bims-proteo Biomed News
on Proteostasis
Issue of 2025–10–12
sixty-two papers selected by
Eric Chevet, INSERM
  1. UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation.
  2. UFM1 at the endoplasmic reticulum: linking ER stress, ribosome quality control, and ER-phagy.
  3. Atg2/TRAPPIII-Ypt1 axis: deciphering the phagophore-ERES connection.
  4. WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2.
  5. Hyperactive 20S proteasome enhances proteostasis and ERAD in C. elegans via degradation of intrinsically disordered proteins.
  6. Structural basis for DCAF2 as a novel E3 ligase for PROTAC-mediated targeted protein degradation.
  7. ER biogenesis without stress: how the ribosome receptor, p180, defines a developmental program beyond the UPR.
  8. Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies.
  9. ASPL couples the assembly of stress granules with their VCP-mediated disassembly.
  10. Structural polymorphism of α-synuclein fibrils alters the pathway of Hsc70-mediated disaggregation.
  11. Toxoplasma gondii VIP1 mediates parasitophorous vacuole-host endoplasmic reticulum interactions to facilitate parasite development.
  12. A genome-to-proteome map reveals how natural variants drive proteome diversity and shape fitness.
  13. Discovery of a CNS active GSK3 degrader using orthogonally reactive linker screening.
  14. The endoplasmic reticulum-associated degradation machinery selectively degrades stress-induced TIN1 during stress recovery.
  15. Non canonical BRCA1 promotes cell survival via modulating PARP13-mediated SEC61G mRNA decay.
  16. PARP10 is critical for stress granule initiation.
  17. Translational regulation in stress biology.
  18. Canonical translation factors eIF1A and eIF5B modulate the initiation step of repeat-associated non-AUG translation.
  19. Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins.
  20. Pre-N and C-terminal extension regions of Arabidopsis HSP90.7 regulate the chaperone activity and ER stress response.
  21. TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and Parkin-independent mitophagy.
  22. Atg16l1 and Xbp1 cooperatively protect from transcription-associated mutagenesis and small intestinal carcinogenesis.
  23. A pyrophosphatase that regulates lipid precursors of N-glycosylation.
  24. Benchmarking Cofolding Methods for Molecular Glue Ternary Structure Prediction.
  25. Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes.
  26. E3 Ubiquitin ligases Cbl-b and c-Cbl maintain the homeostasis of macrophages by regulating the M-CSF/M-CSFR signaling axis.
  27. Ubiquitin-mediated mitophagy regulates the inheritance of mitochondrial DNA mutations.
  28. The integrated stress response suppresses antiviral RNA interference by autophagy-mediated degradation of the RNA-induced silencing complex.
  29. CRISPR activation of the ribosome-associated quality control factor ASCC3 ameliorates fragile X syndrome phenotypes in mice.
  30. PEX14 acts as a molecular link between optineurin and the autophagic machinery to induce pexophagy.
  31. Murine leukemia virus glycoGag antagonizes SERINC5 via ER-phagy receptor RETREG1.
  32. The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity.
  33. SCYL1 deficiency in CALFAN syndrome is associated with ER stress and cell death.
  34. Generalized design of sequence-ensemble-function relationships for intrinsically disordered proteins.
  35. Hijacking a cellular highway: non-lipidated LC3 proteins and PCNT (pericentrin) drive influenza a virus uncoating.
  36. The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly.
  37. PNPLA3-I148M is a neomorph that interferes with two primary hepatic triglyceride clearance pathways.
  38. IRE1α translational suppression potentiates STING-dependent chemoresistance in pancreatic cancer.
  39. Targeting the ATM-TRMT10A-BRCA1 axis confers synthetic lethality to PARP inhibition in metastatic castration-resistant prostate cancer.
  40. Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity.
  41. Structure of a transient protein-folding intermediate by pressure-jump NMR spectroscopy.
  42. Discovery of a tau-aggregate clearing compound that covalently targets P4HB.
  43. UPF1 shuttles between nucleus and cytoplasm independently of its RNA binding and ATPase activities.
  44. The autophagy-specific exocyst subcomplex contributes to phagophore assembly site integrity by promoting phagophore expansion.
  45. Modular Nanoassemblies Mimicking p62 Aggregates for Targeted Organelle Sequestration and Degradation against Breast Cancer.
  46. E3 Ubiquitin Ligases and Asthma Relief.
  47. Genetic modifiers and ascertainment drive variable expressivity of complex disorders.
  48. Mitochondria-associated membranes (MAMs): molecular organization, cellular functions, and their role in health and disease.
  49. Mechanism of USP21 autoinhibition and histone H2AK119 deubiquitination.
  50. Screening of gene function in cell intoxication by CNF1 links Sec61 translocon to Rac1 GTPase activity.
  51. Mechanisms and Determinants of -1 Ribosome Frameshifting and Bypassing.
  52. Structural phylogenetics unravels the evolutionary diversification of communication systems in gram-positive bacteria and their viruses.
  53. Folding-mediated secretion of pure bispecific antibodies.
  54. DCAF12 Ubiquitin Ligase Promotes Lung Cancer Metastasis by Modulating the TRiC/CCT Chaperonin Complex.
  55. VRK2 targeting potentiates anti-PD-1 immunotherapy in hepatocellular carcinoma through MYC destabilization.
  56. Identification of ligands for E3 ligases with restricted expression using fragment-based methods.
  57. AI662270/GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction.
  58. PPP2/PP2A-mediated dephosphorylation of LC3B links PINK1-PRKN/Parkin-mediated mitophagy to SCA12 pathogenesis.
  59. EIPR1 variants cause a neurodevelopmental disorder with endolysosomal and dense core vesicle defects.
  60. SpatPPI: a geometric deep learning model for predicting protein-protein interactions involving intrinsically disordered regions.
  61. The promiscuous ribosomal P-stalk: a new functional portrait.
  62. A chemotherapy based on dual-targeted release of ciprofloxacin in mitochondrial and endoplasmic reticulum.
  1. Nat Commun. 2025 Oct 09. 16(1): 8973
    UBE2J2 sensitizes the ERAD ubiquitination cascade to changes in membrane lipid saturation.
    Aikaterini Vrentzou, Florian Leidner, Claudia C Schmidt, Helmut Grubmüller, Alexander Stein.
      Protein-lipid crosstalk is fundamental to homeostasis in the endoplasmic reticulum (ER). The ER-associated degradation (ERAD) pathway, a branch of the ubiquitin-proteasome system, maintains ER membrane properties by degrading lipid metabolic enzymes. However, the ERAD components that sense membrane properties and their mechanisms remain poorly defined. Using reconstituted systems with purified ERAD factors, we show that membrane composition modulates the ubiquitination cascade at multiple levels. The membrane-anchored E2 UBE2J2 acts as a sensor for lipid packing: in loosely packed membranes, UBE2J2 becomes inactive due to membrane association that impedes ubiquitin loading, while tighter packing promotes its active conformation and interaction with E1. UBE2J2 activity directs ubiquitin transfer by the E3 ligases RNF145, MARCHF6, and RNF139, targeting both themselves and the substrate squalene monooxygenase. Additionally, RNF145 senses cholesterol, altering its oligomerization and activity. These findings reveal that ERAD integrates multiple lipid signals, with UBE2J2 relaying and extending the effect of lipid signals through its cooperation with multiple E3 ligases.
    DOI:  https://doi.org/10.1038/s41467-025-64777-1
  2. Essays Biochem. 2025 Oct 09. pii: EBC20253054. [Epub ahead of print]
    UFM1 at the endoplasmic reticulum: linking ER stress, ribosome quality control, and ER-phagy.
    Masaaki Komatsu, Gaoxin Mao.
      Ubiquitin-fold modifier 1 (UFM1) is a small protein that functions as a ubiquitin-like modifier attached to other proteins to alter their behavior. Although less famous than ubiquitin, UFM1 has gained attention as a key regulator of proteostasis (protein homeostasis) in the cell. Notably, the endoplasmic reticulum (ER) has emerged as the central stage for UFM1's activity. UFM1 was initially recognized for its role in the ER stress response, and we now know it orchestrates two critical quality-control processes at the ER: ribosome-associated quality control and selective autophagy of the ER. Together, these mechanisms ensure that the cell can cope with misfolded proteins and stalled ribosomes, maintaining the health of the ER and the proteins it produces. In this review, we will explore how UFM1 works at the ER, how its components are regulated during stress, how it facilitates both immediate quality control and longer-term ER turnover, and how disruptions in this system lead to disease, especially in the nervous system.
    Keywords:  encephalopathy; endoplasmic reticulum; proteostasis
    DOI:  https://doi.org/10.1042/EBC20253054
  3. Autophagy. 2025 Oct 08. 1-3
    Atg2/TRAPPIII-Ypt1 axis: deciphering the phagophore-ERES connection.
    Rubén Gómez-Sánchez, J Christopher Fromme, Christian Ungermann, Fulvio Reggiori.
      De novo generation of membrane contact sites (MCSs) between the nascent phagophore and the endoplasmic reticulum (ER), particularly the ER exit sites (ERES), are crucial for autophagy as they provide the lipids necessary for the phagophore expansion into an autophagosome. Our recent study provides insights into the mechanism involved in the formation of phagophore-ERES MCSs and uncovers how this event synchronizes the factors involved in phagophore expansion. We revealed that the TRAPPIII complex, the guanine nucleotide exchange factor of the Rab GTPase Ypt1, and the lipid transfer protein Atg2 participate in the phagophore-ERES association. We also show that establishment of phagophore-ERES MCSs leads to TRAPPIII activation and subsequent Ypt1 recruitment onto the phagophore. The presence of active Ypt1 on the growing phagophore enhances local biosynthesis of phosphatidylinositol-3-phosphate (PtdIns3P), triggering the recruitment of the PtdIns3P-effectors Atg18 and Atg21, which play a central role in phagophore expansion. These findings suggest that generation of phagophore-ERES MCSs is one of the signals initiating phagophore expansion.Abbreviations: Atg, autophagy related; ER, endoplasmic reticulum; ERES, ER exit sites; GEF, guanine nucleotide exchange factor; MCS, membrane contact site; PAS, phagophore assembly site; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns3K, phosphatidylinositol 3-kinase; SNARE, soluble NSF attachment protein receptor; TOR, Target of Rapamycin; WIPI, WD-repeat domain, phosphoinositide interacting.
    Keywords:  Atg2; Atg9; TRAPPIII; Ypt1; autophagy; membrane contact sites; phagophore
    DOI:  https://doi.org/10.1080/15548627.2025.2571682
  4. Nat Commun. 2025 Oct 09. 16(1): 8983
    WEE1 inhibitors synergise with mRNA translation defects via activation of the kinase GCN2.
    Jordan C J Wilson, JiaYi Zhu, Vanesa Vinciauskaite, Eloise G Lloyd, Simon Lam, Alexandra Hart, Chen Gang Goh, Fadia Bou-Dagher, Hlib Razumkov, Lena Kobel, Zacharias Kontarakis, John Fielden, Moritz F Schlapansky, Joanna I Loizou, Andreas Villunger, Jacob E Corn, Giulia Biffi, Glenn R Masson, Stefan J Marciniak, Aldo S Bader, Stephen P Jackson.
      Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small-molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors.
    DOI:  https://doi.org/10.1038/s41467-025-64050-5
  5. Sci Adv. 2025 Oct 10. 11(41): eadx3014
    Hyperactive 20S proteasome enhances proteostasis and ERAD in C. elegans via degradation of intrinsically disordered proteins.
    David Salcedo-Tacuma, Nadeem Asad, Md Qamrul Islam, Raymond Anderson, David M Smith.
      Age-related proteinopathies, including Alzheimer's and Parkinson's disease, are driven by toxic accumulation of misfolded and intrinsically disordered proteins (IDPs) that overwhelm cellular proteostasis. The proteasome clears these proteins, but its failure in disease remains unclear. We engineered a Caenorhabditis elegans model with a hyperactive 20S proteasome (α3ΔN) for selective 20S activation. α3ΔN markedly enhanced IDP and misfolded protein degradation, reduced oxidative damage, and improved endoplasmic reticulum-associated degradation (ERAD). Aggregation-prone substrates such as vitellogenins and human alpha-1 antitrypsin (ATZ) were efficiently cleared. Integrated proteomic and transcriptomic analyses reveal systemic adaptations featuring increased protein turnover and oxidative stress resistance independent of superoxide dismutases (SODs). Notably, α3ΔN extended life span and stress resistance independently of canonical unfolded protein response (UPR) signaling via xbp-1. These findings substantiate a "20S pathway" of proteostasis that directly alleviates protein aggregation and oxidative stress, offering a promising therapeutic angle for neurodegenerative diseases.
    DOI:  https://doi.org/10.1126/sciadv.adx3014
  6. Structure. 2025 Oct 03. pii: S0969-2126(25)00349-1. [Epub ahead of print]
    Structural basis for DCAF2 as a novel E3 ligase for PROTAC-mediated targeted protein degradation.
    Evan J McMahon, Alexander G Cioffi, Patrick R Visperas, Yueqing Lin, Michael Shaghafi, Courtney M Daczkowski, Johannes C Hermann, Robert A Everley, Richard M Neve, Daniel A Erlanson, Kevin R Webster, Vikram Narayan, Weiru Wang.
      Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 human E3 ligases. To expand this repertoire, we conducted structural and functional validation of DDB1 (Damage-specific DNA binding protein 1) and Cullin-associated factor (DCAF)2 (DTL/CDT2), a Cullin4-RING ligase substrate adaptor implicated in DNA damage response and cancer, as a novel E3 for TPD. Cryoelectron microscopy (cryo-EM) structures of the DCAF2:DDB1:DDA1 complex (3.3 Å), a ligand bound complex (3.1 Å), and a ternary complex with a covalent proteolysis-targeting chimera (PROTAC) and BRD4 (3.4 Å) reveal PROTAC-mediated substrate recruitment. Using covalent bifunctional tool compounds engaging residue C141 in the WD40 domain, we demonstrate robust ubiquitination in biochemical assays and cellular TPD using the COFFEE (covalent functionalization followed by E3 electroporation) method. These findings position DCAF2 as a promising E3 adaptor for PROTAC strategies and identify C141 as a relevant site for future PROTAC discovery.
    Keywords:  BRD4 degradation; Cullin4-RING ligase; DCAF2; E3 ligase; PROTAC; cryogenic electron microscopy; protein structure; targeted protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.str.2025.09.006
  7. Front Cell Dev Biol. 2025 ;13 1682420
    ER biogenesis without stress: how the ribosome receptor, p180, defines a developmental program beyond the UPR.
    Payam Benyamini, David I Meyer.
      The endoplasmic reticulum (ER) plays a central role in protein and lipid biosynthesis, quality control, and secretion. While its functional roles are well characterized, the mechanisms underlying ER biogenesis remain less defined. Developmental transitions in secretory tissues such as liver, pancreas, mammary gland, and plasma cells illustrate the remarkable capacity to expand their ER network in response to physiological demand. Central to this process is the ribosome receptor p180, a vertebrate-specific integral ER membrane protein whose expression is both necessary and sufficient for rough ER proliferation. Studies in yeast first demonstrated that overexpression of membrane proteins, including HMG-CoA reductase and domains of p180, induces membrane proliferation, thereby establishing yeast as a tractable model for ER biogenesis. In mammalian systems, p180 uniquely links membrane protein expression with biosynthetic scaling, enhancing ribosome binding, mRNA stabilization, lipid biosynthesis, and Golgi biogenesis. Gain- and loss-of-function approaches in human monocytic THP-1 cells confirm that p180 is indispensable for establishing a high-capacity secretory cells phenotype, coordinating the transition from sparse to abundant rough ER and secretory output. Importantly, p180-driven ER proliferation occurs independently of the unfolded protein response (UPR), highlighting distinct yet complementary mechanisms of ER remodeling: p180 as a constitutive biosynthetic scaffold and the UPR as a stress-induced regulator. Together, these findings position p180 as a master determinant of secretory architecture, with implications for development, immunity, and disease. Understanding the molecular underpinnings of p180 function and its integration with lipid metabolism and translation control will advance both basic cell biology and therapeutic strategies targeting secretory dysfunction. Recent work also suggests that p180-mediated ER expansion is dynamically tuned to nutrient availability and growth factor signaling, further linking organelle biogenesis to cellular metabolism. Dysregulation of p180 expression or function may contribute to a variety of pathological states such as cancer, neuronal dysregulation, and atherosclerosis where ER homeostasis is disrupted. Due to its vertebrate-specific origin, p180 also represents an evolutionary concerved lineage that enabled the diversification of complex secretory systems. Ultimately, dissecting the molecular circuits that govern p180 function promises to refine our understanding of organelle plasticity and to identify novel targets for therapeutic intervention.
    Keywords:  P180; endoplasmic reticulum stress; endoplasmic reticulum vesicular trafficking; ribosome receptor; rough ER; rough endoplasmic reticulum; unfolded protein response; upr
    DOI:  https://doi.org/10.3389/fcell.2025.1682420
  8. Proc Natl Acad Sci U S A. 2025 Oct 14. 122(41): e2511840122
    Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies.
    Jasleen Singh, Justin Krish Williams, Quinn Elliott, Rohit Jhawar, Lucas Ferguson, Kathleen Collins, Randy Schekman.
      All eukaryotic cells secrete exosomes, a type of extracellular vesicles derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids, and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here, we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs, small-nucleolar RNAs, and small-nuclear RNAs traffic to MVBs. We show that this RNA-trafficking requires the function of endosomal sorting complexes required for transport (ESCRT) machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes, such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway.
    Keywords:  RNA-trafficking; RNPs; endosomes; exosomes; intraluminal vesicles (ILVs)
    DOI:  https://doi.org/10.1073/pnas.2511840122
  9. Sci Adv. 2025 Oct 10. 11(41): eady3735
    ASPL couples the assembly of stress granules with their VCP-mediated disassembly.
    Gautam Pareek, Dongfang Li, Bo Wang, Jinjun Wu, Brian D Freibaum, Joseph L Basalla, Tharun Selvam Mahendran, Anurag Singh, Amanda Nourse, Honghu Quan, Ravi Kalathur, Mitra S Rana, Brian Maxwell, Yong-Dong Wang, James Messing, Yonghui Ni, Stanley Pounds, Rachayata Dharmat, Jingjun Lu, Xiujie Li-Harms, Alexandre F Carisey, Shondra M Pruett-Miller, J Paul Taylor, Priya Banerjee, Hong Joo Kim, Mondira Kundu.
      Stress granules (SGs) are dynamic RNA-protein assemblies that form in response to cellular stress and must be efficiently disassembled to restore normal cell function. Valosin-containing protein (VCP), an enzyme implicated in neurodegenerative diseases, is essential for SG disassembly, but whether and how this process is coordinated with SG assembly remains unclear. Here, we identify the VCP cofactor, Alveolar soft part sarcoma locus (ASPL) as a key regulator linking SG assembly and disassembly. ASPL promotes SG assembly by facilitating biomolecular condensation of Ras guanosine triphosphatase-activating protein-binding protein (G3BP) and stabilizing its interactions with other SG proteins. ASPL also facilitates phosphorylation and activation of VCP by UNC-51-like kinases 1 and 2 (ULK1/2), enabling G3BP extraction and efficient SG disassembly. Pathogenic VCP mutations that disrupt ASPL binding impair SG disassembly, a defect rescued by phosphomimetic mutations or ASPL depletion. Our findings suggest that disruptions in the ASPL-VCP interaction uncouple SG assembly and disassembly, representing a potential mechanism underlying VCP-associated neurodegenerative diseases.
    DOI:  https://doi.org/10.1126/sciadv.ady3735
  10. EMBO J. 2025 Oct 06.
    Structural polymorphism of α-synuclein fibrils alters the pathway of Hsc70-mediated disaggregation.
    Svenja Jäger, Jessica Tittelmeier, Thi Lieu Dang, Tracy Bellande, Virginie Redeker, Alexander K Buell, Janosch Hennig, Ronald Melki, Carmen Nussbaum-Krammer, Bernd Bukau, Anne S Wentink.
      Pathological aggregation of α-synuclein into amyloid fibrils is a hallmark of synucleinopathies, including Parkinson's disease. Despite this commonality, synucleinopathies display divergent disease phenotypes that have been attributed to disease-specific three-dimensional structures of α-synuclein fibrils, each with unique toxic gain-of-function profiles. The Hsc70 chaperone is remarkable in its ability to disassemble pre-existing amyloid fibrils of different proteins in an ATP and co-chaperone-dependent manner. We find, however, using six well-defined conformational polymorphs of α-synuclein fibrils, that the activity of the Hsc70 disaggregase machinery is sensitive to differences in the amyloid conformation, confirming that fibril polymorphism directly affects interactions with the proteostasis network. Amyloid conformation influences not only how efficiently fibrils are cleared by the Hsc70 machinery but also the balance between depolymerization and fragmentation during disaggregation. We further show that, in vitro, the active processing of fibrils by the Hsc70 machinery inadvertently produces seeding competent species that further promote protein aggregation. Amyloid conformation thus is an important feature that can tilt the balance between beneficial or detrimental protein quality control activities in a disease-context.
    Keywords:  Alpha-synuclein; Disaggregation; Hsp70; Molecular chaperones; Polymorphism
    DOI:  https://doi.org/10.1038/s44318-025-00573-3
  11. Nat Microbiol. 2025 Oct 10.
    Toxoplasma gondii VIP1 mediates parasitophorous vacuole-host endoplasmic reticulum interactions to facilitate parasite development.
    Julia D Romano, Ruth Buh, Tanner Grudda, Julia R Box, John Beltran, Shahbaz M Khan, Isabelle Coppens.
      Membrane contact sites (MCS) are areas of close apposition between organelles without membrane fusion, allowing for exchange of biomolecules. The endoplasmic reticulum (ER) forms many MCS via two proteins, vesicle-associated membrane protein-associated proteins A and B (VAPA and VAPB). The obligate intracellular parasite Toxoplasma gondii resides within mammalian cells in a parasitophorous vacuole (PV), which closely contacts the host ER at distances compatible with MCS. However, the proteins mediating this interaction remain largely unknown. Here, using molecular and microscopy approaches, we show that VAPA and VAPB localize at the PV membrane and, with motile sperm domain-containing protein 2 (MOSPD2), mediate ER-PV interactions. Cells deficient in VAPA, VAPB and MOSPD2 do not recruit host ER at the PV, and parasites show growth defects. We identify a parasite protein that localizes at the PV membrane, called TgVIP1, which harbours an FFAT-like motif that binds VAPA and VAPB. These findings lay the basis for understanding how and why Toxoplasma exploits ER-PV interactions and may uncover new drug targets.
    DOI:  https://doi.org/10.1038/s41564-025-02144-y
  12. Science. 2025 Oct 09. 390(6769): eadu3198
    A genome-to-proteome map reveals how natural variants drive proteome diversity and shape fitness.
    Christopher M Jakobson, Johannes Hartl, Pauline Trébulle, Michael Mülleder, Daniel F Jarosz, Markus Ralser.
      Understanding how genetic variation translates into complex phenotypes remains a fundamental challenge. In this work, we address this by mapping genome-to-proteome relationships in 800 progeny of a cross between two yeast strains adapted to distinct environments. Despite the modest genetic distance between the parents, we observed notable proteomic diversity and mapped more than 6400 genotype-protein associations, with more than 1600 linked to individual genetic variants. Proteomic adaptation emerged from a conserved network of cis- and trans-regulatory variants, often originating from proteins not traditionally linked to gene regulation. This atlas allowed us to forecast organismal fitness effects across diverse conditions. By connecting genomic and proteomic landscapes at unprecedented resolution, our study provides a framework for predicting the phenotypic outcomes of natural genetic variation.
    DOI:  https://doi.org/10.1126/science.adu3198
  13. Nat Commun. 2025 Oct 06. 16(1): 8857
    Discovery of a CNS active GSK3 degrader using orthogonally reactive linker screening.
    Andreas Holmqvist, Nur Mehpare Kocaturk, Christina Duncan, Jennifer Riley, Steven Baginski, Graham Marsh, Joel Cresser-Brown, Hannah Maple, Kristiina Juvonen, Gajanan Sathe, Nicola Morrice, Calum Sutherland, Kevin D Read, William Farnaby.
      Bifunctional targeted protein degraders, also known as Proteolysis Targeting Chimeras (PROTACs), are an emerging drug modality that may offer a new approach for treating neurodegenerative diseases. Identifying chemical starting points for PROTACs remains a largely empirical process and the design rules for identifying Central Nervous System (CNS) active PROTACs have yet to be established. Here we demonstrate a concept of using orthogonally reactive linker reagents, that allow the construction of screening libraries whereby the E3 ligase binder, the target protein binder and the linker can be simultaneously varied and tested directly in cellular assays. This approach enabled the discovery of Glycogen Synthase Kinase 3 (GSK3) PROTACs which are CNS in vivo active in female mice. Our findings provide opportunities to investigate the role of GSK3 paralogs in cellular and in vivo disease models and for the rapid discovery of in vivo quality bifunctional chemical probes for CNS disease concepts.
    DOI:  https://doi.org/10.1038/s41467-025-63928-8
  14. Plant Physiol. 2025 Oct 06. pii: kiaf489. [Epub ahead of print]
    The endoplasmic reticulum-associated degradation machinery selectively degrades stress-induced TIN1 during stress recovery.
    Yi Wang, Zhihui Ma, Jiarui Wu, Congcong Zhang, Yongwu Chen, Liangguang Lin, Juan Mao, Jianjun Zhang, Linchuan Liu, Pengcheng Wang, Jianming Li.
      The unfolded protein response (UPR) signaling pathway is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER) and stimulates production of ER chaperones to restore ER proteostasis. However, how UPR-induced proteins return to their pre-stress levels upon removal of ER stress remains unknown. TUNICAMYCIN-INDUCED1 (TIN1) is an Arabidopsis (Arabidopsis thaliana) protein that is normally expressed in pollen but is rapidly induced by ER stresses in vegetative tissues. Here we show that the ER stress-induced TIN1 is rapidly degraded in the UPR recovery phase. We found that TIN1 degradation depends on its asparagine-linked glycans and requires both EMS-mutagenized bri1 suppressor 5 (EBS5) and EBS6 for its recruitment to the ER-associated degradation (ERAD) complex. Loss-of-function mutations in the core component of this Arabidopsis ERAD complex greatly stabilize TIN1. Interestingly, two other UPR-induced proteins that are coexpressed with TIN1 remained stable upon ER stress removal, suggesting that rapid degradation during the stress-recovery phase likely applies to a subset of UPR-induced proteins. Further investigation is needed to uncover the mechanisms by which the ERAD machinery selectively degrades UPR-induced ER proteins.
    Keywords:  ER-associated degradation; N-glycan; TIN1; UPR; stress recovery
    DOI:  https://doi.org/10.1093/plphys/kiaf489
  15. Oncogenesis. 2025 Oct 06. 14(1): 35
    Non canonical BRCA1 promotes cell survival via modulating PARP13-mediated SEC61G mRNA decay.
    Binghe Sun, Yuting Li, Yue Wu, Cong Wang, Jiawei Zhu, Wanying Zhang, Dake Li, Wancai Que, Guo Chen.
      BRCA1, a well-known tumor suppressor, maintains genomic integrity by facilitating homologous recombination (HR) repair and protecting DNA replication forks. However, its roles beyond DNA repair and replication remain largely unexplored. Here, we demonstrate that BRCA1 interacts with the RNA-binding protein PARP13 in the cytoplasm of ovarian cancer cells, with DNA damage enhancing this interaction via DNA-PK. Notably, BRCA1/PARP13 association is essential for cell survival but does not influence DNA repair efficacy following DNA damage. Mechanistically, PARP13 binds and destabilizes the mRNA of the endoplasmic reticulum (ER) membrane protein SEC61G. Upon DNA damage, BRCA1 disrupts PARP13-mediated SEC61G mRNA decay, leading to SEC61G upregulation. Elevated SEC61G levels cause calcium leakage from the ER into the cytosol, activating the pro-survival kinase Akt. These findings identify the BRCA1-PARP13-SEC61G axis as a non-canonical DNA damage response (DDR) pathway and highlight mRNA stability and ER calcium signaling as potential therapeutic targets to overcome chemoresistance. The schematic illustrates the mechanism by which non-canonical BRCA1 promotes cell survival through regulating PARP13-mediated SEC61G mRNA decay. Under DNA damage-free conditions (left panel), SEC61G maintains cellular calcium homeostasis between the endoplasmic reticulum and cytosol to support normal physiological functions. Upon DNA damage induction (right panel), enhanced interaction between BRCA1 and PARP13 attenuates the mRNA degradation activity of PARP13 toward SEC61G, leading to upregulated SEC61G protein expression. The aberrant accumulation of SEC61G triggers endoplasmic reticulum calcium leakage, which subsequently activates the AKT signaling pathway to enhance cell survival capacity.
    DOI:  https://doi.org/10.1038/s41389-025-00578-x
  16. Life Sci Alliance. 2025 Dec;pii: e202403026. [Epub ahead of print]8(12):
    PARP10 is critical for stress granule initiation.
    Aravinth Kumar Jayabalan, Krishna Bhambhani, Anthony Kl Leung.
      Stress granules (SGs) are cytoplasmic biomolecular condensates enriched with RNA and translation factors. They form in response to stress, in part through phosphorylation of the translation initiation factor eIF2α, and are implicated in viral infection, tumorigenesis, and neurodegeneration. Although ADP-ribosylation plays a key role in SG assembly, the enzyme responsible for this ADP-ribosylation during SG assembly remains unidentified. Here, we systematically knock down the human ADP-ribosyltransferase family and identify PARP10 as pivotal for SG assembly. Live-cell imaging reveals PARP10's crucial role in regulating initial SG assembly kinetics. Further, we pinpoint the core SG component, G3BP1, as a PARP10 substrate and find that PARP10 regulates SG assembly via G3BP1 or a synthetic mimic that recapitulates its domain architecture. PARP10 knockdown reduces eIF2α phosphorylation and alters the SG core composition, notably decreasing translation factor presence. Based on our findings, we propose a model in which ADP-ribosylation acts as a rate-limiting step, initiating the formation of SGs.
    DOI:  https://doi.org/10.26508/lsa.202403026
  17. Nat Cell Biol. 2025 Oct 07.
    Translational regulation in stress biology.
    Naomi R Genuth, Andrew Dillin.
      Organisms must constantly respond to stress to maintain homeostasis, and the successful implementation of cellular stress responses is directly linked to lifespan regulation. In this Review we examine how three age-associated stressors-loss of proteostasis, oxidative damage and dysregulated nutrient sensing-alter protein synthesis. We describe how these stressors inflict cellular damage via their effects on translation and how translational changes can serve as both sensors and responses to the stressor. Finally, we compare stress-induced translational programmes to protein synthesis alterations that occur with age and discuss whether these changes are adaptive or deleterious to longevity and healthy ageing.
    DOI:  https://doi.org/10.1038/s41556-025-01765-z
  18. Nucleic Acids Res. 2025 Sep 23. pii: gkaf994. [Epub ahead of print]53(18):
    Canonical translation factors eIF1A and eIF5B modulate the initiation step of repeat-associated non-AUG translation.
    Hayato Ito, Kodai Machida, Yuzo Fujino, Mayuka Hasumi, Soyoka Sakamoto, Yoshitaka Nagai, Hiroaki Imataka, Hideki Taguchi.
      Nucleotide repeat expansions, such as the GGGGCC repeats in C9orf72, associated with C9-ALS, are linked to neurodegenerative diseases. These repeat sequences undergo a noncanonical translation known as repeat-associated non-AUG (RAN) translation. Unlike canonical translation, RAN translation initiates from non-AUG codons and occurs in all reading frames. To identify potential regulators of RAN translation, we employed a bottom-up approach using a human factor-based reconstituted cell-free translation system to recapitulate RAN translation. This approach revealed that omission of either eIF1A or eIF5B enhanced the translation in all reading frames of C9orf72-mediated RAN translation (C9-RAN), suggesting that eIF1A and eIF5B act as repressors of RAN translation. eIF1A and eIF5B are known to contribute to the fidelity of translation initiation. In HEK293T cells, double knockdown of eIF1A and eIF5B further promoted C9-RAN compared to single knockdowns, indicating that these factors regulate C9-RAN through distinct initiation steps. Furthermore, under eIF1A knockdown conditions, the enhancement of RAN translation via the integrated stress response (ISR) was not observed in HEK293T cells, indicating that eIF1A is involved in the ISR-mediated non-AUG translation.
    DOI:  https://doi.org/10.1093/nar/gkaf994
  19. Nat Commun. 2025 Oct 10. 16(1): 9035
    Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins.
    Xinyu Gong, Yuqian Zhou, Yingli Wang, Yubin Tang, Haobo Liu, Xindi Zhou, Yuchao Zhang, Hanbo Guo, Zhenpeng Guo, Lifeng Pan.
      Macroautophagy maintains cellular and organismal homeostasis, and entails de novo synthesis of double-membrane autophagosome. The effective formation of autophagosome requires the recruitment of the ATG12~ATG5-ATG16L1 complex to the pre-autophagosomal structure by relevant ATG16L1-binding autophagic factors including FIP200. However, the molecular mechanism governing the specific interaction of ATG16L1 with FIP200 remains elusive. Here, we uncover that ATG16L1 contains a FIP200-interacting region (FIR), which not only can directly bind FIP200 Claw domain, but also can serve as an atypical ATG8-interacting motif to selectively recognize mammalian ATG8 family proteins (ATG8s). We determine the high-resolution crystal structures of ATG16L1 FIR in complex with FIP200 Claw and GABARAPL1, respectively, and elucidate the molecular mechanism underlying the interactions of ATG16L1 with FIP200 and ATG8s. To distinguish the precise contribution of FIP200 from ATG8s for binding to ATG16L1 FIR in autophagy, we develop a ATG16L1 mutant that can exclusively interact with ATG8s but not FIP200. Finally, using relevant cell-based functional assays, we demonstrate that the interaction of ATG16L1 with FIP200 is indispensable for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the interactions of ATG16L1 with FIP200 and ATG8s, and are valuable for further understanding the function of ATG16L1 in autophagy.
    DOI:  https://doi.org/10.1038/s41467-025-64097-4
  20. J Biol Chem. 2025 Oct 08. pii: S0021-9258(25)02658-4. [Epub ahead of print] 110806
    Pre-N and C-terminal extension regions of Arabidopsis HSP90.7 regulate the chaperone activity and ER stress response.
    Jenan Noureddine, Adheip Monikantan Nair, Kenneth Andrei Espinosa, Linlin Fan, Joyce Cheng, Rongmin Zhao.
      The ER-localized molecular chaperone HSP90.7 plays a critical role in maintaining protein homeostasis in plants, particularly under stress conditions. However, the functional roles of its pre-N and C-terminal extension (CTE) regions remain poorly understood. In this study, we integrated molecular dynamics simulations, in vitro biochemical assays, and in vivo mutant analysis to investigate the roles of these regions. Deletion of either region did not affect normal seedling development but conferred pronounced hypersensitivity to endoplasmic reticulum (ER) stress. Molecular dynamics simulations revealed that both the pre-N and CTE form regulatory contacts with HSP90.7's N-terminal, middle, and C-terminal domains, likely modulating the chaperone's global stability and interdomain communication. Consistent with these findings, removing the pre-N region increased ATPase activity and altered ATP-binding kinetics, consistent with prior reports for mammalian GRP94, whereas deleting the CTE diminished ATP-independent holdase function. Thus, our findings highlight a conserved regulatory role of the pre-N across ER-localized HSP90s. Together, our results underscore the significance of the pre-N and CTE regions for HSP90.7's functional cycle and establish their specialized roles in ER homeostasis and plant stress resilience.
    Keywords:  Calcium homeostasis; Endoplasmic reticulum (ER); Glucose response protein 94 (GRP94); Molecular chaperone; Plant development; Plant molecular biology; heat shock protein 90 (HSP90)
    DOI:  https://doi.org/10.1016/j.jbc.2025.110806
  21. Sci Adv. 2025 Oct 10. 11(41): eadw4153
    TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and Parkin-independent mitophagy.
    Elena Levi-D'Ancona, Emily M Walker, Jie Zhu, Yamei Deng, Vaibhav Sidarala, Ava M Stendahl, Emma C Reck, Belle A Henry-Kanarek, Anne C Lietzke, Biaoxin Chai, Mabelle B Pasmooij, Dre L Hubers, Venkatesha Basrur, Sankar Ghosh, Linsey Stiles, Alexey I Nesvizhskii, Orian S Shirihai, Scott A Soleimanpour.
      Innate immune signaling is activated in immunometabolic diseases, including type 2 diabetes, yet its impact on glucose homeostasis is controversial. Here, we report that the E3 ubiquitin ligase TRAF6 integrates innate immune signals following diet-induced obesity to promote glucose homeostasis through the induction of mitophagy. Whereas TRAF6 was dispensable for pancreatic β cell function at baseline, TRAF6 was pivotal for insulin secretion, mitochondrial respiration, and mitophagy following metabolic stress in mouse and human islets. TRAF6 was critical for the recruitment and function of the ubiquitin-mediated (Parkin-dependent) mitophagy machinery. Glucose intolerance induced by TRAF6 deficiency following metabolic stress was reversed by concomitant Parkin deficiency by relieving obstructions in receptor-mediated (Parkin-independent) mitophagy. Our results establish that TRAF6 is vital for traffic through Parkin-mediated mitophagy and implicates TRAF6 in the cross-regulation of ubiquitin- and receptor-mediated mitophagy. Together, we illustrate that β cells engage innate immune signaling to adaptively respond to a diabetogenic environment.
    DOI:  https://doi.org/10.1126/sciadv.adw4153
  22. Oncogene. 2025 Oct 07.
    Atg16l1 and Xbp1 cooperatively protect from transcription-associated mutagenesis and small intestinal carcinogenesis.
    Nassim Kakavand, Hang Xiang, Georg Laue, Taous Mekdoud, Lina Welz, Miguel Gomes Silva, Joana P Bernardes, Go Ito, Silke van den Bossche, Julia Kugler, Florian Tran, Alexander Ossysek, Simon Imm, Finn Hinrichsen, Moritz Jesinghaus, Arthur Kaser, Richard Blumberg, Timon E Adolph, Stefan Schreiber, Markus Tschurtschenthaler, Philip Rosenstiel, Konrad Aden.
      Atg16l1 plays a critical role in autophagy, and Xbp1 is part of the endoplasmic reticulum (ER) homeostasis. Both, Atg16l1 and Xbp1 are known risk genes for inflammatory bowel disease (IBD). Previous studies have shown that dysfunctional Atg16l1 and Xbp1 are epithelial-derived drivers of small intestinal inflammation. Despite a clear link between Crohn's disease and small intestinal adenocarcinoma, the molecular impact of autophagy and ER stress in this malignant transformation is not known. Using a model of impaired ribonucleotide excision repair (RER), a key homeostatic repair mechanism in highly proliferative cells, we investigated the impact of Atg16l1 on epithelial DNA damage responses and small intestinal carcinogenesis with and without functional ER homeostasis. We used conditional mouse models for deficient RER (Rnaseh2bΔIEC), bearing a co-deletion of disrupted autophagy (Atg16l1/Rnaseh2bΔIEC) or ER stress resolution (Xbp1/Rnaseh2bΔIEC), and triple-conditional knock-out mice for both, Xbp1 and Atg16l1 (Atg16l1/Xbp1/Rnaseh2bΔIEC). We assessed the degree of DNA damage and the incidence of small intestinal carcinoma. We report that defective epithelial RER induces autophagy, and that dysfunctional autophagy increases RER-induced DNA damage and causes the loss of RER-induced proliferative arrest but no spontaneous carcinogenesis in the gut. We demonstrate that dysfunctional Atg16l1 drastically increases the incidence of spontaneous intestinal adenocarcinomas in mice with defective epithelial RER and impaired ER homeostasis. We provide experimental evidence that the same epithelial mechanisms suppressing gut inflammation also critically protect from small intestinal carcinogenesis. Our findings set a molecular framework for the increased risk of intestinal carcinogenesis in patients with IBD, which links perturbations of ER homeostasis and autophagy defects with accumulating DNA damage. In a model of transcription-associated mutagenesis, deficiency of the IBD risk gene Atg16l1 does not induce small intestinal cancer. In contrast, double deficiency of Xbp1 and Atg16l1 drives spontaneous tumor formation highlighting a cooperative role of Xbp1 and Atg16l1 in tumor suppression.
    DOI:  https://doi.org/10.1038/s41388-025-03591-x
  23. J Cell Biol. 2025 Nov 03. pii: e202509041. [Epub ahead of print]224(11):
    A pyrophosphatase that regulates lipid precursors of N-glycosylation.
    Anant K Menon.
      The oligosaccharide used for protein N-glycosylation in the ER is built as a glycolipid. A recent study by Li, Suzuki, and colleagues (https://doi.org/10.1083/jcb.202501239) identifies a long-sought enzyme that hydrolyzes this lipid as part of a possible homeostatic/quality control mechanism.
    DOI:  https://doi.org/10.1083/jcb.202509041
  24. J Chem Inf Model. 2025 Oct 10.
    Benchmarking Cofolding Methods for Molecular Glue Ternary Structure Prediction.
    Yiyan Liao, Jintao Zhu, Juan Xie, Luhua Lai, Jianfeng Pei.
      Molecular glues (MGs) represent an emerging therapeutic paradigm capable of inducing or stabilizing protein-protein interactions (PPIs), with broad applications in creating neomorphic interactomes and targeted protein degradation. However, current discovery efforts remain largely confined to experimental screening, while in silico rational design of MGs remains a formidable challenge. A critical step toward rational design lies in accurate ternary complex modeling, which is less explored and highly challenging due to the involvement of small-molecule-induced de novo PPIs. Here, we tested the ability of recently developed cofolding models, including AlphaFold 3, Boltz-1, Chai-1, Protenix, and RoseTTAFold All-Atom. Although these models were not specifically trained on ternary complex structures, whether their capability to learn diverse interatomic interactions can generalize well to such ternary systems remains an open question. We systematically curated a data set, named MG-PDB, with 221 noncovalent MG-engaged ternary complexes. MGBench was further introduced as a comprehensive benchmark set, which comprises 88 ternary structures excluded from cofolding models' training data through rigorous time-based partitioning. Our benchmark results demonstrated that AlphaFold 3 achieved the best overall performance among cofolding methods, in terms of both PPI interface prediction (50.6% success rate) and MG-protein interaction recovery (32.9% success rate). However, our homology study showed that most of their successful predictions actually stemmed from memorization. Further analysis revealed three phenomena of current cofolding methods for MG ternary structure prediction. First, these methods struggle to accurately model large interaction interfaces. Second, their predictive accuracy is notably reduced for domain-domain complexes compared to domain-motif interactions. Lastly, they face specific challenges in modeling MG degrader complexes with sufficient accuracy. We showcased they relied on the existing interaction patterns and highlighted the need for further improvements in novel E3 ligase systems. These findings reveal fundamental gaps in existing methods to learn atomic-level interaction rules for MG-engaged ternary complex modeling. As fully open resources, MG-PDB and MGBench establish the essential benchmark for MG ternary complex modeling, providing the definitive standard for evaluating future cofolding methods.
    DOI:  https://doi.org/10.1021/acs.jcim.5c01860
  25. J Cell Biol. 2025 Dec 01. pii: e202407013. [Epub ahead of print]224(12):
    Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes.
    Kevin A Swift, Iryna Pustova, William Kasberg, Jenna Bowman, Krithi Gopinath, Erin Voss, Hayden Nelson, Anjon Audhya.
      Protein trafficking within the endosomal system involves several distinct membrane remodeling events, including those with opposing orientations that lead to the production of intraluminal vesicles (ILVs) and recycling tubules. Components of the endosomal sorting complex required for transport (ESCRT) machinery have been implicated in both pathways, although few studies have directly examined their native dynamics in mammalian cells. Here, we demonstrate that the endogenous ESCRT-III subunit Ist1 exists in at least two different pools on endosomes. High-speed, live-cell imaging further showed that one pool of Ist1 forms transiently on endosomes, while the other is relatively stable. However, upon growth factor stimulation, the stable pool of Ist1 becomes more mobile, and the transient pool accumulates more rapidly on endosomes. Our data indicate that ESCRT-III dynamics are distinct from that of other ESCRT complexes and additionally suggest an intrinsic amount of time is required for ESCRT-mediated ILV biogenesis, irrespective of environmental conditions.
    DOI:  https://doi.org/10.1083/jcb.202407013
  26. Cell Death Dis. 2025 Oct 07. 16(1): 716
    E3 Ubiquitin ligases Cbl-b and c-Cbl maintain the homeostasis of macrophages by regulating the M-CSF/M-CSFR signaling axis.
    Fei Xu, Chensheng Tan, Kun Tang, Guodong Qiao, Yu Shao, Xiaoping Li, Ji Zhou, Peijie Zhu, Mengyun Wu, Jiamin Cai, Xiu Gao, Yufeng Wang, Beibei Huang, Wenjun Wang, Tian Xia, Xuena Xu, Jiaoyang Li, Zhengrong Chen, Yufang Shi, Chuangli Hao, Yi Yang, Jinping Zhang.
      The Casitas B-lineage lymphoma (Cbl) family proteins are E3 ubiquitin ligases implicated in the regulation of various immune cells. However, their function in macrophages remains unclear. Here, we identify both Cbl-b and c-Cbl (Cbls) as inhibitors of macrophage proliferation and promoters of macrophage apoptosis. Mechanically, we identify that Cbls functions upstream of AKT and Erk to mediate the ubiquitination and degradation of M-CSFR. M-CSF stimulation promotes dimerization and autophosphorylation activation of M-CSFR on the macrophage membrane, thereby activating downstream PI3K-AKT and Erk signaling pathways, leading to different biological effects such as macrophage proliferation and survival. At the same time, the Y559 site of the M-CSFR undergoes autophosphorylation, which can promote receptor recruitment and phosphorylation of Cbls. This promotes Cbls to induce K63-linked polyubiquitination at the K791 site of M-CSFR, leading to internalization and degradation of M-CSFR through lysosomal pathways, preventing excessive activation of the signaling pathway. Furthermore, Cbls deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and in vivo and dKO mice spontaneously develop a macrophage-dominated pulmonary enlargement. Together, these data demonstrate that Cbls play critical roles in the regulation of macrophage homeostasis by inhibiting M-CSFR-mediated AKT and Erk activation.
    DOI:  https://doi.org/10.1038/s41419-025-08047-4
  27. Science. 2025 Oct 09. 390(6769): 156-163
    Ubiquitin-mediated mitophagy regulates the inheritance of mitochondrial DNA mutations.
    Michele Frison, Brandon S Lockey, Yu Nie, Zoe Golder, Eleni Theiaspra, Cameron D Ryall, Camilla Lyons, Stephen P Burr, Malwina Prater, Lyuba V Bozhilova, Angelos Glynos, James B Stewart, Nick S Jones, Marcos R Chiaratti, Patrick F Chinnery.
      Mitochondrial synthesis of adenosine triphosphate is essential for eukaryotic life but is dependent on the cooperation of two genomes: nuclear and mitochondrial DNA (mtDNA). mtDNA mutates ~15 times as fast as the nuclear genome, challenging this symbiotic relationship. Mechanisms must have evolved to moderate the impact of mtDNA mutagenesis but are poorly understood. Here, we observed purifying selection of a mouse mtDNA mutation modulated by Ubiquitin-specific peptidase 30 (Usp30) during the maternal-zygotic transition. In vitro, Usp30 inhibition recapitulated these findings by increasing ubiquitin-mediated mitochondrial autophagy (mitophagy). We also found that high mutant burden, or heteroplasmy, impairs the ubiquitin-proteasome system, explaining how mutations can evade quality control to cause disease. Inhibiting USP30 unleashes latent mitophagy, reducing mutant mtDNA in high-heteroplasmy cells. These findings suggest a potential strategy to prevent mitochondrial disorders.
    DOI:  https://doi.org/10.1126/science.adr5438
  28. Proc Natl Acad Sci U S A. 2025 Oct 14. 122(41): e2511857122
    The integrated stress response suppresses antiviral RNA interference by autophagy-mediated degradation of the RNA-induced silencing complex.
    Qiyu Wang, Lingyue Chen, Jiayin Wang, Zhijian Chen, Zixian Ma, Xuan Xie, Xiaohui Zeng, Yuanyuan Bie, Zhaowei Wang.
      The small interfering RNA (siRNA) pathway is a highly conserved antiviral defense mechanism in vertebrates and invertebrates. Although the core components of this pathway are well characterized, its upstream regulatory networks remain poorly understood. Here, we identify the integrated stress response (ISR) as a negative regulator of the siRNA pathway, and demonstrate that the picorna-like virus CrPV (Cricket Paralysis Virus) exploits this mechanism for immune evasion. Mechanistically, the picorna-like virus triggers the ISR through transcriptional suppression of ppp1r15, a key regulator of eukaryotic initiation factor 2α (eIF2α) dephosphorylation. ISR activation subsequently induces the autophagy-lysosomal pathway by up-regulating Atg1 transcription in an ATF4-dependent manner. This process leads to selective degradation of Argonaute 2 (Ago2) and other core components of the RNA-induced silencing complex (RISC), thereby suppressing the host RNA interference (RNAi) machinery and enhancing viral replication. Our findings uncover an unconventional immune evasion strategy employed by a picorna-like virus and establish a previously unrecognized crosstalk between the ISR and siRNA pathways.
    Keywords:  RNA interference; autophagy; integrated stress response; picorna-like virus
    DOI:  https://doi.org/10.1073/pnas.2511857122
  29. Sci Transl Med. 2025 Oct 08. 17(819): eadq3551
    CRISPR activation of the ribosome-associated quality control factor ASCC3 ameliorates fragile X syndrome phenotypes in mice.
    Ji Geng, Xiying Wang, Jie Pan, Danish Khan, Sopida Pimcharoen, Yongjie Zhang, Nima Mosammaparast, Susumu Hirose, Leonard Petrucelli, Onn Brandman, Lei S Qi, Bingwei Lu.
      Loss of fragile X messenger ribonucleoprotein (FMRP) causes fragile X syndrome (FXS), an inherited neurodevelopmental disorder resulting in intellectual disability and autism spectrum disorder; however, the molecular function of FMRP remains uncertain. Here, using cell lines and fibroblasts and induced pluripotent stem cell-derived neurons from healthy individuals and patients with FXS, we showed that FMRP regulates collided ribosomes by recruiting activating signal cointegrator 1 complex subunit 3 (ASCC3), an early-acting ribosome-associated quality control (RQC) factor to collided ribosomes, and either positively or negatively regulating translation, depending on transcript context. Disease-associated ASCC3 variants that perturbed ASCC3-FMRP interaction were also found to be defective in ribosome association and handling of collided ribosomes. In cells of a patient with FXS and the Fmr1 KO mouse model, ASCC3 abundance was reduced, and overexpression of ASCC3 in the brains of fetal Fmr1 KO mice promoted neuronal migration. In addition, CRISPR-mediated activation of ASCC3 by lateral ventricular injection of adeno-associated virus (AAV) ameliorated synaptic defects and improved locomotor activity, cognitive deficits, obsessive-compulsive-like behavior, and social interaction deficits after 1 month in 2-month-old Fmr1 KO mice compared with untreated Fmr1 KO controls. In conclusion, these data implicated FMRP in the handling of collided ribosomes to maintain protein homeostasis during neurodevelopment and synaptogenesis and demonstrated proof of concept that targeting RQC may offer alternative treatment strategies for FXS.
    DOI:  https://doi.org/10.1126/scitranslmed.adq3551
  30. J Cell Biol. 2025 Nov 03. pii: e202411184. [Epub ahead of print]224(11):
    PEX14 acts as a molecular link between optineurin and the autophagic machinery to induce pexophagy.
    Hongli Li, Suyuan Chen, Celien Lismont, Bram Vandewinkel, Mohamed A F Hussein, Cláudio F Costa, Dorien Imberechts, Yiyang Liu, Jorge E Azevedo, Wim Vandenberghe, Steven Verhelst, Hans R Waterham, Pieter Vanden Berghe, Myriam Baes, Marc Fransen.
      Pexophagy, the selective degradation of peroxisomes, is essential for removing excess or dysfunctional peroxisomes, and its dysregulation has been linked to various diseases. Although optineurin (OPTN), an autophagy receptor involved in mitophagy, aggrephagy, and xenophagy, has also been implicated in pexophagy in HEK-293 cells, the underlying mechanisms remain unclear. Using proximity labeling, we identified PEX14, a peroxisomal membrane protein, as a neighboring partner of OPTN. Microscopy analyses revealed that clustering of peroxisomes with OPTN is a key feature of OPTN-mediated pexophagy. Biochemical studies demonstrated that PEX14 and OPTN interact through their coiled-coil and ubiquitin-binding domains, respectively. Further analyses showed that the C-terminal half of overexpressed OPTN triggers pexophagy, likely by oligomerizing with endogenous OPTN. The colocalization of PEX14-OPTN complexes with LC3, together with the suppression of OPTN-mediated peroxisome degradation by bafilomycin A1, supports a model in which PEX14 acts as a docking site for OPTN on the peroxisomal membrane, enabling the recruitment of the autophagic machinery for OPTN-mediated pexophagy.
    DOI:  https://doi.org/10.1083/jcb.202411184
  31. PLoS Pathog. 2025 Oct 09. 21(10): e1013023
    Murine leukemia virus glycoGag antagonizes SERINC5 via ER-phagy receptor RETREG1.
    Iqbal Ahmad, Jing Zhang, Rongrong Li, Wenqiang Su, Weiqi Liu, You Wu, Ilyas Khan, Xiaomeng Liu, Lian-Feng Li, Sunan Li, Yong-Hui Zheng.
      Serine incorporator 5 (SERINC5) is a host restriction factor that inhibits the infectivity of certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV), by incorporating into the viral envelope and blocking viral entry. To counteract this, HIV-1 and MLV encode accessory proteins-Nef and glycoGag, respectively-that downregulate SERINC5 expression in producer cells. Here, we demonstrate that glycoGag employs more complex and effective mechanisms than Nef to antagonize SERINC5. Despite being a type II integral membrane protein, glycoGag primarily localizes to the cytoplasm, while Nef is mainly associated with the plasma membrane. Additionally, glycoGag is rapidly degraded by proteasomes, in contrast to the greater stability of Nef, and becomes stabilized after binding to SERINC5. While both proteins downregulate SERINC5 at the cell surface, glycoGag also targets SERINC5 at the endoplasmic reticulum (ER). We further show that this ER-specific downregulation is mediated by reticulophagy regulator 1 (RETREG1), an ER-phagy receptor, through micro-ER-phagy. These findings reveal that retroviruses hijack a selective autophagy pathway to counteract host restriction and promote productive infection.
    DOI:  https://doi.org/10.1371/journal.ppat.1013023
  32. Cell Death Dis. 2025 Oct 06. 16(1): 703
    The deubiquitinase USP9X and E3 ligase WWP1 orchestrate IGF2BP2 ubiquitination homeostasis to drive TNBC progression and cisplatin sensitivity.
    Tian Xia, Jianyi Zhao, Zhengyu Zhang, Weilin Lu, Yuxin Wang, Xinrui Dong, Mingyi Sang, Linjie Ju, Xu Zhang, Jifu Wei, Qiang Ding.
      The functional impact of post-translational modifications (PTMs) on many N6-methyladenosine (m6A) regulatory proteins remains unclear. Our previous study demonstrated that the m6A reader IGF2BP2 drives triple-negative breast cancer (TNBC) progression through epigenetic regulation. Here, we found that IGF2BP2 ubiquitination homeostasis was dynamically regulated by the opposing actions of USP9X (deubiquitinase) and WWP1 (E3 ligase). We further identified USP9X as a cisplatin-binding protein, whose inactivation upon cisplatin treatment shifts this balance toward WWP1-mediated IGF2BP2 degradation in TNBC. This suppressed IGF2BP2-mediated stabilization and translation of m6A-modified MYC/CDK6 mRNAs, thereby inhibiting TNBC progression. Notably, combined USP9X inhibitor WP1130 and low-dose cisplatin showed synergistic therapeutic efficacy against TNBC in both in vivo and in vitro models. Overall, our findings established that the USP9X/WWP1 axis maintained IGF2BP2 ubiquitination homeostasis to regulate m6A-dependent oncogenic functions in TNBC. Crucially, cisplatin uniquely disrupts this balance through USP9X binding, impairing IGF2BP2's m6A recognition capacity and revealing a novel UPS-mediated drug response mechanism specific to TNBC treatment.
    DOI:  https://doi.org/10.1038/s41419-025-08038-5
  33. Dis Model Mech. 2025 Oct 08. pii: dmm.052371. [Epub ahead of print]
    SCYL1 deficiency in CALFAN syndrome is associated with ER stress and cell death.
    John Hellicar, Tal Dattner, Tian Sun, Lily Percival, Ruby Chrisp, Andrea Pietrobattista, Tomasz Witkos, Aleksander Mironov, Lina Leghlam, Carolin Jentsch, Stefan Koelker, Georg F Hoffmann, Christian Staufner, Wanjin Hong, Dominic Lenz, Martin Lowe.
      CALFAN syndrome is a rare genetic disorder affecting the nervous system and liver, with skeletal abnormalities also reported. It is caused by mutations in the gene encoding SCYL1, a ubiquitously expressed protein localized to the secretory pathway. SCYL1 interacts with trafficking components including ARF GTPases and the COPI vesicle coat complex and appears to function in retrograde secretory trafficking. Despite this knowledge, the mechanisms that underlie CALFAN pathology remain poorly understood. Here, using CALFAN patient and SCYL1 knockout fibroblasts we reveal an accumulation of the abundant secretory cargo procollagen type I in the endoplasmic reticulum (ER) upon SCYL1 deficiency. Surprisingly, we failed to observe procollagen-I trafficking defects in the SCYL1-deficient cells. Nevertheless, ER accumulation of procollagen-I correlated with ER distension and induction of ER stress in the patient fibroblasts, which also underwent increased cell death. The phenotypes were observed at elevated temperature, mimicking the induction of pathology under febrile conditions in CALFAN patients. Our data suggest that ER stress induction is a pathological mechanism in CALFAN syndrome, and that targeting this process may represent a therapeutic strategy.
    Keywords:  CALFAN syndrome; ER stress; Golgi apparatus; Procollagen; SCYL1; Secretory pathway
    DOI:  https://doi.org/10.1242/dmm.052371
  34. Nat Comput Sci. 2025 Oct 06.
    Generalized design of sequence-ensemble-function relationships for intrinsically disordered proteins.
    Ryan K Krueger, Michael P Brenner, Krishna Shrinivas.
      The design of folded proteins has advanced substantially in recent years. However, many proteins and protein regions are intrinsically disordered and lack a stable fold, that is, the sequence of an intrinsically disordered protein (IDP) encodes a vast ensemble of spatial conformations that specify its biological function. This conformational plasticity and heterogeneity makes IDP design challenging. Here we introduce a computational framework for de novo design of IDPs through rational and efficient inversion of molecular simulations that approximate the underlying sequence-ensemble relationship. We highlight the versatility of this approach by designing IDPs with diverse properties and arbitrary sequence constraints. These include IDPs with target ensemble dimensions, loops and linkers, highly sensitive sensors of physicochemical stimuli, and binders to target disordered substrates with distinct conformational biases. Overall, our method provides a general framework for designing sequence-ensemble-function relationships of biological macromolecules.
    DOI:  https://doi.org/10.1038/s43588-025-00881-y
  35. Autophagy. 2025 Oct 08.
    Hijacking a cellular highway: non-lipidated LC3 proteins and PCNT (pericentrin) drive influenza a virus uncoating.
    Yingying Cong, Fulvio Reggiori.
      MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) proteins have long been thought to carry out their cellular and organismal functions, including macroautophagy/autophagy, exclusively in their lipidated form, also referred to as Atg8ylation. They are anchored mainly to the phosphatidylethanolamine present in membranes through the action of two ubiquitin-like conjugation systems. Our recent work, however, uncovered a role of non-lipidated LC3s during influenza A virus (IAV) infection. We revealed that LC3s, together with the centrosomal scaffold protein PCNT (pericentrin), form a dynein adaptor complex that facilitates IAV uncoating at late endosomes (LEs). We also showed that co-opting the LC3s-PCNT complex is an alternative strategy to aggresome processing machinery (APM) hijacking via HDAC6, allowing IAV to exploit the force generated by dynein-dependent motors for virion uncoating and genome delivery in the host cytoplasm. Notably, the function of LC3s in IAV uncoating does not require their Atg8ylation or the core autophagy machinery, and PCNT's role is independent from its centrosomal localization. These findings redefine LC3s as multifunctional adaptor proteins and reveal how viruses can co-opt centrosome assembly machinery components for host invasion.
    Keywords:  Atg8ylation; IAV cell entry; LC3-I; centrosome; dynein
    DOI:  https://doi.org/10.1080/15548627.2025.2572527
  36. J Biol Chem. 2025 Oct 04. pii: S0021-9258(25)02637-7. [Epub ahead of print] 110785
    The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly.
    Alexandra Rita Turi da Fonte Dias, Ingrid Hoffmann.
      MYCBP2 (PAM) is a large signalling hub that plays a key role in various processes, including neuronal connectivity and growth, cell division, and protein ubiquitination. Together with the substrate specificity factor FBXO45, MYCBP2 forms an E3 ligase complex that is involved in mitotic cell fate decision. During extended mitotic arrest caused by anti-microtubule drugs, cells may either experience cell death or escape mitosis through mitotic slippage. E3 ligase mediated ubiquitination is antagonized by deubiquitinating enzymes (DUBs). In this study, we show that despite their opposing activities, DUB-E3 ligase complexes can form and cooperate. We identify an E3 ligase complex consisting of MYCBP2 and a new substrate specificity factor, SPRYD3. Interestingly, SPRYD3-MYCBP2 promotes bipolar spindle formation by facilitating non-canonical ubiquitination on the DUB USP11 cysteine 318. We find that this process promotes bipolar spindle formation and mitotic slippage in presence of microtubule targeting drugs.
    Keywords:  E3 ligase; MYCBP2; SPRYD3/ ubiquitination; USP11; deubiquitinase; mitotic cell fate; spindle assembly
    DOI:  https://doi.org/10.1016/j.jbc.2025.110785
  37. Cell Rep. 2025 Oct 04. pii: S2211-1247(25)01142-8. [Epub ahead of print]44(10): 116371
    PNPLA3-I148M is a neomorph that interferes with two primary hepatic triglyceride clearance pathways.
    David J Sherman, Lei Liu, Edward L LaGory, Preston Fordstrom, Danielle Pruitt, Jose Barreda, Joy G Cagmat, Jiansong Xie, John Ferbas, Ingrid C Rulifson, Raymond J Deshaies.
      The common variant PNPLA3-I148M, globally, is the most significant genetic risk factor for fatty liver disease. However, it is unclear precisely how I148M drives disease risk. Using human hepatoma cells expressing endogenous I148M, we find that the variant impairs cellular secretion of apolipoprotein B (ApoB), the scaffolding protein of very-low-density lipoprotein (VLDL). This is not due to loss-of-function of wild-type PNPLA3. Expression of human I148M in primary hepatocytes and mice also hinders VLDL secretion. Lipidomic profiling reveals a shift from polyunsaturated phosphatidylcholine to polyunsaturated triglycerides in I148M cells, reducing membrane fluidity and, concomitantly, VLDL biogenesis. ApoB secretion is substantially rescued in I148M cells overexpressing ABHD5/CGI-58, an I148M-binding partner that normally activates ATGL/PNPLA2-mediated triglyceride lipolysis. Conversely, knocking down CGI-58 or PNPLA2 mimics I148M. We propose that I148M is a neomorph that exacerbates fatty liver risk by simultaneously impeding two major CGI-58-dependent pathways for liver triglyceride clearance: lipolysis and secretion.
    Keywords:  CP: Metabolism; PNPLA3; lipid droplets; lipolysis; membrane fluidity; phospholipid; polyunsaturated fatty acids; secretory pathway; steatotic liver disease; triglyceride; very-low-density lipoproteins
    DOI:  https://doi.org/10.1016/j.celrep.2025.116371
  38. Cell Death Dis. 2025 Oct 06. 16(1): 680
    IRE1α translational suppression potentiates STING-dependent chemoresistance in pancreatic cancer.
    Yuan Luo, Mengqi Sun, Lei Chang, Zinan He, Xinghang Zhou, Yaming Yuan, Huijuan Sun, Shiqi Luo, Jinyan Huang, Hongkun Wu, Wenjun Liu, Zhangsen Zhou, Yuanhui Mao, Yewei Ji, Tingbo Liang.
      Chemotherapy remains a standard treatment for pancreatic ductal adenocarcinoma (PDAC); however, its effectiveness is limited, and the underlying mechanisms are poorly understood. STING plays diverse and critical roles in cancer, yet the role of PDAC cell-intrinsic STING signaling and its regulation under chemotherapy remain unclear. Here, we report that chemotherapy induces cancer cell-intrinsic STING signaling and that STING deletion in PDAC enhances cell death under chemotherapy while suppressing tumor growth in both immune-deficient and immune-competent mice. Interestingly, chemotherapy selectively inhibits translation of IRE1α, an ER membrane protein and a canonical mediator of ER stress. Loss of IRE1α in PDAC amplifies STING signaling and increases resistance to chemotherapy. Mechanistically, IRE1α interacts with STING via their transmembrane regions, reducing STING stability in PDAC cells. Our study reveals that PDAC cells downregulate IRE1α to reinforce STING-mediated pro-survival response; however, this adaptation also makes them more vulnerable to proteostasis imbalance and ER stress-induced cell death. Notably, we demonstrate that combining ER stress inducers with STING signaling inhibition enhances chemotherapy efficacy both in vitro and in vivo.
    DOI:  https://doi.org/10.1038/s41419-025-07999-x
  39. Sci Adv. 2025 Oct 10. 11(41): eadw7989
    Targeting the ATM-TRMT10A-BRCA1 axis confers synthetic lethality to PARP inhibition in metastatic castration-resistant prostate cancer.
    Ying Yang, Qiang Liu, Xinyan Li, Hua Zhang, Xin Xu, Qi Ma, Somaira Nowsheen, Khaled Aziz, Ye-Xiong Li, Zhenkun Lou, Qiuzi Zhong, Min Deng.
      Metastatic castration-resistant prostate cancer (mCRPC) progresses aggressively and resists existing therapies. Although poly(ADP-ribose) polymerase inhibitors (PARPis) benefit a subset of patients with mCRPC and BRCA1/2 deficiencies, therapeutic options remain limited for those without such mutations. Here, we uncover a critical role for the ATM-TRMT10A-BRCA1 signaling axis in regulating homologous recombination (HR) repair and PARPi sensitivity. We demonstrate that ATM phosphorylates TRMT10A at serine-28 after DNA damage, promoting BRCA1 recruitment and efficient HR repair. TRMT10A deletion disrupts HR repair, sensitizing cells to PARPis. Moreover, TRMT10A is up-regulated in mCRPC through stabilization by USP10. Targeting USP10 with spautin-1 induces TRMT10A degradation and enhances tumor sensitivity to PARPis in cell-derived xenografts and patient-derived xenograft models. These findings identify TRMT10A as a therapeutic vulnerability in mCRPC and demonstrate that combined inhibition of PARP and USP10 offers a promising synthetic lethal strategy for a broader group of patients lacking classical BRCA mutations.
    DOI:  https://doi.org/10.1126/sciadv.adw7989
  40. Cell Rep. 2025 Oct 03. pii: S2211-1247(25)01082-4. [Epub ahead of print]44(10): 116311
    Arsenic regulates ALKBH1 abundance and substrate specificity to promote translation and tumorigenicity.
    Emma Wilkinson, Yan-Hong Cui, Zizhao Yang, Michelle Verghese, Seungwon Yang, Ming Sun, Houxiang Zhu, Jack Peterson, Jiangbo Wei, Yingming Zhao, Mengjie Chen, Chuan He, Yu-Ying He.
      The mechanisms that underlie arsenic tumorigenicity remain incompletely understood. Here, we show that arsenic regulates the abundance and the demethylase activity of ALKBH1, which, in turn, promotes arsenic-induced skin tumorigenesis. At the molecular level, knockdown of ALKBH1 in arsenic-exposed keratinocytes increases m6A enrichment in mRNA, and ALKBH1 demethylates m6A in vitro in an arsenic-dependent manner. Arsenic binds to the ALKBH1 protein at cysteine residues to regulate the m6A RNA demethylase activity of ALKBH1. m6A-immunoprecipitation (IP)-sequencing demonstrates that ALKBH1 regulates m6A demethylation on the NR2C2 transcript to inhibit NR2C2 protein translation in a YTHDF1-dependent manner. Functionally, knockdown of ALKBH1 increases NR2C2 expression, leading to decreased mTOR activation and global translation. Our work uncovers a previously unknown mechanism into arsenic tumorigenicity, adds understanding into the functional effect of arsenic binding to proteins, and implicates ALKBH1 as a potential druggable target and biomarker for arsenic tumorigenicity.
    Keywords:  CP: Cancer; arsenic; m(6)A mRNA methylation; skin cancer; translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116311
  41. Proc Natl Acad Sci U S A. 2025 Oct 14. 122(41): e2519493122
    Structure of a transient protein-folding intermediate by pressure-jump NMR spectroscopy.
    Elahe Masoumzadeh, Joseph M Courtney, Cyril Charlier, Jinfa Ying, Philip Anfinrud, Adriaan Bax.
      Protein folding, as commonly portrayed, involves exploration of a rough, high-dimensional landscape, ending with a final descent into a low-energy folded state. During that journey, the protein may visit shallow basins corresponding to metastable structures, potentially of biological importance. Structural characterization of transiently populated metastable states is challenging due to their low population, which limits traditional NMR, and also makes crystallization for X-ray diffraction difficult without stabilizing mutations, covalent modifications, or the addition of antibodies. Here, we report the structural characterization of the on-pathway folding intermediate of a pressure-sensitized ubiquitin mutant. The obtained non-native β-sheet registry was previously shown to be necessary in the PINK1 mitophagy pathway. We used fast pressure jumps to repeatedly initiate folding and advanced NMR measurements to probe the evolving ensemble of protein conformations. The results reported here demonstrate that the non-native β-sheet hydrogen bond registry can act as a metastable trap during protein folding. This work provides a template for future investigation of metastable conformations and protein folding with rich structural detail.
    Keywords:  CS-Rosetta; metastable protein-folding intermediate; molecular dynamics; retracted strand; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2519493122
  42. Cell Chem Biol. 2025 Oct 06. pii: S2451-9456(25)00301-0. [Epub ahead of print]
    Discovery of a tau-aggregate clearing compound that covalently targets P4HB.
    Louis P Conway, Michelle A Estrada, Weichao Li, Stephen Walker, Benjamin Mielich-Süss, Anurupa Shrestha, Matthew Townsend, Jürgen Korffmann, Greg Potts, Janice Lee, Kenneth P Robinson, Shiyao Wang, Brian Bierie, John R Koenig, Phil Cox, Paul Richardson, Manisha Jhala, Becca McCloud, Sujatha Gopalakrishnan, Kevin Woller, Anil Vasudevan, Scott E Warder, Shaun M McLoughlin, Christopher G Parker.
      The improper folding and aggregation of tau are linked to several neurodegenerative diseases affecting millions worldwide. However, the pathogenesis of tauopathies remains poorly understood, resulting in limited effective treatments. Here, we employ an integrated chemoproteomic phenotypic strategy to identify druggable targets and corresponding chemical probes for the treatment of tauopathies. We identified and optimized an indole-amine compound that potently and extensively clears tau aggregates in two human tauopathy models. Mechanistic and chemoproteomic studies implicate protein disulfide isomerase 1 (P4HB) as the primary target, forming covalent adducts upon metabolic activation. Knockdown of P4HB reduced tau aggregates in three tauopathy models, including an ex vivo murine neuron preclinical model. Functional characterization revealed the compound induces mild endoplasmic reticulum (ER)-stress responses as assessed by RNA sequencing and whole proteomic profiling. Our findings highlight P4HB as a potential therapeutic target for treatment of tauopathies.
    Keywords:  chemoproteomics; neurodegenerative disease; phenotypic screening; target identification; tauopathy
    DOI:  https://doi.org/10.1016/j.chembiol.2025.09.006
  43. RNA. 2025 Oct 07. pii: rna.080476.125. [Epub ahead of print]
    UPF1 shuttles between nucleus and cytoplasm independently of its RNA binding and ATPase activities.
    Sofia Nasif, Andrea Brigitte Eberle, Karin Schranz, Remo Hadorn, Sutapa Chakrabarti, Oliver Mühlemann.
      The ATP-dependent RNA helicase Up-frameshift 1 (UPF1) is an essential protein in mammalian cells and a key factor in nonsense-mediated mRNA decay (NMD), a translation-dependent mRNA surveillance process. UPF1 is mainly cytoplasmic at steady state but accumulates in the nucleus after inhibiting CRM1-mediated nuclear export by Leptomycin B (LMB), indicating that UPF1 shuttles between the nucleus and the cytoplasm. Consistent with its dual localization, there is evidence for nuclear functions of UPF1, for instance in DNA replication, DNA damage response, and telomere maintenance. However, whether any of UPF1's biochemical activities are required for its nuclear-cytoplasmic shuttling remains unclear. To investigate this, we examined two UPF1 mutants: the well-described ATPase-deficient UPF1-DE (D636A/E637A) and a newly generated RNA-binding mutant UPF1-NKR (N524A/K547A/R843A). Biochemical assays confirmed that the UPF1-NKR mutant cannot bind RNA or hydrolyze ATP in vitro but retains interaction with UPF2, UPF3B, and SMG6. Overexpression of UPF1-NKR exerted a dominant-negative effect on endogenous UPF1 and inhibited NMD. Subcellular localization studies revealed that UPF1-DE accumulates in cytoplasmic granules (P-bodies), even in the presence of LMB, whereas UPF1-NKR shuttles normally. This indicates that UPF1's shuttling does not require its RNA-binding or ATPase activities. Notably, the UPF1-DE.NKR double mutant restored nuclear-cytoplasmic shuttling and prevented accumulation in P-bodies, suggesting that the shuttling defect of UPF1-DE arises from its tight binding to RNA. Overall, our findings demonstrate that UPF1's shuttling is independent of its ATPase and RNA-binding activities, with RNA binding itself being a key determinant of its cytoplasmic retention.
    Keywords:  NMD; UPF1; nuclear-cytoplasmic shuttling
    DOI:  https://doi.org/10.1261/rna.080476.125
  44. Proc Natl Acad Sci U S A. 2025 Oct 14. 122(41): e2426476122
    The autophagy-specific exocyst subcomplex contributes to phagophore assembly site integrity by promoting phagophore expansion.
    Ruchika Kumari, Titikhya Nath, Jeremy Pflaum, Inchara R, Sannannagari Vinay, Florian Wilfling, Ravi Manjithaya.
      Autophagy is a crucial intracellular pathway for maintaining cellular homeostasis. It involves forming double-membrane vesicles called autophagosomes, which deliver cytosolic cargoes to the lysosomes/vacuoles for degradation. Biogenesis of autophagosomes is a membrane-intensive process wherein the membrane expansion steps are poorly understood. The tethering complex, exocyst, canonically implicated in secretion, also participates in autophagosome biogenesis in yeast, plants, and mammals. However, the contribution of the exocyst complex in autophagosome biogenesis is unclear. In this study, using yeast temperature-sensitive mutants of the exocyst, we observed the accumulation of multiple abortive PAS incapable of autophagosome biogenesis. These dysfunctional abortive structures were enriched with core autophagy proteins involved in initiation and membrane expansion. However, the membrane expansion ability required for cargo capture was severely compromised in these mutants. Further investigations, including a comprehensive epistasis analysis, revealed that the exocyst plays a role downstream of the Atg1 complex. However, it was required at a stage before phosphatidylinositol 3-kinase (PI3K complex I), Atg2-Atg18, and Atg12-Atg5-Atg16 complexes. Taken together, we show that productive PAS formation and membrane expansion during autophagosome biogenesis are exquisitely orchestrated by the autophagy-specific exocyst subcomplex, which excludes Exo70.
    Keywords:  PAS; autophagy; exocyst; membrane expansion; phagophore
    DOI:  https://doi.org/10.1073/pnas.2426476122
  45. ACS Nano. 2025 Oct 08.
    Modular Nanoassemblies Mimicking p62 Aggregates for Targeted Organelle Sequestration and Degradation against Breast Cancer.
    Yuai Li, Yunting Zhang, Jingwen Wang, Yujie Che, Tao Gong, Zhirong Zhang, Renhe Liu, Yao Fu.
      Selective autophagy relies on multivalent recognition by receptors like SQSTM1/p62 to form aggregates that cluster disperse organelles, undergoing liquid-liquid phase separation to facilitate their clearance and maintain cellular homeostasis. Inspired by this, we present the multivalent nanoparticle-based organelle targeting chimera (NanoTACOrg) to efficiently degrade organelles by flexibly clustering organelles for sequestration and facilitating targeted recruitment of autophagosomes. NanoTACOrg, assembled with a PLGA core, lysosomal escape modules, organelle-targeting modules, and LC3B binding modules, is programmed to selectively degrade various organelles, including mitochondria, endoplasmic reticulum, and Golgi apparatus. After endocytosis and lysosomal escape, NanoTACOrg targets subcellular compartments and mimics p62 aggregate-driven organelle clustering and degradation, without exhibiting the "hook effect". Specifically, NanoTACMito-mediated mitochondrial degradation disrupts oxidative phosphorylation (OXPHOS) while enhancing compensatory glycolysis, thus sensitizing tumor cells to the glucose transporter 1 (GLUT1) inhibitor BAY-876. BAY-876 loaded NanoTACMito potently inhibits tumor growth, recurrence, and metastasis, demonstrating superior therapeutic efficacy by simultaneously targeting OXPHOS and glycolysis. These findings highlight the potential of NanoTACOrg as a versatile and effective platform for cancer therapy, particularly through organelle-specific degradation and metabolic reprogramming.
    Keywords:  metabolic plasticity; multivalent binding; nanoparticle-mediated targeted degradation; organelle; tumor metastasis
    DOI:  https://doi.org/10.1021/acsnano.5c10801
  46. Am J Respir Cell Mol Biol. 2025 Oct 10.
    E3 Ubiquitin Ligases and Asthma Relief.
    Ajay P Nayak.
      
    Keywords:  PELI1, asthma, E3 ligases
    DOI:  https://doi.org/10.1165/rcmb.2025-0532ED
  47. Cell. 2025 Oct 07. pii: S0092-8674(25)01080-3. [Epub ahead of print]
    Genetic modifiers and ascertainment drive variable expressivity of complex disorders.
    Matthew Jensen, Corrine Smolen, Anastasia Tyryshkina, Lucilla Pizzo, Jiawan Sun, Serena Noss, Deepro Banerjee, Matthew Oetjens, Hermela Shimelis, Cora M Taylor, Vijay Kumar Pounraja, Hyebin Song, Laura Rohan, Emily Huber, Laila El Khattabi, Ingrid van de Laar, Rafik Tadros, Connie R Bezzina, Marjon van Slegtenhorst, Janneke Kammeraad, Paolo Prontera, Jean-Hubert Caberg, Harry Fraser, Siddharth Banka, Anke Van Dijck, Charles Schwartz, Els Voorhoeve, Patrick Callier, Anne-Laure Mosca-Boidron, Nathalie Marle, Mathilde Lefebvre, Kate Pope, Penny Snell, Amber Boys, Paul J Lockhart, Myla Ashfaq, Elizabeth McCready, Margaret Nowacyzk, Lucia Castiglia, Ornella Galesi, Emanuela Avola, Teresa Mattina, Marco Fichera, Maria Grazia Bruccheri, Giuseppa Maria Luana Mandarà, Francesca Mari, Flavia Privitera, Ilaria Longo, Aurora Curró, Alessandra Renieri, Boris Keren, Perrine Charles, Silvestre Cuinat, Mathilde Nizon, Olivier Pichon, Claire Bénéteau, Radka Stoeva, Dominique Martin-Coignard, Sophia Blesson, Cedric Le Caignec, Sandra Mercier, Marie Vincent, Christa L Martin, Katrin Mannik, Alexandre Reymond, Laurence Faivre, Erik Sistermans, R Frank Kooy, David J Amor, Corrado Romano, Joris Andrieux, Santhosh Girirajan.
      Variable expressivity of disease-associated variants implies a role for secondary variants that modify clinical features. We assessed the effects of modifier variants on the clinical outcomes of 2,455 individuals with primary variants. Among 124 families with the 16p12.1 deletion, distinct rare and common variant classes conferred risks for specific developmental features, including short tandem repeats for neurological defects. Network analysis suggested distinct mechanisms involving 16p12.1 genes and secondary variants specific to each proband. Within disease and population cohorts of 976 individuals with the 16p12.1 deletion, we found opposing effects of secondary variants on clinical features across ascertainments. Additional analysis of 1,479 probands with other primary variants, such as the 16p11.2 deletion and CHD8 variants, and 1,528 probands without primary variants showed that phenotypic associations differed by primary variant context and were influenced by synergistic interactions between primary and secondary variants. Our study provides a paradigm to dissect the personalized genomic architecture of complex disorders.
    Keywords:  ascertainment; autism spectrum disorder; complex disease; copy-number variants; modifiers; multi-hit model; neurodevelopmental; rare variants; variable expressivity
    DOI:  https://doi.org/10.1016/j.cell.2025.09.012
  48. FEBS Open Bio. 2025 Oct 10.
    Mitochondria-associated membranes (MAMs): molecular organization, cellular functions, and their role in health and disease.
    Viet Bui, Maryline Santerre, Natalia Shcherbik, Bassel E Sawaya.
      Mitochondria-associated membranes (MAMs) are specialized contact sites between the endoplasmic reticulum (ER) and mitochondria that maintain cellular homeostasis through precisely orchestrated molecular mechanisms. These dynamic interfaces are maintained at 10-50 nm distances by complex tethering proteins, including the core IP3R-GRP7 5-VDAC1 complex and regulatory proteins, such as the sigma-1 receptor. MAMs coordinate multiple essential cellular processes: lipid synthesis and transfer, calcium signaling, metabolic regulation, and quality control through autophagy and mitophagy. Recent advances in super-resolution microscopy and proteomics have revealed that MAM dysfunction drives pathogenesis across various diseases. In Alzheimer's disease, disrupted MAM spacing directly affects Aβ production and mitochondrial function, while in Parkinson's disease, α-synuclein accumulation at MAMs impairs phosphatidylserine metabolism and mitochondrial dynamics. Beyond neurodegeneration, MAMs play crucial roles in metabolic disorders, cancer progression, and viral infections. This review provides mechanistic insights into MAM biology, from molecular organization to disease pathogenesis, integrating structural analyses with dynamic visualization approaches. We examine emerging therapeutic strategies targeting MAM-associated pathways and highlight their potential in treating complex diseases.
    Keywords:  ER–mitochondria contact sites; calcium signaling; cellular stress responses; lipid metabolism; mitochondria‐associated membranes; neurodegeneration
    DOI:  https://doi.org/10.1002/2211-5463.70121
  49. Sci Adv. 2025 Oct 10. 11(41): eady2604
    Mechanism of USP21 autoinhibition and histone H2AK119 deubiquitination.
    Sanim Rahman, Chad W Hicks, Alexander Gwizdala, Cynthia Wolberger.
      Monoubiquitinated histone H2A lysine 119 (H2AK119ub) is a modification associated with transcriptional silencing and heterochromatin formation. Ubiquitin-specific protease 21 (USP21), one of four major H2AK119-specific deubiquitinating enzymes (DUBs), plays critical roles in diverse cellular processes. However, the mechanisms by which USP21 specifically deubiquitinates H2AK119ub and is regulated are unknown. We determined the cryo-EM structure of the USP21 catalytic domain bound to an H2AK119ub nucleosome, which revealed a recognition mode that differs from that of other H2AK119-specific DUBs. We unexpectedly found that the N-terminal IDR of USP21 inhibits the enzyme's activity. Using AlphaFold-Multimer to perform a virtual screen of USP21 interactors, we identified kinases that phosphorylate the USP21 IDR and thereby relieve autoinhibition. AlphaFold3 modeling of USP21 suggests a structural model for autoinhibition. AlphaFold analysis suggests that phosphorylation-regulated autoinhibition may be a feature of various USP enzymes. These findings shed light on the mechanisms of H2AK119 deubiquitination and reveal a previously unexplored mode of phosphorylation-dependent DUB autoregulation.
    DOI:  https://doi.org/10.1126/sciadv.ady2604
  50. mBio. 2025 Oct 06. e0258524
    Screening of gene function in cell intoxication by CNF1 links Sec61 translocon to Rac1 GTPase activity.
    Eléa Paillares, Nathalie Deboosere, Stéphane Descorps-Declere, Maud Marechal, Daniel Gillet, Caroline Demangel, Amel Mettouchi, Priscille Brodin, Emmanuel Lemichez.
      The CNF1 toxin from extraintestinal pathogenic Escherichia coli (ExPEC) deamidates glutamine 61 of Rac1 small GTPase, as well as its equivalents in RhoA and Cdc42 into glutamic acid. This post-translational modification of Rho proteins abrogates the hydrolysis of GTP into GDP, thereby enhancing signal transduction. Meanwhile, the sustained GTP-loading of Rac1 Q61E sensitizes it to ubiquitin-mediated proteasomal degradation catalyzed by the HACE1 E3 ligase rate-limiting factor, leading to a cellular depletion of Rac1 over time. We report data from a quantitative genome-wide screen of siRNAs inhibiting CNF1-mediated cellular depletion of Rac1 in primary human cells. As best hits, we identified a group of three siRNAs targeting the Sec61A1 subunit of the Sec61 translocon, as well as HACE1 and the Lu/BCAM host cell receptor of CNF1. We extend these findings by identifying a group of siRNAs targeting genes involved in ER and Golgi homeostasis and trafficking. Functional studies showed that both chemical and genetic inhibition of Sec61A1 dampens GTP-loading and membrane association of Rac1 in CNF1-intoxicated cells, while the proper deamidation of RhoA provides a control of CNF1 cytosolic action. Finally, we extend these findings by showing that inhibition of N-glycosylation of neo-synthesized proteins in the ER abrogates Rac1 GTP-loading in CNF1-treated cells. Collectively, these data point to a control of Rac1 signaling operated by protein biosynthesis and N-glycosylation in the ER.IMPORTANCEThe remarkable evolutionary convergence of bacterial effectors from pathogens toward the host small GTPase Rac1, the master regulator of the actin cytoskeleton, confers to these microbes an enhanced capacity to invade host cells and tissues. The CNF1 toxin, a colonization factor of the gastrointestinal tract produced by pathogenic strains of Escherichia coli, has been instrumental in deciphering the regulation and function of Rac1. By performing a whole-genome screen based on CNF1 action, we establish the key requirement of Sec61 translocon-dependent protein biosynthesis and N-glycosylation at the endoplasmic reticulum for proper activation of Rac1 in intoxicated cells. Our data connect the Sec61 translocon and N-glycosylation of neo-synthesized proteins at the endoplasmic reticulum in the control of the activity of Rac1 and other Rho GTPases.
    Keywords:  CNF1 toxin; HACE1; Rac1; Rho GTPases; Sec61 translocon; mycolactone
    DOI:  https://doi.org/10.1128/mbio.02585-24
  51. Cold Spring Harb Perspect Biol. 2025 Oct 06. pii: a041946. [Epub ahead of print]
    Mechanisms and Determinants of -1 Ribosome Frameshifting and Bypassing.
    Panagiotis Poulis, Marina V Rodnina.
      Ribosomal frameshifting is a recoding mechanism that allows the ribosome to alter its reading frame during translation, often in response to specific messenger RNA (mRNA) elements or cellular conditions. While essential for the life cycle of many viruses, frameshifting also occurs spontaneously or in response to transfer RNA (tRNA) depletion, raising important questions about its regulation and biological relevance. This review explores the structural and kinetic principles that govern -1 frameshifting, highlighting the role of ribosome conformational dynamics, slippery sequences, and mRNA secondary structures. We discuss how programmed, hungry, and spontaneous frameshifting arise from distinct molecular pathways, yet converge on shared mechanistic features. The review also examines translational bypassing as a related form of recoding that involves large-scale ribosome sliding over noncoding regions and relies on a distinct set of RNA and ribosome conformational cues to ensure accurate take-off and landing. These insights expand our understanding of translation fidelity and recoding plasticity.
    DOI:  https://doi.org/10.1101/cshperspect.a041946
  52. Nat Struct Mol Biol. 2025 Oct 10.
    Structural phylogenetics unravels the evolutionary diversification of communication systems in gram-positive bacteria and their viruses.
    David Moi, Charles Bernard, Martin Steinegger, Yannis Nevers, Mauricio Langleib, Christophe Dessimoz.
      Recent advances in artificial-intelligence-based protein structure modeling have yielded remarkable progress in predicting protein structures. Because structures are constrained by their biological function, their geometry tends to evolve more slowly than the underlying amino acids sequences. This feature of structures could in principle be used to reconstruct phylogenetic trees over longer evolutionary timescales than sequence-based approaches; however, until now, a reliable structure-based tree-building method has been elusive. Here, we introduce a rigorous framework for empirical tree accuracy evaluation and tested multiple approaches using sequence and structure information. The best results were obtained by inferring trees from sequences aligned using a local structural alphabet-an approach robust to conformational changes that confound traditional structural distance measures. We illustrate the power of structure-informed phylogenetics by deciphering the evolutionary diversification of a particularly challenging family: the fast-evolving RRNPPA quorum-sensing receptors. We were able to propose a more parsimonious evolutionary history for this critical protein family that enables gram-positive bacteria, plasmids and bacteriophages to communicate and coordinate key behaviors. The advent of high-accuracy structural phylogenetics enables a myriad of applications across biology, such as uncovering deeper evolutionary relationships, elucidating unknown protein functions or refining the design of bioengineered molecules.
    DOI:  https://doi.org/10.1038/s41594-025-01649-8
  53. Nat Biotechnol. 2025 Oct 07.
    Folding-mediated secretion of pure bispecific antibodies.
    Cholpon Tilegenova, Tun Liu, Qian Zhao, Mahati Are, Yu Zhao, Woo Suk Choi, Anusarka Bhaumik, Ruth Steele, Nicholas A Manieri, Bengi Turegun, Alex Ni, Rosa M F Cardoso, Paul Shaffer, Desmond Clark, Robin Ernst, Wenyu Li, Tracy Taylor, Suresh Kumar Swaminathan, Bhargavi Ramaraju, Kevin Liaw, Steven A Jacobs, Sujata Sharma, Wan Cheung Cheung, Adam Zwolak.
      Bispecific antibodies (bsAbs) can enable therapeutic mechanisms, such as dual antigen targeting or receptor agonism, that are impossible using monoclonal antibodies. BsAbs with IgG-like format (bsIgG) are comprised of two unique heavy chains, each having a cognate light chain. Co-expression of these four unique polypeptides often leads to several mispaired species that are difficult to separate from the target bsIgG due to their similar biophysical properties. Here we describe a set of mutations called ProAla that exploit a the unfolded protein response pathway of cells. ProAla heavy chains are engineered with higher folding energy barriers such that only the cognate light and heavy chains can induce folding, chaperone release and secretion. The structures of the ProAla Fab and Fc regions are identical in structure to normal antibodies, enabling maintenance of half-life and function. Mispaired polypeptides fail to secrete from the cell due to enhanced interaction with the endoplasmic reticulum chaperone BiP, resulting in increased purity of secreted bsIgGs.
    DOI:  https://doi.org/10.1038/s41587-025-02842-2
  54. Adv Sci (Weinh). 2025 Oct 05. e09695
    DCAF12 Ubiquitin Ligase Promotes Lung Cancer Metastasis by Modulating the TRiC/CCT Chaperonin Complex.
    Zhenyi Wang, Huanhuan Huang, Kaizong Huang, Xiaowen Cui, Leilei Wu, Renyu Lin, Zhe Zhao, Hua Chen, Cheng Zheng, Weilin Jin, Song Chen, Jiayan Chen, Yaping Xu, Dongping Wei.
      Metastasis is the primary challenge in lung cancer treatment. Although proteostasis supports tumor growth, the mechanism by which ubiquitin ligases reprogram chaperone networks to drive metastasis is poorly understood. In this study, it is revealed that DDB1-CUL4-associated factor (DCAF12), a substrate receptor for CUL4-RING ubiquitin ligases, regulates metastatic progression through ubiquitin-mediated proteostatic reprogramming. DCAF12 depletion suppresses tumor cell migration and stemness in vitro and reduces pulmonary/hepatic metastasis in vivo. Mechanistically, DCAF12 catalyzes the non-degradative ubiquitination of TRiC/CCT subunits, enhancing chaperonin assembly and folding of cytoskeletal effectors (β-actin/tubulin) and oncogenic clients (STAT3/Raptor/mLST8), thereby activating the YAP, STAT3, and mTOR pathways. Both genetic knockdown and pharmacological blockade (via HSF1A) of this axis potently inhibit metastasis. Clinically, DCAF12 overexpression is correlated with YAP/STAT3 activation, advanced metastasis, and poor survival. Three key insights are revealed: 1) ubiquitination-mediated TRiC/CCT regulation as a metastatic switch, 2) DCAF12 as an oncogenic proteostasis hub, and 3) therapeutic potential validated through multimodal targeting. These findings establish the DCAF12-TRiC/CCT axis as a mechanistically novel target that simultaneously disrupts cytoskeletal dynamics and oncogenic signaling, making it a promising therapeutic strategy for metastatic lung cancer.
    Keywords:  DCAF12; TRiC/CCT complex; metastasis; proteostasis; ubiquitination
    DOI:  https://doi.org/10.1002/advs.202509695
  55. Nat Commun. 2025 Oct 10. 16(1): 9027
    VRK2 targeting potentiates anti-PD-1 immunotherapy in hepatocellular carcinoma through MYC destabilization.
    Chen Su, Zhibin Liao, Jie Mo, Furong Liu, Weijian Wang, Haoquan Zhang, Hongwei Zhang, Yachong Liu, Yonglong Pan, He Zhu, Xiaoping Chen, Zhanguo Zhang, Peng Zhu, Bixiang Zhang.
      Dysregulation of MYC proto-oncogene, bHLH transcription factor (MYC) represents a common yet mechanistically unresolved driver of hepatocellular carcinoma (HCC). While MYC remains an elusive therapeutic target, developing strategies to promote its degradation emerges as a promising alternative approach. Here we show that vaccinia-related kinase 2 (VRK2) functions as a direct MYC-interacting kinase that stabilizes the oncoprotein through phosphorylation at Serine (Ser)281/293. This phosphorylation enables VRK2 to compete with the Skp1-Cullin-F-box protein complex containing FBXO24 (SCF-FBXO24) E3 ligase, thereby blocking MYC polyubiquitination and proteasomal degradation. The stabilized MYC-VRK2 complex amplifies transcriptional activation of protumorigenic programs, including the immune checkpoint programmed cell death ligand 1 (PD-L1) and VRK2 itself, establishing a self-reinforcing oncogenic circuit. Therapeutic inhibition of VRK2 in HCC models reduces MYC protein levels, suppresses tumor progression, and synergizes with anti- programmed cell death-1 (PD-1) immunotherapy. Our results reveal VRK2-mediated stabilization of MYC as a critical nexus linking hepatocarcinogenesis to immune evasion, proposing VRK2 kinase inhibition as a mechanism-based therapeutic strategy for MYC-driven HCC.
    DOI:  https://doi.org/10.1038/s41467-025-64079-6
  56. RSC Chem Biol. 2025 Oct 02.
    Identification of ligands for E3 ligases with restricted expression using fragment-based methods.
    Alex G Waterson, Brian D Lehmann, Zhenwei Lu, John L Sensintaffar, Edward T Olejniczak, Bin Zhao, Tyson Rietz, William G Payne, Jason Phan, Stephen W Fesik.
      Heterobifunctional molecules that induce targeted degradation have emerged as powerful tools in chemical biology, target validation, and drug discovery. Despite their promise, the field is constrained by the relative paucity of ligands available for E3 ligases. Expanding the ligand repertoire for E3 ligases and other components of ubiquitin-proteasome system could significantly broaden the scope of the targeted degradation field. In this study, we report the identification of ligands for non-essential E3 ligases that are preferentially expressed in cancer tissues relative to normal tissues. Using a protein-observed NMR-based fragment screen, an ideal technique for this purpose, we identified fragment ligands and characterized their binding modes by X-ray crystallography. These ligands represent promising starting points for further optimization toward the discovery of tumor-selective degraders that may enhance the therapeutic window targeting proteins for which inhibition or degradation is associated with systemic toxicity.
    DOI:  https://doi.org/10.1039/d5cb00198f
  57. JCI Insight. 2025 Oct 08. pii: e188904. [Epub ahead of print]10(19):
    AI662270/GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction.
    Suling Ding, Wen Liu, Zhiwei Zhang, Xiyang Yang, Dili Sun, Jianfu Zhu, Xiaowei Zhu, Shijun Wang, Mengshi Xie, Hongyu Shi, Junbo Ge, Xiangdong Yang.
      The unfolded protein response (UPR), triggered by endoplasmic reticulum (ER) stress, comprises distinct pathways orchestrated by conserved molecular sensors. Although several of these components have been suggested to protect cardiomyocytes from ischemic injury, their precise functions and mechanisms remain elusive. In this study, we observed a marked increase in glucose-regulated protein 94 (GRP94) expression at the border zone of cardiac infarct in a mouse model. GRP94 overexpression ameliorated post-infarction myocardial damage and reduced infarct size. Conversely, GRP94 deficiency exacerbated myocardial dysfunction and infarct size. Mechanistically, GRP94 alleviated hypoxia-induced mitochondrial fragmentation, whereas its depletion exacerbated this fragmentation. Molecular investigations revealed that GRP94 specifically facilitated the cleavage of Opa1 into L-Opa1, but not S-Opa1. The study further elucidated that under hypoxic conditions, the binding shift of Yy1 from lncRNA Oip5os1 to AI662270 promoted Yy1's binding on the GRP94 promoter, thereby enhancing GRP94 expression. AI662270 attenuated mitochondrial over-fragmentation and ischemic injury after myocardial infarction similarly to GRP94. Moreover, coimmunoprecipitation coupled with LC-MS/MS identified the interaction of GRP94 with Anxa2, which regulates Akt1 signaling to maintain L-Opa1 levels. Overall, these findings unveiled what we believe is a novel role for the AI662270/GRP94 axis in linking ER stress to mitochondrial dynamics regulation, proposing new therapeutic avenues for managing cardiovascular conditions through ER stress modulation.
    Keywords:  Cardiology; Cell biology; Cell stress; Hypoxia; Noncoding RNAs
    DOI:  https://doi.org/10.1172/jci.insight.188904
  58. Autophagy. 2025 Oct 08.
    PPP2/PP2A-mediated dephosphorylation of LC3B links PINK1-PRKN/Parkin-mediated mitophagy to SCA12 pathogenesis.
    Ningning Li, Hongyu Hou, Dan Su, Bojun Yang, Rui Ni, Guoqiang Ma, Shan Sun, Qilian Ma, Qiang Peng, Siqian Chen, Kin Yip Tam, Hongfeng Wang, Zheng Ying.
      Atg8-family proteins are autophagosome-associated proteins and play important roles in macroautophagy/autophagy, a conserved process for degrading defective or excessive cellular components. Post-translational modifications of mammalian Atg8-family proteins, including phosphorylation, regulate multiple steps in the autophagic process. In this context, several Atg8-family protein-associated kinases have been found to regulate autophagy, yet the phosphatases in the dephosphorylation of Atg8-family proteins remain unknown. Here, we report that the heterotrimeric PPP2/PP2A (protein phosphatase 2) is a novel regulator in modulating LC3B dephosphorylation. Mechanistically, we find that PPP2-mediated LC3B dephosphorylation reduces the interaction between LC3B and the mitophagy receptor OPTN, thereby impeding the mitochondrial recruitment of phagophores during PINK1-PRKN/Parkin-mediated mitophagy. Interestingly, we find that overexpression of the β2 isoform of PPP2R2B (protein phosphatase 2 regulatory subunit Bbeta; PPP2R2Bβ2), which mimics the spinocerebellar ataxia type 12 (SCA12) pathological condition, harms neuronal survival by enhancing PPP2-mediated LC3B dephosphorylation and reducing mitochondrial recruitment of phagophores upon mitochondrial damage. Importantly, pharmacological induction of mitophagy by the small molecule compound deferiprone (DFP) relieves PPP2R2Bβ2-mediated neuronal toxicity. Overall, our results not only uncover a mechanism by which protein dephosphorylation negatively regulates mitophagy but also provide insights into the pathogenesis of PPP2R2Bβ2-mediated SCA12.
    Keywords:  LC3B; PINK1-PRKN/Parkin-mediated mitophagy; PPP2/PP2A; PPP2R2Bβ2; dephosphorylation; mitochondrial quality control
    DOI:  https://doi.org/10.1080/15548627.2025.2572528
  59. Brain. 2025 Oct 07. pii: awaf371. [Epub ahead of print]
    EIPR1 variants cause a neurodevelopmental disorder with endolysosomal and dense core vesicle defects.
    Saikat Ghosh, Jaskaran Singh, Nadirah S Damseh, Mariasavina Severino, Raffaella De Pace, Adriana E Golding, Michal Jarnik, Poonam Thakran, Laurence Faivre, Jade Heitz, Anne-Sophie Denommé-Pichon, Antonio Vitobello, Lama AlAbdi, Firdous Abdulwahab, Safia Sumayli, Mashael Alqahtani, Huma Arshad Cheema, Iram Javed, JiHye Kim, Hanns Lochmuller, Hagar Mor-Shaked, Jennifer E Neil, Ganeshwaran H Mochida, Giovanni Zifarelli, Peter Bauer, Ehsan Barkhordari, Ehsan Ghayoor Karimiani, Henry Houlden, Bassam Abu-Libdeh, Shimon Edvardson, Orly Elpeleg, Reza Maroofian, Shunmoogum A Patten, Juan S Bonifacino.
      EIPR1 (EARP-interacting protein 1, formerly known as TSSC1) is a WD40-domain protein that interacts with the EARP (endosome-associated recycling protein) and GARP (Golgi-associated retrograde protein) complexes in the process of delivering endosome-derived transmembrane cargos to the plasma membrane and the trans-Golgi network (TGN), respectively. Additionally, EIPR1 cooperates with EARP in the biogenesis of dense core vesicles. While these properties of EIPR1 were established in cultured cells and model organisms, the physiological and pathological importance of EIPR1 in humans remains to be determined. Here we report the identification of five EIPR1 homozygous missense variants [NM_003310.5:c.835C>G p.(Arg279Gly), NM_003310.5:c.813C>G p.(His271Gln), NM_003310.5:c.694C>T p.(Arg232Trp), NM_003310.5:c.47G>A p.(Arg16His) and NM_003310.5:c.419T>A p.(Val140Asp)] in eight individuals from six unrelated families with a neurological disorder featuring a spectrum of global neurodevelopmental delay, microcephaly, ataxia, spasticity, delayed myelination, callosal hypoplasia, cerebellar atrophy, walking and speech impairments, dysmorphic facies, and neutropenia. Cellular studies using a heterologous transfection system demonstrate that these variants reduce EIPR1 protein levels and its physical interaction with EARP and GARP complexes. Furthermore, we show that the Arg279Gly and His271Gln variants reduce the ability of EIPR1 to promote EARP association with endosomes in non-neuronal cells and dense core vesicle biogenesis in iPSC-derived neurons. Additionally, skin fibroblasts from one of the Arg279Gly affected individuals shows reduced recycling of internalized transferrin to the plasma membrane (an EARP-deficiency phenotype) and impaired retrograde transport of internalized Shiga toxin B-subunit to the TGN (a GARP-deficiency phenotype) compared to fibroblasts from an unaffected parent. Moreover, these patient fibroblasts exhibit enlarged lysosomes, increased levels of the lysosomal membrane protein LAMP1, and increased levels of the autophagic markers LC3B-II and SQSTM1, all phenotypes previously associated with GARP deficiency. Knockout of the orthologous eipr1 in zebrafish results in neurodevelopmental and locomotor defects consistent with the clinical phenotype of the human patients. Injection of WT human EIPR1 mRNA into eipr1 KO zebrafish rescues these defects, whereas mRNAs encoding the human EIPR1 variants Arg279Gly or His271Gln fail to do so, confirming the impaired activity of these variants. These findings identify EIPR1 as a novel genetic locus associated with a neurodevelopmental disorder and underscore its critical role in endosomal recycling and dense core vesicle biogenesis, processes essential for the development and function of the nervous system.
    Keywords:  EARP; GARP; Golgi; TSSC1; dense core vesicle; endosomes
    DOI:  https://doi.org/10.1093/brain/awaf371
  60. Genome Biol. 2025 Oct 06. 26(1): 339
    SpatPPI: a geometric deep learning model for predicting protein-protein interactions involving intrinsically disordered regions.
    Zeyu Xu, Yanhao Zhu, Jiyun Han, Juntao Liu.
      Intrinsically disordered proteins and regions (IDRs) lack stable 3D structures, posing challenges for interaction prediction. We present SpatPPI, a geometric deep learning model tailored for IDPPI prediction. SpatPPI leverages structural cues from folded domains to guide the dynamic adjustment of IDRs via geometric modeling, adaptive conformation refinement, and a two-stage decoding mechanism. It captures spatial variability without requiring supervised input and achieves state-of-the-art performance on benchmark datasets. Molecular dynamics simulations further validate its high adaptability to conformational changes in IDRs and strong capacity to generate distinct and structure-aware embeddings. A freely accessible server is available at http://liulab.top/SpatPPI/server .
    Keywords:  Conformational dynamics; Geometric deep learning; Intrinsically disordered proteins; Protein–protein interaction; Residue interaction characteristics
    DOI:  https://doi.org/10.1186/s13059-025-03820-2
  61. Trends Cell Biol. 2025 Oct 07. pii: S0962-8924(25)00219-3. [Epub ahead of print]
    The promiscuous ribosomal P-stalk: a new functional portrait.
    Marek Tchórzewski, Barbara Michalec-Wawiórka.
      The canonical role of the ribosome is to translate the genetic code into functional proteins. Recent discoveries, however, redefine the eukaryotic ribosome, as a regulatory hub, that senses cellular cues and transmits signals to downstream pathways. The P-stalk, an integral component of the ribosomal GTPase-associated center, once viewed as translational supporter, is now emerging as a key regulatory ribosomal module. It has recently been recognized as an activator of the integrated stress response, reshaping the Gcn1/Gcn20→Gcn2 axis into the new Gcn1/Gcn20/P-stalk→Gcn2 order. The P-stalk's structural plasticity allows also the ribosome to rewire gene expression in response to cellular demands, including cytokine response. In this review, an updated functional portrait of the P-stalk is presented, encompassing both ribosome-dependent and -independent activities.
    Keywords:  Gcn2; ribosomal P-stalk; specialized ribosomes; stress response; translation
    DOI:  https://doi.org/10.1016/j.tcb.2025.09.003
  62. Bioorg Chem. 2025 Oct 03. pii: S0045-2068(25)00948-4. [Epub ahead of print]165 109068
    A chemotherapy based on dual-targeted release of ciprofloxacin in mitochondrial and endoplasmic reticulum.
    Xuan Wang, Yan-Ping Liu, Chong-Yang Wang, Ming-Hua Zheng, Jing-Yi Jin.
      Organelle-targeting drugs represent the next generation of precision chemotherapy. To achieve this, a specific organelle-targeting group is essential for various nano-medicines and prodrugs. Simultaneously targeting multiple organelles enhances drug efficacy by enabling lower dosage, minimizing side effects, and potentially altering the action mechanism of drugs through synchronous effects across organelles. However, designing a molecular platform capable of delivering drugs to multiple organelles remains challenging, which hinders the advancement of precise chemotherapy. Here, we conjugated a pyronine unit as a warhead to an antibiotic (ciprofloxacin, Cip) via a covalent linkage, developing a molecular platform termed CPY. This platform simultaneously targets mitochondria (Mito) and the endoplasmic reticulum (ER), releasing Cip in situ through a glutathione-mediated SNAr pathway. CPY induced apoptosis in tumor cells and exhibited anti-tumor activity in a xenograft tumor model. These results demonstrate a feasible strategy for repurposing conventional non-antitumor drugs into chemotherapeutic agents.
    Keywords:  Ciprofloxacin; Endoplasmic reticulum; Glutathione-triggered prodrug; Mitochondria; Precise chemotherapy; Targeting release
    DOI:  https://doi.org/10.1016/j.bioorg.2025.109068
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