bims-proteo Biomed News
on Proteostasis
Issue of 2026–05–31
fifty-four papers selected by
Eric Chevet, INSERM
  1. Oxidation-Induced C-Terminal Amidation Marks Proteins for Degradation.
  2. A transport-independent role for SLC25A12 in mitochondrial stress signalling.
  3. A multi-subunit autophagic capture complex facilitates degradation of ER-stalled MHC class I in pancreatic cancer.
  4. Molecular glues and PROTACs: Unlocking targeted protein degradation in plant biology.
  5. A multilayered stress-response circuit: The mammalian mitochondrial UPR.
  6. Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets.
  7. The ubiquitin-proteasome system and autophagy as guardians of the cellular proteome.
  8. Prohibitin 2 links damaged mitochondria to intracellular bacteria to trigger xenophagy independent of mitophagy.
  9. Enzyme-Induced Supramolecular Proteolysis-Targeting Chimeras Enable Tumor-Targeted Protein Degradation.
  10. Mammalian Lysophagy: mechanisms and pathophysiological implications.
  11. The molecular basis for nuclear pore destruction by a proximity-inducing molecular glue.
  12. Design principles of a membrane-spanning ubiquitin ligase.
  13. Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97.
  14. Disruption of ER-mitochondria contact sites by coronavirus replication organelles sustains viral replication via NSP3 stabilization.
  15. Mapping the GDF15 arm of the integrated stress response in human cells and tissues.
  16. Metastable Protein-Protein Interactions as a Design Principle for PROTACs: Insights from the RIPK1-VHL System.
  17. p97/VCP as a dual regulator of UPS and autophagy in ovarian cancer: a novel therapeutic target.
  18. Spatiotemporally Resolved Protein Tagging for Dynamic and Tissue-Specific Secretome Profiling In Vivo.
  19. Type II tRNA cleavage by SLFN14 endoribonuclease variants linked to inherited thrombocytopenia drives global translational repression.
  20. Membrane ATG8ylation in secretory autophagy.
  21. FAM134B-mediated ER-phagy degrades APP and suppresses Alzheimer's disease pathology.
  22. Allosteric network of dynamic coupling within BAP1-UCH revealed by methyl NMR.
  23. CHOP promotes the transition to chronic integrated stress response signaling with suppression of hepatocyte identity.
  24. Neuroproteasomes regulate endogenous tau paired helical filament formation in an APOE genotype- and age-dependent manner.
  25. VAPB confers selective neuroprotection by driving autophagic degradation of pathogenic aggregates in ALS.
  26. Design principles of human membrane protein topology.
  27. Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages.
  28. Mechanism of RACK1-dependent ZAKα activation at stalled and collided ribosomes.
  29. USP7 inhibition perturbs proteostasis and tumorigenesis in triple-negative breast cancer.
  30. Neuronal regulation of infection recovery prevents pathogenic stress and tissue damage.
  31. CHCHD2 and CHCHD10 promoted autophagic clearance of protein aggregates via GABARAPs.
  32. Blm10/PA200-Activated 20S Proteasomes Promote α-Synuclein Degradation and Bypass Proteasome Inhibition in Parkinson's Disease Models.
  33. Nuclear Receptor Liver Receptor Homolog-1 drives Intestinal Stem Cell regeneration and UPR/ER stress response after gut injury.
  34. Selective autophagy fine-tunes plant immunity to promote cell survival during viral infection.
  35. Chaperone-mediated autophagy is a tumor-suppressive mechanism in hepatocellular carcinoma.
  36. The C9orf72/SMCR8 complex maintains microglial homeostasis via RAB8A ESCRT-mediated lysosomal repair.
  37. The benchmarking and application of tag-degraders in vivo to validate therapeutic targets.
  38. Uncovering the initial response: Intra-mitochondrial surveillance activates the UPRmt.
  39. Hydrophobic Tag Degraders Overcome Endocrine-Resistant Breast Cancer by Recruiting HSP27-Mediated E3 Ligase Complex for ERα Proteasomal Degradation.
  40. Phenylalanyl-tRNA synthetase FARS-1/FARSA balances longevity and immunity by downregulating endogenous mitochondrial double-stranded RNAs.
  41. Yeast Ribosomal Protein Rpl32 Modulates Ribosome Biogenesis, Ribosome Stability and the Cell Cycle.
  42. Native long-read RNA sequencing of human monocytes reveals activation-induced alternative splicing toward functional isoforms.
  43. Mitochondrial respiration modulates Hsf1 activation and the heat shock response.
  44. RNA-binding protein diversity and NLS arginines regulate FUS mixing in mRNA-rich compartments.
  45. Protocol for the in vivo quantification of mitochondria-associated ER membranes in Caenorhabditis elegans.
  46. Metabolic rewiring driven by phosphoglycolate phosphatase deletion inhibits ferroptosis.
  47. Population-scale chemical response revealed by a barcoded yeast collection.
  48. Navigating high-order protein fitness landscapes via deep learning on directed evolution trajectories.
  49. Fusion-positive rhabdomyosarcoma oncofusions share a common interactome.
  50. Mitochondria-lysosome coupling contributes to lysosome acidification and aging.
  51. Protein Stability, Turnover Kinetics, and Abundance Constrain the Scaling of Protein Interaction Networks.
  52. AI-discovered protein fragments as generalizable regulators of biomolecular condensates.
  53. The revised three-step detour pathway in dolichol biosynthesis is evolutionarily conserved in budding yeast.
  54. Small-molecule modulators of HIPK4 activity and proteostasis.
  1. Bioessays. 2026 Jun;48(6): e70147
    Oxidation-Induced C-Terminal Amidation Marks Proteins for Degradation.
    Ian McLauchlan, Jack White, Mohamed Eldeeb.
      Proteostasis hinges on the precise recognition and elimination of damaged proteins through the ubiquitin-proteasome system (UPS), where E3 ubiquitin ligases recognize degradation signals, or degrons. While degrons have traditionally been viewed as genetically encoded or enzymatically generated motifs, emerging evidence suggests that nonenzymatic chemical damage can also create functional degradation signals. Oxidative stress-induced peptide backbone fragmentation, including hydroxyl radical-mediated cleavage, generates proteins bearing C-terminal amide groups (CTAPs), a chemically derived degron class recognized by the SCF-FBXO31 E3 ligase complex. Genome-wide CRISPR screening identified FBXO31 as a selective CTAP reader required for ubiquitination and proteasomal clearance of amidated substrates. Disruption of this pathway impairs proteostasis and, in disease-associated FBXO31 mutants, promotes aberrant degradation of non-amidated proteins such as SUGT1, contributing to cytotoxicity and neurodevelopmental dysfunction. Collectively, these findings redefine degron biology via establishing chemically induced modified amino acid degrons (MAADs) as mechanistic links between oxidative damage and selective protein degradation.
    Keywords:  C‐degron; C‐terminal amide‐bearing proteins; modified amino acid degrons; oxidative stress; posttranslational modifications; proteasome; protein degradation; proteostasis; ubiquitin‐proteasome system
    DOI:  https://doi.org/10.1002/bies.70147
  2. Nat Cell Biol. 2026 May 27.
    A transport-independent role for SLC25A12 in mitochondrial stress signalling.
    Liu Jiang, Lan Li, Jialiang Guan, Ying Liu.
      Mitochondria are central hubs for energy production and cellular adaptation to stress. When mitochondria are damaged, cells activate protective signalling pathways to restore homeostasis and ensure survival. One such pathway, known as the integrated stress response (ISR), reduces overall protein synthesis while enhancing the production of stress-responsive proteins. The mitochondrial carriers SLC25A12 and SLC25A13 transport similar metabolites but are expressed in different tissues and linked to distinct genetic diseases. Here we show that SLC25A12 plays a previously unrecognized role in stress signalling that is independent of its transport activity. SLC25A12 interacts with the mitochondrial protease OMA1, enabling activation of ISR during mitochondrial damage. This signalling function is disrupted by a disease-linked mutation but preserved in transport-deficient variants. Our findings reveal SLC25A12 as a dual-function mitochondrial protein, acting as both a metabolite transporter and a regulator of stress signalling, and suggest that defective ISR activation may contribute to certain SLC25A12-associated pathologies.
    DOI:  https://doi.org/10.1038/s41556-026-01973-1
  3. Mol Cell. 2026 May 29. pii: S1097-2765(26)00311-4. [Epub ahead of print]
    A multi-subunit autophagic capture complex facilitates degradation of ER-stalled MHC class I in pancreatic cancer.
    Marine Berquez, Alexander L Li, Matthew A Luy, Anthony C Venida, Simon McDowall, Thomas O'Loughlin, Gilles Rademaker, Abhilash Barpanda, Jingjie Hu, Julian Yano, Arun P Wiita, Luke A Gilbert, Peter M Bruno, Rushika M Perera.
      Pancreatic ductal adenocarcinoma (PDAC) evades immune surveillance in part through autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I), though the basis for this vulnerability is unclear. Using synchronized endoplasmic reticulum (ER) exit assays, we show that PDAC cells retain MHC-I in the ER and inefficiently traffic it to the plasma membrane. We identify an autophagic capture complex composed of the ER-phagy receptor TEX264 and the cargo receptor NBR1 that targets MHC-I for degradation. Suppression of either receptor restores total and surface MHC-I levels. Capture is linked to antigen loading, as impaired peptide loading increases MHC-I binding to the TEX264-NBR1 complex, while high-affinity peptides reduce binding and promote increased surface localization. A genome-wide CRISPRi screen identified the ER-localized E3 ligase NFXL1 as a mediator of MHC-I ubiquitylation and capture. Elevated NFXL1 correlates with reduced MHC-I expression and poor prognosis, highlighting a targetable pathway regulating PDAC immunogenicity.
    Keywords:  ER-phagy; MHC-I; autophagy; lysosome; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.molcel.2026.05.005
  4. J Exp Bot. 2026 May 27. pii: erag256. [Epub ahead of print]
    Molecular glues and PROTACs: Unlocking targeted protein degradation in plant biology.
    Carla Donderis-Fagoaga, Lidia Orea-Ordóñez, Jorge Lozano-Juste.
      Targeted protein degradation (TPD) has emerged as a chemical strategy to modulate proteostasis, offering important advantages over traditional small molecules. By inducing proximity between a protein of interest (POI) and the cellular degradation machinery, protein degraders enable selective and dynamic degradation of the POI. Unlike classical small molecules (i.e. inhibitors), the event-driven mode of action of chemical degraders offers new therapeutic opportunities for previously intractable diseases. Molecular glues (MGs) and proteolysis-targeting chimeras (PROTACs) can target proteins previously considered undruggable, including those lacking catalytic activity or deep binding pockets, such as transcription factors and scaffold proteins. TPD has gained substantial attention in drug discovery, with several candidates advancing in clinical trials, validating chemically induced proximity as therapeutic strategy. However, in plants, the development of synthetic degraders remains largely unexplored. Here, we review the molecular basis of TPD, with emphasis on MGs and PROTACs. We discuss critical aspects of PROTAC design, including E3 ligase suitability, target selection, and linker optimization. We also summarize engineered tag-based systems and emerging non-proteasomal modalities. Finally, we provide a critical evaluation of the opportunities and limitations of protein degraders and provide a theoretical and practical framework to facilitate the expansion of TPD in plant biology and agriculture.
    Keywords:  Cereblon; E3 ligase; HaloTag; Molecular glue; PROTAC; Targeted Protein Degradation; Von Hippel-Lindau; dTAG; proteasome; small molecule; ternary complex; ubiquitin
    DOI:  https://doi.org/10.1093/jxb/erag256
  5. FEBS J. 2026 May 29.
    A multilayered stress-response circuit: The mammalian mitochondrial UPR.
    Paulina Czechowicz, Anna Więch-Walów, Sylwia Kozioł, Jakub Dudzik, James F Collawn, Rafal Bartoszewski.
      Mitochondrial proteotoxic stress activates the mammalian UPRmt through a multilayered mechanistic architecture rather than a linear pathway. At its core lies an import-gated sensing logic: reduced preprotein import and mito-nuclear stoichiometric imbalance activates the integrated stress response (ISR) toward the translation of ATF4, CHOP, and the mitochondria-targeted transcription factor ATF5. These factors cooperatively reprogram transcription to expand the chaperone-protease capacity while transiently reducing the nuclear-encoded OXPHOS load. Parallel translational mechanisms that include eIF2α-dependent repression, stress-granule triage, and miRNA-driven selective silencing reduce the mitochondrial precursor import and maintain proteostatic symmetry between the cytosol and mitochondria. Within the organelle, LONP1- and CLPP-dependent proteolysis, mitoribosome pausing, and tRNA-processing checkpoints further dampen nascent chain pressure. Epigenetic licensing by demethylases and acetyltransferases links metabolic and bioenergetic status to promoter accessibility at UPRmt loci. Together, these import-gated, translational, and epigenetic control layers form a coherent mechanistic circuit ensuring that mitochondrial recovery is matched to folding, assembly, and metabolic capacity. We propose a unified framework explaining how these layers cooperate to determine adaptive versus maladaptive outcomes.
    Keywords:  Integrated stress response (ISR); Mitochondrial protein import stress; Mitochondrial proteostasis; Mitochondrial stress signaling; Mitochondrial unfolded protein response (UPRmt)
    DOI:  https://doi.org/10.1111/febs.70607
  6. Nat Cell Biol. 2026 May 26.
    Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets.
    Arda Mizrak, Jacob Kæstel-Hansen, Jessica Matthias, J Wade Harper, Nikos S Hatzakis, Robert V Farese, Tobias C Walther.
      Numerous metabolic enzymes translocate from the endoplasmic reticulum (ER) membrane bilayer to the lipid droplet (LD) monolayer, where they perform essential functions. Mislocalization of certain LD-targeted membrane proteins, including HSD17B13 and PNPLA3, is implicated in metabolic dysfunction-associated steatotic liver disease. However, the mechanisms governing the trafficking and accumulation of ER proteins on LDs remain poorly understood. Here using minimal fluorescence photon fluxes nanoscopy and highly inclined and laminated optical single-molecule tracking combined with machine learning, we show that HSD17B13, GPAT4 and the model cargo 'LiveDrop' diffuse at comparable speeds in the ER and on LDs, but become nano-confined upon reaching the LD surface. Mechanistic dissection of LiveDrop targeting revealed that this confinement, along with protein accumulation on LDs, depends on specific residues within its targeting motif. These residues mediate preferential interactions with nanoscale membrane domains, suggesting that LD-targeted proteins selectively partition into distinct lipid-protein environments that transiently alter local motion and concentrate them at the LD surface. Single-molecule trajectories further revealed bidirectional trafficking of LiveDrop across seipin-containing ER-LD bridges, providing direct evidence for lateral protein transfer across membrane contact sites. These findings establish nanodomain-based confinement as a key mechanism driving selective protein accumulation on LDs and reveal how membrane bridges between organelles facilitate protein sorting.
    DOI:  https://doi.org/10.1038/s41556-026-01963-3
  7. FEBS Lett. 2026 May 28.
    The ubiquitin-proteasome system and autophagy as guardians of the cellular proteome.
    Ivan Dikic.
      Maintaining a functional proteome is essential for cellular health and organismal longevity. Disruption of proteostasis is a hallmark of aging and a central driver of diverse pathologies, including neurodegeneration, cancer, and metabolic disease. The ubiquitin-proteasome system (UPS) and autophagy represent the two principal degradative pathways safeguarding proteome integrity, particularly under conditions of stress. While historically viewed as mechanistically distinct, it is now clear that UPS and autophagy operate as an interconnected and adaptive network. This Perspective discusses three core principles that govern their coordination: (1) a shared molecular language of ubiquitin signals and shuttle proteins that determines cargo routing; (2) spatial compartmentalization through organelle-specific quality control modules and phase-separated degradation hubs; and (3) temporal regulation by stress-responsive signaling pathways that reprogram proteolytic output. Understanding this dynamic partnership not only reveals fundamental organizing principles of cellular homeostasis but also identifies new therapeutic nodes for diseases driven by proteostasis collapse.
    Keywords:  ER quality control; autophagy; phase separation; proteasome; proteostasis; ubiquitin
    DOI:  https://doi.org/10.1002/1873-3468.70371
  8. Cell Rep. 2026 May 28. pii: S2211-1247(26)00541-3. [Epub ahead of print]45(6): 117463
    Prohibitin 2 links damaged mitochondria to intracellular bacteria to trigger xenophagy independent of mitophagy.
    Jing Yang, Yitian Ying, Huiwen Ye, Meiyi Chen, Xia Liu, Ganlu Wei, Jiayue She, Xinyu Lin, Muyang Wan, Xun Tan.
      Mitophagy and xenophagy, two selective autophagy pathways sharing common E3 ligases, have been proposed to intersect in host defense against invading pathogens. Here, we show that mitochondrial damage, but not mitophagy, is essential for triggering xenophagy via the inner mitochondrial membrane protein prohibitin 2 (PHB2). Upon bacteria-induced disruption of the outer mitochondrial membrane, PHB2 bridges mitochondria to bacteria by binding bacterial surface proteins, while concurrently interacting with either auto-ubiquitinated E3 ligase ARIH1 or Parkin, two well-characterized mitophagy-associated E3 ligases. This interaction positions polyubiquitin chains near PHB2-targeted bacteria to recruit selective autophagy receptors for initiating xenophagy, leading to the co-autophagic degradation of bacteria and mitochondria, a process unaffected by mitophagy inhibition. Our findings establish an uncovered mechanism of mitochondria-dependent antibacterial autophagy, positioning mitochondrial PHB2 as both a bacterial sensor and an E3 ligase scaffold, and unveiling a previously unidentified process governing the recruitment of mitophagy-associated E3 ligases to intracellular bacteria.
    Keywords:  ARIH1; CP: cell biology; CP: molecular biology; Listeria; PHB2; Salmonella; Staphylococcus aureus; mitochondria; mitophagy; parkin; ubiquitin; xenophagy
    DOI:  https://doi.org/10.1016/j.celrep.2026.117463
  9. JACS Au. 2026 May 25. 6(5): 2912-2923
    Enzyme-Induced Supramolecular Proteolysis-Targeting Chimeras Enable Tumor-Targeted Protein Degradation.
    Chunjing Liang, Xiaoqing Ma, Xiaxue Chen, Cunxun Dai, Xiaodi Sun, Jie Li, Ruyun Dong, Peng Dong, Ming Wang.
      Targeted protein degradation (TPD) using proteolysis-targeting chimeras (PROTACs) offers a promising approach to modulating intracellular protein homeostasis and eliminating disease-associated proteins. However, conventional PROTACs often exhibit limited control over tissue and spatial selectivity for in vivo protein degradation as well as the hook effect, thereby compromising their therapeutic potency. Herein, we report enzyme-induced supramolecular PROTACs (eiSupTACs) that harness tumor-specific biochemistry and bioorthogonal chemistry to assemble multivalent degraders for tumor-targeted protein degradation. eiSupTACs are constructed from peptide- and protein-targeting ligand conjugates functionalized with cysteine and 2-cyanobenzothiazole motifs, which undergo bioorthogonal assembly into supramolecular nanoparticles upon activation by tumor-overexpressed cathepsin B and elevated intracellular glutathione. eiSupTACs present multiple recruiting ligands for both E3 ligases and protein targets, facilitating proteasome-mediated degradation selectively within cancer cells. We demonstrate potent and selective degradation of bromodomain protein 4 and glutathione peroxidase 4 (GPX4) in multiple cancer cell lines, with minimal activity in nonmalignant cells. Notably, the degradation of GPX4 via eiSupTACs induces ferroptosis and suppresses tumor growth in a murine xenograft model. This study establishes a modular, tumor-selective TPD platform, providing a generalizable strategy for spatiotemporally resolved protein homeostasis modulation for targeted therapy.
    Keywords:  SupTAC; ferroptosis; in situ assembly; targeted protein degradation; tumor
    DOI:  https://doi.org/10.1021/jacsau.6c00250
  10. Autophagy. 2026 May 25.
    Mammalian Lysophagy: mechanisms and pathophysiological implications.
    Fujian Ji, Mingran Dai, Zimu Wang, Enyong Dai, Rui Kang, Daniel J Klionsky, Daolin Tang, Yan Huang, Yu Sun, Liu Tong.
      Lysophagy is a form of selective macroautophagy/autophagy that preserves lysosomal integrity by eliminating damaged lysosomes. Lysosomal membrane permeabilization can arise from diverse physiological and pathological insults, including proteotoxic stress, crystalline particles, pathogens and chemical perturbations, and occurs along a continuum ranging from transient nanoscale lesions to catastrophic rupture. Cells respond to lysosomal injury through a hierarchical quality-control network in which membrane repair, lysophagic removal and lysosomal regeneration operate in a coordinated manner. Damage recognition involves sensing of exposed lumenal glycans and membrane lipids, followed by ubiquitin-dependent tagging that recruits selective autophagy receptors and activates the core autophagy machinery to form lysophagosomes. Lysophagy is closely integrated with membrane repair pathways, metabolic signaling and innate immune responses that together determine lysosomal fate. Dysregulated lysosomal quality control has been implicated in diverse diseases, including neurodegeneration, infection, cancer and chronic inflammatory disorders. In this review, we summarize current mechanistic insights and emerging experimental approaches for studying lysosomal quality control and lysophagy in mammalian cells.
    Keywords:  Autophagy receptor; disease; lysophagy; lysosomes; repair
    DOI:  https://doi.org/10.1080/15548627.2026.2679642
  11. Cell Chem Biol. 2026 May 27. pii: S2451-9456(26)00151-0. [Epub ahead of print]
    The molecular basis for nuclear pore destruction by a proximity-inducing molecular glue.
    Stephen M Hinshaw, Linjie Yuan, Md Abdullah Al Noman, Michael J Martinez, Gianna M Colombo, Brendan G Dwyer, Wenzhi Ji, Bryan A Romero, Ines Forrest, Joey Lu, Jing Bian, Trae B Dunn, Ethan R Garvin, Daniel Fernandez, Tinghu Zhang, Nathanael S Gray, Steven M Corsello.
      Molecular glues that induce new protein interactions can be potent therapeutics. We and others recently discovered that the small molecule PRLX-93936 (PRLX), which was originally developed as an erastin derivative with antitumor activity, is a molecular glue that alters the substrate specificity of the TRIM21 ubiquitin ligase. PRLX causes TRIM21 to bind the nuclear pore protein NUP98, triggering nuclear pore complex (NPC) degradation. We present here the structural and biochemical basis of NUP98 recognition, finding that ternary complex assembly depends on the creation of a composite TRIM21-small molecule surface competent for NUP98 binding. A scarcity of direct small molecule-NUP98 contacts likely explains how multiple structurally diverse TRIM21 ligands can induce NPC degradation. We also report the discovery of an enhanced molecular glue, MAN-021, and describe its structure. Our findings provide a basis for rational development of next-generation small molecules with enhanced or differentiated activities.
    Keywords:  E3 ligase; NUP98; TRIM21; crystal structure; drug discovery; molecular glue; ternary complex
    DOI:  https://doi.org/10.1016/j.chembiol.2026.04.016
  12. Mol Cell. 2026 May 26. pii: S1097-2765(26)00307-2. [Epub ahead of print]
    Design principles of a membrane-spanning ubiquitin ligase.
    Carys Williams, Laura M Nocka, George Hedger, Pragya Parashara, Els Pardon, Naomi R Latorraca, Ganesh V Pusapati, Parijat Sarkar, Dorothy Lartey, Lei Gao, Ljiljana Milenkovic, Rod Chalk, Jan Steyaert, Susan Marqusee, Loïc Carrique, J Fernando Bazan, Sarah L Rouse, Jennifer H Kong, Christian Siebold, Rajat Rohatgi.
      Receptor-type E3 ubiquitin ligases enable extracellular signals to control ubiquitylation in the cytoplasm, playing widespread roles in development, metabolism, and immunity. Using cryoelectron microscopy, integrated with biophysical and functional studies, we visualized a human E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex attenuates Hedgehog signaling by ubiquitylating Smoothened (SMO), a G-protein-coupled receptor (GPCR) that transduces morphogen signals. A long helix in the MMM complex engages SMO using an intramembrane degron and extends into the cytoplasm to suspend an activated and precisely oriented RING domain below the plasma membrane. This architecture enables ubiquitylation of the cytoplasmic surface of SMO, reducing SMO abundance at primary cilia. Our structure provides insights into MEGF8 mutations, which cause multi-organ birth defects, and defines a paradigm for how transmembrane E3 ligases control the cell surface abundance of GPCRs and other signaling receptors.
    Keywords:  E3 ligase; GPCR; Hedgehog signaling; Smoothened; birth defects; cryoelectron microscopy; morphogens; primary cilia; transmembrane receptor; ubiquitylation
    DOI:  https://doi.org/10.1016/j.molcel.2026.05.001
  13. Res Sq. 2026 May 13. pii: rs.3.rs-9588629. [Epub ahead of print]
    Npl4 decodes polyubiquitin length and gates D1-D2 coupling in human VCP/p97.
    Nils Walter, Laxmikanta Khamari, Jingxuan Tang, Stephani Moon.
      VCP/p97 binds the Npl4 - Ufd1 heterodimer adaptor to extract polyubiquitinated substrates for proteasomal degradation, but how it decodes K48-linked chain length and how D1-coupled events license downstream D2 power strokes remain unclear. Here we introduce smUbiRAD, or single-molecule ubiquitin recognition and dynamics, and identify a sharp chain-length threshold: Npl4 binds transiently to short chains but switches to long-lived, multivalent engagement on tetra- and penta-ubiquitin. Ufd1 and p97 further stabilize these complexes mainly by suppressing Npl4 dissociation without affecting initial encounter. In fully assembled p97-Ufd1-Npl4-substrate complexes, D1 ATP hydrolysis-rather than D2-drives rapid Npl4 exchange. These results support a model in which D1-powered conformational changes promote cofactor Npl4, but not Ufd1, turnover and gate iterative coupling to downstream D2-driven substrate processing. Finally, we show that multisystem proteinopathy variants R155H and A232E bias p97 toward a high-affinity resting state and accelerate Npl4 exchange, implicating hyperactive cofactor cycling as a disease-linked dysregulation.
    DOI:  https://doi.org/10.21203/rs.3.rs-9588629/v1
  14. EMBO J. 2026 May 28.
    Disruption of ER-mitochondria contact sites by coronavirus replication organelles sustains viral replication via NSP3 stabilization.
    Xinyue Zheng, Liqiao Hu, Xubing Long, Zhilei Zhao, Rongrong Chen, Rong Bai, Buyun Tian, Maoge Zhou, Binbin Ding, Tao Xu, Zonghong Li.
      Coronaviruses establish infection by reorganizing the host endoplasmic reticulum (ER) to form double-membrane vesicles (DMVs), which function as viral replication platforms. However, the role of other cellular organelles in this process remains incompletely understood. Here, we uncover a self-reinforcing cycle between viral replication organelles and mitochondrial damage that sustains coronavirus replication. We show that DMV formation disrupts ER-mitochondria contact sites (ERMCs), causing mitochondrial damage. This injury initiates a feed-forward mechanism wherein mitochondria release the matrix enzyme ECHS1 into the cytosol. Cytosolic ECHS1 then binds and stabilizes the DMV inducer protein NSP3 by blocking its K963 ubiquitination via the host E3 ligase RBBP6, thereby promoting further DMV formation. Disrupting this cycle, either through enhanced ER-mitochondria tethering or targeted interference with ECHS1-NSP3 binding, effectively suppresses viral replication. Our findings reveal that coronaviruses exploit an inter-organellar feedback loop linking mitochondrial damage to DMV formation, identifying new potential therapeutic targets for inhibition of coronaviral replication.
    DOI:  https://doi.org/10.1038/s44318-026-00816-x
  15. Commun Biol. 2026 May 27.
    Mapping the GDF15 arm of the integrated stress response in human cells and tissues.
    Janell L M Smith, Kamaryn T Tanner, Jack Devine, Anna S Monzel, Taivan Batjargal, Maxwell Z Wilson, Alan A Cohen, Martin Picard.
      Mitochondrial stress activates the integrated stress response (ISR) and triggers cell-cell communication through the secretion of the metabokine growth differentiation factor 15 (GDF15). However, the gene network underlying the ISR remains poorly defined across metabolically diverse cellular states and tissues. Using RNAseq data from fibroblasts subjected to eleven metabolic perturbations, including genetic and pharmacological mitochondrial OxPhos defects, we show that the ISR has multiple arms. To quantify the GDF15 arm of ISR activation in human cells, we developed an ISRGDF15 index. We validate the ISRGDF15 index in datasets from optogenetic and small molecule activation of ISR kinases, demonstrating its rapid kinetics preceding to GDF15 gene expression. We then deploy the ISRGDF15 index across 44 postmortem human tissues, confirm its correlation with age, and report that the ISRGDF15 is upregulated in the heart of individuals with acute causes of death in the emergency room, whereas it was upregulated in the brain of individuals who died after protracted hospital inpatient stays. These data highlight distinct arms of the ISR and clarify genes related to the GDF15 ISR arm, yielding an ISRGDF15 index that can be used to investigate tissue-specific and age-related ISR activation in both in vitro cultures and human tissues.
    DOI:  https://doi.org/10.1038/s42003-026-10312-x
  16. JACS Au. 2026 May 25. 6(5): 2935-2948
    Metastable Protein-Protein Interactions as a Design Principle for PROTACs: Insights from the RIPK1-VHL System.
    Yue Wu, Zhen Zhang, Nina J Hawkins, Weiping Tang, Xuhui Huang.
      Proteolysis-Targeting Chimeras (PROTACs) eliminate disease-relevant proteins by stabilizing short-lived, metastable protein-protein interactions (PPIs) between a target protein and an E3 ligase. These transient encounter complexes are central to PROTAC activity, yet they are difficult to predict and are often invisible to structure-based or AI-only modeling approaches that favor stable, native interactions. Here, we show that explicitly accounting for metastable PPIs provides an effective design principle for rational PROTAC development. Using large-scale molecular dynamics (MD) simulations combined with integrative generalized master equation (IGME) modeling, we mapped the ensemble of metastable PPIs between receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the von Hippel-Lindau (VHL) E3 ligase, and identified four distinct PPIs suitable for degrader engagement. We find that different PPIs preferentially accommodate different linker architectures, providing a mechanistic explanation for the counterintuitive experimental observation that PROTACs with very short and very long linkers can consistently yield potent degradation. Guided by this insight, we established a virtual screening workflow for linker design and identified the benzylic position on the VHL ligand as a viable linkage site. Rationally designed novel PROTACs featuring this site achieve complete degradation with subnanomolar to low-nanomolar potency. Our results demonstrate that incorporating protein dynamics into degrader design substantially expands the accessible PROTAC design space.
    Keywords:  Markov state model; PROTAC; RIPK1; rational drug design; target protein degradation
    DOI:  https://doi.org/10.1021/jacsau.6c00260
  17. J Mol Histol. 2026 May 25. pii: 178. [Epub ahead of print]57(3):
    p97/VCP as a dual regulator of UPS and autophagy in ovarian cancer: a novel therapeutic target.
    Ilkay Corumluoglu, Ebru Alimogullari.
      Ovarian cancer is among the most lethal gynecologic malignancies worldwide, with high mortality rates largely attributable to late diagnosis and the development of therapeutic resistance. Cellular proteostasis in cancer cells is tightly regulated by the ubiquitin-proteasome system (UPS) and autophagy, two major protein quality control pathways. Key proteins involved in these pathways, including p97/valosin-containing protein (p97/VCP), ubiquitin (Ub), p62/SQSTM1, and LC3B, have been implicated in cancer progression; however, their coordinated regulation in ovarian cancer remains incompletely understood. In this study, we investigated the expression and regulation of UPS- and autophagy-related proteins in the human ovarian cancer cell line MDAH-2774 under pharmacological modulation of autophagy. Cells were treated with rapamycin (10 µM), an autophagy inducer, or chloroquine (50 µM), an autophagy flux inhibitor, for defined incubation periods. Protein expression levels and cellular localization of p97/VCP, Ub, p62, and LC3-II were analyzed using Western blotting, immunofluorescence staining, and siRNA-mediated p97/VCP silencing. Our results demonstrated that p97/VCP and ubiquitin were expressed at relatively high levels in ovarian cancer cells, whereas autophagy markers p62 and LC3B showed reduced basal expression, suggesting dysregulated autophagic activity. Rapamycin treatment markedly increased p97/VCP expression, supporting its involvement in autophagy induction. In contrast, chloroquine treatment significantly reduced p97/VCP levels while inducing a pronounced accumulation of Ub, p62, and LC3-II (p < 0.05), consistent with impaired autophagic flux and disrupted proteasomal degradation. Furthermore, siRNA-mediated suppression of p97/VCP significantly decreased the expression of p97/VCP, LC3-II, ubiquitin, and the autophagy-modulating responses to rapamycin and chloroquine compared with control siRNA (p < 0.05). p97/VCP suppression was associated with alterations in endoplasmic reticulum-associated degradation (ERAD) and a concomitant reduction in unfolded protein response (UPR)-related protein levels, indicating increased cellular stress. Collectively, these findings highlight p97/VCP as a central regulator of proteostasis in ovarian cancer through its coordinated roles in both the UPS and autophagy pathways. Dysregulation or pharmacological inhibition of p97/VCP may exacerbate proteotoxic stress by disrupting the balance between autophagy and UPR signaling, potentially contributing to tumor progression and therapy resistance. Further mechanistic studies are warranted to elucidate the underlying signaling networks and to evaluate p97/VCP-mediated proteostasis pathways as potential therapeutic targets in ovarian cancer.
    Keywords:  Autophagy; Chloroquine; Ovarian cancer; Proteasome; Proteostasis; Rapamycin; p97/VCP
    DOI:  https://doi.org/10.1007/s10735-026-10838-8
  18. Angew Chem Int Ed Engl. 2026 May 26. e5498199
    Spatiotemporally Resolved Protein Tagging for Dynamic and Tissue-Specific Secretome Profiling In Vivo.
    Qizhen Zheng, Rui Yao, Tianyu Ma, Lijuan Li, Ying Jiang, Ming Wang.
      The dynamic and tissue-specific nature of protein secretion underlies a wide range of physiological and pathological processes, yet tools for profiling the in vivo secretome with high spatial and temporal resolution remain limited. Here, we present STePTag (Spatio-Temporal Protein Tagging), a conditional proximity labeling (PL) system for profiling secreted proteins in live animals. STePTag integrates the rapid labeling kinetics of the PL enzyme TurboID with a destabilized dihydrofolate reductase (DHFR) domain, enabling tight post-translational control of labeling activity through the small-molecule stabilizer trimethoprim (TMP). Targeting STePTag to the endoplasmic reticulum (ER) confines labeling to the secretory pathway and enables robust labeling within 10 min of TMP administration. Furthermore, we design tissue-specific lipid nanoparticles (tsLNPs) to enable programmable, in vivo delivery of STePTag to the mouse liver. Using this approach, we identified 93 liver-derived secretory proteins under physiological conditions and uncovered 40 dynamically regulated proteins in a model of acetaminophen-induced acute liver injury (ALI), including Aldh1a1, which we functionally validated as a protective factor in ALI. Together, STePTag provides a versatile platform for spatiotemporally resolved secretome profiling, enabling the discovery of context-dependent biomarkers and tissue-derived signaling molecules in native physiological environments.
    Keywords:  Proximity Labeling; Secretome; Spatiotemporal; Tissue‐specific lipid nanoparticles; TurboID
    DOI:  https://doi.org/10.1002/anie.5498199
  19. PLoS Biol. 2026 May 29. 24(5): e3003830
    Type II tRNA cleavage by SLFN14 endoribonuclease variants linked to inherited thrombocytopenia drives global translational repression.
    Chengchao Ding, Xinyi Ashley Liu, Fushun Zhang, Saori Uematsu, Shu-Bing Qian, Yan Xiang.
      Schlafens proteins (SLFNs) are interferon-inducible regulators of RNA metabolism that influence antiviral defense and cell fate. Human SLFN14 is a ribosome-associated endoribonuclease whose pathogenic variants cause autosomal dominant inherited thrombocytopenia (IT), but the molecular basis of this disorder remains unclear. Here, using HEK293T cells expressing human SLFN14 variants, we show that SLFN14 represses global protein synthesis through selective cleavage of type II tRNAs. IT-linked mutations alter SLFN14 RNA substrate specificity, enhancing depletion of type II tRNAs while reducing rRNA cleavage. This shift promotes ribosome stalling at codons decoded by type II tRNAs, triggering global translational arrest, stress signaling, and cell death. These findings reveal how altered RNA targeting by SLFN14 can drive disease and highlight selective tRNA targeting as a mechanism than regulates translation and cell fate.
    DOI:  https://doi.org/10.1371/journal.pbio.3003830
  20. Autophagy. 2026 May 24. 1-16
    Membrane ATG8ylation in secretory autophagy.
    Jayanta Debnath, Andrew M Leidal.
      Mammalian Atg8-family (ATG8) proteins are crucial for macroautophagic/autophagic degradation in the lysosome and facilitate non-degradative processes including multiple distinct forms of unconventional protein secretion. These secretion pathways, collectively termed secretory autophagy, depend upon ATG8 conjugated to membranes to both specify and traffic molecules for extracellular release. Here, we review the current understanding of how membrane ATG8ylation supports secretory autophagy, and propose a cell biological framework for classifying the growing repertoire of secretory autophagy pathways based on membrane ATG8ylation at discrete intracellular vesicular intermediates. Finally, we detail the emerging roles of these pathways in physiology and disease.Abbreviations: Aβ, amyloid-β; Acb1, acyl-coA-binding 1; ALS, amyotrophic lateral sclerosis; APP, amyloid beta precursor protein; APEX2, ascorbate peroxidase; ATG, autophagy related; AWOL, autophagosome-mediated exit without lysis; BafA1, bafilomycin A1; BirA*, mutant BirA biotin ligase; BMI, body-mass index; CASM, ATG8 conjugation at single membranes; DAMPs, danger/damage-associated molecular patterns; DBI, diazepam binding inhibitor, acyl-CoA binding protein; DSS, dextran sodium sulfate; ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum intermediate compartment; ESCRT, endosomal complexes required for transport; EVs, extracellular vesicles; EVPs, extracellular vesicles and particles; HMGB1, high mobility group box 1; IDE, insulin degrading enzyme; IFNB, interferon beta; ILV, intralumenal vesicles; LANDO, LC3-associated endocytosis; LAP, LC3-associated phagocytosis; LIR, LC3 interacting region; LDELS, LC3-dependent EV loading and secretion; LLOMe, L-leucyl-L-leucine methyl ester hydrobromide; M2, influenza A virus matrix 2, MAD, migratory autolysosome disposal; miRNAs, microRNAs; M-MDSC, monocytic myeloid derived suppressor cells; MVEs, multivesicular endosomes; PAMPs, pathogen-associated molecular patterns; P-bodies, processing bodies; PE, phosphatidylethanolamine; PD, Parkinson disease; PS, phosphatidylserine; RBPs, RNA binding proteins; R-EV, RAB22A-induced extracellular vesicle; SLC2A1, solute carrier family 2 member 1; TFRC, transferrin receptor; TGN, trans-Golgi network; TMED10, transmembrane p24 trafficking protein 10; THU, TMED10-channeled unconventional secretion; SALI, secretory autophagy during lysosome inhibition; SCF, SKP1-CUL1-F-box; SNAREs, soluble NSF attachment protein receptors.
    Keywords:  ATG8ylation; extracellular vesicles and particles; noncanonical autophagy; secretory autophagy; unconventional protein secretion
    DOI:  https://doi.org/10.1080/15548627.2026.2676796
  21. EMBO J. 2026 May 26.
    FAM134B-mediated ER-phagy degrades APP and suppresses Alzheimer's disease pathology.
    Yuting Zhang, Jun Sun, Yang Cai, Ziyan Xu, Xiang Li, Wei Wei, Pai Liu, Qiming Sun, Zhi-Hao Wang, Yixian Cui.
      Endoplasmic reticulum autophagy (ER-phagy) is a selective autophagy pathway in which receptor proteins target ER membranes and proteins for degradation, yet its role in Alzheimer's disease (AD) remains unclear. Here, we identify FAM134B/RETREG1 as a specific ER-phagy receptor mediating amyloid precursor protein (APP) degradation. FAM134B directly interacts with ER-localized wild-type and familial mutant APP via their C-terminal domains and recruits LC3 through its LC3-interacting region (LIR) to promote APP delivery to phagophores for lysosomal degradation. In AD, epigenetic silencing at the FAM134B promoter suppresses its transcription by limiting TFEB/TFE3 binding despite their nuclear enrichment. This transcriptional suppression impairs ER-phagy, leading to APP accumulation and exacerbated AD pathology. AAV-mediated hippocampal expression of wild-type, but not LIR-mutant, FAM134B in 5XFAD mice restores ER-phagy, enhances APP clearance, reduces Aβ deposition, preserves synaptic and myelin integrity, and improves cognitive performance. These findings establish FAM134B downregulation as an upstream pathogenic event in AD, suggesting ER-phagy enhancement as a promising strategy to suppress Aβ generation at its source.
    DOI:  https://doi.org/10.1038/s44318-026-00818-9
  22. Nat Commun. 2026 May 26.
    Allosteric network of dynamic coupling within BAP1-UCH revealed by methyl NMR.
    Chih-Hsuan Lai, Yuan-Chao Lou, Chi-Fon Chang, Wei-Lin Lu, Manoj Kumar Sriramoju, Yong-Sheng Wang, Kuen-Phon Wu, Carlo Camilloni, Shang-Te Danny Hsu.
      BRCA1-associated protein 1 (BAP1) is a tumor suppressor whose deubiquitinase (DUB) activity is essential for transcriptional regulation and is frequently compromised in cancer by mutations clustered in its ubiquitin carboxyl-terminal hydrolase (UCH) domain. The structural and dynamic bases of these oncogenic mutations remain elusive. Here, we introduce 22 cancer mutations and additional methyl mutations on the other 22 cancer mutation sites of BAP1-UCH to map their effects on the methyl chemical shifts. This analysis reveals an allosteric coupling network centered on a conserved leucine (L49) shared across human UCH paralogs. Strikingly, a single-carbon side-chain truncation in the L49V variant abolishes DUB activity, coinciding with disruption of correlated μs-ms timescale motions within a phenylalanine cluster that undergoes concerted motions within the L49 hub. These findings uncover how BAP1-UCH sustains its catalytic competence through a delicately tuned dynamic network and how minute alterations can collapse this allosteric balance, providing a mechanistic link between subtle structural perturbations and oncogenesis.
    DOI:  https://doi.org/10.1038/s41467-026-73600-4
  23. bioRxiv. 2026 May 15. pii: 2026.05.13.724984. [Epub ahead of print]
    CHOP promotes the transition to chronic integrated stress response signaling with suppression of hepatocyte identity.
    Theo F Velarde, Kaihua Liu, Zewei Zhang, Reed C Adajar, Chaoxian Zhao, Huojun Cao, D Thomas Rutkowski.
      The transcription factor CHOP promotes cell death during ER stress, but it is strongly induced even by moderate stresses that do not result in appreciable cell death. Its role during less severe stresses-especially in intact tissues in vivo -is poorly understood. Here, we both deleted and restored CHOP specifically in hepatocytes and challenged animals with ER stress in vivo . We found that CHOP influenced stress-dependent hepatocyte gene expression through two previously unappreciated mechanisms. It directly suppressed the expression of transcriptional master regulators of hepatocyte identity and metabolism. And more broadly, it exacerbated ER stress through the promotion of protein synthesis, which led to persistent activation of the integrated stress response (ISR) despite dephosphorylation of eIF2α. This shift to second-phase ISR signaling was phenocopied by deletion of the protective UPR sensor ATF6α, suggesting that it reflects a transition from an acute stress response to a chronic one. Our findings show that CHOP augments the capacity of the ISR and UPR to continue to mount a protective response even after eIF2α phosphorylation has been suppressed. In vivo , where ISR signaling intersects with hepatocyte gene regulatory networks, this transition favors lipid dysregulation, highlighting a pathway through which CHOP impacts tissue function independent of cell death.
    DOI:  https://doi.org/10.64898/2026.05.13.724984
  24. Nat Neurosci. 2026 May 29.
    Neuroproteasomes regulate endogenous tau paired helical filament formation in an APOE genotype- and age-dependent manner.
    Victoria Paradise, Kalin D Konrad-Vicario, Chi Nguyen, Nyle A Sharif, Xiao Wang, Rijuta D Mukim, Malavika Sabu, Bianca T Corjuc, Joanna Bafia, Jack Fu, Gabriella C Maldonado, Michael Strickland, Sarah L Grauman, Helen Figueroa, Bradley T Hyman, David M Holtzman, Tal Nuriel, Kapil V Ramachandran.
      In Alzheimer's disease (AD), endogenous tau undergoes a pathogenic transition to form paired helical filaments (PHFs), but the cellular mechanisms driving this process have been elusive. Here, we identify the neuron-specific plasma membrane proteasome ('neuroproteasome') as a critical determinant of tau proteostasis. Selective inhibition of neuroproteasome function rapidly triggers the de novo formation of endogenous, sarkosyl-insoluble tau PHFs in primary neurons and mouse brain, which share key biochemical and ultrastructural features with PHFs from human AD brains. The APOE gene has three isoforms (E2, E3 and E4), with APOE4 being the largest genetic risk factor for AD. Neuroproteasome abundance at the plasma membrane is differentially modulated by ApoE isoforms (E2 > E3 > E4) and declines with age. ApoE4 neurons accumulate tau aggregates following modest neuroproteasome disruption, whereas ApoE2 neurons remain resistant. Our findings delineate a neuron-specific mechanism linking genetic and age-related risk factors to the formation of AD-relevant tau pathology, and position neuroproteasome function as a potential target to preserve proteostasis.
    DOI:  https://doi.org/10.1038/s41593-026-02297-x
  25. Acta Neuropathol Commun. 2026 May 29.
    VAPB confers selective neuroprotection by driving autophagic degradation of pathogenic aggregates in ALS.
    Priyanka Tripathi, Haihong Guo, Alfred Yamoah, Ramkumar Mathur, Panagiotis Doukas, Alice Dreser, Christopher Marvin Jesse, Eleonora Aronica, Andreas Hermann, Harry Steinbusch, Gary Anthony Brook, Joachim Weis, Anand Goswami.
      During the progression of amyotrophic lateral sclerosis (ALS), only specific motor neurons (MNs) preferentially deteriorate, while others are spared until the disease reaches its end stage. Resilient MNs possess several protective factors, yet the precise molecular mechanism(s) underlying selective neuronal vulnerability remains poorly understood. Vesicle-associated membrane protein (VAMP)-binding protein B (VAPB) is an endoplasmic reticulum (ER) protein involved in protein quality control (PQC) mechanisms, including unfolded protein response (UPR) as well as autophagy. A dominantly inherited P56S mutation in the VAPB gene has been linked to ALS8, atypical ALS, and late-onset spinal muscular atrophy (SMA). The P56S VAPB mutation causes ER-associated inclusions, disorganization, and ER stress, contributing to MN degeneration through toxic gain and loss of function. Over-expression of VAPB protein confers neuroprotection in a mouse model of ALS, and increased levels of neuronal VAPB inversely correlate with the absence of pathological aggregates. We hypothesize that VAPB is crucial for motor neuron survival by promoting autophagic degradation of ALS-associated aggregates, while lack of VAPB confers neuronal vulnerability. We analyzed the brain and spinal cord from sporadic (s) and familial (f) ALS patients, comparing patterns of VAPB immunoreactivity using immunohistochemistry, complemented by Western and dot blot analysis. Pathophysiological insights from these studies were further explored using cell culture models, including MNs derived from induced pluripotent stem cells (iPSCs). Consistent with our hypothesis we observed that MNs/neurons resistant to ALS exhibited elevated levels of VAPB and were devoid of pathogenic aggregates. Similarly, ALS-resistant oculomotor neurons showed increased VAPB immunoreactivity compared to normal controls. VAPB was often found to be sequestered within toxic aggregates alongside autophagy-related proteins in the lumbar spinal cord MNs. Notably, a compensatory increase in VAPB immunoreactivity was observed at the C-bouton synapse, suggesting a potential alternative mechanism of neuroprotection. Supporting these findings, in vitro experiments indicated that VAPB overexpression promoted autophagy and assisted in clearing ALS-associated RNA-binding protein aggregates. In summary, VAPB promotes selective neuronal survival by facilitating the autophagic clearance of toxic aggregates. Abnormal VAPB accumulations likely disrupt these neuroprotective processes.
    Keywords:  ALS8; Autophagy; RBPs; Selective MN vulnerability; VAPB
    DOI:  https://doi.org/10.1186/s40478-026-02298-8
  26. J Cell Biol. 2026 Aug 03. pii: e202604059. [Epub ahead of print]225(8):
    Design principles of human membrane protein topology.
    Haoxi Wu, Ramanujan S Hegde.
      We have curated and annotated the topologic determinants for all human membrane proteins made at the endoplasmic reticulum. This census of 4,863 proteins allowed us to systematically analyze the physical properties of their 20,546 transmembrane domains (TMDs) and flanking soluble regions. Single-pass proteins house the majority of large exoplasmic and cytosolic domains, whereas multipass proteins overwhelmingly contain short loops and tails. All classes of TMDs have positively charged cytosolic flanks, but negatively charged exoplasmic flanks feature primarily on TMDs inserted by Oxa1 family insertases. The TMD pair, a topologic unit of two TMDs with a short exoplasmic loop, is the dominant building block of multipass proteins. TMD pairs accommodate high-hydrophilicity and charge-containing TMDs crucial for multipass protein functions. We interpret these context-dependent TMD features in light of current mechanistic models for membrane protein biogenesis and function. Our findings have implications for the evolution of membrane proteomes and for engineering new membrane proteins.
    DOI:  https://doi.org/10.1083/jcb.202604059
  27. Nat Commun. 2026 May 27.
    Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages.
    Sara C Buch-Larsen, Ivo A Hendriks, Kyuto Tashiro, Jonas D Elsborg, Sergey Y Vakhrushev, Jesper V Olsen, Bernhard Lüscher, Glen Liszczak, Ivan Ahel, Michael L Nielsen.
      ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.
    DOI:  https://doi.org/10.1038/s41467-026-73677-x
  28. Mol Cell. 2026 May 29. pii: S1097-2765(26)00288-1. [Epub ahead of print]
    Mechanism of RACK1-dependent ZAKα activation at stalled and collided ribosomes.
    Anna Constance Vind, José Francisco Martínez, Zhenzhen Wu, Andrii Bugai, Kelly Mordente, Giancarlo Abis, Sébastien Chamois, Sofia Ramalho, Catarina Pechincha, Laura Ryder, Qiuyan Chen, Mads Rasmussen, Xinyao Shi, Dandan He, Jesper Q Svejstrup, Peter Haahr, David Gatfield, Maria R Conte, Torben Heick Jensen, Melanie Blasius, Simon Bekker-Jensen.
      Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38- and JNK-activating MAP3 kinase ZAKα senses translational impairment. Combining AlphaFold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKα dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKα via direct interactions with the ribosomal proteins RACK1 and RPS27 as well as 18S rRNA helix-26. In this conformation, the RNA-binding S (sensing) and C-terminal domain of ZAKα span across the mRNA exit channel. Loss of ribosome processivity and mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKα to stalled and collided ribosomes is associated with sequestration of the sterile alpha-motif (SAM) domain on RACK1, which allows for transient ZAKα dimerization, activation loop trans-autophosphorylation, and RSR activation. Our findings highlight how ZAKα senses both stalled and collided ribosomes in human cells through overlapping mechanisms.
    Keywords:  ZAK-alpha; ribosome collision; ribosome stalling; ribotoxic stress response; translation surveillance
    DOI:  https://doi.org/10.1016/j.molcel.2026.04.034
  29. NPJ Breast Cancer. 2026 May 23.
    USP7 inhibition perturbs proteostasis and tumorigenesis in triple-negative breast cancer.
    Ahhyun Kim, Priya Gopalakrishnan, Marina Suárez-Pizarro, Claire C Chen, Xianxi Wang, Sydney M McCoy, Nikita Umesh, Angie Mordant, Natalie K Barker, Laura E Herring, Rasha T Kakati, Philip M Spanheimer, Michael J Emanuele, Claudia A Benavente.
      The deubiquitinase USP7 is a critical regulator of tumorigenesis, known for stabilizing the MDM2-p53 pathway. Emerging evidence highlights USP7's p53-independent roles in proliferation and tumorigenesis. Our study reveals that USP7 is broadly upregulated in breast cancer, including triple-negative breast cancer (TNBC), a subtype characterized by near-universal TP53 loss. This provides a model to investigate p53-independent functions of USP7. Using TNBC models, we define p53-independent roles of USP7 in regulating proteostasis and tumorigenesis. Importantly, genetic and pharmacologic USP7 inactivation impaired tumor progression in TNBC models. To explore USP7's role in p53-mutant TNBCs, we performed deep quantitative proteomics across TNBC cell lines, identifying shared USP7 targets involved in cell proliferation, genome stability, and proteostasis. Acute USP7 inactivation allowed us to infer proximally controlled proteins that are likely direct targets. Surprisingly, many of the proteins downregulated by USP7 inhibition are E3 ubiquitin ligases. Thus, a key USP7 function in TNBC is to antagonize the degradation of ubiquitinating enzymes, since these enzymes are often susceptible to auto-ubiquitination and degradation. Notably, we identified TOPORS, a dual ubiquitin- and SUMO-ligase, among novel USP7 substrates. TOPORS interacts with the BRCA1-A DNA damage repair complex, suggesting a USP7-TOPORS-BRCA1-A axis that might further explain the continued proliferation of genomically unstable TNBCs. Collectively, these data nominate USP7 as a potential therapeutic vulnerability in TNBC.
    DOI:  https://doi.org/10.1038/s41523-026-00974-5
  30. bioRxiv. 2026 May 12. pii: 2026.05.08.723879. [Epub ahead of print]
    Neuronal regulation of infection recovery prevents pathogenic stress and tissue damage.
    Phillip Wibisono, Sagi Levy, Jingru Sun.
      Neural regulation of innate immunity is increasingly recognized, yet how the nervous system controls immune resolution after pathogen clearance remains poorly understood. Using Caenorhabditis elegans as a genetically tractable model, we identify the AIA interneurons as critical regulators of both infection-phase homeostasis and post-infection recovery. Acute silencing or genetic ablation of AIA neurons during Salmonella enterica infection results in reduced host survival with heightened activation of conserved immune and stress pathways, including PMK-1/p38 MAPK, insulin/IGF-1 signaling, and the XBP-1-mediated unfolded protein response (UPR). AIA-deficient animals exhibit excessive immune and stress response gene expression and increased intestinal tissue damage, demonstrating that immune hyperactivation is detrimental. Strikingly, selective silencing of AIA neurons during the recovery phase after pathogen clearance significantly impairs survival, revealing that neural activity is required not only for defense but also for resolution. This recovery defect is rescued by knockdown of xbp-1 , but not pmk-1 or daf-16 , indicating that unresolved ER stress is the principal driver of post-infection mortality. Consistently, AIA silencing during recovery sustains UPR activation and exacerbates epithelial barrier damage. Together, our findings establish AIA interneurons as central coordinators of immune homeostasis that limit pathological stress responses during infection and actively promote infection resolution. These findings provide mechanistic insight into how the nervous system not only restrains excessive immune and stress responses during infection but also actively resolves stress signaling and preserves tissue integrity during post-infection recovery.
    DOI:  https://doi.org/10.64898/2026.05.08.723879
  31. Autophagy. 2026 May 25.
    CHCHD2 and CHCHD10 promoted autophagic clearance of protein aggregates via GABARAPs.
    Zhou Wei, Maggie Zhang, Willcyn Tang, Brijesh Kumar Singh, Zhang Zhiwei, Zhou Lei, Jaron Goh Kim Wee, Faith Tan Rui En, Huang Jingxiu, Sun Qiaoyang, Xiao Bin, Gupta Priyanka, Alfred Sun Xuyang, Zeng Li, Shen Han-Ming, Tan Eng King.
      Mutations in mitochondrial protein CHCHD2 and its paralog CHCHD10 were identified in patients with Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) or Alzheimer disease (AD). CHCHD2 and CHCHD10 mutations caused neurodegeneration in model animals as seen in patients, but their pathophysiological roles remain elusive. Here we reported a direct role of CHCHD2 and CHCHD10 in autophagy. We identified a protein complex composing of CHCHD2-CHCHD10-C1QBP/p32-Atg8-family proteins (ATG8s), in which each molecule interacted with another. CHCHD2, CHCHD10 and C1QBP/p32 associated with ATG8s, preferentially, GABARAPs. Disease-associated CHCHD2 and CHCHD10 mutations exhibited varied interaction with ATG8s. By binding to GABARAPs, CHCHD2 and CHCHD10 underwent autophagic degradation, and recruited the ULK1 complex. Autophagy initiation defects occurred upon transient knockdown of CHCHD2, and also in human iPSC-derived CHCHD2-/- or CHCHD2T61I dopaminergic neurons. Importantly, CHCHD2 and CHCHD10 promoted autophagy. CHCHD2 reduced protein aggregates in cells and toxic SNCA/α-synuclein species in mouse striatum. Our study thus revealed mitochondrial proteins CHCHD2 and CHCHD10 as both autophagy substrates and autophagy activators and laid groundwork for therapy targeting patients with neurodegeneration.
    Keywords:  Aggregates; CHCHD10; CHCHD2; GABARAPs; autophagy; neurodegeneration
    DOI:  https://doi.org/10.1080/15548627.2026.2678427
  32. Aging Cell. 2026 Jun;25(6): e70566
    Blm10/PA200-Activated 20S Proteasomes Promote α-Synuclein Degradation and Bypass Proteasome Inhibition in Parkinson's Disease Models.
    Tariq T Ali, Anton Zhornyak, Madiha Merghani, Zora Buschenlange, Eri Sakata, Tiago F Outeiro, Blagovesta Popova, Gerhard H Braus.
      Protein homeostasis is essential for maintaining normal cellular function. However, protein homeostasis efficiency declines with age, leading to the accumulation of aberrant protein structures associated with neurodegenerative diseases such as Parkinson's disease (PD). PD is characterized by the aggregation of alpha-synuclein (αSyn) into cytoplasmic inclusions. This process is accompanied by elevated phosphorylation at serine 129 (S129). The accumulation of αSyn into aggregates and their propagation disrupts key proteostasis pathways, including the ubiquitin-proteasome system (UPS) or autophagy, contributing to cellular dysfunction and neuronal death. This study identified the proteasome activator Blm10 and its human ortholog PA200 as modulators of αSyn degradation and toxicity. The conserved Blm10/PA200 protein plays a key role in regulating proteasome activity and assembly. The αSyn expression increases Blm10 protein stability through autophagy inhibition, in a manner dependent on αSyn phosphorylation at S129 in yeast. Overexpression of BLM10 or PA200 reduces αSyn aggregation and enhances αSyn turnover via activation of the 20S proteasome in yeast and mammalian cells. Blm10 and PA200-capped 20S proteasomes efficiently degrade both monomeric as well as oligomeric αSyn in vitro. Notably, capped proteasomes retain proteolytic activities in the presence of αSyn, indicating resistance to αSyn-induced inhibition, in contrast to 20S or 26S proteasomes. These results reveal a distinct proteasome subtype that bypasses UPS impairment and restores proteolytic capacity under proteotoxic stress. Our findings establish Blm10/PA200 as critical regulators of αSyn proteostasis and highlight its protective role in maintaining protein homeostasis and cell viability under conditions of αSyn toxicity.
    Keywords:  20S proteasome; Parkinson disease; alpha‐synuclein; autophagy; posttranslational modifications; proteasomal chaperones; protein homeostasis; yeast
    DOI:  https://doi.org/10.1111/acel.70566
  33. bioRxiv. 2026 May 17. pii: 2026.05.15.725547. [Epub ahead of print]
    Nuclear Receptor Liver Receptor Homolog-1 drives Intestinal Stem Cell regeneration and UPR/ER stress response after gut injury.
    Hsi-Ju Chen, Kristina N Braverman, Bohan Yu, James R Bayrer.
      Stem cell renewal and crypt survival are tightly controlled processes critical for gut repair. Defining key regulators of intestinal healing is critical for the development of new epithelial-targeted therapies. We previously showed that the nuclear receptor LRH-1 (NR5A2) maintains intestinal epithelial health and protects against inflammatory damage. Here, using lineage tracing and selective LRH-1 knockout in the Atoh1 + secretory lineage we show LRH-1 is vital for intestinal stem cell (ISC) regeneration in complementary in vivo and ex vivo injury-recovery models. Transcriptomic profiling and pathway analysis reveal downregulation of ER stress and unfolded protein response (UPR) programs. Using a new in vivo model to ascertain how LRH-1 directly impacts intestinal cell responses, we identify key ER stress response genes Ire1α and Xbp1 as potential LRH-1 targets. Together our results uncover a novel mechanism whereby LRH-1 sustains the IRE1α-XBP1 arm of the UPR to support injury-induced dedifferentiation and ISC regeneration. Our findings highlight LRH-1 as a promising therapeutic target for restoring epithelial integrity in inflammatory intestinal disorders.
    DOI:  https://doi.org/10.64898/2026.05.15.725547
  34. Science. 2026 May 28. 392(6801): eadu9554
    Selective autophagy fine-tunes plant immunity to promote cell survival during viral infection.
    Marion Clavel, Anita Bianchi, Roksolana Kobylinska, Roan Groh, Xi Zhang, Juncai Ma, Ranjith K Papareddy, Nenad Grujic, Lorenzo Picchianti, Ethan Stewart, Michael Schutzbier, Karel Stejskal, Juan Carlos de la Concepcion, Victor Sanchez de Medina Hernandez, Yoav Voichek, Pieter Clauw, Joanna Gunis, Gerhard Durnberger, Jens Christian Muelders, Annett Grimm, Arthur Sedivy, Mathieu Erhardt, Victoria Vyboishchikov, Peng Gao, Esther Lechner, Emilie Vantard, Thomas Potuschak, Jakub Jez, Elisabeth Roitinger, Pascal Genschik, Byung-Ho Kang, Yasin Dagdas.
      RNA viruses co-opt host endomembranes to form replication complexes, often triggering cellular stress and immune responses. Here, we show that Arabidopsis thaliana activates selective autophagy to respond to viruses targeting mitochondria, chloroplasts, and the endoplasmic reticulum. Rather than degrading viral components, autophagy selectively removes the immune regulator Enhanced Disease Susceptibility 1 (EDS1) to prevent cell death. This targeted mechanism is mediated by oligomeric metabolic enzymes that moonlight as selective autophagy receptors, linking organelle stress to immune homeostasis. Our findings establish selective autophagy as an essential immune rheostat that fine-tunes defense responses and safeguards cellular integrity to promote host survival during viral infections.
    DOI:  https://doi.org/10.1126/science.adu9554
  35. Cell Rep. 2026 May 28. pii: S2211-1247(26)00468-7. [Epub ahead of print]45(6): 117390
    Chaperone-mediated autophagy is a tumor-suppressive mechanism in hepatocellular carcinoma.
    Khushbu Patel, Begoña Zapateria, Alexander J Ledet, Saba S Hazaveh, Floralba Gjergjova, Antonio Diaz, Simone Sidoli, Amaia Lujambio, Ana María Cuervo, Fernando Macian, Esperanza Arias.
      Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway essential for proteostasis and stress adaptation that declines with aging and metabolic disease, conditions closely linked to hepatocellular carcinoma (HCC). Using genetically engineered mouse models with systemic, hepatocyte-specific, or T cell-specific deletion of the CMA regulator LAMP2A in an MYC-driven, TP53-deficient HCC context, we demonstrate that CMA exerts cell-type-dependent tumor-suppressive functions. Hepatocyte-intrinsic CMA loss promotes early malignant transformation, whereas T cell-specific CMA deficiency impairs early immune-mediated tumor control but is also required to sustain tumor growth. Proteomic profiling identifies the cohesin complex component STAG2 as a putative CMA substrate that accumulates in CMA-dysregulated hepatocytes, a finding validated in human HCC tissues, shown to drive cell cycle dysregulation and proliferation, contributing to hepatocarcinogenesis. These results establish CMA as a dual hepatocyte- and immune-dependent mechanism that suppresses liver tumorigenesis and positions STAG2 as a CMA-controlled node with therapeutic relevance in HCC.
    Keywords:  CP: cancer; CP: immunology; cancer; cell-type specificity; liver disease; lysosomal degradation; malignant transformation; proteostasis; stromal antigen 2; tumor suppression
    DOI:  https://doi.org/10.1016/j.celrep.2026.117390
  36. EMBO J. 2026 May 29.
    The C9orf72/SMCR8 complex maintains microglial homeostasis via RAB8A ESCRT-mediated lysosomal repair.
    Shan Li, Shidong Xu, Feng Li, Qirui Zhao, Penghui Zhang, Qinghua Guan, Xiangxiang Sun, Jundong Bi, Hu Xiao, Yiyuli Tang, Cheng Peng, Qingfeng Chen, Yonghua Wang, Mei Yang.
      Microglia are critical regulators of neuroinflammation and neurodegeneration. Haploinsufficiency of C9orf72, the most frequently mutated gene in amyotrophic lateral sclerosis and frontotemporal dementia, has been linked to autophagy-lysosomal pathway defects, but the role of C9orf72 in microglia remains unclear. Here, we identify the C9orf72/SMCR8 complex as a key regulator of microglial homeostasis through promoting lysosomal membrane repair. Loss of C9orf72 and SMCR8 in mice causes age‑dependent neuroinflammation and microgliosis, with microglia adopting a disease-associated state. In aged brain and spinal cord tissue, microglia display lysosomal damage marked by galectin‑3 accumulation. Using a lysosomotropic agent to induce lysosomal damage in microglia, we find that C9orf72/SMCR8-deficient cells accumulate damaged lysosomes and show defective recruitment of phosphorylated RAB8A and the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery to damaged lysosomes. Notably, mutant microglia accumulate GTP‑bound RAB8A, which becomes hyperphosphorylated and mislocalized to RAB7-positive, LAMP1-negative vesicles. The GTPase-activating activity of the C9orf72/SMCR8 complex is essential for lysosomal repair. Our findings reveal that the C9orf72/SMCR8 complex coordinates RAB8A-ESCRT-mediated lysosomal repair to safeguard microglial homeostasis and limit neuroinflammation.
    DOI:  https://doi.org/10.1038/s44318-026-00817-w
  37. Nat Commun. 2026 May 29.
    The benchmarking and application of tag-degraders in vivo to validate therapeutic targets.
    Charlene M Magtoto, Stephen Mieruszynski, Hao Dong, Ashley P Ng, Ladina Di Rago, Andrew J Kueh, Martin Brzozowski, Christoph Grohmann, Joel R Walker, Ngee Kiat Chua, Laura F Dagley, Alessio Ciulli, Guillaume Lessene, Marco J Herold, Joan K Heath, John Silke, Rebecca Feltham.
      Transitioning a candidate therapeutic target from bench to bedside requires significant time and financial investment, yet clinical success remains low often due to poor on-target toxicity assessment during preclinical validation. Tag-degraders provide a tool to improve target validation by enabling degradation of any protein of interest via a degron-tag. This drug-based, reversible, and dose-dependent method of protein removal can mimic degrader-based drug treatments and assess the implications of target protein depletion in vivo. However, each degrader has a distinct pharmacokinetic profile that will influence its effectiveness across tissues. To create a resource to enable the most appropriate choice of tag-degrader, we benchmark the dTAG, HaloPROTAC, and NanoTAC systems in vivo by employing a transgenic mouse expressing a reporter protein targetable by these tag-degraders. Through various treatment regimes, we characterise each degrader profile across a panel of 20 tissues and organs, highlighting the superior degradation by dTAG molecules, and identify differences between degradation in whole tissues versus single cell populations. Using an FKBPF36V knock-in mouse expressing 65K-FKBPF36V, we reveal target specific degradation kinetics, and a critical requirement for 65K in mice. Together, this resource will assist researchers in choosing the right degrader and tag for their own applications.
    DOI:  https://doi.org/10.1038/s41467-026-73681-1
  38. Mol Cell. 2026 May 26. pii: S1097-2765(26)00308-4. [Epub ahead of print]
    Uncovering the initial response: Intra-mitochondrial surveillance activates the UPRmt.
    Asli Aras Taskin, Sahana Shankar, Carlotta Peselj, Annette Flotho, Maria Gomez-Fabra Gala, Daniel Poveda-Huertes, Lisa Myketin, Duygu Mutlu, Adinayarana Marada, Sebastian Schuck, Mandy Jeske, Sabrina Büttner, Marcin Luzarowski, Chris Meisinger, F-Nora Vögtle.
      The mitochondrial unfolded protein response (UPRmt) protects mitochondria from proteotoxic stress. Current models induce acute and severe mitochondrial disruption and propose cytosolic detection following the release of mitochondrial damage signals into the cytosol. However, this mode of toxicity contrasts sharply with physiological stress, such as the gradual accumulation of reactive oxygen species (ROS) during aging or chronic respiratory chain defects. Here, we employ a chemogenetic strategy in yeast to induce low levels of hydrogen peroxide (H2O2) in the mitochondrial matrix and show that mild oxidative stress activates the UPRmt independently of cytosolic damage. We identify the presequence proteases MPP and Oct1 as early ROS targets, thereby linking redox imbalance to UPRmt activation: oxidative stress induces glutathionylation of critical cysteines, impairing protease activity and causing the accumulation of unprocessed precursors in proteotoxic matrix aggregates. These aggregates are detected by intra-mitochondrial surveillance, activating UPRmt signaling. Thus, mitochondrial self-surveillance initiates rapid protective signaling as a primary response to mitochondrial dysfunction.
    Keywords:  mitochondria-nucleus communication; mitochondrial protein biogenesis; mitochondrial unfolded protein response; oxidative stress; presequence processing; reactive oxygen species
    DOI:  https://doi.org/10.1016/j.molcel.2026.05.002
  39. Angew Chem Int Ed Engl. 2026 May 26. e25741
    Hydrophobic Tag Degraders Overcome Endocrine-Resistant Breast Cancer by Recruiting HSP27-Mediated E3 Ligase Complex for ERα Proteasomal Degradation.
    Lilan Xin, Zemin Song, Yali Cui, Chao Wang, Yan Chen, Jing Liu, Jian Min, Zhiye Hu, Ruijing Xiao, Xin Liu, Zhangxiao Guo, Hongli Wang, Zheyang Hu, Jian Huang, Kaiwei Liang, Chune Dong, Hai-Bing Zhou.
      Hydrophobic tag (HyT)-mediated protein degradation has emerged as a pivotal tool for targeted protein degradation (TPD), yet its underlying degradation mechanism remains incompletely elucidated. Herein, we designed structurally optimized HyT-based degraders by covalently conjugating hydrophobic amino acid tags to ERα-targeting ligands via alkane linkers of varying lengths, identifying the lead compound VI-10h. VI-10h exhibited potent antiproliferative activity and efficient ERα degradation in endocrine-resistant breast cancer cells (LCC2, MCF-7D538G, MCF-7Y537S, and MCF-7EGFR) and superior antitumor activity compared to the clinical drug fulvestrant (Ful) in MCF-7 and tamoxifen-resistant LCC2 xenograft models. To elucidate the HyT-mediated degradation mechanism, we synthesized biotin-conjugated HyTs (biotin-Lys and biotin-Trp) and performed pull-down assays combined with mass spectrometry. Our results unveiled that VI-10h selectively recruits heat shock protein 27 (HSP27) as a non-canonical E3 ligase adaptor protein, forms an ERα-HSP27-RING1 ternary complex to promote ERα degradation, disrupts estrogen-dependent oncogenic networks, and circumvents the drug resistance associated with conventional CRBN- or VHL-dependent E3 ligase-recruiting degraders. This study clarifies a HyT-mediated ERα degradation mechanism and supports the feasibility of using HyT degraders to overcome resistance to conventional E3 ligase-recruiting strategies and endocrine-resistant breast cancer, thereby establishing a molecular design strategy for next-generation targeted degraders.
    Keywords:  E3 ligase; ERα; HSP27; endocrine resistance; hydrophobic tag
    DOI:  https://doi.org/10.1002/anie.202525741
  40. Mol Cell. 2026 May 25. pii: S1097-2765(26)00284-4. [Epub ahead of print]
    Phenylalanyl-tRNA synthetase FARS-1/FARSA balances longevity and immunity by downregulating endogenous mitochondrial double-stranded RNAs.
    Jooyeon Sohn, Moonhyeon Jeon, Hanseul Lee, JinA Lim, Seokjin Ham, JiSoo Park, Jongsun Lee, Yoonji Jung, Gee-Yoon Lee, Hyemin Min, Eunseok Kang, Yerim Jang, Yunhee Kong, Dajeong Bong, Yoosik Kim, Seung-Jae V Lee.
      Endogenous double-stranded RNAs (dsRNAs) are immunogenic self-molecules that drive aberrant immune activation under pathological conditions. Here, we show that dsRNAs and their regulation by RNA-binding proteins are key determinants of the fine balance between aging and immunity in Caenorhabditis elegans and cultured human cells. We find elevated levels of dsRNAs with organismal aging and cellular senescence. We identify a moonlighting function for phenylalanyl-tRNA synthetase, FARS-1/FARSA, as a key factor necessary and sufficient for extending lifespan by downregulating dsRNAs, in particular, mitochondrial dsRNAs. FARS-1/FARSA possesses a previously unrecognized dsRNA-binding domain and mediates dsRNA downregulation with the RNA helicase, RHA-2/DHX37, independently of its canonical role in translation. Notably, increased dsRNA expression resulting from genetic inhibition of fars-1/FARSA upregulates immune response-related genes and enhances innate immunity against pathogens. Our study establishes that FARS-1/FARSA is an evolutionarily conserved dsRNA-binding protein that delays aging and promotes longevity by suppressing dsRNA accumulation.
    Keywords:  Caenorhabditis elegans; FARS-1/FARSA; RNA-binding protein; double-stranded RNA; immunity; longevity; mitochondria; senescence
    DOI:  https://doi.org/10.1016/j.molcel.2026.04.030
  41. Mol Biol Cell. 2026 May 27. mbcE25100479
    Yeast Ribosomal Protein Rpl32 Modulates Ribosome Biogenesis, Ribosome Stability and the Cell Cycle.
    Stephen M Doris, Elizabeth S Noblin, Raphyel Rosby, Susan A Gerbi.
      How does the cell coordinate its two major activities of cell growth and cell division? To explore this, we have genetically depleted yeast ribosomal protein Rpl32 of the 60S ribosomal subunit, which is an essential protein for cell proliferation. 3-4 hours after Rpl32 depletion, the cell cycle arrests at G1. We have undertaken a kinetic analysis of the early cellular events to deduce the pathway from Rpl32 depletion to G1 arrest. Rpl32 depletion blocks pre-rRNA processing of the initial 35S pre-rRNA, thus preventing ribosomal biogenesis and nuclear export of 60S ribosomal subunits. Interestingly, the L25-GFP reporter transiently accumulates in a focal spot that resembles the nucleolar body/Cajal body. Amazingly, the inhibition of ribosome biogenesis in the nucleus is signalled to the cytoplasm where mature 18S and 25S rRNAs are degraded in a ribophagy-independent manner; Rpl32 protease-degradation uses de-ubiquitination. Nonetheless, the ribosomes that remain after degradation are sufficient for translation whose efficiency is unchanged through 6 hours after Rpl32 depletion, and the cell size and vacuole increase in size. The level of cyclin 1 mRNA is rapidly diminished after Rpl32 depletion and is a likely factor for the arrest of the cell cycle at G1.
    DOI:  https://doi.org/10.1091/mbc.E25-10-0479
  42. Nat Commun. 2026 May 29.
    Native long-read RNA sequencing of human monocytes reveals activation-induced alternative splicing toward functional isoforms.
    Alejandra Bodelón, Maurice J H van Haaren, Paula Sobrevals Alcaraz, Lyanne J P M Sijbers, Rianne C Scholman, Lucas W Picavet, Aafke de Ligt, Daphne van Ginneken, Remco G A Erkens, Harmjan R Vos, Jorg J A Calis, Sebastiaan J Vastert, Jorg van Loosdregt.
      Alternative splicing is a key mechanism for expanding transcriptomic and proteomic complexity, yet its role in innate immune activation remains incompletely understood. Here, we applied Oxford Nanopore native RNA-sequencing to generate an isoform-level transcriptome of primary human monocytes before and after activation with lipopolysaccharide. We identify over 24,000 expressed isoforms, including thousands of previously unannotated variants. Activation induced widespread isoform-specific expression changes, leading to extensive isoform switching events, which were validated using matched short-read RNA-Seq. These activation-induced shifts enhanced transcript immune-regulatory functions: activated monocytes preferentially express longer, coding-competent isoforms with complete open reading frames, fewer retained introns, and increased domain complexity. By integrating matched Ribo-seq and proteomic data, we demonstrate that these isoform modulations are associated with enhanced translation of immune effector proteins. Together, our findings position alternative splicing as a dynamic and functional regulator of monocyte activation, emphasizing the need for isoform-level resolution to fully understand immune cell function.
    DOI:  https://doi.org/10.1038/s41467-026-73661-5
  43. bioRxiv. 2026 May 11. pii: 2026.05.07.723568. [Epub ahead of print]
    Mitochondrial respiration modulates Hsf1 activation and the heat shock response.
    D W McDonald, A Dea, R Sava, Y J Kim, L Joos, D Pincus, M L Duennwald.
      Cells employ a bevy of transcriptional and post-translational stress responses to tolerate the burden of misfolded proteins induced by stress. In particular, the heat shock response facilitates the upregulation of molecular chaperones and protein remodeling factors that mediate proteostasis in response to accumulated misfolded proteins in the nucleus and cytosol. However, in response to stress neurons struggle to induce a canonical heat shock response, highlighting our poor understanding of how neurons maintain proteostasis. Specifically, the ability of post-mitotic respiring cells to regulate the heat shock response in comparison to their rapidly dividing, predominantly glycolytic counterparts has been under-studied. In this study, we employ yeast models that are easily manipulated to generate energy via glycolysis or mitochondrial respiration by changing the carbon source in the media. Using this model, we demonstrate that Hsf1 activity, the heat shock response and proteostasis are impaired in respiring cells. Interestingly, our data show that reduced Hsf1 activity regulates viability of respiring cells, with respiring cells poorly tolerating constitutively activated Hsf1. Finally, we describe alternative post-translational programming of the molecular chaperones Hsp70 and Hsp104 that plausibly enables respiring cells to mediate proteostasis despite a dampened heat shock response. Our findings offer new insights into possible proteostatic strategies employed by cells in different metabolic conditions.
    DOI:  https://doi.org/10.64898/2026.05.07.723568
  44. Cell Rep. 2026 May 28. pii: S2211-1247(26)00508-5. [Epub ahead of print]45(6): 117430
    RNA-binding protein diversity and NLS arginines regulate FUS mixing in mRNA-rich compartments.
    Diane Valenti, Vandana Joshi, Serhii Pankivskyi, Hoang Hung Cai, Loic Hamon, Bénédicte Desforges, Amandine Molliex, Julien Duez, Julian Bursztyka, Laurianne Davignon, Alexandre Maucuer, Guillaume Bollot, David Pastré.
      Despite being prone to condensation, many RNA-binding proteins (RBPs) do not form large condensates in cells. This issue is still widely researched, particularly because aggregation of RBPs, such as FUS, is the hallmark of some neurodegenerative diseases. Elevated RNA levels and protein chaperone activity have already emerged as key factors preventing aberrant phase separation. Here, we explored the role of RBP diversity in mRNA-rich condensates. While FUS and its partners form distinct compartments when probed one by one, increasing RBP diversity buffers FUS spatial segregation. In addition, we found that frequently mutated arginine residues in the nuclear localization signal (NLS) at the C-terminal end promote FUS mixing with multiple RBPs. Therefore, we anticipate that pathological NLS mutations in FUS not only alter its active nuclear import but also regulate FUS interactions with its partners in mRNA-rich compartments with putative consequences for the onset and progression of FUS-related neurodegenerative diseases.
    Keywords:  ALS; CP: molecular biology; FUS pathological mutations; LCDs; MLOs; NLS domain; RGG domains; RNA; RNA-binding proteins; amino acid sequence; biomolecular condensates; homotypic/heterotypic interactions; membrane less organelles; phase separation; stress granules
    DOI:  https://doi.org/10.1016/j.celrep.2026.117430
  45. STAR Protoc. 2026 May 22. pii: S2666-1667(26)00245-5. [Epub ahead of print]7(2): 104592
    Protocol for the in vivo quantification of mitochondria-associated ER membranes in Caenorhabditis elegans.
    Fivos Borbolis, Konstantinos Palikaras.
      Mitochondria-associated membranes (MAMs) are specialized contact sites between the endoplasmic reticulum and mitochondria, with multiple functional aspects. Here, we present a confocal microscopy-based protocol for quantifying the area of MAM-like domains in intestinal cells of the nematode Caenorhabditis elegans using organelle-specific fluorescent reporters. We describe steps for worm synchronization, microscope setup, sample preparation, and image acquisition. We then detail procedures for manual single-image analysis and scalable automated batch processing, enabling robust quantification of ER-mitochondria contacts across experimental conditions. For complete details on the use and execution of this protocol, please refer to Roussos et al.1.
    Keywords:  Cell Biology; Model Organisms; microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2026.104592
  46. Sci Adv. 2026 May 29. 12(22): eaeb2368
    Metabolic rewiring driven by phosphoglycolate phosphatase deletion inhibits ferroptosis.
    Marian Brenner, Sina Höhlein, Paul Wirth, Leon Neidt, Melina Lappe, Elisa Hopke, Eirini Sfakianaki, Martina Fischer, Kerstin Hadamek, Angelika Keller, Sebastian Bothe, José Pedro Friedmann Angeli, Anna M Schmoker, Arminja N Kettenbach, Agnes Fekete, Werner Schmitz, Ingrid Tessmer, Elisabeth Jeanclos, Antje Gohla.
      Modulating ferroptosis, a form of cell death driven by uncontrolled lipid peroxidation, is of interest in numerous diseases. Here, we found that the deletion of phosphoglycolate phosphatase (PGP), an essential enzyme that safeguards high glycolytic flux, suppresses ferroptosis. Using metabolomic and isotopic labeling experiments together with lipid and proteomic profiling, we find that PGP loss drives a rewiring of the pentose phosphate pathway and of cellular energy and lipid metabolism that triggers a multifactorial antioxidant response. Paradoxically, our attempts to block PGP pharmacologically led to the realization that the recently described PGP inhibitor compound 1 (CP1) exerts a strong ferroptosis-sensitizing effect. Using genetic, biochemical, and biophysical approaches, we characterize CP1 as a direct, species-independent, dual inhibitor of PGP and ferroptosis suppressor protein 1 (FSP1), and further find that CP1 triggers FSP1 self-assembly. In sum, we identify PGP as a target protein for ferroptosis control and introduce a small-molecule FSP1 inhibitor with unique features to the armamentarium of pharmacological ferroptosis modulators.
    DOI:  https://doi.org/10.1126/sciadv.aeb2368
  47. Nat Commun. 2026 May 28.
    Population-scale chemical response revealed by a barcoded yeast collection.
    Abhishek Dutta, Marion Garin, Victor Loegler, Gauthier Brach, Anne Friedrich, Mami Yoshimura, Hiroyuki Hirano, Hiroyuki Osada, Charles Boone, Yoko Yashiroda, Jing Hou, Joseph Schacherer.
      Natural genetic variation shapes how microbial populations adapt to environmental and chemical challenges, but scalable approaches to map genotype-phenotype relationships across diverse genetic backgrounds remain limited. Here, we developed a systematically barcoded collection of 520 Saccharomyces cerevisiae natural isolates that captures the ecological, geographical and genetic diversity of the species. Using pooled barcode sequencing, we profiled fitness responses to over 600 bioactive and natural compounds, revealing broader and more polarized bioactivity than the standard yeast gene-deletion collection. Fitness-based clustering defined six major compound groups with reproducible, population-structured sensitivity patterns. Genome-wide association analysis identified significant genetic variants across 106 compounds, linking natural polymorphisms to chemical responses and involving genes in genome maintenance, ribosome biogenesis, vesicular trafficking and stress tolerance. Together, our barcoded natural population provides a scalable framework for chemical-genetic screening, enabling systematic dissection of how genetic diversity shapes microbial fitness and adaptation.
    DOI:  https://doi.org/10.1038/s41467-026-73532-z
  48. Proc Natl Acad Sci U S A. 2026 Jun 02. 123(22): e2520561123
    Navigating high-order protein fitness landscapes via deep learning on directed evolution trajectories.
    Chengzhi Song, Liang Ma, Lingfeng Xue, Yingfan Xu, Qihan Zhang, Yuxi Liu, Chen Song, Yihan Lin.
      Accurately predicting the fitness effects of high-order mutations is a grand challenge in understanding and engineering proteins. Existing models, including pretrained protein language models, struggle to capture the multiresidue interactions that govern these effects. Here, we introduce DENet, a deep learning framework that harnesses the rich comutation information within directed evolution (DE) trajectories to reconstruct high-resolution fitness landscapes for deciphering and engineering of complex protein variants. Applied to the cancer target KRAS, DENet-guided screening systematically identified high-order mutants with potent activities and uncovered hidden allosteric mechanisms. For MEK1, DENet nominated complex variants with >1,000-fold increased drug resistance, revealed synergistic tail mutations, and retrospectively identified over 75% of known clinical mutations, largely outperforming existing models. To broaden the framework's applicability, we developed an in silico strategy that simulates directed evolution to infer comutation information from widely available single-mutant datasets. DENet provides a quantitative framework for navigating complex fitness landscapes, uniting the rational engineering of multimutation proteins with the elucidation of their mechanisms and clinical implications.
    Keywords:  DENet; directed evolution; drug resistance; epistasis; protein engineering
    DOI:  https://doi.org/10.1073/pnas.2520561123
  49. Nat Commun. 2026 May 28.
    Fusion-positive rhabdomyosarcoma oncofusions share a common interactome.
    S P Zimmerman, C D Delaney, B K Lau, L B DeGraw, G H Rupprecht, M J A Groot Koerkamp, T de Souza, J Drost, R A Schoot, M T Meister, J F Shern, L M Wagner, G G Wang, K C Wood, C M Linardic, C M Counter.
      Fusion-positive rhabdomyosarcoma (FP-RMS) arises from at least seven distinct oncofusions sharing a common PAX3/7 N-terminal DNA-binding domain fused to divergent C-terminal partners. How different oncofusions produce the same cancer was unknown. Here we show they are functionally interchangeable, associate with a shared protein network we term the common interactome, bind overlapping target genes, and drive a similar core transcriptional program. The common interactome contains the C-terminal partners of known oncofusions and a newly identified translocation, suggesting oncofusions arise by PAX3/7 DNA-binding domain fusing to interactome members. As loss of common interactome proteins impaired oncogenic activity we screened the interactome for shared vulnerabilities. This identified thymidylate synthase as preferentially required for FP-RMS growth. Accordingly, the antifolate pralatrexate suppressed growth across all seven oncofusions, in multiple human FP-RMS cell lines, and a patient-derived xenograft. These findings demonstrate that divergent FP-RMS oncofusions are functionally fungible through a shared interactome that defines common vulnerabilities.
    DOI:  https://doi.org/10.1038/s41467-026-73749-y
  50. Mol Cell. 2026 May 29. pii: S1097-2765(26)00310-2. [Epub ahead of print]
    Mitochondria-lysosome coupling contributes to lysosome acidification and aging.
    Qingqing Liu, Seungmin Yoo, Zhixin A Zhang, Liying Li, Hetian Su, Lingraj Vannur, Alexandra C Wooldredge, Jun-Wei B Hughes, Pierre-Yves Desprez, Nan Hao, Gordon Lithgow, Julie K Andersen, Malene Hansen, Judith Campisi, Chuankai Zhou.
      Nearly all cellular processes are pH dependent. The acidic pH inside the lysosome (vacuole in yeast) is essential for cellular content degradation, signaling, and autophagy. Defects in lysosome/vacuole acidification are a conserved hallmark of aging and age-related diseases. Traditionally, the lysosome/vacuole is thought to import free protons (H⁺) from the surrounding neutral cytosol. Here, we uncovered a conserved lysosome/vacuole acidification mechanism from yeast to human involving lysosomal/vacuolar uptake of H+ pumped out by mitochondrial electron transport chain through mitochondria-lysosomes/vacuoles membrane contacts. Aging/senescence-associated disruption of mitochondria-lysosome/vacuole contacts causes lysosomal/vacuolar de-acidification, which can be reversed by either expressing an engineered linker to connect these two organelles or through an asymmetry-dependent rejuvenation process in daughter cells. Preserving lysosomal acidification in senescent human cells prevents the induction of major senescence-associated secretory phenotype factors and restores autophagic flux. These findings reshape our current understanding of the mechanisms underlying lysosomal/vacuolar (de-)acidification in both young and aged/senescent cells.
    Keywords:  Mito-Vac/Lyso contacts; SASP; aging; autophagy; cellular senescence; mitochondria; proton; vacuolar/lysosomal acidification
    DOI:  https://doi.org/10.1016/j.molcel.2026.05.004
  51. bioRxiv. 2026 May 14. pii: 2026.05.11.724303. [Epub ahead of print]
    Protein Stability, Turnover Kinetics, and Abundance Constrain the Scaling of Protein Interaction Networks.
    Muskan Goel, Daniel Allen Nissley, Xavier Castellanos-Girouard, Charles P Kuntz, Yiqing Wang, M Shahid Mukhtar, Adrian Serohijos, Jonathan P Schlebach.
      The propensity of proteins to form oligomers is ultimately dictated by their structural configuration(s). Proteins that persist in a discrete conformational state may form a limited number of specific interactions while those that sample a broader structural ensemble may instead associate with a wider array of partners. These intrinsic tendencies potentially constrain the way proteins navigate wider interaction networks. In this work, we aggregated and surveyed a wide variety of biophysical, biochemical, and cellular descriptors of the S. cerevisiae proteome to identify biases in the connectivity of its protein-protein interaction network. Using mass spectrometry-based interactome measurements and various protein stability estimates, we find that a disproportionate number of abundant, yet unstable binding proteins act as network hubs. Moreover, we show that these features alone can be used to discriminate between hubs and non-hub proteins with high accuracy (AUROC = 0.898). Interestingly, we find that half-lives of hub proteins depend on whether or not they reside within static complexes and/ or whether they interact with molecular chaperones. Finally, we note that the observed connectivity biases associated with abundant, unstable proteins only pertain to network hubs, but not to the bottlenecks that connect them. Together, our findings reveal how the conformational stability of a protein may constrain its context within protein-protein interaction networks.
    DOI:  https://doi.org/10.64898/2026.05.11.724303
  52. bioRxiv. 2026 May 12. pii: 2026.05.08.723928. [Epub ahead of print]
    AI-discovered protein fragments as generalizable regulators of biomolecular condensates.
    Andrew Savinov, Jibin Sadasivan, Kyle J White, Jack D Rubien, Gene-Wei Li, Lindsay B Case.
      Biomolecular condensates are a major driver of cellular organization; however, we lack a predictable and systematic approach to modulate the multivalent interactions underlying their formation. Here, we demonstrate that the AI-driven FragFold method enables robust and generalizable design of protein fragments to control biomolecular condensate formation. We apply this approach across diverse proteins: G3BP1, SARS-CoV-2 nucleocapsid, TDP-43, and focal adhesion kinase (FAK). Computationally screening 2,235 fragments, we selected 18 candidates for further investigation. Overall, we attain a 50% success rate (9/18 designs) in discovering condensate-controlling protein fragments, experimentally testing just 3-5 candidates per protein. For each condensate-forming protein, the success rate is at least 40%. Furthermore, FragFold-predicted fragment binding modes align with their condensate-inhibitory or -enhancing activities, revealing both known and newly identified interactions underlying condensate formation. In FAK, a condensate-inhibitory fragment uncovered a domain interaction required for phase separation, and mutational analysis validated its importance. Notably, this inhibitory fragment also suppresses FAK condensate formation in living mammalian cells. Together, these results establish AI-guided protein fragment discovery as a generalizable strategy to dissect and control the molecular interactions that govern biomolecular condensates.
    DOI:  https://doi.org/10.64898/2026.05.08.723928
  53. Proc Natl Acad Sci U S A. 2026 Jun 02. 123(22): e2613147123
    The revised three-step detour pathway in dolichol biosynthesis is evolutionarily conserved in budding yeast.
    Kazuki Hanaoka, Kuya Matsunaga, Souichirou Shimizu, Soshi Sakai, Harald Pichler, Kouichi Funato.
      The identification of SRD5A3, a causative gene for congenital disorders of glycosylation (CDGs), together with its yeast ortholog DFG10, established the prevailing model that dolichol is synthesized from polyprenol in a single step. Subsequently, a recent discovery of DHRSX in CDG patients revised this view and led to the proposal of a three-step detour pathway for dolichol biosynthesis. However, it remains unclear whether this pathway represents a conserved mechanism or reflects evolutionary diversity in eukaryotes. Here, we identified TDA5 as a yeast ortholog of DHRSX. Deletion of TDA5 caused glycosylation defects, reduced dolichol levels, and accumulated polyprenol. All these phenotypes were rescued by expression of DHRSX, but not by DFG10 or SRD5A3. These findings show that Tda5 serves the same function as DHRSX in yeast, thereby demonstrating conservation of the three-step detour pathway in yeast and supporting a broader eukaryotic framework for dolichol biosynthesis.
    Keywords:  DHRSX; TDA5; congenital disorders of glycosylation (CDGs); dolichol; yeast
    DOI:  https://doi.org/10.1073/pnas.2613147123
  54. bioRxiv. 2026 May 14. pii: 2026.05.12.724395. [Epub ahead of print]
    Small-molecule modulators of HIPK4 activity and proteostasis.
    Zaile Zhuang, Riley K Togashi, Patrick Kearney, Ian Pass, Steven M Swick, Fu-Yue Zeng, Andrey A Bobkov, Lynn M Fujimoto, Shubhankar Dutta, Athina Zerva, Nicolai D Raig, Debasmita Saha, Atoosa Emami, Martin P Schwalm, Bradley K Moon, Samuel T Howard, Stefan Knapp, Thomas Hanke, Thomas D Y Chung, James K Chen.
      Homeodomain-interacting protein kinase 4 (HIPK4) is a dual-specificity kinase that is predominantly expressed in differentiating spermatids, required for sperm development, and a promising target for nonhormonal male contraception. Genetic and functional studies have established an essential role for HIPK4 in spermiogenesis, where it acts at least in part through regulation of the F-actin-scaffolded acroplaxome during spermatid head shaping. The direct molecular targets of HIPK4 and their downstream effectors remain poorly defined, and small-molecule probes would be versatile tools for further investigating HIPK4 functions. Synthetic HIPK4 ligands could also be valuable leads for the development of nonhormonal male contraceptives. Here, we report the discovery of a cyanoquinoline-based series of HIPK4 inhibitors with nanomolar potency. Our lead compounds are selective for HIPK4, both within the HIPK family and across the broader kinome, establishing this scaffold as a useful starting point for probe and lead development. Unexpectedly, we found that a subset of these cyanoquinolines also perturbs HIPK4 proteostasis in a cell type-specific manner. In spermatids, these compounds induce the formation of detergent-insoluble HIPK4 aggregates and promote interactions between this kinase and the autophagy receptor Tax1-binding protein 1 (TAX1BP1). Together, our findings establish cyanoquinoline ligands as a new chemotype for probing HIPK4 biology and advancing male contraceptive discovery.
    DOI:  https://doi.org/10.64898/2026.05.12.724395
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