bims-proteo Biomed News
on Proteostasis
Issue of 2025–11–09
53 papers selected by
Eric Chevet, INSERM
  1. Secretome translation shaped by lysosomes and lunapark-marked ER junctions.
  2. CNX:FAM134B-driven ERLAD of ATZ polymers proceeds via enhanced formation of VAPA:ORP1L:RAB7 contact sites between ER and endolysosomes.
  3. Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1.
  4. PROTAC repurposing uncovers a noncanonical binding surface that mediates chemical degradation of nuclear receptors.
  5. How Pathogens Maintain Proteostasis During Infection.
  6. Beyond the Secretory Pathway: New Insights Into Protein Release.
  7. Zinc-Related IZH1 as a Novel Regulator of Endoplasmic Reticulum Homeostasis in Yeast.
  8. Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia-like bacterium Simkania negevensis.
  9. Golgiphagy mediated by TM9SF3 acts as quality control for stressed Golgi.
  10. Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.
  11. Rational design of potent small-molecule SMARCA2/A4 degraders acting via the recruitment of FBXO22.
  12. Identification of ATG7 as a protein ATG8ylation substrate.
  13. The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters.
  14. Pmt4 recognizes two separate acceptor sites to O-mannosylate in the S/T-rich regions of substrate proteins.
  15. Mechanistic basis for protein conjugation in a diverged bacterial ubiquitination pathway.
  16. TUSC3 regulates ERMA-mediated Mg2+ uptake for synaptic function and neurodevelopment.
  17. Amino acid insufficiency impairs hepatic vitamin A mobilization in mice.
  18. Lysosomal and mTORC1 signaling dysregulation underpin the pathology of spastic paraplegia type 80.
  19. Protocol for halofuginone-mediated metabolic reprogramming of murine T cells via activation of the GCN2 pathway.
  20. Modeling protein-small molecule conformational ensembles with PLACER.
  21. Improving Proteostasis of Trafficking-Deficient GABAA Receptor Variants by Activating IRE1.
  22. Contact-dependent incorporation of endoplasmic reticulum into retraction fibers and migrasomes.
  23. A Modular Uba1-Nanobody Fusion Enables Selective Ubiquitin Transfer to Tagged E2 Enzymes.
  24. Metabolic environment-driven remodeling of mitochondrial ribosomes regulates translation and biogenesis.
  25. nERdy: network analysis of endoplasmic reticulum dynamics.
  26. Exploiting nanopore sequencing advances for tRNA sequencing of human cancer models.
  27. The ER thioredoxin-related transmembrane protein TMX2 controls redox-mediated tethering of ER-mitochondria contacts.
  28. Endoplasmic reticulum protein FAM134B acts as a regulator of mitochondrial morphology.
  29. Membrane curvature at the ER-PM contact sites.
  30. Thiol isomerase ERp46 catalyzes the disulfide formation of coagulation factor XII enhancing its activity.
  31. IGF2BP2 stabilized by USP7 promotes cancer-associated fibroblast activation and attenuates gemcitabine sensitivity in PDAC.
  32. TRIM21 and OTUD6A orchestrate AKT K27-linked atypical ubiquitination to modulate cancer chemoresistance.
  33. Atomically accurate de novo design of antibodies with RFdiffusion.
  34. Genomic and translational insights into eIF2B-mediated salt tolerance in sea rice HD961.
  35. Multi-dimensional Proteomics Reveal Metformin's Impact on Interconnected Regulatory Networks of Protein Turnover, Ubiquitination, DNA Damage, and Cell Cycle.
  36. HERPUD1 mediates palmitic acid-induced UPR sustaining TNBC aggressiveness and is destabilized by CK2 pharmacological inhibition.
  37. Endoplasmic reticulum-resident protein DNAJC10 inhibits glioblastoma metastasis by suppressing XBP-1s-driven EGFR transcription.
  38. Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis.
  39. Methamphetamine impairs stress resistance and extracellular matrix integrity via modulating the unfolded protein response in Caenorhabditis elegans.
  40. Molecular Insights into the Dual Mechanism of Bepristat 2a on Protein Disulfide Isomerase (PDI) Activity.
  41. Mutant p53 protein accumulation is selectively targetable by proximity-inducing drugs.
  42. TRIB3 Links Endoplasmic Reticulum Stress to Impaired Efferocytosis in Atherosclerosis.
  43. Adaptive ER stress promotes mitochondrial remodelling and longevity through PERK-dependent MERCS assembly.
  44. Goblet cell-expressed microprotein FXYD3 determines gut homeostasis by maintaining mucus barrier integrity.
  45. TMEM166 negatively regulates unfolded protein response to affect hepatocellular carcinoma cell growth and sorafenib resistance.
  46. Click-linking: a cell-compatible protein crosslinking method based on click chemistry.
  47. The Role of UFMylation in the Development and Progression of Gastric Cancer.
  48. E3 ligase Praja1 mediates ubiquitination and degradation of microtubule-associated protein tau.
  49. Independent mechanisms of inflammation and myeloid bias in VEXAS syndrome.
  50. Ubiquitylation-dependent Rap2 activation regulates lamellipodia dynamics during cell migration.
  51. Mass spectrometry detects folding intermediates populated during urea-induced protein denaturation.
  52. Direct measurements of luminal Ca2+ with endo-lysosomal GFP-aequorin reveal functional IP3 receptors.
  53. IGF2BP2 promotes malignant progression of ovarian cancer by regulating protein synthesis through liquid-liquid phase separation.
  1. Nature. 2025 Nov 05.
    Secretome translation shaped by lysosomes and lunapark-marked ER junctions.
    Heejun Choi, Ya-Cheng Liao, Young J Yoon, Jonathan Grimm, Nan Wang, Luke D Lavis, Robert H Singer, Jennifer Lippincott-Schwartz.
      The endoplasmic reticulum (ER) is a highly interconnected membrane network that serves as a central site for protein synthesis and maturation1. A crucial subset of ER-associated transcripts, termed secretome mRNAs, encode secretory, lumenal and integral membrane proteins, representing nearly one-third of human protein-coding genes1. Unlike cytosolic mRNAs, secretome mRNAs undergo co-translational translocation, and thus require precise coordination between translation and protein insertion2,3. Disruption of this process, such as through altered elongation rates4, activates stress response pathways that impede cellular growth, raising the question of whether secretome translation is spatially organized to ensure fidelity. Here, using live-cell single-molecule imaging, we demonstrate that secretome mRNA translation is preferentially localized to ER junctions that are enriched with the structural protein lunapark and in close proximity to lysosomes. Lunapark depletion reduced ribosome density and translation efficiency of secretome mRNAs near lysosomes, an effect that was dependent on eIF2-mediated initiation and was reversed by the integrated stress response inhibitor ISRIB. Lysosome-associated translation was further modulated by nutrient status: amino acid deprivation enhanced lysosome-proximal translation, whereas lysosomal pH neutralization suppressed it. These findings identify a mechanism by which ER junctional proteins and lysosomal activity cooperatively pattern secretome mRNA translation, linking ER architecture and nutrient sensing to the production of secretory and membrane proteins.
    DOI:  https://doi.org/10.1038/s41586-025-09718-0
  2. Autophagy Rep. 2025 ;4(1): 2574355
    CNX:FAM134B-driven ERLAD of ATZ polymers proceeds via enhanced formation of VAPA:ORP1L:RAB7 contact sites between ER and endolysosomes.
    Elisa Fasana, Ilaria Fregno, Maurizio Molinari.
      Membrane contact sites (MCS) between organelles maintain the proximity required for controlled exchange of small molecules and ions yet preventing fusion events that would compromise organelles' identity and integrity. Here, by investigating the intracellular fate of the disease-causing Z-variant of alpha1 antitrypsin (ATZ), we report on a novel function of MCS between the endoplasmic reticulum (ER) and RAB7/LAMP1-positive endolysosomes in ER-to-lysosome-associated degradation (ERLAD). For this function, the VAPA:ORP1L:RAB7 multi-protein complex forming MCS between the ER and endolysosomes engages, in an ERLAD client-driven manner, the misfolded protein segregation complex formed by the lectin chaperone calnexin (CNX), the ER-phagy receptor FAM134B, and the ubiquitin-like protein LC3. Generation of this supramolecular complex facilitates the membrane fusion events regulated by the SNARE proteins STX17 and VAMP8 that ensure efficient delivery of ATZ polymers from their site of generation, the ER, to the site of their intracellular clearance, the degradative RAB7/LAMP1-positive endolysosomes.
    Keywords:  Calnexin (CNX); ER-phagy; ER-to-Lysosome-Associated Degradation (ERLAD); Endoplasmic Reticulum (ER); FAM134B; Membrane Contact Sites (MCS); ORP1L; RAB7; VAPA; endolysosomes
    DOI:  https://doi.org/10.1080/27694127.2025.2574355
  3. Nat Struct Mol Biol. 2025 Nov 06.
    Structural landscape of the degrading 26S proteasome reveals conformation-specific binding of TXNL1.
    Connor Arkinson, Christine L Gee, Zeyuan Zhang, Ken C Dong, Andreas Martin.
      The 26S proteasome targets many cellular proteins for degradation during homeostasis and quality control. Proteasome-interacting cofactors modulate these functions and aid in substrate degradation. Here we solve high-resolution structures of the redox active cofactor TXNL1 bound to the human 26S proteasome at saturating and substoichiometric concentrations by time-resolved cryo-electron microscopy (cryo-EM). We identify distinct binding modes of TXNL1 that depend on the proteasome conformation and ATPase motor states. Together with biophysical and biochemical experiments, we show that the resting-state proteasome binds TXNL1 with low affinity and in variable positions on top of the Rpn11 deubiquitinase. In contrast, in the actively degrading proteasome, TXNL1 uses additional interactions for high-affinity binding, whereby its C-terminal tail covers the catalytic groove of Rpn11 and coordinates the active-site Zn2+. Furthermore, these cryo-EM structures of the degrading proteasome capture the ATPase hexamer in several spiral-staircase arrangements that indicate temporally asymmetric hydrolysis and conformational changes in bursts during mechanical substrate unfolding and translocation. Remarkably, we catch the proteasome in the act of unfolding the β-barrel mEos3.2 substrate while the ATPase hexamer is in a particular staircase register. Our findings advance current models for protein translocation through hexameric AAA+ motors and reveal how the proteasome uses its distinct conformational states to coordinate cofactor binding and substrate processing.
    DOI:  https://doi.org/10.1038/s41594-025-01695-2
  4. Nat Commun. 2025 Nov 06. 16(1): 9805
    PROTAC repurposing uncovers a noncanonical binding surface that mediates chemical degradation of nuclear receptors.
    Andrew D Huber, Wenwei Lin, Young-Hwan Jung, Shyaron Poudel, Guangwei Yang, Jing Wu, Annalise G Carrigan, Vishwajeeth Pagala, Wei Wang, Yingxue Fu, Zuo-Fei Yuan, Stephanie D Byrum, Ka Yang, Rebecca R Florke Gee, Elizabeth D Arnold, Allister J Loughran, Jingheng Wang, Shondra M Pruett-Miller, Junmin Peng, Taosheng Chen.
      Proteolysis-targeting chimeras (PROTACs) containing a target protein ligand linked to an E3 ubiquitin ligase ligand induce target protein degradation through E3 recruitment. Most PROTACs bind a surface cleft of the protein of interest rather than a buried pocket. Using the nuclear receptor PXR, we previously described the inherent difficulties of PROTAC targeting via a deep solvent-inaccessible ligand binding pocket. Here, we discover that the CRBN-dependent MDM2 PROTAC MD-224 is a potent PXR degrader that achieves its activity from binding adjacent to the ligand-binding pocket. Furthermore, because the proximal region is a structural feature common among nuclear receptors, MD-224 also targets additional receptors for proteasomal degradation. Using structure- and activity-guided medicinal chemistry, we ablated MDM2 degradation and generated MD-224 analogs with activities skewed toward different receptors. Thus, we describe (1) PROTAC repurposing as a potential route of degrader discovery and (2) nuclear receptor-targeted degradation through a noncanonical binding site.
    DOI:  https://doi.org/10.1038/s41467-025-64773-5
  5. Mol Microbiol. 2025 Nov 04.
    How Pathogens Maintain Proteostasis During Infection.
    Carissa Chan, Eduardo A Groisman.
      Molecular chaperones play a critical role in proteostasis by aiding the folding of newly synthesized proteins and the refolding of misfolded proteins. Cells must match the protein synthesis rate to the protein folding capacity to avoid the accumulation of unfolded proteins that can form toxic aggregates. The Hsp70 chaperone DnaK binds to ribosomes and decreases protein synthesis in the bacterial pathogen Salmonella enterica serovar Typhimurium when facing cytoplasmic Mg2+ starvation, an infection-relevant stress that disrupts proteostasis. DnaK decreases protein synthesis independently of J-domain cochaperones and nucleotide exchange factor GrpE even though J-domain cochaperones and GrpE are required for DnaK's canonical role in protein folding and refolding. DnaK's activity contrasts with that exhibited by the bacteria-specific chaperone trigger factor, which associates with ribosomes and carries out cotranslational protein folding in Mg2+-abundant conditions. Under infection-relevant conditions, the master regulator of S. typhimurium virulence and Mg2+ homeostasis PhoP promotes the expression of DnaK, but not of J-domain cochaperones, GrpE, or trigger factor, suggesting that the differential expression of chaperones and cochaperones furthers S. typhimurium pathogenesis. Hsp70 chaperones also associate with ribosomes in eukaryotic cells but instead promote protein synthesis, the opposite effect that DnaK binding to ribosomes has in bacteria. Thus, Hsp70 chaperone activity differs across growth conditions and among organisms.
    Keywords:  DnaK; Hsp70; J‐domain cochaperone; PhoP; RpoH; magnesium; protein folding; protein synthesis; trigger factor
    DOI:  https://doi.org/10.1111/mmi.70034
  6. Traffic. 2025 Oct;26(10-12): e70022
    Beyond the Secretory Pathway: New Insights Into Protein Release.
    Ruey-Hwa Chen, Antonio J Costa-Filho, Jayanta Debnath, Thierry Galli, Liang Ge, Deborah Goberdhan, Wei Guo, Kangmin He, Ralf Jacob, Tiebang Kang, Min Goo Lee, Lian Li, Fabio Lolicato, Jun Lu, Vivek Malhotra, Walter Nickel, Vassiliki Nikoletopoulou, Stacey K Ogden, Georgia Maria Sagia, Feng Shao, Anbing Shi, Clotilde Thery, Christel Vérollet, Julien Villeneuve, Frederik Verweij, Yanzhuang Wang, Juan Wang, Shenjie Wu, Yihong Ye, Hang Yin, Li Yu, Min Zhang, Ying Zhang, Xin Zhou, Chiara Zurzolo.
      In eukaryotes, protein secretion plays essential roles in intercellular communications and extracellular niche-building. Protein secretion generally requires a signal sequence that targets cargos to the canonical secretory pathway consisting of the endoplasmic reticulum (ER), the Golgi apparatus, plasma membrane, and vesicles moving between these compartments. However, cytoplasmic proteins lacking signal sequences (e.g., IL1β, Acb1, FGF2) have been detected, and many have defined functions in the extracellular space, suggesting unconventional protein secretion (UcPS) via alternative pathways. In recent years, scientists have uncovered many new UcPS paradigms, reporting a plethora of mechanisms that collectively form a new field. The inaugural Cold Spring Harbor Asia (CSHA) conference on "Molecular Mechanisms and Physiology of Unconventional Secretion" is the first meeting to bring these researchers together, providing a collegial platform for information sharing at this exciting frontier of cell biology research.
    Keywords:  CUPS; autophagy; extracellular vesicle; lysosome; stress adaptation; unconventional protein secretion
    DOI:  https://doi.org/10.1111/tra.70022
  7. Mol Biol Cell. 2025 Nov 05. mbcE25070348
    Zinc-Related IZH1 as a Novel Regulator of Endoplasmic Reticulum Homeostasis in Yeast.
    Jonathan Palmiero, Lynzie Wilkinson, Arianna Forzano, Amira Aly, Victoria Iuzzolino, Chloe LoSauro, Simrat Mangat, Nicolas McGuire, Rebecca Robinson, Michel Becuwe.
      Endoplasmic reticulum (ER) homeostasis is maintained through tightly regulated processes that coordinate lipid metabolism and proteostasis. The ER-resident acyl-CoA diphosphatase FIT2, and its yeast homolog Scs3, are key regulators of this balance; their loss disrupts ER morphology and induces chronic ER stress, though the underlying mechanisms remain unclear. To uncover factors involved in Scs3-dependent ER maintenance, we conducted a genome-wide multicopy suppressor screen in SCS3 knockout yeast cells, which display inositol auxotrophy. This analysis identified IZH1, a zinc-related ER membrane protein homologous to the human PAQR (Progestin and AdipoQ Receptor) family, as a genetic interactor of SCS3. IZH1 overexpression enhanced INO1 expression, partially restored growth of SCS3 knockout cells in inositol-deprived conditions, and reduced ER stress levels without correcting ER morphology defects. Moreover, IZH1 overexpression attenuated unfolded protein response signaling during acute proteotoxic stress and normalized ER-associated degradation kinetics. Together, these findings identify Izh1 as a novel regulator of ER homeostasis and provide new insight into how FIT2/Scs3 influence ER function.
    DOI:  https://doi.org/10.1091/mbc.E25-07-0348
  8. PLoS Pathog. 2025 Nov;21(11): e1013626
    Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia-like bacterium Simkania negevensis.
    Eva-Maria Hörner, Vanessa Lachmayer, Thomas Hermanns, Adriana Moldovan, Kay Hofmann, Vera Kozjak-Pavlovic.
      In the arms race between a pathogen and the host, the defense mechanisms of the host cell, including the ubiquitin system, are often counteracted by bacteria. Simkania negevensis (Sne), an obligate intracellular Chlamydia-like bacterium connected with respiratory diseases, possesses numerous deubiquitinases, but not much is known about its other ubiquitin-modifying enzymes. Sne infects a wide range of hosts, developing inside a tubular vacuole in close contact with the host endoplasmic reticulum (ER) and mitochondria. Our study describes an uncharacterized Sne ubiquitin E3 RING-ligase (SNE_A12920 or SneRING), which primarily generates K63- and K11-linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 enzymes. SneRING is expressed upon infection of various human cell lines, as well as amoebae. We show that a portion of the expressed SneRING co-localizes with mitochondria and ER and that the SneRING interactome includes mitochondrial and ER proteins involved in organelle morphology and stress response. Our work offers an initial characterization of a bacterial RING ligase potentially involved in the host cell remodeling to accommodate the unique intracellular lifestyle of Sne.
    DOI:  https://doi.org/10.1371/journal.ppat.1013626
  9. Dev Cell. 2025 Nov 03. pii: S1534-5807(25)00604-5. [Epub ahead of print]60(21): 2841-2843
    Golgiphagy mediated by TM9SF3 acts as quality control for stressed Golgi.
    Hallvard L Olsvik, Terje Johansen.
      Selective autophagy is important for organelle quality control. In this issue of Developmental Cell, Yang et al. identify the Golgi resident transmembrane protein TM9SF3 as a selective autophagy receptor required for lysosomal degradation of Golgi fragments (Golgiphagy) following nutrient stress, pH disruption, blockade of ER-to-Golgi trafficking, and defects in Golgi-mediated glycosylation functions.
    DOI:  https://doi.org/10.1016/j.devcel.2025.09.019
  10. Cell Rep. 2025 Oct 25. pii: S2211-1247(25)01243-4. [Epub ahead of print]44(11): 116472
    Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.
    Hao-Yang Liu, Susovan Sarkar, Feng Yuan, Grant Ashby, Carl C Hayden, Jon M Huibregtse, Jeanne C Stachowiak.
      Endocytic recycling of transmembrane proteins is essential to cellular function. The intracellular domains of transmembrane proteins are frequently ubiquitylated, a modification that is recognized by adaptor proteins during clathrin-mediated endocytosis. Recent work suggests that transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of others. Here, we show that preferential internalization of poly-ubiquitylated transmembrane proteins can result in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Further, poly-ubiquitylated receptors significantly outcompeted their less ubiquitylated counterparts for uptake of extracellular ligands. These findings suggest that clathrin-coated vesicles may act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving others behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, these findings suggest a mechanism for selective internalization of high priority cargo, with simultaneously exclusion and protection of functional proteins lacking poly-ubiquitylation.
    Keywords:  CP: Cell biology; clathrin; endocytosis; membrane traffic; transmembrane protein; ubiquitylation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116472
  11. Nat Commun. 2025 Nov 03. 16(1): 9679
    Rational design of potent small-molecule SMARCA2/A4 degraders acting via the recruitment of FBXO22.
    Elisia Villemure, Tom Januario, Mingshuo Zeng, Hanna G Budayeva, Benjamin T Walters, Aaron Lictao, Ke Sherry Li, Xiaofen Ye, Caroline L Gilchrist, Bridget Hoag, Nicholas F Endres, Peter L Hsu, John Chan, Tommy K Cheung, Michael R Costa, Jean-Philippe Fortin, Noriko Ishisoko, Brett M Babin, Joyce Liu, Alexandra Frommlet, Joachim Rudolph, Robert L Yauch.
      Target-anchored monovalent degraders are more drug-like than their bivalent counterparts, Proteolysis Targeting Chimeras (PROTACs), while offering greater target specificity control than E3 ligase-anchored monovalent degraders, also known as molecular glues. However, their discovery has typically been serendipitous, and the rules governing their identification remain unclear. This study focuses on the intentional discovery of SMARCA2/A4 monovalent degraders using a library based on SMARCA2/A4 bromodomain-binding ligands. Compound G-6599 emerged as a lead candidate, showing exceptional degradation potency and specificity for SMARCA2/A4. Mechanistic studies reveal that G-6599 operates through the ubiquitin-proteasome pathway and the E3 ligase FBXO22. G-6599 promotes ternary complex formation between SMARCA2 and FBXO22 involving covalent conjugation to a cysteine residue on the latter. Unlike other recently identified FBXO22-dependent degraders, it does not require biotransformation. The selective degradation ability of G-6599, along with its unique mechanism, highlights the therapeutic potential of target-anchored monovalent degraders.
    DOI:  https://doi.org/10.1038/s41467-025-64669-4
  12. Cell Rep. 2025 Nov 03. pii: S2211-1247(25)01256-2. [Epub ahead of print]44(11): 116485
    Identification of ATG7 as a protein ATG8ylation substrate.
    Huazhong Xie, Jiahui Long, Yijun Huang, Yao Wang, Peiqing Liu, Min Li.
      Protein ATG8ylation is a post-translational modification where ubiquitin-like protein LC3/ATG8 forms covalent conjugation with cellular proteins, a process reversed by ATG4. In contrast to the well-characterized ATG8 lipidation/membrane ATG8ylation, research on protein ATG8ylation remains limited. In this study, we identify deconjugation-resistant LC3B Q116A and F80A/L82A mutants as tools for protein ATG8ylation. We demonstrate that protein ATG8ylation depends exclusively on ATG4, ATG3, and ATG7. Tandem affinity purification-mass spectrometry reveals ATG7 as a substrate of protein ATG8ylation with K140 as its modification site. We show that protein ATG8ylation of ATG7 forms a mono-LC3B conjugate, while ATG3 undergoes lysine-dependent, mixed-linkage poly-LC3B chains. ATG7 and ATG3 function as E1 and E2 enzymes in protein ATG8ylation, potentially cooperating with E3 ligases. Notably, endogenous ATG7 ATG8ylation attenuates autophagy by disrupting its interaction with ATG3. These findings highlight ATG7 as both a central catalytic enzyme and key substrate in autophagy regulation through protein ATG8ylation.
    Keywords:  ATG3; ATG4; ATG7; CP: molecular biology; LC3 lipidation; autophagy; deconjugation-resistant; modification site; post-translational modification; protein ATG8ylation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116485
  13. J Cell Sci. 2025 Nov 03. pii: jcs.264084. [Epub ahead of print]
    The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters.
    Pragya Parashara, Lei Gao, Alyssa Riglos, Dorothy Lartey, Sonia B Sidhu, Tessa Marks, Carys Williams, Grace Siauw, Kai-Jing Lee, Anna I L Ostrem, Christian Siebold, Michael Riffle, Maia Kinnebrew, Teresa M Gunn, Jennifer H Kong.
      Mahogunin Ring Finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitination and degradation. While MGRN1 is known to regulate the Hedgehog signaling effector Smoothened (SMO) via the transmembrane adapter Multiple Epidermal Growth Factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify Attractin (ATRN) and Attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN interacts with the RING domain of MGRN1. Functional assays suggest that ATRN and ATRNL1 work with MGRN1 to promote the ubiquitination and degradation of the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 or ATRN leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, resulting in enhanced eumelanin production. These findings expand the known repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to facilitate substrate receptor specificity.
    Keywords:  E3 ubiquitin ligase; GPCR trafficking; MGRN1; Melanocortin receptors; Receptor regulation; Transmembrane adapter proteins
    DOI:  https://doi.org/10.1242/jcs.264084
  14. Nat Commun. 2025 Nov 04. 16(1): 9726
    Pmt4 recognizes two separate acceptor sites to O-mannosylate in the S/T-rich regions of substrate proteins.
    Minge Du, Zuanning Yuan, Amanda Kovach, Meinan Lyu, Huilin Li.
      Protein O-mannosyltransferases (PMTs) add mannose to serine/threonine (S/T)-rich proteins in the endoplasmic reticulum, facilitating proper folding and trafficking through the secretory pathway. These enzymes share a conserved architecture that includes a large transmembrane domain housing the catalytic pocket and a lumenal β-trefoil-folded MIR domain. Although S/T-rich regions in acceptor proteins are generally disordered, it remains unclear how PMTs selectively target these regions over other intrinsically disordered sequences. Here, using cryo-EM and X-ray crystallography, we demonstrate that the Saccharomyces cerevisiae Pmt4 dimer recognizes an S/T-rich peptide at two distinct sites. A groove above the catalytic pocket in the transmembrane domain binds the mannose-accepting S/T site, while the lumenal MIR domain engages an S/T-X-S/T motif. Notably, the substrate peptide is simultaneously bound by the catalytic pocket of one Pmt4 protomer and the MIR domain of the other, revealing an unexpected cooperative dual substrate recognition mechanism. This mechanism likely underpins the invariant dimeric architecture observed in all PMT family members.
    DOI:  https://doi.org/10.1038/s41467-025-64729-9
  15. Nat Struct Mol Biol. 2025 Nov 03.
    Mechanistic basis for protein conjugation in a diverged bacterial ubiquitination pathway.
    Qiaozhen Ye, Minheng Gong, Jiaxi Cai, Lydia R Chambers, Huilin Zhou, Raymond T Suhandynata, Kevin D Corbett.
      Ubiquitination is a fundamental eukaryotic protein post-translational modification pathway, in which ubiquitin or a ubiquitin-like protein (Ubl) is typically conjugated to a lysine of a target protein. Ubiquitination is initiated by adenylation of the Ubl C terminus, followed by sequential formation of Ubl-cysteine thioester intermediates with E1, E2 and (optionally) E3 proteins before formation of the final Ubl-lysine isopeptide linkage. Recent work has revealed two ubiquitination-related bacterial pathways in the context of antiphage immunity. Bioinformatics analyses have hinted at the existence of additional uncharacterized bacterial pathways that include ubiquitination-like machinery. Here, we describe the architecture and biochemical mechanisms of an alternative Bub (bacterial ubiquitination-like) pathway, revealing structural parallels and mechanistic differences when compared to other ubiquitination pathways. We show that Bub operons encode functional E1, E2 and Ubl proteins that are related to their eukaryotic counterparts but likely function through oxyester rather than thioester intermediates. We also identify an enzyme family in Bub operons with a conserved catalytic site and a role in Ubl-target conjugation. The genomic context of Bub operons suggests that they also function in antiphage immunity and we present evidence that one Bub pathway may regulate translation in response to stress. Overall, our results reveal an uncharacterized family of bacterial ubiquitination-related pathways with a distinctive biochemical mechanism.
    DOI:  https://doi.org/10.1038/s41594-025-01696-1
  16. Nat Commun. 2025 Nov 07. 16(1): 9752
    TUSC3 regulates ERMA-mediated Mg2+ uptake for synaptic function and neurodevelopment.
    Gyeongrin Park, Namhoon Kim, Seon-Yong Kim, Hyeonjeong Lee, Cathena Meiling Li, Jae Hong Seol, Se-Young Choi, Yong-Keun Jung.
      Intellectual disability (ID) is characterized by deficits in cognition and adaptive behavior, with few treatment options. Tumor Suppressor Candidate 3 (TUSC3) has been genetically linked to autosomal recessive ID, but its molecular mechanism and therapeutic potential remain unclear. Here we show that TUSC3 is essential for endoplasmic reticulum (ER) Mg²⁺ homeostasis and neuronal function. Using a TUSC3 knockout (KO) mouse model, we find ID-like phenotypes including impairments in learning, memory, stress adaptation, and social behavior. Mechanistically, TUSC3 forms an ER-localized Mg²⁺ transport complex with ERMA and its loss leads to ER Mg²⁺ depletion, PERK-eIF2α pathway activation, synaptic dysfunction, and neuronal vulnerability. Fibroblasts from TUSC3 mutant patients similarly exhibit ER Mg²⁺ deficiency and heightened ER stress. Magnesium supplementation restores ER Mg²⁺ levels, reduces ER stress, and rescues cognitive deficits. Our findings establish ER Mg²⁺ dysregulation as a key driver of neurodevelopmental dysfunction and a promising therapeutic target.
    DOI:  https://doi.org/10.1038/s41467-025-65668-1
  17. Proc Natl Acad Sci U S A. 2025 Nov 11. 122(45): e2501834122
    Amino acid insufficiency impairs hepatic vitamin A mobilization in mice.
    Chintan T Bhavsar, Youn-Kyung Kim, Flavio C Rodriguez-Polanco, Brian A Zalma, Esther M Lopez, Saad A Farooq, Maria Ibrahim, Eileen White, Tracy G Anthony, Loredana Quadro.
      Retinol-binding protein 4 (RBP4) is the sole specific serum carrier of vitamin A, delivering it from the hepatic stores to the peripheral organs in a complex with retinol (ROH) and transthyretin (TTR). Regulators of hepatic mobilization aside from vitamin A status itself are ill-defined. Here, we show that amino acid (AA) insufficiency by diet (low-protein, leucine-devoid diet) or drug (asparaginase, ASNase) elevated liver RBP4 without depleting hepatic retinoid stores. In addition, ASNase reduced liver TTR protein. Furthermore, circulating ROH-RBP4-TTR levels were attenuated, and retinoid levels in peripheral organs were perturbed. To understand the basis for elevated RBP4 in the liver, we isolated total and polysomal mRNA to assess gene-specific translation. ASNase significantly reduced Rbp4 mRNA translation, indicating that elevated hepatic RBP4 protein was not due to increased protein synthesis. In contrast, ASNase reduced Ttr mRNA abundance but not its translation. Global deletion of the AA insufficiency sensor, general control nonderepressible 2 (GCN2), lessened ASNase-induced RBP4 protein accumulation in the liver but did not rescue circulating ROH-RBP4 levels. These effects were replicated by chemical inhibition of GCN2 in ASNase-exposed primary hepatocytes. Finally, hepatocyte-specific knockout of autophagy-related 7, an enzyme involved in autophagy and protein secretion, fully rescued circulating ROH-RBP4-TTR and normalized liver RBP4 and TTR during ASNase. Overall, our findings identify AA insufficiency to modulate hepatic ROH-RBP4 mobilization independent of vitamin A status.
    Keywords:  asparaginase; integrated stress response; retinol; retinol-binding protein 4; transthyretin
    DOI:  https://doi.org/10.1073/pnas.2501834122
  18. Nat Commun. 2025 Nov 07. 16(1): 9833
    Lysosomal and mTORC1 signaling dysregulation underpin the pathology of spastic paraplegia type 80.
    Yiqiang Zhi, Tongtong Zhang, Danping Lu, Shuhuai Lin, Huizhen Su, Yupei Wu, Qiyuan Chang, Shuyuan Wang, Chenning Lv, Honggao Fu, Li-Yu Chen, Wan-Jin Chen, Ning Wang, Zhifei Fu, Xiang Lin, Dan Xu.
      Endosomal sorting complex required for transport (ESCRT) is the major membrane remodeling complex, closely associated with endolysosomal repair and hereditary spastic paraplegias (HSP) diseases. Loss of function mutations in the ESCRT-I component UBAP1 causes a rare type of HSP (spastic paraplegia 80, SPG80), while the underlying pathological mechanism is unclear. Here, we found that UBAP1 but not SPG80 causing mutant was efficiently recruited to damaged lysosomes and mediated lysosome recovery. Loss of UBAP1 results in dysfunction of lysosomes, disconnecting mTOR localization on lysosomes, leading to cytoplasmic mTORC1 activation and TFEB dephosphorylation, as confirmed in vitro and in vivo models. Administration of rapamycin, a specific inhibitor of mTORC1, enhances mTOR lysosomal localization and TFEB phosphorylation. This pharmacological intervention effectively attenuated disease progression and restored lysosomal homeostasis in Ubap1 deficiency mice. Our findings reveal UBAP1's role in lysosome regulation and suggest rapamycin may benefit patients with HSP and other motor neuron disorders.
    DOI:  https://doi.org/10.1038/s41467-025-64800-5
  19. STAR Protoc. 2025 Oct 30. pii: S2666-1667(25)00579-9. [Epub ahead of print]6(4): 104173
    Protocol for halofuginone-mediated metabolic reprogramming of murine T cells via activation of the GCN2 pathway.
    Meghan Kates, Michael St Paul, Pamela S Ohashi, Samuel D Saibil.
      Modifying T cell metabolism and activating conserved stress pathways can enhance T cell efficacy in adoptive cell therapy for cancer treatment. Here, we present a protocol to activate the General Control Non-depressible 2 (GCN2)-mediated branch of the integrated stress response (ISR) in murine T cells using the drug halofuginone. We outline the process of isolating CD8+ T cells from T cell receptor transgenic mice, activating them with bone-marrow-derived dendritic cells, and subsequently activating GCN2 and the ISR with halofuginone. For complete details on the use and execution of this protocol, please refer to St. Paul et al.1.
    Keywords:  Cell Biology; Cell isolation; Immunology; Metabolism
    DOI:  https://doi.org/10.1016/j.xpro.2025.104173
  20. Proc Natl Acad Sci U S A. 2025 Nov 11. 122(45): e2427161122
    Modeling protein-small molecule conformational ensembles with PLACER.
    Ivan Anishchenko, Yakov Kipnis, Indrek Kalvet, Guangfeng Zhou, Rohith Krishna, Samuel J Pellock, Anna Lauko, Gyu Rie Lee, Linna An, Justas Dauparas, Frank DiMaio, David Baker.
      Modeling the conformational heterogeneity of protein-small molecule interactions is important for understanding natural systems and evaluating designed systems but remains an outstanding challenge. We reasoned that while residue-level descriptions of biomolecules are efficient for de novo structure prediction, for probing heterogeneity of interactions with small molecules in the folded state, an entirely atomic-level description could have advantages in speed and generality. We developed a graph neural network called PLACER (protein-ligand atomistic conformational ensemble resolver) trained to recapitulate correct atomic positions from partially corrupted input structures from the Cambridge Structural Database and the Protein Data Bank; the nodes of the graph are the atoms in the system. PLACER accurately generates structures of diverse organic small molecules given knowledge of their atom composition and bonding. When given a description of the larger protein context, it builds up structures of small molecules and protein side chains for protein-small molecule docking. Because PLACER is rapid and stochastic, ensembles of predictions can be readily generated to map conformational heterogeneity. In enzyme design efforts described here and elsewhere, we find that using PLACER to assess the accuracy and preorganization of the designed active sites results in higher success rates and higher activities; we obtain a preorganized retroaldolase with a kcat/KM of 11,000 M-1min-1, considerably higher than any pre-deep learning design for this reaction. We anticipate that PLACER will be widely useful for rapidly generating conformational ensembles of small molecule and small molecule-protein systems and for designing higher activity preorganized enzymes.
    Keywords:  enzyme design; ligand docking; machine learning
    DOI:  https://doi.org/10.1073/pnas.2427161122
  21. ACS Chem Neurosci. 2025 Nov 02.
    Improving Proteostasis of Trafficking-Deficient GABAA Receptor Variants by Activating IRE1.
    Xu Fu, Ya-Juan Wang, KyungA Lee, Lucie Y Ahn, Xi Chen, Brock T Harvey, Meng Wang, Hailey Seibert, Pei-Pei Zhang, Adrian Guerrero, Ashleigh E Schaffer, Christopher I Richards, R Luke Wiseman, Jeffery W Kelly, Ting-Wei Mu.
      Gamma-aminobutyric acid type A receptors (GABAARs) are essential for maintaining the excitation-inhibition balance in the central nervous system. Genetic variations of GABAARs result in a variety of neurological disorders, such as epilepsy. A key pathogenic mechanism involves protein misfolding and defective assembly of GABAARs in the endoplasmic reticulum (ER), resulting in impaired surface expression and loss of function. Here, we investigated three trafficking-deficient variants of the GABAAR α1 subunit (GABRA1), including D219N (ClinVar Variation ID: 127232), G251D (Variation ID: 419523), and P260L. We demonstrated that selective pharmacological activation of the IRE1/XBP1s signaling arm of the unfolded protein response using IXA62, IXA554, and IXA105 increases total and surface protein levels of all three α1 variants without affecting wild-type receptor protein levels in HEK293T cells. Patch-clamping recordings further showed that treatment with IXA62, IXA554, and IXA105 increases the peak GABA-evoked current amplitudes in HEK293T cells expressing α1(D219N) and α1(G251D). Mechanistic analyses revealed that IXA62 and IXA554 remodel the GABAAR-associated proteostasis network by promoting folding and anterograde trafficking while inhibiting degradation in HEK293T cells expressing α1(D219N) variant and human iPSC-derived neurons carrying α1(G251D) variant. These results suggest that selective IRE1/XBP1s activation pharmacologically can be further developed to provide a potential therapeutic avenue for genetic epilepsies caused by GABAAR trafficking defects.
    Keywords:  GABAA receptors; IRE1; epilepsy; misfolding; proteostasis; proteostasis regulators
    DOI:  https://doi.org/10.1021/acschemneuro.5c00227
  22. J Cell Biol. 2025 Dec 01. pii: e202505064. [Epub ahead of print]224(12):
    Contact-dependent incorporation of endoplasmic reticulum into retraction fibers and migrasomes.
    Peiyao Fan, Rui Ji, Yiling Wen, Yaping Dang, Yong Li, Xiaojie Yan, Murong Li, Qiaoxia Zheng, Yifan Ge, Pengli Zheng, Yang Chen.
      Migrating cells form retraction fibers (RFs) at their trailing edge, where migrasomes, ranging from 0.5 to 3 μm, grow at the tips or intersections of RF. Migrasomes play crucial roles when released extracellularly, but before release, they remain physically connected to cell body via RFs, facilitating long-range signal transmission. Since many signaling molecules are highly localized, the mechanism of long-range signal transmission has not been fully understood. Here, we demonstrated that tubular ER extended into RFs and localized to migrasomes, which depended on microtubule-regulated ER extension. Tubular ER adhered to migrasome biogenesis site through ER-plasma membrane contact sites (ER-PM MCSs). Notably, tubular ER functions as cholesterol and calcium reservoir, facilitating the transfer of cholesterol and calcium to migrasomes, potentially at ER-PM MCSs that promoted membrane expansion, stability, and localized secretion of migrasome. Our findings revealed a novel dynamic of tubular ER and provided a new mechanism for long-range site-specific calcium and cholesterol transmission through RFs and migrasomes in migrating cells.
    DOI:  https://doi.org/10.1083/jcb.202505064
  23. J Biol Chem. 2025 Nov 05. pii: S0021-9258(25)02762-0. [Epub ahead of print] 110910
    A Modular Uba1-Nanobody Fusion Enables Selective Ubiquitin Transfer to Tagged E2 Enzymes.
    Charlotte Wijne, Francesca D'Amico, David A Pérez Berrocal, Monique P C Mulder, Hidde L Ploegh, Jin Gan.
      The ubiquitin-activating enzyme Uba1 initiates the ubiquitination cascade by activating ubiquitin and subsequently transferring it to a broad range of E2 conjugating enzymes via its C-terminal ubiquitin-fold domain (UFD). Tools to selectively redirect this transfer to defined E2s, and thus govern control of E2 enzyme usage, are limited. By replacing the ubiquitin-fold domain (UFD) of Uba1 with the high-affinity nanobody VHH05, we create an engineered E1 enzyme (Uba1-VHH05) that selectively engages E2s fused to the 6e-tag, which is the epitope recognized by VHH05. This plug-and-play interface preserves native Uba1 catalytic activity while allowing ubiquitin loading to be directed toward user-defined E2s without altering other components of the cascade. We show that Uba1-VHH05 supports transfer of both wild-type ubiquitin and the activity-based probe Ub-Dha to a range of tagged E2s. This recapitulates endogenous in vitro activities, including polyubiquitin chain formation. This strategy enables precise dissection of E2-specific functions and offers a new tool to generate orthogonal ubiquitin cascades in vitro and ultimately in cells.
    DOI:  https://doi.org/10.1016/j.jbc.2025.110910
  24. Mol Cell. 2025 Nov 06. pii: S1097-2765(25)00853-6. [Epub ahead of print]
    Metabolic environment-driven remodeling of mitochondrial ribosomes regulates translation and biogenesis.
    Fan Zheng, Zanlin Yu, Junming Zhuang, Lingraj Vannur, William Nickols, YongQiang Wang, Liying Li, Sho Joseph Ozaki Tan, Seungmin Yoo, Yifan Cheng, Chuankai Zhou.
      Cytosolic translation activity is fine-tuned by environmental conditions primarily through signaling pathways that target translation initiation factors. Although mitochondria possess their own translation machinery, they lack an autonomous signaling network analogous to their cytosolic counterpart for regulating translation activity. Consequently, our understanding of how mitochondrial translation activity is adjusted under different metabolic environments remains very limited. Here, we report a noncanonical mechanism for regulating mitochondrial translation activity via metabolism-dependent changes in the mitochondrial ribosome (mitoribosome) in S. cerevisiae. These changes arise from a metabolism-modulated mitoribosome assembly pathway that regulates the composition and conformation of the mitoribosome, thereby adjusting its translation activity to meet metabolic demands. Moreover, the translation activity of the mitoribosome feeds back to regulate the biogenesis of nuclear-encoded mitochondrial proteins, influencing mitochondrial functions and aging. Such a ribosomal remodeling-based "gear-switching" mechanism represents an orthogonal mode of translation regulation, compensating for the absence of a translation-modulating signaling network within mitochondria.
    Keywords:  aging; metabolism; mitochondria; mitoribosome; translation activity
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.012
  25. Commun Biol. 2025 Nov 05. 8(1): 1529
    nERdy: network analysis of endoplasmic reticulum dynamics.
    Ashwin Samudre, Guang Gao, Ben Cardoen, Bharat Joshi, Ivan Robert Nabi, Ghassan Hamarneh.
      The endoplasmic reticulum (ER) comprises smooth tubules, ribosome-studded sheets, and peripheral sheets that can present as tubular matrices. ER shaping proteins determine ER morphology, however, understanding their role in tubular matrix formation requires reconstructing the dynamic, convoluted ER network. Existing reconstruction methods are sensitive to parameters or require extensive annotation and training for deep learning. We introduce nERdy, an image processing based approach, and nERdy+, a D4-equivariant neural network, for accurate extraction and representation of ER networks and junction dynamics, outperforming previous methods. Comparison of stable and dynamic representations of the extracted ER structure reports on tripartite junction movement and distinguishes tubular matrices from peripheral ER networks. Analysis of live cell confocal and Stimulated emission depletion microscopy (STED) time series data shows that Atlastin and Reticulon 4 promote dynamic tubular matrix formation and enhance junction dynamics, identifying novel roles for these ER shaping proteins in regulating ER structure and dynamics.
    DOI:  https://doi.org/10.1038/s42003-025-08892-1
  26. NAR Cancer. 2025 Dec;7(4): zcaf044
    Exploiting nanopore sequencing advances for tRNA sequencing of human cancer models.
    Adva Kochavi, Arno Velds, Maya Suzuki, Shinichiro Akichika, Tsutomu Suzuki, Roderick L Beijersbergen, Reuven Agami.
      Transfer RNAs (tRNAs) are essential regulators of protein synthesis, and dysregulation of their abundance and modification status is involved in many human diseases including cancer. Despite the rapid development of novel tRNA sequencing approaches, due to tRNAs' stable secondary structure and abundant modification sites, the human tRNA landscape has remained mostly unexplored. Here, we evaluated the new RNA004 chemistry of Oxford Nanopore Technologies, that is integrated with updated Dorado base-caller models, for tRNA quantification and modification annotation in human cancer models. We demonstrated that this technology identifies variations in tRNA expression across cancer cell lines and in response to external stress conditions, with highly reproducible results. We also show that analysis of base-calling error rate can indicate the presence of known modifications, including the cancer-associated tRNAPhe-Wybutosine modification. Furthermore, implementing the updated Dorado modification-calling feature, we showed the potential of RNA004 tRNA-seq in predicting common tRNA modifications. We also pinpointed possible limitations and challenges associated with both modification calling methods. Overall, RNA004 tRNA-seq can potentially enhance our understanding of the human tRNAome by simultaneously analyzing both tRNA abundance and modifications.
    DOI:  https://doi.org/10.1093/narcan/zcaf044
  27. Cell Rep. 2025 Oct 30. pii: S2211-1247(25)01257-4. [Epub ahead of print]44(11): 116486
    The ER thioredoxin-related transmembrane protein TMX2 controls redox-mediated tethering of ER-mitochondria contacts.
    Junsheng Chen, Megan C Yap, Arthur Bassot, Danielle M Pascual, Tadashi Makio, Jannik Zimmermann, Heather Mast, Rakesh Bhat, Samuel G Fleury, Yuxiang Fan, Adriana Zardini Buzatto, Jack Moore, Klaus Ballanyi, Liang Li, Michael Overduin, M Joanne Lemieux, Hélène Lemieux, Grazia M S Mancini, Bruce Morgan, Paul C Marcogliese, Thomas Simmen.
      Thioredoxin-related transmembrane proteins (TMXs) of the endoplasmic reticulum (ER) determine not only redox conditions within the ER lumen but also the formation and function of ER-mitochondria membrane contact sites (ERMCS). The presence of cytosolic, reactive oxygen species (ROS)-derived redox nanodomains at ERMCS suggests TMXs could also control these. The prime candidate for such a function is TMX2, the sole TMX family protein with a cytosolic thioredoxin domain. Indeed, TMX2 controls the extent of ERMCS through interaction with outer mitochondrial membrane proteins, including TOM70. Assisted by cytosolic peroxiredoxins, TMX2 moderates the sulfenylation of the TOM70 C206 residue. Thereby, TMX2 reduces mitochondrial Ca2+ uptake and metabolism. Accordingly, mutation of the TMX2 gene in cells from a patient with a neurodevelopmental disorder with microcephaly, cortical malformations, and spasticity (NEDMCMS) results in hyperactive mitochondria. In a fly in vivo NEDMCMS model, TMX2 knockdown manifests predominantly in glial cells, where it prevents seizure-like behavior.
    Keywords:  CP: Molecular biology; Ca(2+); ER; MCS; PRDX; TMX2; TOM70; endoplasmic reticulum; membrane contact sites; mitochondria; peroxiredoxin; redox
    DOI:  https://doi.org/10.1016/j.celrep.2025.116486
  28. J Cell Sci. 2025 Nov 03. pii: jcs.263920. [Epub ahead of print]
    Endoplasmic reticulum protein FAM134B acts as a regulator of mitochondrial morphology.
    Sebabrata Maity, Anwesha Dutta Gupta, Izaz Monir Kamal, Rajdeep Das, Rupsha Mondal, Arpit Tyagi, Deepak Sharma, Joy Chakraborty, Saikat Chakrabarti, Oishee Chakrabarti.
      The endoplasmic reticulum (ER) and mitochondria are known to affect myriad cellular mechanisms. More recently, dynamic association between them has been identified in different eukaryotes; these interactions vary in their composition and involvement in regulation of intracellular machineries. FAM134B or RETREG1, originally identified as an oncogene, regulates ER membrane shape and curvature. It is a key ER-phagy or reticulophagy receptor, which promotes autophagy of not only the ER but also simultaneous dual autophagy of ER and mitochondria. While it is known that FAM134B can potentiate contact with mitochondria, its direct involvement in affecting mitochondrial dynamics remains unexplored. Here we show that FAM134B can interact with the canonical fission-promoting protein, DRP1. Functional depletion of FAM134B leads to local Actin rearrangement and reduced DRP1 recruitment onto mitochondria, resulting in hyperfusion. A decrease in FAM134B levels is observed with aging in rat brains, cell and mouse models of Parkinson's disease and patient-derived samples. Our study establishes FAM134B as the ER partner that helps in maintaining mitochondrial morphology and dynamics.
    Keywords:  DRP1; FAM134B; Fission; Mitochondrial hyperfusion
    DOI:  https://doi.org/10.1242/jcs.263920
  29. Trends Cell Biol. 2025 Nov 06. pii: S0962-8924(25)00225-9. [Epub ahead of print]
    Membrane curvature at the ER-PM contact sites.
    Yang Yang, Luis A Valencia, Bianxiao Cui.
      Membrane contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) are essential for lipid transfer, calcium signaling, and membrane organization. While the formation and function of ER-PM contacts are increasingly well-characterized, the spatiotemporal regulation of their localization remains elusive. Emerging evidence using nanopatterned substrates, ultrastructural imaging, and protein localization analyses indicates that membrane curvature can act as a spatial cue for the recruitment of specific tethering proteins, influencing where contact sites form. This opinion article synthesizes recent advances linking membrane topography ER-PM contact organization and highlights systems where curvature actively orchestrates contact position through curvature-sensing proteins. It also outlines key unanswered questions about how membrane curvature integrates into broader signaling networks that govern organelle contact communication.
    Keywords:  ER–PM contact; RyR2; calcium signaling; junctophilin; membrane curvature
    DOI:  https://doi.org/10.1016/j.tcb.2025.10.002
  30. J Thromb Haemost. 2025 Nov 04. pii: S1538-7836(25)00716-0. [Epub ahead of print]
    Thiol isomerase ERp46 catalyzes the disulfide formation of coagulation factor XII enhancing its activity.
    Aizhen Yang, Yaqiong Zhang, Yaowei Sun, Miao Jiang, Yi Lu, Yue Han, Depei Wu, Zhipu Luo, Yi Wu.
       BACKGROUND: Protein disulfide isomerases (PDIs) are a family of thiol oxidoreductases that catalyze the oxidation, reduction, and isomerization of disulfide bonds. While some PDIs enhance arterial thrombosis, their roles in coagulation system remain largely unknown.
    METHODS: The effect of a thiol blocker NEM and a reducing agent DTT on FXII activity was measured by chromogenic assay, activated partial thromboplastin time (aPTT) and thrombin generation assay. Redox states of FXII disulfides were determined by thiol labeling and mass spectrometry. Functional disulfides were evaluated through cysteine mutagenesis of FXII and predicting structural function via molecular dynamics simulations. Thiol modification and kinetic trapping were used to identify ERp46 substrates. The inferior vena cava stenosis model assessed the roles of ERp46 and FXII in venous thrombosis.
    RESULTS: NEM significantly prolonged aPTT and inhibited FXIIa chromogenic activity, while DTT inhibited clotting, thrombin generation and substrate HK cleavage. Of the PDIs tested, only oxidized ERp46 enhanced FXII activity. Screening identified Cys513-Cys529 and Cys540-Cys571 as crucial disulfides for FXII function. Notably, half of Cys540-Cys571 were in partially disulfide-bonded form. ERp46 oxidized Cys540-Cys571 and increased FXII activity. In vivo, ERp46 deficiency reduced venous thrombus growth, with no additive effect observed in mice with combined ERp46 and FXII deficiency. Only wild-type FXII (FXII/WT) protein, not FXII/C540S-C571S mutant, restored venous thrombus growth in FXII deficient mice.
    CONCLUSIONS: These findings reveal a novel redox-regulatory mechanism for FXII activity and identify the critical role of ERp46 in oxidization of Cys540-Cys571 disulfide facilitating the activation of FXII and intrinsic coagulation pathway.
    Keywords:  Coagulation; Disulfide; Endoplasmic reticulum protein 46; Factor XII; Redox; Thrombosis
    DOI:  https://doi.org/10.1016/j.jtha.2025.10.016
  31. Cell Rep. 2025 Oct 25. pii: S2211-1247(25)01247-1. [Epub ahead of print]44(11): 116476
    IGF2BP2 stabilized by USP7 promotes cancer-associated fibroblast activation and attenuates gemcitabine sensitivity in PDAC.
    Fujing Ge, Hongdao Zhu, Xiangning Liu, Yuekang Li, Runqiu Guo, Chenming Zeng, Tao Yuan, Liu Yang, Xin Dong, Yulian Wu, Renhua Gai, Ronggui Hu, Tianhua Zhou, Qiaojun He, Hong Zhu, Bo Yang.
      N6-Methyladenosine (m6A) modification is a prevalent alteration in RNA, influencing stability, localization, and translation. The m6A reader IGF2BP2 stabilizes various mRNA transcripts of oncogenic proteins and has been extensively implicated in various tumors. Intriguingly, in pancreatic ductal adenocarcinoma (PDAC), IGF2BP2 maintains its protein stability despite the elevated autophagic activity, suggesting the existence of regulatory mechanisms that counteract IGF2BP2 degradation in PDAC. Herein, we explore the crosstalk between m6A modification and protein homeostasis, particularly the protein stability of IGF2BP2 under excessive activation of autophagic flux. Mechanistically, we show that deubiquitinase USP7 acts on IGF2BP2, which enhances the stability of IGF2BP2 by reversing its K33-linked polyubiquitin chains, thereby preventing its degradation via the autophagy-lysosome pathway. Accumulated IGF2BP2 stabilizes PDGFA mRNA, activating myofibroblastic cancer-associated fibroblasts (myCAFs) through PDGFR interaction, which promotes resistance to gemcitabine. These findings uncover the USP7/IGF2BP2/PDGFA axis as a critical regulatory pathway in PDAC progression and chemoresistance.
    Keywords:  CP: Cancer; N(6)-methyladenosine; PDGFR; cancer-associated fibroblasts; deubiquitinase; pancreatic ductal adenocarcinoma
    DOI:  https://doi.org/10.1016/j.celrep.2025.116476
  32. Nat Struct Mol Biol. 2025 Nov 04.
    TRIM21 and OTUD6A orchestrate AKT K27-linked atypical ubiquitination to modulate cancer chemoresistance.
    Qiwei Jiang, Peipei Song, Shuishen Zhang, Qin Ren, Meiyan Zhu, Jiawei Ge, Lang Bu, Wei Chen, Xueji Wu, Shiyao Han, Yaqing Su, Lei Wang, Wei Xie, Chao Cheng, Zhenwei Peng, Jianping Guo.
      Ubiquitination regulates various physiological and pathological processes. However, the impact of atypical AKT ubiquitination and its potential role in tumorigenesis remain unclear. Here we show that AKT is modified by K27-linked ubiquitination by the E3 ubiquitin ligase TRIM21, a process antagonized by the deubiquitinase OTUD6A. As such, TRIM21 acts as a tumor suppressor by repressing AKT activity, whereas OTUD6A counteracts AKT suppression. Mechanistically, TRIM21-mediated AKT ubiquitination disrupts SKP2-mediated or TRAF6-mediated K63 ubiquitination, thereby blocking AKT membrane localization and its kinase activity. Upon activation in response to amino acids, S6K1 directly phosphorylates and inactivates OTUD6A, enabling a negative feedback loop regulating AKT activity in a deubiquitination-dependent manner. In agreement with this model, Otud6a deficiency reduces lung tumorigenesis in a KrasG12D-driven lung cancer mouse model and TRIM21 induction alleviates hyperactive AKT-induced tumor growth in vivo. Thus, our findings unveil a fine-tuned regulation of AKT through atypical ubiquitination and suggest the strategy for combating AKT-driven cancers by targeting the TRIM21-OTUD6A axis.
    DOI:  https://doi.org/10.1038/s41594-025-01697-0
  33. Nature. 2025 Nov 05.
    Atomically accurate de novo design of antibodies with RFdiffusion.
    Nathaniel R Bennett, Joseph L Watson, Robert J Ragotte, Andrew J Borst, DéJenaé L See, Connor Weidle, Riti Biswas, Yutong Yu, Ellen L Shrock, Russell Ault, Philip J Y Leung, Buwei Huang, Inna Goreshnik, John Tam, Kenneth D Carr, Benedikt Singer, Cameron Criswell, Basile I M Wicky, Dionne Vafeados, Mariana Garcia Sanchez, Ho Min Kim, Susana Vázquez Torres, Sidney Chan, Shirley M Sun, Timothy T Spear, Yi Sun, Keelan O'Reilly, John M Maris, Nikolaos G Sgourakis, Roman A Melnyk, Chang C Liu, David Baker.
      Despite the central role of antibodies in modern medicine, no method currently exists to design novel, epitope-specific antibodies entirely in silico. Instead, antibody discovery currently relies on immunization, random library screening or the isolation of antibodies directly from patients1. Here we demonstrate that combining computational protein design using a fine-tuned RFdiffusion2 network with yeast display screening enables the de novo generation of antibody variable heavy chains (VHHs), single-chain variable fragments (scFvs) and full antibodies that bind to user-specified epitopes with atomic-level precision. We experimentally characterize VHH binders to four disease-relevant epitopes. Cryo-electron microscopy confirms the binding pose of designed VHHs targeting influenza haemagglutinin and Clostridium difficile toxin B (TcdB). A high-resolution structure of the influenza-targeting VHH confirms atomic accuracy of the designed complementarity-determining regions (CDRs). Although initial computational designs exhibit modest affinity (tens to hundreds of nanomolar Kd), affinity maturation using OrthoRep3 enables production of single-digit nanomolar binders that maintain the intended epitope selectivity. We further demonstrate the de novo design of scFvs to TcdB and a PHOX2B peptide-MHC complex by combining designed heavy-chain and light-chain CDRs. Cryo-electron microscopy confirms the binding pose for two distinct TcdB scFvs, with high-resolution data for one design verifying the atomically accurate design of the conformations of all six CDR loops. Our approach establishes a framework for the computational design, screening and characterization of fully de novo antibodies with atomic-level precision in both structure and epitope targeting.
    DOI:  https://doi.org/10.1038/s41586-025-09721-5
  34. BMC Biol. 2025 Nov 03. 23(1): 333
    Genomic and translational insights into eIF2B-mediated salt tolerance in sea rice HD961.
    Haomin Chen, Mingming Chen, Shan Yang, Hongkai Zhou, Zhihao Xie, Yongxiang Huang, Daming Chen.
       BACKGROUND: Soil salinization threatens global rice production, driving the urgent need for salt-tolerant rice cultivars. Sea rice HD961, renowned for its exceptional salt tolerance, serves as an ideal model for elucidating molecular adaptations to salinity.
    RESULTS: In this study, we generated a high-quality, chromosome-level genome assembly of HD961 using Nanopore long-read sequencing, Illumina short-read polishing, and Hi-C-based scaffolding, providing a robust foundation for translatomic analysis. To explore translational responses to salt stress, we integrated ribosome profiling (Ribo-seq) with the QEZ-seq protocol and RNA sequencing (RNA-seq) under 150 mM NaCl conditions. Our results reveal that salt stress selectively enhances translational efficiency (TE) in genes critical for ion homeostasis, antioxidant defense, and cell wall remodeling, enabling HD961 to maintain cellular balance under stress. Especially, eukaryotic translation initiation factor 2B (eIF2B) emerged as a key regulator, with its upregulation and the formation of stress-induced eIF2B-containing bodies indicating a novel mechanism to optimize protein synthesis. Additionally, ribosome footprint profiling revealed codon-specific modulation of A-site dwell times, with the GCG codon showing a particularly pronounced shift under salt stress, suggesting fine-tuned translational control that prioritizes stress-responsive proteins.
    CONCLUSION: Together, these findings highlight eIF2B-mediated translational regulation as central to HD961's salt tolerance, offering valuable genomic and translatomic resources for breeding salt-tolerant rice and other crops.
    Keywords:  HD961; Ribosome profiling; Salt stress; Translation regulation; eIF2B
    DOI:  https://doi.org/10.1186/s12915-025-02440-3
  35. Mol Cell Proteomics. 2025 Oct 30. pii: S1535-9476(25)00539-0. [Epub ahead of print] 101440
    Multi-dimensional Proteomics Reveal Metformin's Impact on Interconnected Regulatory Networks of Protein Turnover, Ubiquitination, DNA Damage, and Cell Cycle.
    Zhiyuan Wang, Jianlong Li, Jinyan Duan, Bing Shan, Yaoyang Zhang.
      Metformin, a first-line therapy for type 2 diabetes, has also been implicated in regulating diverse physiological and pathological processes, including lifespan extension, cancer, and other disease-related conditions. However, its mechanisms of action remain incompletely understood, with many effects still unexplained. In this study, we investigated the impact of metformin on the cellular ubiquitinome and associated protein turnover. Through an integrated analysis combining ubiquitinome profiling with pulsed metabolic labeling, we found that metformin markedly suppresses global protein ubiquitination, including various types of ubiquitin chain linkages, and concurrently inhibits both protein synthesis and degradation. Notably, metformin induces a marked reduction in the ubiquitination of histone H4, a modification closely associated with DNA damage repair. We further establish a mechanistic link whereby metformin regulates DNA damage repair and cell cycle progression through downregulating ubiquitination. Together, our findings demonstrate that metformin modulates ubiquitination and proteostasis, central processes that regulate numerous cellular functions. By identifying histone H4 ubiquitination as a key target, we elucidate a potential mechanism through which metformin influences DNA repair and cell cycle progression. This comprehensive dataset advances understanding of the drug's multifaceted pharmacological activities and provides a valuable resource for future drug development.
    Keywords:  Cell cycle; DNA damage repair; Metformin; Proteostasis; Ubiquitinome
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101440
  36. Cell Death Dis. 2025 Nov 05. 16(1): 793
    HERPUD1 mediates palmitic acid-induced UPR sustaining TNBC aggressiveness and is destabilized by CK2 pharmacological inhibition.
    Laura Hernández-Torres, Viviana A Cavieres, Omar Cortés, Eloisa Arias-Muñoz, Francisca Cruzat-Arias, Jorge Catalán-Aguilera, Javiera Álvarez-Indo, Macarena Aguilera-Olguín, Ronny Hernández, Eduardo Silva-Pavez, Julio C Tapia, Jorge Cancino, Carlos F Lagos, Tammy P Pástor, Abigail J Galarza, Gonzalo A Mardones, Manuel Varas-Godoy, Nicole Villarreal-Cruz, Franz Villarroel-Espindola, Isabel Saffie, Alfonso González, María José Barrera, Pamela Ehrenfeld, Patricia V Burgos.
      HERPUD1 is a protein of the endoplasmic reticulum (ER) that is sensitive to the unfolded protein response (UPR) induced during ER stress and has been linked to ER stress tolerance in cancer cells. Many tumors, including triple-negative breast cancer (TNBC), which lacks an effective treatment, display UPR activity as a malignancy trait. However, whether HERPUD1 provides an ER-dependent mechanistic link for tumorigenic agents and/or potential therapeutic targets remains unknown. To address these possibilities, we first analyzed HERPUD1 expression in breast cancer (BC) biopsies via immunohistochemistry and immunofluorescence, revealing significantly higher levels in BC, including luminal A and TNBC, compared to non-malignant tissue. In TNBC, in addition to epithelial cells, HERPUD1 associated with inflammatory infiltrates, highlighting its potential role in tumor progression. Palmitic acid (PA), a dietary saturated fatty acid, is an obesity-associated tumor risk factor that induces ER stress and activates UPR. Interestingly, MDA-MB-231 cells, but not other BC cell lines, specifically upregulate HERPUD1 together with XBP1s and ATF4, key UPR factors, in response to PA, whereas TG treatment elevated HERPUD1 across all tested cell lines. HERPUD1 silencing reduced TNBC cell proliferation, migration, and invasion while enhancing doxorubicin (DOX) cytotoxicity, in both 2D and 3D cell culture models. HERPUD1 ablation also elevated UPR activation under TG. In contrast, PA-induced stress led to reduced UPR activation and lower IL-6 and IL-8 levels in the absence of HERPUD1 expression. We identified CK2 as a kinase that regulates HERPUD1 stability via Ser-59 phosphorylation. Strikingly, inhibition of CK2 with CX-4945 not only reduced HERPUD1 levels but also increased the sensitivity of BC cells to DOX. HERPUD1-S59D phosphomimetic mutants showed opposite effects.Our findings establish HERPUD1 as a key mediator of PA-driven aggressiveness, dependent on the lipid-handling capacity of TNBC cells and reveals a mechanistic to lipid stress and tumor progression.
    DOI:  https://doi.org/10.1038/s41419-025-08111-z
  37. Mol Biomed. 2025 Oct 24. 6(1): 84
    Endoplasmic reticulum-resident protein DNAJC10 inhibits glioblastoma metastasis by suppressing XBP-1s-driven EGFR transcription.
    Erdi Zhao, Yue Yu, Yingli Gao, Teng Li, Shiyu Hao, Meiyang Chen, Ming Xu, Sinkemani Arjun, Chunyan Yang, Yancun Yin, Minjing Li.
      Glioblastoma (GBM) is characterized by the highly infiltrative growth of cancer cells into the surrounding brain parenchyma. DnaJ Heat Shock Protein Family (Hsp40) Member C10 (DNAJC10, also known as ERDJ5 and PDIA19), involved in endoplasmic reticulum-associated degradation (ERAD), has been identified as a tumor suppressor in several cancers. However, its precise role and underlying mechanism in GBM remain unclear. We found that DNAJC10 expression is downregulated in GBM patients and correlated with poor survival outcomes. Overexpression of DNAJC10 reduced GBM cell migration and invasion in vitro, while its knockdown promotes these processes. Moreover, DNAJC10 overexpression inhibits infiltrative growth of GBM cells, suppresses tumor propagation and prolongs survival in xenografted mice. Mechanistically, DNAJC10 regulates multiple molecules and pathways involved in cell motility, including the epidermal growth factor receptor (EGFR) pathway. Importantly, DNAJC10 overexpression decreases EGFR transcription by inhibiting spliced X-box binding protein 1 (XBP-1s). DNAJC10 regulates XBP-1s splicing through the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response (UPR). XBP-1s binds the EGFR promoter and enhances recruitment of SET7/9 methyltransferase, H3K4me3, and H3K4me1. Pharmacological inhibition of histone methylation attenuates XBP-1s-induced EGFR transcription, indicating XBP-1s promotes EGFR expression via recruiting SET7/9 for H3K4 methylation. XBP-1s overexpression reverses DNAJC10-mediated EGFR downregulation. Collectively, DNAJC10 suppresses EGFR transcription by inhibiting the UPR IRE1α-XBP-1s axis, reducing SET7/9 recruitment and H3K4 methylation at the EGFR promoter. Targeting DNAJC10 or XBP-1s could be a potential approach for inhibiting GBM infiltration and may represent a novel avenue for GBM treatment.
    Keywords:  EGFR; Glioblastoma; Metastasis; Tumor suppressor; Unfolded protein response (UPR)
    DOI:  https://doi.org/10.1186/s43556-025-00308-0
  38. J Biol Chem. 2025 Nov 05. pii: S0021-9258(25)02766-8. [Epub ahead of print] 110914
    Hsp90 co-chaperone FKBP4 facilitates CCT8 folding and connects Hsp90 to chaperonin-dependent proteostasis.
    Yun-Yu Huang, Ya-Lan Chang, Yun Chen, Wei-Yu Chiang, Ai-Tao Chiang, Pang-Hung Hsu, Shu-Chun Teng.
      Hsp70, Hsp90, and chaperonin complexes are three essential molecular chaperones facilitating protein folding within eukaryotic cells. However, the important interplay among these systems is incompletely understood. FKBP4 is a co-chaperone of Hsp90 and exhibits increased expression in multiple types of cancers. In this study, we employed two proximity-dependent biotin identification (BioID) systems to explore potential clients of the FKBP4-Hsp90 complex. Analysis of BioID mass spectrometry data revealed that the top category of the FKBP4-associated protein is cadherin-binding proteins, and one of the cadherin-binding proteins is a subunit of the chaperonin containing TCP-1 complex, CCT8. Furthermore, knockdown of FKBP4 led to the aggregation of CCT8 and compromised the stability of CCT8 clients, CDK2 and α-tubulin, indicating the dependency of the FKBP4-Hsp90 complex for CCT8 folding. These findings suggest that CCT8 is a client of the FKBP4-Hsp90 complex, implying a functional crosstalk between two of the three protein folding systems in eukaryotic cells.
    Keywords:  CCT8; FKBP4; Hsp90; chaperonin; client; co-chaperone
    DOI:  https://doi.org/10.1016/j.jbc.2025.110914
  39. J Hazard Mater. 2025 Oct 30. pii: S0304-3894(25)03232-7. [Epub ahead of print]500 140312
    Methamphetamine impairs stress resistance and extracellular matrix integrity via modulating the unfolded protein response in Caenorhabditis elegans.
    Yuanpeng Li, Hongshuang Wang, Hongyuan Li, Xiaohui Wang.
      Methamphetamine (METH) contamination of aquatic environments poses a growing threat to animal health, yet the mechanisms by which extracellular matrix (ECM) alterations influence cellular stress defenses remain poorly defined. Using Caenorhabditis elegans, this study demonstrates that 0.5 μM METH exposure compromises ECM integrity and thereby diminishes survival under ultraviolet, osmotic, and heat stress, reducing stress resistance by approximately 17-37 %. Mechanistically, METH-induced proteotoxicity is critically dependent on ECM integrity. Loss of col-109 or col-120 abrogates METH-induced polyglutamine aggregation and largely blocks the activation of both the endoplasmic reticulum (hsp-4) and mitochondrial (hsp-6, hsp-60, atfs-1) unfolded protein responses (UPR). Conversely, modulation of mitochondrial UPR through atfs-1 gain- and loss-of-function alleles bidirectionally regulates ECM gene expression and chondroitinase (chhy-1), revealing reciprocal ECM-UPR crosstalk. At the organismal level, functional UPR pathways preserve cuticle integrity and stress resilience under METH challenge. These results indicate that the ECM-UPR interplay constitutes an evolutionarily conserved stress-sensing axis, whereby ECM perturbation signals through UPR to maintain proteostasis and barrier function. These findings identify ECM and UPR components as promising biomarkers of environmental METH exposure and suggest that targeting this axis could mitigate xenobiotic-induced toxicity.
    Keywords:  Caenorhabditis elegans; Extracellular matrix; Methamphetamine; Stress resistance; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.jhazmat.2025.140312
  40. J Phys Chem Lett. 2025 Nov 06. 11861-11866
    Molecular Insights into the Dual Mechanism of Bepristat 2a on Protein Disulfide Isomerase (PDI) Activity.
    Juhong Wu, Xinyuan Liao, Shoujing Cao, Wanjun Chen, Linan Lin, Robert Flaumenhaft, Cai Yuan, Mingdong Huang, Jinyu Li.
      Protein disulfide isomerase (PDI) is a multifunctional thiol isomerase with extracellular activity crucial for thrombus formation. Inhibition of extracellular PDI suppresses thrombosis without impairing normal hemostasis, highlighting it as a promising antithrombotic target. We previously identified bepristats, a class of small molecules that selectively and reversibly inhibit the substrate oxidoreductase activity of PDI while paradoxically enhancing its ability to reduce GSSG. Here, we elucidate the molecular basis for this dual regulation by bepristat 2a (bep2a) through an integrated strategy combining molecular simulations and site-directed mutagenesis. Our results reveal that bep2a targets the major substrate-binding site with H256 identified as a critical residue for ligand recognition. This interaction is stabilized through a water-mediated hydrogen bond and induces compaction of the protein. In addition, bep2a binding allosterically triggered a conformational rearrangement of W396 and partially unwound the active-site helix. This structural change increases solvent exposure of catalytic cysteines C397 and C400, thereby enhancing the cleavage activity of the a' domain by facilitating nucleophilic attack. Thus, these findings uncover the structural basis for the simultaneous inhibition of PDI substrate oxidoreductase activity and enhancement of its catalytic disulfide cleavage function of bep2a and offer new insights for the design of highly selective PDI modulators.
    DOI:  https://doi.org/10.1021/acs.jpclett.5c03127
  41. Nat Chem Biol. 2025 Nov 03.
    Mutant p53 protein accumulation is selectively targetable by proximity-inducing drugs.
    Ananthan Sadagopan, Maximilian Carson, Eriks J Zamurs, Nicholas Garaffo, Heng-Jui Chang, Stuart L Schreiber, Matthew Meyerson, William J Gibson.
      TP53 mutant cancers are associated with approximately half of cancer deaths. The most common mechanism of p53 inactivation involves missense mutations. Such mutations in TP53 result in a robust upregulation of the p53 protein. Here, we demonstrate an induced proximity approach to selectively kill TP53 mutant cells. This approach uses the increased abundance of p53 protein in TP53 mutant cancer cells to concentrate toxic molecules in these cells. We demonstrate this approach with a molecule that binds the Y220C mutant of p53 and concentrates a PLK1 inhibitor in cells harboring TP53Y220C mutations. The resulting bifunctional molecule promotes formation of a p53Y220C-PLK1 ternary complex, mislocalizes PLK1, inhibits PLK1 activity, elicits selective G2/M arrest and induces apoptosis in TP53Y220C cells while sparing wild-type TP53 cells. These data exemplify a potentially generalizable framework for targeting TP53 missense mutations by leveraging mutant p53 protein abundance to induce cell death, independent of p53's transcriptional activity.
    DOI:  https://doi.org/10.1038/s41589-025-02051-7
  42. Circ Res. 2025 Nov 04.
    TRIB3 Links Endoplasmic Reticulum Stress to Impaired Efferocytosis in Atherosclerosis.
    Aarushi Singhal, Stefan Russo, Umesh Kumar Dhawan, Kunzangla Bhutia, Christopher G Bell, Hedayatullah Hayat, Thomas D Nightingale, Monica de Gaetano, Orina Belton, Eoin Brennan, Patricia B Munroe, Catherine Godson, Mary Barry, Carol C Shoulders, Heather L Wilson, Guillermo Velasco, Endre Kiss-Toth, Manikandan Subramanian.
       BACKGROUND: Defective macrophage efferocytosis is a key driver of chronic nonresolving inflammation in dyslipidemia-associated diseases, such as obesity and atherosclerosis. However, the mechanism by which intracellular lipid accumulation impairs macrophage efferocytosis remains unclear. We hypothesized that lipid-induced endoplasmic reticulum (ER) stress mediates defective macrophage efferocytosis.
    METHODS: Bone marrow-derived macrophages were exposed to 7-ketocholesterol or palmitate to induce ER stress, and efferocytosis was quantified by measuring uptake of fluorescently labeled apoptotic cells with microscopy and flow cytometry. Key pathways were interrogated with pharmacological inhibitors, siRNA, and in vivo models, including obese mice and in Ldlr-/- mice with hematopoietic-specific deletion of TRIB3 (Tribbles pseudokinase-3). Human relevance was assessed by testing efferocytosis in macrophages from individuals carrying the TRIB3 Q84R coronary artery disease risk variant (rs2295490) and by examining carotid endarterectomy samples.
    RESULTS: Activation of the ATF4 (activating transcription factor 4) branch of the ER stress pathway in lipid-loaded foamy macrophages led to upregulation of TRIB3, which triggered the downregulation of Rab27a, resulting in impaired focal exocytosis of intracellular membrane pools towards nascent, apoptotic cell-containing phagosomes. The resultant delay in phagosome closure stalled efferocytosis. In obese mice, this impairment was reversed using an ER stress-relieving chemical chaperone and via macrophage-specific knockdown of ATF4 or TRIB3. In atherosclerotic mice, hematopoietic cell-specific deletion of TRIB3 led to increased lesional efferocytosis, decreased plaque necrosis, and increased collagen, which are characteristic of stable plaques. In humans, TRIB3 expression was higher in vulnerable regions of carotid plaques, and macrophages from individuals carrying the gain-of-function TRIB3 Q84R risk variant expressed more TRIB3 and displayed decreased efferocytosis.
    CONCLUSIONS: Lipid-induced ER stress impairs macrophage efferocytosis via activation of the ATF4-TRIB3-Rab27a signaling axis, leading to exacerbated plaque necrosis. Targeted disruption of TRIB3 signaling in macrophages represents a novel therapeutic approach to promote efferocytosis and stabilize atherosclerotic plaques.
    Keywords:  7-ketocholesterol; atherosclerosis; efferocytosis; endoplasmic reticulum; macrophages
    DOI:  https://doi.org/10.1161/CIRCRESAHA.125.326839
  43. Cell Death Differ. 2025 Nov 01.
    Adaptive ER stress promotes mitochondrial remodelling and longevity through PERK-dependent MERCS assembly.
    Jose C Casas-Martinez, Qin Xia, Penglin Li, Maria Borja-Gonzalez, Antonio Miranda-Vizuete, Emma McDermott, Peter Dockery, Leo R Quinlan, Katarzyna Goljanek-Whysall, Afshin Samali, Brian McDonagh.
      The transfer of information and metabolites between the mitochondria and the endoplasmic reticulum (ER) is mediated by mitochondria-ER contact sites (MERCS), allowing adaptations in response to changes in cellular homeostasis. MERCS are dynamic structures essential for maintaining cell homeostasis through the modulation of calcium transfer, redox signalling, lipid transfer, autophagy and mitochondrial dynamics. Under stress conditions such as ER protein misfolding, the Unfolded Protein Response (UPRER) mediates PERK and IRE1 activation, both of which localise at MERCS. Adaptive UPRER signalling enhances mitochondrial function and calcium import, whereas maladaptive responses lead to excessive calcium influx and apoptosis. In this study, induction of mild acute ER stress with tunicamycin (TM) in myoblasts promoted myogenesis that required PERK for increased MERCS assembly, mitochondrial turnover and function. Similarly, treatment of C. elegans embryos with an acute low concentration of TM, promoted an extension in lifespan and health-span. The adaptive ER stress response following a low dose of TM in both myoblasts and C. elegans, increased MERCS assembly and activated autophagy machinery, ultimately promoting an increase in mitochondrial remodelling. However, these beneficial adaptations were dependent on the developmental stage, as treatment of myotubes or adult C. elegans resulted in a maladaptive response. In both models the adaptations to UPRER activation were dependent on PERK signalling and its interaction with the UPRmt. The results demonstrate PERK is required for the increased mitochondrial ER communication in response to adaptive UPR signalling, promoting mitochondrial remodelling and improved physiological function.
    DOI:  https://doi.org/10.1038/s41418-025-01603-7
  44. Cell Rep. 2025 Nov 03. pii: S2211-1247(25)01273-2. [Epub ahead of print]44(11): 116502
    Goblet cell-expressed microprotein FXYD3 determines gut homeostasis by maintaining mucus barrier integrity.
    Wenjuan Yang, Yue Xue, Peishuo Zhu, Zhinong Jiang, Rukun He, Hengrui Tian, Tao Wang, Peng Xiao, Wenwen Lian, Qian Cao, Yunn-Hwen Gan, Lihua Lai, Qingqing Wang.
      The intestinal mucus layer produced by goblet cells is a critical component of innate immunity. The key host factors and regulatory mechanisms controlling goblet cell function in mucus layer formation remain poorly understood. This study identifies a function for the microprotein FXYD domain-containing transport regulator 3 (FXYD3) in goblet cells in regulating mucus layer formation to maintain intestinal homeostasis. Deficiency of FXYD3 in mouse intestinal epithelial cells results in a damaged mucus barrier, leading to dysbiosis and increased susceptibility to colitis. Mechanistically, FXYD3 interacts with endoplasmic reticulum Ca2+-ATPase SERCA2 to enhance its pump activity. FXYD3 deficiency causes defects in ER Ca2+ homeostasis and mucin glycosylation, impairing mucus layer integrity. Furthermore, metabolites of gut microbiota, propionate, and butyrate promote FXYD3 expression. In ulcerative colitis patients, FXYD3 expression is significantly downregulated and correlates with disease severity. These findings indicate FXYD3 is a key mediator of host-microbiota interactions for intestinal health.
    Keywords:  CP: Immunology; FXYD3; SERCA2; goblet cells; mucin glycosylation; mucus barrier
    DOI:  https://doi.org/10.1016/j.celrep.2025.116502
  45. Cell Death Dis. 2025 Nov 05. 16(1): 794
    TMEM166 negatively regulates unfolded protein response to affect hepatocellular carcinoma cell growth and sorafenib resistance.
    Tao Li, Jinqiu Feng, Dan Xia, Yaxin Lou, Pengli Guo, Shufang Ye, Zongming Zhang, Yingyu Chen.
      Transmembrane protein 166 (TMEM166), an endoplasmic reticulum (ER)-resident membrane protein, exerts anticancer effects by inducing autophagy and apoptosis. Although tissues of various cancers downregulate its expression, the biological function of TMEM166 in hepatocellular carcinoma (HCC) remains unclear. Herein, we report that TMEM166 negatively regulates unfolded protein response (UPR) in HCC. TMEM166 was noted to interact with ACSL3 to maintain ACSL3 stability and facilitate lipid storage. TMEM166 deletion reduced ACSL3 expression and increased lipid utilisation in the mitochondria through fatty acid β-oxidation (FAO), ultimately boosting ATP production. Moreover, TMEM166-knockout (KO) cells demonstrated accelerated protein synthesis via the AMPK-mTOR axis. These effects induced sublethal ER stress and UPR activation in TMEM166-KO cells. Furthermore, TMEM166 KO promoted HCC cell proliferation and sorafenib resistance via UPR activity upregulation. We analysed the clinical significance of TMEM166-regulated UPR in human HCC cells and noted that TMEM166 expression was negatively correlated with the activities of UPR-related transcriptional factors such as ATF4, ATF6 and XBP1s in the cells. This study is the first to elucidate the relationship among TMEM166, ER stress, and HCC and may provide and indicate newer avenues for TMEM166-targeted gene therapy strategies for HCC treatment.
    DOI:  https://doi.org/10.1038/s41419-025-08176-w
  46. Nat Commun. 2025 Nov 06. 16(1): 9811
    Click-linking: a cell-compatible protein crosslinking method based on click chemistry.
    Bruno C Amaral, Andrew R M Michael, Nicholas I Brodie, D Alex Crowder, Kristen H Eiriksson, David C Schriemer.
      Crosslinking mass spectrometry (XL-MS) has the potential to map the human interactome at high resolution and with high fidelity, replacing indirect, error-prone sampling methods such as affinity pulldown MS. However, the sampling depth of XL-MS remains stubbornly low. We present a crosslinking strategy that splits the crosslinking reaction into two sequential and orthogonal coupling events. The method involves pre-stabilizing the spatial proteome with a fixation protocol inspired by immunofluorescence imaging, followed by a stepwise process that begins with extensively labeling surface-accessible lysines in the cell with N-hydroxysuccinimide (NHS)-modified click reagents. We show that a subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors generates crosslinks at levels approaching 30% of the total signal, as demonstrated by a subtractive approach. The method generates no detectable side reactions or obvious distortions of the spatial proteome. Protein-protein interactions (PPIs) are detected at levels approximately 20 times higher than a conventional DSS-based method, outperforming even enrichable crosslinkers.
    DOI:  https://doi.org/10.1038/s41467-025-64888-9
  47. Oncol Res. 2025 ;33(11): 3231-3245
    The Role of UFMylation in the Development and Progression of Gastric Cancer.
    Ying Fang, Anqi Wu, Yu-Sheng Cong, Guoqing Li.
      Gastric Cancer (GC) is a highly prevalent and poorly prognostic gastrointestinal malignancy with low overall treatment efficacy worldwide. Early diagnostic markers and potential therapeutic targets for GC treatment are urgently needed. UFMylation, a novel ubiquitin-like modification is indispensable for numerous fundamental cellular processes. Deficiency in this modification is reported to be associated with several human diseases including cancer. Accumulating evidence suggests that the expression of the key UFMylation components is closely associated with GC cell proliferation, invasion, metastasis, and chemotherapy resistance. Recent clinical studies have further highlighted the prognostic value and therapeutic potential of UFMylation in the clinical management of GC. However, the precise molecular mechanisms through which UFMylation contributes to GC remain largely unclear. This review aims to summarize recent findings on the functional roles of UFMylation in diverse cellular processes, such as endoplasmic reticulum (ER) homeostasis, DNA damage response (DDR), protein translation, and quality control pathways, discuss potential underlying mechanisms in GC development and progression, and to explore potential therapeutic implications targeting the UFMylation pathway in GC.
    Keywords:  Post-translational modifications; UFMylation; gastric cancer
    DOI:  https://doi.org/10.32604/or.2025.066402
  48. FEBS J. 2025 Nov 03.
    E3 ligase Praja1 mediates ubiquitination and degradation of microtubule-associated protein tau.
    Shiho Aoki, Wataru Onodera, Akihiko Takashima, Kotaro Kawasaki, Kazuki Imadegawa, Hikaru Kurahashi, Mizuho Oishi, Toru Asahi, Yoshiyuki Soeda.
      The RING-H2 type E3 ligase Praja family is composed of E3 ubiquitin-protein ligases Praja1 and Praja2, which promote the degradation of substrates through the ubiquitin-proteasome system. Both paralogs contribute to neuronal maturation and differentiation, indicating a significant role in the nervous system. Aggregation-prone proteins associated with neurodegenerative diseases, including TAR DNA-binding protein 43 (TDP-43) and α-synuclein, are degraded and/or suppressed by Praja1. Furthermore, the expression level of the microtubule-associated protein tau (MAPT) gene, which is frequently mutated in Alzheimer's disease, is regulated by Praja2. Although the Praja family has been shown to recognize various aggregation-prone proteins as substrates, it has not been determined whether tau, a key protein that aggregates in tauopathies, is also recognized by Praja proteins. In this study, we show that Praja1, but not Praja2, recognizes tau as a candidate substrate. We observed that the tau protein level in human neuroblastoma SH-SY5Y cells decreased depending on the E3 ligase activity of Praja1. Furthermore, the in vivo/in vitro ubiquitination assay showed that Praja1 ubiquitinates tau, indicating that it is a target substrate. Next, by combining ancestral sequence reconstruction and mutational analysis, we revealed that the Praja1-tau interaction began just after the duplication of the Praja family in the common ancestor of placentals. Lastly, to test whether this interaction is disrupted under pathological conditions, P301L tau was introduced, resulting in a degradation similar to that of wild-type tau. These results reveal an unidentified mechanism of tau proteostasis by Praja1 and may provide insight into the pathogenesis of neurodegenerative diseases, including tauopathy.
    Keywords:  E3 ubiquitin ligase; Gene duplication; Molecular evolution; Neofunctionalization; Praja family; Praja1; Substrate specificity; Tau
    DOI:  https://doi.org/10.1111/febs.70303
  49. Nature. 2025 Nov 03.
    Independent mechanisms of inflammation and myeloid bias in VEXAS syndrome.
    Varun K Narendra, Tandrila Das, Linsey J Wierciszewski, Rebecca J Londoner, Joshua K Morrison, Pia Martindale, Tessa Devine, Kevin Chen, Michael Trombetta, Yuzuka Kanno, Alejandro E Casiano, Elisa de Stanchina, Caleb A Lareau, Scott W Lowe, Alexander D Gitlin.
      Somatically acquired mutations in the E1 ubiquitin-activating enzyme UBA1 within hematopoietic stem and progenitor cells (HSPCs) were recently identified as the cause of the adult-onset autoinflammatory syndrome VEXAS (vacuoles, E1 enzyme, X linked, autoinflammatory, somatic)1. UBA1 mutations in VEXAS lead to clonal expansion within the HSPC and myeloid, but not lymphoid, compartments. Despite its severity and prevalence, the mechanisms whereby UBA1 mutations cause multiorgan autoinflammation and hematologic disease are unknown. Here, we employ somatic gene editing approaches to model VEXAS-associated UBA1 mutations in primary macrophages and HSPCs. Uba1-mutant macrophages exposed to inflammatory stimuli underwent aberrant apoptotic and necroptotic cell death mediated by Caspase-8 and RIPK3-MLKL, respectively. Accordingly, in mice challenged with TNF or LPS, the UBA1 inhibitor TAK-243 exacerbated inflammation in a RIPK3-Caspase-8-dependent manner. In contrast, Uba1 mutation in HSPCs induced an unfolded protein response and myeloid bias independently of RIPK3-Caspase-8. Mechanistically, aberrant cell death of Uba1-mutant macrophages coincided with a kinetic defect in Lys63/Met1 (i.e., linear) polyubiquitylation of inflammatory signaling complexes. Collectively, our results link VEXAS pathogenesis with that of rarer monogenic autoinflammatory syndromes; highlight specific ubiquitin-associated defects stemming from an apical mutation in the ubiquitylation cascade; and support therapeutic targeting of the inflammatory cell death axis in VEXAS.
    DOI:  https://doi.org/10.1038/s41586-025-09815-0
  50. J Cell Sci. 2025 Nov 07. pii: jcs.264375. [Epub ahead of print]
    Ubiquitylation-dependent Rap2 activation regulates lamellipodia dynamics during cell migration.
    Andrew Neumann, Revathi Sampath, Emily Mayerhofer, Valeryia Mikalayeva, Vytenis Arvydas Skeberdis, Ieva Sarapinienė, Rytis Prekeris.
      Cell migration is a complex process hallmarked by front-to-back cell polarity that is established by the highly dynamic actin cytoskeleton. Branched actin polymerization creates a lamellipodium at the leading edge of the cell, while the contractile acto-myosin cytoskeleton is present at the lagging edge. Rap2, a Ras GTPase family member, has previously been reported to localize to the lamellipodium as a result of Rab40/CRL5 E3 ubiquitin ligase induced ubiquitylation. However, how Rap2 functions and how ubiquitylation targets Rap2 to the lamellipodium remained unclear. Here, we demonstrate that Rap2 is recruited to retracting lamellipodia ruffles where it inhibits RhoA, likely through interactions with ARHGAP29, and regulates lamellipodia dynamics, thus facilitating cell migration. Furthermore, using a variety of genetic and pharmacological techniques, we show that Rab40/CRL5-dependent ubiquitylation is required for GEF-dependent Rap2 activation, a necessary step for Rap2 targeting to the lamellipodium membrane. As such, we demonstrate how this unique ubiquitylation and activation of Rap2 regulates lamellipodia actin dynamics during cell migration.
    Keywords:  Cell migration; Rap2; Ubiquitylation
    DOI:  https://doi.org/10.1242/jcs.264375
  51. Chem Sci. 2025 Oct 23.
    Mass spectrometry detects folding intermediates populated during urea-induced protein denaturation.
    Nicklas Österlund, Jacob S Jordan, Eleonora Renzi, Gergo Peter Szekeres, Kevin Pagel.
      Protein folding stability can be probed using urea, a chaotropic agent that disrupts non-covalent interactions at molar concentrations. The denaturation process is typically monitored via optical spectroscopy, which provides ensemble-averaged measurements and may struggle to resolve folding intermediates. In contrast, electrospray ionization mass spectrometry (ESI-MS) captures a non-averaged snapshot of all populated assembly and folding states within a protein conformational ensemble. However, high urea concentrations have traditionally been considered incompatible with ESI. Here, we leverage recent advancements in nano ESI emitter design, utilizing well-defined small-diameter emitters which enables protein charge states to be resolved from solutions containing up to 8 M urea. This approach allows us to directly detect the disruption of native tertiary and quaternary structures and to monitor stability changes in response to solution pH and ligand binding. We demonstrate this using single-domain proteins that follow simple two-state unfolding pathways, as well as more complex multidomain proteins and multimeric protein complexes. Our results show strong agreement with conventional urea-denaturation curves obtained via optical spectroscopy, while also providing enhanced resolution of intermediate folding and assembly states that are challenging to capture using traditional methods.
    DOI:  https://doi.org/10.1039/d5sc05773f
  52. J Cell Biol. 2025 Dec 01. pii: e202410094. [Epub ahead of print]224(12):
    Direct measurements of luminal Ca2+ with endo-lysosomal GFP-aequorin reveal functional IP3 receptors.
    Belén Calvo, Patricia Torres-Vidal, Alba Delrio-Lorenzo, Carla Rodriguez, Francisco J Aulestia, Jonathan Rojo-Ruiz, Beatriz Callejo, Bridget M McVeigh, Marco Keller, Christian Grimm, Viola Oorschot, Vera Moiseenkova-Bell, David I Yule, Javier Garcia-Sancho, Sandip Patel, M Teresa Alonso.
      Endo-lysosomes are considered acidic Ca2+ stores, but direct measurements of luminal Ca2+ within them are limited. Here, we report that the Ca2+-sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca2+ dynamics in this compartment. We show that Ca2+ uptake is ATP-dependent and sensitive to blockers of ER Ca2+ pumps. We find that the Ca2+ mobilizing messenger IP3 evokes robust luminal responses in wild-type cells, but not in IP3R knockout cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channels TPCs and TRPMLs. Stimulation with IP3-forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis was consistent with the presence of IP3Rs within the endo-lysosomal system. Our data reveal a physiologically relevant, IP3-sensitive store of Ca2+ within the endo-lysosomal system.
    DOI:  https://doi.org/10.1083/jcb.202410094
  53. Cell Rep. 2025 Nov 04. pii: S2211-1247(25)01282-3. [Epub ahead of print]44(11): 116511
    IGF2BP2 promotes malignant progression of ovarian cancer by regulating protein synthesis through liquid-liquid phase separation.
    Xueyou Xiong, Xuan Wang, Zhenlong Wang, Feixue Chen, Yaqian Shi, Xiyi Chen, Weijian Gong, Xuemei Jia, Juan Xu.
      Ovarian cancer (OC) is the most lethal gynecological malignancy, and high expression of IGF2BP2 is significantly associated with poor prognosis. Intracellular macromolecules regulate diverse biological functions via liquid-liquid phase separation (LLPS). However, whether IGF2BP2 undergoes LLPS and promotes tumor progression in OC through spatiotemporal coordination remains elusive. Here, we demonstrate that IGF2BP2 forms condensates through LLPS in OC cells, which recruit target RNAs and translation initiation factors to enhance de novo protein synthesis. Both N6-methyladenosine (m6A)-modified RNA binding and acetylation of IGF2BP2 promote its LLPS. However, substitution of five lysines and one glycine with glutamine within the m6A-binding pocket of IGF2BP2 blocks acetylation and disrupts its LLPS, which in turn dampens global protein synthesis and abolishes the oncogenic activity of IGF2BP2 in OC. Our findings reveal that IGF2BP2-driven LLPS promotes OC progression by enhancing protein synthesis, highlighting its potential as a therapeutic target.
    Keywords:  CP: cancer; CP: molecular biology; IGF2BP2; LLPS; liquid-liquid phase separation; m(6)A; ovarian cancer; translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116511
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