bims-proteo Biomed News
on Proteostasis
Issue of 2025–10–19
fifty-six papers selected by
Eric Chevet, INSERM
  1. Differential regulation of translational stress responses by herpesvirus ubiquitin deconjugases.
  2. Temporal regulation of signal recognition particle during translation.
  3. Chemical Probe Approach Reveals Endo-α-mannosidase Triages Misfolded Glycoproteins in the Calnexin/Calreticulin Cycle.
  4. Role of AAA-ATPase Cdc48p in peroxisomal quality control.
  5. The E3 ubiquitin ligase ZNRF3 restricts WNT receptor complex activity by stimulating the selective degradation of WNT-engaged FZD.
  6. Mechanisms of assembly and function of the Hsp70-Hsp40 chaperone machinery.
  7. Response of UBR-box E3 ubiquitin ligases and protein quality control pathways to perturbations in protein synthesis and skeletal muscle size.
  8. Structural basis of K11/K48-branched ubiquitin chain recognition by the human 26S proteasome.
  9. Immunoregulatory Functions of the ER Stress Sensor IRE1 in Myeloid Cell Biology.
  10. Loss of O-GlcNAcylation in cardiac myocytes triggers the integrated stress response contributing to heart failure.
  11. Targeted Histone Deacetylase Degradation via Chemical Induced Proximity by Direct Recruitment of the CUL4 Complex Adaptor Protein DDB1.
  12. Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
  13. Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death.
  14. Cholesterol sensing by the SCAP-FAM134B complex regulates ER-phagy and STING innate immunity.
  15. Structural basis of enhanced stalling efficiency of an engineered ribosome arrest peptide.
  16. Visualization of lysosomal membrane proteins by cryo electron tomography.
  17. Molecular mechanisms governing the formation of distinct Upf1-containing complexes in yeast.
  18. Lon/Pim1 mediated degradation of presequence translocase-associated motor components Pam16 and Pam18 in Saccharomyces cerevisiae.
  19. HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis.
  20. Of condensates and coats - reciprocal regulation of clathrin assembly and the growth of protein networks.
  21. Pharmacologic inhibition of IRE1α-dependent decay protects alveolar epithelial identity and prevents pulmonary fibrosis in mice.
  22. Stress, plasticity, and fibrosis: unfolding the role of the IRE1α/RIDD/Fgfr2 axis.
  23. Interaction proteomics of Polycystins 1 and 2 reveal a novel role for the BLOC-1/BORC lysosomal positioning complex.
  24. Slowing down to take it in: Endocytosis during cellular aging.
  25. Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
  26. Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis.
  27. Plant Heat Shock Protein Hsp90 enhances stress resistance through integrating protein quality control, chloroplast protection, hormone signal network, and immune defense.
  28. Nuclear 2'-O-methylation regulates RNA splicing through its binding protein FUBP1.
  29. Small-molecule allosteric activator of ubiquitin-specific protease 7 (USP7).
  30. Posterior pole of dermapteran oocytes houses a single aggregate of organelles reminiscent of endolysosomal vesicular assemblies described in mouse oocytes.
  31. Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
  32. Protein disulfide isomerases regulate androgen receptor stability and promote prostate cancer cell growth and survival.
  33. Split-site ubiquitination gives ZNFX1 new power in RNA defense.
  34. Cryo-EM structure of the human Derlin-1/p97 complex reveals a hexameric channel in ERAD.
  35. ER Stress Ire1-Xbp1s Pathway Maintains Youthful Epidermal Basal Layer Through the Regulation of Cell Proliferation.
  36. PIM3-mediated phosphorylation stabilizes myeloid leukemia factor 2 to promote metastasis in osteosarcoma.
  37. Preservation of Autophagy May Be a Mechanism Behind Healthy Aging.
  38. Bidirectional regulation of KEAP1 BTB domain-based sensor activity.
  39. Lysosomal proteomics reveals mechanisms of neuronal APOE4-associated lysosomal dysfunction.
  40. The E3 ubiquitin ligase TRIM56 promotes aggregation and activation of Src protein through Lys63-linked polyubiquitination in hepatocellular carcinoma.
  41. Protocol for identifying DNA damage responders through proximity biotinylation of proliferating cell nuclear antigen interactors.
  42. Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids.
  43. From Biogenesis to Breakdown: How Protein Biogenesis and Quality Control Failures Drive Mitochondrial Disease.
  44. Linked dimers of the AAA+ ATPase Msp1 reveal energetic demands and mechanistic plasticity for substrate extraction from lipid bilayers.
  45. Substrate-dependent activation of LONP1 informs on proteolytic regulation and ATPase motor function.
  46. Hijacking Extracellular Targeted Protein Degrader-Drug Conjugates for Enhanced Drug Delivery.
  47. Molecular landscape of the fungal plasma membrane and implications for antifungal action.
  48. Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes.
  49. Stimulated thyroid hormone synthesis machinery drives thyrocyte cell death independent of ER stress.
  50. Translational Remodeling of the Synaptic Proteome During Aging.
  51. The E3 ubiquitin ligase Trip12 semi-selectively attenuates Wnt signaling.
  52. Metabolic Regulation of Copper Homeostasis Governs the Sec61-Dependent Protein Translocation Process in Saccharomyces cerevisiae.
  53. Tomato ripening regulator SlSAD8 disturbs nuclear gene transcription and chloroplast-associated protein degradation.
  54. A generalizable deep learning framework for structure-based protein-ligand affinity ranking.
  55. The interactome era: Integrating RNA-seq, proteomics, and network biology to decode cellular senescence.
  56. Protein palmitoylation and sphingolipid metabolism control regulated exocytosis in cytotoxic lymphocytes.
  1. FEBS J. 2025 Oct 12.
    Differential regulation of translational stress responses by herpesvirus ubiquitin deconjugases.
    Jiangnan Liu, Noemi Nagy, Carlos Mario Ayala-Torres, Maria G Masucci.
      The strategies adopted by viruses to counteract the potential antiviral effects of ribosomal quality control (RQC) that regulates the fidelity of protein translation, ribosome recycling, and the activation of ribosomal and integrated stress responses are poorly understood. Here, we investigated the capacity of the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of human pathogenic herpesviruses to interfere with the triggering of RQC upon the induction of translational stress in cytosolic and endoplasmic reticulum (ER)-associated ribosomes. We found that the vDUBs encoded by Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and Kaposi sarcoma virus (KSHV) share the capacity to counteract the ubiquitination of RPS10, RPS20, and RPS3, and the UFMylation of RPL26 in cells treated with the translation elongation inhibitor anisomycin (ANS), which resulted in the rescue of model RQC and ER-RQC substrates from proteasome- and lysosome-dependent degradation, readthrough of stall-inducing mRNAs, and inhibition of ER-phagy. In contrast, while inhibiting the ubiquitination of RPS10, RPS20, and RPS3, and rescuing RQC substrates almost as efficiently as the homologs, the herpes simplex virus-1 (HSV1) encoded vDUB failed to counteract RPL26 UFMylation. Furthermore, it was unable to rescue the ER-RQC substrate or inhibit ER-phagy, nor did it promote ZAKα phosphorylation or activate the ISR. Our findings pinpoint important differences in the strategies adopted by these human viruses for regulating translational stress responses.
    Keywords:  UFM1; deubiquitinase; herpesvirus; integrated stress; reticulophagy; ribosomal quality control
    DOI:  https://doi.org/10.1111/febs.70278
  2. J Mol Biol. 2025 Oct 09. pii: S0022-2836(25)00548-0. [Epub ahead of print] 169482
    Temporal regulation of signal recognition particle during translation.
    Ruilin Qian, Radoslaw J Gora, Sowmya Chandrasekar, Shu-Ou Shan.
      Signal recognition particle (SRP) is a universally conserved protein targeting machine that directs newly synthesized proteins to the endoplasmic reticulum (ER). SRP recognizes signal sequences on nascent ER proteins as they emerge from the ribosome and, in response, activates interaction with the SRP receptor (SR) at the ER membrane. Early work suggested that SRP loses targeting competence as the nascent chain elongates; however, the underlying molecular mechanism remains unclear. Here we address this question using a combination of steady-state and single-molecule fluorescence spectroscopy measurements. A Förster resonance energy transfer (FRET) assay revealed increased dynamic excursions of the signal sequence from SRP on ribosomes bearing longer nascent chains, leading to a suboptimal conformation of SRP and its impaired interaction kinetics with SR. In addition, the nascent polypeptide associated complex (NAC) amplifies the effects of longer nascent chains to further exclude SRP from ER targeting. Our findings reveal the profound effects of an elongating nascent polypeptide on the conformation and activity of SRP and a key role of NAC in the temporal regulation of SRP, which together impose a limited window for cotranslational ER protein targeting during protein synthesis.
    Keywords:  protein targeting; ribosome; signal recognition particle; signal sequence; single molecule fluorescence
    DOI:  https://doi.org/10.1016/j.jmb.2025.169482
  3. ACS Chem Biol. 2025 Oct 16.
    Chemical Probe Approach Reveals Endo-α-mannosidase Triages Misfolded Glycoproteins in the Calnexin/Calreticulin Cycle.
    Akito Taira, Makoto Hirano, Taiki Kuribara, Chie Watanabe, Satoshi Hiraki, Mitsuaki Hirose, Zalihe Hakki, Spencer J Williams, Yukishige Ito, Kiichiro Totani.
      Protein N-glycosylation contributes to folding and quality control of secretory proteins involved in protein misfolding diseases. A central quality control machinery of nascent glycoproteins in the endoplasmic reticulum (ER) is the calnexin/calreticulin (CNX/CRT) cycle. This cycle assists and checks protein folding by monitoring glycan structure, however how terminally misfolded glycoproteins are discharged from the cycle has remained unclear. Here, we leveraged chemical probes to identify a previously uncharacterized ER endo-α-mannosidase complex (ER-EM) that provides this missing release step. ER-EM selectively cleaves the terminal Glc-Man disaccharide from glucosylated high-mannose glycans only when the glycan is attached to a hydrophobic aglycone─an intrinsic marker of misfolded proteins─thereby converting Glc1Man9GlcNAc2 to Man8AGlcNAc2 glycans that cannot bind CNX/CRT. This activity is allosterically stimulated by hydrophobic ligands and shares the same aglycone preference as the folding sensor UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1), creating a two-tier surveillance system in which UGGT1 reglucosylates incompletely folded proteins, whereas ER-EM ejects those that fail to mature. Proteomic and native-gel analyses revealed that ER-EM is an ∼ 800 kDa assembly composed of at least carboxylesterase 1D (Ces1d), ERp57 and UGGT1; the lack of activity of recombinant Ces1d alone underscores that the catalytic function arises only through the concerted action of this multisubunit complex. ER-EM therefore acts as a folding-status-dependent triage factor that liberates terminally misfolded glycoproteins from the CNX/CRT cycle and targets them for degradation, adding a critical new branch to the ER quality-control network.
    DOI:  https://doi.org/10.1021/acschembio.5c00532
  4. Cell Rep. 2025 Oct 10. pii: S2211-1247(25)01176-3. [Epub ahead of print]44(10): 116405
    Role of AAA-ATPase Cdc48p in peroxisomal quality control.
    Ismaila Francis Yusuf, Tomasz Jeziorek, Andrea Droste, Wolfgang Girzalsky, Ralf Erdmann.
      Most peroxisomal matrix proteins contain a type 1 peroxisomal targeting signal (PTS1), which is recognized by the cytosolic receptor Pex5p for delivery into peroxisomes. Following cargo translocation, the receptor is monoubiquitinated and recycled to the cytosol by the AAA-ATPases Pex1p and Pex6p. Defects in recycling trigger a quality control process by which the receptor is polyubiquitinated, extracted, and targeted to the proteasome for degradation by the RADAR (receptor accumulation and degradation in the absence of recycling) pathway. Although the RADAR pathway is evolutionarily conserved, it is unknown whether it is active in Saccharomyces cerevisiae. Here, we identify and characterize the RADAR pathway in S. cerevisiae and discover that the AAA-ATPases Msp1p and predominantly Cdc48p, together with its cofactors Ufd1p/Npl4p, are constituents of this pathway. We conclude that peroxisomes contain an endoplasmic reticulum-associated degradation-like protein quality control system (RADAR) in which Cdc48p and Ufd1p/Npl4p extract misfolded, polyubiquitinated receptors from the peroxisomal membrane for proteasomal degradation.
    Keywords:  AAA-ATPase; ATPases associated with diverse cellular activities; CHX; CP: Cell biology; CP: Molecular biology; RADAR; UPS; cycloheximide chase; peroxins; peroxisomal import receptor; peroxisome; protein degradation; protein stability; quality control; ubiquitin proteasomal system
    DOI:  https://doi.org/10.1016/j.celrep.2025.116405
  5. Sci Signal. 2025 Oct 14. 18(908): eadv1529
    The E3 ubiquitin ligase ZNRF3 restricts WNT receptor complex activity by stimulating the selective degradation of WNT-engaged FZD.
    Bo Lu, Feng Cong.
      Ligands of the WNT family induce formation of the WNT receptor signalosome and promote stabilization of the transcriptional coactivator β-catenin. The homologous transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 inhibit WNT-dependent stabilization of β-catenin by stimulating the degradation of the WNT receptor FZD, whereas the secreted R-spondin proteins promote the stabilization of FZD by inducing the degradation of ZNRF3 and RNF43. Here, we report that the R-spondin-induced stabilization of β-catenin in HEK293 cells was not mimicked by FZD overexpression, highlighting a gap in our understanding of this important regulatory mechanism. Contrary to the conventional view that ZNRF3 constitutively mediates the ubiquitylation and degradation of FZD, we found that ZNRF3-induced FZD degradation depended on endogenous WNT and that ZNRF3 selectively degraded WNT-engaged FZD. WNT enhanced the association between FZD and the intracellular adaptor protein DVL, and DVL subsequently recruited ZNRF3 to FZD to promote FZD degradation. Our data suggest that WNT signaling actively restricts itself through ZNRF3-dependent degradation of WNT-engaged FZD and that R-spondin enhances WNT signaling by prolonging the action of the WNT-engaged FZD complex, rather than by simply increasing the abundance of FZD on the cell surface.
    DOI:  https://doi.org/10.1126/scisignal.adv1529
  6. Mol Cell. 2025 Oct 14. pii: S1097-2765(25)00783-X. [Epub ahead of print]
    Mechanisms of assembly and function of the Hsp70-Hsp40 chaperone machinery.
    Yajun Jiang, Ziad Ibrahim, Youlin Xia, Mary Clay, Alexander Myasnikov, Kalyan Immadisetty, Zhilian Xia, Liang Tang, Paolo Rossi, Pritha Ganguly, Jiangshu Liu, Darcie Miller, Meixia Che, Santiago M Palacios, Günter Kramer, Bernd Bukau, Charalampos G Kalodimos.
      Hsp70 and Hsp40 molecular chaperones form a central machinery that remodels client proteins involved in numerous biological processes. Here, we integrated cryo-electron microscopy and nuclear magnetic resonance spectroscopy to determine the architecture of the full-length Hsp70-Hsp40 machinery. The structure of the complex in a physiologically inhibited state reveals distinct regulatory mechanisms. In the active state, the Hsp40 glycine-phenylalanine (G/F)-rich region acts as a pseudo-substrate for Hsp70, directly modulating refolding. This region also maintains Hsp40 in an autoinhibited state; upon binding to Hsp70, the inhibition is disrupted, exposing a cryptic client-binding site that enables client engagement and refolding. Transitions between these states are central to controlling refolding efficiency. Disrupting either the autoinhibited state or the G/F-Hsp70 interaction impairs function and elicits a compensatory heat shock response in cells. Our findings uncover the regulatory dynamics of a fundamental chaperone system, with broad implications for understanding protein homeostasis and the cellular response to stress.
    Keywords:  Hsp40; Hsp70; NMR; cryo-EM; heat shock response; molecular chaperones; protein folding
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.023
  7. Am J Physiol Cell Physiol. 2025 Oct 13.
    Response of UBR-box E3 ubiquitin ligases and protein quality control pathways to perturbations in protein synthesis and skeletal muscle size.
    Leslie M Baehr, Luis Gustavo Oliveira de Sousa, Craig A Goodman, Adam P Sharples, David S Waddell, Sue C Bodine, David C Hughes.
      The N-degron pathway contributes to proteolysis by targeting N-terminal residues of destabilized proteins via E3 ligases that contain a UBR-box domain. Emerging evidence suggests the UBR-box family of E3 ubiquitin ligases (UBR1-7) are involved in the positive regulation of skeletal muscle mass. The purpose of this study was to explore the role of UBR-box E3 ubiquitin ligases under enhanced protein synthesis and skeletal muscle growth conditions. Cohorts of adult male mice were electroporated with constitutively active Akt (Akt-CA) or UBR5 RNAi constructs with a rapamycin diet intervention for 7 and 30 days, respectively. In addition, the UBR-box family was studied during the regrowth phase post nerve crush induced inactivity. Skeletal muscle growth with Akt-CA or regrowth following inactivity increased protein abundance of UBR1, UBR2, UBR4, UBR5 and UBR7. This occurred with corresponding increases in Akt-mTORC1/S6K and MAPK/p90RSK signaling and protein synthesis. The increases in UBR-box E3s, ubiquitination, and proteasomal activity occurred independently of mTORC1 activity and were associated with increases in markers related to autophagy, ER-stress, and protein quality control pathways. Finally, while UBR5 knockdown (KD) evokes atrophy, it occurs together with hyperactivation of mTORC1 and protein synthesis. In UBR5 KD muscles, we identified an increase in protein abundance for UBR2, UBR4 and UBR7, which may highlight a compensatory response to maintain proteome integrity. Future studies will seek to understand the role of UBR-box E3s towards protein quality control in skeletal muscle plasticity.
    Keywords:  N-degron pathway; UBR5; hypertrophy; proteasome system; protein turnover
    DOI:  https://doi.org/10.1152/ajpcell.00602.2025
  8. Nat Commun. 2025 Oct 15. 16(1): 9094
    Structural basis of K11/K48-branched ubiquitin chain recognition by the human 26S proteasome.
    Piotr Draczkowski, Szu-Ni Chen, Ting Chen, Yong-Sheng Wang, Hsin-An Shih, Jessica Y C Huang, Ming-Chieh Tsai, Shu-Yu Lin, Steven Lin, Rosa Viner, Yuan-Chih Chang, Kuen-Phon Wu, Shang-Te Danny Hsu.
      Beyond the canonical K48-linked homotypic polyubiquitination for proteasome-targeted proteolysis, K11/K48-branched ubiquitin (Ub) chains are involved in fast-tracking protein turnover during cell cycle progression and proteotoxic stress. Here, we report cryo-EM structures of human 26S proteasome in a complex with a K11/K48-branched Ub chain. The structures revealed a multivalent substrate recognition mechanism involving a hitherto unknown K11-linked Ub binding site at the groove formed by RPN2 and RPN10 in addition to the canonical K48-linkage binding site formed by RPN10 and RPT4/5 coiled-coil. Additionally, RPN2 recognizes an alternating K11-K48-linkage through a conserved motif similar to the K48-specific T1 binding site of RPN1. The insights gleaned from these structures explain the molecular mechanism underlying the recognition of the K11/K48-branched Ub as a priority signal in the ubiquitin-mediated proteasomal degradation.
    DOI:  https://doi.org/10.1038/s41467-025-64719-x
  9. J Leukoc Biol. 2025 Oct 17. pii: qiaf145. [Epub ahead of print]
    Immunoregulatory Functions of the ER Stress Sensor IRE1 in Myeloid Cell Biology.
    Alonso Lira, Javier López, Fabiola Osorio.
      Myeloid cells- including monocytes, macrophages, dendritic cells (DCs), and granulocytes constitute a versatile arm of the immune system, serving as frontline sentinels that detect and react to environmental cues and orchestrate tailored immune responses. Their ability to respond promptly to distinct threats depends on dynamic processes that include differentiation, antigen presentation, and secretion of pro-inflammatory and antimicrobial mediators. These functions are tightly linked to the integrity of the endoplasmic reticulum (ER), an essential organelle responsible for the synthesis, folding, and modification of proteins involved in immune signaling. Disruption of ER homeostasis is commonly induced by infection, inflammation, autoimmunity, or cancer settings, leading to ER stress and activation of the unfolded protein response (UPR), a three-pronged signaling pathway aiming to restore the fidelity of the cellular proteome. Among UPR branches, the IRE1/XBP1 pathway has emerged as a central regulator of myeloid cell function, integrating proteostatic stress with immune modulation. Despite growing evidence positioning the IRE1/XBP1s axis as a pivotal immunological target bearing biomedical potential, the context-dependent outcomes of this UPR branch in myeloid cells, ranging from protective to maladaptive, remain incompletely understood. In this review, we explore the multifaceted roles of IRE1 in shaping myeloid cell responses across physiological and pathological states, highlighting molecular mechanisms and their impact on immune homeostasis and disease pathogenesis.
    Keywords:  IRE1; Unfolded protein response; XBP1; homeostasis; infections; myeloid cells; pathology
    DOI:  https://doi.org/10.1093/jleuko/qiaf145
  10. J Biol Chem. 2025 Oct 14. pii: S0021-9258(25)02670-5. [Epub ahead of print] 110818
    Loss of O-GlcNAcylation in cardiac myocytes triggers the integrated stress response contributing to heart failure.
    Kyriakos N Papanicolaou, Wenxi Zhang, Aidan Dunphy, Justin C Zhong, Clara Y Cho, Dan R Turner, Deepthi Ashok, D Brian Foster, Brian O'Rourke, Natasha E Zachara.
      Heart failure (HF) is a significant global health problem, affecting an estimated 64 million people worldwide. At the core of HF is the progressive dysfunction and irreversible loss of cardiac myocytes. O-GlcNAc transferase (OGT) is a conserved enzyme that catalyzes the addition of N-acetyl glucosamine (GlcNAc) to serine or threonine residues of intracellular proteins. This dynamic protein modification, termed O-GlcNAcylation, has been implicated in nutrient sensing, metabolic regulation and stress adaptation. The integrated stress response (ISR) is a pathway that enables cells to rapidly respond to acute environmental changes and cell damage. During ISR, the translation factor eIF2α is phosphorylated shutting down general translation but favoring the rapid production of stress-adaptive proteins. However, prolonged activation of the ISR can be detrimental to cells. In this study, we found that inhibiting OGT activates the GCN2/eIF2α/Atf4 signaling axis of the ISR. Activation of this pathway could be blocked by ISRIB, a small molecule that opposes the activity of phosphorylated eIF2α. Mice with inducible deletion of OGT in adult cardiomyocytes developed HF, and treatment with ISRIB significantly delayed the progression to HF. Our study reveals the regulatory impact of O-GlcNAcylation on the ISR and highlights a new potential strategy for alleviating HF.
    Keywords:  Atf4; GCN2; ISRIB; OGT; PERK; cardiomyocytes; cardiomyopathy; eIF2α; mTOR; translation initiation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110818
  11. ACS Med Chem Lett. 2025 Oct 09. 16(10): 2070-2077
    Targeted Histone Deacetylase Degradation via Chemical Induced Proximity by Direct Recruitment of the CUL4 Complex Adaptor Protein DDB1.
    Shiyang Zhai, Nicola Willemsen, Tao Sun, Mateo Malenica, Shixin Deng, Matthias Geyer, Finn K Hansen.
      Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) has emerged as a powerful strategy for disease treatment. By recruiting E3 ligases, these molecules enable selective degradation of pathogenic proteins. Cereblon (CRBN), a key component of the CUL4-DDB1-CRBN E3 ligase complex, is the most commonly recruited E3 ligase in PROTACs, including those targeting histone deacetylases (HDACs). In this study, we designed SZ-2, a bifunctional molecule derived from the DDB1 ligand MM-02-57 and the HDAC inhibitor vorinostat, to simultaneously bind DDB1 and HDACs. SZ-2 effectively induced degradation of HDAC1 and HDAC2 and demonstrated potent anti-multiple myeloma activity, highlighting its potential as a novel therapeutic agent.
    Keywords:  Cancer; Damage-specific DNA binding protein 1 (DDB1); Histone deacetylase (HDAC); Proteolysis targeting chimeras (PROTACs); Targeted Protein Degradation (TPD)
    DOI:  https://doi.org/10.1021/acsmedchemlett.5c00506
  12. Methods Mol Biol. 2026 ;2976 47-60
    Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
    Jennifer E Palmer, David C Rubinsztein.
      Autophagy is a conserved lysosomal degradation pathway that recycles protein aggregates and damaged organelles to maintain cytoplasmic quality control. Measuring the amount of the lipid-conjugated autophagic protein LC3B-II is a useful way to test whether a particular perturbation affects autophagy. However, the level of LC3B-II is affected by factors that alter either the rate of autophagosome biogenesis or degradation. Consequently, the same steady-state LC3B-II level can be reached by opposing autophagic fluxes. It is thus essential when measuring LC3B-II to perform the assay both in the absence and presence of a lysosomal inhibitor, enabling measurement of the rate of synthesis independent of its degradation. LC3B-II is also a small protein that can be challenging to detect by western blotting. In this chapter, we will provide a method for the efficient western blotting of LC3B-II and guidance as to the interpretation of the results.
    Keywords:  ATG8; Autophagy; Bafilomycin A1; LC3B; SDS-PAGE; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_5
  13. EMBO Rep. 2025 Oct 16.
    Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death.
    Gina Gotthardt, Janik Weckesser, Georg Tascher, Sara Barros da Gama, Hannah J Uckelmann, Shibo Sun, Martin P Schwalm, Thorsten Mosler, Giulio Ferrario, José Pedro Friedmann Angeli, Christian Münch, Stefan Knapp, Stefan Müller.
      Covalent inhibitors are an attractive targeting strategy that has expanded the development of degraders to target poorly druggable proteins including the E3 ligase RNF4. We show that RNF4 is a potential vulnerability of AML. High RNF4 expression levels correlate with poor patient survival and depletion of RNF4 results in increased sensitivity of AML cells to antileukemic drugs. Therefore, we aimed to develop chemical degraders (PROTACs) of RNF4 using a known covalent RNF4 ligand (CCW16), containing a chloro-N-acetamide group, as well as established E3 ligands targeting CRBN or VHL. However, while CCW16 and CCW16-derived PROTACs react potently with cysteines in recombinant RNF4, in cells, CCW16 forms covalent bonds with a large number of proteins, including peroxiredoxins. Consequently, CCW16 based PROTACs do not trigger degradation of RNF4, but induce the ferroptosis marker heme oxygenase-1 and impair cell viability in a distinct, RNF4-independent, ferroptotic cell death pathway. We hypothesize that other chloro-N-acetamide-containing E3 ligase ligands would also induce ferroptosis. Indeed, the RNF114 ligand EN219 also strongly induces ferroptosis, suggesting that ligands harboring this electrophile induce undesired off-target toxicity.
    Keywords:  AML; CCW16; Covalent PROTACs; Ferroptosis; RNF4
    DOI:  https://doi.org/10.1038/s44319-025-00593-4
  14. Nat Cell Biol. 2025 Oct;27(10): 1739-1756
    Cholesterol sensing by the SCAP-FAM134B complex regulates ER-phagy and STING innate immunity.
    Boran Li, Dongheng Zhou, Xinyi Wang, Xiao Jiang, Yongjuan Sang, Youjing Dai, Yu Yao, Yi Zhang, Chen Chen, Shulin Li, Wang Ni, Quan Zhou, Aifu Lin, Xinyang Hu, Liang Ge, Zhiying Wu, Pinglong Xu, Dante Neculai, Wei Liu, Qiming Sun.
      The endoplasmic reticulum (ER) is central to cholesterol biosynthesis and trafficking, yet paradoxically maintains low cholesterol levels, enabling it to sense fluctuations that impact various signalling pathways. However, the role of ER cholesterol in cellular signalling remains unclear. Here we show that the ER-phagy receptor FAM134B interacts directly with both cholesterol and SCAP, a key regulator of cholesterol biosynthesis. When ER cholesterol is high, FAM134B and SCAP are sequestered by cholesterol-tightened interactions, halting ER-phagy, STING activation and cholesterol synthesis. Under low cholesterol conditions, FAM134B dissociates from SCAP, allowing SCAP to activate SREBP2 and upregulate cholesterol synthesis, while FAM134B either facilitates ER-phagy through oligomerization or aids STING trafficking to activate innate immune responses. These findings reveal that the SCAP-FAM134B complex senses ER cholesterol levels, regulating both ER-phagy and immune signalling, with implications for diseases linked to cholesterol imbalance.
    DOI:  https://doi.org/10.1038/s41556-025-01766-y
  15. Nucleic Acids Res. 2025 Oct 14. pii: gkaf978. [Epub ahead of print]53(19):
    Structural basis of enhanced stalling efficiency of an engineered ribosome arrest peptide.
    Manoj Kumar Sriramoju, Tzu-Ping Ko, Piotr Draczkowski, Shang-Te Danny Hsu.
      Ribosome stalling is a critical regulatory mechanism in protein synthesis, controlling the rate and fidelity of translation. Arrest peptides, short sequences within nascent chains, can induce ribosome stalling, providing insights into the dynamics of translation and potential therapeutic targets. In this study, we investigated the molecular mechanisms of ribosome stalling induced by an engineered ribosomal arrest peptide (eRAP). We used cryo-electron microscopy and biochemical assays to characterize the interactions between eRAP, the ribosome, and accessory factors such as the trigger factor. Our results reveal intricate details of the eRAP-induced ribosome stalling, including the conformational changes in the ribosomal tunnel and the nascent chain. We also observed interactions between eRAP and specific ribosomal components, highlighting the role of key amino acids in mediating ribosome stalling. Furthermore, comparison with other stalling mechanisms, such as those induced by antibiotics or natural nascent peptides, elucidates the unique features of eRAP-induced stalling. Overall, our findings provide a comprehensive understanding of ribosome stalling by arrest peptides, shedding light on the fundamental processes of translation and offering potential avenues for therapeutic interventions targeting translation regulation.
    DOI:  https://doi.org/10.1093/nar/gkaf978
  16. Nat Commun. 2025 Oct 17. 16(1): 9234
    Visualization of lysosomal membrane proteins by cryo electron tomography.
    Bridget M McVeigh, José J De Jesús-Pérez, Dirk H Siepe, Prerana Gogoi, Shrawan Kumar Mageswaran, Marian Kalocsay, Elaine M Mihelc, Vera Y Moiseenkova-Bell.
      Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.
    DOI:  https://doi.org/10.1038/s41467-025-64314-0
  17. Cell Rep. 2025 Oct 10. pii: S2211-1247(25)01186-6. [Epub ahead of print]44(10): 116415
    Molecular mechanisms governing the formation of distinct Upf1-containing complexes in yeast.
    Iuliia Iermak, Nicole R Wilson Eisele, Katharina Kurscheidt, Matina-Jasemi Loukeri, Jérôme Basquin, Fabien Bonneau, Lukas M Langer, Achim Keidel, Elena Conti.
      Upf1 is a master regulator of nonsense-mediated mRNA decay (NMD), an mRNA surveillance and degradation pathway conserved from yeast to human. In Saccharomyces cerevisiae, Upf1 exists in two distinct complexes with factors that mediate NMD activation or 5'-3' mRNA degradation. We combined endogenous purifications and biochemical reconstitutions of yeast Upf1 complexes with structural analyses and biochemical assays to elucidate the molecular mechanisms driving the organization of the Upf1-5'-3' and Upf1-2-3 complexes. We show that yeast Upf1 is in a constitutive complex, whereby its CH, RecA, and C-terminal domains interact with the mRNA decapping factor Dcp2, NMD-associated proteins Nmd4 and Ebs1, and the 5'-3' exoribonuclease Xrn1, respectively. Together, the interacting surfaces and closed conformation of Upf1 in the Upf1-5'-3' complex sterically obstruct the binding of Upf2-3. Our work points to a major restructuring upon recruitment of these factors during NMD and provides insights into evolutionary divergence amongst species.
    Keywords:  AlphaFold; CP: Molecular biology; Saccharomyces cerevisiae; Upf1; biochemical reconstitution; cryo-EM; endogenous purification; nonsense-mediated decay
    DOI:  https://doi.org/10.1016/j.celrep.2025.116415
  18. Biochem J. 2025 Oct 10. pii: BCJ20243016. [Epub ahead of print]
    Lon/Pim1 mediated degradation of presequence translocase-associated motor components Pam16 and Pam18 in Saccharomyces cerevisiae.
    Yerranna Boggula, Arpan Chatterjee, Gaurav Simaiya, Amita Pal, Akash Srinivasan, Naresh Babu Sepuri.
      Mitochondrial protein homeostasis depends mainly on the efficient import and folding of nuclear-encoded proteins, and defects in this process can lead to proteotoxicity, which is harmful to the cell. Mitochondrial chaperones and proteases are essential defense mechanisms that ensure dysfunctional proteins' proper concentration, folding, and degradation. Lon protease 1 (Pim1 in yeast) is the mitochondrial matrix protease known to prevent protein aggregation by degrading unfolded proteins. Here, we show that two essential components of ATP-dependent presequence translocase and associated motor (PAM complex)- Pam18 and Pam16 are specifically targeted for degradation by the proteolytically active Lon/Pim1, both in vitro and in vivo. Further, overexpression of Pam18 and Pam16 exacerbates the growth defect of the delta pim1 strain. Hence, our study reveals, for the first time, that components involved in protein import are substrates of Pim1, which could have potential implications for regulating mitochondrial protein import and proteostasis.
    Keywords:  Lon/Pim1 protease; Mitochondria; Protein turnover; Proteolysis; Proteostasis; Saccharomyces cerevisiae; mitochondrial protein import; presequence translocase-associated motor
    DOI:  https://doi.org/10.1042/BCJ20243016
  19. Cell Rep. 2025 Oct 09. pii: S2211-1247(25)01153-2. [Epub ahead of print]44(10): 116382
    HUWE1 stimulates mTORC1 activity by enhancing Rheb interaction with mTORC1 and supports de novo pyrimidine synthesis.
    Takayuki Ikeda, Sungki Hong, Shota Yoshida, Swayam Prakash Srivastava, Abhiram Kunamneni, Yao Yao, Om Khuperkar, Venkatesha Basrur, Henry Kuang, Maureen Kachman, Abraham Raskind, Heidi Baum, Arya Joshi, Vinamra Swaroop, Aaron Thurman, Oliviamae Kopasz-Gemmen, Siddharth Kandukuri, F N U Pradeepa, Hideto Yonekura, Jiandie D Lin, Ken Inoki.
      The mechanistic target of rapamycin complex 1 (mTORC1), a central regulator of cell growth, is activated by Rheb small GTPase. Our recent studies have demonstrated that polyubiquitinated Rheb enhances its interaction with mTORC1, resulting in the activation of mTORC1. Here, we demonstrate that the HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1), an E3 ubiquitin ligase, preferentially interacts with ubiquitinated Rheb and facilitates Rheb's binding to mTORC1 and its subsequent activation. The ablation of HUWE1 results in reduced ubiquitination of Rheb and decreased mTORC1 activity in cultured cells and mouse liver. HUWE1 is also necessary for Rheb to interact with carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase (CAD), a key enzyme in pyrimidine biosynthesis, and for CAD activation through the activation of the mTORC1-S6K1 pathway. Moreover, HUWE1 maintains CAD expression by increasing its transcript in cells and liver tissues. Therefore, HUWE1 acts as a key organizer of the ubiquitinated Rheb complex, playing a vital role in enhancing mTORC1 activity and pyrimidine synthesis by increasing both CAD activity and expression.
    Keywords:  CAD; CP: Cell biology; CP: Molecular biology; HUWE1; Rheb; mTOR; mTORC1; pyrimidine; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2025.116382
  20. Nat Commun. 2025 Oct 15. 16(1): 9139
    Of condensates and coats - reciprocal regulation of clathrin assembly and the growth of protein networks.
    Brandon T Malady, Andromachi Papagiannoula, Advika Kamatar, Susovan Sarkar, Gavin T Lebrun, Liping Wang, Carl C Hayden, Eileen M Lafer, David J Owen, Sigrid Milles, Jeanne C Stachowiak.
      Clathrin-mediated endocytosis is essential for membrane traffic, impacting a diverse range of cellular processes including cell signaling homeostasis, cell adhesion, and receptor recycling. During endocytosis, invagination of the plasma membrane is coordinated by a network of proteins that recruit and assemble the clathrin coat. Recent work demonstrated that clathrin accessory proteins which arrive early at endocytic sites, such as Eps15 and Fcho2, form phase-separated condensates that recruit downstream machinery, promoting maturation of clathrin-coated vesicles. However, the mechanisms by which protein condensates regulate and are regulated by clathrin assembly remain unclear. Using in vitro reconstitution and nuclear magnetic resonance spectroscopy, we demonstrate that protein condensates provide a platform for recruitment and assembly of clathrin triskelia. This condensate driven assembly is enhanced in the presence of the accessory protein, AP2, which is incorporated within condensates. In turn, clathrin assembly restricted condensate growth, exhibiting surfactant-like behavior that stabilized protein-protein interactions while imposing the preferred curvature of the clathrin lattice. This mutual regulation promotes assembly of clathrin-coated vesicles while preventing uncontrolled expansion of protein condensates. More broadly, reciprocal regulation of protein condensates and clathrin coats may provide a framework for understanding how disordered and structured protein assemblies can work together to build cellular architectures.
    DOI:  https://doi.org/10.1038/s41467-025-64816-x
  21. J Clin Invest. 2025 Oct 15. pii: e184522. [Epub ahead of print]135(20):
    Pharmacologic inhibition of IRE1α-dependent decay protects alveolar epithelial identity and prevents pulmonary fibrosis in mice.
    Vincent C Auyeung, Tavienne L Steinberg, Alina Olivier, Luka Suzuki, Mary E Moreno, Imran S Khan, Michael S Downey, Maike Thamsen, Lu Guo, Dustin J Maly, Bradley J Backes, Dean Sheppard, Feroz R Papa.
      Stress-induced epithelial plasticity is central to lung regeneration, fibrosis, and malignancy, but how cellular stress leads to differentiation is incompletely understood. Here, we found a central role for IRE1α, a conserved mediator of the unfolded protein response (UPR), in stimulating the plasticity of alveolar type 2 (AT2) cells. In single-cell RNA-seq, IRE1α activity was associated with loss of AT2 identity and progression toward a damage-associated transitional state unique to fibrosis. AT2 plasticity required destructive regulated IRE1α-dependent decay (RIDD), which we demonstrated by deploying PAIR2, a kinase modulator that inhibits RIDD while preserving IRE1α's adaptive XBP1 mRNA splicing activity. In vivo, selective inhibition of RIDD with PAIR2 reduced AT2 differentiation into profibrotic transitional cells and protected mice from bleomycin-induced pulmonary fibrosis. Mechanistically, we identified the Fgfr2 mRNA as a direct and regulated substrate for IRE1α's RNase in primary AT2 cells and in a biochemically reconstituted cell-free system. Loss of Fgf signaling caused AT2 differentiation, while gain of signaling protected cells from IRE1α-induced differentiation. We propose that IRE1α downregulates Fgf signaling through RIDD, provoking loss of AT2 identity and differentiation towards a profibrotic phenotype. Thus, IRE1α's RIDD activity emerges as a novel target for treatment of pulmonary fibrosis and potentially other diseases driven by aberrant epithelial cell plasticity.
    Keywords:  Cell stress; Fibrosis; Protein kinases; Pulmonology; Stem cells; Therapeutics
    DOI:  https://doi.org/10.1172/JCI184522
  22. J Clin Invest. 2025 Oct 15. pii: e196740. [Epub ahead of print]135(20):
    Stress, plasticity, and fibrosis: unfolding the role of the IRE1α/RIDD/Fgfr2 axis.
    SeungHye Han.
      Recent advances in sequencing technologies have enabled the identification of intermediate cell states during alveolar epithelial differentiation, which expand during repair following injury and in fibrotic lungs. Although ER stress has been implicated in pulmonary fibrosis, the underlying mechanisms remain elusive. The featured study by Auyeung and colleagues looked for links between the unfolded protein response sensor inositol-requiring enzyme 1α (IRE1α), intermediate epithelial cell states, and fibrotic remodeling in the lung. They identified Regulated IRE1-Dependent Decay (RIDD) as a key effector of IRE1α signaling that drives differentiation of alveolar epithelial type 2 cells to damage-associated intermediate cells and contributes to pulmonary fibrosis, likely by degrading Fgfr2 mRNA. These findings unveil therapeutic targets and open new avenues for investigating the interplay between cellular stress responses, epithelial differentiation, and fibrotic disease.
    DOI:  https://doi.org/10.1172/JCI196740
  23. Mol Cell Proteomics. 2025 Oct 12. pii: S1535-9476(25)00190-2. [Epub ahead of print] 101091
    Interaction proteomics of Polycystins 1 and 2 reveal a novel role for the BLOC-1/BORC lysosomal positioning complex.
    Fatima Lukmani, Jonathan M Shillingford, Mackenzie Brauer, Dhairya Patel, Jonathan St- Germain, James A Shayman, Brian Raught, Gagan D Gupta.
      PKD1 and PKD2 are the most commonly mutated genes in autosomal dominant polycystic kidney disease (ADPKD). However, the precise roles of the encoded Polycystin 1/2 (PC1, PC2) proteins, and how their functions are disrupted in ADPKD, remain unclear. Here, we characterize the protein interaction networks of PC1 and PC2 in cycling and ciliated cells using proximity-dependent biotinylation (BioID), identifying a common set of 172 proteins that interact with the C-terminus of PC1 and the full length PC2 protein, enriched in autophagy regulators, endoplasmic reticulum (ER) tethers, ER stress proteins, and other proteins previously linked to ADPKD. Notably, we also find that PC1 specifically interacts with ciliary and lysosomal proteins, including components of the biogenesis of lysosome-related organelles complex (BLOC-1) and BLOC-one-related-complex (BORC). BLOC-1/BORC co-localizes with PC1 at lysosomes and cilia, and is required for proper ciliary PC1 localization. Additionally, PC1 mutant kidney cells derived from an ADPKD patient display defects in BLOC-1/BORC distribution. Renal cells depleted of PC1 exhibit abnormal lysosomal distribution, similar to those depleted of BLOC-1/BORC components. Lastly, shRNA knockdown of BLOC-1/BORC components promoted cystogenesis in a 3D in vitro cyst model, and this could be attenuated by heterologous expression of the C-terminus of PC1. This rich dataset thus links the BLOC-1/BORC complex to PC1 function and can be further mined for additional mechanistic insights into the PC1/2 ADPKD proteins.
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101091
  24. J Cell Biol. 2025 Nov 03. pii: e202510010. [Epub ahead of print]224(11):
    Slowing down to take it in: Endocytosis during cellular aging.
    Derek C Prosser.
      Aging cells functionally decline and accumulate damage through poorly understood mechanisms. In this issue, Antentor et al. (https://doi.org/10.1083/jcb.202412064) find that increased vacuolar pH in older yeast cells slows clathrin-mediated endocytosis. These findings have broad implications in aging-related plasma membrane protein quality control.
    DOI:  https://doi.org/10.1083/jcb.202510010
  25. Nat Cell Biol. 2025 Oct;27(10): 1688-1707
    Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
    Rabia R Khawaja, Ernesto Griego, Kristen Lindenau, Asma Salek, Jessica Gambardella, Aurora Scrivo, Hannah R Monday, Mathieu Bourdenx, Jesús Madero-Pérez, Zohaib N Khan, Bhakti Chavda, Ronald Cutler, Sarah Graff, Simone Sidoli, Gaetano Santulli, Laura Santambrogio, Inmaculada Tasset, Susmita Kaushik, Li Gan, Pablo E Castillo, Ana Maria Cuervo.
      Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.
    DOI:  https://doi.org/10.1038/s41556-025-01771-1
  26. J Cell Biol. 2025 Dec 01. pii: e202503068. [Epub ahead of print]224(12):
    Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis.
    Chris T Ho, Elliot B Evans, Kimberly Lukasik, Ellen C O'Shaughnessy, Aneri Shah, Chih-Hsuan Hsu, Brenda Temple, James E Bear, Stephanie L Gupton.
      Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role of the actin binding protein coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrin-dependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain-enriched E3 ubiquitin ligase that binds the netrin receptor DCC, and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was unable to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.
    DOI:  https://doi.org/10.1083/jcb.202503068
  27. J Exp Bot. 2025 Oct 13. pii: eraf447. [Epub ahead of print]
    Plant Heat Shock Protein Hsp90 enhances stress resistance through integrating protein quality control, chloroplast protection, hormone signal network, and immune defense.
    Jieting Wu, Yuxin Li, Haoran Yin, Lei Zhao, Chengbin Xu, Xiaofan Fu, Jing Shang, Shuhan Liu, Haijuan Guo, Fang Ma.
      Given the groundbreaking progress in identifying unique functions of plant Hsp90 in stress responses in recent years, and the absence of systematic integration of these new mechanisms in protein quality control, hormone network regulation, chloroplast protection, and immune defense in existing reviews, this article aims to fill this gap. Recent studies have revealed four key mechanisms: (1) Hsp90 forms complexes with E3 ligases to promote polyubiquitination of heat-induced protein aggregates, cooperating with the 26S proteasome for clearance, a pathway hijacked by viruses; (2) Hsp90 stabilizes auxin receptor TIR1, reconstructs root auxin gradients via polarized PIN1, activates ABA biosynthesis, and enhances insect resistance through JA signaling; (3) Hsp90C maintains photosystem renewal, protects chloroplast DNA via CND1 translocation, and stabilizes thylakoid proteins via its CTE domain; (4) Hsp90 escorts CERK1 to initiate PTI, activates NLRs for ETI, and interacts with ATG8 to enhance autophagic pathogen clearance. This review integrates key new discoveries since 2012, and identifies core research gaps to address, namely regulation of Hsp90 abundance "optimal threshold", its combined stress response mechanism, transformation of knowledge from model plants to major food crops, and provides a clear direction framework for subsequent research.
    Keywords:  Biotic interactions; Hormone; Hsp90 (Heat shock protein 90); Immune; Plant cell; Stress
    DOI:  https://doi.org/10.1093/jxb/eraf447
  28. Sci Adv. 2025 Oct 17. 11(42): eady3894
    Nuclear 2'-O-methylation regulates RNA splicing through its binding protein FUBP1.
    Boyang Gao, Bochen Jiang, Zhongyu Zou, Bei Liu, Weijin Liu, Li Chen, Lisheng Zhang, Chuan He.
      2'-O-methylation (Nm) is an abundant RNA modification exists on different mammalian RNA species. However, potential Nm recognition by proteins has not been extensively explored. Here, we used RNA affinity purification, followed by mass spectrometry to identify Nm-binding proteins. The Nm-binding protein candidates exhibit enriched binding at known Nm sites. Some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay validated preference of FUBP1 to Nm-modified RNA. As FUBP1 predominantly binds intronic regions, we profiled Nm sites in chromatin-associated RNA (caRNA) and found Nm enrichment within introns. Depletion of Nm led to skipped exons, suggesting Nm-dependent splicing regulation. The caRNA Nm sites overlap with FUBP1-binding sites, and Nm depletion reduced FUBP1 occupancy on modified regions. Furthermore, FUBP1 depletion induced exon skipping in Nm-modified genes, supporting its role in mediating Nm-dependent splicing regulation. Overall, our findings identify FUBP1 as an Nm-binding protein and uncover previously unrecognized nuclear functions for RNA Nm modification.
    DOI:  https://doi.org/10.1126/sciadv.ady3894
  29. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2510496122
    Small-molecule allosteric activator of ubiquitin-specific protease 7 (USP7).
    Isabella Jaen Maisonet, Mona Sharafi, Emilie J Korchak, Andres Salazar-Chaparro, Ariana S Bratt, Tvesha Parikh, Anthony C Varca, Binita Shah, Michael Darnowski, Mia Chung, Wei Pin Teh, Jianwei Che, Irina Bezsonova, Sara J Buhrlage.
      Ubiquitin-specific protease 7 (USP7) is a deubiquitylase essential for cell homeostasis, DNA repair, and regulation of both tumor suppressors and oncogenes. Recently, haploinsufficiency of USP7 has been associated with Hao-Fountain syndrome (HAFOUS), a rare neurodevelopmental disorder. Although a range of USP7 inhibitors have been developed over the last decade, in the context of HAFOUS, USP7 activators may represent a more relevant approach. To address this challenge, we report the identification and characterization of a small-molecule activator of USP7 called MS-8. Structural and functional studies show that MS-8 allosterically activates USP7, mimicking the endogenous autoactivation mechanism of the enzyme. We observed that MS-8 engages and activates mutant USP7 in a cellular context, impacting downstream proteins. Taken together, our study provides validation of the USP7 activator that paves the way toward activation-driven USP7 pharmacology.
    Keywords:  DUB; Hao–Fountain syndrome; USP7; deubiquitylases; small-molecule activator
    DOI:  https://doi.org/10.1073/pnas.2510496122
  30. Dev Biol. 2025 Oct 11. pii: S0012-1606(25)00295-7. [Epub ahead of print]529 121-129
    Posterior pole of dermapteran oocytes houses a single aggregate of organelles reminiscent of endolysosomal vesicular assemblies described in mouse oocytes.
    Magda Sochaczewska, Malgorzata Sekula, Waclaw Tworzydlo, Szczepan M Bilinski.
      Oocytes of derived dermapterans (the Eudermaptera), in addition to "conventional" organelles, i.e. mitochondria, elements of endoplasmic reticulum, Golgi complexes, lysosome-like bodies and reserve materials (yolk spheres, lipid droplets), contain unexpectedly rich set of noncanonical organelles. It includes assemblies of endoplasmic reticulum and fine granular "nuage" material, ribosome-associated vesicles, and extensive arrays of parallel arranged endoplasmic reticulum cisternae. During vitellogenesis (yolk formation), all lysosome-like bodies are gradually translocated towards the posterior oocyte pole, where they form relatively large, morphologically distinct compartment that we termed the posterior pole lysosomal compartment. This compartment is not surrounded by a limiting membrane and stains positively with Proteostat, a dye used to detect proteins implicated in the formation of biomolecular condensates. In the light of these results, we hypothesize that posterior pole lysosomal compartment is reminiscent of endolysosomal vesicular assembly/ies participating in the sequestration/degradation of aggregated proteins in mammalian oocytes.
    Keywords:  Biomolecular condensates; Endolysosomal vesicular assemblies (ELVAs); Germ plasm; Nuage; Oogenesis; Ribosome-associated vesicles
    DOI:  https://doi.org/10.1016/j.ydbio.2025.10.009
  31. Acta Neuropathol. 2025 Oct 14. 150(1): 43
    Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
    Zachary M Augur, Garrett M Fogo, Mason R Arbery, Yi-Chen Hsieh, Nalini R Rao, Kritika Goyal, Emily Dexter, David A Bennett, Jeffrey N Savas, Andrew M Stern, Tracy L Young-Pearse.
      The autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) are the primary protein degradative mechanisms maintaining proteostasis in neurons. However, the impact of human genetic variation on these pathways and the role of BAG3 are poorly understood, particularly in the context of Alzheimer's disease, where proteostatic dysfunction is a defining hallmark. We utilized a large panel of iPSCs from deeply phenotyped cohorts to interrogate genetic contributions to baseline autophagic flux and UPS activity in human neurons, and protein turnover was assessed using SILAC-based quantitative proteomics. Across this panel of neurons, we observed substantial inter-individual differences in autophagic flux, which was inversely correlated with UPS activity. This reciprocal relationship extended to tau homeostasis, where higher autophagic flux resulted in reduced accumulation of aggregated, phosphorylated tau. Proteomic analyses revealed that global protein turnover dynamics stratified based on degradation pathway activity and could predict pathway-specific substrate dependencies. Interestingly, Bcl-2-associated athanogene 3 (BAG3), an important member of the chaperone-assisted selective autophagy pathway, emerged as a dynamically regulated autophagy chaperone, responsive to pharmacological inhibition of both the UPS and ALP. BAG3 knockout in neurons decreased autophagic flux and increased levels of high-molecular-weight phosphorylated tau. Notably, familial AD mutations and Aβ exposure induced BAG3 expression in neurons, while elevated BAG3 levels in human brain tissue were associated with higher neuropathological burden and disease progression. Our findings identify BAG3 as a key modulator of proteostasis in human neurons. Its regulation across genetic backgrounds and pathological stimuli suggests a central role in maintaining degradation activities in Alzheimer's disease and related disorders.
    Keywords:  Alzheimer’s disease; Amyloid-beta; Autophagy; BAG3; Neurons; Ubiquitin proteasome system; iPSC
    DOI:  https://doi.org/10.1007/s00401-025-02947-7
  32. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2509222122
    Protein disulfide isomerases regulate androgen receptor stability and promote prostate cancer cell growth and survival.
    Jianling Xie, Kaikai Shen, Wenken Liang, Zijian Kuang, Raj K Shrestha, Adrienne R Hanson, Scott L Townley, Meiling He, Sishu Yu, Peiwen Zhou, Liangzhen Zhu, Zhiwen Gong, Xiang Ao, Sushma R Rao, Qing Zhang, Kaijie Chen, Jinfen Wei, Shashikanth Marri, Marten F Snel, Swati Irani, Liye Chen, Ling Wang, Daniel P McDougal, John B Bruning, Minglin Ou, Shaobo Wang, Christopher G Proud, Hongli Du, Lisa M Butler, Luke A Selth.
      Cancer cells exhibit accelerated protein production to accommodate their high rates of growth and proliferation. Elevated protein synthesis creates a dependency on endoplasmic reticulum (ER)-resident proteins and chaperones, which are required to maintain proteostasis. In this study, we identified the protein disulfide isomerases (PDIs) PDIA1 and PDIA5, which play a critical role in folding of client proteins in the ER, as important regulators of prostate cancer growth and response to therapy. PDIA1 and PDIA5 are upregulated in prostate cancer and induced by the androgen receptor (AR) signaling axis. Genetic or pharmacological disabling of PDIA1/PDIA5 caused redox stress, mitochondrial dysfunction, growth inhibition, and death of prostate cancer cells in vitro and in vivo. The critical functions of these enzymes in redox homeostasis and cell survival were observed in both AR-driven and AR-independent models of prostate cancer. Loss of PDIA1/PDIA5 activity led to ubiquitination and degradation of the AR, revealing a feedback loop between these chaperones and the AR pathway. Mechanistically, PDIA1/PDIA5 regulated AR stability by mediating disulfide bond formation, an activity that required cysteines 669 and 844 in AR's ligand-binding domain. Importantly, targeting PDIAs sensitized prostate cancer cells to the AR antagonist, enzalutamide. This study reveals a mechanism governing AR proteostasis in prostate cancer and positions PDIA1/5 as viable therapeutic targets.
    Keywords:  PDIA1; PDIA5; androgen receptor; prostate cancer; protein degradation
    DOI:  https://doi.org/10.1073/pnas.2509222122
  33. Mol Cell. 2025 Oct 16. pii: S1097-2765(25)00782-8. [Epub ahead of print]85(20): 3734-3736
    Split-site ubiquitination gives ZNFX1 new power in RNA defense.
    Stacy M Horner.
      Recent work by Grabarczyk et al.1 uncovers the molecular mechanism by which ZNFX1, an interferon-stimulated gene, employs a novel split-site E3 ligase domain structure to ubiquitinate both protein lysine residues and RNA 2' hydroxyls. This activity enables ZNFX1 to compact pathogenic RNA into dense, ubiquitin-coated particles, revealing a new modality for interferon-induced antiviral defense.
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.022
  34. Commun Biol. 2025 Oct 17. 8(1): 1481
    Cryo-EM structure of the human Derlin-1/p97 complex reveals a hexameric channel in ERAD.
    Qian Wang, Deqiang Yao, Bing Rao, Ying Xia, Wenguo Li, Shaobai Li, Mi Cao, Yafeng Shen, An Qin, Yu Cao.
      The ER-associated degradation (ERAD) pathway retrotranslocates misfolded proteins from the ER lumen to the cytoplasm for proteasomal degradation. Derlin-1 and p97 are central to this process, forming a canonical 4:6 complex with tetrameric Derlin-1. Using cryo-electron microscopy, we identify a novel human Derlin-1/p97 complex with a 6:6 stoichiometry, where hexameric Derlin-1 assembles as three dimers. This hexameric channel forms a significantly larger trans-ER membrane tunnel, potentially accommodating bulkier substrates. Structural comparisons revealed conformational flexibility in Derlin-1, suggesting the "U"-shaped tetramer may act as an intermediate in hexamer formation. The formation of this hexameric channel is mediated by interactions with p97 and appears dependent on p97's ATPase activity, which provides the driving force for the transition between the tetrameric channel conformation to the intermediate "U"-shaped conformation. These findings highlight the dynamic nature of the Derlin-1/p97 complex and its implications for understanding ERAD retrotranslocation.
    DOI:  https://doi.org/10.1038/s42003-025-08880-5
  35. Aging Cell. 2025 Oct 12. e70258
    ER Stress Ire1-Xbp1s Pathway Maintains Youthful Epidermal Basal Layer Through the Regulation of Cell Proliferation.
    Daniel Semmy, Kota Abe, Mizuki Honda, Hiroko Omori, Shohei Ogamino, Tobias Clausen Mercurio, Kyosuke Asakawa, Emi K Nishimura, Shinya Oki, Yasuyuki Ohkawa, Tohru Ishitani.
      The endoplasmic reticulum (ER) stress-response is an adaptive cellular mechanism activated by an accumulation of unfolded proteins within the ER. Although recent evidence shows that the ER stress-response is activated in aged tissues, and therefore ER stress is considered a candidate driver of aging, the spatiotemporal regulation and roles of the ER stress-response during aging remain unclear. To address this research gap, we introduced an Ire1-Xbp1s ER stress-response pathway-sensitive reporter into the ultra-short-lived vertebrate Nothobranchius furzeri that allows for the analysis of its aging processes within a short period of time. Using this reporter in N. furzeri, we confirmed the previously reported age-dependent activation of ER stress in various tissues and identified an unexpected role of the Ire1-Xbp1s ER stress-response pathway in regulating epidermal tissue homeostasis and aging. The Ire1-Xbp1s ER stress-response pathway is active in the young epidermal basal layer but declines with aging. Photo-isolation chemistry-based spatial transcriptomics and functional assays revealed that the Ire1-Xbp1s pathway maintains young epidermal cell proliferation by activating the cell cycle regulator Vcp, whereas the age-dependent decline in glucose metabolism reduces Ire1-Xbp1s activity, consequently downregulating cell proliferation. Collectively, our study elucidates a previously unidentified role of the ER stress-response in skin aging, which can offer insights into therapeutic targets for promoting healthy skin.
    Keywords:  Ire1 protein; cell proliferation; endoplasmic reticulum stress; skin aging; unfolded protein response
    DOI:  https://doi.org/10.1111/acel.70258
  36. J Clin Invest. 2025 Oct 15. pii: e191040. [Epub ahead of print]135(20):
    PIM3-mediated phosphorylation stabilizes myeloid leukemia factor 2 to promote metastasis in osteosarcoma.
    Cuiling Zeng, Xin Wang, Jinkun Zhong, Yu Zhang, Ju Deng, Wenqiang Liu, Weixuan Chen, Xinhao Yu, Dian Lin, Ruhua Zhang, Shang Wang, Jianpei Lao, Qi Zhao, Li Zhong, Tiebang Kang, Dan Liao.
      Osteosarcoma is the most common primary malignant bone cancer, characterized by a high incidence of lung metastasis and a lack of therapeutic targets. Here, by combining an in vivo CRISPR activation screen with the interactome of STUB1, a tumor suppressor in osteosarcoma, we identified that myeloid leukemia factor 2 (MLF2) promotes osteosarcoma metastasis. Mechanistically, MLF2 disrupted the interaction between BiP and IRE1α, thereby activating the IRE1α/XBP1-S-MMP9 axis. The E3 ligase STUB1 ubiquitinated MLF2 at Lys119 and targeted it for proteasomal degradation, whereas PIM3-mediated phosphorylation of MLF2 at Ser65 enhanced its stabilizing interaction with USP21. Our findings demonstrate that the PIM3/MLF2 axis is a critical regulator of osteosarcoma lung metastasis. We propose PIM3 as a potential therapeutic target for patients with osteosarcoma lung metastasis.
    Keywords:  Cancer; Cell biology; Oncology; Protein kinases; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI191040
  37. Aging Cell. 2025 Oct 13. e70246
    Preservation of Autophagy May Be a Mechanism Behind Healthy Aging.
    Arsun Bektas, Shepherd H Schurman, Julián Candia, Olaya Santiago-Fernández, Susmita Kaushik, Ana Maria Cuervo, Luigi Ferrucci.
      Autophagy is intricately linked with protective cellular processes, including mitochondrial function, proteostasis, and cellular senescence. Animal studies have indicated that autophagy becomes dysfunctional with aging and may contribute to T cell immunosenescence. In humans, it remains unclear whether autophagy is impaired in CD4+ T cells as people age. To answer this question, we examined basal and inducible autophagic activity in a series of experiments comparing CD4+ T cells from younger (23-35 years old) and older (67-93 years old) healthy donors. We used immunofluorescence to detect LC3 (a marker of autophagosomes and autolysosomes) and LAMP2 (a marker of endolysosomes) in conjunction with bafilomycin A1 (which inhibits the acidification of lysosomes) and CCCP (a mitochondrial uncoupler) to manipulate autophagic flux. We found a significantly higher autophagy flux in CD4+ T cells from older compared to younger donors and a higher number of LC3+ compartments among older donors. Since the overall amount of autophagosomes degraded was comparable between the two groups, we concluded that autophagosome biogenesis was reduced in the older group. Rather than a decline, our findings in healthy older donors point toward a compensatory enhancement of human CD4+ T cell autophagy with age, which may be a mechanism behind healthy aging.
    Keywords:  CD4+ T cells; autophagy; healthy aging
    DOI:  https://doi.org/10.1111/acel.70246
  38. Redox Biol. 2025 Oct 08. pii: S2213-2317(25)00398-2. [Epub ahead of print]87 103885
    Bidirectional regulation of KEAP1 BTB domain-based sensor activity.
    Takafumi Suzuki, Kenji Takagi, Tatsuro Iso, Huaichun Wen, Anqi Zhang, Tetsuya Hatakeyama, Hiraku Oshima, Tsunehiro Mizushima, Masayuki Yamamoto.
      The KEAP1-CUL3 ubiquitin ligase regulates protein stability of transcriptional factor NRF2 and plays critical roles in cellular stress response. The BTB domain of KEAP1 functions as a sensor for electrophilic chemicals. However, the precise mechanisms by which electrophiles are recognized and inhibit BTB activity remain unclear. Here, we show that electrophilic modification alters the spatial arrangement of the BTB homodimer, regulating its ligase activity. Co-crystal structural analyses and functional studies using potent NRF2-inducing CDDO-derivatives, synthetic electrophilic compounds structurally related to clinically approved molecules such as Omaveloxolone, revealed that the key sensor residue, Cys151, resides in a structurally elaborate environment within the BTB domain. Modification of Cys151 by NRF2 inducers changes the spatial configuration of the CUL3-binding sites in the BTB homodimer, reducing KEAP1-CUL3 complex affinity. In contrast, a Cys151-targeting NRF2 inhibitor induces an opposite rearrangement of the BTB homodimer. This study elucidates the molecular mechanism by which the BTB domain finely regulates KEAP1-CUL3 ubiquitin ligase activity.
    Keywords:  BTB domain; KEAP1; NRF2; Stress sensor; Ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.redox.2025.103885
  39. Autophagy. 2025 Oct 16.
    Lysosomal proteomics reveals mechanisms of neuronal APOE4-associated lysosomal dysfunction.
    Einar K Krogsaeter, Justin McKetney, Leopoldo Valiente-Banuet, Angelica Marquez, Alexandra Willis, Zeynep Cakir, Erica Stevenson, Gwendolyn M Jang, Antara Rao, Emmy Li, Anton Zhou, Anjani Attili, Timothy S Chang, Martin Kampmann, Yadong Huang, Nevan J Krogan, Danielle L Swaney.
      APOE4 is the primary risk factor for Alzheimer disease (AD). Early AD pathological events first affect the neuronal endolysosomal system, which in turn causes neuronal protein aggregation and cell death. Despite the crucial influence of lysosomes upon AD pathophysiology, and that APOE4 localizes to lysosomes, the influence of APOE4 on lysosomal function remains unexplored. We find that expression of APOE4 in neuronal cell lines results in lysosomal alkalinization and impaired lysosomal function. To identify driving factors for these defects, we performed quantitative lysosomal proteome profiling. This revealed that APOE4 expression results in differential regulation of numerous lysosomal proteins, correlating with APOE allele status and disease severity in AD brains. In particular, APOE4 expression results in the depletion of lysosomal LGALS3BP and the accumulation of lysosomal TMED5. We additionally validated that these lysosomal protein changes can be targeted to modulate lysosomal function. Taken together, this work thereby reveals that APOE4 causes widespread lysosomal defects through remodeling the lysosomal proteome, with the lysosomal TMED5 accumulation and LGALS3BP depletion manifesting as lysosomal alkalinization in APOE4 neurons.
    Keywords:  APOE4; Alzheimer disease; LGALS3BP; LysoIP; TMED5; lysosomes; pH
    DOI:  https://doi.org/10.1080/15548627.2025.2576613
  40. Cell Death Dis. 2025 Oct 16. 16(1): 731
    The E3 ubiquitin ligase TRIM56 promotes aggregation and activation of Src protein through Lys63-linked polyubiquitination in hepatocellular carcinoma.
    Lihui Zhu, Xiuling Cui, Hongwei Xu, Min Yang, Lihui Han.
      Elevated activity of proto-oncogene tyrosine kinase Src is associated with tumorigenesis and progression of hepatocellular carcinoma (HCC). It is well recognized that activation of Src is mainly driven by its intermolecular autophosphorylation. However, the precise mechanism involved in the activation of Src remains to be fully understood. Here we identified tripartite motif-containing protein (TRIM) 56, a member of E3 ubiquitin ligase family, as a novel regulator of Src activation. The data revealed that TRIM56 directly interacted with Src and catalyzed the polyubiquitination and subsequent aggregation of Src, resulting in Src activation and HCC progression. Mechanistically, TRIM56 interacted with the SH3 domain of Src protein via its B-box1 domain and catalyzed the Lys63-linked polyubiquitination of Src at the Lys184 residue, leading to the aggregation and activation of Src. Altogether, here we demonstrated that TRIM56 acted as a tumor promoter in HCC and it exerted a novel regulatory effect on Src activation. Thus, this study suggested a promising therapeutic strategy for HCC patients by targeting TRIM56.
    DOI:  https://doi.org/10.1038/s41419-025-08074-1
  41. STAR Protoc. 2025 Oct 10. pii: S2666-1667(25)00542-8. [Epub ahead of print]6(4): 104136
    Protocol for identifying DNA damage responders through proximity biotinylation of proliferating cell nuclear antigen interactors.
    Roberta Borosta, István Szepesi-Nagy, István Kató, Gergely Róna.
      TurboID-based proximity labeling is a powerful approach to capture protein-protein interactions within their native cellular environment. Here, we present a step-by-step protocol for fusing proliferating cell nuclear antigen (PCNA) to TurboID and generating stable cell lines via lentiviral transduction. We describe steps for cell synchronization, DNA damage induction, and proximity labeling, followed by fractionation, affinity purification, and mass spectrometry to identify biotinylated proteins. For complete details on the use and execution of this protocol, please refer to Rona et al.1.
    Keywords:  Cell Biology; Cell separation/fractionation; Signal transduction
    DOI:  https://doi.org/10.1016/j.xpro.2025.104136
  42. Nat Commun. 2025 Oct 13. 16(1): 9091
    Capturing disease severity in LIS1-lissencephaly reveals proteostasis dysregulation in patient-derived forebrain organoids.
    Lea Zillich, Matteo Gasparotto, Andrea Carlo Rossetti, Olivia Fechtner, Camille Maillard, Anne Hoffrichter, Eric Zillich, Ammar Jabali, Fabio Marsoner, Annasara Artioli, Ruven Wilkens, Christina B Schroeter, Andreas Hentschel, Stephanie H Witt, Nico Melzer, Sven G Meuth, Tobias Ruck, Philipp Koch, Andreas Roos, Nadia Bahi-Buisson, Fiona Francis, Julia Ladewig.
      LIS1-lissencephaly is a neurodevelopmental disorder marked by reduced cortical folding and severe neurological impairment. Although all cases result from heterozygous mutations in the LIS1 gene, patients present a broad spectrum of severity. Here, we use patient-derived forebrain organoids representing mild, moderate, and severe LIS1-lissencephaly to uncover mechanisms underlying this variability. We show that LIS1 protein levels vary across patient lines and partly correlate with clinical severity, indicating mutation-specific effects on protein function. Integrated morphological, transcriptomic, and proteomic analyses reveal progressive changes in neural progenitor homeostasis and neurogenesis that scale with severity. Mechanistically, microtubule destabilization disrupts cell-cell junctions and impairs WNT signaling, and defects in protein homeostasis, causing stress from misfolded proteins, emerge as key severity-linked pathways. Pharmacological inhibition of mTORC1 partially rescues these defects. Our findings demonstrate that patient-derived organoids can model disease severity, enabling mechanistic dissection and guiding targeted strategies in neurodevelopmental disorders.
    DOI:  https://doi.org/10.1038/s41467-025-64980-0
  43. Mol Cell Biol. 2025 Oct 17. 1-27
    From Biogenesis to Breakdown: How Protein Biogenesis and Quality Control Failures Drive Mitochondrial Disease.
    Phoebe J Leeming, Julia Mercuri-Svik, Diana Stojanovski.
      Mitochondria rely on the coordinated function of over 1000 proteins, most of which are nuclear-encoded, synthesized in the cytosol, and imported into distinct mitochondrial sub-compartments. Thirteen additional proteins are synthesized within the organelle itself, forming core components of the oxidative phosphorylation (OXPHOS) system. Once inside, mitochondrial precursors undergo precise maturation, folding, and assembly, supported by specialized factors that ensure their function. These processes are safeguarded by an intricate network of chaperones, proteases, and disaggregases that maintain proteome integrity. Protein biogenesis and quality control are deeply interconnected, operating continuously to preserve mitochondrial function. Disruption at any stage, whether in import, folding, assembly, or degradation, can lead to proteotoxic stress and mitochondrial dysfunction, underlying a wide spectrum of mitochondrial diseases. Despite progress in characterizing many of these pathways in human cells, large gaps in knowledge remain. A complete understanding of protein biogenesis and surveillance mechanisms is essential to uncover how their dysregulation drives disease. This knowledge will be foundational for interpreting pathogenic mutations, predicting disease mechanisms, and ultimately guiding therapeutic strategies aimed at restoring mitochondrial proteostasis and health.
    Keywords:  Mitochondria; mitochondrial disease; protein import; protein quality control
    DOI:  https://doi.org/10.1080/10985549.2025.2566671
  44. FEBS Lett. 2025 Oct 13.
    Linked dimers of the AAA+ ATPase Msp1 reveal energetic demands and mechanistic plasticity for substrate extraction from lipid bilayers.
    Deepika Gaur, Brian Acquaviva, Baylee A Smith, Nathan Walker, Isabella Walter, Matthew L Wohlever.
      Msp1 is a membrane-anchored AAA+ (ATPases Associated with diverse cellular Activities) enzyme that extracts membrane proteins from lipid bilayers. To understand how the subunits in the homohexamer convert ATP hydrolysis into mechanical work, we developed covalently linked dimers combining wild-type and catalytically inactive (E193Q) subunits. These assembled into pseudohexameric trimers of dimers and retained ATPase activity, indicating that E193Q does not act as a dominant negative for ATP hydrolysis. Conversely, substrate extraction was impaired in some constructs, suggesting position-specific effects. Surprisingly, constructs with a twofold difference in ATPase rates showed minimal differences in substrate extraction across lipid environments, suggesting excess ATPase capacity. These findings clarify how Msp1 coordinates hydrolysis, its energetic requirements, and substrate access to the pore.
    Keywords:  ATPase associated with diverse cellular activities (AAA+); lipid bilayer; membrane protein; mitochondria; proteostasis
    DOI:  https://doi.org/10.1002/1873-3468.70187
  45. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2415153122
    Substrate-dependent activation of LONP1 informs on proteolytic regulation and ATPase motor function.
    Jeffrey T Mindrebo, Gabriel C Lander.
      AAA+ enzymes use energy from ATP hydrolysis to remodel diverse cellular targets. Structures of substrate-bound AAA+ complexes suggest that these enzymes employ a conserved hand-over-hand mechanism to thread substrates through their central pore. However, the fundamental aspects of the mechanisms governing motor function and substrate processing within specific AAA+ families remain unresolved. We used cryoelectron microscopy to structurally interrogate reaction intermediates from in vitro biochemical assays to inform the underlying regulatory mechanisms of the human mitochondrial AAA+ protease, LONP1. Our results demonstrate that substrate binding, rather than nucleotide binding, activates the assembly and allosterically regulates proteolytic activity. The N-terminal domain plays a critical role in this process and facilitates the initial stages of substrate selection and engagement. Moreover, structures of LONP1 actively degrading a substrate in the presence of ATP provide important context to the conventional understanding of the hand-over-hand translocation mechanism, suggesting that ATP hydrolysis is likely not limited to a single position in the right-handed spiral during the hand-over-hand translocation mechanism.
    Keywords:  AAA+ motors; ATP hydrolysis; LONP1; allostery; cryo-EM
    DOI:  https://doi.org/10.1073/pnas.2415153122
  46. J Am Chem Soc. 2025 Oct 15.
    Hijacking Extracellular Targeted Protein Degrader-Drug Conjugates for Enhanced Drug Delivery.
    Fangzhu Zhao, Yan Wu, Kaitlin Schaefer, Yun Zhang, Kun Miao, Zi Yao, Snehal D Ganjave, Kaan Kumru, Trenton M Peters-Clarke, Alex Inague, James A Olzmann, Kevin K Leung, James A Wells.
      Antibody-based therapeutics encompass diverse modalities for targeting tumor cells. Among these, antibody-drug conjugates (ADCs) and extracellular targeted protein degradation (eTPD) specifically depend on efficient lysosomal trafficking for activity. A major limitation of ADCs is their reliance on antigens with efficient internalization, while eTPD approaches, although capable of trafficking diverse targets to lysosomes, lack cytotoxic potency. To address this, we developed degrader-drug conjugates (DDCs), leveraging the endocytic and recycling activities of eTPD to enhance lysosomal delivery. We utilized fast internalizers, the low-density lipoprotein receptor (LDLR) and the chemokine receptor (CXCR7), to enhance lysosomal delivery. LDLR-based degraders enabled efficient and selective degradation of diverse extracellular membrane proteins, while DDCs with cytotoxic payload enhanced cytotoxicity compared to conventional ADCs in vitro. This dual modality addresses key challenges of inadequate internalization in conventional ADCs and cytotoxic potency in current eTPD strategies. Our findings demonstrate that DDCs provide additional optionality for developing next-generation antibody therapeutics with broader utility and improved efficacy in cancer treatment.
    DOI:  https://doi.org/10.1021/jacs.5c15047
  47. Nat Commun. 2025 Oct 14. 16(1): 9125
    Molecular landscape of the fungal plasma membrane and implications for antifungal action.
    Jennifer Jiang, Mikhail V Keniya, Anusha Puri, Xueying Zhan, Jeff Cheng, Huan Wang, Gigi Lin, Yun-Kyung Lee, Nora Jaber, Caifeng Zhao, Cynthia Pang, Yasmine Hassoun, Haiyan Zheng, Erika Shor, Zheng Shi, Sang-Hyuk Lee, Min Xu, David S Perlin, Wei Dai.
      Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their native structure and spatial distribution remain poorly characterized. Herein, we employ an integrative approach to investigate the organization of plasma membrane protein complexes in Candida glabrata, focusing on two abundant and essential membrane proteins, the β-(1,3)-glucan synthase (GS) and the proton pump Pma1. We show that treatment with caspofungin, an echinocandin antifungal that targets GS, disrupts the native distribution of membrane protein complexes and alters membrane biophysical properties. Perturbation of the sphingolipid biosynthesis further modulates drug susceptibility, revealing that the lipid environment plays an integral role in membrane protein organization and GS-echinocandin interactions. Our work highlights the importance of characterizing membrane proteins in their native context to understand their functions and inform the development of novel antifungal therapies.
    DOI:  https://doi.org/10.1038/s41467-025-64171-x
  48. Nat Commun. 2025 Oct 13. 16(1): 9080
    Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes.
    Chuqi Liu, Dexiang Liu, Xiang Zheng, Jiali Guan, Xinyan Zhou, Haikun Zhang, Shen Wang, Qiubai Li, Gan Lu, Jun He, Cong Ma.
      Tumor-derived exosomes carry programmed death-ligand 1 (PD-L1), which binds programmed cell death protein 1 (PD-1) on T cells, suppressing immune responses locally and systemically. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. Here, we identify Munc13-4 as a crucial regulator of this process. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Mechanistically, Munc13-4 collaborates with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. Cryogenic electron microscopy (cryo-EM) analysis of the Munc13-4-Rab27a complex provide structural insights into exosome secretion. Importantly, PD-L1 sorting relies on a ternary complex composed of Munc13-4, PD-L1 and HRS, which is regulated by interferon gamma (IFNγ) signaling. A designed peptide that disrupts Munc13-4-PD-L1 interaction impedes PD-L1 sorting, enhances antitumor immunity, and suppresses tumor growth, highlighting the therapeutic potential of targeting this pathway.
    DOI:  https://doi.org/10.1038/s41467-025-64149-9
  49. J Clin Invest. 2025 Oct 14. pii: e187044. [Epub ahead of print]
    Stimulated thyroid hormone synthesis machinery drives thyrocyte cell death independent of ER stress.
    Crystal Young, Xiaohan Zhang, Xiaofan Wang, Aaron P Kellogg, Kevin Pena, August Z Cumming, Xiao-Hui Liao, Dennis Larkin, Hao Zhang, Emma Mastroianni, Helmut Grasberger, Samuel Refetoff, Peter Arvan.
      It is now recognized that patients and animal models expressing genetically-encoded misfolded mutant thyroglobulin (TG, the protein precursor for thyroid hormone synthesis) exhibit dramatic swelling of the endoplasmic reticulum (ER) with ER stress and cell death in thyrocytes - seen both in homozygotes (with severe hypothyroidism) and heterozygotes (with subclinical hypothyroidism). The thyrocyte death phenotype is exacerbated upon thyroidal stimulation (by thyrotropin, TSH), as cell death is inhibited upon treatment with exogenous thyroxine. TSH stimulation might contribute to cytotoxicity by promoting ER stress, or by an independent mechanism. Here we've engineered knockout mice completely lacking Tg expression. Like other animals/patients with mutant TG, these animals rapidly develop severe goitrous hypothyroidism; however, thyroidal ER stress is exceedingly low - lower even than that seen in wildtype mice. Nevertheless, mice lacking TG exhibit abundant thyroid cell death, which depends upon renegade thyroidal iodination - it is completely suppressed in a genetic model lacking effective iodination, or in Tg-KO mice treated with propylthiouracil (iodination inhibitor), or iodide deficiency. Thyrocytes in culture are killed not in the presence of H2O2 alone, but rather upon peroxidase-mediated iodination, with cell death blocked by propylthiouracil. Thus, in the thyroid gland bearing Tg mutation(s), TSH-stimulated iodination activity triggers thyroid cell death.
    Keywords:  Apoptosis; Cell stress; Endocrinology; Genetics; Thyroid disease
    DOI:  https://doi.org/10.1172/JCI187044
  50. Aging Cell. 2025 Oct 16. e70262
    Translational Remodeling of the Synaptic Proteome During Aging.
    Cinzia Caterino, Martino Ugolini, William Durso, Kristina Jevdokimenko, Marco Groth, Konstantin Riege, Matthias Görlach, Eugenio Fornasiero, Alessandro Ori, Steve Hoffmann, Alessandro Cellerino.
      An important hallmark of aging is the loss of proteostasis, which can lead to the formation of protein aggregates and mitochondrial dysfunction in neurons. Although it is well known that protein synthesis is finely regulated in the brain, especially at synapses, where mRNAs are locally translated in an activity-dependent manner, little is known as to the changes in the synaptic proteome and transcriptome during aging. Therefore, this work aims to elucidate the relationship between the transcriptome and proteome at the soma and synaptic levels during aging. Proteomic and transcriptomic data analysis reveal that, in young animals, proteins and transcripts are correlated and synaptic regulation is driven by changes in the soma. During aging, there is a decoupling between transcripts and proteins and between somatic and synaptic compartments. Furthermore, the soma-synapse gradient of ribosomal genes changes upon aging, that is, ribosomal transcripts are less abundant and ribosomal proteins are more abundant in the synaptic compartment of old mice with respect to younglings. Additionally, transcriptomics data highlight a difference in the splicing of certain synaptic mRNA with aging. Taken together, our data provide a valuable resource for the study of the aging synapse.
    Keywords:  RNA‐Seq; aging; alternative splicing; bioinformatics; synaptosomes
    DOI:  https://doi.org/10.1111/acel.70262
  51. iScience. 2025 Oct 17. 28(10): 113575
    The E3 ubiquitin ligase Trip12 semi-selectively attenuates Wnt signaling.
    Jessica Ensing, Amber D Ide, Carla Gilliland, Visakuo Tsurho, Emma Rudisel, Isabella Caza, Amber N Stratman, Nathan J Lanning, Stephanie Grainger.
      Wnt signaling is critical for the development and maintenance of many cell lineages, including hematopoietic stem cells (HSCs). The tight regulation of Wnt signals is essential, as overactivation can drive tumorigenesis. Numerous Wnt ligands and Frizzled (Fzd) receptors exist, and the negative regulation of particular Fzd signals is the focus of this study. We previously identified the requirement of Wnt9a-Fzd9b pairing for early HSC proliferation. However, the mechanisms controlling activation and signal termination are unclear. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) targets the third intracellular loop of Fzd9b at K437, promoting lysosomal degradation by destabilizing Fzd9b membrane expression. Our data further indicates that Trip12 is semi-selective for Fzd9b. The Trip12-mediated reduction of Fzd9b surface expression dampens Wnt9a/Fzd9b signaling, affecting HSC proliferation in zebrafish. Our findings reveal a receptor-specific regulatory mechanism, with implications for targeted Wnt pathway therapies.
    Keywords:  Biochemistry; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2025.113575
  52. Mol Cell Biol. 2025 Oct 14. 1-30
    Metabolic Regulation of Copper Homeostasis Governs the Sec61-Dependent Protein Translocation Process in Saccharomyces cerevisiae.
    Vandana Anjana, Smriti Anand, Prateeksha Thakur, Rajshree Pal, Santoshi Acharjee, Sugandh Sharma, Sharayu Subhash Awachat, Ritika Manjhi, Devika Rejidev, Ranu Singh, Raghuvir Singh Tomar.
      The concentration of cellular labile pool of copper must be strictly regulated because disruption in copper homeostasis results in diseases. In Saccharomyces cerevisiae, elevated levels of labile copper impair cell viability by inhibiting Sec61-mediated protein translocation into the endoplasmic reticulum. We investigated how metabolic pathways, specifically mitochondrial respiration and autophagy, contribute to copper homeostasis and the translocation of secretory proteins. We show that copper selectively inhibits protein translocation in yeast cells grown in minimal medium but not in a rich medium, highlighting a critical role of nutrients in modulating copper toxicity. Supplementation of specific amino acids suppresses the copper-induced defects in protein translocation and cell death, identifying amino acids as suppressors of the copper toxicity. Using a panel of gene deletion mutants affecting mitochondrial functions, autophagy, peroxisomes, and lipid droplets, we demonstrate that metabolic pathways regulate subcellular concentration of copper and translocation of secretory proteins. Further, disruption of redox and pH homeostasis, and pharmacological inhibition of respiration, reveals that correct subcellular concentration of copper is essential to prevent inhibitory effects on protein translocation. Together, our findings provide mechanistic insights into how metabolic status influences cellular copper homeostasis and the secretory pathway of proteins, with broader implications for understanding diseases of copper metabolism.
    Keywords:  Copper; amino acids; autophagy; copper homeostasis; mitochondria; protein translocation
    DOI:  https://doi.org/10.1080/10985549.2025.2569577
  53. Nat Plants. 2025 Oct 17.
    Tomato ripening regulator SlSAD8 disturbs nuclear gene transcription and chloroplast-associated protein degradation.
    Chan Xu, Rui Li, Xiaoyan Chen, Zhengyang Fu, Xuechun Cui, Juanni Yao, Yanna Shi, Wei Deng, Zhengguo Li, Yulin Cheng.
      Fruit ripening is a tightly regulated developmental process, in which nuclear gene transcription represents a crucial component of the mechanisms1. Chloroplast-associated protein degradation, a recently discovered pathway for chloroplast protein degradation, has also been reported to control fruit ripening2. Here we report a negative regulator of tomato ripening, termed SlSAD8, which disturbs both nuclear gene transcription and chloroplast-associated protein degradation. As an atypical stearoyl-ACP desaturase (SAD) protein exhibiting dual localization in plastids and the nucleus, SlSAD8 negatively regulates ripening initiation and chloroplast-to-chromoplast transition during fruit ripening. In the nucleus, SlSAD8 interacts with ripening-initiation-associated transcription factor SlNAM1, thereby disturbing the transcriptional activation of ethylene biosynthesis genes. Additionally, SlSAD8 interacts with plastid-transition-associated E3 ligase SlSP1 in the plastid, disturbing the chloroplast-associated protein degradation pathway to elevate chloroplast protein levels. Our findings uncover an unusual ripening regulator that targets distinct subcellular compartments to manipulate gene expression, providing insights into the intricate regulatory networks of fruit ripening.
    DOI:  https://doi.org/10.1038/s41477-025-02134-2
  54. Proc Natl Acad Sci U S A. 2025 Oct 21. 122(42): e2508998122
    A generalizable deep learning framework for structure-based protein-ligand affinity ranking.
    Benjamin P Brown.
      Rapid and accurate estimation of protein-ligand binding affinities is crucial for early-stage drug discovery, yet hindered by a trade-off between the accuracy of gold-standard physics-based methods and the speed of simpler empirical scoring functions. Machine learning (ML) promised to bridge this gap, but its potential is unrealized due to limited model generalizability. Current ML models often fail when predicting affinities for novel proteins or chemical series unseen during training. We hypothesize that this failure stems from a competition within these models during training, where the learning of spurious correlations from structural motifs prevalent in the training data competes with the learning of transferable, physicochemical principles governing molecular interaction. Here, we introduce COnvolutional Representation of Distance-dependent Interactions with Attention Learning (CORDIAL), a deep learning framework designed with an inductive bias toward learning the distance-dependent physicochemical interaction signatures between proteins and ligands, explicitly avoiding direct parameterization of their chemical structures. This interaction-only approach proves effective. Through leave-superfamily-out validation that simulates encounters with novel protein families, we demonstrate that CORDIAL maintains predictive performance and calibration. This contrasts with diverse contemporary ML models, whose predictive ability is degraded under these conditions. Our results highlight the value of encoding appropriate task-specific physicochemical principles into ML architectures and offer a validated strategy for developing generalizable models for structure-based drug discovery.
    Keywords:  computer-aided drug design; deep learning; generalizability; virtual screening
    DOI:  https://doi.org/10.1073/pnas.2508998122
  55. Ageing Res Rev. 2025 Oct 11. pii: S1568-1637(25)00262-4. [Epub ahead of print]113 102916
    The interactome era: Integrating RNA-seq, proteomics, and network biology to decode cellular senescence.
    Mohd Shahzaib, Domenico Aprile, Tiziana Squillaro, Nicola Alessio, Gianfranco Peluso, Giovanni Di Bernardo, Umberto Galderisi.
      Cellular senescence is a dynamic state in which cells permanently withdraw from the cell cycle while continuing to reshape their internal and external environment. It is characterized by persistent DNA damage responses, chromatin reorganization, and the secretion of a complex mixture of cytokines and proteases collectively known as the senescence-associated secretory phenotype (SASP). Transcriptomic and proteomic studies have defined key markers, including CDKN2A, CDKN1A, TP53, and SASP factors, but these approaches provide only static inventories. They do not explain how regulatory programs are executed through protein interactions that assemble, dissolve, and reorganize over time. Interactomics now fills this gap. Advances such as affinity purification mass spectrometry (AP-MS), proximity labeling (BioID/TurboID), and cross-linking mass spectrometry (XL-MS) reveal that senescence is driven not by single molecules but by the rewiring of protein-protein interactions (PPIs). These dynamic networks stabilize DNA damage response hubs, restructure chromatin and the nuclear lamina, regulate SASP secretion, and remodel metabolism. By integrating interactomic data with transcriptomic and proteomic profiles, it is now possible to uncover therapeutic vulnerabilities and guide the design of senolytics, senomorphics, and strategies that block senescence escape. Important challenges remain. Weak or transient interactions are often lost, background signals can obscure specificity, and membrane complexes are under-represented. Emerging single-cell and spatial technologies are beginning to overcome these limitations, revealing how senescence differs across tissues, contexts, and disease states. In essence, senescence is not just a change in gene expression but a reorganization of the cell's communication networks. Interactomics offers the framework needed to decode this complexity and to design precision therapies for aging and age-related disease.
    Keywords:  Cellular senescence; DNA damage response; Interactomics; Protein-protein interactions; SASP
    DOI:  https://doi.org/10.1016/j.arr.2025.102916
  56. Sci Immunol. 2025 Oct 17. 10(112): eado3825
    Protein palmitoylation and sphingolipid metabolism control regulated exocytosis in cytotoxic lymphocytes.
    Artem Kalinichenko, Jakob Huemer, Theresa Humer, Matthias Haimel, Michael Svaton, Nicolas Socquet-Juglard, Giovanna Perinetti Casoni, Celine Prakash, Maximilian von der Linde, Julia Pazmandi, Cheryl van de Wetering, Javier Nunez-Fontarnau, Anton Kamnev, Sarah Giuliani, Martin G Jaeger, Elisa Hahn, Sarah Dobner, Andrea Rukavina, Elise Sylvander, Jacqueline Seigner, Christina Rashkova, Birgit Hoeger, Michael W Traxlmayr, Manfred Lehner, Yenan T Bryceson, Janna Saarela, Thomas Hannich, Irinka Castanon, Georg Winter, Loïc Dupré, Kaan Boztug.
      Regulated exocytosis controls key cellular functions ranging from neurotransmitter release to the secretion of immune mediators, and its disruption is associated with numerous pathologies. The cytotoxic activity of lymphocytes is particularly dependent on regulated and polarized lytic granule delivery toward infected or malignant cells. Although genetic and mechanistic studies have identified factors regulating exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Through a genome-scale CRISPR knockout screen in a human natural killer cell line, we characterized a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation among the most prominent pathways. By combining global protein palmitoylation and lipidomic studies, we found that ZDHHC17 drives palmitoylation of the core SNARE complex protein SNAP23 to target cytotoxic granules to GM1-rich lipid rafts whose assembly is controlled by serine palmitoyltransferase. In summary, our study identifies previously unrecognized factors essential for cytotoxic function in human lymphocytes and uncovers how lipid metabolism and protein palmitoylation are involved in the process of regulated exocytosis.
    DOI:  https://doi.org/10.1126/sciimmunol.ado3825
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