bims-proteo Biomed News
on Proteostasis
Issue of 2025–11–30
71 papers selected by
Eric Chevet, INSERM
  1. State-selective small molecule degraders that preferentially remove aggregates and oligomers.
  2. GlueFinder: A Data-Driven Framework for the Rational Discovery of Molecular Glues.
  3. Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability.
  4. IRE1 Regulates TOR Signaling via RIDD of RAPTOR1b to Coordinate Growth and Stress Adaptation.
  5. Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation.
  6. TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress.
  7. Inhibitors supercharge kinase turnover through native proteolytic circuits.
  8. ER exit sites mediated by the COPII adaptor sec24D selectively recruit lipid raft-preferring proteins for rapid ER export.
  9. Development of FBXO22 Degraders and the Recruitment Ligand 2-Pyridinecarboxyaldehyde (2-PCA).
  10. Cadherin Regulation of Endoplasmic Reticulum-Plasma Membrane Contact Sites.
  11. Phosphorylation-coupled autoregulation of TANGO1 and Sec16A maintains functional ER exit sites.
  12. SINE compounds activate exportin 1 degradation through an allosteric mechanism.
  13. The deubiquitinating enzyme Otu1 releases substrates from the conserved initiation complex of the Cdc48/p97 ATPase for proteasomal degradation.
  14. In-situ cross-linking mass spectrometry reveals compartment-specific proteasomal interactions and structural heterogeneity.
  15. WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response.
  16. Portioning organelles for autophagic clearance.
  17. Timing of transcription controls the selective translation of newly synthesized mRNAs during acute environmental stress.
  18. Transcriptome-wide mRNP condensation precedes stress granule formation and excludes new mRNAs.
  19. Drug repurposing screen identifies an HRI activating compound that promotes adaptive mitochondrial remodeling in MFN2-deficient cells.
  20. Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex.
  21. The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation.
  22. Homocysteine disrupts lysosomal function by V-ATPase inhibition.
  23. Ribosome remodeling drives translation adaptation during viral infection and cellular stress.
  24. Structural basis for the recruitment and selective phosphorylation of Akt by mTORC2.
  25. Proteostasis network response to environmental chronic stress: linking survival to protein aggregation in a human neuroblastoma cellular model.
  26. Sec14L6 is a phosphoinositide transporter that regulates phosphoinositide homeostasis and biogenesis of lipid droplets.
  27. Toxoplasma effector TgROP1 establishes membrane contact sites with the endoplasmic reticulum during infection.
  28. KIF5A binds RNA to orchestrate synaptic mRNA localization and stress granules in ALS.
  29. A late cytoplasmic surveillance pathway ensures ribosome integrity.
  30. The luminal domain region of Seipin/Fld1 is dispensable for establishing functional ER sites for lipid droplet biogenesis.
  31. Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response.
  32. Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins.
  33. Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.
  34. Multiplex design and discovery of proximity handles for programmable proteome editing.
  35. Disruption of the PIKfyve complex unveils an adaptive mechanism to promote lysosomal repair and mitochondrial homeostasis.
  36. FPhotocleavable PROTACs as tools for dynamic regulation of protein function.
  37. Stress granules and protein aggregates reveal intracellular resource competition.
  38. 4E-BP1 differentially regulates translation of a subset of human mRNAs.
  39. TRAP mediated conformational changes of the human Sec61 channel.
  40. The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.
  41. NAD+ restores proteostasis through splicing-dependent autophagy.
  42. EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression.
  43. Faf1 accelerates p97-mediated protein unfolding by promoting ubiquitin engagement.
  44. Distinct responses to non-autonomous UPRER mediated by glutamatergic and octopaminergic neurons.
  45. The functional landscape of the human ubiquitinome.
  46. Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC).
  47. Liquid-liquid phase separation of ATXN2L enhances mRNA translation in hepatocellular carcinoma.
  48. Annexin A2 stabilizes the endoplasmic reticulum and actin cytoskeleton and influences the formation of reovirus factories.
  49. Integrated Stress Response Signatures Drive Monocyte Dysfunction in GBA1 - and LRRK2 -Linked Parkinson's Disease.
  50. ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling.
  51. Proteome Landscapes Decode Organelle Vulnerabilities in cortical and dopaminergic-like induced neurons Across Lysosomal Storage Disorders.
  52. Integrin-specific signaling drives ER stress-dependent atherogenic endothelial activation.
  53. Unbiased RNA Degrader Identification Uncovers an LC3B-Recruiting Chimera for COL15A1 mRNA Degradation.
  54. FLASH-AWAY: Intrabody-Directed Targeting of Optogenetic Tools for Protein Degradation.
  55. Recycling of ribosomes at stop codons drives the rate of translation and the transition from proliferation to RESt.
  56. The Dynamic UPR Rheostat Orchestrates Single-Cell Plasticity in Glioblastoma.
  57. Defining the role of β-cell IRE1α/XBP1 pathway and its gene regulatory network components in non-obese diabetic mice.
  58. tRNA-dependent conformational dynamics and folding coordinate translational regulation by a full-length T-box riboswitch.
  59. Harnessing the E3 ligase SPOP for targeted degradation of the NUP98::KDM5A fusion oncoprotein.
  60. Design of Tissue-Selective PROTACs Through Recruiting E3 Ligase Scaffolding Protein MAGEA11.
  61. Targeting prohibitins activates the ISR through DELE1-HRI by impairing protein import into the mitochondrial matrix.
  62. In vivo cell-type specific secretome profiling.
  63. Targeted protein degradation in the transmembrane and extracellular space.
  64. The ESCRT-0 protein HRS regulates hepatocellular lipid droplet catabolism.
  65. Investigating the neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.
  66. The implications of alternative splicing regulation for maximum lifespan.
  67. RNA innate immunity constitutes a barrier for interspecies chimerism.
  68. UFMylation of 14-3-3ε coordinates MAVS signaling complex assembly to promote antiviral innate immune induction.
  69. Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function.
  70. Cnpy1 is a candidate endoplasmic reticulum chaperone of Vomeronasal type 2 GPCRs.
  71. Inhibition of oligomeric BAX by an anti-apoptotic dimer.
  1. Nat Commun. 2025 Nov 25. 16(1): 10486
    State-selective small molecule degraders that preferentially remove aggregates and oligomers.
    Jakub Luptak, Dean Clift, Aamir Mukadam, Jonathan Benn, Tyler Rhinesmith, Stephen H McLaughlin, Amy C Dodds, Jerson E Lapetaje, Matylda Sczaniecka-Clift, David J France, William A McEwan, Leo C James.
      TRIM21 is a unique E3 ligase that uses a clustering-based activation mechanism to degrade complex multimeric substrates. This activity underpins the targeted protein degradation technology Trim-Away and genetically encoded degraders that selectively target aggregated tau protein and prevent tauopathy. Here we describe small molecules that mimic TRIM21's natural epitope and function as either effective inhibitors or potent and selective degraders called TRIMTACs. TRIMTACs mediate degradation as rapidly as PROTACs but can also selectively degrade specific protein pools depending on assembly state. We demonstrate the utility of this state-specific degradation by selectively removing the pro-inflammatory signalling protein Myd88 when assembled into the Myddosome and the cell-death protein RIPK3 when polymerised into the Necrosome. We further show that TRIMTACs can inhibit seeded tau aggregation under conditions where a PROTAC is ineffective. These results highlight that TRIM21's clustering-based activation can be exploited by small molecule degraders to carry out state-selective degradation of therapeutic targets.
    DOI:  https://doi.org/10.1038/s41467-025-65454-z
  2. bioRxiv. 2025 Oct 17. pii: 2025.10.17.683126. [Epub ahead of print]
    GlueFinder: A Data-Driven Framework for the Rational Discovery of Molecular Glues.
    Jeffrey Skolnick, Bharath Srinivasan, Hongyi Zhou.
      Molecular glues can drive targeted protein degradation by stabilizing ternary complexes between proteins of interest and E3 ubiquitin ligases, but rational design has lagged due to limited rules for interface recognition and an overreliance on a few ligases (e.g., VHL or Cereblon). We introduce GlueFinder, a systematic, unbiased platform that leverages structural bioinformatics to mine the Protein Data Bank for ligand binding pockets adjacent to the protein interface which are ligandable sites near protein-protein interfaces that can nucleate glue-mediated complex formation. After validating its performance on a benchmark of experimentally solved dimeric structures with known and predicted glues, we applied GlueFinder to three therapeutically important targets, EGFR, HER2, and KRAS, and predicted candidate glues that recruit 24, 111, and 148 distinct E3 ligases to these targets, respectively. We further demonstrate that GlueFinder can promote the formation of non-native EGFR complexes, possibly enabling ternary assemblies that would not form on their own. Together, these results establish a general, computation-guided strategy for molecular glue discovery that decouples design from legacy degrader scaffolds and specific ligase dependencies, expands the usable E3 ligase repertoire, and enables rational targeting of interfacial binding pockets. GlueFinder thus broadens both the scope and precision of targeted protein degradation and moves the field toward mechanism-driven, systematic glue development across diverse therapeutic contexts.
    Keywords:  E3 ligase; Molecular glue; degraders; induced proximity; rational design
    DOI:  https://doi.org/10.1101/2025.10.17.683126
  3. Cell Rep. 2025 Nov 25. pii: S2211-1247(25)01357-9. [Epub ahead of print]44(12): 116585
    Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability.
    Bertina Telusma, Jean-Claude Farré, Danica S Cui, Suresh Subramani, Joseph H Davis.
      Cells remodel their proteomes in response to changing environments by coordinating protein synthesis and degradation. In yeast, degradation occurs via proteasomes and vacuoles, with bulk and selective autophagy supplying vacuolar cargo. Although these pathways are known, their relative contributions to proteome-wide remodeling remain unreported. To assess this, we developed a method (nPL-qMS) to pulse-label the methylotrophic yeast Komagataella phaffii (Pichia pastoris) with isotopically labeled nutrients that, when coupled to quantitative proteomics, enables global monitoring of protein degradation following an environmental perturbation. Genetic ablations revealed that autophagy drives most proteome remodeling upon nitrogen starvation, with minimal non-autophagic contributions. Cytosolic protein complexes, including ribosomes, are degraded through bulk autophagy, whereas degradation of peroxisomes and mitochondria uses selective autophagy. Notably, these pathways are independently regulated by environmental cues. Our approach expands known autophagic substrates, highlights autophagy's major role in fungal proteome remodeling, and provides rich resources and methods for future proteome remodeling studies.
    Keywords:  CP: Microbiology; CP: Molecular biology; autophagy; mitophagy; pexophagy; protein degradation; proteome quality control; quantitative mass spectrometry
    DOI:  https://doi.org/10.1016/j.celrep.2025.116585
  4. bioRxiv. 2025 Oct 31. pii: 2025.10.30.685559. [Epub ahead of print]
    IRE1 Regulates TOR Signaling via RIDD of RAPTOR1b to Coordinate Growth and Stress Adaptation.
    Brandon C Reagan, Joo Yong Kim, Evan Angelos, Josh V Vermaas, Federica Brandizzi.
      Plant growth and stress resilience depend on integrating diverse signals into coordinated cellular responses. The endoplasmic reticulum (ER) stress sensor IRE1 maintains ER homeostasis and modulates Target of Rapamycin (TOR) signaling. Here, we reveal that TOR misregulation in an ire1ab mutant reduces sensitivity to the stress hormone abscisic acid (ABA), mediated by TOR-dependent phosphorylation of the ABA receptor PYL1. Further, we show that IRE1's endonuclease activity is required for TOR regulation, acting independently of the canonical IRE1/bZIP60 unfolded protein response. Instead, it occurs via Regulated IRE1- Dependent Decay (RIDD) of specific transcripts. We identify RAPTOR1b as a direct RIDD target, establishing a mechanistic link between ER stress sensing and TOR signaling. RIDD- mediated degradation of RAPTOR1b mRNA is required for appropriate ABA responses and stress adaptation. These findings uncover a noncanonical IRE1-TOR signaling axis that fine- tunes growth and stress responses through selective mRNA decay.
    DOI:  https://doi.org/10.1101/2025.10.30.685559
  5. bioRxiv. 2025 Nov 01. pii: 2025.10.31.685944. [Epub ahead of print]
    Lipid bilayer thinning near a ubiquitin ligase selects ER membrane proteins for degradation.
    Rudolf Pisa, Tom A Rapoport.
      Misfolded or unassembled membrane proteins in the endoplasmic reticulum (ER) are polyubiquitinated, translocated into the cytosol, and degraded by the proteasome, a poorly understood process that is conserved in all eukaryotes. Here, we use S. cerevisiae to elucidate how ER membrane proteins are selected for degradation. We show that hydrophilic residues in a trans-membrane (TM) segment cause the TM to partition into a thinned membrane region next to the ubiquitin ligase Hrd1, which then leads to substrate polyubiquitination and degradation. In the case of single-pass membrane proteins, the Hrd1-associated Der1 protein contributes to partitioning and degradation. In contrast, multi-pass proteins require Hrd1 to function on its own. Our results provide a general mechanism by which ER membrane proteins are targeted for degradation.
    DOI:  https://doi.org/10.1101/2025.10.31.685944
  6. Nat Commun. 2025 Nov 28. 16(1): 10743
    TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress.
    Nandini Shukla, Maxwell L Neal, Jean-Claude Farré, Fred D Mast, Rajasri Sarkar, Linh Truong, Theresa Simon, Leslie R Miller, John D Aitchison, Suresh Subramani.
      Peroxisomes are versatile organelles mediating energy homeostasis and redox balance. While peroxisome dysfunction is linked to numerous diseases, the mechanisms regulating peroxisome dynamics during cellular stress remain elusive. Using yeast, we show that proteotoxic stress, including loss of endoplasmic reticulum (ER) or cytosolic chaperone function, impaired ER protein translocation, disrupted N-linked glycosylation, or reductive stress, triggers peroxisome proliferation. This occurs through increased de novo biogenesis from the ER, as well as growth and division, rather than impaired pexophagy. Peroxisome biogenesis is essential for cellular recovery from proteotoxic stress. Through comprehensive testing of major signaling pathways, we determine this response to be mediated by activation of the heat shock response and inhibition of Target of Rapamycin (TOR) signaling. Notably, the effects of proteotoxic stress and TOR inhibition on peroxisomes are also observed in human fibroblasts. Our findings reveal a critical and conserved role of peroxisomes in cellular response to proteotoxic stress.
    DOI:  https://doi.org/10.1038/s41467-025-65776-y
  7. Nature. 2025 Nov 26.
    Inhibitors supercharge kinase turnover through native proteolytic circuits.
    Natalie S Scholes, Martino Bertoni, Arnau Comajuncosa-Creus, Katharina Kladnik, Xuefei Guo, Fabian Frommelt, Matthias Hinterndorfer, Hlib Razumkov, Polina Prokofeva, Martin P Schwalm, Florian Born, Sandra Roehm, Hana Imrichova, Brianda L Santini, Eleonora Barone, Caroline Schätz, Miquel Muñoz I Ordoño, Severin Lechner, Andrea Rukavina, Iciar Serrano, Miriam Abele, Anna Koren, Stefan Kubicek, Stefan Knapp, Nathanael S Gray, Giulio Superti-Furga, Bernhard Kuster, Yigong Shi, Patrick Aloy, Georg E Winter.
      Targeted protein degradation is a pharmacological strategy that relies on small molecules such as proteolysis-targeting chimeras (PROTACs) or molecular glues, which induce proximity between a target protein and an E3 ubiquitin ligase to prompt target ubiquitination and proteasomal degradation1. Sporadic reports indicated that ligands designed to inhibit a target can also induce its destabilization2-4. Among others, this has repeatedly been observed for kinase inhibitors5-7. However, we lack an understanding of the frequency, generalizability and mechanistic underpinnings of these phenomena. Here, to address this knowledge gap, we generated dynamic abundance profiles of 98 kinases after cellular perturbations with 1,570 kinase inhibitors, revealing 160 selective instances of inhibitor-induced kinase destabilization. Kinases prone to degradation are frequently annotated as HSP90 clients, therefore affirming chaperone deprivation as an important route of destabilization. However, detailed investigation of inhibitor-induced degradation of LYN, BLK and RIPK2 revealed a differentiated, common mechanistic logic whereby inhibitors function by inducing a kinase state that is more efficiently cleared by endogenous degradation mechanisms. Mechanistically, effects can manifest by ligand-induced changes in cellular activity, localization or higher-order assemblies, which may be triggered by direct target engagement or network effects. Collectively, our data suggest that inhibitor-induced kinase degradation is a common event and positions supercharging of endogenous degradation circuits as an alternative to classical proximity-inducing degraders.
    DOI:  https://doi.org/10.1038/s41586-025-09763-9
  8. Nat Commun. 2025 Nov 27. 16(1): 10694
    ER exit sites mediated by the COPII adaptor sec24D selectively recruit lipid raft-preferring proteins for rapid ER export.
    Ivan Castello-Serrano, Shikha Dagar, Rossana Ippolito, Kandice R Levental, Ilya Levental.
      The determinants of sub-cellular trafficking for many membrane proteins are poorly understood. Lipid-driven membrane nanodomains known as lipid rafts have been widely implicated in post-Golgi traffic, but their involvement in protein sorting in the endoplasmic reticulum has not been widely considered. To assess the role of membrane domains in the early secretory pathway, we use the Retention Using Selective Hooks system to synchronize and quantitatively assess trafficking rates and destinations of model proteins with tunable raft affinities. We find that raft-preferring constructs exit the ER faster than raft-excluded and have distinct preferences for ER exit sites marked by specific isoforms of sec24 cargo adaptors. Namely, raft-excluded cargo localizes to sec24A-positive sites while raft-preferring cargo localizes to sec24D ERES, dependent on p24-family cargo adapters TMED2/10. Finally, sec24D, but not sec24A, ERES accumulate a fluorescent cholesterol analog. These observations suggest that association with raft-like domains affects protein export from the ER.
    DOI:  https://doi.org/10.1038/s41467-025-65726-8
  9. J Am Chem Soc. 2025 Nov 26.
    Development of FBXO22 Degraders and the Recruitment Ligand 2-Pyridinecarboxyaldehyde (2-PCA).
    Tian Qiu, Zhe Zhuang, Woong Sub Byun, Zuzanna Kozicka, Kheewoong Baek, Jianing Zhong, Abby M Thornhill, Julia K Ryan, Katherine A Donovan, Eric S Fischer, Benjamin L Ebert, Nathanael S Gray.
      Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce the proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands are required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we described AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioacetal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced the FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in the TPD.
    DOI:  https://doi.org/10.1021/jacs.5c14141
  10. bioRxiv. 2025 Oct 15. pii: 2025.10.15.682666. [Epub ahead of print]
    Cadherin Regulation of Endoplasmic Reticulum-Plasma Membrane Contact Sites.
    Sonam Lhamo, Navaneetha Krishnan Bharathan, William Giang, Kandice R Levental, Cory L Simpson, Stephanie Zimmer, Andrew P Kowalczyk.
      The spatial organization and dynamics of the endoplasmic reticulum (ER) govern when and where ER tubules engage with other organelles and the plasma membrane. We previously found that ER tubules are closely associated with desmosomes, but the mechanisms of ER recruitment to these adhesive intercellular junctions were unclear. Here, we demonstrate that adherens junctions recruit ER tubules to intercellular junctions in a manner dependent upon E-cadherin association with α-catenin and vinculin. During cell-cell junction assembly, adherens junctions and ER appear nearly simultaneously at nascent cell-cell contacts, followed by desmosome formation. ER recruitment to cell-cell contacts allows the formation of ER-plasma membrane contact sites (ER-PMCS) and the assembly of a complex comprising adherens junctions, ER-PMCS, and desmosomes. Ablating adherens junctions disrupts this tripartite complex and perturbs global cellular lipid levels. Collectively, our findings identify cadherins as key organizers of ER-PMCS positioning and suggest that this complex integrates cellular mechanical elements with plasma membrane homeostasis.
    DOI:  https://doi.org/10.1101/2025.10.15.682666
  11. Nat Commun. 2025 Nov 25. 16(1): 10434
    Phosphorylation-coupled autoregulation of TANGO1 and Sec16A maintains functional ER exit sites.
    Miharu Maeda, Masashi Arakawa, Masaki Wakabayashi, Yukie Komatsu, Kota Saito.
      Secretory proteins are synthesized in the endoplasmic reticulum (ER) and begin their transport from specialized domains on the ER called ER exit sites (ERES), where COPII proteins assemble. We previously demonstrated that the interaction between TANGO1 and Sec16A is critical for ERES formation. In this study, we reveal that the phosphorylation of TANGO1 and Sec16A is regulated by a FAM83A/CK1α-mediated negative feedback loop. Conversely, their dephosphorylation is regulated in a spatially distinct manner by different phosphatase complexes: PPP6R3/PPP6C for Sec16A and PPP1R15B/PPP1C for TANGO1. Excessive phosphorylation of either TANGO1 or Sec16A leads to ERES disassembly, while excessive dephosphorylation impairs secretion. Our findings demonstrate that maintaining a balanced phosphorylation state of TANGO1 and Sec16A through autoregulation by FAM83A/CK1α and the phosphatases PP1 and PP6 is essential for sustaining proper secretory activity at the ERES.
    DOI:  https://doi.org/10.1038/s41467-025-65409-4
  12. Nat Chem Biol. 2025 Dec;21(12): 2002-2013
    SINE compounds activate exportin 1 degradation through an allosteric mechanism.
    Casey E Wing, Ho Yee Joyce Fung, Bert Kwanten, Tolga Cagatay, Ashley B Niesman, Maarten Jacquemyn, Mehdi Gharghabi, Brecht Permentier, Binita Shakya, Rhituparna Nandi, Joseph M Ready, Trinayan Kashyap, Sharon Shacham, Yosef Landesman, Rosa Lapalombella, Dirk Daelemans, Yuh Min Chook.
      Overexpression of exportin 1 (XPO1/CRM1) in cancer cells mislocalizes numerous cancer-related nuclear export cargoes. Covalent selective inhibitors of nuclear export (SINEs), including the cancer drug selinexor, restore proper nuclear localization by blocking XPO1-cargo interaction. These inhibitors also induce XPO1 degradation through the Cullin-RING E3 ligase (CRL) substrate receptor ASB8. Here we present cryo-electron microscopy structures revealing ASB8 binding to a cryptic XPO1 site that is exposed upon SINE conjugation. Unlike typical molecular glue degraders that directly bridge CRLs and substrates, SINEs bind XPO1 independently of ASB8, triggering an allosteric mechanism that enables high-affinity ASB8 recruitment, leading to XPO1 ubiquitination and degradation. ASB8-mediated degradation is also triggered by the endogenous itaconate derivative 4-octyl itaconate, suggesting that synthetic XPO1 inhibitors exploit a native cellular mechanism. This allosteric XPO1 degradation mechanism expands known modes of targeted protein degradation beyond molecular glue degraders and proteolysis-targeting chimeras of CRL4.
    DOI:  https://doi.org/10.1038/s41589-025-02058-0
  13. bioRxiv. 2025 Nov 11. pii: 2025.11.08.687396. [Epub ahead of print]
    The deubiquitinating enzyme Otu1 releases substrates from the conserved initiation complex of the Cdc48/p97 ATPase for proteasomal degradation.
    Hao Li, Haipeng Guan, Tom A Rapoport.
      Many eukaryotic proteins are modified with a polyubiquitin chain and then recruited to either the Cdc48 ATPase (p97 or VCP in mammals) or the 26S proteasome by conserved cofactors. They can then shuttle between the Cdc48 ATPase and the 26S proteasome before being degraded. How substrates avoid being trapped on the Cdc48 ATPase complex is incompletely understood, as they can undergo repeated cycles of translocation through the ATPase pore. Here, we show that the deubiquitinating enzyme (DUB) Otu1 (Yod1 in mammals) can break this futile cycle. Otu1 trims the ubiquitin chain of the substrate before its translocation through the Cdc48 pore is initiated, allowing transfer to the proteasome and subsequent degradation. Thus, Otu1 differs from other DUBs in that it stimulates, rather than retards, protein degradation. A cryo-EM structure shows that the mammalian homolog Yod1 binds to p97 simultaneously with other Cdc48 cofactors. As in the yeast system, polypeptide translocation through the ATPase pore is initiated by the unfolding of a ubiquitin molecule, suggesting that the mechanism of substrate processing is conserved in all eukaryotes.
    DOI:  https://doi.org/10.1101/2025.11.08.687396
  14. Nat Commun. 2025 Nov 28. 16(1): 10725
    In-situ cross-linking mass spectrometry reveals compartment-specific proteasomal interactions and structural heterogeneity.
    Lili Zhao, Runtao Zhao, Zhou Gong, Fuxiang Liang, Nan Zhao, Bowen Zhong, Maili Liu, Yukui Zhang, Qun Zhao, Lihua Zhang, Chun Tang.
      The proteasome is an essential cellular machine that degrades ubiquitinated substrate proteins to maintain proteostasis. Studies of purified proteasomes, however, cannot fully recapitulate its complexity. Here, we employ in-situ cross-linking mass spectrometry (XL-MS) combined with nuclear-cytoplasmic fractionation to characterize human 26S proteasome within intact cells. Our analysis reveals extensive compositional and conformational heterogeneity between subcellular compartments, along with distinct interactomes and dynamic states. Notably, we identify additional ubiquitin-associated proteasomal subunits and uncover compartment-specific ubiquitin-binding and ubiquitination patterns. Further we identify previously unreported proteasome-interacting proteins, including deubiquitinase USP15, and reveal a hybrid proteasome variant wherein translation initiation factor EIF3M substitutes for subunit Rpn9. Leveraging cross-linking-derived distance restraints, we model transient interactions and dynamic subcomplexes of the proteasome. Together, our work establishes a robust framework for in-situ structural analysis of large protein complexes and provides mechanistic insights into how compartment-specific architectures and interactions regulate proteasome function.
    DOI:  https://doi.org/10.1038/s41467-025-65752-6
  15. Nat Commun. 2025 Nov 24.
    WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response.
    Rinskje B Tjeerdsma, Timothy F Ng, Maurits Roorda, Daniëlle Bianchi, Sora Yang, Clara Bonnet, Michael VanInsberghe, Marieke Everts, Femke J Bakker, H Rudolf de Boer, Nathalie Moatti, Nicole Hustedt, Jay Z Yin, Lisa Hoeg, Matthew Leibovitch, Frank Sicheri, Alexander van Oudenaarden, Steven de Jong, Jeroen van den Berg, Marvin E Tanenbaum, Thijn R Brummelkamp, Daniel Durocher, Marcel A T M van Vugt.
      The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Additionally, a genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinase GCN2 or its co-factor GCN1 rescued WEE1i-mediated cytotoxicity. Conversely, loss of the collided ribosome sensor ZNF598 increased sensitivity to WEE1i. Mechanistically, WEE1i induced paradoxical GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. ISR activation was independent of WEE1 presence, pointing at off-target GCN2 engagement by multiple chemically distinct WEE1i. ISR activation was observed in cancer cells as well as non-transformed cells, and required GCN1 and ongoing translation. Consequently, WEE1i induce multiple independent cellular effects: DNA damage, premature mitotic entry and sensitization to DNA-damaging chemotherapeutics in an ISR-independent fashion, as well as ISR activation independently of CDK1/2 activation. Importantly, low-dose WEE1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i trigger toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.
    DOI:  https://doi.org/10.1038/s41467-025-66514-0
  16. Autophagy. 2025 Nov 28.
    Portioning organelles for autophagic clearance.
    Mikhail Rudinskiy, Maurizio Molinari.
      The lysosomal/vacuolar clearance of portions of organelles including the endoplasmic reticulum (ER), mitochondria, the Golgi apparatus and the nucleus, organellophagy, is mediated by autophagy receptors anchored at the surface of their respective organelles. Organellophagy receptors are activated, induced or derepressed in response to stimuli such as nutrient or oxygen deprivation, accumulation of toxic or aged macromolecules, membrane depolarization, pathogen invasion, cell differentiation and many others. Their activation drives the portioning of the homing organelle, and the engagement of Atg8/LC3/GABARAP (LC3) proteins via LC3-interacting regions (LIRs) that results in autophagic clearance. In our latest work, we elaborate on the fact that all known mammalian and yeast organellophagy receptors expose their LIR embedded within intrinsically disordered regions (IDRs), i.e. cytoplasmic stretches of amino acids lacking a fixed three-dimensional structure. Our experiments reveal that the IDR modules of organellophagy receptors are interchangeable, required and sufficient to induce the fragmentation of the organelle that displays them at the limiting membrane, independent of LC3 engagement. LC3 engagement drives lysosomal delivery. Building on these findings, we propose harnessing practical and therapeutic potential of controlled organelle fragmentation and organellophagy through ORGAnelle TArgeting Chimeras (ORGATACs).
    Keywords:  Endoplasmic reticulum (Er)phagy; ORGAnelle TArgeted chimeras (ORGATACs); intrinsically disordered regions (IDRs); mitophagy; organellophagy receptors; targeted organelle degradation
    DOI:  https://doi.org/10.1080/15548627.2025.2597458
  17. Mol Cell. 2025 Nov 24. pii: S1097-2765(25)00900-1. [Epub ahead of print]
    Timing of transcription controls the selective translation of newly synthesized mRNAs during acute environmental stress.
    Mostafa Zedan, Alexandra P Schürch, Stephanie Heinrich, Pablo A Gómez-García, Sarah Khawaja, Léona Dörries, Karsten Weis.
      When cells encounter stress, they rapidly mount an adaptive response by switching from pro-growth to stress-responsive gene expression programs. How cells selectively silence pre-existing, pro-growth transcripts yet efficiently translate transcriptionally induced stress mRNA and whether these transcriptional and post-transcriptional responses are coordinated are poorly understood. Here, we show that following acute glucose withdrawal in S. cerevisiae, pre-existing mRNAs are not first degraded to halt protein synthesis, nor are they sequestered away in P-bodies. Rather, their translation is quickly repressed through a sequence-independent mechanism that differentiates between mRNAs produced before and after stress, followed by their decay. Transcriptional induction of endogenous transcripts and reporter mRNAs during stress is sufficient to escape translational repression, while induction prior to stress leads to repression. Our results reveal a timing-controlled coordination of the transcriptional and translational responses in the nucleus and cytoplasm, ensuring a rapid and wide-scale reprogramming of gene expression following environmental stress.
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.004
  18. Mol Cell. 2025 Nov 24. pii: S1097-2765(25)00899-8. [Epub ahead of print]
    Transcriptome-wide mRNP condensation precedes stress granule formation and excludes new mRNAs.
    Hendrik Glauninger, Jared A M Bard, Caitlin J Wong Hickernell, Karen M Velez, Edo M Airoldi, Weihan Li, Robert H Singer, Sneha Paul, Jingyi Fei, Tobin R Sosnick, Edward W J Wallace, D Allan Drummond.
      Stress-induced messenger ribonucleoprotein (mRNP) condensation is conserved across eukaryotes, resulting in stress granule formation under intense stresses, yet the mRNA composition and function of these condensates remain unclear. Exposure of ribosome-free mRNA following stress is thought to cause condensation and stress granule formation through mRNA-sequence-dependent interactions, leading to disproportionate condensation of long mRNAs. Here, we show that, by contrast, virtually all mRNAs condense in response to multiple stresses in budding yeast with minor length dependence and often without stress granule formation. New transcripts escape mRNP condensation, enabling their selective translation. Inhibiting translation initiation causes formation of mRNP condensates distinct from stress granules and processing bodies (P bodies), and these translation-initiation-inhibited condensates (TIICs) are omnipresent, even in unstressed cells. Stress-induced mRNAs are excluded from TIICs due to the timing of their expression, indicating determinants of escape that are independent of sequence. Together, our results reveal a previously undetected level of translation-linked molecular organization and stress-responsive regulation.
    Keywords:  TIICs; biomolecular condensation; condensates; mRNP granules; stress granules; stress response; translation initiation; translational regulation
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.003
  19. Proc Natl Acad Sci U S A. 2025 Dec 02. 122(48): e2517552122
    Drug repurposing screen identifies an HRI activating compound that promotes adaptive mitochondrial remodeling in MFN2-deficient cells.
    Prerona Bora, Mashiat Zaman, Samantha Oviedo, Sergei Kutseikin, Nicole Madrazo, Prakhyat Mathur, Meera Pannikkat, Sophia Krasny, Rama Aldakhlallah, Alan Chu, Kristen A Johnson, Danielle A Grotjahn, Timothy E Shutt, R Luke Wiseman.
      Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth type 2A (CMT2A). These mutations can disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR)-the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2-deficient cells. These results demonstrate the potential for pharmacologic ISR activation through the OMA1-DELE1-HRI signaling pathway as a potential strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.
    Keywords:  drug repurposing; integrated stress response; mitochondrial dysfunction
    DOI:  https://doi.org/10.1073/pnas.2517552122
  20. bioRxiv. 2025 Oct 20. pii: 2025.10.20.683548. [Epub ahead of print]
    Tryptoline Stereoprobe Elaboration Identifies Inhibitors of the GRPEL1-HSPA9 Chaperone Complex.
    Rachel E Hayward, Raymond F Berkeley, Zijian Gao, Maximilian Garhammer, Marc A Morizono, Evert Njomen, Haoxin Li, Kristen E DeMeester, Victor Cociorva, Mark A Herzik, F Ulrich Hartl, Bruno Melillo, Benjamin F Cravatt.
      Activity-based protein profiling has identified hundreds of proteins from diverse classes that react at specific cysteine residues with stereochemically defined electrophilic compounds (stereoprobes) in human cells. The structure-activity relationships underlying these stereoprobe-protein interactions, however, remain poorly understood. Here we show that the protein interaction landscape of tryptoline acrylamide stereoprobes can be profoundly altered by structural modifications distal to the acrylamide reactive group. The majority of stereoprobe liganding events occurred at non-orthosteric sites and mostly evaded assignment by the machine learning-based co-folding model Boltz-2, which instead tended to misplace the stereoprobes in orthosteric pockets (an outcome we term "orthostery burnout"). We found that stereoprobes reacting with C124 in the nucleotide exchange factor GRPEL1 disrupt interactions with the mitochondrial HSP70 chaperone HSPA9/mortalin, leading to impairments in mitochondrial protein import and induction of mitophagy. Our results highlight tryptoline acrylamides as a versatile source of covalent ligands targeting non-orthosteric sites on proteins, including tool compounds that perturb the mitochondrial HSP70 chaperone system.
    DOI:  https://doi.org/10.1101/2025.10.20.683548
  21. Elife. 2025 Nov 28. pii: RP102714. [Epub ahead of print]13
    The Shigella flexneri effector IpaH1.4 facilitates RNF213 degradation and protects cytosolic bacteria against interferon-induced ubiquitylation.
    Luz Saavedra-Sanchez, Mary S Dickinson, Shruti S Apte, Yifeng Zhang, Maarten De Jong, Samantha Skavicus, Nicholas S Heaton, Neal M Alto, Jorn Coers.
      A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFNγ). The IFNγ receptor is expressed on most human cells, and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFNγ-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri, uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFNγ-primed host cells. Linear (M1-) and lysine-linked ubiquitylation of S. flexneri requires RNF213 but is independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as an inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella.
    Keywords:  E3 ligase; RNF213; Shigella; autophagy; immunology; infectious disease; inflammation; microbiology; ubiquitin; xenophagy
    DOI:  https://doi.org/10.7554/eLife.102714
  22. J Cell Biol. 2026 Jan 05. pii: e202503081. [Epub ahead of print]225(1):
    Homocysteine disrupts lysosomal function by V-ATPase inhibition.
    Yang Yang, Qianjin Kong, Chaolian Liu, Fengyang Wang, Meijiao Li, Shalan Li, Yuehui Shi, Leonard Krall, Xin Wang, Shan He, Kai Jiang, Xuna Wu, Mei Yang, Chonglin Yang.
      Lysosomes are degradation and signaling organelles central to metabolic homeostasis. It remains unclear whether and how harmful metabolites compromise lysosome function in the etiopathology of metabolic disorders. Combining Caenorhabditiselegans and mouse models, we demonstrate that homocysteine, an intermediate in methionine-cysteine metabolism and the cause of the life-threatening disease homocystinuria, disrupts lysosomal functions. In C. elegans, mutations in cystathionine β-synthase cause strong buildup of homocysteine and developmental arrest. We reveal that homocysteine binds to and homocysteinylates V-ATPase, causing its inhibition and consequently impairment of lysosomal degradative capacity. This leads to enormous enlargement of lysosomes with extensive cargo accumulation and lysosomal membrane damage in severe cases. Cbs-deficient mice similarly accumulate homocysteine, displaying abnormal or damaged lysosomes reminiscent of lysosomal storage diseases in multiple tissues. These findings not only uncover how a metabolite can damage lysosomes but also establish lysosomal impairment as a critical contributing factor to homocystinuria and homocysteine-related diseases.
    DOI:  https://doi.org/10.1083/jcb.202503081
  23. bioRxiv. 2025 Oct 24. pii: 2025.10.24.684008. [Epub ahead of print]
    Ribosome remodeling drives translation adaptation during viral infection and cellular stress.
    Hsin-Yu Tsai, Luochen Liu, Rebecca H Fleming, Julian Mintseris, Derrick Ekanayake, Xin Gu, Steven P Gygi, Amy S Y Lee.
      The ribosome is the highly conserved molecular machine that decodes mRNAs during protein synthesis. While traditionally thought to consist of a uniform set of proteins, here we discover that ribosome composition is reprogrammed to adapt to intrinsic and external cellular perturbations. During infection by non-segmented negative-sense viruses, viral entry into cells recruits the large ribosomal subunit protein rpL40 to a noncanonical site on the small subunit of 80S ribosomes near the mRNA entry site. These specialized ribosomes preferentially bind viral mRNAs to drive enhanced viral protein synthesis that is critical for replication under host pressures. Unexpectedly, we find that viruses have co-opted this translation pathway from a previously unrecognized endogenous ribosome remodeling program in which metabolic stress alters ribosome structure to promote mRNA translation required for cell survival. Thus, ribosome remodeling is a conserved mechanism enabling dynamic protein synthesis across pathogen and cellular adaptation.
    DOI:  https://doi.org/10.1101/2025.10.24.684008
  24. Science. 2025 Nov 27. eadv7111
    Structural basis for the recruitment and selective phosphorylation of Akt by mTORC2.
    Martin S Taylor, Maggie Chen, Matthew Hancock, Maximilian Wranik, Bryant D Miller, Timothy R O'Meara, Brad A Palanski, Scott B Ficarro, Brian J Groendyke, Yufei Xiang, Kazuma T Kondo, Karen Y Linde-Garelli, Michelle J Lee, Dibyendu Mondal, Daniel Freund, Samantha Congreve, Kaay Matas, Maximiliaan Hennink, Kera Xibinaku, Max L Valenstein, Trevor van Eeuwen, Jarrod A Marto, Andrej Sali, Yi Shi, Nathanael S Gray, David M Sabatini, Nam Chu, Kacper B Rogala, Philip A Cole.
      The mTOR protein kinase forms two multiprotein complexes, mTORC1 and mTORC2, that function in distinct signaling pathways. mTORC1 is regulated by nutrients, and mTORC2 is a central node in phosphoinositide-3 kinase (PI3K) and small guanosine triphosphate Ras signaling networks commonly deregulated in cancer and diabetes. Although mTOR phosphorylates many substrates in vitro, in cells, mTORC1 and mTORC2 have high specificity: mTORC2 phosphorylates the protein kinases Akt and PKC, but not closely related kinases that are mTORC1 substrates. To understand how mTORC2 recognizes substrates, we created semisynthetic probes to trap the mTORC2-Akt complex and determine its structure. Whereas most protein kinases recognize amino acids adjacent to the phosphorylation site, local sequence contributes little to substrate recognition by mTORC2. Instead, the specificity determinants were secondary and tertiary structural elements of Akt that bound the mTORC2 component mSin1 distal to the mTOR active site and were conserved amongst at least 18 related substrates. These results reveal how mTORC2 recognizes its canonical substrates and may enable the design of mTORC2-specific inhibitors.
    DOI:  https://doi.org/10.1126/science.adv7111
  25. Cell Mol Life Sci. 2025 Nov 27. 82(1): 430
    Proteostasis network response to environmental chronic stress: linking survival to protein aggregation in a human neuroblastoma cellular model.
    Emiliano Montalesi, Daniela Caissutti, Camilla Moliterni, Antonella Ferrante, Rita Pepponi, Tina Garofalo, Maurizio Sorice, Roberta Misasi, Niccolò Candelise.
      Proteins tend to misfold upon stressful events that alter their homeostasis, potentially leading to protein aggregation. A tight regulation of synthesis, folding and degradation, defined as proteostasis network (PN), is required to ensure the functionality of the cell. PN is of utmost importance in post-mitotic cells such as neurons, where protein quality must be preserved for their entire lifetime. Most neurodegenerative disorders are associated with dysregulation of this network. Here, we describe the alteration in key components of the PN during chronic stress and link them with the increase in the amyloid burden and with the aggregation of the protein TDP-43, a major player in Amyotrophic Lateral Sclerosis and other neurodegenerative diseases. Neuroblastoma SH-SY5Y cells were treated with a panel of environmental stressors and analyzed after 24 h and 72 h. Treatments resulted in altered PN functionality, including proteasome impairment, halted protein synthesis, engulfed bulk and selective autophagy, in the absence of overt cell death. Thioflavin staining showed increased amyloid burden throughout treatments, associated with phosphorylated TDP-43 (pTDP-43). Biochemical analyses further revealed the cleavage and increased insolubility of pTDP-43. Our results suggest that TDP-43 is a central player during the integrated stress response to chr onic insults and that increased amyloid burden may reflect the global wellfare of a cellular system, pointing toward the alteration of the PN as the main drive for the onset of sporadic neurodegenerative disorders.
    Keywords:  Cell fate; Cellular homeostasis; Prion-like; Protein misfolding; Protein quality control; Protein solubility
    DOI:  https://doi.org/10.1007/s00018-025-05884-6
  26. Nat Commun. 2025 Nov 26. 16(1): 10518
    Sec14L6 is a phosphoinositide transporter that regulates phosphoinositide homeostasis and biogenesis of lipid droplets.
    Tiantian Zhou, Juan Xiong, Xuewen Hu, Yuanjiao Du, Anbing Shi, Weiping Chang, Jichao Qin, Lin Deng, Wei-Ke Ji.
      Lipid droplets (LDs) are evolutionarily conserved organelles essential for cellular metabolism. They form and grow at the endoplasmic reticulum (ER), requiring lipid transfer between these compartments, yet the underlying molecular mechanisms remain elusive. We identify Sec14L6, a unique Sec14 family member, as a lipid transporter regulating phosphoinositide (PIP) homeostasis and LD biogenesis, promoting adipogenic differentiation of mesenchymal stem cells. Sec14L6 directly binds the LD biogenesis factor ACSL3, which facilitates the association of Sec14L6 with LD surface. Furthermore, the ER membrane protein PGRMC1 recruits Sec14L6 to the ER. Targeted lipidomics revealed profound PIP dysregulation in Sec14L6-KO cells: LDs accumulated phosphoinositide-4-phosphate (PI4P) and PI(4,5)P₂, while these PIPs were reduced within the ER. In vitro assays demonstrated that Sec14L6 transports PI4P and PI(4,5)P₂. Sec14L6 knockout significantly impaired LD formation; this defect was rescued by wild-type Sec14L6, but not by lipid-transfer-deficient mutants. Our study reveals an essential role for Sec14L6 in PIP homeostasis and promotes LD biogenesis through lipid transfer between the ER and LDs.
    DOI:  https://doi.org/10.1038/s41467-025-65540-2
  27. Nat Microbiol. 2025 Nov 25.
    Toxoplasma effector TgROP1 establishes membrane contact sites with the endoplasmic reticulum during infection.
    Chahat Mehra, Jesús Alvarado Valverde, Ana Margarida Nogueira Matias, Francesca Torelli, Tânia Catarina Medeiros, Julian Straub, James D Asaki, Peter J Bradley, Katja Luck, Steffen Lawo, Moritz Treeck, Lena Pernas.
      Membrane contact sites (MCS) are essential for organelle communication in eukaryotic cells. Pathogens also establish MCS with host organelles, but the mechanisms underlying these interactions and their role in infection remain poorly understood. Here, using a fluorescence sensor and CRISPR-based loss-of-function screening, together with imaging and proteomics, we identify the parasite effector mediating MCS between host endoplasmic reticulum (ER) and the vacuole containing the intracellular parasite Toxoplasma gondii. TgROP1 acts as a tether and mimics a canonical FFAT motif to bind the host ER proteins VAPA and VAPB. The loss of VAPA/B abolished host ER-Toxoplasma MCS and decreased pathogen growth. These findings indicate that targeting of host MCS tethers is a strategy exploited by pathogens during infection, which could inform future treatment design.
    DOI:  https://doi.org/10.1038/s41564-025-02193-3
  28. bioRxiv. 2025 Nov 02. pii: 2025.10.31.685813. [Epub ahead of print]
    KIF5A binds RNA to orchestrate synaptic mRNA localization and stress granules in ALS.
    Phuong Le, Neeraj K Lal, Shuhao Xu, Sara Mumford, Megan Huang, Dong Yang, Orel Mizrahi, Benjamin Hoover, Brian Yee, Yuan Mei, Katherine Rothamel, Hsuan-Lin Her, Steven M Blue, Neil A Shneider, Gene W Yeo.
      Neuronal health depends on the precise transport and local translation of mRNAs to maintain synaptic function across highly polarized cellular architecture. While kinesin motor proteins are known to mediate mRNA transport, the specificity and direct involvement of individual kinesins as RNA-binding proteins (RBPs) remain unclear. Here, we demonstrate that KIF5A, a neuron-specific kinesin implicated in amyotrophic lateral sclerosis (ALS), functions as an RBP. We show that KIF5A directly binds mRNAs encoding synaptic ribosomal proteins and is required for their synaptic localization and for maintaining normal synaptic composition and function. Additionally, we show ALS-linked KIF5A mutations confer gain-of-function properties, enhancing mRNA binding, increasing synaptic ribosomal protein accumulation, inducing neuronal hyperexcitability, and impairing stress responses. These findings reveal a previously unrecognized mechanism by which mutant KIF5A disrupts synaptic homeostasis. Our work positions a kinesin motor protein as an RBP with critical roles in mRNA transport, local translation, and stress response.
    Highlights: KIF5A interacts with mRNA encoding synaptic ribosomal proteinsKIF5A is required for normal synaptic composition and functionKIF5A binds to G3BP1 and G3BP1 stress granule associated proteinsKIF5A mutant ALS patient-derived motor neurons have abnormal synaptic function and stress response.
    DOI:  https://doi.org/10.1101/2025.10.31.685813
  29. bioRxiv. 2025 Oct 29. pii: 2025.10.29.685433. [Epub ahead of print]
    A late cytoplasmic surveillance pathway ensures ribosome integrity.
    Ruta Chitale, Kaoling Guan, Shilpa Rao, Caroline Wang, Can Cenik, David W Taylor, Arlen W Johnson.
      Errors in ribosome assembly can produce defective subunits that can lead to aberrant translation events. How faulty ribosomes are recognized and whether they are recognized during assembly or translation remains poorly understood. We utilized a mutation in the ribosomal protein uL16 to track defective 60S subunits through the biogenesis and translation pathways. This mutation deletes a critical loop in the P site and arrests pre- 60S particles during late cytoplasmic maturation. However, simultaneous mutations in the late biogenesis factors Nmd3 and Tif6 bypass this block, releasing defective ribosomes into the translational pool. Cryo-EM and selective ribosome profiling reveal that these ribosomes can form peptide bonds, but stall predominantly at the first few codons. We show that the uL16 mutant ribosomes are detected and targeted for degradation during biogenesis and that they escape degradation if they enter translation. We identify Reh1 as a non-canonical ribosome assembly factor that is required for this surveillance pathway.
    DOI:  https://doi.org/10.1101/2025.10.29.685433
  30. Nat Commun. 2025 Nov 27. 16(1): 10642
    The luminal domain region of Seipin/Fld1 is dispensable for establishing functional ER sites for lipid droplet biogenesis.
    Amit Khatri, Ritika Chaudhary, R Mankamna Kumari, Vineet Choudhary.
      Lipid droplet (LD) biogenesis occurs in the endoplasmic reticulum (ER), the mechanisms of which is not completely known. Seipin (Fld1 in yeast) is a crucial ER membrane protein that defines LD biogenesis sites. Here, we show that truncated seipin, Fld1-∆LR in yeast, and the human equivalent hSeipin-∆LR, mutants lacking the conserved luminal domain region (LR), functionally complement the LD biogenesis defect of fld1∆ mutants. Fld1-∆LR foci colocalize with factors: Nem1, Ldb16, Pex30 and Yft2, which are important for LD biogenesis and these sites become enriched in diacylglycerol upon stimulation of LD formation. Fld1-∆LR forms a homo-oligomeric complex facilitated by protein-protein interactions. We show that mutating the 31st proline abrogates the functioning of Fld1-∆LR. We demonstrate the critical regulatory role of LR of seipin in partitioning triacylglycerol into LDs. We conclude that LR of seipin is dispensable for establishing functional ER sites to recruit proteins for LD biogenesis.
    DOI:  https://doi.org/10.1038/s41467-025-65645-8
  31. bioRxiv. 2025 Oct 17. pii: 2025.10.16.682859. [Epub ahead of print]
    Cryo-EM reveals how ASX-173 inhibits human asparagine synthetase to activate the integrated stress response.
    Kirk A Staschke, Nicholas T Walda, Lucciano A Pearce, Ronald C Wek, Wen Zhu, Yuichiro Takagi.
      Targeting asparagine metabolism is a promising strategy for treating asparaginase-resistant acute lymphoblastic leukemia (ALL), sarcoma, and potentially other solid tumors. Here, we characterize the molecular mechanism by which a cell-penetrable small molecule, ASX-173, inhibits human asparagine synthetase (ASNS), the enzyme that catalyzes intracellular asparagine biosynthesis. ASX-173 reduces cellular asparagine levels, induces the integrated stress response (ISR), and reduces cell growth in HEK-293A cells. A cryo-EM structure reveals that ASX-173 engages a unique, hydrophobic pocket formed by AMP, Mg 2+ , and pyrophosphate in the C-terminal synthetase domain of ASNS, thereby enabling multivalent, high-affinity binding. Based on in vitro kinetic and thermal shift assays, we find that ASX-173 binds to the ASNS/Mg 2+ /ATP complex and is therefore a rare example of an uncompetitive enzyme inhibitor with potential therapeutic use. These findings provide a structural and mechanistic basis for targeting ASNS with small molecules, which have application in treating cancer and other human diseases.
    DOI:  https://doi.org/10.1101/2025.10.16.682859
  32. bioRxiv. 2025 Oct 13. pii: 2025.10.13.682107. [Epub ahead of print]
    Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins.
    Frederick Rehfeld, Claire Lundstrom, He Zhang, Joshua T Mendell.
      Selenocysteine, incorporated into polypeptides at recoded termination codons, plays an essential role in redox biology. Using GPX1 and GPX4, selenoenzymes that mitigate oxidative stress, as reporters, we performed genome-wide knockout screens to identify regulators of selenocysteine incorporation. This revealed that selenoprotein production is limited by ribosome collisions that occur at inefficiently decoded selenocysteine codons. Accordingly, slowed translation elongation reduced collisions and enhanced selenocysteine decoding. Oxidative stress also slowed translation elongation and augmented selenoprotein production. We identified translation elongation factor EEF1G as a sensor of oxidized glutathione that couples the cellular redox state to translation elongation rate. Oxidative stress sensing by EEF1G slows translation, enhancing production of detoxifying selenoproteins to restore homeostasis. These findings reveal how programmed ribosome collisions enable gene regulation in response to stress.
    DOI:  https://doi.org/10.1101/2025.10.13.682107
  33. bioRxiv. 2025 Oct 17. pii: 2025.10.17.683119. [Epub ahead of print]
    Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.
    Maria K Janowska, Vanesa Racigh, Christoper N Woods, María Silvina Fornasari, Rachel E Klevit.
      Zinc is an essential metal that supports diverse cellular functions. Zinc exerts its biological activity through protein binding, serving as catalytic cofactors and structural stabilizers of many enzymes, transcription factors, and ubiquitin E3 ligases, among others. Despite total cellular zinc concentrations reaching hundreds of micromolar, free zinc levels are tightly buffered. Elevated free zinc promotes mismetalation and protein aggregation. While zinc is redox-inert, its cysteine-based protein ligands are readily oxidized. Oxidative modification of cysteines leads to zinc dissociation and a rapid increase in free zinc. With ~3000 proteins in the human zinc proteome, uncontrolled zinc release could be highly deleterious. Metallothioneins buffer zinc under basal conditions, but their re-synthesis following oxidative inactivation occurs on the scale of hours, raising the question of how free zinc is managed in the interim. Histidine, the second most prevalent zinc-coordinating residue, is resistant to oxidative modification. We characterized zinc binding by the small heat shock protein HSPB5 (αB-crystallin), a cysteine-free, histidine-rich protein chaperone that responds to cellular stress and found: (1) HSPB5 binds zinc with high affinity and rapid reversibility; (2) zinc binding requires the disordered HSPB5 N-terminal region; (3) zinc binding increases HSPB5 disorder; and (4) prolonged zinc exposure promotes formation of assemblies of oligomers cross-bridged by zinc. We propose that HSPB5 has evolved specialized zinc-dependent properties distinct among human sHSPs, enabling it to function not only as a protein chaperone but also as a conditional zinc reservoir under oxidative stress.
    Keywords:  HSPB5; alphaB crystallin; chaperone; intrinsic disorder; metalloproteins; small heat shock proteins; zinc
    DOI:  https://doi.org/10.1101/2025.10.17.683119
  34. bioRxiv. 2025 Oct 13. pii: 2025.10.13.681693. [Epub ahead of print]
    Multiplex design and discovery of proximity handles for programmable proteome editing.
    Chase C Suiter, Green Ahn, Melodie Chiu, Yi Fu, Shayan Sadre, Jessica J Simon, David S Lee, Douglas M Fowler, Dustin J Maly, David Baker, Jay Shendure.
      Although we now have a rich toolset for genome editing, an equivalent framework for manipulating the proteome with a comparable flexibility and specificity remains elusive. A promising strategy for "proteome editing" is to use bifunctional molecules (e.g. PROteolysis-Targeting Chimeras or PROTACs1) that bring a target protein into proximity with a degradation or stabilization effector, but their broader application is constrained by a limited repertoire of well-characterized target or effector "handles". We asked whether coupling de novo protein design to a multiplex screening framework could address this gap by accelerating the discovery of effector handles for intracellular protein degradation, stabilization, or relocalization. Using LABEL-seq2, a sequencing-based assay that enables multiplex, quantitative measurement of protein abundance, we screened 9,715 de novo designed candidate effector handles for their ability to recruit a target protein to components of the ubiquitin-proteasome system3 (UPS) (FBXL12, TRAF2, UCHL1, USP38) or the autophagy pathway4 (GABARAP, GABARAPL2, MAP1LC3A). In a single experiment, we discovered hundreds of de novo designed effector handles that reproducibly drove either intracellular degradation (n = 277) or stabilization (n = 204) of a reporter protein. Validation of a subset of these hits in an orthogonal assay confirmed that sequencing-based measurements from the primary screen reliably reflected changes in intracellular abundance of the target protein. Successful effector handles were discovered for both the UPS (n = 194) and autophagy (n = 287) pathways, which provide complementary routes for programmable proteome editing. Autophagy-recruiting effector handles generalized to endogenous targets, as substituting the reporter-specific target handle with a high-affinity MCL1 binder5 reduced endogenous levels of this intracellular oncoprotein6. Moreover, directing autophagy-recruiting effector handles to the outer mitochondrial membrane dramatically perturbed mitochondrial networks in a manner consistent with synthetic tethering and sequestration7,8. Beyond generating a diverse repertoire of protein abundance or localization effector handles, our results establish a scalable, low-cost platform that links deep learning-guided protein design to functional cellular readouts, and chart a course toward a general framework for programmable proteome editing.
    DOI:  https://doi.org/10.1101/2025.10.13.681693
  35. Nat Commun. 2025 Nov 28. 16(1): 10761
    Disruption of the PIKfyve complex unveils an adaptive mechanism to promote lysosomal repair and mitochondrial homeostasis.
    Candice Kutchukian, Maria Casas, Rose E Dixon, Eamonn J Dickson.
      Lysosomes are essential organelles that regulate cellular homeostasis through complex membrane interactions. Phosphoinositide lipids play critical roles in orchestrating these functions by recruiting specific proteins to organelle membranes. The PIKfyve/Fig4/Vac14 complex regulates PI(3,5)P₂ metabolism, and intriguingly, while loss-of-function mutations cause neurodegeneration, acute PIKfyve inhibition shows therapeutic potential in neurodegenerative disorders. We demonstrate that PIKfyve/Fig4/Vac14 dysfunction triggers a compensatory response where reduced mTORC1 activity leads to ULK1-dependent trafficking of ATG9A and PI4KIIα from the TGN to lysosomes. This increases lysosomal PI(4)P, facilitating cholesterol and phosphatidylserine transport at ER-lysosome contacts to promote membrane repair. Concurrently, elevated lysosomal PI(4)P recruits ORP1L to ER-lysosome-mitochondria three-way contacts, enabling PI(4)P transfer to mitochondria that drives ULK1-dependent fragmentation and increased respiration. These findings reveal a role for PIKfyve/Fig4/Vac14 in coordinating lysosomal repair and mitochondrial homeostasis, offering insights into cellular stress responses.
    DOI:  https://doi.org/10.1038/s41467-025-65798-6
  36. Bioorg Med Chem. 2025 Nov 19. pii: S0968-0896(25)00432-8. [Epub ahead of print]133 118491
    FPhotocleavable PROTACs as tools for dynamic regulation of protein function.
    Hanqiao Xu, Hidetomo Yokoo, Yosuke Demizu.
      Proteolysis-targeting chimeras (PROTACs) have emerged as valuable tools for targeted protein degradation. However, conventional designs generally lack mechanisms for temporal regulation. In this study, we explored photocleavable PROTACs incorporating a nitrobenzyl linker, aiming to achieve light-triggered inactivation of BET protein degradation. Docking simulations indicated that the linker did not disrupt the formation of the ternary complex, and LC-MS/MS analyses indicated that photocleavage occurred rapidly within cells. Cellular experiments demonstrated that the compounds promoted efficient degradation of the BET protein at low concentrations, and recovery assays with UV irradiation indicated that protein expression could be restored after transient depletion. These results suggest that photocleavable PROTACs offer a practical approach to controlling degradation with temporal precision and may provide useful chemical biology tools for investigating the dynamic functions of BET proteins.
    Keywords:  Nitrobenzyl linker; Photocleavable PROTAC; Targeted protein degradation; Temporal control; UV irradiation
    DOI:  https://doi.org/10.1016/j.bmc.2025.118491
  37. bioRxiv. 2025 Nov 10. pii: 2025.11.08.687377. [Epub ahead of print]
    Stress granules and protein aggregates reveal intracellular resource competition.
    Hannah E Buchholz, Sean A Martin, Jane E Dorweiler, Derek C Prosser, Emily M Sontag, Anita L Manogaran.
      Stress granules are biomolecular condensates that form in response to environmental stress and disassemble once normal conditions are restored. However, when disassembly fails, stress granules can persist and solidify. While stress granule solidification has been well documented, the cellular mechanisms underlying the transition from reversible to persistent stress granules remain unclear. Persistent stress granules can seed the formation of pathological aggregates, such as TDP-43 in amyotrophic lateral sclerosis 1, 2 . Although amyloid and tau aggregates are hallmarks of Alzheimer's disease, a subset of patients also develop TDP-43 deposits, suggesting a possible role for stress granule solidification in Alzheimer's disease progression 3-5 . Despite theoretical models explaining why persistence and ensuing solidification occurs, strong in vivo evidence is lacking 6 . Here we show that competition for limited chaperone resources drive stress granule persistence. In the presence of TDP-43 aggregates or yeast amyloid proteins called prions, stress granule disassembly is slowed or halted disassembly. Using yeast prions as a model, we show that the addition of chaperones, specifically the AAA+ ATPase molecular chaperone, Hsp104, resulted in resumption of stress granule disassembly. Our results demonstrate that the competition for shared resources, such as molecular chaperones, can limit stress granule disassembly. We suspect that the presence of pathological aggregates results in resource competition within the aging brain, contributing to the persistence of stress granules and their subsequent solidification and aggregation.
    DOI:  https://doi.org/10.1101/2025.11.08.687377
  38. J Biol Chem. 2025 Nov 25. pii: S0021-9258(25)02828-5. [Epub ahead of print] 110976
    4E-BP1 differentially regulates translation of a subset of human mRNAs.
    Mehreen Mahbub, Baishakhi Saha, Dixie J Goss.
      Elevated levels of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) influence cap-independent translation by forming a complex with eukaryotic initiation factor 4E (eIF4E) (eIF4E•4E-BP1). Certain mRNAs-including those encoding hypoxia-inducible factor-1α (HIF-1α), fibroblast growth factor-9 (FGF-9), and two isoforms of tumor suppressors, p53 (p53A, and p53B)-contain structured 5'untranslated regions (UTRs) that enable translation either via cap-independent translation enhancer (CITE)-like or internal ribosomal entry site (IRES)-like mechanisms. However, how 4E-BP1 modulates these mechanisms remains unclear. Using fluorescence-based anisotropy assays, we showed that the eIF4E•4E-BP1 binds more tightly to the 5' m7G-cap of these mRNAs than eIF4E alone. Luciferase reporter assays further demonstrated that 4E-BP1 inhibits translation of CITE-like (HIF-1α, and p53A) more effectively than IRES-like (FGF-9, and p53B) mRNAs. Although binding affinity of eIF4E•4E-BP1 to each of these mRNAs increased, only CITE-like mRNA translation was significantly inhibited. Importantly, eIF4GI557-1599 and its binding partner eIF4A, overcomes this inhibition selectively for CITE-like mRNAs, while eIF4E alone has only partial effects. In contrast, IRES-like mRNAs, despite binding to eIF4E•4E-BP1, exhibited only a modest translational repression and were minimally affected by eIF4GI557-1599•eIF4A or eIF4E. These findings reveal that 4E-BP1 selectively represses cap-independent translation in a transcript-specific manner. Moreover, fluorescence anisotropy revealed that 4E-BP1 enhances eIF4GI557-1599 recruitment to certain mRNAs, potentially facilitating 43S pre-initiation complex (43S PIC) assembly during stress. These results provide new mechanistic insights into selective translational control with implications for stress response and cancer progression via non-canonical translation.
    Keywords:  4E-BP1; 5’ cap-independent translation enhancer (CITE); Cap-independent translation initiation; eIF4A; eIF4E; eIF4GI(557-1599); internal ribosome entry site (IRES)
    DOI:  https://doi.org/10.1016/j.jbc.2025.110976
  39. Biochim Biophys Acta Biomembr. 2025 Nov 24. pii: S0005-2736(25)00082-3. [Epub ahead of print] 184488
    TRAP mediated conformational changes of the human Sec61 channel.
    Nidhi Sorout, Volkhard Helms.
      The integral membrane pore Sec61 catalyzes the translocation of many secretory precursor proteins into the endoplasmic reticulum, as well as the insertion of transmembrane proteins into the cell and organellar membranes. Precursor proteins that possess weak signal peptides frequently require presence of the accessory membrane protein TRAP. Structural biology has recently established that TRAP shares several contact sites with Sec61, though not by an extended binding interface. However, how TRAP mechanistically supports the translocation of precursor proteins is still partially unresolved. Here, atomistic molecular dynamics simulations revealed that TRAP binding keeps Sec61 in a partially opened state, with looser packing of its transmembrane helices. TRAP maintained a partially opened Sec61 lateral gate and pore ring, shifting the plug helix towards an open conformation. These observations corroborate the existing model of how TRAP may support translocation of client precursor proteins with weak signal peptides.
    Keywords:  Conformational dynamics; Molecular dynamics simulation; Protein translocation; Sec61 complex; Signal peptide; TRAP complex
    DOI:  https://doi.org/10.1016/j.bbamem.2025.184488
  40. Nat Commun. 2025 Nov 28. 16(1): 10731
    The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.
    Yin Wang, Yifei Liao, Yizhe Sun, Bidisha Mitra, Rui Guo, Brenda Iturbide Piedras, Shaowen White, Hsin-Yao Tang, John M Asara, Italo Tempera, Paul M Lieberman, Benjamin E Gewurz.
      Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.
    DOI:  https://doi.org/10.1038/s41467-025-65760-6
  41. Autophagy. 2025 Nov 28.
    NAD+ restores proteostasis through splicing-dependent autophagy.
    Ruixue Ai, Evandro F Fang.
      Autophagy preserves neuronal integrity by clearing damaged proteins and organelles, but its efficiency declines with aging and neurodegeneration. Depletion of the oxidized form of nicotinamide adenine dinucleotide (NAD+) is a hallmark of this decline, yet how metabolic restoration enhances autophagic control has remained obscure. Meanwhile, alternative RNA splicing errors accumulate in aging brains, compromising proteostasis. Here, we identify a metabolic - transcriptional mechanism linking NAD+ metabolism to autophagic proteostasis through the NAD+ -EVA1C axis. Cross-species analyses in C. elegans, mice, and human samples reveal that NAD+ supplementation corrects hundreds of age- or Alzheimer-associated splicing errors, notably restoring balanced expression of EVA1C isoforms. Loss of EVA1C impairs the memory and proteostatic benefits of NAD+, underscoring its essential role in neuronal resilience. Mechanistically, NAD+ rebalances EVA1C isoforms that interact with chaperones BAG1 and HSPA/HSP70, reinforcing their network to facilitate chaperone-assisted selective autophagy and proteasomal degradation of misfolded proteins such as MAPT/tau. Thus, NAD+ restoration coordinates RNA splicing fidelity with downstream proteostatic systems, establishing a metabolic - transcriptional checkpoint for neuronal quality control. This finding expands the paradigm of autophagy regulation, positioning metabolic splice-switching as a crucial mechanism to maintain proteostasis and suggesting new strategies to combat aging-related neurodegenerative diseases.
    Keywords:  Aging; NAD+ precursors; alzheimer disease; machine learning; rna splicing; tauopathy
    DOI:  https://doi.org/10.1080/15548627.2025.2596679
  42. bioRxiv. 2025 Nov 13. pii: 2025.11.12.688098. [Epub ahead of print]
    EMT activates ER-to-Golgi trafficking through upregulation of REEP2 to promote lung cancer progression.
    Kevin Fulp, Oluwafunminiyi Obaleye, Shike Wang, Xin Liu, Jiang Yu, Jonathan M Kurie, Jun Xu, William K Russell, Xiaochao Tan, Guan-Yu Xiao.
      Membrane trafficking governs the transport of molecules to both intracellular and extracellular locations, thereby maintaining cell homeostasis. During cancer progression, alterations in membrane trafficking are frequently observed. However, the mechanisms underlying the dysregulation of membrane trafficking in cancer progression remain largely unresolved. Recent evidence has demonstrated that epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma (LUAD) employs a membrane trafficking program to coordinate cancer cell invasion and immunosuppression in the tumor microenvironment (TME). To further dissect the pro-tumorigenic membrane trafficking program, here we conducted a CRISPR interference (CRISPRi) in vivo screen for membrane trafficking regulators in a syngeneic mouse model with a complete immune system. This screen identified REEP2, an endoplasmic reticulum (ER) shaping protein, as a novel regulator of the EMT-dependent membrane trafficking program, which is associated with a poor prognosis in LUAD patients and is required for LUAD metastasis in a syngeneic orthotopic LUAD mouse model. Mechanistically, the EMT activator ZEB1 upregulates REEP2 expression through miR-183- and miR-193a-mediated regulation that promotes the transportation of secretory cargoes from the ER exit site (ERES) to the Golgi, thereby augmenting the secretion of pro-tumorigenic factors. The REEP2-driven secretion promotes cancer cell proliferation, migration, and the infiltration of myeloid-derived suppressor cells (MDSCs) in the TME. These findings identify REEP2 as a critical mediator of the EMT-driven pro-metastatic membrane trafficking program, revealing a specific vulnerability in mesenchymal LUAD.
    DOI:  https://doi.org/10.1101/2025.11.12.688098
  43. bioRxiv. 2025 Oct 28. pii: 2025.10.27.684972. [Epub ahead of print]
    Faf1 accelerates p97-mediated protein unfolding by promoting ubiquitin engagement.
    Zengwei Liao, Connor Arkinson, Andreas Martin.
      P97/VCP is a protein unfoldase of the AAA+ ATPase family that plays essential roles in numerous cellular processes, including ER-associated degradation and DNA replication. P97 utilizes various cofactors to process different substrates. For unfolding of proteins that are modified with K48-linked ubiquitin chains, p97 works with the heterodimeric cofactor Ufd1-Npl4, and the cofactor Faf1 was shown to enhance this activity in the context of replisome disassembly, yet the underlying mechanisms remain unknown. Here, we employ an in vitro reconstituted system with human components for biochemical experiments, mutational studies, FRET-based assays, and cryo-EM structure determination to reveal that Faf1 plays a generic role in accelerating ubiquitin-dependent substrate processing by promoting the unfolding of an initiator ubiquitin and its engagement by the ATPase motor. Faf1 thereby uses its p97-bound C-terminal UBX domain to anchor a long helix that braces the UT3 domain of Ufd1 and apparently stabilizes the Ufd1-Npl4 cofactor for ubiquitin unfolding. Our findings demonstrate how p97 works simultaneously with several cofactors to facilitate the unfolding of ubiquitinated proteins, indicating more complex regulatory mechanisms for substrate selection than for the simpler Cdc48 ortholog in yeast.
    DOI:  https://doi.org/10.1101/2025.10.27.684972
  44. Commun Biol. 2025 Nov 24. 8(1): 1650
    Distinct responses to non-autonomous UPRER mediated by glutamatergic and octopaminergic neurons.
    Aeowynn J Coakley, Adam J Hruby, Jing Wang, Andrew Bong, Tripti Nair, Carmen M Ramos, Athena Alcala, Daniel Hicks, Maxim Averbukh, Naibedya Dutta, Darius Moaddeli, Camilla Pearson, Cynthia J Siebrand, Mattias de Los Rios Rogers, Arushi Sahay, Sean P Curran, Peter J Mullen, Bérénice A Benayoun, Gilberto Garcia, Ryo Higuchi-Sanabria.
      The capacity to deal with stress declines during the aging process, and preservation of cellular stress responses is critical to healthy aging. The unfolded protein response of the endoplasmic reticulum (UPRER) is one such conserved mechanism, which is critical for the maintenance of several major functions of the ER during stress, including protein folding and lipid metabolism. Hyperactivation of the UPRER by overexpression of the major transcription factor, xbp-1s, solely in neurons drives lifespan extension as neurons send a neurotransmitter-based signal to other tissues to activate UPRER in a non-autonomous fashion. Previous work identified serotonergic, dopaminergic, and tyraminergic neurons in this signaling paradigm. To further expand our understanding of the neural circuitry that underlies the non-autonomous signaling of ER stress, we activated UPRER solely in glutamatergic, octopaminergic, and GABAergic neurons in C. elegans and paired whole-body transcriptomic analysis with functional assays. We found that UPRER-induced signals from glutamatergic neurons increased expression of canonical protein homeostasis pathways and octopaminergic neurons promoted pathogen response pathways, while more modest changes were detected in GABAergic UPRER activation. These findings provide further evidence for the distinct role neuronal subtypes play in driving the diverse response to ER stress.
    DOI:  https://doi.org/10.1038/s42003-025-09036-1
  45. bioRxiv. 2025 Oct 08. pii: 2025.10.08.681129. [Epub ahead of print]
    The functional landscape of the human ubiquitinome.
    Julian van Gerwen, Maximilian Fottner, Shengbo Wang, Bede Busby, Ellen Boswell, Paul Schnacke, Andrea C Carrano, Malina A Bakowski, Emily R Troemel, Romain Studer, Marta Strumillo, Maria-Jesus Martin, J Wade Harper, Kathrin Lang, Andrew R Jones, Eric J Bennett, Juan Antonio Vizcaíno, Inigo Barrio-Hernandez, Pedro Beltrao.
      Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Perturbation proteomics revealed that these highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize functional ubi-sites with roles in cellular signaling, we constructed a functional score for more than 100,000 ubi-sites by integrating evolutionary, proteomic, and structural features using machine learning. Our score identifies ubi-sites regulating diverse protein functions and rationalizes mechanisms of genetic disease. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying protein ubiquitination.
    DOI:  https://doi.org/10.1101/2025.10.08.681129
  46. Elife. 2025 Nov 26. pii: e101496. [Epub ahead of print]14
    Specific proteolysis mediated by a p97-directed proteolysis-targeting chimera (PROTAC).
    Constanza Salinas-Rebolledo, Javier Blesa, Guillermo Valenzuela-Nieto, David Schwefel, Natalia López González Del Rey, Maxs Méndez-Ruette, Janine Burkhalter, Elizabeth Carrazana, Francisca Díaz-Tejeda, Ignacio Arias Catalán, Claudio Cappelli Leon, Natalia Salvadores, Luis Federico Bátiz, Ronald Jara, José A Obeso, Pedro Chana-Cuevas, Gopal P Sapkota, Alejandro Rojas-Fernandez.
      The p97 protein is a member of the AAA + family of ATPases. This protein is encoded by the VCP gene. It is a mechanoenzyme that uses energy from ATP hydrolysis to promote protein unfolding and segregation actively. The unfolded products are subsequently presented to the 26S proteasome for degradation. p97 substrate recognition is mediated by adaptors, which interact with substrates directly or indirectly through ubiquitin modifications, resulting in substrate funnelling into the central pore of the p97 hexamer and unfolding. Here, we engineered synthetic adaptors to target specific substrates to p97, using the extraordinary intracellular binding capabilities of camelid nanobodies fused to the UBX domain of the p97 adaptor protein Fas-associated factor-1 (FAF1). In such a way, we created a p97-directed proteolysis-targeting chimera (PROTAC), representing a novel and unique E3 ubiquitin ligase-independent strategy to promote specific proteolysis. All functional assays were performed in human cell lines to evaluate the system's efficacy and specificity in a physiologically relevant context.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.101496
  47. Cell Rep. 2025 Nov 21. pii: S2211-1247(25)01360-9. [Epub ahead of print]44(12): 116588
    Liquid-liquid phase separation of ATXN2L enhances mRNA translation in hepatocellular carcinoma.
    Shenghong Wang, Peilan Guo, Wenqiang Ma, Honglin Huang, Qianqian Zhang, Slawomir Wolczynski, Nafis Rahman, Jiali Liu, Suk-Ying Tsang, Xuguang Du, Fengchao Wang, Jie Gao, Xiangdong Li.
      RNA-binding proteins (RBPs) serve as key regulators of hepatocellular carcinoma (HCC); however, the specific mechanisms by which RBPs function in this context require clarification. Here, we identify an RBP, ATXN2L (ataxin 2-like), that is significantly upregulated in HCC tissues and correlates with a poor prognosis. Knockdown of ATXN2L in HCC cells or knockout of Atxn2l in mice suppresses HCC progression. Mechanistically, enhanced liquid-liquid phase separation (LLPS) activity of ATXN2L results in the formation of larger ATXN2L-positive granules, which facilitate the recruitment of several eukaryotic initiation factors (eIFs) and their downstream targets, such as ADAM9 (ADAM metallopeptidase domain 9), thereby promoting mRNA translation. Moreover, the promotion of ATXN2L granules on ADAM9 translation is further enhanced via co-localization with stress granules (SGs). Together, our findings reveal that ATXN2L functions as a critical translational regulator in HCC progression through LLPS activity, which could serve as an effective therapeutic target for HCC treatment.
    Keywords:  ADAM9; ATXN2L; CP: molecular biology; HCC; LLPS; SGs; mRNA translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116588
  48. J Virol. 2025 Nov 24. e0138925
    Annexin A2 stabilizes the endoplasmic reticulum and actin cytoskeleton and influences the formation of reovirus factories.
    Raquel Tenorio, Gwen M Taylor, Ana Cayuela, C O S Sorzano, Isabel Fernández de Castro, Sara Y Fernández-Sánchez, Alexa N Roth, Xayathed Somoulay, Gavin S Treadaway, Terence S Dermody, Cristina Risco.
      Early in infection, mammalian orthoreoviruses (reoviruses) build neo-organelles called viral factories (VFs). These structures incorporate fragments of endoplasmic reticulum (ER) and serve as sites of viral genome replication and particle assembly. Two reovirus nonstructural proteins, σNS and µNS, remodel the ER to produce the vesicles and tubules that form the VF matrix. Using co-immunoprecipitation assays followed by mass spectrometry, we identified annexin A2 (ANXA2), which binds actin and cellular membranes, as a host factor that interacts with reovirus nonstructural proteins. In the absence of ANXA2, VF formation was accelerated in the early phases of reovirus infection, and yields of reovirus were increased. Moreover, organization of the ER network and structure of the actin cytoskeleton were altered in the absence of ANXA2, suggesting that ANXA2 binding to actin is required to maintain ER network morphology. These findings provide evidence that interactions of reovirus nonstructural proteins directly or indirectly with ANXA2 are required for ER remodeling and formation of the membranous fragments that serve as the matrix to assemble reovirus factories.IMPORTANCEReovirus uses ER fragments to build the membranous scaffold of viral factories (VFs). Host proteins that participate in the ER remodeling that precedes factory biogenesis are not known. We identified actin-binding protein ANXA2 as a cellular factor required for maintenance of ER morphology. The absence of ANXA2 destabilizes the actin cytoskeleton and consequently the ER, which accelerates VF biogenesis and enhances reovirus replication. Uncovering the cellular factors used by viruses to form VFs deepens an understanding of viral cell biology and highlights new targets for antiviral drug development.
    Keywords:  actin; annexin A2; endoplasmic reticulum; reovirus; viral factories; µNS; σNS
    DOI:  https://doi.org/10.1128/jvi.01389-25
  49. medRxiv. 2025 Oct 13. pii: 2025.10.08.25337448. [Epub ahead of print]
    Integrated Stress Response Signatures Drive Monocyte Dysfunction in GBA1 - and LRRK2 -Linked Parkinson's Disease.
    Daniele Mattei, Erica Brophy, Mikaela Rosen, Oriol Narcis Majos, Aloysius Domingo, Elena Meijia, Beomjin Jang, Tarek Khashan, Mengxi Yang, Deborah Raymond, Casey Young, Jack Humphrey, Elisa Navarro, Amanda Allan, Katherine Leaver, Viktoriya Katsnelson, Alexander Chung, Benjamin Muller, Matthew Swan, Vicki L Shanker, Mariel Pullman, Adina Wise, Roberto Ortega, Kelly Astudillo, Steven Frucht, Susan Bressman, Giulietta Riboldi, Laurie J Ozelius, Rachel Saunders-Pullman, Towfique Raj.
      Monocytes are increasingly implicated in Parkinson's disease (PD) pathogenesis, with idiopathic cases showing mitochondrial and lysosomal dysfunction. However, the impact of PD-associated mutations on monocytes remains unclear. To address this, we investigated transcriptomic and functional disturbances in peripheral monocytes from patients with GBA1 - and LRRK2 -associated PD and idiopathic PD. Transcriptomic data revealed shared and mutation-specific signatures, including those related to immune dysregulation, and consistent defects in lysosomal and mitochondrial pathways. Network and pathway analyses further uncovered downregulation in protein translation and enrichment of integrated stress response (ISR) signatures, alongside aberrant expression of genes linked to ER stress, proteostasis, mitophagy and type-I interferon signaling. These data suggest that monocyte immune dysfunction in PD may be, at least in part, a consequence of impaired proteostasis, organelle stress and maladaptive ISR activation. We further interrogated these signatures in functional assays in patient-derived macrophages, which revealed impaired mitochondrial potential, proteolysosomal dysfunction, and defective phagocytosis. Our findings define convergent molecular and functional abnormalities in genetic PD monocytes, implicating proteostasis failure and maladaptive ISR activation as upstream drivers of immune dysfunction, highlighting novel targetable pathways for therapeutic intervention.
    DOI:  https://doi.org/10.1101/2025.10.08.25337448
  50. bioRxiv. 2025 Nov 01. pii: 2025.10.31.685804. [Epub ahead of print]
    ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling.
    Joung Hyuck Joo, William Kasberg, Spencer Douglas, Uwemedimo Udoh, Alexandre Carisey, James Messing, Yong-Dong Wang, Shilpa Narina, Shondra M Pruett-Miller, Myriam Labelle, Jennifer Lippincott-Schwartz, Chi-Lun Chang, Mondira Kundu.
      Cancer cells adapt to nutrient stress by remodeling the repertoire of proteins on their surface, enabling survival and progression under starvation conditions. However, the molecular mechanisms by which nutrient cues reshape the cell surface proteome to influence cell behavior remain largely unresolved. Here, we show that acute glucose starvation, but not amino acid deprivation or mTOR inhibition, selectively impairs ER-to-Golgi export of specific cargoes, such as E-cadherin, in a SEC24C-dependent manner. Quantitative cell surface proteomics reveal that glucose deprivation remodels the cell surface proteome, notably reducing surface expression of key adhesion molecules. This nutrient-sensitive reprogramming enhances cell migration in vitro and promotes metastasis in vivo . Mechanistically, we show that AMPK and ULK1 signaling orchestrate this process independent of autophagy, with ULK1-mediated phosphorylation of SEC31A driving SEC24C-dependent COPII reorganization. These findings establish ER-to-Golgi trafficking as a nutrient-sensitive regulatory node that links metabolic stress to cell surface remodeling and metastatic potential.
    DOI:  https://doi.org/10.1101/2025.10.31.685804
  51. bioRxiv. 2025 Oct 08. pii: 2025.10.08.681047. [Epub ahead of print]
    Proteome Landscapes Decode Organelle Vulnerabilities in cortical and dopaminergic-like induced neurons Across Lysosomal Storage Disorders.
    Felix Kraus, Yuchen He, Yizhi Jiang, Delong Li, Yohannes A Ambaw, Federico M Gasparoli, Joao A Paulo, Tobias C Walther, Robert Farese, Steven P Gygi, Florian Wilfling, J Wade Harper.
      Lysosomes maintain cellular homeostasis by degrading proteins delivered via endocytosis and autophagy and recycling building blocks for organelle biogenesis. Lysosomal Storage Disorders (LSDs) comprise a broad group of diseases affecting lysosomal degradation, ion flux, and lipid catabolism. Within this group, sphingolipidoses genes involved in glycosphingolipid breakdown are known (GBA1) or candidate (SMPD1, ASAH1) risk factors for Parkinsons Disease, though disease mechanisms remain unclear. Using our previously reported LSD mutant proteomic landscape in HeLa cells, we observed pronounced variability in endolysosomal proteome signatures among sphingolipid pathway mutants, with ASAH1 knockout cells showing altered lysosomal lipid composition, impaired endocytic trafficking, and disrupted ultrastructure by cryo-electron tomography. To extend these findings in a more physiologic context, we generated a human embryonic stem (ES) cell library comprising 23 LSD gene knockouts and profiled proteomic changes during differentiation into cortical and midbrain dopaminergic neurons over a 7 to 10 week period. LSD mutants exhibited lineage-specific alterations in organellar proteomes, revealing diverse vulnerabilities. Notably, GBA1 knockout and ASAH1knockout dopaminergic neurons showed disruptions in synaptic and mitochondrial compartments, correlating with impaired dopaminergic neuronal firing and disrupted presynaptic protein localization. This LSD mutant toolkit and associated proteomic landscape provides a resource for defining molecular signatures of LSD gene loss and highlights convergence of lysosomal dysfunction, synaptic integrity, and mitochondrial health as potential links between sphingolipidoses and PD risk.
    DOI:  https://doi.org/10.1101/2025.10.08.681047
  52. Redox Biol. 2025 Oct 30. pii: S2213-2317(25)00424-0. [Epub ahead of print]88 103911
    Integrin-specific signaling drives ER stress-dependent atherogenic endothelial activation.
    Cyrine Ben Dhaou, Zaki Al-Yafeai, G Ali Cruz-Marquez, Matthew L Scott, Brenna Pearson-Gallion, Elizabeth Cockerham, Hanna Li, Jonette M Peretik, Yuning Hong, Nirav Dhanesha, Arif Yurdagul, Oren Rom, Md Shenuarin Bhuiyan, A Wayne Orr.
      Atherogenic endothelial activation is driven by both the local arterial microenvironment, marked by altered extracellular matrix (ECM) composition and disturbed blood flow, and soluble proinflammatory cues such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, augments these proinflammatory stimuli. Although endoplasmic reticulum (ER) stress is a hallmark of atheroprone regions, its regulation by extracellular matrix and its precise role in endothelial inflammatory activation are not well defined. Here, we show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, but not in those adhered to basement membrane proteins. This matrix-specific ER stress response requires activation of the integrin family of ECM receptors, as endothelial cells deficient for integrin activation (talin1 L325R mutation) fail to activate ER stress in response to disturbed flow and oxLDL, while direct stimulation of integrins using CHAMP peptides is sufficient to induce ER stress. Silencing fibronectin-binding integrins (α5, αv) using siRNA blocks ER stress induction in vitro, and endothelial-specific deletion of α5 or αv reduces ER stress at atheroprone regions in vivo. Mechanistically, integrin-dependent ER stress is not associated with increased protein synthesis, unfolded protein accumulation, or superoxide production. Scavenging superoxide with TEMPOL does not alleviate ER stress. However, pharmacological inhibition of ER stress using TUDCA suppresses proinflammatory and metabolic gene expression (bulk RNA-seq), without affecting NF-κB activation. Instead, TUDCA prevents activation of the JNK-c-Jun signaling axis, which we show to be essential for proinflammatory gene induction. Blocking this pathway using a JNK inhibitor (SP600125) or dominant-negative c-Jun (TAM67) abrogates inflammatory gene expression following oxLDL or disturbed flow. Together, these findings identify a novel mechanism by which fibronectin-integrin signaling promotes ER stress in response to mechanical and metabolic stressors, amplifying endothelial inflammation through JNK-c-Jun signaling.
    Keywords:  Atherosclerosis; ER stress; Inflammation; Integrin; Oxidized LDL; Shear stress
    DOI:  https://doi.org/10.1016/j.redox.2025.103911
  53. J Am Chem Soc. 2025 Nov 26.
    Unbiased RNA Degrader Identification Uncovers an LC3B-Recruiting Chimera for COL15A1 mRNA Degradation.
    Xiaoxuan Su, Yuquan Tong, Patrick R A Zanon, Jielei Wang, Toru Tanaka, Jessica L Childs-Disney, Matthew D Disney.
      Heterobifunctional RNA degraders, such as ribonuclease-targeting chimeras (RiboTACs), eliminate structured RNAs by recruiting endogenous effector proteins. Here, we expand the effector toolbox by co-opting microtubule-associated protein 1 light chain 3B (MAP1LC3B, also known as LC3B), a core autophagy component recently implicated in mRNA decay. An RNA-binding fragment (F1) with a mapped transcriptome-wide binding profile was conjugated to the LC3B ligand ispinesib to generate the degrader F1-ispinesib. Integration of RNA-seq and Chemical Cross-Linking and Isolation by Pull-down combined with NGS Sequencing (Chem-CLIP-seq) data identified collagen type XV alpha 1 chain (COL15A1) mRNA as a target selectively degraded by F1-Ispinesib. The induced COL15A1 mRNA decay was LC3B- and autophagy-dependent, confirmed by genetic and pharmacological inhibition. Cellular engagement of LC3B was validated by NanoBRET and photocatalytic proximity labeling. In human aortic smooth muscle cells, F1-ispinesib phenocopied siRNA-mediated COL15A1 knockdown, reducing protein levels, suppressing proliferation, and enhancing migration. A synthetic LC3B recruitment model also recapitulated the on-target COL15A1 mRNA decay, further supporting the degradation mechanism. These findings establish LC3B as a recruitable effector protein for small-molecule-directed RNA decay and provide a general URID framework for discovering degraders that harness diverse RNA-regulatory proteins.
    DOI:  https://doi.org/10.1021/jacs.5c14363
  54. ACS Synth Biol. 2025 Nov 23.
    FLASH-AWAY: Intrabody-Directed Targeting of Optogenetic Tools for Protein Degradation.
    Zirui Zhuang, Ran Li, Bing Wang, Yuxin Meng, Yuancong Li, Junjun Nan, Xin Zhao, Ji Jing.
      Protein homeostasis, or proteostasis, is essential for cellular proteins to function properly. The buildup of abnormal proteins (such as damaged, misfolded, or aggregated proteins) is associated with many diseases, including cancer. Therefore, maintaining proteostasis is critical for cellular health. Currently, genetic methods for modulating proteostasis, such as RNA interference and CRISPR knockout, lack spatial and temporal precision. They are also not suitable for depleting already-synthesized proteins. Similarly, molecular tools like PROTACs and molecular glue face challenges in drug design and discovery. To directly control targeted protein degradation within cells, we introduce an intrabody-based optogenetic toolbox named Flash-Away. Flash-Away integrates the light-responsive ubiquitination activity of the RING domain of TRIM21 for protein degradation, coupled with specific intrabodies for precise targeting. Upon exposure to blue light, Flash-Away enables rapid and targeted degradation of selected proteins. This versatility is demonstrated through successful application to diverse protein targets, including actin, MLKL, and ALFA-tag fused proteins. This innovative light-inducible protein degradation system offers a powerful approach to investigate the functions of specific proteins within physiological contexts. Moreover, Flash-Away presents potential opportunities for clinical translational research and precise medical interventions, advancing the prospects of precision medicine.
    Keywords:  TRIM21; intrabody; optogenetic; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1021/acssynbio.4c00822
  55. Mol Cell. 2025 Nov 24. pii: S1097-2765(25)00898-6. [Epub ahead of print]
    Recycling of ribosomes at stop codons drives the rate of translation and the transition from proliferation to RESt.
    Annarita Miluzio, Alessandra Scagliola, Ivan Ferrari, Hasan Yilmaz, Giacomo D'Andrea, Laura Cassina, Stefania Oliveto, Sara Ricciardi, Alessandra Boletta, Stefano Biffo.
      Translation is made of initiation, elongation, and termination. The role of termination in relaying extracellular outputs to the translation machinery is unknown. We show, in mice, that the controlled recycling of ribosomes post-termination is a major checkpoint that integrates mitogenic signals and antiviral responses. In detail, the recycling of ribosomes at stop codons, maximal translation, and cellular proliferation strictly depend on eIF6 phosphorylation, both in vitro and in vivo. Lack of eIF6 phosphorylation, as observed during viral infection or prolonged starving, causes accumulation of ribosomes at stop codons and a massive translational remodeling. The outcome is a cellular status that we named RESt, for reversible energetic stop. RESt is marked by pro-survival and pro-inflammatory NF-κB signaling and a switch to respiration. Acute RESt is rescued by eIF6 phosphorylation, but chronic RESt invivo leads to senescence. Thus, the recycling rate of ribosomes post-termination is a physiologically controlled event impacting initiation.
    Keywords:  60S ribosomes; ABCE1; ATF4; implantation; integrated stress response; metabolism; protein synthesis; quiescence; stop codon; translation termination
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.002
  56. J Mol Neurosci. 2025 Nov 27. 75(4): 157
    The Dynamic UPR Rheostat Orchestrates Single-Cell Plasticity in Glioblastoma.
    Zekeriya Duzgun.
      Glioblastoma (GBM) adapts to microenvironmental stress through the unfolded protein response (UPR), yet whether the three canonical arms IRE1/XBP1, ATF6, and PERK operate as a graded control system at single-cell resolution remains unclear. We reanalyzed publicly available scRNA-seq datasets spanning discovery (n = 871 cells) and validation cohorts (n = 11,877 cells) to quantify arm-specific activities and their coordination across tumor cell states and pseudotime. We introduce arm-resolved metrics, including a Rheostat Index (per-cell dispersion of arm scores) and balance (normalized Shannon entropy), and map dynamic dominance switching (early ATF6 → late IRE1; rare PERK dominance) along lineage trajectories. IRE1 and ATF6 consistently exhibit tight coupling, whereas PERK remains semi-independent, indicating an adaptive division of labor. Rheostat tuning is associated with hypoxia and glycolytic programs and reorganizes across platforms (SMART-seq, 10x) and datasets. To minimize artificial correlations, we employ non-overlapping target sets and validate results using transcription factor activity inference. Statistical analyses prioritize patient-level inference via pseudobulk summaries and random-effects models to mitigate pseudoreplication. Overall, our results support a graded, arm-resolved UPR rheostat that governs GBM cellular plasticity and stress tolerance. These findings motivate therapeutic strategies that rebalance the rheostat attenuating IRE1/ATF6 survival signaling while permitting PERK-mediated death programs rather than globally suppressing the UPR. Our transcriptomic analyses infer arm-resolved coordination; functional validation will require perturbation studies and protein-level readouts.
    Keywords:  Cellular plasticity; Glioblastoma; Hypoxia; PERK/IRE1/ATF6 balance; Single-cell RNA-seq; Unfolded protein response
    DOI:  https://doi.org/10.1007/s12031-025-02447-z
  57. Nat Commun. 2025 Nov 26. 16(1): 10574
    Defining the role of β-cell IRE1α/XBP1 pathway and its gene regulatory network components in non-obese diabetic mice.
    Hugo Lee, Khagani Eynullazada, Qiaodan Ou, Junha Shin, Sushmita Roy, Feyza Engin.
      The unfolded protein response sensor, IRE1α, acts through its regulated IRE1α-dependent decay (RIDD) activity or transcription factor XBP1 to determine cell fate and survival. While blunting RIDD activity prevents diabetes in type 1 diabetes preclinical model non-obese diabetic mice, β-cell-specific function of XBP1 at different stages of disease remains unknown. Here we show that deletion of Xbp1 in β-cells (Xbp1β-/-) of non-obese diabetic mice before insulitis is protective against diabetes. Histological and transcriptomic analyses indicate that following a transient loss of maturity, β-cells of Xbp1β-/- mice exhibit reduced insulitis, apoptosis, and antigenicity phenocopying Ire1αβ-/- mice with no changes in RIDD activity. Comparative transcriptome and regulatory network analyses reveal a largely shared component between the Ire1αβ-/- and Xbp1β-/- mice as well as network components unique to Xbp1β-/-, indicative of IRE1α-independent roles of XBP1. Our findings define the role of β-cell IRE1α/XBP1 and identify previously unrecognized regulatory networks and nodes of this pathway.
    DOI:  https://doi.org/10.1038/s41467-025-65635-w
  58. Nat Commun. 2025 Nov 25. 16(1): 10438
    tRNA-dependent conformational dynamics and folding coordinate translational regulation by a full-length T-box riboswitch.
    Xiaolin Niu, Shanshan Cai, Junzheng Wang, Chunlai Chen, Xianyang Fang.
      T-box riboswitches regulate gene expression by sensing tRNA aminoacylation status, but their dynamic mechanisms remain elusive. Here, we present single-molecule FRET studies of a full-length translational ileS T-box, showing that its decoding domain folds independently, and the initial tRNA anticodon binding promotes proper folding of the discriminator domain. Subsequent uncharged tRNA binding stabilizes the Antisequestrator (AntiS) conformation and GAG linker, resulting in translation initiation. Conversely, charged tRNA binds to a distinct T-box conformation, of which the linker is highly flexible and stem III is away from AntiS, rendering irreversible transition to the Sequestrator conformation if downstream sequences are transcribed, leading to translation inhibition. Collectively, both steps of tRNA binding are in a conformational selection manner, and the GAG linker, especially G96, acts as the main linchpin in tRNA 3΄-termini sensing. An elaborate model is proposed to understand how cotranscriptional folding, stepwise tRNA binding and dynamic conformational transitions coordinate T-box regulation.
    DOI:  https://doi.org/10.1038/s41467-025-65388-6
  59. Cell Rep. 2025 Nov 25. pii: S2211-1247(25)01374-9. [Epub ahead of print]44(12): 116602
    Harnessing the E3 ligase SPOP for targeted degradation of the NUP98::KDM5A fusion oncoprotein.
    Ecem Kirkiz, Gabriel Kaufmann, Simone Bergqvist, Pablo Fernández-Pernas, Thomas Eder, Laura Quell, Melanie Allram, Gabriele Manhart, Wencke Walter, Torsten Haferlach, Florian Grebien.
      Nucleoporin 98-rearranged (NUP98-r) acute myeloid leukemia (AML) is associated with poor outcomes and remains a major therapeutic challenge due to the absence of strategies that directly eliminate NUP98 fusion oncoproteins. Targeted degradation of cancer-driving oncofusions is an attractive approach, but the molecular mechanisms controlling NUP98 oncofusion stability are unknown. Using a CRISPR-Cas9 screen, we identify the E3 ligase Speckle-type POZ protein (SPOP) as a direct regulator of NUP98 fusion oncoprotein stability and a novel tumor suppressor in NUP98-r AML. Loss of SPOP increases NUP98 oncofusion levels and promotes leukemia cell proliferation. Exploiting this specificity, we demonstrate that induced proximity of SPOP and NUP98::lysine-specific demethylase 5A (KDM5A) through a biological proteolysis-targeting chimera (bioPROTAC) induces full clearance of the fusion oncoprotein, driving terminal differentiation and apoptosis of NUP98-r leukemia cells in vitro and in vivo. This study identifies SPOP as a direct regulator of NUP98 oncofusion stability and outlines a strategy to redirect the ubiquitin-proteasome system against oncogenic fusions.
    Keywords:  AML; CP: cancer; NUP98; PROTAC; SPOP; condensate; fusion protein; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116602
  60. bioRxiv. 2025 Nov 03. pii: 2025.11.03.686228. [Epub ahead of print]
    Design of Tissue-Selective PROTACs Through Recruiting E3 Ligase Scaffolding Protein MAGEA11.
    Isabella E Jacobsen, Rui Shi, Cole R Scholtz, William C K Pomerantz, Gunda I Georg.
      Proteolysis targeting chimeras (PROTACs) are an emerging therapeutic modality that induces protein degradation by recruiting E3 ligases. Most reported PROTACs recruit ubiquitously expressed E3 ligases, such as cereblon and the von Hippel-Lindau tumor suppressor. Of the additional 600+ E3 ligases, recruiting those with tissue-restricted expression is attractive for increasing the specificity of PROTACs. To this end, tissue-specific E3 ligases or E3 ligase-associated proteins that can be recruited for targeted protein degradation need to be identified. This work describes the first reported PROTAC that recruits the tissue-specific E3 ligase scaffolding protein MAGEA11. As an initial demonstration, a library of bromodomain and extra-terminal domain (BET)-targeting PROTACs that recruit MAGEA11 was synthesized. The library was screened in osteosarcoma U2OS cells, identifying lead compound 105B. 105B potently degrades BET proteins in U2OS osteosarcoma cell lines (BRD4 DC 50 = 0.130 nM, D max = 78%) and KYSE180 esophageal squamous cell carcinoma cell lines (DC 50 = 40 nM, D max = 70%), but shows no degradation in non-cancerous, MAGEA11-deficient HEK293T cells. Mechanistic studies confirmed 105B's dependence on the ubiquitin-proteasome system and engagement of both MAGEA11 and BRD4. 105B decreased levels of BET-regulated gene products c-Myc, RUNX2, and KRT14; however, improvements are still necessary to affect selective cytotoxicity. This work reports the first example of a PROTAC recruiting a tissue-specific E3 ligase for cancer-restricted degradation of BET proteins and highlights the need for further development of MAGEA11-recruiting degraders.
    DOI:  https://doi.org/10.1101/2025.11.03.686228
  61. Cell Death Differ. 2025 Nov 25.
    Targeting prohibitins activates the ISR through DELE1-HRI by impairing protein import into the mitochondrial matrix.
    Ismael Sánchez-Vera, Ana M Cosialls, Nekane Maritorena-Hualde, Max-Hinderk Schuler, Rodolfo Lavilla, Gabriel Pons, Lucas T Jae, Daniel Iglesias-Serret, Joan Gil.
      Prohibitins (PHBs) are predominantly located at the inner mitochondrial membrane, displaying significant roles in tumor progression, invasion, and apoptotic resistance, often overexpressed in primary tumors. Importantly, we developed a synthetic molecule, fluorizoline, that induces apoptosis by selectively targeting PHBs in various cancer cell lines and primary samples from different hematological neoplasms. Fluorizoline induces apoptosis by activating the pro-apoptotic branch of the integrated stress response (ISR) pathway in HeLa and HAP1 cells, specifically via the ATF4-CHOP-NOXA axis. We identified compensatory mechanisms for four ISR-related kinases, with HRI emerging as the primary kinase responsible for the activation of the ISR and apoptosis induction, implicating mitochondrial stress in ISR activation. Here, we investigate the mitochondrial stress response signaling pathway responsible for activating HRI after targeting PHBs either by fluorizoline treatment or by PHBs downregulation in HeLa and HAP1 cancer cell lines. In this study, we describe how PHBs regulate the localization of the mitochondrial stress sensor DELE1, leading to ISR activation and apoptosis induction in HeLa and HAP1 cells. Our findings demonstrate that DELE1 promotes ISR activation upon fluorizoline treatment and PHBs downregulation. Although fluorizoline treatment activates the cleavage of long DELE1 (L-DELE1) to its cleaved form (S-DELE1), OMA1 was found to be dispensable for activating the ISR upon fluorizoline treatment. Furthermore, our findings indicate a potential impairment of the mitochondrial protein import machinery upon targeting PHBs, as the import of other mitochondrial proteins beyond DELE1 is also disrupted. These findings reveal a previously unknown physiological role of PHBs in preserving the mitochondrial protein import pre-sequence pathway, possibly due to the interaction between PHBs and DNAJC19. This novel insight underscores the potential of targeting PHBs, such as with fluorizoline, to overwhelm mitochondrial stress in cancer.
    DOI:  https://doi.org/10.1038/s41418-025-01618-0
  62. Methods Cell Biol. 2026 ;pii: S0091-679X(24)00244-9. [Epub ahead of print]200 1-19
    In vivo cell-type specific secretome profiling.
    Jie Liu, Toren Finkel, Shihui Liu.
      Serum is a complex mixture of proteins that originate from a wide range of cells and tissues. At present, it is difficult to know what set of proteins any given tissue contributes to the circulating proteome. Here, we describe a method of using proximity biotinylation of proteins that normally transit through the endoplasmic reticulum to profile secreted proteins from cells in culture. We also describe a mouse model that enables the elucidation of the in vivo tissue-specific secretome. As examples, we demonstrate how we can readily identify in vivo endothelial-specific secretion, and how this model allows for the characterization of muscle-derived serum proteins that either increase or decrease with exercise. Our "Secretome Mouse" model is broadly applicable to other cell and tissue types.
    Keywords:  BioID; Biotin; Endothelial cell; Muscle; Proximity biotinylation; Secretome
    DOI:  https://doi.org/10.1016/bs.mcb.2024.12.001
  63. Science. 2025 Nov 27. 390(6776): eadx5094
    Targeted protein degradation in the transmembrane and extracellular space.
    Rongfeng Zhu, Heng Zhang, Peng R Chen.
      Transmembrane and extracellular proteins play crucial roles in diverse cellular functions and communication, affecting the progression and treatment of various diseases by mediating vital cellular processes. Whereas targeted protein degradation (TPD) represents an advancing therapeutic modality that leverages cellular degradation machinery to eliminate proteins of interest, present strategies have been largely confined to intracellular targets. Now, emerging strategies toward transmembrane and extracellular proteins are rapidly expanding the horizon of this powerful technology. Here, we review TPD in the transmembrane and extracellular space (meTPD) and discuss platform technologies, features, applications, and limitations. We focus on the conceptual innovations used in developing the present meTPD technology as well as its potential value for biological research and therapeutic interventions.
    DOI:  https://doi.org/10.1126/science.adx5094
  64. bioRxiv. 2025 Nov 13. pii: 2025.11.13.686840. [Epub ahead of print]
    The ESCRT-0 protein HRS regulates hepatocellular lipid droplet catabolism.
    Mathilda M Willoughby, Ankit Shroff, Bridget E Crossman, Micah B Schott.
      Lipid droplets (LDs) are dynamic organelles that regulate lipid storage and metabolism pathways central to metabolic liver disease. LD turnover occurs in part through lysosomal catabolism (i.e. lipophagy) whereby LDs are thought to follow two distinct trafficking pathways: autophagosome-dependent macro lipophagy and the autophagosome-independent micro lipophagy. However, the molecular machinery that regulates these two distinct pathways, especially that of microlipophagy in mammalian cells, is poorly understood. In yeast, microlipophagy has been shown to rely on a protein family known as the endosomal sorting complex required for transport (ESCRT). Here, we used an ESCRT-specific RNAi library in hepatocytes which identified the ESCRT-0 protein hepatocyte growth factor receptor substrate (HRS) as a critical regulator of LD homeostasis. HRS depletion leads to significant LD accumulation which is not due to increased LD formation but from impaired LD catabolism. HRS-deficient cells retain lipolysis activity; however, they exhibit decreased LD targeting via microlipophagy, accompanied by compensatory increases in autophagosome targeting to LDs. In agreement with these findings, HRS knockdown suppressed mTOR signaling, boosted autophagosome formation, and reduced the degradation of autophagic cargo. Despite maintaining lysosome numbers, HRS knockdown raised lysosomal pH causing decreased autophagic degradative capacity and contributing to LD accumulation. Overall, these findings identify HRS as a modulator of LD turnover in mammalian cells, regulating lipophagy through lysosomal function.
    Significance Statement: The regulatory molecular mechanisms of lipophagy are not clearly defined. This study identifies novel ESCRT proteins as regulators of LD homeostasis in several cell lines.In hepatocytes, we identified HRS specifically regulates LD catabolism, whereby HRS-dependent regulation of LDs is dual-faceted, affecting LD-lysosomal targeting and lysosomal function.Our findings are significant because they provide mechanistic insights into the role of ESCRT proteins in LD metabolism. Elucidating ESCRT-mediated lipophagy can potentially aid in developing novel targets to prevent aberrant lipid trafficking and utilization, particularly in the liver where LDs can accumulate and cause irreversible liver damage.
    DOI:  https://doi.org/10.1101/2025.11.13.686840
  65. Nat Commun. 2025 Nov 26. 16(1): 10545
    Investigating the neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.
    Sébastien Küry, Janelle E Stanton, Geeske M van Woerden, Amélie Bosc-Rosati, Tzung-Chien Hsieh, Lise Bray, Marielle Oloudé, Cory Rosenfelt, Marie Pier Scott-Boyer, Victoria Most, Tianyun Wang, Jonas J Papendorf, Charlotte de Konink, Wallid Deb, Virginie Vignard, Maja Studencka-Turski, Thomas Besnard, Anna M Hajdukowicz, Franziska G Thiel, Sophie Wolfgramm, Laëtitia Florenceau, Silvestre Cuinat, Sylvain Marsac, Yann Verrès, Audrey Dangoumau, Léa Poirier, Ingrid M Wentzensen, Annabelle Tuttle, Cara Forster, Johanna Striesow, Richard Golnik, Damara Ortiz, Laura Jenkins, Jill A Rosenfeld, Alban Ziegler, Clara Houdayer, Dominique Bonneau, Erin Torti, Amber Begtrup, Kristin G Monaghan, Sureni V Mullegama, Catharina M L Nienke Volker-Touw, Koen L I van Gassen, Renske Oegema, Mirjam S de Pagter, Katharina Steindl, Anita Rauch, Ivan Ivanovski, Kimberly McDonald, Emily Boothe, Andrew Dauber, Janice Baker, Noelle Andrea V Fabie, Raphael A Bernier, Tychele N Turner, Siddharth Srivastava, Kira A Dies, Lindsay C Swanson, Carrie Costin, Alali Abdulrazak, Rebekah K Jobling, John Pappas, Rachel Rabin, Dmitriy Niyazov, Anne Chun-Hui Tsai, Karen Kovak, David B Beck, May Christine V Malicdan, David R Adams, Lynne Wolfe, Rebecca D Ganetzky, Colleen C Muraresku, Davit Babikyan, Zdeněk Sedláček, Miroslava Hančárová, Andrew T Timberlake, Hind Al Saif, Berkley Nestler, Kayla King, M J Hajianpour, Gregory Costain, D'Arcy Prendergast, Chumei Li, David Geneviève, Antonio Vitobello, Arthur Sorlin, Christophe Philippe, Tamar Harel, Ori Toker, Ataf Sabir, Derek Lim, Mark J Hamilton, Lisa J Bryson, Elaine Cleary, Sacha Weber, Trevor L Hoffman, Anna M Cueto-González, Eduardo F Tizzano, David Gómez-Andrés, Marta Codina-Solà, Athina Ververi, Efterpi Pavlidou, Alexandros Lambropoulos, Kyriakos Garganis, Marlène Rio, Jonathan Levy, Sarah J Langas, Anne M McRae, Mathieu K Lessard, Maria Daniela D'Agostino, Isabelle De Bie, Meret Wegler, Rami Abou Jamra, Susanne B Kamphausen, Viktoria Bothe, Lorraine Potocki, Eric Olinger, Yves Sznajer, Elsa Wiame, Michelle L Thompson, Molly C Schroeder, Catherine Gooch, Raphael A Smith, Arti Pandya, Larissa M Busch, Uwe Völker, Elke Hammer, Kristian Wende, Benjamin Cogné, Bertrand Isidor, Jens Meiler, Clémentine Ripoll, Stéphanie Bigou, Frédéric Laumonnier, Peter W Hildebrand, Evan E Eichler, Kirsty McWalter, Peter M Krawitz, Florence Roux-Dalvai, Ype Elgersma, Julien Marcoux, Marie-Pierre Bousquet, Arnaud Droit, Jeremie Poschmann, Andreas M Grabrucker, Francois V Bolduc, Stéphane Bézieau, Frédéric Ebstein, Elke Krüger.
      Neurodevelopmental proteasomopathies are a group of disorders caused by variants in proteasome subunit genes, that disrupt protein homeostasis and brain development through poorly characterized mechanisms. Here, we report 26 distinct variants in PSMC5, encoding the AAA⁺ ATPase subunit PSMC5/RPT6, in individuals with syndromic neurodevelopmental conditions. Combining genetic, multi-omics and biochemical approaches across cellular models and Drosophila, we unveil the essential role of proteasomes in sustaining key cellular processes. Loss of PSMC5/RPT6 function impairs proteasome activity, leading to protein aggregation, disruption of mitochondrial homeostasis, and dysregulation of lipid metabolism and immune signaling. It also compromises synaptic balance, neuritogenesis, and neural progenitor cell stemness, causing deficits in higher-order functions, including learning and locomotion. Pharmacological targeting of integrated stress response kinases reveals a mechanistic link between proteotoxic stress and spontaneous type I interferon activation. These findings expand our understanding of proteasome-dependent quality control in neurodevelopment and suggest potential therapeutic strategies for neurodevelopmental proteasomopathies.
    DOI:  https://doi.org/10.1038/s41467-025-65556-8
  66. Nat Commun. 2025 Nov 24. 16(1): 10317
    The implications of alternative splicing regulation for maximum lifespan.
    Wei Jiang, Sika Zheng, Liang Chen.
      Mammalian maximum lifespan (MLS) varies over a hundred-fold, yet the molecular mechanisms underlying this diversity remain unclear. We present a cross-species analysis of alternative splicing (AS) across six tissues in 26 mammals, identifying hundreds of conserved AS events significantly associated with MLS, with the brain containing twice as many tissue-specific events as peripheral tissues. MLS-AS events are enriched in pathways related to mRNA processing, stress response, neuronal functions, and epigenetic regulation, and are largely distinct from genes whose expression correlates with MLS, indicating that AS captures unique lifespan-related signals. The brain exhibits certain associations divergent from peripheral tissues and reduced overlap with body mass (BM)-associated splicing; neither is observed at the gene expression level. While MLS- and age-associated AS events show limited overlap, the shared events are enriched in intrinsically disordered protein regions, suggesting a role in protein flexibility and stress adaptability. Furthermore, MLS-associated AS events display stronger RNA-binding protein (RBP) motif coordination than age-associated ones, highlighting a more genetically programmed adaptation for lifespan determination, in contrast to the more variable splicing changes seen with chronological aging. These findings suggest alternative splicing as a distinct, transcription-independent axis of lifespan regulation, offering insights into the molecular basis of longevity.
    DOI:  https://doi.org/10.1038/s41467-025-65339-1
  67. Cell. 2025 Nov 24. pii: S0092-8674(25)01244-9. [Epub ahead of print]
    RNA innate immunity constitutes a barrier for interspecies chimerism.
    Yingying Hu, Hai-Xi Sun, Masahiro Sakurai, Zhou Luo, Amanda E Jones, Tianlei Cheng, Jia Huang, Lizhong Liu, Canbin Zheng, Jie Li, Yue Lu, Benjamin Ravaux, Bingbing He, Yi Ding, Tianbin Liu, Yan Wu, Zhijian J Chen, John M Abrams, Elizabeth H Chen, Ying Gu, Jun Wu.
      Creating interspecies chimeras with human pluripotent stem cells (hPSCs) offers a promising strategy for modeling human development and generating donor organs; however, poor human cell integration remains a major barrier. Most existing efforts to improve human chimerism focus on genetically modifying donor hPSCs, while altering the host embryo remains largely unexplored. Using an interspecies PSC competition model, we discovered that RNA innate immunity in "winner" mouse cells drives the competitive elimination of hPSCs. Disrupting RNA-sensing pathways reduced the competitiveness and viability of mouse PSCs, and mouse embryos lacking Mavs-a key gene in RNA innate immunity-led to markedly improved human cell survival and chimerism. We also found that contact-dependent horizontal RNA transfer likely underlies this immune activation. Overall, our study uncovers a previously unrecognized role for RNA innate immunity in cell competition and demonstrates that targeting host immune pathways represents a powerful approach to improve human chimerism in animals.
    Keywords:  MAVS; RLR pathway; RNA innate immunity; cell competition; horizontal RNA transfer; human pluripotent stem cells; interspecies PSC co-culture; interspecies chimeras; mouse epiblast stem cells; tunneling nanotube
    DOI:  https://doi.org/10.1016/j.cell.2025.10.039
  68. bioRxiv. 2025 Oct 21. pii: 2025.10.21.683613. [Epub ahead of print]
    UFMylation of 14-3-3ε coordinates MAVS signaling complex assembly to promote antiviral innate immune induction.
    Isaura Vanessa Gutierrez, Moonhee Park, Lauren Sar, Ryan E Rodriguez, Hannah M Schmidt, Daltry L Snider, Gabriella Torres, K Matthew Scaglione, Stacy M Horner.
      Post-translational modifications regulate RIG-I signaling in diverse ways. We previously showed that UFMylation, the covalent attachment of the ubiquitin-fold modifier UFM1 to proteins, enhances RIG-I signaling by promoting its interaction with its membrane-targeting adaptor 14-3-3ε. Here, we map UFM1 conjugation to lysines K50 and K215 on 14-3-3ε and demonstrate how these UFMylation events control RIG-I signaling. Using in vitro and cellular UFMylation assays, we reveal that K50R/K215R mutations abolish UFMylation and reduce type I and III interferon induction following RIG-I activation. Unexpectedly, these mutations do not disrupt 14-3-3ε-RIG-I interaction. Instead, they paradoxically enhance RIG-I interaction with MAVS while simultaneously reducing 14-3-3ε-MAVS interaction. These findings establish UFMylation of 14-3-3ε as an important control that shapes MAVS complex architecture to ensure optimal RIG-I signaling and highlights the broader regulatory role of UFMylation in antiviral innate immunity.
    Importance: Post-translational modifications provide regulatory control of antiviral innate immune responses. Our study reveals that UFMylation of 14-3-3ε is required for RIG-I-mediated innate immune signaling. We demonstrate that conjugation of UFM1 to specific lysine residues on 14-3-3ε enhances downstream signaling events that facilitate interferon induction. It does this by stabilizing 14-3-3ε association with the MAVS signaling complex and coordinating productive complex architecture. By identifying the precise sites of UFMylation on 14-3-3ε and their functional consequences, we provide insights into the regulatory layers governing antiviral innate immunity. These findings complement emerging evidence that UFMylation serves as a versatile modulator across diverse immune pathways. Furthermore, our work highlights how protein chaperones like 14-3-3ε can be dynamically modified to orchestrate complex signaling cascades, suggesting potential therapeutic approaches for targeting dysregulated innate immunity.
    DOI:  https://doi.org/10.1101/2025.10.21.683613
  69. bioRxiv. 2025 Oct 08. pii: 2025.10.08.681214. [Epub ahead of print]
    Translation regulation of ATF4 by the termination complex Hbs1-Pelo is required for visual system development and function.
    Katherine Querry, Inês Lago-Baldaia, Narayanan Nampoothiri V P, Christopher Garbark, Abby J Carney, Vilaiwan M Fernandes, Deepika Vasudevan.
      Inherited retinal diseases are a class of genetically heterogenous disorders characterized by mutations in genes required for retinal function, resulting in progressive loss of vision in human patients. One such deletion mutation in the translation termination factor, HBS1L, results in a suite of developmental anomalies in human patients, including progressive vision loss defects. HBS1L, and its interaction partner Pelota (Pelo), are required for recycling stalled ribosomes on mRNAs, but the specific mRNA target causative of vision defects seen with HBS1L deletion remains unknown. Further, the specific cell types in the visual system that require HBS1L for proper development and function are also unknown. Here, we discover that loss of the Drosophila HBS1L homolog, Hbs1 , results in reduced expression of the stress responsive Activating Transcription Factor 4 (ATF4) which is encoded by an mRNA containing multiple upstream open reading frames (uORFs) in its 5' leader. We corroborate these results in cultured human cells, where we find that HBS1L and Pelo promote translation reinitiation at the ATF4 ORF by facilitating proper translation termination at the preceding uORFs. Like human HBS1L deficiency patients, loss of function Drosophila mutants for Hbs1 , pelo , and ATF4 show vision defects as measured by electroretinograms (ERG). Depleting Hbs1 in lamina neurons replicated the ERG defects seen in Hbs1 mutants, suggesting that Hbs1-Pelo is required for proper lamina neuron function. Further confocal analysis of Hbs1 mutants revealed 'vacuolization' defects in the lamina layer, which are indicative of defective synapse formation between lamina neurons and photoreceptors. Strikingly, restoring ATF4 expression in the lamina partially rescued ERG defects in Hbs1 mutants, indicating that ATF4 is likely a relevant mRNA target regulated by Hbs1-Pelo in these cells. Together, our data support a model wherein Hbs1-Pelo mediated translation regulation of ATF4 in lamina neurons underlies the inherited retinal disease caused by HBS1L deletion.
    DOI:  https://doi.org/10.1101/2025.10.08.681214
  70. bioRxiv. 2025 Oct 07. pii: 2025.10.06.680635. [Epub ahead of print]
    Cnpy1 is a candidate endoplasmic reticulum chaperone of Vomeronasal type 2 GPCRs.
    G V S Devakinandan, Nandana Nanda, Abdul Rishad, Syed Dastagir Hussain, Sishir Subedi, Adish Dani.
      Mouse vomeronasal sensory neurons are continuously generated from stem cells and differentiate to express V1R or V2R G-protein coupled receptors (GPCRs), along with their respective Gαi2 or Gαo G-protein subunits. We have previously reported that Gαo-type neurons exhibit elevated expression of endoplasmic reticulum (ER) chaperones and a distinctive hypertrophic, gyroid ER architecture. Here we identify full-length mouse Cnpy1 with its expression and localization exclusive to the ER of Gαo neurons. Cnpy1 deletion resulted in mice that were deficient in Gαo neuronal activation upon exposure to vomeronasal stimuli and a marked reduction in male-male aggressive behavior. In Cnpy1 -/- mice, Gαo neuron develop normally till birth, but undergo selective, progressive apoptosis during postnatal development, despite normal trafficking of V2R GPCRs to dendritic tips. Immunoprecipitation and mass spectrometry revealed that Cnpy1 associates with V2R GPCRs and other ER chaperones. Together, these findings identify Cnpy1 as a component of an ER chaperone complex essential for Gαo neuron signaling and survival.
    DOI:  https://doi.org/10.1101/2025.10.06.680635
  71. Cell. 2025 Nov 21. pii: S0092-8674(25)01242-5. [Epub ahead of print]
    Inhibition of oligomeric BAX by an anti-apoptotic dimer.
    Catherine E Newman, Micah A Gygi, Haleh Alimohamadi, Thomas M DeAngelo, Christina M Camara, Julian Mintseris, Ezra Yu, Edward P Harvey, Zachary J Hauseman, Lixin Fan, Yun-Xing Wang, Elizabeth W-C Luo, Marina Godes, Jacob Gehtman, Ann M Cathcart, Steven P Gygi, John R Engen, Gregory H Bird, Gerard C L Wong, Thomas E Wales, Loren D Walensky.
      BAX is a pro-apoptotic BCL-2 protein that resides in the cytosol as a monomer until triggered by cellular stress to form an oligomer that permeabilizes mitochondria and induces apoptosis. The paradigm for apoptotic blockade involves heterodimeric interactions between pro- and anti-apoptotic monomers. Here, we find that full-length BCL-w forms a distinctive, symmetric dimer (BCL-wD) that dissociates oligomeric BAX (BAXO), inhibits mitochondrial translocation, promotes retrotranslocation, blocks membrane-porating activity, and influences apoptosis induction of cells. Structure-function analyses revealed discrete conformational changes upon BCL-w dimerization and reciprocal structural impacts upon BCL-wD and BAXO interaction. Small-angle X-ray scattering (SAXS) analysis demonstrated that BAXO disrupts membranes by inducing negative Gaussian curvature, which is reversed by positive Gaussian curvature exerted by BCL-wD. Systematic truncation and mutagenesis dissected the core features of BCL-wD activity-dimerization, BAXO engagement, and membrane interaction. Our studies reveal a downstream layer of apoptotic control mediated by protein and membrane interactions of higher-order BCL-2 family multimers.
    Keywords:  BAX; BCL-2 family proteins; BCL-w; anti-apoptotic; apoptosis; cell death; chemical crosslinking mass spectrometry; dimer; hydrogen deuterium exchange mass spectrometry; membrane curvature; mitochondria; mitochondrial retrotranslocation; mitochondrial translocation; oligomer; pro-apoptotic; small-angle X-ray scattering
    DOI:  https://doi.org/10.1016/j.cell.2025.10.037
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