mechanism by which proteins can harness chaperone promiscuity for optimal folding.
    DOI:  https://doi.org/10.1126/sciadv.aeg5464
  19. PLoS Comput Biol. 2026 Jul 31. 22(7): e1014588
    Prolines in linkers of multi-domain proteins: Intrinsic regulators of misfolding and aggregation.
    Simona Manasra, Yaakov Levy.
      Proline is a chemically unique amino acid that influences protein structure and slows peptide-bond formation during translation. While its enrichment in intrinsically disordered regions is well known, the functional importance of its organization along protein sequences remains unclear. Here, we performed a proteome-wide analysis of inter-domain linkers in human proteins to investigate how proline organization relates to domain architecture and folding demands. We find that clustered and consecutive prolines are enriched in linkers following topologically complex domains that require the formation of long-range contacts. This suggests that proline clusters may act as intrinsic sequence-encoded pauses during translation, helping coordinate co-translational folding. We further show that linkers with enriched proline clusters frequently flank aggregation-prone domains and that proteins containing such linkers tend to have longer cellular half-lives. Together, our results demonstrate that the organization of proline residues, beyond their overall abundance, is an important determinant of protein folding, aggregation, and stability, revealing a general mechanism by which amino acid sequences regulate protein behavior.
    DOI:  https://doi.org/10.1371/journal.pcbi.1014588
  20. bioRxiv. 2026 Jul 20. pii: 2026.07.15.738672. [Epub ahead of print]
    Autophagy prevents ER stress-induced Tight Junction barrier disruption via claudin-2 homeostasis.
    Priya Arumugam, Kushal Saha, Ashwinkumar Subramenium Ganapathy, Alexandra Wang, Leonard Harris, Gregory Yochum, Prashant Nighot.
      Defective intestinal epithelial tight junction (TJ) barrier function and endoplasmic reticulum (ER) stress are central pathological features of inflammatory bowel disease (IBD), yet the molecular mechanisms ER stress to TJ disruption remains poorly understood. Here, we investigated the role of autophagy in regulating intestinal TJ homeostasis during ER stress. ER stress was elevated in inflamed Crohn's disease tissue and chronic dextran sulfate sodium (DSS) colitis. In human intestinal epithelial Caco-2 monolayers, murine colon, and human colonic explants, induction of ER stress with tunicamycin, thapsigargin, or brefeldin A disrupted TJ barrier integrity, as demonstrated by reduced transepithelial electrical resistance and increased paracellular permeability. ER stress selectively increased the pore-forming TJ protein claudin-2 and altered occludin localization without significantly affecting other claudins. Pharmacologic activation of autophagy with rapamycin attenuated ER stress, restored TJ barrier function, reduced claudin-2 accumulation, and preserved occludin localization. Conversely, CRISPR-Cas9-mediated deletion of autophagy gene ATG7 exacerbated ER stress, apoptosis, and TJ barrier dysfunction in vitro, while intestinal epithelial-specific Atg7 knockout mice exhibited enhanced ER stress-induced intestinal permeability in-vivo. Mechanistically, prolonged ER stress impaired autophagic flux through IRE1α kinase signaling, resulting in accumulation of p62 and claudin-2. Inhibition of IRE1α kinase activity restored autophagy, reduced claudin-2 levels, and preserved TJ barrier function. We further identified adaptor-associated kinase 1 (AAK1) as a downstream mediator of IRE1α signaling during ER stress, with increased AP2M1 phosphorylation and altered claudin-2 trafficking. Claudin-2 overexpression alone induced ER stress and lysosomal damage, suggesting a feed-forward mechanism amplifying epithelial injury. Finally, enteric rapamycin administration reduced ER stress and restored autophagy in murine DSS colitis. Collectively, these findings identify an IRE1α-AAK1-autophagy axis as a critical regulator of intestinal TJ barrier integrity during ER stress.
    DOI:  https://doi.org/10.64898/2026.07.15.738672
  21. bioRxiv. 2026 Jul 13. pii: 2026.07.11.737968. [Epub ahead of print]
    Retrograde trafficking inhibitors allosterically trap Get3 to block tail-anchored protein biogenesis.
    Juliet A Lee, Xilin Gu, Charlene Chan, Vida Storm Robertson, Victor Garcia-Ruiz, Yancheng E Li, Anna Huyen Ngo, Philip Alabi, Vladimir Denic, Jason K Sello, William M Clemons.
      Retro-1 and Retro-2 are structurally distinct small molecules that protect cells from diverse toxins and viruses by disrupting retrograde trafficking, yet their mechanism of action has remained elusive. We show that both compounds target Get3, the ATPase chaperone of the guided entry of tail-anchored proteins (GET) pathway, which mediates biogenesis of tail-anchored SNARE proteins required for retrograde transport to the ER membrane. Cryo-electron microscopy reveals that Retro compounds bind a cryptic pocket in Get3, allosterically stabilizing Get3 in a stalled complex with upstream pathway components. Our work uncovers the GET pathway as an unsuspected vulnerability in pathogen entry, provides clear routes toward compound optimization, and establishes stabilization of dynamic protein complexes as a therapeutic strategy.
    DOI:  https://doi.org/10.64898/2026.07.11.737968
  22. Autophagy. 2026 Jul 31. 1-18
    Trr1 (thioredoxin reductase 1) modulates ERphagy in aging via liquid-liquid phase separation.
    Yuan Gao, Jingyi Zhao, Xiangdong Hu, Per Sunnerhagen, Beidong Liu.
      Aging is associated with the deterioration of various biological processes including disrupted proteostasis and impaired macroautophagy/autophagy. Biomolecules can undergo liquid-liquid phase separation (LLPS) to form biomolecular condensates that exert specific biological functions. Trr1 (thioredoxin reductase 1) is a pivotal enzyme in the thioredoxin antioxidant system. Deletion of TRR1 results in impaired autophagy; however, the underlying mechanism is largely unexplored. In this study, we explored whether LLPS of Trr1 affected autophagy. Trr1 formed dynamic LLPS condensates during replicative aging in yeast. Phase separation of Trr1 occurred in response to endoplasmic reticulum (ER) stress generated by cellular aging, rather than to oxidative stress. Furthermore, Trr1 condensates participated at the phagophore assembly site during endoplasmic reticulophagy and promoted autophagosome development by affecting lipidation of the Atg8 protein. Additionally, maintaining the liquid-like dynamic nature of Trr1 condensates was essential for cellular fitness. Our findings revealed an unconventional role of Trr1 through LLPS in aging. The function of phase-separated condensates of Trr1 in mitigating aging-associated ER stress offers insights into the mechanisms underlying healthy cellular aging. These findings highlight a potential target for developing interventions to combat aging and associated diseases.Abbreviations: Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum; ERAD: endoplasmic reticulum-associated degradation; ERphagy: endoplasmic reticulophagy; FRAP: fluorescence recovery after photobleaching; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; PAS: phagophore assembly site; PLDs: prion-like domains; RFP: red fluorescent protein; RLS: replicative lifespan; Trr1: thioredoxin reductase 1; Trx: thioredoxin; UPR: unfolded protein response; IDRs: intrinsically disordered regions.
    Keywords:  Aging; ERphagy; endoplasmic reticulum stress; liquid-liquid phase separation; thioredoxin reductase 1
    DOI:  https://doi.org/10.1080/15548627.2026.2702873
  23. Trends Biochem Sci. 2026 Jul 31. pii: S0968-0004(26)00212-4. [Epub ahead of print]
    Linkerology in PROTACs: learnings for proximity-inducing therapeutics.
    Rubén Prieto-Díaz, Aina Urbina, María Rodríguez-Ríos, Alessio Ciulli.
      Targeted protein degradation has rapidly evolved from a chemical biology concept into a therapeutic modality, with the first proteolysis-targeting chimera (PROTAC) degrader now approved as a medicine. Linkers play a central role in governing ternary complex formation and conferring drug-like properties on bifunctional compounds. However, linker optimisation remains one of the least rationalised steps in degrader design. In this review, we introduce a linkerology framework that integrates a data-driven analysis of PROTAC linker chemotypes with historical context, representative case studies, emerging proximity-based modalities, and clinical-stage trends. We reveal persistent biases in the explored linker space and identify linker features that are preferentially retained in clinical-stage degraders. Together, these insights position linkerology as a central discipline in PROTAC design and provide guidance for the development of next-generation proximity-based therapeutics.
    Keywords:  PROTACs; TPD; bifunctional molecules; induced proximity; linkers; proximity-based therapeutics
    DOI:  https://doi.org/10.1016/j.tibs.2026.07.004
  24. J Am Chem Soc. 2026 Jul 29. 148(29): 30697-30701
    Multidomain Relaxation Dispersion NMR Resolves an Intermediate-Gated Binding Pathway for Selective Recognition of Linear Diubiquitin by HOIL-1L.
    Erik Walinda, Naoki Ishii, Daichi Morimoto, Kenji Sugase.
      Molecular recognition is governed not only by the structural complementarity of the final complex, but by the kinetic pathway through which it forms. Polyubiquitin chains, in which sequence-identical domains are covalently linked yet must be discriminated by dedicated receptors to encode distinct cellular signals, exemplify this challenge. For NMR relaxation dispersion studies of such systems, spectral overlap between identical domains prevents conventional uniform isotope labeling from resolving the per-domain exchange contributions. Here we show, using domain-selective 15N labeling combined with R2 relaxation dispersion, that the binding of linear (Met1-linked) diubiquitin to HOIL-1L NZF proceeds through an apparent three-state pathway as detected by relaxation dispersion, in which rapid ligand-dependent pre-equilibration populates a conformationally preorganized intermediate that is selectively captured by NZF in a slower, chain-type-selective step. This kinetic hierarchy offers a mechanistic basis for understanding the discrimination between linear and Lys63-linked ubiquitin chains that static structures alone cannot reveal. This strategy should be broadly applicable to multidomain recognition systems─including epigenetic reader complexes and multivalent signaling adaptors─where the pathway between known structural end points remains hidden.
    DOI:  https://doi.org/10.1021/jacs.6c09447
  25. J Cell Biol. 2026 Oct 05. pii: e202510026. [Epub ahead of print]225(10):
    Mechanical forces stimulate Golgi export.
    Chandini Bhaskar Naidu, Javier Vera Lillo, Eugenia Almacellas, Anabel-Lise Le Roux, Sabine Bardin, Nicolas Mateos, Jessica Angulo-Capel, Adam Wolowczyk, Yugo Terashima, Yuichi Wakana, Pere Roca-Cusachs, Maria F Garcia-Parajo, Franck Perez, Bruno Goud, Jean-Baptiste Manneville, Stéphanie Miserey, Felix Campelo.
      Cells face diverse mechanical stimuli that vary with cell type, state, and pathological conditions. Mechanobiology investigates how cells sense and respond to these forces. While most work has focused on the cell surface and nucleus as primary mechanosensors, how intracellular organelles adapt to extracellular mechanical forces remains largely unknown. Here, we show that extracellular mechanical signals influence the secretory function of the Golgi apparatus. By subjecting adherent cells to mechanical challenges-cell spreading on different ligands, altered substrate stiffness, or equibiaxial strain-we reveal that extracellular forces modulate Golgi-to-cell surface carrier biogenesis, thereby regulating exocytosis. Together with changes in Golgi membrane tension, we identify molecular determinants of the mechanotransduction pathway, including microtubule acetylation, diacylglycerol production, and protein kinase D activity. In turn, inhibition of Golgi export suppresses this mechanoresponse and causes impaired cell spreading. These findings uncover a bidirectional mechanotransduction axis in which extracellular mechanics tune Golgi secretory output, providing a framework for investigating organelle-based mechanoadaptation in physiology and disease.
    DOI:  https://doi.org/10.1083/jcb.202510026
  26. bioRxiv. 2026 Jul 14. pii: 2026.06.16.732723. [Epub ahead of print]
    Golgi CATCHR complexes function as organizing hubs for vesicle tethering and fusion.
    Walter S Aragon-Ramirez, Amrita Khakurel, Irina Pokrovskaya, Farhana Taher Sumya, Vladimir V Lupashin.
      Approximately one-third of all human proteins transit through the secretory pathway, where the Golgi apparatus orchestrates protein modification, sorting, and distribution through highly selective vesicle budding and fusion events. Central to these processes are the Complexes Associated with Tethering Containing Helical Rods (CATCHR), multisubunit tethering complexes that coordinate vesicle docking and fusion through interactions with coiled-coil tethers (CCTs), Rab GTPases, SNAREs, and Sec1/Munc18 (SM) proteins and other trafficking factors. To define the molecular organization of Golgi CATCHR complexes, we generated the first comprehensive proximity-interaction map of the COG, GARP, and EARP tethering complexes using functional, near-endogenously expressed TurboID-tagged subunits. Comparative proximity proteomics revealed that each CATCHR complex assembles a distinct trafficking module composed of characteristic CCTs, Rab-associated proteins, SNAREs, and SM proteins, establishing a system-level framework for the spatial organization of Golgi and endosomal membrane trafficking. The COG complex preferentially associated with Golgi CCTs and the STX5-SCFD1 fusion machinery, GARP with CCDC186, and STX16-VPS45 pathway, and EARP with GRIPAP1, the VPS33B-VIPAS39 (CHEVI) complex, and RAB11-dependent recycling machinery. Beyond validating known interactions, our study identifies CCDC186 as a vesicle tether, establishes WWOX as a previously unrecognized regulator of Golgi homeostasis and glycosylation, and provides evidence that Golgi CATCHR complexes function as central organizing hubs that assemble specialized trafficking modules to coordinate vesicle tethering and membrane fusion.
    DOI:  https://doi.org/10.64898/2026.06.16.732723
  27. J Cell Biol. 2026 Aug 03. pii: e202409149. [Epub ahead of print]225(8):
    PARP1 hyperactivity stalls translation and triggers stress granule formation following DNA damage.
    Maria Tereshchenko, Rebecca Earnshaw, Christopher Chin Sang, Minjung Won, Jeanine L Van Nostrand, Ryan C Russell, Hyun O Lee.
      Cells respond to various stressors by inhibiting global translation and forming stress granules (SGs), cytoplasmic organelles enriched in certain RNA-binding proteins, and RNA. Genotoxic stress also induces SG assembly, but it is unclear how nuclear stress signals are transmitted to trigger cytoplasmic responses. We show that DNA-damaging agents that activate a nuclear poly(ADP-ribose) polymerase (PARP), PARP1, stall translation and induce SGs. We find that PARP1 activation depletes NAD+, which depletes cellular ATP, activating ATP-sensor AMPK and inhibiting mTORC1 via Raptor phosphorylation. Subsequent hypophosphorylation of 4EBP1 inhibits translation. These effects are suppressed by PAR-metabolism regulators, XRCC1, PARG, and Nudix5, and reversed by NAD+ precursor supplementation. Cells lacking SG scaffolds, G3BP1 and G3BP2, show reduced viability after genotoxic stress, which is rescued by G3BP1 overexpression. These findings link PARP1 activity to translational control and SG formation, which may protect against cell death following DNA damage. These mechanisms provide insight into PARP1- and stress granule-associated diseases, including cancer and neurodegeneration.
    DOI:  https://doi.org/10.1083/jcb.202409149
  28. bioRxiv. 2026 Jul 20. pii: 2026.07.19.739413. [Epub ahead of print]
    Molecular basis of ubiquitin-independent recognition and degradation of ODC/Antizyme by the 26S proteasome.
    Diego R Ramos-Ortiz, Hao-Hsuan Hsieh, Ken C Dong, Andreas Martin.
      The ubiquitin-proteasome system represents the main pathway for targeted protein degradation in eukaryotic cells. The majority of substrates is recruited for degradation through ubiquitin modifications, and the underlying principles are well established. However, the requirements for ubiquitin-independent substrates are still poorly understood. Here, we reveal the mechanisms for the antizyme-mediated degradation of the yeast ornithine decarboxylase (yODC), the first reported ubiquitin-independent substrate of the 26S proteasome. Using biochemical studies and cryo-EM structure determination, we show how antizyme binding makes the yODC monomer prone for degradation by exposing an interface that is normally buried in the catalytically active ODC dimer. Together with a surface on antizyme, yODC forms a two-part interface that binds the N-terminal coiled coil of two ATPase subunits, Rpt4 and Rpt5, for delivery to the 26S proteasome motor. This positions the N-terminal unstructured region of yODC for insertion into the ATPase channel to initiate degradation, which we found does not depend on a specific sequence. Interestingly, binding of the globular yODC/antizyme complex to the Rpt4/Rpt5 coiled coil allosterically stabilizes a proteasome conformation that facilitates substrate engagement by the ATPase motor and may represent a primed pre-initiation state with a general role in ubiquitin-dependent and -independent degradation.
    DOI:  https://doi.org/10.64898/2026.07.19.739413
  29. Oncogene. 2026 Jul 29.
    HERC4 synergizes with USP16 to promote lung adenocarcinoma growth by augmenting protein translation via stabilizing RPS15.
    Qiuyun Jiang, Yiyang Liu, Wenshuang Sun, Ziyang Liu, Na Tao, Yunshu Zhao, Pingfei Li, Yujie Ou, Yuening Sun, Hongbiao Huang, Jia Yu, Wenhua Liang, Xinliang Mao.
      HERC4 is a well-known HERC family ubiquitin ligase in several types of cancer but its role in lung cancer remains elusive. In the present study, we found that HERC4 is highly dysregulated in lung adenocarcinoma (LUAD) and promotes LUAD cell growth. Mechanically, HERC4 interacts with translation-related proteins and specifically stabilizes RPS15, a component of 40S ribosomal subunit, by promoting its deubiquitination in a manner independent of E3 ligase activity. HERC4 collaborates with USP16, a deubiquitinase that also interacts with translation-related proteins, to stabilize RPS15 by preventing its K48-linked ubiquitination. Further studies revealed that the interaction between HERC4 and USP16 is important to regulate RPS15 and to promote LUAD cell proliferation. Knockdown of HERC4 or USP16 prevents the recruitment of translation-related proteins to ribosomes, increases their nuclear retention and reduces global translational efficacy. Overexpression of RPS15 partially rescues reduced protein translation efficiency and cell survival triggered by HERC4/USP16 knockdown. Moreover, knockdown of HERC4 or USP16 upregulates p53 and downregulates p38 via RPS15 dysregulation. In conclusion, the present study reveals a novel ubiquitination modulation on ribosomal stability and protein translation. HERC4 synergizes with USP16 to deubiquitinate and stabilize RPS15, thereby potentiating global protein translation and promoting LUAD growth. The HERC4/USP16-RPS15 axis may represent a potential therapeutic target for LUAD treatment.
    DOI:  https://doi.org/10.1038/s41388-026-03902-w
  30. Cold Spring Harb Perspect Biol. 2026 Jul 29. pii: a041869. [Epub ahead of print]
    Translational Recoding in Tumors.
    Chen Weller, Osnat Bartok, Panagiotis Poulis, Yardena Samuels.
      Protein synthesis is tightly regulated in cells; however, in cancer, ribosomes deviate from canonical translation, generating altered protein products. These deviations arise from cell-intrinsic alterations, as well as extrinsic pressures within the tumor microenvironment, collectively reshaping the translational landscape and reducing translation fidelity. Translational recoding in cancer expands proteome diversity and promotes tumor fitness by enhancing stress adaptation, metabolic, and phenotypic plasticity. At the same time, recoding events generate peptides that are often presented as tumor-specific antigens, thereby eliciting immune responses against cancer. Accordingly, therapeutic strategies that modulate translational fidelity and induce recoding are emerging to enhance tumor immunogenicity and improve immunotherapy responses. Here, we examine the drivers and consequences of translational recoding in cancer, its dual role in promoting tumor adaptation while shaping immune surveillance, and its potential as a targetable vulnerability in cancer therapy.
    DOI:  https://doi.org/10.1101/cshperspect.a041869
  31. Bio Protoc. 2026 Jul 20. 16(14): e5758
    A Practical Experimental Protocol for Identification and Validation of UFMylation Substrate in Human Cells.
    Qian Liang, Yaoyao Fang, Juexi Dong, Xingling Yi, Yu-Sheng Cong.
      UFMylation is an evolutionarily conserved ubiquitin-like modification that covalently conjugates UFM1 to lysine residues of substrates via a sequential E1-E2-E3 enzymatic cascade. UFMylation plays a pivotal role in maintaining cellular homeostasis, and its dysregulation is closely linked to multiple major diseases, including malignant tumors, hematopoietic defects, neurodegenerative disorders, and congenital developmental defects, highlighting its important biological significance. However, few substrates of UFMylation have been reported to date, limiting our deep understanding of the mechanistic functions of this modification. This major bottleneck stems from two major technical limitations: the overwhelming abundance of ribosomal protein L26 (RPL26)-UFM1 conjugates masks signals from low-abundance substrates, and conventional methods rely on cumbersome cotransfection of multiple pathway components with poor efficiency and specificity in UFMylated peptides enrichment. To address these challenges, we have developed an effective and specific experimental protocol for UFMylation detection and large-scale substrate identification. This protocol employs CRISPR-Cas9-mediated gene editing to generate UFSP1/UFSP2 double-knockout (UFSP1KO/UFSP2KO , DKO) HEK293T cells, which completely abrogate de-UFMylation and thus significantly elevate global protein UFMylation levels upon exogenous introduction of mature UFM1-ΔC2. In addition, exogenous co-expression of the E3 ligase core components UFL1 and DDRGK1 can further improve the sensitivity of substrate detection. This protocol enables large-scale identification of UFMylation substrates with modification sites via high-efficiency enrichment with the K-ε-VG antibody and LC-MS/MS analysis. Key features • Employs UFSP1/UFSP2 DKO HEK293T cells with exogenous mature UFM1-ΔC2 to enhance UFMylation. • Simplified transfection via single-factor assays using UFM1-ΔC2, UFL1, and DDRGK1. • Combines K-ε-VG antibody enrichment with LC-MS/MS for large-scale substrate identification. • Verifies UFMylation sites via site-directed mutagenesis and UFSP2-mediated de-UFMylation.
    Keywords:  DDRGK1; Substrate; UFL1; UFMylation; UFSP1/2
    DOI:  https://doi.org/10.21769/BioProtoc.5758
  32. bioRxiv. 2026 Jul 17. pii: 2026.07.01.735856. [Epub ahead of print]
    K63-linked ubiquitin chains mark inactive Smoothened for packaging into ciliary extracellular vesicles.
    Mingli Zhu 祝明莉, Yash Raj, Gary A Bradshaw, David U Mick, Marian Kalocsay, Maxence V Nachury.
      Signaling receptors can exit cilia either by retrieval back into the cell or by secretion in extracellular vesicles (EVs), a process known as ectocytosis. The mechanisms that govern ectocytosis remain poorly understood. Here, we leverage quantitative proteomic profiling to identify the Hedgehog signaling receptor Smoothened (SMO) as a major cargo of cilia-derived EVs. Surprisingly, ligands that promote ciliary accumulation of SMO strongly suppressed its packaging into EVs, indicating that ectocytosis selectively packages specific conformational states of SMO. We further find that SMO packaged into EVs is extensively modified with K63-linked ubiquitin chains. Preventing SMO ubiquitination or selectively removing K63-linked ubiquitin chains from SMO markedly reduced its secretion into EVs and caused SMO accumulation within cilia. Together, these results identify K63-linked ubiquitin chains as a sorting signal for ciliary ectocytosis and indicate that ubiquitin-dependent packaging of inactive SMO contributes to the dynamic redistribution of SMO during Hedgehog signaling.
    DOI:  https://doi.org/10.64898/2026.07.01.735856
  33. Cell Rep. 2026 Jul 30. pii: S2211-1247(26)00844-2. [Epub ahead of print]45(8): 117766
    Nutrient stress activates RAB5B-mediated autophagy to remodel the synaptic proteome.
    Melina Overhoff, Lotte Ickert, Sebastian Marten, Andrew Hill, Frederik Tellkamp, Ali Chitsaz, Saskia L Ludwig, Philipp Antczak, Felix C Koehler, Roman-Ulrich Müller, Marcus Krüger, Natalia L Kononenko.
      Synaptic proteostasis is crucial for neuronal function, yet how synapses adapt to metabolic stress remains unclear. We show that nutrient stress, particularly serum withdrawal, induces autophagy-dependent remodeling of the synaptic proteome, whereas mTORC1 inhibition produces limited effects. Nutrient stress activates synaptic autophagy within 1-2 h and promotes the recruitment of the LC3 lipidation machinery via RAB5B-positive endosomal compartments in a dynein-dependent manner. Live imaging reveals enhanced RAB5B-ATG16L1 co-trafficking and increased ATG5 mobility upon serum withdrawal, indicating spatiotemporally controlled delivery of autophagy precursors to synaptic compartments. Functionally, nutrient deprivation dampens neuronal activity, while a fasting-mimicking diet induces synaptic proteome remodeling overlapping with starvation-associated autophagy cargo. In contrast, restriction of mTORC1-activating amino acids fails to induce comparable remodeling. Together, these findings identify a RAB5B-mediated trafficking pathway that links nutrient sensing to synaptic degradation, revealing how neurons maintain proteostasis under metabolic challenge.
    Keywords:  CP: metabolism; CP: neuroscience; autophagy; endosomes; nutrient stress; proteostasis; synapse; trafficking
    DOI:  https://doi.org/10.1016/j.celrep.2026.117766
  34. J Cell Biol. 2026 Sep 07. pii: e202605024. [Epub ahead of print]225(9):
    Spatiotemporal regulation of DNA repair proteins between Golgi and nucleus maintains genome stability.
    George Galea, Karolina Kuodyte, Muzamil Majid Khan, Peter J Thul, Beate Neumann, Emma Lundberg, Rainer Pepperkok.
      The Golgi complex serves as a critical hub for cellular homeostasis, yet its communication with the nucleus remains largely unexplored. By analyzing and siRNA-validating localization data from the Human Protein Atlas, we uncovered substantial proteome interconnectivity between the Golgi and nucleus, including an unexpected enrichment for DNA repair factors. We identify a cluster of DNA damage response (DDR) proteins occupying distinct sub-Golgi compartments that redistribute dynamically between the Golgi and nucleus in response to genotoxic stress, with the type of DNA lesion shaping the direction of redistribution. Focusing on the homologous recombination (HR) regulator RAD51C, we show that DNA damage triggers ataxia telangiectasia mutated (ATM)-dependent release of a giantin-tethered Golgi RAD51C pool, with subsequent importin-β-dependent nuclear import, where repair-associated foci form. Giantin depletion prematurely releases RAD51C, producing aberrant nuclear foci lacking key DDR markers, reducing ATM activation and HR efficiency, elevating genome instability, and accelerating proliferation. The Golgi thus acts as a spatiotemporal coordination node for DDR factors safeguarding genomic stability.
    DOI:  https://doi.org/10.1083/jcb.202605024
  35. bioRxiv. 2026 Jul 16. pii: 2026.07.16.738907. [Epub ahead of print]
    Substrate biasing in UCHL5 proteoforms.
    Rishi S Patel, Nipuni M Pannala, Chih-Hsuan Lai, Manoj Kumar Sriramoju, Yong-Sheng Wang, Ting Chen, Dabin Jung, Payam Kelich, Soroush Asadi, Elizaveta Shestoperova, Shalini Iyer, Emad Tajkhorshid, Daniel Flaherty, Eric Strieter, Shang-Te Danny Hsu, Chittaranjan Das.
      Proteolytic deubiquitinating enzymes bridge a gap in substrate recognition through complex regulatory mechanisms. A growing portion of these are accomplished through proteoforms that uniquely control association and diverse sets of cleavage capabilities that relay distinct physiological outcomes. This study describes substrate biasing governed by UCHL5 proteoforms. It demonstrates that N-terminal ubiquitination activates the enzyme towards monoubiquitin substrates, a feature that is conserved across UCHL5 homologs. Crystallographic and spectroscopic data suggest that the N-terminal ubiquitin binds intramolecularly in an allosteric binding site and inhibits branched chain substrate cleavage. Association with Rpn13/Adrm1 relieves this inhibition and reestablishes its ability to debranch, potentially controlling nonspecific debranching compared to retention of needed activity on the 26S proteasome. Collectively, this study describes the molecular basis for substrate selectivity in a deubiquitinating enzyme, an unexplored area in the enzymes that counteract ubiquitin E3 ligases.
    DOI:  https://doi.org/10.64898/2026.07.16.738907
  36. Anal Chem. 2026 Jul 27.
    DNA-Alkyne-Chain Probes Enable N-Glycosite-Resolved GlycoBarcoding of Cell-Surface Proteins.
    Huimin Bao, Yao Yao, Rui Zhang, Jianpu Tang, Tailing Xue, Cao Fang, Dayong Yang.
      Cell surface glycoproteins are vital mediators of intercellular communication and primary targets for therapeutic intervention. However, the comprehensive profiling of the "surface glycome" on living cells remains hindered by the low abundance of these proteins and the inherent complexity of glycosylation. Here, we present GlycoCSP, a site-specific glycoproteomics strategy that utilizes alkyne-functionalized DNA scaffolds to achieve high-resolution GlycoBarcoding of cell surface proteins. By leveraging the spatial reach of extended DNA chains and a high-density alkyne array, GlycoCSP ensures specific labeling and robust covalent capture of surface glycoproteins. This platform integrates protein-level enrichment with an orthogonal tandem release proteolysis, enabling the precise mapping of N-glycosylation signatures at the site level. Applying GlycoCSP to live cells, we identified 2,016 extracellular N-glycosylation sites across 1,420 proteins, validated by deamidation mass shifts and the canonical N-X-S/T/C motif. Comparative analysis across breast cancer cell lines revealed that site-specific glycosylation occupancy provides a distinct layer of surfaceome heterogeneity that is independent of protein abundance. By providing a mass spectrometry-readable framework for decoding the surface glycode, GlycoCSP enables the unbiased discovery of glycosylation-dependent biomarkers and therapeutic targets previously inaccessible to conventional proteomics.
    DOI:  https://doi.org/10.1021/acs.analchem.6c02112
  37. J Med Chem. 2026 Jul 27.
    A Structure-guided Active Site Affinity Ligand Unmasks a Stress-Sensitizing Role for BLVRB in the Endoplasmic Reticulum.
    Rahul Raghavan Thekke Veedu, Jawaad Sheriff, Natasha M Nesbitt, Natalia Marchenko, Lisa Pennacchia, Tiziana Ginex, Patrick Hearing, Dale F Kreitler, Wadie F Bahou.
      Biliverdin IXb reductase (BLVRB) is an NAD(P)H-dependent oxidoreductase that regulates hematopoiesis and cellular stress, although measurement and sequelae of cellular active site engagement remain undefined. Here, we report the development of a nanoBRET platform enabling real-time BLVRB target engagement. Structure-guided design and chemical syntheses of BODIPY-labeled pyrazolopyrimidinone inhibitors generate cell-permeable acceptor ligands retaining high-affinity binding to the BLVRB active site. In vitro and cellular nanoBRET assays demonstrate specific energy transfer and inform equilibrium binding affinities, target engagement, and residence time analyses for diverse panels of BLVRB inhibitors. NanoBRET demonstrates strong concordance with enzymatic inhibition and is validated by crystallographic structures confirming active site binding. Live-cell imaging using affinity ligands reveals predominant endoplasmic reticulum localization and transient suppression of the ER stress chaperone GRP78/BiP without eliciting a canonical unfolded protein response. These studies inform a redox-regulated mechanism whereby spatiotemporal BLVRB active site engagement functions as a stress sensitizer modulating ER proteostasis.
    DOI:  https://doi.org/10.1021/acs.jmedchem.6c01466
  38. J Cheminform. 2026 Jul 24. pii: 103. [Epub ahead of print]18(1):
    Computational mapping of productive POI-E3 ligase conformations to guide de novo degrader design: application to WEE1 and PKMYT1 PROTACs.
    Husam Nassar, Matthias Schmidt, Hany S Ibrahim, Dina Robaa, Wolfgang Sippl.
      Targeted protein degradation has emerged as a promising therapeutic strategy, yet rational degrader design remains challenged by the dynamic nature of protein of interest (POI)-E3 ligase interactions. While X-ray crystallography and cryo-EM provide valuable structural snapshots, they are insufficient for capturing the conformational heterogeneity underpinning efficient ubiquitination and degradation. Here, we present a unified computational workflow to systematically generate and evaluate POI-E3 ligase conformational states for CRBN- and VHL-mediated proteolysis-targeting chimeras (PROTACs). The workflow integrates warhead connectivity analysis, conformational clustering, ubiquitination accessibility assessment and molecular dynamics simulations to identify productive POI-E3 ligase geometries. Analysis of experimental structures revealed that PROTAC linkers do not exceed 15 Å, providing a practical attachment-atom distance based filter for docking-derived models. Furthermore, POI-E3 ligase conformations differing by more than 7.5 Å Cα RMSD exhibited distinct ubiquitination profiles, offering quantitative guidance for defining structurally and functionally divergent states. Experimental ternary complexes consistently positioned one or more solvent-exposed POI lysine residues within 50 Å of the E2 catalytic Cys111, establishing a mechanistically grounded criterion for ubiquitination competence. Validation against 34 experimentally determined PROTAC ternary complexes achieved a 97% recovery rate and identified multiple ubiquitination competent conformations beyond experimental snapshots. The workflow was subsequently applied to model productive WEE1-CRBN and PKMYT1-CRBN conformations, for which no experimental structures are available. PROTAC induced-fit docking demonstrated that active PROTACs selectively engage productive POI-E3 ligase geometries with linker-compatible attachment atom distances. Overall, this study provides a quantitative, structure-based framework for guiding the rational design of ubiquitination-based degraders. The code and example data supporting this workflow are openly available at https://github.com/Husam-PSE/PROTACMap .Scientific contributionThis study offers a generalizable computational framework for identifying productive POI-E3 ligase conformations. It demonstrates that effective degradation depends on the interplay between conformational diversity, feasible warhead connectivity and preserved ubiquitination competence, rather than solely on ternary complex stability or binding affinity. Our computational approach was applied on WEE1 and PKMYT1 PROTACs for which no experimental ternary structure is available.
    Keywords:  PKMYT1; PROTACs; Targeted protein degradation; Ubiquitination; WEE1
    DOI:  https://doi.org/10.1186/s13321-026-01268-5
  39. Small Methods. 2026 Jul 29. e70912
    Profiling Multiplexed Protein-Specific Sialylation in Cancer Drug Resistance Using Proximity Ligation Sequencing.
    Ning Zhang, Gaoyu Song, Zhiyuan Peng, Kangjia Du, Cong Cai, Shuo Miao, Yiyuan Tang, Ya Wang, Juan Li.
      Aberrant glycosylation, particularly enhanced sialylation of membrane proteins, acts as a critical regulator of receptor signaling, immune evasion, and therapeutic resistance in cancer. However, systematic and multiplexed interrogation of protein-specific sialylation in living cells remains technically challenging. Here, we report SATP-seq, a next-generation sequencing-based strategy that integrates a sialic acid-reactive probe (SA) with a targeted protein probe (TP) to enable ensemble profiling of protein-specific sialylation. By combining metabolic glycan labeling with nanobody- or aptamer-mediated protein recognition, SATP-seq achieves dual recognition of sialic acids and protein epitopes through DNA-programmed proximity ligation. The resulting ligation products encode sialylation states into unique DNA barcodes, converting glycosylation information into sequencable signals for multiplexed and quantitative analysis within a single sequencing run. Application of SATP-seq to gefitinib-sensitive PC9 and gefitinib-resistant PC9GR cells enables parallel profiling of seven membrane glycoproteins and reveals resistance-associated remodeling of protein-specific sialylation. Notably, differential sialylation of EGFR and CD47 emerges between sensitive and resistant cells, suggesting coordinated reprogramming of proliferative and immune-regulatory pathways. Enzymatic desialylation, EGFR knockdown, and proteomic validation collectively confirm the specificity and biological relevance of these alterations. Together, these findings establish multiplexed protein-specific sialylation profiling as a scalable approach for dissecting glycosylation-driven mechanisms of cancer drug resistance.
    Keywords:  aptamer; cancer drug resistance; nanobody; protein‐specific sialylation; proximity Ligation Sequencing
    DOI:  https://doi.org/10.1002/smtd.70912
  40. bioRxiv. 2026 Jul 16. pii: 2026.07.14.738309. [Epub ahead of print]
    Overlapping upstream ORFs repress translation and expand proteome diversity in Arabidopsis.
    Hsin-Yen Larry Wu, Yu-Hsuan Cheng, Isaiah D Kaufman, Qiaoyun Ai, Polly Yingshan Hsu.
      Upstream open reading frames (uORFs) are widespread cis-regulatory elements that modulate translation initiation of downstream main ORFs (mORFs). Among them, overlapping uORFs (ouORFs) that overlap with mORFs are predicted to exert the strongest translational repression, yet they remain largely unexplored because of the difficulty of their identification. Here, we developed complementary computational approaches to systematically identify translated ouORFs from super-resolution ribosome profiling data in Arabidopsis. We identified 965 translated ouORFs alongside 7,180 canonical non-overlapping uORFs (nuORFs). We found that ouORFs exert substantially stronger translational repression than nuORFs, and that this repression depends primarily on Kozak context rather than uORF length. In addition, genes containing ouORFs or nuORFs have weaker mORF Kozak contexts than genes without uORFs, which may further reduce mORF translation. Moreover, ouORF translation promotes initiation downstream of the annotated mORF start codon, generating N-terminally truncated protein isoforms with altered domain composition and subcellular localization. Using ATPS2 as an example, we demonstrate that ouORF translation regulates alternative translation initiation to control the balance between chloroplast and cytosolic protein isoforms. Together, our findings establish ouORFs as a versatile class of translational regulatory elements that coordinate both protein abundance and protein diversity, providing the first genome-wide characterization of translated ouORFs in plants.
    Keywords:  N-terminal truncation; alternative protein isoforms; alternative translation initiation; overlapping uORF; subcellular localization; upstream ORF
    DOI:  https://doi.org/10.64898/2026.07.14.738309
  41. Cell Rep. 2026 Jul 31. pii: S2211-1247(26)00822-3. [Epub ahead of print]45(8): 117744
    CRISPR activation screen uncovers MARCKSL1 as a gauge for extracellular vesicle secretion.
    Yingjie Zhong, Anna E George, Roman I Koning, Nalan Liv, Cecilia de Heus, Rayman T N Tjokrodirijo, Manon Messchendorp, Jimmy J L Akkermans, Birol Cabukusta, George M C Janssen, Peter A van Veelen, Ruud H M Wijdeven, Jacques Neefjes, Frederik J Verweij, Ilana Berlin.
      Extracellular vesicles (EVs) are central mediators of intercellular communication that originate from diverse membrane reservoirs. EV biogenesis can occur via multiple pathways, including outward budding of the plasma membrane (PM) and fusion of mature endosomes with the PM. The present study investigates how cells balance these competing routes using a genome-wide CRISPR activation screen for factors that alter surface expression of CD63-an EV-associated tetraspanin that shuttles between the PM and endosomes. This unbiased strategy identifies the membrane adaptor MARCKSL1 known to be upregulated across diverse tumor types. Overexpression of MARCKSL1 elevates CD63 abundance at the PM and boosts EV secretion. Proximity-based proteomics implicates the cytoskeleton-PM linker Radixin and the SNARE-associated protein STXBP3 as MARCKSL1 binding partners that respectively promote either PM-derived or endosome-derived EV secretion. Collectively, our findings reveal new insights into PM remodeling and position MARCKSL1 as a gauge between distinct EV biogenesis platforms.
    Keywords:  CD63; CP: cell biology; MARCKSL1; RDX; STXBP3; ectosomes; exosomes; extracellular vesicles; microvesicles; plasma membrane dynamics; vesicle budding
    DOI:  https://doi.org/10.1016/j.celrep.2026.117744
  42. Nat Cell Biol. 2026 Jul 29.
    The glycolytic enzyme PGAM1 functions as a metabolic-autophagy checkpoint to coordinate growth and stress tolerance.
    Yi Zhang, Pengwei Zhao, Hangfei Liang, Zhaojie Liu, Shuling Dong, Yingcong Chen, Weijing Yao, Yuting Chen, Liting Yang, Zhonglan Shi, Lei Zhang, Yibin Pan, Fanghong Zheng, Qi Lin, Shichao Wang, Jinheng Pan, Mingzhu Fan, Shan Feng, Cheng Ma, Lifeng Pan, Ying Xing, Fan Yang, Shuiping Liu, Du Feng, Qiming Sun, Guixin Zhu, Cong Yi.
      Cell survival requires tight coordination between growth-promoting metabolism and cellular quality-control pathways, yet how these processes are integrated remains unclear. Here we identify the conserved glycolytic enzyme PGAM1 as a metabolic-autophagy checkpoint that links glycolysis to autophagy initiation independently of its catalytic activity. Using complementary yeast and mammalian systems we show that PGAM1 functions as a molecular scaffold that recruits phosphatidylinositol 3-kinase complex I to the phagophore assembly site, thereby licensing autophagosome biogenesis. This autophagy-regulatory function is genetically essential, evolutionarily conserved and functionally separable from glycolysis. It is regulated by Atg1/ULK1-mediated phosphorylation that enhances Atg14 binding under starvation. Functionally, PGAM1 coordinates anabolic growth and stress-induced survival to maintain cellular homeostasis. In cancer, PGAM1 upregulation enhances both glycolytic flux and autophagy capacity. Disruption of either function markedly impairs tumour growth, establishing PGAM1 as a homeostatic checkpoint that is hijacked in cancer to drive both proliferation and stress tolerance.
    DOI:  https://doi.org/10.1038/s41556-026-02034-3
  43. J Am Chem Soc. 2026 Jul 27.
    Discovery of Noninhibitory Macrocyclic Ligands for Protein Tyrosine Phosphatase 1B Using a Function-Based, Iterative Screening Strategy.
    Jiajun Dong, Bo Li, Chung-Wei Fu, Thomas Kodadek.
      Chemically induced proximity is a powerful modality for manipulating protein function. Most of the effort in this field has focused on targeted protein degradation, but recruitment of other types of post-translational modification enzymes to a target protein is also of interest. To construct such reagents, one would ideally like to have ligands that engage the enzyme without inhibiting its activity. In this study, we describe a screening platform for the discovery of noninhibitory macrocyclic ligands for a protein tyrosine phosphatase, using PTP1B as an exemplary model target. This workflow involves sequential screens of small libraries of bead-displayed macrocycles in which only one position of the macrocycle is varied in each round of screening while the others are held as invariant placeholders. The beads co-display a high KM substrate for the phosphatase, allowing ligand-dependent recruitment of the enzyme to the bead surface to be coupled to dephosphorylation of the co-displayed substrate. This is detected by staining with a labeled anti-phosphotyrosine antibody. Finally, we demonstrate that the same general approach can be applied to proteins lacking enzymatic activity by screening against biotin ligase-target protein fusions and employing a proximity labeling-like assay to register screening hits.
    DOI:  https://doi.org/10.1021/jacs.6c07205
  44. FEBS J. 2026 Jul 29.
    Inhibition of mitochondrial protein translation sensitizes tumor cells to death receptor 5 agonistic antibody.
    Gabriele Casagrande Raffi, Jian Chen, Elsemieke Passchier, Cor Lieftink, Chrysa Papagianni, Jeanette H W Leusen, Roderick L Beijersbergen, Liqin Wang, Rene Bernards.
      While many antagonistic antibodies are in routine clinical use, only a single agonistic antibody has received regulatory approval to date. While antibodies that activate Death Receptor 5 (DR5) were thought to have utility in the treatment of cancer by enhancing extrinsic apoptosis signaling, to date all clinical studies with these DR5 agonists have failed to deliver significant clinical benefit. A notable example of this is the DR5 agonistic antibody conatumumab. Here, we provide two potential avenues to improve the activity of DR5 agonists. First, we show that a dimeric IgA version (dIgA2) of the conatumumab antibody has a higher toxicity to cancer cells and a shorter half-life in vivo compared to the original IgG version of the antibody. Moreover, we conducted a genome-wide CRISPR screen to identify genes for which inactivation enhances the sensitivity of cancer cells to the dIgA2 DR5 antibody. We found that inhibition of mitochondrial protein translation synergizes with DR5 agonists. Consequently, antibiotics that inhibit mitochondrial protein translation also synergize with DR5 agonists. Finally, we show that these antibiotics activate the Integrated Stress Response (ISR) and upregulate DR5 through the EIF2a-ATF4 axis, which sensitizes cancer cells to DR5 activation. These data suggest a potential combination strategy for the effective use of DR5 agonistic antibodies.
    Keywords:  CRISPR screening; apoptosis; dimeric IgA; integrated stress response; mitochondria
    DOI:  https://doi.org/10.1111/febs.70669
  45. bioRxiv. 2026 Jul 21. pii: 2026.07.21.739842. [Epub ahead of print]
    Non-Canonical Activation of HSF1 Stimulates mTORC1-Mediated Translation in HCMV-Infected Monocytes.
    Michael J Miller, Shima Moradpour, Brittany W Geiler, Jamil Mahmud, Wayne A Decatur, Gary C Chan.
      Human cytomegalovirus (HCMV) is a major cause of organ disease among immunonaïve and immunocompromised individuals. HCMV infection stimulates the survival of normally short-lived circulating monocytes, allowing these blood cells to mediate the dissemination of the virus from the initial point of infection to distant organ sites. We previously showed that HCMV induces a non-canonical phosphorylation of Akt within infected monocytes that activates the stress response transcription factor Heat Shock Factor 1 (HSF1). In this study, we demonstrate that HSF1 is necessary for the survival of HCMV-infected monocytes using both pharmacological and genetic approaches. In contrast, HSF1 inhibition had minimal effect on the viability of uninfected cells, indicating the specific involvement of HSF1 on the survival of infected monocytes. Surprisingly, the aberrant activation of HSF1 by HCMV did not trigger nuclear relocalization, suggesting that HSF1's regulation of monocyte viability occurs within the cytoplasm. Indeed, we found that HCMV-activated, cytoplasmic HSF1 directly binds to mTOR, a critical component of the mTORC1 complex involved in the regulation of mRNA translation. SUnSET (Surface Sensing of Translation) assays revealed HCMV-activated HSF1 increases mRNA translation through mTORC1. Ribosomal profiling identified the increased translation of a selected subset of pro-survival transcripts, including cIAP2, which we validated to selectively stimulate the survival of HCMV-infected monocytes. Taken together, these data demonstrate that the non-canonical activation of HSF1 in infected monocytes drives mTORC1-dependent translation of antiapoptotic transcripts, ensuring the survival and dissemination of infected monocytes.
    IMPORTANCE: HCMV is a primary driver of morbidity and mortality in individuals with compromised or immature immune systems. Spread of HCMV throughout the body relies on the infection of peripheral blood monocytes, which spread the virus to end-organ tissues. However, the naturally short lifespan of monocytes must be overcome to allow for viral spread to occur. Here, we demonstrate that HCMV uniquely regulates the cellular stress response to promote the long-term survival of infected monocytes. Specifically, HCMV activates the cellular stress response transcription factor HSF1 to block the progression of apoptosis. In contrast to traditional heat shock stress where HSF1 translocates into the nucleus to mediate transcription, HCMV infection retains activated HSF1 in the cytoplasm where it binds to mTOR to promote protein synthesis of prosurvival factors necessary for the survival of infected monocytes. Overall, our study provides insight into the complex regulator mechanisms through which HCMV usurps host stress responses to promote viral dissemination.
    DOI:  https://doi.org/10.64898/2026.07.21.739842
  46. Cancer Res Commun. 2026 Jul 29.
    E3 Ubiquitin Ligase Mindbomb1 Is a Novel Negative Regulator of Tumor-Suppressive BMP Signaling.
    Charisa L Cottonham, Gloria E Hernandez, Tommy K Cheung, Christopher Rose, Rajini Srinivasan, Robert Piskol, Xiaosai Yao, Christian W Siebel.
      Mindbomb1 (MIB1), an E3 ubiquitin ligase required for NOTCH activation, has been identified as a strongly selective cancer dependency in genome-wide loss-of-function screens in over 1000 cancer cell lines. However, MIB1-correlating dependencies in these screens unexpectedly linked MIB1 not to Notch but rather to the family of Transforming Growth Factor-β (TGF-β) and Bone Morphogenic Protein (BMP) signaling molecules. Here, using genetic and pharmacologic methods, we show that MIB1 loss phenocopies BMP tumor suppressive function. MIB1-dependent cancer cell lines were sensitive to BMP ligands, and MIB1 loss selectively enhanced BMP signaling, as evidenced by SMAD1/5/9 phosphorylation and transcriptional responses that correlated with increased BMP receptor protein levels. Growth inhibition and enhanced signaling were both reversed by inhibitors of BMP type 1 receptors, demonstrating that these MIB1 effects directly reflect a function for MIB1 as a negative regulator of BMP signaling. Unlike wild-type MIB1, re-expression of RING-domain-deleted MIB1 failed to rescue growth inhibition in MIB1-depleted cells, suggesting that negative regulation depends on E3 ligase activity. Supporting this regulatory role, we found that MIB1 and the BMP type II receptor BMPR2 physically associate in both cancer and endothelial cells. By revealing this previously unknown role for MIB1 as a Notch-independent negative regulator of BMP signaling, our study expands the significance of MIB1 in cancer biology, with implications for targeting BMP signaling in cancer and other diseases.
    DOI:  https://doi.org/10.1158/2767-9764.CRC-25-0760
  47. bioRxiv. 2026 Jun 18. pii: 2026.06.15.732391. [Epub ahead of print]
    A Stress-Adaptive Lipid Kinase Axis Defines Metabolic Vulnerabilities in Neuroendocrine Prostate Cancer.
    Yang Zheng, Caleb Cheng, Yizhi Cao, Gabriel Cruz, Yuping Zhang, Radha Paturu, Somnath Mahapatra, Jing Hu, Rahul Mannan, Hüseyin Karabürk, Rupam Bhattacharyya, Yitong Yin, Yi Zhao, Wenyan Liu, Xuhong Cao, Hui Xue, Chungen Li, Zhen Wang, Stephanie J Miner, Ulka Vaishampayan, Vaibhav Sahai, Lois S Weisman, Ke Ding, Costas A Lyssiotis, Yuzhuo Wang, Yuanyuan Qiao, Arul M Chinnaiyan.
      Neuroendocrine prostate cancer (NEPC) persists in a profoundly hypoxic microenvironment, yet the mechanisms enabling tumor adaptation to this metabolically challenging niche remain undefined. Here, we identify the lipid kinase PIKfyve as overexpressed in NEPC, functioning as a central node in a stress-lipid kinase axis that drives adaptation to persistent endoplasmic reticulum (ER) stress. Mechanistically, NEPC requires PIKfyve-mediated lysosomal degradation and lipid recycling to maintain metabolic homeostasis under hypoxia. PIKfyve inhibition disrupts lysosomal function, leading to ER stress accumulation and activation of a compensatory, sterol regulatory element-binding protein (SREBP)-dependent de novo lipogenesis program essential for NEPC survival. This stress-lipid axis creates a synthetic vulnerability between PIKfyve and fatty acid synthase (FASN), where dual inhibition synergistically amplifies ER stress, triggers the terminal unfolded protein response, and induces tumor cell death. These findings reveal a metabolic adaptation in NEPC and provide preclinical evidence that co-targeting PIKfyve and FASN can overcome hypoxia-associated stress adaptation.
    Keywords:  ER stress; PIKfyve; lipid metabolism; lysosome; neuroendocrine prostate cancer
    DOI:  https://doi.org/10.64898/2026.06.15.732391
  48. bioRxiv. 2026 Jul 23. pii: 2026.07.20.739556. [Epub ahead of print]
    Leveraging Supramolecular Polymers to Induce the Targeted Protein Degradation of α-Synuclein.
    Windfield S Swetman, Malay Mondal, Ashe M Davis, Vijay Rangachari, Tristan D Clemons.
      Halting the progression of neurodegenerative diseases remains one of the foremost challenges in medicinal chemistry due to the complex biology that drives disease progression. For example, a hallmark of synucleinopathies, such as Parkinson's disease, is the misfolding and aggregation of the protein α-Synuclein (α-Syn), driving the formation of toxic oligomers and fibrils that avoid natural intracellular clearance mechanisms, participate in unusual protein-protein interactions, and ultimately contribute to the death of dopaminergic neurons. The field of targeted protein degradation (TPD) has emerged as an innovative therapeutic route to selectively degrade proteins of interest that leverage natural intracellular protein degradation machinery. First generation TPD therapeutics have traditionally been designed as bifunctional, chimeric compounds in which a short covalent linker tethers a ligand designed to bind target proteins to a ligand that initiates an either proteosome- or lysosome-dependent protein degradation cascade. While initial studies have indicated the promise of these approaches, translation to the clinical setting has been challenging due to difficulties in achieving cellular internalization, long-term stability, and establishment of a generalizable strategy. To overcome these obstacles, this work has focused on adding modularity and dynamic capability to this classical model by leveraging a multivalent macromolecular approach to TPD. Specifically, peptide amphiphiles (PAs) were designed to self-assemble into high-aspect-ratio supramolecular nanofibers and present peptide epitopes on the surface of the fibers to target simultaneous binding of α-Syn and recruitment of enzymes that facilitate entry into the lysosome-dependent chaperone-mediated autophagy protein degradation pathway. In vitro application of these bioactive PA nanofibers has demonstrated the ability to independently internalize in cells and reduce α-Syn protein levels selectively and effectively. While further optimization of this model has the potential to be a viable therapeutic against α-Syn aggregation, the modularity of these supramolecular nanofibers through facile monomer design and incorporation illustrates the potential of establishing a platform technology for targeting a diverse range of pathologic proteins.
    DOI:  https://doi.org/10.64898/2026.07.20.739556
  49. Curr Biol. 2026 Jul 29. pii: S0960-9822(26)00873-0. [Epub ahead of print]
    Evolution of a core ribosomal innovation in octopus.
    Rishav Mitra, Richard Han, Trey J Scott, Anik G Grearson, Jessica A Willi, Christina G Liu, Hyeongju Kim, Michael C Jewett, Nicholas W Bellono, Amy S Y Lee.
      Much of biology focuses on how genetic changes mediate new functions, but less attention is given to adaptations within the ancient molecular machines that execute the central dogma. Octopuses exhibit complex nervous systems and sophisticated behaviors that rival vertebrates but via an entirely divergent evolutionary history. Here, we serendipitously discovered that octopus ribosomes contain a structural break in the core ribosomal RNA that is unique among all animals. This break site enhances translation fidelity to reduce miscoding and subsequent protein aggregation, even when engineered into evolutionarily distant bacterial ribosomes. Furthermore, high-fidelity translation by octopus ribosomes supports proteomic stability during extensive RNA editing observed in cephalopods, suggesting synergy between distinct non-canonical modes of gene regulation. This adaptation emerged in recently derived octopuses with expanded nervous systems, thereby revealing a mechanism that could broadly support the evolution of novel organismal traits.
    Keywords:  28S rRNA; RNA editing; cephalopod; octopus; protein aggregation; proteostasis; rRNA break; ribosome; ribosome evolution; translation fidelity
    DOI:  https://doi.org/10.1016/j.cub.2026.07.008
  50. iScience. 2026 Aug 21. 29(8): 116804
    Proteostatic defect drives biophysical remodeling that triggers cell competition.
    Wonjae Song, Mai Yukitake, Kei Kozawa, Nanami Sato, Jiaying Wen, Zehao Dai, Susumu Ishikawa, Akifumi Shiomi, Hirofumi Shintaku, Seiichiro Ishihara, Hisashi Haga, Yasuyuki Fujita.
      Cell competition is a fundamental tissue-surveillance process in which less-fit "loser" cells are actively eliminated by "winner" neighbors. Here, we establish a mammalian epithelial model of ribosomal protein insufficiency using Madin-Darby canine kidney (MDCK) cells with tetracycline-inducible shRNA targeting ribosomal protein large subunit 24 (Rpl24). When Rpl24-knockdown cells are co-cultured with normal cells, Rpl24-knockdown cells undergo apoptosis through cell competition with surrounding normal cells. Rpl24 knockdown disrupts protein homeostasis, leading to cytoplasmixc protein aggregates. The chemical chaperone 4-phenylbutyric acid (4-PBA) diminishes aggregate accumulation and markedly reduces competitive cell death. Proteostasis disruption also remodels cellular biophysics; Rpl24-knockdown cells exhibit lower homeostatic density, increased cell area, and reduced cell-surface tension. Importantly, these biophysical alterations are reversed by 4-PBA. Together, our findings reveal that ribosomal protein insufficiency links proteostatic stress to biophysical "loser" traits, establishing proteostasis-dependent biophysical remodeling as a key determinant of competitive cell elimination.
    Keywords:  Ribosomal Protein Rpl24; apoptosis; cell competition; cellular biophysical alterations; mammalian cell culture; p53; proteostasis
    DOI:  https://doi.org/10.1016/j.isci.2026.116804
  51. Biomedicines. 2026 Jul 15. pii: 1585. [Epub ahead of print]14(7):
    Live Visualization of Endoplasmic Reticulum Redox Potential in Zebrafish Embryos Reveals Region-Specific Heterogeneity.
    Monika Verma, Niraj Rajesh Bhatt, Koushika Chandrasekaran, Aseem Chaphalkar, Kriti Verma, Shreyansh Umale, Shweta Verma, Chetana Sachidanandan, Kausik Chakraborty.
      Objective: Redox homeostasis is an integral part of many cellular processes, and its perturbation is associated with conditions such as diabetes, aging, and neurodegenerative disorders. Redox homeostasis or redox potential in organelles is maintained within a particular range to facilitate the organelle-specific cellular redox reactions. Previous studies using yeast, cell systems, and nematodes have demonstrated that the Endoplasmic Reticulum (ER) has a more oxidizing environment, while the cytosol exhibits a reducing redox potential. However, we know very little about how universal this phenomenon is. Methods: We created transgenic zebrafish (Danio rerio) lines with roGFP sensors targeted to the ER and cytosol for studying physiological redox potential at the systems level. In the process, we also characterized the ER-targeting signal sequence in D. rerio for the first time. Results: Measurements of the redox state in live embryos found that the endoplasmic reticulum exhibits consistent deviations from its expected oxidizing redox state in multiple regions of the developing embryos. The ER is far more reduced than expected in certain tissues of the embryo, including certain regions of the brain. We confirmed this heterogeneity using another transgenic line expressing ER-targeted roGFPiE, a redox-sensitive GFP better suited to measuring changes in ER redox potential. We also observed the resilient nature of the ER redox state following tunicamycin (Tm) and Azetidine-2-carboxylic acid-induced proteostasis perturbations and only mild changes with Tm. Conclusions: While our study provides a first glimpse of the diversity in ER redox homeostasis, these unanticipated redox states of the ER will require new biological definitions.
    Keywords:  oxidizing cytosol; redox homeostasis; redox potential; reducing ER; roGFP; signal sequence; zebrafish
    DOI:  https://doi.org/10.3390/biomedicines14071585
  52. bioRxiv. 2026 Jul 22. pii: 2026.07.21.739776. [Epub ahead of print]
    A domain-swapped proPC1/3 structure reveals a conformational checkpoint for ER export.
    Pankaj Kumar, Phillip A Hyon, Rishi Nixon, Ashley V Bullington, Ciara A Payne, Iris Lindberg, Daniel L Kober.
      Prohormone convertase 1/3 (PC1/3, encoded by PCSK1 ) is a serine protease expressed in neuroendocrine cells that is required to produce insulin, glucagon-like peptide-1, adrenocorticotrophic hormone, and other peptide hormones. PC1/3 is synthesized as the zymogen proPC1/3, which undergoes autocatalytic maturation in the endoplasmic reticulum (ER) before trafficking to secretory granules. However, how autocatalytic maturation licenses the ER exit of PC1/3 remains unknown. Here, we determined the structure of an immature, catalytically inactive human proPC1/3 S382A , which exits the ER as a domain-swapped homodimer. This domain-swapped conformation allows proPC1/3 S382A to complete a conserved calcium pocket normally formed after autocatalysis and thereby become competent for ER exit. By defining this calcium pocket as a conformational checkpoint for ER export, our findings provide insights into the maturation of PC1/3 which may apply broadly to the PCSK family. Finally, our structure provides explanations for the functional consequences of many deleterious PCSK1 mutations.
    DOI:  https://doi.org/10.64898/2026.07.21.739776
  53. bioRxiv. 2026 Jul 13. pii: 2026.07.12.737973. [Epub ahead of print]
    Faf2 is required for neural differentiation in embryonic neural progenitor cells.
    Anneke Dixie Kakebeen, Leon Dunphy, Heather K Hazen, Lee A Niswander.
      Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation.
    Highlights: FAF2 is required to regulate ER homeostasis in neural progenitor cellsFAF2 knockout blocks differentiation of neural progenitor cells to neurons at the cell morphological level, but does not inhibit the mounting of transcriptional programs associated with neural differentiation.Excess ER stress due to FAF2 knockout contributes to blocked neural differentiation.
    DOI:  https://doi.org/10.64898/2026.07.12.737973
  54. bioRxiv. 2026 Jul 23. pii: 2026.07.22.739993. [Epub ahead of print]
    A de novo designed enzyme for photo-proximity labeling of E3 ligase neighborhoods in live cells.
    Nam Hyeong Kim, Zhi Lin, Sangwon Nam, Danielle L Swaney, Nevan Krogan, Yong Ho Kim, Michael J Therien, William F DeGrado, James A Wells.
      Photocatalytic proximity labeling proteomics (photo-PLP) has emerged as a powerful technology for rapid capture of protein interactomes in situ. Typically, photo-PLP relies on chemical conjugation of the photocatalyst to the target of interest which creates practical challenges for derivatized photocatalyst synthesis and bioconjugation specificity. Integrating the precision of genetically encodable enzymes with the versatility of chemically defined photocatalysts provides a modular approach to further expand the scope of neighborhood mapping. Here, we present EYClamp, a de novo designed proximity labeling enzyme harnessing the off-the-shelf photocatalyst Eosin Y (EY) as a cofactor. Using a domain-swapped dimer architecture, we designed a scaffold that binds EY with high affinity ( K d = 10 nM) and lengthens its triplet excited-state lifetime by 29-fold. EYClamp enables efficient, multi-scale photocatalytic proximity labeling in live cells with aryl-diazirine-, aryl-azide- and phenol-biotin. We genetically fused EYClamp to a panel of six important E3 ligases. Using EYClamp, we identified over 1,500 candidate neighbors for KEAP1, MDM2, ASB7 and STUB1, providing a broad and unbiased view of these important neighborhoods. Critical functional networks were revealed including ASB7 engagement with HP1a/CUL5 complex for heterochromatin remodeling. Our EYClamp provides a genetically encodable "plug-and-play" solution for photo-PLP interactome discovery of the large family of E3 ligases and establishes domain-swapping as a promising strategy for de novo photoenzyme design.
    DOI:  https://doi.org/10.64898/2026.07.22.739993
  55. Angew Chem Int Ed Engl. 2026 Jul 28. e1973691
    Selective and Potent First-in-Class CRBN-Dependent Molecular Glue Degraders of WW Domain-Binding Protein 4.
    Yuhang Liu, Lu Chen, Bo Peng, Pingping Zeng, Linhui Cao, Lu Huang, Lixin Zhou, Shuke Yang, Jun Wang, Yanli Sun, Lu Chen, Yixuan Feng, Taiting Shi, Qi Chen, Kehao Zhao, Jing Lu, Baishan Jiang, Wenchao Lu.
      Targeted protein degradation via molecular glues represents a powerful modality for modulating "undruggable" proteins. Herein, through proteomic profiling of a CRBN-binding library and rigorous structure-activity relationship (SAR) refinement, we report the discovery of dWBP4-1: a first-in-class, highly selective, CRBN-dependent molecular glue degrader of the spliceosome-associated scaffold protein WBP4. dWBP4-1 induces rapid, nanomolar degradation of WBP4 via a canonical G-loop-mediated mechanism, exhibiting exceptional proteome-wide selectivity with negligible transcriptomic or alternative splicing perturbation. Leveraging this highly specific target-glue interaction, we mapped the minimal WBP4 degron to a 41-amino-acid sequence to establish a compact, inducible chemical-genetic platform termed wTAG. When fused to diverse proteins of interest, wTAG enables robust, monotonic degradation devoid of the hook effect. While the wTAG system is highly versatile, we delineate its boundaries when applied to challenging targets like Cyclin D1, where factors such as steric hindrance, lysine availability, complex sequestration, and tag accessibility (N- vs. C-terminal fusion) must be carefully interrogated. Collectively, this study highlights the discovery of a highly selective WBP4 molecular glue and translates its underlying degron into a robust tool for precise protein control.
    Keywords:  WBP4; molecular glue degrader; scaffold protein; targeted protein degradation; wTAG
    DOI:  https://doi.org/10.1002/anie.1973691
  56. Plant J. 2026 Aug;127(3): e71077
    COG7 links Golgi integrity to stress signaling and senescence.
    María Fernanda Gómez-Méndez, Yahir Oseguera, Genesis Oseguera, Jingzhe Guo, Rachel Wu, Tyler Inskeep, Jinzheng Wang, Heeseung Choi, Katayoon Dehesh.
      The mechanisms linking Golgi function to stress adaptation and senescence remain poorly understood. Here, we identify the conserved oligomeric Golgi (COG) subunit COG7 as a non-redundant determinant of Golgi integrity and stress adaptation in Arabidopsis thaliana. Functional disruption of COG7 reduces Golgi size, enhances Rapid Stress Response Element (RSRE)-dependent stress signaling, and accelerates dark-induced senescence. Complementation analyses reveal functional specialization within the COG complex, as only COG3, COG5, and COG6 partially restore stress signaling and senescence phenotypes. At the molecular level, cog7 exhibits altered glycosylation, increased ubiquitination, and elevated autophagy. However, disruption of glycosylation pathways or dark-induced candidate glycosyltransferases does not affect RSRE activation, proteostasis-associated responses, or senescence progression, indicating that glycosylation changes are downstream consequences rather than drivers of the stress phenotype. Similarly, CAMTA3-dependent RSRE activation is genetically separable from senescence and proteostasis pathways. Together, these findings show that Golgi dysfunction generates multiple parallel outputs rather than a single linear stress pathway and establish COG7 as a central regulator linking Golgi integrity to stress signaling, proteostasis, and senescence during dark-induced stress.
    Keywords:  COG complex; Golgi apparatus; autophagy; proteostasis; senescence; stress signaling
    DOI:  https://doi.org/10.1111/tpj.71077
  57. bioRxiv. 2026 Jul 19. pii: 2026.07.16.739079. [Epub ahead of print]
    Toxoplasma TgATG2 works with TgATG9 and TgProp1 to drive autophagic flux for parasite extracellular survival and persistence.
    Silvia Masci, Pariyamon Thaprawat, Federica Piro, Tracey L Schultz, Vern B Carruthers, Manlio Di Cristina.
      Chronic infection by Toxoplasma gondii depends on long-term survival of bradyzoites within tissue cysts, a parasite stage highly resistant to current therapies and a major barrier to eradication. Autophagy has emerged as critical pathway for bradyzoite persistence, yet the core machinery driving autophagosome formation in T. gondii remains poorly defined. Here, we identify TGME49_304630 as TgATG2, a previously uncharacterized, unusually large ATG2-like protein with conserved structural features of lipid-transfer factors. TgATG2 associates with TgATG9 and TgPROP1, key components of the parasite autophagy pathway, supporting its role in a membrane expansion complex required for autophagosome biogenesis. Using independent genetic knockouts, we show that TgATG2 is dispensable for intracellular tachyzoite replication but required for parasite fitness during extracellular stress and, most critically, for bradyzoite autophagy and viability. TgATG2 ablation disrupts autophagic activity in bradyzoites, causing progressive loss of viability and compromised cyst integrity. To overcome limitations of previous indirect assays, we developed a bradyzoite-specific dual-fluorescence TgATG8 reporter that quantitatively measures autophagic flux in T. gondii and confirmed TgATG2 as a major contributor. Importantly, TgATG2-deficient parasites are severely impaired during chronic infection in mice, with reduced brain cyst burdens, abnormal cyst morphology, and markedly diminished ex vivo bradyzoite viability. Together, these findings establish TgATG2 as a central component of the T. gondii autophagy machinery, demonstrate that autophagosome biogenesis is critical for parasite persistence in vivo, and define a molecular vulnerability and quantitative platform for targeting autophagy-dependent parasite survival.
    DOI:  https://doi.org/10.64898/2026.07.16.739079
  58. bioRxiv. 2026 Jul 16. pii: 2026.07.15.738729. [Epub ahead of print]
    ER shaping proteins guide spindle elongation and division during rapid cleavage mitoses.
    Katherine R Rollins, Austin R Clark, Prakriti Kandel, Schuyler B van Engelenburg, J Todd Blankenship.
      The ER is a complex network of membranes that inhabits much of the cytoplasm of cells - however, this network undergoes a massive condensation and rapid remodeling during cell division. In Drosophila cleavage divisions, this results in a tight association of the ER with centrosomes and mitotic spindle poles. Previous work has shown that this relationship between the ER and centrosomes must be finely tuned to enable successful spindle elongation, and that overaccumulation of the ER in these stages can result in failed centrosome maturation. During interphase, the ER exists in tubular and sheet-like arrangements, with a variety of "shaping" proteins enforcing these topologies. Here, we examine the contributions of these ER shaping proteins to the rapid changes that occur during cleavage mitoses in the Drosophila embryo. A screen of ER shaping proteins revealed that disruption of Reep-family proteins leads to mitotic failures at characteristic cleavage stages. Compromising ReepA , the Drosophila ortholog of the Reep1-4 subfamily, had a lesser impact on early embryonic mitoses. However, ReepB (the ortholog of the Reep5-6 subfamily) disruption, significantly affects ER mitotic coat morphologies, resulting in a 'frilled ER' phenotype and a reduction of ER adherence to the spindle space accompanied by division failures. Overexpressing ReepA does not rescue ReepB mitotic or ER morphology defects and instead introduces local condensates of abnormal ER structures. These data suggest that dedicated Reep proteins guide ER mitotic properties at specific early developmental stages. Using a cell-based in vitro analysis of Drosophila Reeps, we identify differential "tubulating" properties of ReepA and ReepB. Together these data suggest that the minutes-scale ER remodeling required for early mitoses is governed by shaping proteins, and that ReepB family members are especially important in some of the most rapid cleavage divisions that occur in early embryo.
    DOI:  https://doi.org/10.64898/2026.07.15.738729
  59. Nat Struct Mol Biol. 2026 Jul 30.
    Protein thiol alterations drive pathologic liquid-liquid phase separation in the aging brain.
    Thibaut Vignane, Martín Hugo, Christian Hoffmann, Polina Reichert, Antonia Katsouda, Davide D'Andrea, Han Wang, Johannes V Tromm, Marko Miler, Ferran Comas, Dunja Petrovic, Suyuan Chen, Jan Lj Miljkovic, Danail Stoychev, Erik Lacko, Vladimir M Jovanovic, Suwarna Chakraborty, Sunil J Tripathi, Edwin Vázquez-Rosa, Jordan L Morris, Suvagata Roy Chowdhury, Julien Prudent, Albert Sickmann, Natalija Polovic, Andrew A Pieper, Ilan Davis, Kostas Tokatlidis, Bindu D Paul, Michael P Murphy, Christian Münch, Andreas Papapetropoulos, Dragomir Milovanovic, Milos R Filipovic.
      Cellular homeostasis relies on regulation of processes, including protein post-translational modifications (PTMs) and biomolecular condensation. Aging disrupts the equilibrium of these processes, increasing susceptibility to disease and mortality. Here we used chemoproteomic techniques to generate an atlas of cysteine PTMs in the mouse brain and showed that age-related increases in thiol oxidation promoted the formation of biomolecular condensates. By contrast, protein persulfidation, regulated by hydrogen sulfide production, inhibited biomolecular condensation, preserving protein function. Age-induced alterations in cysteine PTMs influenced the phase separation properties of synapsin 1 and G3BP2, leading to impaired neurotransmitter release and defective stress granule formation and resolution, features associated with aging and neurodegenerative diseases. Mice deficient in cystathionine γ-lyase, the enzyme responsible for hydrogen sulfide production, exhibited reduced lifespans and spontaneously developed protein aggregates with age. Our results highlight the therapeutic potential of protein persulfidation in reversal of dysregulated biomolecular condensation and suggest that sulfide donors could be used to mitigate age-related diseases.
    DOI:  https://doi.org/10.1038/s41594-026-01857-w
  60. PLoS Biol. 2026 Jul;24(7): e3003833
    Structural basis for substrate recognition and inhibition of human glucose-6-phosphate transporter SLC37A4.
    Hui Li, Xiaomin Peng, Yuwen Huang, Wencheng Wu, Nan Li, Ziqi Cheng, Xuepeng Wei.
      Glucose 6 phosphate (G6P) homeostasis is essential for maintaining blood glucose levels and coordinating anabolic and catabolic pathways. A key step in this process is the delivery of G6P into the endoplasmic reticulum (ER), where it is hydrolyzed by glucose 6 phosphatase to glucose and inorganic phosphate (Pi). This transport step is carried out by the ER carrier SLC37A4 (also known as the G6P transporter, G6PT), which imports G6P into the ER lumen while exporting Pi to the cytosol, and loss-of-function mutations in SLC37A4 cause glycogen storage disease type Ib. Despite its central role in G6P homeostasis, how SLC37A4 recognizes G6P and couples its transport to Pi antiport has remained unclear. Here we report cryo-electron microscopy structures of human SLC37A4 in three states: the apo form at 2.8 Å resolution, a G6P-bound state at 3.2 Å resolution and a chlorogenic acid (CHA) bound state at 3.3 Å resolution. SLC37A4 adopts the canonical Major Facilitator Superfamily fold and harbors a central, positively charged cavity that accommodates anionic substrates. In the G6P-bound structure, SLC37A4 adopts an outward-open conformation facing the ER lumen, in which G6P binds to the electropositive pocket. In the CHA-bound structure, SLC37A4 adopts an inward-facing conformation, with CHA bound at a cytosolic site that locks the transporter in an arrested state and prevents the conformational transitions required for G6P/Pi exchange. Combined with thermostability and transport-based analyses of G6P binding and disease variants, these structures support a rocker switch mechanism in which electrostatic neutralization of the central positively charged cavity by anionic substrate drives the conformational changes underlying G6P/Pi exchange. Together, these findings define the structural basis of G6P/Pi exchange by SLC37A4, provide a molecular rationale for pathogenic mutations in glycogen storage disease type Ib, and provide a framework for targeting SLC37A4 to modulate G6P homeostasis.
    DOI:  https://doi.org/10.1371/journal.pbio.3003833
  61. J Cell Sci. 2026 Jul 15. pii: jcs265083. [Epub ahead of print]139(14):
    The LRRK2 G2019S variant rewires Rab GTPase phosphorylation after lysosomal damage and promotes cell death in human macrophages.
    Rebecca Morrison, Simeon R Mihaylov, Chak Hon Luk, Cyril Lemerle, Angela Rodgers, Natalia Athanasiadi, Huw R Morris, Enrica Pellegrino, Sila K Ultanir, Maximiliano G Gutierrez.
      Variants in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, yet how these variants alter immune cell function remains unclear. Because LRRK2 is activated by lysosomal damage in macrophages, we investigated how the pathogenic G2019S variant affects macrophage responses to lysosomal damage. Here, we show that LRRK2 G2019S has an effect during lysosomal damage through kinase-dependent and kinase-independent mechanisms. Phosphoproteomic analysis revealed that lysosomal damage induces selective rewiring of LRRK2-dependent Rab GTPase phosphorylation, characterised by increased Rab12 phosphorylation and reduced Rab35 phosphorylation without global kinase hyperactivation. Strikingly, LRRK2 G2019S macrophages showed increased susceptibility to apoptosis following lysosomal damage. This increase in cell death occurred independently of the kinase activity, indicating a distinct kinase-independent role of LRRK2 in regulating cell survival. We generated isogenic induced pluripotent stem cells from patients carrying the LRRK2 G2019S variant and confirmed that LRRK2 G2019S macrophages are more susceptible to cell death in a kinase-independent manner. Together, our findings support a model in which the LRRK2 G2019S variant selectively changes the phosphorylation of Rab GTPases in macrophages and increases cell death after lysosomal damage in macrophages.
    Keywords:  Apoptosis; LRRK2; Lysosomal damage; Macrophage; Parkinson's disease; Rab GTPase
    DOI:  https://doi.org/10.1242/jcs.265083
  62. bioRxiv. 2026 Jul 17. pii: 2026.07.16.739020. [Epub ahead of print]
    A machine learning model predicts protein stability of annotated and alternate protein isoforms.
    Océane Marescal, Iain M Cheeseman.
      The regulation of protein stability is essential for cellular homeostasis and is determined by a combination of intrinsic sequence motifs and extrinsic recognition enzymes. Despite growing knowledge of the protein degradation machinery, the ability to predict a protein's stability from its amino acid sequence remains challenging. Here we develop a machine learning model to predict protein stability from N-terminal amino acid sequences. Using our model and experimental validation, we identify known and novel sequence motifs governing protein stability. We additionally use this model to predict the stability of alternative translational isoforms with distinct N-termini produced from the same mRNA. Despite differing by a limited number of amino acids, we identify N-terminal isoforms with drastically different stabilities relative to their annotated counterparts, highlighting the potential of N-terminal extensions and truncations to regulate protein function. Together, this model provides a valuable tool for evaluating additional protein datasets and protein design strategies.
    DOI:  https://doi.org/10.64898/2026.07.16.739020
  63. bioRxiv. 2026 Jul 21. pii: 2026.07.16.739042. [Epub ahead of print]
    Altered neuronal start codon stringency favors cap-independent repeat-associated non-AUG translation.
    Clare M Wieland, Shannon E Wright, Sydney Willey, Ishita Purwar, Samantha J Grudzien, Amy Krans, Erinn Laimon, Melissa J Asher, Adrian Isaacs, Amanda L Garner, Peter K Todd.
      Intronic GGGGCC repeat expansions in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This expansion supports a non-canonical form of translational initiation known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat proteins that contribute to neurodegeneration. Here, we find that the efficiency of RAN translation and its dependency on the 5' 7-methylguanosine mRNA cap are variable across cell types, with both rodent neurons and human iNeurons favoring cap-independent RAN translation from two distinct repeats (CGG and GGGGCC) across multiple reading frames. Treatment with an eIF4E inhibitor that blocks global cap-dependent translation enhances RAN translation specifically in neurons. Intriguingly, cap-independent RAN translation exhibits less reliance on near-cognate codons for initiation than cap-dependent RAN translation. This finding led us to identify a surprising global increase in start codon stringency in neurons as a contributor to the relatively higher cap-independent RAN translation in this cell type. This effect correlates with a cytoplasmic redistribution of eIF1 in neurons and is reversed with neuronal overexpression of the eukaryotic initiation factor eIF5, which relaxes start codon stringency and selectively enhances cap-dependent RAN translation. Taken together, these findings reveal several neuron-specific features of translational regulation that favor cap-independent RAN translation with implications for nucleotide repeat expansion disorder pathogenesis and neuronal translational regulation.
    GRAPHICAL ABSTRACT:
    DOI:  https://doi.org/10.64898/2026.07.16.739042
  64. bioRxiv. 2026 Jul 14. pii: 2026.07.13.738256. [Epub ahead of print]
    Intermolecular disulfide bond formation promotes Hsp42 higher-order assembly and shapes client selection in yeast.
    Long Duy Duong, Daniel Escobar-Osorio, Alexander B Saltzman, Kevin A Morano.
      Cellular redox homeostasis plays a critical role in regulating protein function, including chaperone activity, through reversible oxidation of cysteine and methionine residues. Previously, we found that budding yeast cells experiencing redox imbalance due to inactivated thioredoxin reductase ( trr1 Δ) activate the heat shock response and induce hyperaccumulation of the small heat shock protein/sequestrase Hsp42 with misfolded proteins. Building on that finding, this study identified cysteine 127 (C127) within Hsp42 as a redox-active residue that becomes oxidized in trr1 Δ cells, upon treatment with the powerful oxidant hydrogen peroxide, or by exposure to the cysteine crosslinker divinyl sulfone (DVSF). In trr1 Δ cells, C127 oxidation promoted intermolecular disulfide bond formation and contributed to Hsp42 homo-oligomerization. We show that stable oligomerization requires both the prion-like domain (PrLD) and C127 oxidation. While Hsp42-GFP formed prominent persistent foci in trr1 Δ cells, replacement of C127 with non-thiol reactive serine decreased foci formation. Furthermore, the C127S mutation diminished Hsp42 oligomerization and sedimentability. Immunoprecipitation coupled with mass spectrometry analysis revealed that Hsp42 in trr1 Δ cells preferentially associated with mitochondrial precursor proteins accumulated in the cytoplasm, as well as oxidation-reduction enzymes. The observed client selectivity was altered by the C127S mutation that diversified the spectrum of Hsp42-associated proteins. Collectively, these findings identify Cys127 as a redox-active switch that regulates Hsp42 assembly, foci formation, stability, and client specificity in response to oxidative stress.
    DOI:  https://doi.org/10.64898/2026.07.13.738256
  65. Oncogene. 2026 Jul 30.
    OTUD6A promotes de novo pyrimidine synthesis by deubiquitinating and stabilizing CAD in cervical cancer.
    Liming Liao, Lizhou Zeng, Yiliang Zhao, Zihao Guo, Tong Zeng, Ronghui Yang, Yu Xu, Xuesong Wu, Hongkun Wu, Mengyao Chen, Tao Zhu, Yapeng Ji, Binghui Li, Chuanzhen Yang.
      Carbamoyl-phosphate synthetase II, aspartate transcarbamylase and dihydroorotase (CAD) is a multifunctional, rate-limiting enzyme involved in de novo pyrimidine synthesis. Its activity is tightly regulated, primarily through phosphorylation and allosteric mechanisms. However, the contribution of other post-translational modifications to CAD regulation remains largely unexplored. Here we identify ubiquitination as a novel regulatory mechanism controlling CAD stability. We show that CAD undergoes K29-linked ubiquitination and proteasomal degradation following ubiquitination at lysine residues K1325 and K1411 within its carbamoyl-phosphate synthetase II (CPS II) domain. Inhibiting CAD ubiquitination by mutating these lysine sites promotes its stability, de novo pyrimidine synthesis and tumor growth. Notably, CAD protein expression is elevated in cervical cancer and is associated with poor prognosis. Furthermore, we identify OTU domain-containing protein 6 A (OTUD6A) as a deubiquitylase that directly interacts with the CPS II domain of CAD, leading to its deubiquitylation and stabilization. OTUD6A overexpression enhances de novo pyrimidine synthesis and tumor growth in a CAD-dependent manner. OTUD6A is also upregulated in cervical cancer, positively correlating with CAD protein levels and poor prognosis of cervical cancer patients. Collectively, our results reveal the OTUD6A-CAD axis as a critical regulator of pyrimidine metabolism in cervical cancer, highlighting a potential vulnerability for therapeutic targeting.
    DOI:  https://doi.org/10.1038/s41388-026-03909-3
  66. Cell Rep. 2026 Jul 25. pii: S2211-1247(26)00817-X. [Epub ahead of print]45(8): 117739
    Human GBP4 promotes TRIM21-BIP-dependent autophagy to restrict M. tuberculosis infection by preventing SORT1-mediated progranulin degradation.
    Qinglong Guo, Jing Bi, Yu Fu, Li Song, Haojia Wu, Xi Chen, Yaqi Gong, Carl G Feng, Guoliang Zhang.
      Interferons (IFNs) are potent antimicrobial cytokines. However, effector mechanisms mediating their function in humans are poorly understood, partly because IFNs can induce numerous effector molecules. While guanylate-binding proteins (GBPs) are IFN-inducible, their role in cell-autonomous resistance to intracellular pathogens is incompletely understood. We demonstrate that human GBP1-5 significantly inhibits intracellular Mycobacterium tuberculosis (Mtb) survival, but only GBP4 mediates autophagy. GBP4 facilitates Mtb containment by promoting lysosomal acidification and autophagy via type Ⅰ IFN-dependent TFEB and FOXO3a activation. Mechanistically, GBP4 binds progranulin (GRN), reducing SORT1-mediated GRN lysosomal degradation. Moreover, GBP4 facilitates the GBP4-GRN-BIP-AKT complex assembly while inhibiting the GBP4-BIP-TRIM21 complex formation, thereby enhancing GRN-mediated BIP accumulation and AKT degradation. This leads to AKT inhibition, and concomitant TFEB and FOXO3a activation. GRN, BIP, AKT, and TRIM21 are essential for GBP4-mediated mycobactericidal activity. Our study uncovers a key role for GBP4 in regulating cell-autonomous resistance in human macrophages and may facilitate the development of host-directed therapies against tuberculosis (TB).
    Keywords:  AKT; BIP; CP: microbiology; CP: molecular biology; GRN; IFNs; Mycobacterium tuberculosis; TRIM21; autophagy flux; human GBP4; lysosomal acidification
    DOI:  https://doi.org/10.1016/j.celrep.2026.117739
  67. Nat Methods. 2026 Jul 30.
    Inference of secreted protein signaling activities in intercellular communication.
    Beibei Ru, Lanqi Gong, Emily Yang, Seongyong Park, George Zaki, Kenneth Aldape, Lalage Wakefield, Peng Jiang.
      The human genome encodes ~1,900 secreted proteins, many of which mediate intercellular communication. Secreted proteins do not act cell-autonomously, limiting systematic approaches to characterize their functions. Here we introduce SecAct (Secreted Activity, https://secact.ccr.cancer.gov ), a computational framework that infers the signaling activities of 1,170 human secreted proteins from spatial, single-cell and bulk transcriptomic data. The inference model harnesses precomputed intercellular signaling signatures trained on 1,258 spatial transcriptomics samples spanning 37 cancer types. Transcriptomics data from antisecreted protein therapies validate SecAct's accuracy in predicting the repression of secreted protein activity following treatment. For spatial and single-cell transcriptomics data, SecAct provides interactive modules for analyzing secreted protein-mediated cell-cell communication. Applying SecAct to 54 cancer immunotherapy cohorts comprising 5,174 patients, we identified secreted proteins associated with tumor immunity. In vivo experiments validated lymphocyte antigen 86 (LY86), whose function in cancer was previously unknown, as an antitumor regulator.
    DOI:  https://doi.org/10.1038/s41592-026-03172-0
  68. iScience. 2026 Aug 21. 29(8): 116785
    LRMDA promotes intestinal mucosal barrier integrity and limits colitis and colon cancer.
    Liping Yang, Yuyao Guo, Bikun Xiao, Di Wang, Bo Zhong, Zhi-Dong Zhang, Dandan Lin.
      Intestinal homeostasis relies on the integrity of the mucosal barrier, whose disruption contributes to inflammatory bowel disease (IBD), enteric infection, and colorectal cancer (CRC). Here, we report that leucine-rich melanocyte differentiation-associated protein (LRMDA) is markedly downregulated in inflamed mucosal tissues from patients with IBD and in colorectal tumor tissues. Genetic ablation of LRMDA in mice exacerbates dextran sulfate sodium (DSS)-induced colitis, enhances colorectal tumorigenesis in both azoxymethane (AOM)/DSS and AOM/Vil-Cre;Trp53 fl/fl models, and increases susceptibility to enteric pathogen infection. LRMDA deficiency is associated with impaired mucin-related gene expression, reduced mucus layer thickness, and compromised intestinal barrier integrity. Ultrastructural analyses further suggest that LRMDA may support mucin granule transport and secretion in goblet cells. Collectively, these findings identify LRMDA as a previously unrecognized contributor to intestinal mucosal barrier integrity and intestinal homeostasis under inflammatory and tumorigenic conditions.
    Keywords:  LRMDA; colitis; colon cancer; intestinal barrier integrity; mucin
    DOI:  https://doi.org/10.1016/j.isci.2026.116785
  69. Nat Biotechnol. 2026 Jul 29.
    An AI-enabled structural atlas decodes kinase specificity across the human proteome.
    David R Vanderwall, Edward L Huttlin, Julian Mintseris, Tomer M Yaron-Barir, Jared L Johnson, Kevin D Dong, Alex J Bott, Yuchen He, Christina B Schroeter, Geordon A Frere, Mohamed Uduman, Harin Lee, Sean Landry, Sean A Beausoleil, Joao A Paulo, Lewis C Cantley, Steven P Gygi.
      Of the 1.8 million serine/threonine/tyrosine residues in the human proteome, only 6% bear experimental validation of phosphorylation, and only 5% of these have been mapped to a kinase. Here we present KinoPlex, a computational framework that integrates predicted protein structures and kinase recognition motifs to assign phosphorylation potential and kinase specificity to all serine/threonine/tyrosine residues. Using ~20,000 AlphaFold models and positive-unlabeled transfer learning, we identified ~567,000 residues as structurally phospho-competent. We intersected these with kinase position-specific scoring matrices to quantify motif specificity, yielding ~250,000 high-confidence candidates with sequence recognition potential and optimal structural presentation. The structural atlas uncovered fundamental organizing principles guiding kinase substrate recognition and dynamics of phosphorylation, including a phenomenon we call sequence-structure selective coupling, whereby kinases achieve specificity through structural scarcity of their preferred motif (negative-selecting kinases) or promiscuity through its structural accessibility (positive-selecting kinases), rather than by motif discrimination alone. Deep phosphoproteomics in K562 cells validates KinoPlex predictions and kinase enrichment capacities.
    DOI:  https://doi.org/10.1038/s41587-026-03239-5
  70. Sci Bull (Beijing). 2026 Jul 23. pii: S2095-9273(26)00841-8. [Epub ahead of print]
    GABPA recruits the Integrator endonuclease complex to promote transcription elongation.
    Hang Yang, Jinjing Geng, Hanghong Yu, Danyi Lu, Hantian Zhao, Wusa Baqie, Yuhang Chen, Fei Xavier Chen, Chunxia Zhang.
      Transcription factors (TFs) coordinate gene regulatory programs essential for cell identity, yet how individual TFs modulate distinct steps of the transcription cycle remains incompletely understood. GA-binding protein alpha (GABPA), an erythroblast transformation-specific (ETS)-family TF, is crucial for naïve pluripotency during mouse preimplantation development, but its molecular functions remain elusive. Using an acute protein degradation system, we dissected GABPA activity with high temporal resolution, thereby identifying its primary transcriptional targets and underlying mechanisms while minimizing the secondary effects associated with conventional gene knockout approaches. We found that GABPA is essential for mouse embryonic stem cell (mESC) viability through a previously unrecognized mechanism independent of its canonical heterotetrameric partner, GABPB. Mechanistically, GABPA physically interacts with the INTS4/9/11 endonuclease module of the Integrator complex to promote the productive elongation of RNA polymerase II (Pol II) at genes involved in ribonucleoprotein complex biogenesis. Acute loss of GABPA leads to the accumulation of promoter-proximal Pol II and a reduction in Ser2-phosphorylated Pol II across gene bodies, indicating defective transcriptional elongation, whereas prolonged depletion leads to a broader collapse of transcription-associated chromatin features and the general transcription machinery. Together, our study redefines GABPA as a multifaceted transcriptional regulator that acts independently of GABPB and provides a framework for the temporally resolved analysis of TF function in stem cell biology.
    Keywords:  GABPA; Integrator; RNA Pol II; Transcription elongation
    DOI:  https://doi.org/10.1016/j.scib.2026.07.060
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