bims-proteo Biomed News
on Proteostasis
Issue of 2025–11–23
fifty-six papers selected by
Eric Chevet, INSERM
  1. A degron-mimicking molecular glue drives CRBN homo-dimerization and degradation.
  2. Role of p62 nuclear condensates in regulating ubiquitin-mediated proteasomal degradation.
  3. RAF inhibitors activate the integrated stress response by direct activation of GCN2.
  4. A UBH-UBX module amplifies p97/VCP's unfolding power to facilitate protein extraction and degradation.
  5. Chromosome duplication causes premature aging via defects in ribosome quality control.
  6. Divergent proteome tolerance against gain and loss of chromosome arms.
  7. Hydroxyurea induces an oxidative stress response that triggers ER expansion and cytoplasmic protein aggregation.
  8. HSP90 buffers deleterious genetic variations in BRCA1.
  9. Structural basis of regulated N-glycosylation at the secretory translocon.
  10. Deacetylation of HSC70 by SIRT2 promotes chaperone mediated autophagy.
  11. Structures of human organellar SPFH protein complexes.
  12. TUSC3 serves as a rate-limiting gatekeeper of a glycan-mediated ER Triage Checkpoint for BMP4/Dpp.
  13. The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.
  14. Deubiquitinases cleave ubiquitin-fused ribosomal proteins and physically counteract their targeting to the UFD pathway.
  15. Plasma membrane-endoplasmic reticulum coupling probed with genetically-encoded voltage sensors.
  16. Enrichment-Free, Targeted Covalent Drug Discovery in Live Cells.
  17. Lysosome-Mediated Targeted Protein Degradation: Emerging Chimeric Platforms for Extracellular and Intracellular Therapeutics.
  18. Beyond the nucleus: the ATM-CHEK2 axis senses mtROS to orchestrate Mitophagy.
  19. Mechanism of cooperative strigolactone perception by the MAX2 ubiquitin ligase-receptor-substrate complex.
  20. ZAK activation at the collided ribosome.
  21. Getting sticky: How nuclear speckles tune the condensation-prone proteome.
  22. Limiting ER-associated degradation capacity triggers acute and chronic effects on insulin biosynthesis.
  23. From The Integrated Stress Response to Oxidative Stress; A Historical Perspective.
  24. REEP1 Accumulation Disrupts ER Integrity and Drives Spinal Motoneuron Degeneration in Distal Hereditary Motor Neuropathy.
  25. Pyrazolone-based ERO1 inhibitors in ERO1-driven Triple-Negative Breast Cancer and SEPN1-Related Myopathy: Structure-activity relationship and therapeutic potential.
  26. Computational design of pH-sensitive binders.
  27. From stress to homeostasis: Mass spectrometry-based insights into the unfolded protein response (UPR) and proteostasis.
  28. Coronaviral nsp6 hijacks ERAD machinery to facilitate lipolysis and supply membrane components for DMV growth.
  29. The canonical ER stress IRE1α/XBP1 pathway mediates skeletal muscle wasting during pancreatic cancer cachexia.
  30. Tubulation of membrane sheets by curvature-inducing proteins.
  31. Multiplex neurodegeneration proteotoxicity platform reveals DNAJB6 promotes non-toxic FUS condensate gelation and inhibits neurotoxicity.
  32. Recent Insights into Mechanisms Regulating the Proteasome: Implications for Human Disease.
  33. The lactate sensor NDRG3 decelerates ER-to-Golgi transport through interaction with the long isoform of syntaxin-5.
  34. Design and evaluation of an HSP70-targeting PROTAC in synergy with an HSF1 inhibitor for enhanced antitumor activity.
  35. DnaJB1 chaperone inhibits tau aggregation by recognizing its N-terminus.
  36. Giant DNA viruses encode a hallmark translation initiation complex of eukaryotic life.
  37. Distinct cis-acting elements combinatorically mediate co-localization of mRNAs encoding for co-translational interactors in cytoplasmic clusters in S. cerevisiae.
  38. Age-Related Alterations in the Expression of Mesencephalic Astrocyte-derived Neurotrophic Factor in the Brain and Their Impact on Neurobehavioral Functions.
  39. Integrative profiling of condensation-prone RNAs during early development.
  40. Disrupted glycosylphosphatidylinositol anchoring induces ER stress and restricts enterovirus infection.
  41. The translational landscape of reactive astrocytes reveals the impact of eIF2B-mediated dysregulation in VWM disease.
  42. Chemical inhibition of exon junction complex assembly impairs mRNA localization and neural stem cells ciliogenesis.
  43. ER protein CLCC1 promotes nuclear envelope fusion in herpesviral and host processes.
  44. Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.
  45. Molecular insights on the mechanism of α1-antitrypsin condensate formation and maturation.
  46. Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 Axis in PEX1-depleted HeLa cells.
  47. Update on VAP, a ubiquitous signpost for the ER.
  48. UFL1-mediated UFMylation antagonizes IFT88 ubiquitination and degradation to maintain ciliary homeostasis.
  49. A ubiquitin-like protein controls assembly of a bacterial type VIIb secretion system.
  50. Structural Studies of Nedicistrovirus IRES-Driven, Initiation Factor-independent Translation Shed Light on Key Steps of Eukaryotic Translation Elongation.
  51. Programmable initiation of mRNA translation by trans-RNA.
  52. Design, Synthesis, and Cellular Efficacy of Inositol-Requiring Enzyme Type 1 (IRE1α) Inhibitors.
  53. KLF5 enables dichotomous lineage programs in pancreatic cancer via the AAA+ ATPase coactivators RUVBL1 and RUVBL2.
  54. PROTACS- targeted protein degradation as a path to precision cancer therapeutics.
  55. Cholesterol Transport from ER to Outer Mitochondria by ERLIN2 in Steroid Metabolism.
  56. Leveraging protein language models to identify complex trait associations with previously inaccessible classes of functional rare variants.
  1. Nat Commun. 2025 Nov 19. 16(1): 10157
    A degron-mimicking molecular glue drives CRBN homo-dimerization and degradation.
    Gerasimos Langousis, Pablo Gainza, Moritz Hunkeler, Despoina Kapsitidou, Etienne J Donckele, Stefano Annunziato, Lars Wiedmer, Katherine F M Jones, Bradley DeMarco, Chao Quan, Richard D Bunker, Kevin J Lumb, Bernhard Fasching, John C Castle, Sharon A Townson, Débora Bonenfant.
      Cereblon (CRBN) is an E3 ubiquitin ligase widely harnessed for targeted protein degradation (TPD). We report the discovery of a molecular glue degrader (MGD), MRT-31619, that drives homo-dimerization of CRBN and promotes its fast, potent, and selective degradation by the ubiquitin proteasome system. Interestingly, the cryo-electron microscopy (cryo-EM) structure of the CRBN homodimer reveals a unique mechanism whereby two molecular glues assemble into a helix-like structure and drive ternary complex formation by mimicking a neosubstrate G-loop degron. This CRBN chemical knockout offers a valuable tool to elucidate the molecular mechanism of MGDs, to investigate its endogenous substrates and understand their physiological roles.
    DOI:  https://doi.org/10.1038/s41467-025-65094-3
  2. Essays Biochem. 2025 Nov 17. pii: EBC20253033. [Epub ahead of print]
    Role of p62 nuclear condensates in regulating ubiquitin-mediated proteasomal degradation.
    Chen Lulu-Shimron, Aaron Ciechanover, Victoria Cohen-Kaplan.
      The ubiquitin-proteasome system (UPS) is essential for maintaining cellular proteostasis by selective proteasomal degradation of ubiquitinated proteins. Proper function of the UPS ensures turnover of proteins that have completed their role and removal of damaged proteins. Recent studies have identified p62/Sequestosome-1 as a key modulator of UPS efficiency, particularly through its ability to form dynamic, membraneless condensates via liquid-liquid phase separation. Within the nucleus, these structures recruit and concentrate components of the UPS, including its proteolytic arm - the 26S proteasome and ubiquitinated substrates. This organization enhances substrate recognition and degradation efficiency. Nuclear p62 condensates play an essential role in controlling the turnover of oncogenic proteins. Specifically, they facilitate the proteasomal degradation of the transcription factor c-Myc and prevent its nuclear accumulation by recruiting both c-Myc and its E3 ligase complex SCFFbxw7. Additionally, nuclear p62 condensates contribute to the maintenance of promyelocytic leukemia (PML) nuclear bodies and protect them from stress-induced disassembly by stabilizing the PML protein through sequestration and subsequent degradation of RING Finger Protein 4 (RNF4) - its major E3 ligase. Under stress conditions such as oxidative stress, heat shock, or DNA damage, p62 nuclear condensates rapidly assemble and recruit molecular chaperones and ubiquitin ligases, thereby promoting the clearance of misfolded and damaged proteins. Loss of nuclear p62 or disruption of its condensate-forming domains affects UPS function and compromises proteostasis. These findings highlight the role of p62 condensates in coordinating nuclear protein quality control and protecting cells from proteotoxic and oncogenic stress.
    Keywords:  LLPS condensates; p62; proteasome; protein degradation; ubiquitin
    DOI:  https://doi.org/10.1042/EBC20253033
  3. Nat Commun. 2025 Nov 17. 16(1): 10033
    RAF inhibitors activate the integrated stress response by direct activation of GCN2.
    Rebecca Gilley, Andrew M Kidger, Graham Neill, Eve Morrison, Paul Severson, Dominic P Byrne, Niall S Kenneth, Gideon Bollag, Chao Zhang, Taiana Maia de Oliveira, Patrick A Eyers, Richard Bayliss, Glenn R Masson, Simon J Cook.
      Paradoxical activation of wild type RAF by chemical RAF inhibitors (RAFi) is a well-understood 'on-target' biological and clinical response. In this study, we show that a range of RAFi drive ERK1/2-independent activation of the Unfolded Protein Response (UPR), including expression of ATF4 and CHOP, that requires the translation initiation factor eIF2α. RAFi-induced ATF4 and CHOP expression was not reversed by inhibition of PERK, a known upstream activator of the eIF2α-dependent Integrated Stress Response (ISR). Rather, RAFi exposure activated GCN2, an alternate eIF2α kinase, leading to eIF2α-dependent (and ERK1/2-independent) ATF4 and CHOP expression. The GCN2 kinase inhibitor A-92, GCN2 RNAi, GCN2 knock-out or ISRIB (an eIF2α antagonist) all reversed RAFi-induced expression of ATF4 and CHOP indicating that RAFi require GCN2 to activate the ISR. RAFi also activated full-length recombinant GCN2 in vitro and in cells, generating a characteristic 'bell-shaped' concentration-response curve, reminiscent of RAFi-driven paradoxical activation of WT RAF dimers. Activation of the ISR by RAFi was abolished by a GCN2 kinase dead mutation. A M802A GCN2 gatekeeper mutant was activated at lower RAFi concentrations, demonstrating that RAFi bind directly to the GCN2 kinase domain; this is supported by mechanistic structural models of RAFi interaction with GCN2. Since the ISR is a critical pathway for determining cell survival or death, our observations may be relevant to the clinical use of RAFi, where paradoxical GCN2 activation is a previously unappreciated off-target effect that may modulate tumour cell responses.
    DOI:  https://doi.org/10.1038/s41467-025-65376-w
  4. Nat Commun. 2025 Nov 19. 16(1): 10162
    A UBH-UBX module amplifies p97/VCP's unfolding power to facilitate protein extraction and degradation.
    Xin-Yu Huo, Di Liu, Rong Zou, Zhao-Peng Li, Yunxia Li, Lifeng Pan, Yaoyang Zhang, Zai-Rong Zhang.
      The p97-UFD1L-NPLOC4 ATPase unfolds numerous proteins for proteasomal degradation, but whether it suffices to pull proteins out of lipid bilayer remains unclear. Here, we identify a conserved ubiquitin-binding helix (UBH) in many UBX-containing p97 adapters, including FAF2, across yeast, plants, and metazoans. The UBH-UBX substantially facilitates the engagement of ubiquitinated substrates with p97-UFD1L-NPLOC4, and enhances p97 motor's working ATPase and unfolding activities by approximately twofold. Using purified p97-UFD1L-NPLOC4-FAF2UBH-UBX, we reconstitute membrane protein extraction from the ER and mitochondria, establishing p97-UFD1L-NPLOC4-FAF2 (p97-UNF) as a power-enhanced unfoldase. Deleting UBH or disrupting UBH-ubiquitin interaction impairs substrate targeting, reduces p97-UNF's working ATPase and unfolding activities, and abolishes membrane protein extraction and degradation. We propose that UBH-UBX module amplifies p97's mechanical output power, enabling the removal of challenging substrates from large assemblies and ensuring rapid responses to protein misfolding or regulatory signals in diverse physiological processes.
    DOI:  https://doi.org/10.1038/s41467-025-65166-4
  5. PLoS Biol. 2025 Nov;23(11): e3003509
    Chromosome duplication causes premature aging via defects in ribosome quality control.
    Leah E Escalante, James Hose, Jamie M Ahrens, Hollis Howe, Norah Paulsen, Sofia J Liss, Michael Place, Audrey P Gasch.
      Down syndrome, caused by an extra copy of Chromosome 21, causes lifelong problems. One of the most common phenotypes among people with Down syndrome is premature aging, including early tissue decline, neurodegeneration, and shortened life span. Yet the reasons for premature systemic aging are a mystery and difficult to study in humans. Here we show that chromosome amplification in wild yeast also produces premature aging and shortens life span. Chromosome duplication disrupts nutrient-induced cell-cycle arrest, entry into quiescence, and cellular health during chronological aging, across genetic background and independent of which chromosome is amplified. Using a genomic screen, we discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). We show that aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates compared to euploid cells. Although they maintain proteasome activity, aneuploids also show signs of ubiquitin dysregulation and sequestration into foci. Remarkably, inducing ribosome stalling in euploids produces similar aging phenotypes, while up-regulating limiting RQC subunits or poly-ubiquitin alleviates many of the aneuploid defects. We propose that the increased translational load caused by having too many mRNAs accelerates a decline in translational fidelity, contributing to premature aging.
    DOI:  https://doi.org/10.1371/journal.pbio.3003509
  6. Mol Cell. 2025 Nov 20. pii: S1097-2765(25)00864-0. [Epub ahead of print]85(22): 4268-4278.e6
    Divergent proteome tolerance against gain and loss of chromosome arms.
    Yi Di, Wenxue Li, Joan Josep Castellano, Wenjie Jin, Joanna N Modi, Barbora Salovska, Delyar Khosroabadi, Wei Hu, Alison M Taylor, Yansheng Liu.
      How aneuploid cells tolerate chromosome arm gains or losses remains an open question. Using an isogenic human lung cell model with either chromosome 3p loss or 3q gain, combined with quantitative mass spectrometry and isotopic labeling, we reveal distinct proteostasis mechanisms for gain- and loss-type aneuploidy. Surprisingly, while compensation for 3q gain is primarily driven by increased degradation of excess protein complex subunits, 3p loss is neither counteracted by global protein degradation nor selectively reduced degradation. Rather, there is a relative upregulation in protein synthesis of those 3p-encoded proteins that participate in stable protein complexes to maintain functional complex stoichiometry. Additionally, 3p-encoded proteins that are in a complex show increased thermal stability in loss-type aneuploidy, potentially via their interactions with other proteins from euploid chromosomes. Together, our findings uncover distinct proteomic buffering strategies that enable cells to tolerate either excessive or deficient single-arm aneuploidy.
    Keywords:  aneuploidy; chromosome 3; dynamic SILAC; gain and loss; lung cancer; protein degradation; protein synthesis; proteomic buffering; proteomics; proteostasis
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.023
  7. PLoS Biol. 2025 Nov 19. 23(11): e3003493
    Hydroxyurea induces an oxidative stress response that triggers ER expansion and cytoplasmic protein aggregation.
    Ana Sánchez-Molina, Manuel Bernal, Joel D Posligua-García, Antonio J Pérez-Pulido, Laura de Cubas, Elena Hidalgo, M-Henar Valdivieso, Silvia Salas-Pino, Rafael R Daga.
      The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is essential for the correct folding of proteins entering the secretory pathway and forming membranes. However, the precise mechanisms by which disruptions in protein folding within the ER activate proteostatic mechanisms remain to be fully elucidated. In this study, we demonstrate that in Schizosaccharomyces pombe the antineoplastic agent hydroxyurea (HU) induces a transient perinuclear ER expansion, Bip1 accumulation, and the clustering of nuclear pore complexes in a specific region of the nuclear envelope. This striking phenotype is mimicked by diamide (DIA), a specific inducer of thiol stress, and can be prevented or rapidly reversed by dithiothreitol, a reducing agent, suggesting that ER expansion results from disulfide stress. Furthermore, HU or DIA treatments resulted in the accumulation of misfolded proteins in cytoplasmic foci containing Hsp104 disaggregase and Hsp70/Ssa1 chaperones. Our data show that HU impacts redox-dependent protein folding, impairs the secretory pathway, and activates specific proteostatic mechanisms in both the ER and the cytoplasm.
    DOI:  https://doi.org/10.1371/journal.pbio.3003493
  8. Mol Cell. 2025 Nov 19. pii: S1097-2765(25)00867-6. [Epub ahead of print]
    HSP90 buffers deleterious genetic variations in BRCA1.
    Brant Gracia, Xing-Han Zhang, Patricia Montes, Tin Chanh Pham, Min Huang, Junjie Chen, Georgios Ioannis Karras.
      Protein-folding chaperone heat shock protein 90 (HSP90) buffers genetic variation in diverse organisms, but the clinical significance of HSP90 buffering in human disease remains unclear. Here, we show that HSP90 buffers mutations in the BRCT domain of BRCA1. HSP90-buffered BRCA1 mutations result in protein variants that retain interactions with partner proteins and strongly rely on HSP90 for protein stability and function in cell survival. Moreover, HSP90-buffered BRCA1 variants confer poly (ADP-ribose) polymerase (PARP) inhibitor resistance in cancer cells. Low-level HSP90 inhibition overcomes this resistance, revealing a cryptic and mutant-specific HSP90-contingent synthetic lethality. Furthermore, by stabilizing metastable variants across the entirety of the BRCT domain, HSP90 reduces the clinical severity of BRCA1 mutations, allowing them to accumulate in populations. We estimate that HSP90 buffers 18% of known human BRCA1-BRCT missense mutations. Our work extends the clinical significance of HSP90 buffering to a prevalent class of variations in BRCA1, pioneering its importance in therapy resistance and cancer predisposition.
    Keywords:  BRCA1; HSP90; HSP90 inhibition; PARP inhibition; breast cancer; mutational buffer; polytherapy; protein folding; structural mutations; synthetic lethality
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.026
  9. Nature. 2025 Nov 19.
    Structural basis of regulated N-glycosylation at the secretory translocon.
    Melvin Yamsek, Mengxiao Ma, Roshan Jha, Yu Wan, Qianru Li, Frank Zhong, Katherine DeLong, Zhe Ji, Rajat Rohatgi, Robert J Keenan.
      Most human secretory pathway proteins are N-glycosylated by oligosaccharyltransferase (OST) complexes as they enter the endoplasmic reticulum (ER)1-3. Recent work revealed a substrate-assisted mechanism by which N-glycosylation of the chaperone glucose-regulated protein 94 (GRP94) is regulated to control cell surface receptor signalling4. Here we report the structure of a natively isolated GRP94 folding intermediate tethered to a specialized CCDC134-bound translocon. Together with functional analysis, the data reveal how a conserved N-terminal extension in GRP94 inhibits OST-A and how structural rearrangements within the translocon shield the tethered nascent chain from inappropriate OST-B glycosylation. These interactions depend on a hydrophobic CCDC134 groove, which recognizes a non-native conformation of nascent GRP94. Our results define a mechanism of regulated N-glycosylation and illustrate how the nascent chain remodels the translocon to facilitate its own biogenesis.
    DOI:  https://doi.org/10.1038/s41586-025-09756-8
  10. Autophagy Rep. 2025 ;4(1): 2580781
    Deacetylation of HSC70 by SIRT2 promotes chaperone mediated autophagy.
    Byunghyun Ahn, Wenzhe Chen, Wenbiao Shi, Ruben Shrestha, Fenghua Hu, Hening Lin.
      Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation essential for cellular proteostasis. CMA is activated during cellular stress, such as starvation, and involves the chaperone protein HSC70 (HSPA8) recognizing substrates containing KFERQ-like motifs. However, the regulatory mechanisms governing CMA activation remain poorly understood. Here, we demonstrate that the NAD+ -dependent deacetylase SIRT2 promotes CMA activation by deacetylating HSC70 at lysine 557 (K557). Our findings reveal that SIRT2 activity is upregulated during starvation, enhancing its interaction with HSC70 and facilitating the deacetylation of K557. Deacetylation of HSC70 at K557 increases its binding affinity to CMA substrates, thereby promoting their lysosomal degradation. Mutation of K557 to a deacetylation-mimetic arginine (K557R) enhances CMA activity under both nutrient-rich and starvation conditions, while the acetylation-mimetic glutamine mutant (K557Q) impairs substrate binding and CMA activation. Furthermore, the inhibition or knockdown of SIRT2 reduces CMA activity, which is rescued by HSC70 K557R expression. These findings identify SIRT2-mediated deacetylation of HSC70 as a regulatory mechanism for CMA activation during nutrient deprivation and highlight the role of protein lysine acetylation in proteostasis. This study provides insights into the interplay between SIRT2, HSC70, and CMA, with potential implications for diseases linked to proteostasis dysregulation, including neurodegenerative disorders and cancer.
    Keywords:  Chaperone-mediated autophagy; HSC70; KFERQ motif; SIRT2; amino acids starvation; deacetylation; heat shock chaperones; lysosomes; protein degradation; sirtuin
    DOI:  https://doi.org/10.1080/27694127.2025.2580781
  11. Nat Commun. 2025 Nov 17. 16(1): 10064
    Structures of human organellar SPFH protein complexes.
    Jingjing Gao, Dawafuti Sherpa, Nikita Kupko, Haruka Chino, Jianwei Zeng, Sichen Shao.
      Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are found in all kingdoms of life and in multiple eukaryotic organelles. SPFH proteins assemble into homo- or hetero-oligomeric rings that form domed structures. Most SPFH assemblies also abut a cellular membrane, where they are implicated in diverse functions ranging from membrane organization to protein quality control. However, the precise architectures of different SPFH complexes remain unclear. Here, we report single-particle cryo-EM structures of the endoplasmic reticulum (ER)-resident Erlin1/2 complex and the mitochondrial prohibitin (PHB1/2) complex, revealing assemblies of 13 heterodimers of Erlin1 and Erlin2 and 11 heterodimers of PHB1 and PHB2, respectively. We also describe key interactions underlying the architecture of each complex and conformational heterogeneity of the PHB1/2 complex. Our findings elucidate the distinct stoichiometries and properties of human organellar SPFH complexes and highlight common principles of SPFH complex organization.
    DOI:  https://doi.org/10.1038/s41467-025-65078-3
  12. bioRxiv. 2025 Oct 03. pii: 2025.10.03.680295. [Epub ahead of print]
    TUSC3 serves as a rate-limiting gatekeeper of a glycan-mediated ER Triage Checkpoint for BMP4/Dpp.
    Antonio Galeone, Emilio Solazzo, Francesco Lavezzari, Seung-Yeop Han, Bruna My, Riccardo Rizzo, Giuseppe Gigli, Hamed Jafar-Nejad, Thomas Vaccari.
      Trimming of the three glucose residues decorating nascent N -glycoproteins is a critical step for their entry into the endoplasmic reticulum quality control (ERQC) cycle and recognition by ER chaperones. However, the functional relevance of the second glucose (G2) and the regulatory step upstream of its removal by ER glucosidase II (GCS2) remains poorly understood. Here, we report that TUSC3, a component of the oligosaccharyltransferase (OST) complex, regulates G2 to G1 trimming on N- glycosylated bone morphogenetic protein 4 (BMP4) and its Drosophila homolog Dpp to promote their ERQC entry. Loss- and gain-of-function genetic experiments and biochemical assays in mammalian cells and flies indicate that TUSC3 serves as a dosage-sensitive gatekeeper that influences the decision between proper folding and secretion versus elimination by ER-associated degradation for BMP4 molecules, thereby tuning BMP signaling. Together, these data reveal an unrecognized role for an OST component in early glycoprotein maturation, relevant to a major developmental signaling pathway.
    DOI:  https://doi.org/10.1101/2025.10.03.680295
  13. Mol Cell Proteomics. 2025 Nov 17. pii: S1535-9476(25)00570-5. [Epub ahead of print] 101471
    The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.
    Calvin P Lin, Nathan Lee, Francis X Alipranti, Harry Li, Elizabeth A Komives.
      The E3 ligase substrate receptor ankyrin and SOCS box protein 9 (ASB9) was shown to bind over 10 different proteins including metabolic enzymes such as creatine kinase, filament proteins such as vimentin, and histones. In previous work, we characterized the ASB9-Cullin 5 E3 ligase (ASB9-CRL5) ubiquitylation of creatine kinase and showed that ubiquitylation required the ring-between-ring ligase, ARIH2. Here, we characterized the ASB9-CRL5 ubiquitylation of histones and show that histones H3 and H4 are polyubiquitylated by the ASB9-CRL5 whereas histones H2A and H2B are much poorer substrates. Many, but not all lysines in the histones are ubiquitylated suggesting some substrate specificity. Binding experiments show that the ligase-histone interaction is highly electrostatic and the neddylated ASB9-CRL5 binds with the highest affinity. Histones in nucleosomes or in complex with the chaperone Asf1, are not ubiquitylated. Only K48 and K63 polyubiquitin chains were observed, suggesting that the ubiquitylation probably drives histone degradation. The presence of ASB9 in specific cell types correlates with situations in which free histones H3 and H4 need to be degraded. In this work, we demonstrate that the ASB9-CRL5 is the ligase that facilitates degradation of histones H3 and H4. In addition, this work represents the first example of Cullin-5 mediated ubiquitylation that does not require a ring-between-ring "helper" ligase.
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101471
  14. Mol Cell. 2025 Nov 20. pii: S1097-2765(25)00889-5. [Epub ahead of print]
    Deubiquitinases cleave ubiquitin-fused ribosomal proteins and physically counteract their targeting to the UFD pathway.
    Stephanie Patchett, Seyed Arad Moghadasi, Ankita Shukla, Farid El Oualid, Beatrix M Ueberheide, Shaun K Olsen, Tony T Huang.
      In eukaryotes, each ribosomal subunit includes a ribosomal protein (RP) that is encoded as a fusion protein with ubiquitin (Ub). In yeast, each Ub-RP fusion requires processing by deubiquitylating enzymes (DUBs) to generate ribosome assembly-competent RPs and contribute to the cellular Ub pool. However, how Ub-RP fusions are processed by DUBs in human cells remains unclear. Here, we discovered that Ub-RPs are substrates of the Ub-fusion degradation (UFD) pathway in human cells via lysine 29 and 48 (K29/K48)-specific ubiquitylation and proteasomal degradation. We identified a pool of DUBs that catalytically process Ub-RPs, as well as DUBs that physically occlude Ub-RP interaction with UFD pathway Ub E3 ligases to prevent their degradation in a non-catalytic manner. Our results suggest that DUBs both process and stabilize Ub-RPs, whereas the UFD pathway regulates levels of Ub-RPs that cannot be fully processed by DUBs to fine-tune protein homeostasis.
    Keywords:  Cezanne; DUBs; HUWE1; OTUD7B; RPL40; RPS27a; TRIP12; UBA52; UBA80; UFD; deubiquitinases; deubiquitinating enzymes; ubiquitin fusion degradation pathway
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.028
  15. bioRxiv. 2025 Oct 04. pii: 2025.10.03.680358. [Epub ahead of print]
    Plasma membrane-endoplasmic reticulum coupling probed with genetically-encoded voltage sensors.
    Masoud Sepehri Rad, Meyer B Jackson.
      The endoplasmic reticulum (ER) forms an elaborate contiguous network extending through the cytoplasm of eukaryotic cells. The ER is surrounded by a membrane that separates its lumen from the cytoplasm. The ER membrane harbors channels and pumps capable of controlling ion flux and creating a voltage gradient. Because the ER membrane potential is difficult to study experimentally little is known about how voltage influences its many vital functions. Here we introduce optical probes of ER membrane potential derived from the hybrid voltage sensor (hVoS) family of genetically-encoded voltage sensors. Probes were targeted to the ER using motifs from three ER proteins, Sec61β, cytochrome P450, and cytochrome b 5 type A. As shown recently with other types of ER voltage sensors, patch-clamp fluorometry recording with our new probes demonstrated that voltage steps applied to the plasma membrane elicit a voltage change at the ER membrane. These probes exhibited subtle differences in their responses suggesting they target different ER compartments. The steeper voltage dependence of Sec61β-hVoS (mCerulean3-Sec61β) signals suggested that this probe targets an ER compartment rich in voltage-gated ion channels. The ER voltage change is slow, but its onset is virtually synchronous with the plasma membrane voltage step. This suggests a direct electrical coupling into the ER lumen through plasma membrane-ER contacts. Analysis with the aid of an equivalent circuit provided an estimate of the resistance of these contacts. The rapid, direct transmission of voltage changes from the plasma membrane to the ER provides a mechanism for regulating ER function that could be especially important in excitable cells. The sensors introduced here provide researchers with powerful tools for imaging ER voltage and assessing its impact on cellular function.
    Significance: The ER is encompassed by a membrane, and the voltage across this membrane is likely to play important roles in many cellular functions. It is difficult to study this voltage due to the inaccessibility of organelle membranes. We introduced new hybrid genetically-encoded optical voltage sensors that target the ER. These probes showed that a voltage step applied to the plasma membrane changes the voltage at the ER membrane in a manner consistent with direct electrical coupling to the cell surface through contacts. This coupling will enable voltage changes at the plasma membrane to influence ER function. The probes introduced here will enable researchers to image ER voltage and probe plasma membrane-ER signaling.
    DOI:  https://doi.org/10.1101/2025.10.03.680358
  16. ACS Chem Biol. 2025 Nov 18.
    Enrichment-Free, Targeted Covalent Drug Discovery in Live Cells.
    Kevin D Dong, Qing Yu, Ka Yang, Bertrand J Wong, Hong Yue, Sipei Fu, Rebecca L Whitehouse, Eric S Fischer, Steven P Gygi.
      Live-cell activity-based protein profiling (ABPP) with mass spectrometry enables the proteome-wide quantification of compound reactivity, yet resulting datasets often suffer from low data completeness for high-priority targets and do not give users the option to measure compound-induced protein changes within the same screening assay. To address these limitations, we developed CysDig, an enrichment-free chemoproteomics platform for the targeted covalent drug discovery in live cells. Using the CysDig platform, we screened 288 cysteine-reactive electrophiles against 300 functionally annotated cysteine sites. From this screen, we identified covalent binders that liganded dozens of sites and identified multiple instances of acute compound-induced protein degradation of ACAT1. We validated a molecule that engaged with the active site of HECT E3 ligase HUWE1 and showed that chemical inhibition stabilized known substrates. Together, these findings establish CysDig as a powerful, targeted platform for live-cell covalent drug screening, expanding the current repertoire of available approaches for ligand discovery in live cells.
    DOI:  https://doi.org/10.1021/acschembio.5c00581
  17. Drug Dev Res. 2025 Dec;86(8): e70197
    Lysosome-Mediated Targeted Protein Degradation: Emerging Chimeric Platforms for Extracellular and Intracellular Therapeutics.
    Shayan Asadi, Mina Taheri-Torbati, Prashant Kesharwani, Amirhossein Sahebkar.
      Targeted protein degradation (TPD) is an emerging drug discovery approach aimed at enabling the selective removal of disease-associated proteins. While proteolysis-targeting chimeras (PROTACs) have advanced intracellular degradation via the ubiquitin-proteasome system, their limitation to cytosolic proteins excludes ~40% of the human proteome that is extracellular or membrane-bound. Lysosome-targeting chimeras (LYTACs) address this gap by harnessing lysosomal trafficking receptors, thereby mediating the degradation of extracellular and membrane proteins. More recently, methylarginine-targeting chimeras (MrTACs) have extended lysosomal strategies to certain intracellular targets, bypassing proteasomal dependence. This review critically examines the mechanistic underpinnings, design strategies, and bioanalytical challenges associated with lysosome-mediated degradation platforms. Emphasis is placed on their therapeutic implications, analytical evaluation, and potential for expanding druggable targets. Together, these emerging lysosomal chimeras offer a paradigm shift in TPD, with far-reaching applications in precision medicine and chemical biology.
    Keywords:  chimeric degraders; extracellular protein clearance; lysosome‐targeting chimeras (LYTACs); precision medicine; targeted protein degradation
    DOI:  https://doi.org/10.1002/ddr.70197
  18. Autophagy. 2025 Nov 19.
    Beyond the nucleus: the ATM-CHEK2 axis senses mtROS to orchestrate Mitophagy.
    Qi-Qiang Guo, Xiao-Yu Song, Liu Cao.
      Mitochondrial reactive oxygen species (mtROS) are typically viewed as harmful byproducts of stress. However, our recent study establishes their fundamental role as essential signaling molecules that activate a protective adaptive response. We discovered that mtROS serve as the specific trigger to activate the ATM-CHEK2/CHK2 DNA damage response pathway, which in turn coordinates the key steps of PINK1-PRKN/Parkin-dependent mitophagy. Upon activation by mtROS, CHEK2 phosphorylates ATAD3A to initiate PINK1 import arrest, OPTN to enhance cargo recognition, and BECN1 (beclin 1) to promote autophagosome formation. This work reveals a novel mtROS-driven signaling cascade, expanding the function of the ATM-CHEK2 pathway beyond the nucleus and positioning it as a central integrator of cellular homeostasis by responding to both genomic and mitochondrial stress.
    Keywords:  Dna damage response pathway; OPTN; PINK1; mitophagy; mtROS
    DOI:  https://doi.org/10.1080/15548627.2025.2592883
  19. Nat Commun. 2025 Nov 21. 16(1): 10291
    Mechanism of cooperative strigolactone perception by the MAX2 ubiquitin ligase-receptor-substrate complex.
    Alexandra I Vancea, Brandon Huntington, Wieland Steinchen, Christos G Savva, Umar F Shahul Hameed, Stefan T Arold.
      Strigolactones are plant hormones that regulate development and mediate interactions with soil organisms, including the germination of parasitic plants such as Striga hermonthica. Strigolactone perception by receptors initiates the degradation of transcriptional repressors via E3 ubiquitin ligases, but the mechanistic link between hormone binding and substrate ubiquitination has remained unclear. We determine cryogenic electron microscopy structures of the receptor-ligase-substrate complex, composed of Arabidopsis ASK1 and substrate, and Striga F-box and receptor proteins. Strigolactone hydrolysis by the receptor, which covalently retains the D-ring, is a prerequisite for complex formation. The substrate engages the complex through two domains, forming a dynamic interface that stabilises the receptor-ligase assembly and repositions the ASK1, suggesting a mechanism for efficient ubiquitination. Here, we show how dynamic, multivalent interactions within the receptor-ligase-substrate complex translate hormone perception into targeted protein degradation, providing insight into how plants integrate hormonal signals into developmental decisions.
    DOI:  https://doi.org/10.1038/s41467-025-65205-0
  20. Nature. 2025 Nov 19.
    ZAK activation at the collided ribosome.
    Vienna L Huso, Shuangshuang Niu, Marco A Catipovic, James A Saba, Timo Denk, Eugene Park, Jingdong Cheng, Otto Berninghausen, Thomas Becker, Rachel Green, Roland Beckmann.
      Ribosome collisions activate the ribotoxic stress response mediated by the MAP3K ZAK, which in turn regulates cell-fate consequences through downstream phosphorylation of the MAPKs p38 and JNK1. Despite the critical role of ZAK during cellular stress, a mechanistic and structural understanding of ZAK-ribosome interactions and how these lead to activation remain elusive. Here we combine biochemistry and cryo-electron microscopy to discover distinct ZAK-ribosome interactions required for constitutive recruitment and for activation. We find that upon induction of ribosome collisions, interactions between ZAK and the ribosomal protein RACK1 enable its activation by dimerization of its SAM domains at the collision interface. Furthermore, we discover how this process is negatively regulated by the ribosome-binding protein SERBP1 to prevent constitutive ZAK activation. Characterization of novel SAM variants as well as a known pathogenic variant of the SAM domain of ZAK supports a key role of the SAM domain in regulating kinase activity on and off the ribosome, with some mutants bypassing the ribosome requirement for ZAK activation. Collectively, our data provide a mechanistic blueprint of the kinase activity of ZAK at the collided ribosome interface.
    DOI:  https://doi.org/10.1038/s41586-025-09772-8
  21. Mol Cell. 2025 Nov 20. pii: S1097-2765(25)00890-1. [Epub ahead of print]85(22): 4116-4118
    Getting sticky: How nuclear speckles tune the condensation-prone proteome.
    Michaela Müller-McNicoll.
      Recent work by Faraway et al.1 uncovers interstasis-a feedback mechanism whereby the stiffening of nuclear condensates caused by the accumulation of condensation-prone resident proteins entraps mRNAs encoding these proteins, thereby limiting their translation to restore proteome balance.
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.029
  22. J Clin Invest. 2025 Nov 18. pii: e187341. [Epub ahead of print]
    Limiting ER-associated degradation capacity triggers acute and chronic effects on insulin biosynthesis.
    Anoop Arunagiri, Leena Haataja, Maroof Alam, Noah F Gleason, Emma Mastroianni, Chao-Yin Cheng, Sami Bazzi Onton, Jeffrey Knupp, Ibrahim Metawea, Anis Hassan, Dennis Larkin, Deyu Fang, Billy Tsai, Ling Qi, Peter Arvan.
      In pancreatic β-cells, misfolded proinsulin is a substrate for Endoplasmic Reticulum-Associated protein Degradation (ERAD) via HRD1/SEL1L. β-cell HRD1 activity is alternately reported to improve, or impair, insulin biogenesis. Further, while β-cell SEL1L deficiency causes HRD1 hypofunction and diminishes islet insulin content; reports conflict as to whether β-cell ERAD deficiency increases or decreases proinsulin levels. Here we've examined β-cell-specific Hrd1-KO mice (chronic deficiency), plus rodent (and human islet) β-cells treated acutely with HRD1 inhibitor. β-Hrd1-KO mice developed diabetes with decreased islet proinsulin yet a relative increase of misfolded proinsulin re-distributed to the ER; upregulated biochemical markers of β-cell ER stress and autophagy; electron microscopic evidence of ER enlargement and decreased insulin granule content; and increased glucagon-positive islet cells. Misfolded proinsulin was also increased in islets treated with inhibitors of lysosomal degradation. Preceding any loss of total proinsulin, acute HRD1 inhibition triggered increased nonnative proinsulin, increased phospho-eIF2ɑ with inhibited proinsulin synthesis, and increased LC3b-II (the abundance of which requires expression of SigmaR1). We posit a subset of proinsulin molecules undergoes HRD1-mediated disposal. When HRD1 is unavailable, misfolded proinsulin accumulates, accompanied by increased phospho-eIF2ɑ that limits further proinsulin synthesis, plus SigmaR1-dependent autophagy activation, ultimately lowering steady-state β-cell proinsulin (and insulin) levels - triggering diabetes.
    Keywords:  Beta cells; Cell biology; Endocrinology; Insulin
    DOI:  https://doi.org/10.1172/JCI187341
  23. J Biol Chem. 2025 Nov 19. pii: S0021-9258(25)02810-8. [Epub ahead of print] 110958
    From The Integrated Stress Response to Oxidative Stress; A Historical Perspective.
    Mehdi Amiri, Phoenix Toboz, Michael A Bellucci, Soroush Tahmasebi, Nahum Sonenberg.
      Oxidative stress has exerted fundamental evolutionary pressure since the emergence of aerobic life. Its impact on the physiology and function of all organisms is profound and consequential for cell survival. The integrated stress response (ISR) plays a critical role in counteracting oxidative stress via translational control of a subset of mRNAs. Here, we summarize the fundamental discoveries that shaped our understanding of the ISR pathway's role in cellular adaptation to oxidative stress from studies of protein synthesis in reticulocyte lysates to the regulation of glutathione metabolism downstream of the ISR pathway. We describe recent advances in studying mRNA translation changes in response to oxidative stress based on high throughput translatome analyses.
    Keywords:  Glutathione; ISR; Oxidative Stress; eIF2; mRNA Translation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110958
  24. Adv Sci (Weinh). 2025 Nov 21. e11483
    REEP1 Accumulation Disrupts ER Integrity and Drives Spinal Motoneuron Degeneration in Distal Hereditary Motor Neuropathy.
    Andrea Bock, Mona Schurig, Miles Willoughby, Andrea Mirecki, Eric Seemann, Kateryna Lohachova, Istvan Katona, Sonnhild Mittag, Lutz Liebmann, Patricia Franzka, Mehdi Heidari Horestani, Mukhran Khundadze, Thorsten Mosler, Timothy Louie, Marianne de Visser, Marian A J Weterman, Michael Kiehntopf, Christian Beetz, Sandor Nietzsche, Otmar Huber, Joachim Weis, Michael M Kessels, Ramachandra M Bhaskara, Britta Qualmann, Ivan Đikić, Christian A Hübner.
      REEP1 contributes to the shaping of the endoplasmic reticulum (ER) through conserved transmembrane hairpins and a long C-terminal amphipathic helix. REEP1 loss-of-function causes hereditary spastic paraplegia due to degeneration of cortical motoneuron axons. Patients with deletion of REEP1 exon5 (Δexon5), which deletes part of its amphipathic helix, however, develop muscle atrophy due to degeneration of spinal motoneuron axons (distal hereditary motor neuropathy/dHMN). It is known that REEP1 knockout mice exhibit simplified ER structures in cortical motoneurons. Here, we show that these neurons are progressively lost while spinal motoneurons remain intact. Conversely, Δexon5 knockin (KI) mice lose spinal motoneurons preceded by ER fragmentation, whereas cortical motoneurons remain intact. Mechanistically, REEP1 undergoes ubiquitination and proteasomal degradation, a process compromised in the Δexon5 variant due to impaired ubiquitination, which thus accumulates in peripheral nerves. Proteomic analysis identifies HUWE1 as the E3 ligase responsible for REEP1 turnover. Modeling and liposome shaping assays reveal that the Δexon5 variant retains its capacity to induce membrane curvature. Consistently, other REEP1 variants associated with dHMN also show compromised ubiquitination and preserved transmembrane hairpins. Therefore, it is proposed that accumulation of shaping-competent REEP1 variants in the ER drives ER fragmentation and spinal motoneuron degeneration in dHMN.
    Keywords:  HSP; REEP1; dHMN; endoplasmic reticulum; membrane shaping; ubiquitination
    DOI:  https://doi.org/10.1002/advs.202511483
  25. Pharmacol Res. 2025 Nov 19. pii: S1043-6618(25)00462-1. [Epub ahead of print] 108037
    Pyrazolone-based ERO1 inhibitors in ERO1-driven Triple-Negative Breast Cancer and SEPN1-Related Myopathy: Structure-activity relationship and therapeutic potential.
    Michele Retini, Alessandro Cherubini, Alice Marrazza, Ersilia Varone, Serena Germani, Giorgia Maria Renna, Adriano Recchia, Andrea Guidarelli, Stefano Fumagalli, Chiara Grasselli, Michele Mari, Giovanni Piersanti, Giovanni Bottegoni, Roberto Tonelli, Marco Gobbi, Bo-Ram Jin, Jaehyung Cho, Orazio Cantoni, Ester Zito.
      Endoplasmic reticulum oxidoreductin 1 alpha (ERO1A) is a disulfide oxidase that facilitates oxidative protein folding by reoxidizing protein disulfide isomerase (PDI), a process essential for maintaining endoplasmic reticulum (ER) homeostasis. Under ER stress, ERO1A expression is upregulated via the unfolded protein response (UPR), promoting cell survival. However, sustained ERO1A activity can impair proteostasis and contribute to disease. Notably, ERO1A is overexpressed in triple-negative breast cancer (TNBC), where it supports tumor growth and adaptation to hypoxia, and in SEPN1-related myopathy, a rare congenital muscle disorder linked to ER and oxidative stress. To investigate ERO1A as a therapeutic target, we conducted a structure-activity relationship (SAR) study of EN460-based pyrazolone inhibitors. Forty derivatives and three EN460 salts were synthesized to optimize potency and solubility. In vitro and cell-based assays revealed that effective inhibition required covalent binding to Cys397, interactions with Arg287 and Trp200, and distortion of the phenyl ring. While sulfonic acid substitution improved solubility, it abolished activity by disrupting key interactions. The most potent compound, I29, featuring a mono ortho-fluorine substitution, demonstrated improved inhibitory activity (IC₅₀ = 2.6µM) and efficacy in preclinical models of TNBC and SEPN1-related myopathy. These findings highlight ERO1A's pathological role in cancer and congenital muscle disease and support its inhibition as a promising therapeutic strategy for conditions characterized by chronic ER and oxidative stress.
    Keywords:  Breast cancer; ERO1A; SEPN1-related Myopathy; Stress of the endoplasmic reticulum (ER stress); UPR (Unfolded protein response)
    DOI:  https://doi.org/10.1016/j.phrs.2025.108037
  26. bioRxiv. 2025 Sep 29. pii: 2025.09.29.678932. [Epub ahead of print]
    Computational design of pH-sensitive binders.
    Green Ahn, Brian Coventry, Ella Haefner, Shayan Sadre, Jenny Hu, Mimosa Van, Buwei Huang, Isaac Sappington, Adam J Broerman, Mauriz A Lichtenstein, Matthias Glögl, Inna Goreshnik, Dionne Vafeados, David Baker.
      pH gradients are central to physiology, from vesicle acidification to the acidic tumor microenvironment. While therapeutics have been developed to exploit these pH changes to modulate activity across different physiological environments, current approaches for generating pH-dependent binders, such as combinatorial histidine scanning and display-based selections, are largely empirical and often labor-intensive. Here we describe two complementary principles and associated computational methods for designing pH-dependent binders: (i) introducing histidine residues adjacent to positively charged residues at binder-target interfaces to induce electrostatic repulsion and weaken binding at low pH, and (ii) introducing buried histidine-containing charged hydrogen-bonding networks in the binder core such that the protein is destabilized under acidic conditions. Using these methods, we designed binders that dissociate at acidic pH against ephrin type-A receptor 2, tumor necrosis factor receptor 2, interleukin-6, proprotein convertase subtilisin/kexin type 9, and the interleukin-2 mimic Neo2. Fusions of the designs to pH-independent binders of lysosomal trafficking receptors function as catalytic degraders, inducing target degradation at substoichiometric levels. Our methods should be broadly useful for designing pH-sensitive protein therapeutics.
    DOI:  https://doi.org/10.1101/2025.09.29.678932
  27. Adv Biol Regul. 2025 Oct 30. pii: S2212-4926(25)00054-5. [Epub ahead of print] 101127
    From stress to homeostasis: Mass spectrometry-based insights into the unfolded protein response (UPR) and proteostasis.
    Lea A Barny, Lars Plate.
      The unfolded protein response (UPR) is a central regulator of proteostasis, coordinating cellular adaptation to endoplasmic reticulum (ER) stress. It is comprised of three signaling branches: ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1), and PERK (protein kinase RNA-like ER kinase), which mediate transcriptional and translational reprogramming of the proteostasis network. These pathways display both functional redundancy and branch-specific activities. Dysregulated UPR signaling contributes to diverse pathologies: in cancer, UPR activation supports uncontrolled proliferation and treatment resistance, whereas in aging, proteostasis decline and diminished UPR responsiveness are hallmarks. Traditional approaches, including transcriptomics and western blotting, have been widely used to monitor UPR activity, but they offer limited insight into its regulation at the protein level. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics allows comprehensive, branch-specific profiling of UPR signaling. Recent advances, including data-independent acquisition (DIA) MS and automated sample preparation, have further improved sensitivity, reproducibility, and detection of low-abundance UPR target proteins. Proteomics thus provides a systematic and scalable framework to interrogate UPR regulation across cell types and disease models. When integrated with complementary datasets, protein-level measurements can uncover context-dependent molecular signatures of UPR activity, offering insights into disease mechanisms and guiding the rational design of targeted pharmacological interventions. Future work integrating high-resolution LC-MS/MS proteomics with tissue and single-cell analyses will further clarify the role of the UPR in health and disease.
    Keywords:  Activating transcription factor 6; Bottom-up proteomics; ER stress; Inositol requiring enzyme 1; Protein kinase R-like ER kinase; Proteomics automation; Proteostasis
    DOI:  https://doi.org/10.1016/j.jbior.2025.101127
  28. Nat Commun. 2025 Nov 18. 16(1): 10094
    Coronaviral nsp6 hijacks ERAD machinery to facilitate lipolysis and supply membrane components for DMV growth.
    Shu-Rui Liu, Yuzheng Zhou, Jinwei Li, Zhaohuan Wang, Yu Ye, Jiafan Miao, Keda Shi, Birong Zheng, Binbin Ding, Jian Pan, Chun-Mei Li, Yiping Li, Panpan Hou, Deyin Guo.
      The positive-strand RNA ( + RNA) viruses extensively remodel cellular endomembranes to facilitate viral replication, with coronaviruses forming a specialized viral replication organelle (RO) known as double-membrane vesicles (DMVs). These DMVs serve as platforms for viral replication and shield viral RNA from host immune recognition. However, the biogenesis, structural organization, and physiological properties of DMVs remain poorly understood. In this study, we demonstrate that the coronavirus non-structural protein 6 (nsp6) anchors DMVs to lipid droplets (LDs), hijacks the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery to degrade PLIN2, and redirects fatty acids (FAs) from LDs to DMVs, thereby supplying lipids for DMV growth. Furthermore, nsp6 anchors ERAD-derived vesicles to DMVs, directly refurnishing membrane components for DMV expansion. Disruption of lipolysis or ERAD impairs DMV formation and inhibits coronaviral replication. We further validated the antiviral effects of ERAD inhibition in female mice in vivo. Our findings elucidate the mechanisms and functional significance of virus-induced organelle remodeling and DMV biogenesis. Given the conservation of viral ROs across +RNA viruses, these structures represent a promising and attractive target for the development of broad-spectrum antiviral therapies.
    DOI:  https://doi.org/10.1038/s41467-025-65118-y
  29. EMBO Mol Med. 2025 Nov 17.
    The canonical ER stress IRE1α/XBP1 pathway mediates skeletal muscle wasting during pancreatic cancer cachexia.
    Aniket S Joshi, Meiricris Tomaz da Silva, Anh Tuan Vuong, Bowen Xu, Ravi K Singh, Ashok Kumar.
      Cancer cachexia is a debilitating syndrome characterized by the progressive loss of skeletal muscle mass with or without fat loss. Recent studies have implicated dysregulation of the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) pathways in skeletal muscle under various conditions, including cancer. In this study, we demonstrate that the IRE1α/XBP1 branch of the UPR promotes activation of the ubiquitin-proteasome system, autophagy, JAK-STAT3 signaling, and fatty acid metabolism in the skeletal muscle of the KPC mouse model of pancreatic cancer cachexia. Moreover, we show that the IRE1α/XBP1 pathway is a key contributor to muscle wasting. Skeletal muscle-specific deletion of the XBP1 transcription factor significantly attenuates tumor-induced muscle atrophy. Mechanistically, transcriptionally active XBP1 binds to the promoter regions of genes such as Map1lc3b, Fbxo32, and Il6, which encode proteins known to drive muscle proteolysis. Pharmacological inhibition of IRE1α using 4µ8C in KPC tumor-bearing mice attenuates cachexia-associated molecular changes and improves muscle mass and strength. Collectively, our findings suggest that targeting IRE1α/XBP1 pathway may offer a therapeutic strategy to counteract muscle wasting during pancreatic cancer-induced cachexia.
    Keywords:  ER Stress; Fatty Acid Oxidation; JAK-STAT; Muscle Wasting; Unfolded Protein Response
    DOI:  https://doi.org/10.1038/s44321-025-00337-w
  30. Biochim Biophys Acta Biomembr. 2025 Nov 18. pii: S0005-2736(25)00078-1. [Epub ahead of print] 184484
    Tubulation of membrane sheets by curvature-inducing proteins.
    Emad Ghazizadeh, Mahdi Zeidi, Wylie Stroberg.
      The endoplasmic reticulum (ER) is a highly dynamic organelle that undergoes continuous remodeling between tubular and sheet-like structures, driven by curvature-inducing proteins and membrane mechanics. Understanding the physical principles underlying ER shape transitions is crucial for elucidating its role in cellular homeostasis and disease. In this study, we use a mesoscopic model of membrane-protein interactions to investigate how intrinsic curvature, protein concentration, and membrane stiffening collectively regulate ER tubulation. Our results demonstrate that the critical concentration for tubulation depends nonlinearly on intrinsic curvature due to a competition between adsorption and remodeling ability. Additionally, increased membrane stiffness upon protein adsorption enhances tubulation efficiency at lower intrinsic curvatures and changes tubule geometry at higher intrinsic curvatures. Phase diagrams are constructed to map the conditions necessary for membrane remodeling, revealing critical protein concentration thresholds for ER transformation. These findings provide a quantitative framework for ER shape regulation, offering insights into how different curvature-inducing proteins coordinate ER morphogenesis.
    Keywords:  Curvature-inducing proteins; Endoplasmic reticulum; Mesoscale simulation; Molecular dynamics; Phase field; Tubular ER
    DOI:  https://doi.org/10.1016/j.bbamem.2025.184484
  31. Nat Commun. 2025 Nov 21. 16(1): 10285
    Multiplex neurodegeneration proteotoxicity platform reveals DNAJB6 promotes non-toxic FUS condensate gelation and inhibits neurotoxicity.
    Samuel J Resnick, Seema Qamar, Pushya Krishna, Vladislav Korobeynikov, Hannes Ausserwoger, Alyssa Miller, Pietro Esposito, Juan A Varela, Jenny Sheng, Lei Haley Huang, Jonathon Nixon-Abell, Schuyler Melore, Chyi Wei Chung, Nino F Läubli, Sofia Kapsiani, Xuecong Li, Jingshu Wang, Nancy Zhang, Mahabub Maraj Alam, Alondra S Burguete, Theresa C Swayne, Yanyan Chen, Ya-Cheng Liao, Neil A Shneider, Michele Vendruscolo, Tuomas P J Knowles, Clemens F Kaminski, Francesco Simone Ruggeri, Gabriele S Kaminski Schierle, Peter St George-Hyslop, Alejandro Chavez.
      Neurodegenerative disorders (NDDs) are a family of diseases that remain poorly treated despite their growing global health burden. To gain insight into the mechanisms and modulators of neurodegeneration, we developed a yeast-based multiplex genetic screening platform. Using this platform, 32 NDD-associated proteins are probed against a library of 132 molecular chaperones from both yeast and humans, and an unbiased set of ~900 human proteins. We identify both broadly active and specific modifiers of our various cellular models. To illustrate the translatability of this platform, we extensively characterize a potent hit from our screens, the human chaperone DNAJB6. We show that DNAJB6 modifies the toxicity and solubility of multiple amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD)-linked RNA-binding proteins (RBPs). Biophysical examination of DNAJB6 demonstrated that it co-phase separates with, and alters the behavior of FUS containing condensates by locking them into a loose gel-like state which prevents their fibrilization. Domain mapping and a deep mutational scan of DNAJB6 revealed key residues required for its activity and identified variants with enhanced activity. Finally, we show that overexpression of DNAJB6 prevents motor neuron loss and the associated microglia activation in a mouse model of FUS-ALS.
    DOI:  https://doi.org/10.1038/s41467-025-65178-0
  32. Mol Cell Biol. 2025 Nov 20. 1-18
    Recent Insights into Mechanisms Regulating the Proteasome: Implications for Human Disease.
    Rintaro Sumikama, Yuri Shibata, Rihan Wu, Shigeo Murata.
      Since its discovery several decades ago, the proteasome has been recognized as one of the most complex and highly evolved proteolytic systems. Through the selective and rapid degradation of ubiquitinated proteins, it plays a pivotal role in maintaining cellular proteostasis and governing essential biological processes such as cell cycle regulation and signal transduction. Recent advances in cryo-electron microscopy (cryo-EM), together with developments in mass spectrometry and large-scale genetic screening, have provided unprecedented insights into proteasome biology. These approaches have not only revealed the proteasome as a precisely engineered molecular machine optimized for substrate specificity and efficient degradation, but have also facilitated the identification of previously unrecognized regulatory factors and post-translational modifications that fine-tune its activity. Moreover, accumulating evidence has demonstrated that proteasome capacity is tightly regulated at multiple levels, including transcriptional control, assembly dynamics, and subcellular localization, to meet diverse cellular demands and preserve proteostasis. Importantly, dysregulation of these processes is linked to human diseases, underscoring the proteasome's central role in cellular physiology and its promise as a therapeutic target. Ongoing research is uncovering new regulatory layers and structural complexities, highlighting the proteasome's indispensable and versatile role in health and disease.
    Keywords:  Proteasome; human disease; proteostasis; ubiquitin
    DOI:  https://doi.org/10.1080/10985549.2025.2587085
  33. Proc Natl Acad Sci U S A. 2025 Nov 25. 122(47): e2511307122
    The lactate sensor NDRG3 decelerates ER-to-Golgi transport through interaction with the long isoform of syntaxin-5.
    Pia E Ferle, Niklas Krause, Judith Koliwer, Jörn Michael Völker, Fabia Becker, Alexander Hillebrand, Leonie F Schröder, Stefanie Jäger, Seby Edassery, Dali Liu, Nevan J Krogan, Jeffrey N Savas, Gabriele Fischer von Mollard, Michael Schwake.
      BET1, GOSR2, and STX5 variants can cause fatal inherited diseases, including epilepsies, muscular dystrophy, and multisystemic disorders. Together with Sec22b, they form a SNARE complex that mediates fusion of ER-derived vesicles with the ER-Golgi-intermediate compartment and the cis-Golgi. The Sec1/Munc18 protein SCFD1/Sly1 accelerates ER-to-Golgi SNARE complex assembly and membrane fusion, but much less is known about downregulation of ER-to-Golgi trafficking under cellular stress conditions. Here, we identify the lactate and hypoxia sensor protein NDRG3 as a binding partner of the ER-to-Golgi SNARE complex. NDRG3 binds via its C-terminal domain to the N-terminal domain of the long isoform of Stx5, thereby impairing ER-to-Golgi trafficking under hypoxic conditions and elevated intracellular lactate levels. In NDRG3-deficient cells, hypoxia- and lactate-induced inhibition of ER-to-Golgi trafficking is abolished. Our work identifies NDRG3 as a negative regulator of ER-to-Golgi SNARE complex function, mechanistically linking hypoxia and lactate to membrane trafficking in the secretory pathway.
    Keywords:  SNARE; congenital muscular dystrophy; hypoxia; lactate; syntaxin-5
    DOI:  https://doi.org/10.1073/pnas.2511307122
  34. RSC Med Chem. 2025 Nov 18.
    Design and evaluation of an HSP70-targeting PROTAC in synergy with an HSF1 inhibitor for enhanced antitumor activity.
    Wei-Hao Huang, Teng-Yu Mao, Guo-Yao Dai, Jian-Mei Ye, Jia-Bao Li, Shuo-Bin Chen, Jia-Heng Tan, Zhi-Shu Huang, Shi-Liang Huang.
      Heat shock protein (HSP) 70 represents a clinically promising anti-tumor target, yet the development of effective inhibitors faces numerous technical challenges. To address this, we developed novel non-ATP site Proteolysis-targeting Chimeras (PROTACs) that selectively degrade HSP70 by engaging the E3 ubiquitin ligase CRBN. However, the PROTACs exhibited limited degradation activity, potentially due to heat shock response-mediated HSP70 upregulation. To circumvent this resistance mechanism, we explored combination therapy with the heat shock factor 1 (HSF1) inhibitor DTHIB to disrupt the heat shock feedback loop, markedly enhancing HSP70 degradation. The combination strategy showed synergistic and selective anti-tumor activity across a panel of cancer cell lines. This success relied on the distinct profile of C4, which preferentially targets cytosolic HSP70 and, unlike conventional inhibitors, effectively circumvents compensatory HSP70 upregulation.
    DOI:  https://doi.org/10.1039/d5md00726g
  35. bioRxiv. 2025 Oct 02. pii: 2025.10.02.680111. [Epub ahead of print]
    DnaJB1 chaperone inhibits tau aggregation by recognizing its N-terminus.
    Pawel M Wydorski, Paulina Macierzynska, Sofia Bali, Jaime Vaquer-Alicea, Lukasz A Joachimiak.
      A network of protein folding and degradation machineries maintains protein homeostasis by preventing the accumulation of misfolded proteins and by facilitating their clearance. These systems are also crucial for the inhibition of protein aggregation in neurodegenerative diseases where misfolded proteins often aggregate into β-rich amyloid fibrils. How these machineries selectively recognize pathological aggregates over normal conformations remains unclear. Here, we present the molecular logic for how a Hsp70 co-chaperone from the J-domain protein family, DnaJB1, binds pathological aggregates of the microtubule-associated protein tau through the recognition of the flexible N-terminus that comprises the disordered fuzzy coat of fibrils. We show that this interaction contributes to the regulation of tau assembly in cellular models of tau aggregation and depends on the presence of the negatively charged residues. We determined that DnaJB1 inhibits tau aggregation in vitro through these interactions, and found that this weak, transient binding can be enhanced by the presence of polyanionic factors such as heparin. As prospective client-binding sites, we identified the charged hinge between the two β-sandwich C-terminal domains I and II, as well as the conserved J-domain of this chaperone. This work presents novel biochemical and structural insights into how the molecular chaperone DnaJB1 recognizes full-length forms of tau protein in a pathological context.
    DOI:  https://doi.org/10.1101/2025.10.02.680111
  36. bioRxiv. 2025 Sep 30. pii: 2025.09.30.678621. [Epub ahead of print]
    Giant DNA viruses encode a hallmark translation initiation complex of eukaryotic life.
    J Maximilian Fels, Aidan B Hill, Richard Han, Jasmine M Garcia, Hugo Bisio, Chantal Abergel, Philip J Kranzusch, Amy S Y Lee.
      In contrast to living organisms, viruses were long thought to lack protein synthesis machinery and instead depend on host factors to translate viral transcripts. Here, we discover that giant DNA viruses encode a distinct and functional IF4F translation initiation complex to drive protein synthesis, thereby blurring the line between cellular and acellular biology. During infection, eukaryotic IF4F on host ribosomes is replaced by an essential viral IF4F that regulates viral translation, virion formation, and replication plasticity during altered host states. Structural dissection of viral IF4F reveals that the mRNA cap-binding subunit mediates exclusive interactions with viral mRNAs, constituting a molecular switch from translating host to viral proteins. Thus, our study establishes that viruses express a eukaryotic translation initiation complex for protein synthesis, illuminating a series of evolutionary innovations to a core process of life.
    DOI:  https://doi.org/10.1101/2025.09.30.678621
  37. J Mol Biol. 2025 Nov 14. pii: S0022-2836(25)00612-6. [Epub ahead of print] 169546
    Distinct cis-acting elements combinatorically mediate co-localization of mRNAs encoding for co-translational interactors in cytoplasmic clusters in S. cerevisiae.
    Qamar Shhade, Marianna Estrada, Rawad Hanna, Muhammad Makhzumy, Hagit Bar-Yosef, Guenter Kramer, Bernd Bukau, Ayala Shiber.
      Many newly synthesized proteins assemble co-translationally, providing a vital mechanism to prevent subunit misfolding in the crowded cytoplasm. Initial evidence indicates that the spatial organization of mRNAs aids this assembly, but it is unclear how these mRNAs are organized and how common this mechanism is. We used single-molecule Fluorescence in situ Hybridization in Saccharomyces cerevisiae to examine the spatial organization of mRNAs encoding subunits of various cytosolic complexes involved in critical cellular functions, such as fatty acid synthesis, glycolysis, translation and various amino acid biosynthesis. We found that mRNAs of the same protein complex often co-localize in specific cytoplasmic clusters. Additionally, we observed that the mRNAs encoding enzymes of biosynthetic pathways are organized in cytosolic clusters. Focusing on mRNAs encoding fatty acid synthase complex subunits, we discovered that non-coding cis elements significantly influence mRNA localization in an additive manner. Specifically, 5' and 3' UTRs, together with further upstream and downstream regions, facilitate co-localization. Inhibiting mRNA co-localization impaired growth when complex activity was essential, highlighting the importance of mRNA spatial organization for cellular survival. Transiently disrupting mRNA translation also affected clustering, indicating that both the nascent chains and mRNA sequence targeting cues are combinatorically contributing to spatial organization. Proteomics analysis demonstrates the impact of cis-elements on the abundance of the encoded subunits, as well as the entire pathway. In summary, we provide evidence that mRNA co-localization in cytoplasmic foci is coordinated by complementary mechanisms crucial for co-translational assembly, allowing efficient regulation of protein complex formation and entire pathways.
    Keywords:  complex assembly; ribosome; spatial mRNA organization; translation
    DOI:  https://doi.org/10.1016/j.jmb.2025.169546
  38. Mech Ageing Dev. 2025 Nov 18. pii: S0047-6374(25)00101-0. [Epub ahead of print] 112125
    Age-Related Alterations in the Expression of Mesencephalic Astrocyte-derived Neurotrophic Factor in the Brain and Their Impact on Neurobehavioral Functions.
    Di Hu, Wen Wen, Hui Li, Zuohui Zhang, Hong Lin, Jia Luo.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic protein localized in the endoplasmic reticulum (ER) and pivotally involved in maintaining ER homeostasis. The ER is central to protein synthesis, folding, degradation and secretion (proteostasis), and experiences considerable stress in neurodegenerative diseases (NDDs), which activates the unfolded protein response (UPR). Aging, the primary risk factor for NDDS, is associated with impaired ER function. MANF is shown to be protective in various experimental models of NDDs. We hypothesized that the expression of MANF in the brain declines with age, which may increase the vulnerability to NDDs. We measured MANF levels in the brain and plasma of 1-, 4-, 11-, and 22-month-old male and female mice. A progressive decline of MANF levels was observed, with the lowest levels detected in 22 months. Reduced MANF expression was found in aged mice across several brain areas, including the cerebral cortex, olfactory bulb, thalamus, hypothalamus, hippocampus, and cerebellum. There was a sex difference in MANF levels in aged mice. Aging also altered the expression of UPR and MANF interacting proteins. Using cerebellar Purkinje cell (PC)-specific MANF deficient mice, we showed that MANF deficiency impaired motor coordination in female, but not male mice. MANF deficiency weakened spatial learning and memory in both male and female mice. Male MANF deficient mice displayed increased sociability, whereas female mice exhibit social withdrawal. Taken together, MANF expression in the brain declined with age and MANF deficiency impacted neurobehaviors in a sex-specific manner.
    Keywords:  Aging; endoplasmic reticulum stress; neurobehavioral deficits; neurodegeneration; proteostasis; unfolded protein response
    DOI:  https://doi.org/10.1016/j.mad.2025.112125
  39. Cell Genom. 2025 Nov 19. pii: S2666-979X(25)00321-0. [Epub ahead of print] 101065
    Integrative profiling of condensation-prone RNAs during early development.
    Tajda Klobučar, Jona Novljan, Ira A Iosub, Boštjan Kokot, Iztok Urbančič, D Marc Jones, Anob M Chakrabarti, Nicholas M Luscombe, Jernej Ule, Miha Modic.
      Complex RNA-protein networks play a pivotal role in the formation of many types of biomolecular condensates. How RNA features contribute to condensate formation, however, remains incompletely understood. Here, we integrate tailored transcriptomics assays to identify a distinct class of developmental condensation-prone RNAs termed "smOOPs" (semi-extractable, orthogonal-organic-phase-separation-enriched RNAs). These transcripts localize to larger intracellular foci, form denser RNA subnetworks than expected, and are heavily bound by RNA-binding proteins (RBPs). Using an explainable deep learning framework, we reveal that smOOPs harbor characteristic sequence composition, with lower sequence complexity, increased intramolecular folding, and specific RBP-binding patterns. Intriguingly, these RNAs encode proteins bearing extensive intrinsically disordered regions and are highly predicted to be involved in biomolecular condensates, indicating an interplay between RNA- and protein-based features in phase separation. This work advances our understanding of condensation-prone RNAs and provides a versatile resource to further investigate RNA-driven condensation principles.
    Keywords:  OOPS; RIC-seq; RNA-RNA interactions; RNA-protein networks; condensation; condensation-prone RNAs; deep learning; phase separation; semi-extractability
    DOI:  https://doi.org/10.1016/j.xgen.2025.101065
  40. PLoS Pathog. 2025 Nov;21(11): e1013685
    Disrupted glycosylphosphatidylinositol anchoring induces ER stress and restricts enterovirus infection.
    Shangrui Guo, Xinyu Li, Meng Xun, Yingli He, Andrew W Tai, Hongliang Wang.
      Many positive-sense RNA viruses, including viruses from the Picornaviridae, Coronaviridae and Flaviviridae family, exploit endoplasmic reticulum (ER)-derived membrane structures as sites of genome replication. Here we use a pooled CRISPR genetic screening strategy to identify glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer genes as host factors for echovirus 7 infection. In addition to supporting the biogenesis of CD55, which is a GPI anchor protein and an entry factor for some echoviruses, the GPI anchor synthesis machinery also supports several other enterovirus infections by enhancing viral replication and replication organelle biogenesis. Disruption of GPI anchor transfer machinery compromises ER integrity and causes ER stress. Consistent with these findings, ER-resident sensor, inositol-requiring protein 1α (IRE1α) is activated and regulated IRE1-dependent decay of mRNA (RIDD) is detected to reduce ER stress. Interestingly, enterovirus viral RNA, but not Hepatitis C Virus RNA, is degraded during this process due to specific sequences in the Untranslated Region (UTR). This study revealed novel links between GPI anchoring, ER stress and enterovirus infection, and illuminates new host targets for antiviral therapy.
    DOI:  https://doi.org/10.1371/journal.ppat.1013685
  41. NAR Mol Med. 2025 Apr;2(2): ugaf009
    The translational landscape of reactive astrocytes reveals the impact of eIF2B-mediated dysregulation in VWM disease.
    Shir Mandelboum, Liat Lev-Ari, Andrea Atzmon, Melisa Herrero, Naama Brezner, Daniel Benhalevy, Orna Elroy-Stein.
      A devastating genetic recessive neurodegenerative disorder, vanishing white matter disease (VWMD), stems from mutations in eIF2B-a master regulator of mRNA translation initiation and mediator of cellular stress response. While astrocytes, the brain's essential support cells, are known to be central to VWMD pathology, the molecular mechanisms underlying their dysfunction remain poorly understood. Our study reveals that even a mild mutation in eIF2B5 profoundly disrupts astrocyte mRNA translation regulation upon cytokine-mediated activation, affecting nearly one-third of all expressed genes. Through innovative integration of RNA-seq and Ribo-seq analyses using primary cell cultures of astrocytes isolated from eIF2B5R132H/R132H mice, we discovered attempts to compensate for impaired protein production by increasing mRNA levels. However, this compensation proves insufficient to maintain critical cellular functions. Our comprehensive analysis uncovered significant disruptions in cellular energy production and protein synthesis machinery. We also predicted previously unknown defects in cholesterol biosynthesis within mutant astrocytes. Moreover, a meta-analysis of translation initiation scores pinpointed, for the first time, a short list of specific pivotal gene candidates that may drive disease progression. This powerful combination of transcriptome and translatome illuminates the complex pathophysiology of VWMD and identifies promising new biomarkers and therapeutic target opportunities.
    DOI:  https://doi.org/10.1093/narmme/ugaf009
  42. Nucleic Acids Res. 2025 Nov 13. pii: gkaf1200. [Epub ahead of print]53(21):
    Chemical inhibition of exon junction complex assembly impairs mRNA localization and neural stem cells ciliogenesis.
    Tommaso Villa, Oriane Pourcelot, David Dierks, Marion Faucourt, Cindy Burel, Floric Slimani, Léa Guyonnet, Nathalie Spassky, Schraga Schwartz, Edouard Bertrand, Olivier Bensaude, Hervé Le Hir.
      The exon junction complex (EJC) is formed by the essential eIF4A3, MAGOH, and Y14 core proteins. It is universally deposited during splicing at exon-exon junctions. The EJC is known to impact almost every post-transcriptional regulatory step throughout the life of messenger RNAs (mRNAs) including their modifications, splicing, decay, and trafficking. Its dysregulation leads to neurodevelopmental pathologies. Here, we show that EJC-i, a compound known to block the ATPase activity of eIF4A3, inhibits de novo EJC assembly. EJC-i and targeted knockdown of either eIF4A3 or Y14 core EJC subunits lead to very similar phenotypes by impacting the destiny of mRNAs due to alterations in alternative splicing, nonsense-mediated mRNA decay, genome-wide m6A methylation, and proper localization of specific transcripts, in particular to the centrosome. Both EJC impairment methods disrupt the centrosome function, which might be responsible for mitotic arrest at prometaphase. As a small molecule that readily diffuses into cells, EJC-i is a particularly easy-to-use and versatile tool to investigate EJC functions in live cells or whole organisms that are not prone to genetic manipulation. Indeed, this property was used to disrupt ciliogenesis in primary neural stem cells.
    DOI:  https://doi.org/10.1093/nar/gkaf1200
  43. Nat Commun. 2025 Nov 21. 16(1): 10256
    ER protein CLCC1 promotes nuclear envelope fusion in herpesviral and host processes.
    Bing Dai, Adrian W Sperl, Lucas Polack, Isabel Mejia, Haley Dame, Tien Huynh, Chloe Deveney, Nathalie Lavoie, Chanyoung Lee, John G Doench, Matthew D Daugherty, Ekaterina E Heldwein.
      Herpesvirales are an ancient viral order that causes lifelong infections in species from mollusks to humans. They export their capsids from the nucleus to the cytoplasm by a noncanonical nuclear egress route that involves capsid budding at the inner nuclear membrane followed by fusion of this temporary envelope with the outer nuclear membrane. Here, using a whole-genome CRISPR screen, we identify ER protein CLCC1 as important for the fusion stage of nuclear egress in herpes simplex virus 1. We also find that the genomes of Herpesvirales that infect mollusks and fish encode CLCC1 genes acquired from host genomes by horizontal gene transfer. In uninfected cells, loss of CLCC1 causes a nuclear blebbing defect, suggesting a role in host nuclear export. We hypothesize that CLCC1 facilitates an ancient cellular membrane fusion mechanism that Herpesvirales have hijacked or co-opted for capsid export and propose a mechanistic model.
    DOI:  https://doi.org/10.1038/s41467-025-65115-1
  44. Nature. 2025 Nov 19.
    Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.
    Sarah E Pierce, Steven Erwood, Keyede Oye, Meirui An, Nicholas Krasnow, Emily Zhang, Aditya Raguram, Davis Seelig, Mark J Osborn, David R Liu.
      Precise genome-editing technologies such as base editing1,2 and prime editing3 can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration4,5 or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.
    DOI:  https://doi.org/10.1038/s41586-025-09732-2
  45. PLoS Comput Biol. 2025 Nov;21(11): e1013634
    Molecular insights on the mechanism of α1-antitrypsin condensate formation and maturation.
    Ignacio Sanchez-Burgos, Andres R Tejedor, Rosana Collepardo-Guevara, Jorge Bernardino de la Serna, Jorge R Espinosa.
      The deficiency of [Formula: see text]-antitrypsin protein is a genetic disorder characterized by the accumulation of misfolded protein aggregates within hepatocytes, leading to liver dysfunction. In the lung, it is found in macrophages, bronchial and epithelial alveolar cells type 2, leading to pulmonary emphysema. Despite extensive research, the precise mechanism underlying the formation of [Formula: see text]-antitrypsin inclusion bodies remain elusive. In this study, we combine equilibrium and non-equilibrium molecular dynamics simulations to elucidate the intricate process of [Formula: see text]-antitrypsin condensate formation and maturation. Our mechanistic model explains cluster accumulation-specifically the onset of this pathogenesis-through the emergence of phase-separated liquid-like protein droplets, which subsequently undergo inter-protein β-sheet transitions between misfolded variants, resulting in solid-like clusters. We find that this mechanism only applies to the misfolded variant, Z-[Formula: see text]-antitrypsin, which phase-separates driven by its disordered C-terminus. In contrast, the native protein, M-[Formula: see text]-antitrypsin, shows much lower propensity to phase-separate and later form kinetically trapped aggregates. Furthermore, we explore how Z-[Formula: see text]-antitrypsin exhibits an increased capacity to form condensates near external walls with different types of interactions. Such conditions can be similar to those found within the endoplasmic reticulum membrane, where phase separation and hardening take place. Overall, our results shed light on the molecular basis of [Formula: see text]-antitrypsin-related disorders and provide valuable microscopic insights for the development of therapeutic strategies targeting protein misfolding and aggregation-related disorders.
    DOI:  https://doi.org/10.1371/journal.pcbi.1013634
  46. Autophagy. 2025 Nov 20.
    Regulation of pexophagy by a novel TBK1-MARCHF7-PXMP4-NBR1 Axis in PEX1-depleted HeLa cells.
    Yong Hwan Kim, Joon Bum Kim, Ji-Eun Bae, Na Yeon Park, Seong Hyun Kim, Jae-Young Um, Dong-Seok Lee, Kyu Sun Lee, Peter K Kim, Doo Sin Jo, Dong-Hyung Cho.
      Peroxisomes are essential for lipid metabolism and redox balance, with pexophagy playing a critical role in maintaining cellular homeostasis. However, the regulatory mechanisms of pexophagy remain unclear. Through functional screening, we identified MARCHF7 as a novel E3 ligase regulating pexophagy. MARCHF7 depletion impaired pexophagic flux under PEX1 knockdown conditions. MARCHF7 binds to PXMP4 and promotes its ubiquitination at lysine 20 in PEX1-deficient cells. Depletion of PXMP4 impairs pexophagy, and reconstitution with the PXMP4 lysine 20 ubiquitination-defective mutant failed to rescue pexophagy. PEX1 depletion also induces TBK1 phosphorylation at serine 172, activating TBK1, which subsequently phosphorylates MARCHF7. This activation is driven by ROS accumulation, which reduces PXMP4 ubiquitination and prevents peroxisome loss. Furthermore, downregulation of MARCHF7 or PXMP4 impairs NBR1 recruitment to peroxisomes, suggesting that ubiquitinated PXMP4 acts as a recognition signal for NBR1. Collectively, our findings establish the TBK1-MARCHF7-PXMP4-NBR1 axis as a key regulatory pathway for pexophagy in response to PEX1 depletion.
    Keywords:  MARCHF7; PEX1; PXMP4; TBK1; peroxisome; pexophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2593585
  47. Biol Chem. 2025 Nov 21.
    Update on VAP, a ubiquitous signpost for the ER.
    Tim P Levine.
      The small protein family of VAMP-associated proteins (VAPs) have the unique position in cell biology as intracellular signposts for the Endoplasmic Reticulum (ER). VAP is recognised by a wide range of other proteins that use it to target the ER, either simply being recruited from the cytoplasm, or being recruited from separate organelles. The latter process makes VAP a component of many bridges between the ER and other compartments at membrane contact sites. The fundamental observations that identify VAP as the ER signpost have largely remained unchanged for over two decades. This review will describe how increased understanding of the special role of VAP in recent years has led to new discoveries: what constitutes the VAP family, how proteins bind to VAP, and which cellular functions connect to the ER using VAP. It will also describe the pitfalls that have led to difficulties determining how some proteins bind VAP and suggest some possibilities for future research.
    Keywords:  FFAT motif; cyclic AMP (cAMP); lipid transfer protein (LTP); non-vesicular transport; nucleolus; short linear motif (SLiM)
    DOI:  https://doi.org/10.1515/hsz-2025-0199
  48. Cell Death Differ. 2025 Nov 21.
    UFL1-mediated UFMylation antagonizes IFT88 ubiquitination and degradation to maintain ciliary homeostasis.
    Runa Wang, Guizhi Guo, Renshuai Zhang, Lin Li, Yingxin Gong, Long Yin, Shuhua Li, Changfeng Wei, Jun Zhou, Min Liu, Jie Ran.
      UFMylation, a post-translational modification involving the covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to target proteins, has been implicated in a wide spectrum of human diseases. However, the underlying molecular mechanisms are poorly understood. Herein, we demonstrate that UFM1-specific ligase 1 (UFL1), the sole ligase for UFMylation, is indispensable for ciliary homeostasis. Genetic ablation of UFL1 in mice results in severe defects in ciliary structure and function in multiple tissues. Mechanistic investigation reveals that intraflagellar transport 88 (IFT88), a protein essential for ciliary assembly and maintenance, undergoes UFMylation at lysine 572. The UFMylation antagonizes IFT88 ubiquitination by Praja ring finger ubiquitin ligase 2 (PJA2), thereby preventing its proteasomal degradation. The lysine 572-to-arginine mutant of IFT88 exhibits increased stability and efficacy in rescuing ciliary defects induced by UFL1 depletion. Our findings identify a critical role for IFT88 UFMylation in ciliary homeostasis and offer novel insights into human ciliopathies.
    DOI:  https://doi.org/10.1038/s41418-025-01625-1
  49. Sci Adv. 2025 Nov 21. 11(47): eady9587
    A ubiquitin-like protein controls assembly of a bacterial type VIIb secretion system.
    Gabriel U Oka, Nathanaël Benoit, Axel Siroy, Francesca Gubellini, Esther Marza, Rémi Fronzes.
      Type VII secretion systems (T7SS) are protein translocation machines crucial for virulence and bacterial competition in Gram-positive bacteria. Despite their importance, the structural basis for assembly of type VIIb secretion systems (T7SSb), a widely distributed variant in Firmicutes, remains poorly understood. We present the cryo-electron microscopy structure of the T7SSb core complex from Bacillus subtilis, revealing how the ubiquitin-like protein YukD, coordinates assembly of the secretion machinery. YukD interacts extensively with the central channel component YukB and facilitates its association with the pseudokinase YukC, forming a stable building block for channel assembly. Time-lapse microscopy and competition assays demonstrate that YukD is essential for proper T7SSb complex formation and contact-dependent bacterial killing. Our findings reveal how bacteria have adapted a ubiquitin-like protein as a structural regulator for assembling a large secretion complex.
    DOI:  https://doi.org/10.1126/sciadv.ady9587
  50. bioRxiv. 2025 Sep 29. pii: 2025.09.29.679340. [Epub ahead of print]
    Structural Studies of Nedicistrovirus IRES-Driven, Initiation Factor-independent Translation Shed Light on Key Steps of Eukaryotic Translation Elongation.
    Swastik De, Clara G Altomare, Irina S Abaeva, Prikshat Dadhwal, Priyanka Garg, Francisco Acosta-Reyes, Zuben P Brown, Tatyana V Pestova, Christopher U T Hellen, Joachim Frank.
      We utilized the Nedicistrovirus (NediV) intergenic region (IGR) IRES-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl-tRNA-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound post-proofreading complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading.
    DOI:  https://doi.org/10.1101/2025.09.29.679340
  51. Nat Biotechnol. 2025 Nov 21.
    Programmable initiation of mRNA translation by trans-RNA.
    Longfei Jia, Tan-Trung Nguyen, Saori Uematsu, Yifei Gu, Shengcho Shi, Yaser Hashem, Shu-Bing Qian.
      Several approaches exist to silence genes, but few tools are available to activate individual mRNAs for translation inside cells. Guiding ribosomes to specific start codons without altering the original sequence remains a formidable task. Here we design capped trans-RNAs capable of directing ribosomes to specific initiation sites on individual mRNAs when the trans-cap is positioned near the target start codon. Structural and biochemical data suggest that the capped trans-RNA facilitates ribosome loading and scanning on the target mRNA through a synergistic mechanism involving alternative cap recognition. The trans-RNA also acts independently of the cap on the target mRNA, enabling translation of circular RNAs lacking internal ribosome entry sites. We apply trans-RNAs in vivo to achieve programmable alternative translation of endogenous genes in mouse liver. Finally, we provide the evidence for the existence of natural transcripts that, similarly to exogenous trans-RNAs, activate translation of endogenous mRNAs.
    DOI:  https://doi.org/10.1038/s41587-025-02897-1
  52. ACS Med Chem Lett. 2025 Nov 13. 16(11): 2224-2231
    Design, Synthesis, and Cellular Efficacy of Inositol-Requiring Enzyme Type 1 (IRE1α) Inhibitors.
    Giulia Murbach, Adam D Mitrevski, Patricia A Fontan, Marcelo M Nociari, David H Thompson.
      A library of 66 small molecules targeting IRE1α were designed using a molecular docking approach and prepared by a two-step reaction sequence using diverse substrates. All compounds utilized a 1-amino-4-bromonaphthalene core that was modified via Suzuki coupling with boronic acids to form intermediates that were carbamoylated to form urea-linked inhibitor candidates. We developed a 33 DoE approach for the Suzuki coupling reaction that was optimized with 216 reactions via HTE. By screening the purified compounds in a tunicamycin-induced ER stress assay with ARPE-19 cells and quantifying their kinase inhibition activity by RT-qPCR, we identified 14 derivatives with the potential for IRE1α inhibition. IC50 assays showed that six of the compounds displayed IRE1α inhibition alike KIRA6, a standard in IRE1α inhibition, with three of the leads possessing improved IC50. Viability screens indicated that the best IRE1α inhibitors were not cytotoxic in the working concentrations and displayed improved protection from apoptosis compared to KIRA6.
    Keywords:  ARPE-19; DESI-MS; DoE; ER stress; HTE; IRE1α inhibitors; KIRA6; Molecular docking; Suzuki coupling
    DOI:  https://doi.org/10.1021/acsmedchemlett.5c00418
  53. Nat Commun. 2025 Nov 15. 16(1): 9996
    KLF5 enables dichotomous lineage programs in pancreatic cancer via the AAA+ ATPase coactivators RUVBL1 and RUVBL2.
    Patrick J Cunniff, Nicole Sivetz, Damianos Skopelitis, Olaf Klingbeil, Daniel Toobian, Diogo Maia-Silva, Mikala Egeblad, Christopher R Vakoc.
      Lineage plasticity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and contributes to tumor heterogeneity and therapeutic resistance. Here, we identify KLF5 as a dynamic master regulator of epithelial lineage identity in PDAC, with dichotomous roles in promoting either classical or basal-like transcriptional programs. Through unbiased proteomic and genetic screens, we uncover the AAA+ ATPases RUVBL1 and RUVBL2 as essential coactivators of KLF5 across both lineage states. We demonstrate that ATP hydrolysis by RUVBL1/2 is required for the stable interaction with an intrinsically disordered region of KLF5, enabling its recruitment to lineage-specific enhancers and driving transcriptional regulation of identity-defining genes. Notably, small-molecule inhibitors of RUVBL1/2 ATPase activity, which have anti-PDAC activity in vivo, suppress KLF5-dependent transcription. These findings define a previously unrecognized mechanism of ATP hydrolysis-dependent transcriptional coactivation and highlight a potential therapeutic strategy for modulating aberrant lineage programs in cancer.
    DOI:  https://doi.org/10.1038/s41467-025-66007-0
  54. Adv Cancer Res. 2025 ;pii: S0065-230X(25)00049-1. [Epub ahead of print]168 269-369
    PROTACS- targeted protein degradation as a path to precision cancer therapeutics.
    Hannah R Gatley, Paul B Fisher, Swadesh K Das.
      New strategies and mechanisms for managing and treating cancer are essential; a novel therapeutic approach has now emerged in the scope of targeted protein degradation called Proteolysis-Targeting Chimeras (PROTACs). This technology specifically targets and degrades disease-causing proteins via the ubiquitin-proteasome system, leading to expanded research in both academia and industry over the past two decades. The diversity of PROTAC classes based on the E3 ligase recruiting ligand and the target protein allows for a universal molecular structure that can be tailored for a specific target. As such, PROTACs have been widely evaluated across a multitude of cancer variants and reported to effectively target a wide range of proteins across multiple cellular pathways. Overall, the ability of PROTAC technology to degrade both 'druggable' and 'undruggable' targets has resulted in a rapid expansion of research in the brief time since its initial discovery. Continued intense efforts will help further shape this evolving field of 'dynamic protein management' to transition PROTACs into clinical settings.
    Keywords:  Cancer; Proteolysis-targeting chimera (PROTAC); Ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.1016/bs.acr.2025.08.001
  55. Mol Cell Biol. 2025 Nov 18. 1-16
    Cholesterol Transport from ER to Outer Mitochondria by ERLIN2 in Steroid Metabolism.
    Himangshu S Bose, William E Burak, Randy M Whittal.
      Cholesterol trafficking from the endoplasmic reticulum (ER) through the mitochondria-associated ER membrane (MAM) and finally to mitochondria is essential for mammalian survival. ER lipid raft-associated protein 2 (ERLIN2) scaffolds raft-like microdomains in the trans-Golgi network, endosomes, and plasma membrane. We found that ERLIN2 assists in rolling cholesterol trafficking-associated lipid vesicles by facilitating the intermediate folding of cholesterol trafficker steroidogenic acute regulatory protein (StAR) from the ER to MAM prior to delivery to the outer mitochondrial membrane. Each ERLIN2-StAR interaction is short. The absence of ERLIN2 ablates mitochondrial cholesterol transport. Over time, StAR association with ERLIN2 increases from the ER to MAM, thereby enhancing mitochondrial cholesterol transport. Thus, ERLIN2 is central for regulating mitochondrial cholesterol trafficking required for mitochondrial steroid metabolism.
    Keywords:  Steroids; cholesterol; endoplasmic reticulum; mitochondria associated-ER membrane (MAM); pregnenolone; steroidogenic acute regulatory protein (StAR)
    DOI:  https://doi.org/10.1080/10985549.2025.2583172
  56. Cell Genom. 2025 Nov 19. pii: S2666-979X(25)00324-6. [Epub ahead of print] 101068
    Leveraging protein language models to identify complex trait associations with previously inaccessible classes of functional rare variants.
    Seon-Kyeong Jang, Zitian Wang, Richard Border, Dinh Tuan, Angela Wei, Ulzee An, Sriram Sankararaman, Vasilis Ntranos, Jonathan Flint, Noah Zaitlen.
      Protein language models (PLMs) improve variant effect predictions, but their role in gene discovery for complex traits remains unclear. We introduce an allelic series-based regression test that uses PLM-derived variant effect predictions as proxies for effect sizes, identifying ∼46% more associations than standard burden tests. Extending this to isoform-level analysis, we find 26 gene-trait pairs with stronger associations in non-canonical versus canonical transcripts, highlighting isoform-specific effects. Finally, we identify evolutionary plausible variants (EPVs), missense variants assigned higher likelihoods than the wild-type alleles by PLMs, representing 0.45% of missense variants. EPVs show higher allele frequencies than synonymous variants, consistent with differential selection pressures, and are linked to nine traits, including protective associations with low-density lipoprotein (LDL) and bone mineral density. Together, our results demonstrate how PLMs can enhance rare-variant interpretation and gene-trait association discovery in exome data.
    Keywords:  complex traits; exome sequence; gene-based test; missense variants; protein language model; rare-variant association
    DOI:  https://doi.org/10.1016/j.xgen.2025.101068
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