bims-proteo Biomed News
on Proteostasis
Issue of 2025–03–23
forty-four papers selected by
Eric Chevet, INSERM
  1. TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress.
  2. HaloPROTAC3 treatment activates the unfolded protein response of the endoplasmic reticulum in non-engineered mammalian cell lines.
  3. The primate-specific Nedd4-1(NE) localizes to late endosomes in response to amino acids to suppress autophagy.
  4. Genetic protection from type 1 diabetes resulting from accelerated insulin mRNA decay.
  5. IRE1α-mediated UPR activation in gastrointestinal cancers: adaptive mechanisms and therapeutic potential.
  6. Unlocking protein networks with Predictomes: The SPOC advantage.
  7. Molecular glues that inhibit deubiquitylase activity and inflammatory signaling.
  8. Targeting biomolecular condensates: The rise of engineered chaperones.
  9. ATG2A-WDR45/WIPI4-ATG9A complex-mediated lipid transfer and equilibration during autophagosome formation.
  10. PERRC: Protease Engineering with Reactant Residence Time Control.
  11. Lipid juggling: Any1 scrambles membranes for endosome biogenesis.
  12. DR5 disulfide bonding functions as a sensor and effector of protein folding stress.
  13. The E3 ligase HRD1 enhances plant antiviral immunity by targeting viral movement proteins.
  14. License to drive: Receptor-mediated ER exit of proteins and lipids.
  15. A phenopushing platform to identify compounds that alleviate acute hypoxic stress by fast-tracking cellular adaptation.
  16. ER O-glycosylation in synovial fibroblasts drives cartilage degradation.
  17. Quantum Mechanics-Based Ranking of Predicted Proteolysis Targeting Chimeras-Mediated Ternary Complexes.
  18. Proteostasis regulation of GABAA receptors in neuronal function and disease.
  19. Lysosomal storage-independent Fabry disease variants with α-galactosidase A misprocessing-induced ER stress and the unfolded protein response.
  20. METTL3 knockout accelerates hepatocarcinogenesis via inhibiting endoplasmic reticulum stress response.
  21. Lack of AtMC1 catalytic activity triggers autoimmunity dependent on NLR stability.
  22. Lactylation orchestrates ubiquitin-independent degradation of cGAS and promotes tumor growth.
  23. Structurally heterogeneous ribosomes cooperate in protein synthesis in bacterial cells.
  24. TBK1 is a signaling hub in coordinating stress-adaptive mechanisms in head and neck cancer progression.
  25. A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing.
  26. Transfer RNA acetylation regulates in vivo mammalian stress signaling.
  27. Chaperone-mediated autophagy degrades SERPINA1E342K/α1-antitrypsin Z variant and alleviates cell stress.
  28. MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers.
  29. AMPK-dependent Parkin activation suppresses macrophage antigen presentation to promote tumor progression.
  30. USP8 and Hsp70 regulate endoreplication by synergistically promoting Fzr deubiquitination and stabilization.
  31. TMBIM-2 orchestrates systemic mitochondrial stress response via facilitating Ca2+ oscillations.
  32. Proteostasis Disruption in Inherited Bone Marrow Failure Syndromes.
  33. Conquering PROTAC molecular design and drugability.
  34. Structural recognition and stabilization of tyrosine hydroxylase by the J-domain protein DNAJC12.
  35. Deep indel mutagenesis reveals the impact of amino acid insertions and deletions on protein stability and function.
  36. Transcriptional heterogeneity shapes stress-adaptive responses in yeast.
  37. Natural human tRNAAla anticodon variants mistranslate the genetic code.
  38. Defective gating of the Sec61 translocon results in reorganisation of the actin cytoskeleton and perturbed formation of the actomyosin contractile ring.
  39. Deciphering the folding code of collagens.
  40. Murine Leukemia Virus GlycoGag Antagonizes SERINC5 via ER-phagy Receptor RETREG1.
  41. Turnover atlas of proteome and phosphoproteome across mouse tissues and brain regions.
  42. POT, an optogenetics-based endogenous protein degradation system.
  43. GraphBAN: An inductive graph-based approach for enhanced prediction of compound-protein interactions.
  44. Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles.
  1. bioRxiv. 2025 Jan 02. pii: 2024.12.31.630809. [Epub ahead of print]
    TOR and heat shock response pathways regulate peroxisome biogenesis during proteotoxic stress.
    Nandini Shukla, Maxwell L Neal, Jean-Claude Farré, Fred D Mast, Linh Truong, Theresa Simon, Leslie R Miller, John D Aitchison, Suresh Subramani.
      Peroxisomes are versatile organelles mediating energy homeostasis and redox balance. While peroxisome dysfunction is linked to numerous diseases, the molecular mechanisms and signaling pathways regulating peroxisomes during cellular stress remain elusive. Using yeast, we show that perturbations disrupting protein homeostasis including loss of ER or cytosolic chaperone function, impairments in ER protein translocation, blocking ER N-glycosylation, or reductive stress, cause peroxisome proliferation. This proliferation is driven by increased de novo biogenesis from the ER as well as increased fission of pre-existing peroxisomes, rather than impaired pexophagy. Notably, peroxisome biogenesis is essential for cellular recovery from proteotoxic stress. Through comprehensive testing of major signaling pathways, we determine this response to be mediated by activation of the heat shock response and inhibition of Target of Rapamycin (TOR) signaling. Finally, the effects of proteotoxic stress and TOR inhibition on peroxisomes are also captured in human fibroblasts. Overall, our findings reveal a critical and conserved role of peroxisomes in cellular response to proteotoxic stress.
    DOI:  https://doi.org/10.1101/2024.12.31.630809
  2. Mol Biol Cell. 2025 Mar 19. mbcE24080342
    HaloPROTAC3 treatment activates the unfolded protein response of the endoplasmic reticulum in non-engineered mammalian cell lines.
    Aleksandra S Anisimova, G Elif Karagöz.
      Proteins fused to HaloTag, an engineered haloalkane dehalogenase, can be depleted by a heterobifunctional degrader compound HaloPROTAC3. The binding of HaloPROTAC3 to both the HaloTag and the E3 ligase von Hippel-Lindau (VHL) brings them into proximity and mediates the degradation of the HaloTag fusion proteins. Here, we generated a colon cancer cell line HCT116 expressing HaloTag fused to the RNA-binding protein IGF2BP3 to study its function. HaloPROTAC3 treatment depleted 75% of HaloTag-IGF2BP3 in 5 hours. Transcriptomics revealed that HaloPROTAC3 treatment resulted in the destabilization of IGF2BP3 target mRNAs and activated the unfolded protein response (UPR). Surprisingly, we found that HaloPROTAC3 results in UPR activation in non-engineered mammalian cells. Our data demonstrate that HaloPROTAC3 causes mild endoplasmic reticulum stress independent of IGF2BP3 function and shall guide future studies using the HaloPROTAC3 protein depletion strategy.
    DOI:  https://doi.org/10.1091/mbc.E24-08-0342
  3. Nat Commun. 2025 Mar 18. 16(1): 2682
    The primate-specific Nedd4-1(NE) localizes to late endosomes in response to amino acids to suppress autophagy.
    G Kefalas, A Priya, A Astori, A Persaud, L Jing, A M Sydor, H H Y Yao, N Warner, Y Zhang, J H Brumell, A M Muise, S Sari, H C Su, M J Lenardo, W H A Kahr, B Raught, D Rotin.
      The ubiquitin ligase Nedd4 (Nedd4-1), comprised of C2-WW(n)-HECT domains, regulates protein trafficking. We recently described a primate-specific Nedd4-1 splice isoform with an extended N-terminus replacing the C2 domain, called Nedd4-1(NE). Here, we show that while canonical Nedd4-1 is primarily localized to the cytosol, Nedd4-1(NE) localizes to late endosomes. This localization is mediated by the NE region, is dependent on amino acid availability, is independent of mTORC1, and is inhibited by the autophagy inducer IKKβ. We further demonstrate that VPS16B, which regulates late endosome to lysosome maturation, is a unique Nedd4-1(NE) substrate that co-localizes with Nedd4-1(NE) in the presence of nutrients. Importantly, a potentially pathogenic homozygous variant identified in the NE region (E70Q) of a patient with lymphangiectasia and protein-losing enteropathy leads to reduced VPS16B ubiquitination by Nedd4-1(NE). Finally, we report that Nedd4-1(NE) inhibits autophagy, likely by disrupting late endosome to autophagosome maturation. This work identified an mTORC1-independent, IKK-driven mechanism to regulate Nedd4-1(NE) localization to late endosomes in primates in response to nutrient availability, and uncovered suppression of autophagy by this ubiquitin ligase.
    DOI:  https://doi.org/10.1038/s41467-025-57944-x
  4. Cell. 2025 Mar 11. pii: S0092-8674(25)00206-5. [Epub ahead of print]
    Genetic protection from type 1 diabetes resulting from accelerated insulin mRNA decay.
    René van Tienhoven, Denis O'Meally, Tristan A Scott, Kevin V Morris, John C Williams, John S Kaddis, Arnaud Zaldumbide, Bart O Roep.
      Insulin gene (INS) variation and beta-cell stress are associated with the risk of development of type 1 diabetes (T1D) and autoimmunity against insulin. The unfolded protein response alleviating endoplasmic reticulum (ER) stress involves activation of inositol-requiring enzyme 1α (IRE1α) that impedes translation by mRNA decay. We discover that the IRE1α digestion motif is present in insulin mRNA carrying SNP rs3842752 (G>A). This SNP in the 3' untranslated region of INS associates with protection from T1D (INSP). ER stress in beta cells with INSP led to accelerated insulin mRNA decay compared with the susceptible INS variant (INSS). Human islets with INSP showed improved vitality and function and reversed diabetes more rapidly when transplanted into diabetic mice than islets carrying INSS only. Surrogate beta cells with INSP expressed less ER stress and INS-DRiP neoantigen. This explanation for genetic protection from T1D may act instead of or in concert with the previously proposed mechanism attributed to INS promoter polymorphism.
    Keywords:  ER stress; beta cell function; defective ribosomal product; genetic risk; immune tolerance; immunogenicity; insulin gene; neoantigen; type 1 diabetes; unfolded protein response
    DOI:  https://doi.org/10.1016/j.cell.2025.02.018
  5. Drug Discov Today. 2025 Mar 15. pii: S1359-6446(25)00048-0. [Epub ahead of print] 104335
    IRE1α-mediated UPR activation in gastrointestinal cancers: adaptive mechanisms and therapeutic potential.
    Valappan Veetil Soumya, Baby Jisna, Davis Anu, Chevookaren Francis Binoy, Thekkekara Devassy Babu.
      The endoplasmic reticulum (ER) plays a crucial part in protein synthesis, folding and quality control. Disruptions in these processes lead to ER stress (ERS) and activate the unfolded protein response (UPR) to restore cellular homeostasis. In gastrointestinal cancers, inositol-requiring enzyme 1α (IRE1α) is a key regulator of the UPR, helping cancer cells adapt to hostile conditions such as hypoxia, oxidative stress and chemotherapy. Elevated IRE1α activity supports tumor survival, progression and metastasis by mitigating ERS-induced apoptosis. However, targeting IRE1α signaling presents a promising therapeutic strategy, potentially impairing cancer cell adaptation to stress and enhancing treatment efficacy. Targeting IRE1α offers promising therapeutic opportunities for gastrointestinal cancer.
    Keywords:  Endoplasmic reticulum stress; gastrointestinal cancer; inositol-requiring enzyme 1α; therapeutic potential; unfolded protein response
    DOI:  https://doi.org/10.1016/j.drudis.2025.104335
  6. Mol Cell. 2025 Mar 20. pii: S1097-2765(25)00141-8. [Epub ahead of print]85(6): 1050-1051
    Unlocking protein networks with Predictomes: The SPOC advantage.
    Arne Elofsson.
      In this issue of Molecular Cell, Schmid and Walter present "Predictomes,"1 a machine-learning-based platform that utilizes AlphaFold-Multimer (AF-M) to identify high-confidence protein-protein interactions (PPIs). Their SPOC classifier is better than existing methods at separating true and false interactions.
    DOI:  https://doi.org/10.1016/j.molcel.2025.02.010
  7. Nat Struct Mol Biol. 2025 Mar 17.
    Molecular glues that inhibit deubiquitylase activity and inflammatory signaling.
    Francesca Chandler, Poli Adi Narayana Reddy, Smita Bhutda, Rebecca L Ross, Arindam Datta, Miriam Walden, Kieran Walker, Stefano Di Donato, Joel A Cassel, Michael A Prakesch, Ahmed Aman, Alessandro Datti, Lisa J Campbell, Martina Foglizzo, Lillie Bell, Daniel N Stein, James R Ault, Rima S Al-Awar, Antonio N Calabrese, Frank Sicheri, Francesco Del Galdo, Joseph M Salvino, Roger A Greenberg, Elton Zeqiraj.
      Deubiquitylases (DUBs) are crucial in cell signaling and are often regulated by interactions within protein complexes. The BRCC36 isopeptidase complex (BRISC) regulates inflammatory signaling by cleaving K63-linked polyubiquitin chains on type I interferon receptors (IFNAR1). As a Zn2+-dependent JAMM/MPN (JAB1, MOV34, MPR1, Pad1 N-terminal) DUB, BRCC36 is challenging to target with selective inhibitors. Here, we discover first-in-class inhibitors, termed BRISC molecular glues (BLUEs), which stabilize a 16-subunit human BRISC dimer in an autoinhibited conformation, blocking active sites and interactions with the targeting subunit, serine hydroxymethyltransferase 2. This unique mode of action results in selective inhibition of BRISC over related complexes with the same catalytic subunit, splice variants and other JAMM/MPN DUBs. BLUE treatment reduced interferon-stimulated gene expression in cells containing wild-type BRISC and this effect was abolished when using structure-guided, inhibitor-resistant BRISC mutants. Additionally, BLUEs increase IFNAR1 ubiquitylation and decrease IFNAR1 surface levels, offering a potential strategy to mitigate type I interferon-mediated diseases. Our approach also provides a template for designing selective inhibitors of large protein complexes by promoting rather than blocking protein-protein interactions.
    DOI:  https://doi.org/10.1038/s41594-025-01517-5
  8. Cell Chem Biol. 2025 Mar 20. pii: S2451-9456(25)00063-7. [Epub ahead of print]32(3): 381-383
    Targeting biomolecular condensates: The rise of engineered chaperones.
    Yevheniia Bushman, Duhita A Mirikar, Andrew W Truman.
      Molecular chaperones like Hsp70s are key players in protein quality control (PQC), capable of eliminating toxic intracellular condensates. In this issue of Cell Chemical Biology, Zhang et al.1 present a computational approach to design novel J-domain protein (JDP) constructs that bind to Hsp70 and enhance its chaperone activity.
    DOI:  https://doi.org/10.1016/j.chembiol.2025.02.005
  9. Autophagy. 2025 Mar 21. 1-3
    ATG2A-WDR45/WIPI4-ATG9A complex-mediated lipid transfer and equilibration during autophagosome formation.
    Yang Wang, Goran Stjepanovic.
      Macroautophagy/autophagy is a highly conserved cellular process, spanning from yeast to humans, and plays a vital role in maintaining cellular homeostasis. Dysregulation of autophagy has been linked to a wide range of diseases. A hallmark of autophagy is the formation of double-membrane vesicles called autophagosomes. Autophagosome biogenesis requires a large number of phospholipids, with the endoplasmic reticulum (ER) being the main lipid source. The ATG2A-WDR45/WIPI4-ATG9A complex serves as the core machinery responsible for lipid transfer and equilibration during this process. In our recent study, we resolved the cryo-electron microscopy (cryo-EM) structures of the ATG2A-WDR45/WIPI4 and ATG2A-WDR45/WIPI4-ATG9A complexes, providing critical insights into their architecture and function. Additionally, molecular dynamics simulations were employed to investigate the mechanism by which ATG2A mediates lipid extraction from donor membranes. Our findings offer structural and mechanistic insights into the spatially coupled processes of lipid transfer and re-equilibration, which are essential for phagophore membrane expansion.Abbreviation: ATG: autophagy related; ATG2A: autophagy related 2A; ATG2A[NR]: ATG2A N-terminal region; ATG9A: autophagy related 9A; cryo-EM: cryo-electron microscopy; cryo-ET: cryo-electron tomography; ER: endoplasmic reticulum; PtdIns3P: phosphatidylinositol-3-phosphate; SpAtg2[NR]: Schizosaccharomyces pombe Atg2 N-terminal region; SUVs: small unilamellar vesicles; TGN: trans-Golgi network; TMEM41B: transmembrane protein 41B; VMP1: vacuole membrane protein 1; WDR45/WIPI4: WD repeat domain 45.
    Keywords:  ATG2A-ATG9A complex; Cryo-EM; autophagosome formation; phospholipid scramblases; phospholipid transfer
    DOI:  https://doi.org/10.1080/15548627.2025.2473388
  10. bioRxiv. 2025 Mar 04. pii: 2025.03.02.641063. [Epub ahead of print]
    PERRC: Protease Engineering with Reactant Residence Time Control.
    Sage Nelson, Jokent Gaza, Seyednima Ajayebi, Ronald Masse, Raymond Pho, Cianna Scutero, Samantha Martinusen, Lawton Long, Amor Menezes, Alberto Perez, Carl Denard.
      Proteases with engineered specificity hold great potential for targeted therapeutics, protein circuit construction, and biotechnology applications. However, many proteases exhibit broad substrate specificity, limiting their applications. Engineering protease specificity remains challenging because evolving a protease to recognize a new substrate, without counterselecting against its native substrate, often results in high residual activity on the original substrate. To address this, we developed Protease Engineering with Reactant Residence Time Control (PERRC), a platform that exploits the correlation between endoplasmic reticulum (ER) retention sequence strength and ER residence time. PERRC allows precise control over the stringency of protease evolution by adjusting counterselection to selection substrate ratios. Using PERRC, we evolved an orthogonal tobacco etch virus protease variant, TEVESNp, that selectively cleaves a substrate (ENLYFES) that differs by only one amino acid from its parent sequence (ENLYFQS). TEVESNp exhibits a remarkable 65-fold preference for the evolved substrate, marking the first example of an engineered orthogonal protease driven by such a slight difference in substrate recognition. Furthermore, TEVESNp functions as a competent protease for constructing orthogonal protein circuits in bacteria, and molecular dynamic simulations analysis reveals subtle yet functionally significant active site rearrangements. PERRC is a modular dual-substrate display system that facilitates precise engineering of protease specificity.
    DOI:  https://doi.org/10.1101/2025.03.02.641063
  11. J Cell Biol. 2025 Apr 07. pii: e202502158. [Epub ahead of print]224(4):
    Lipid juggling: Any1 scrambles membranes for endosome biogenesis.
    Sinead Iduna Schwabl, Konstantin Adrian Siegmann, David Teis.
      Multivesicular bodies (MVBs) are crucial for membrane protein degradation and lipid homeostasis. A recent study by Gao and colleagues (https://doi.org/10.1083/jcb.202410013) identifies Any1 as a phospholipid scramblase that plays an important role in MVB biogenesis by coordinating membrane remodeling with lipid transfer through Vps13 at organelle contact sites.
    DOI:  https://doi.org/10.1083/jcb.202502158
  12. Mol Cancer Res. 2024 Mar 19.
    DR5 disulfide bonding functions as a sensor and effector of protein folding stress.
    Mary E Law, Zaafir M Dulloo, Samantha R Eggleston, Gregory P Takacs, Grace M Alexandrow, Young Il Lee, Mengxiong Wang, Brian Hardy, Hanyu Su, Bianca Forsyth, Parag Das, Pran K Datta, Chi-Wu Chiang, Abhisheak Sharma, Siva Rama Raju Kanumuri, Olga A Guryanova, Jeffrey K Harrison, Boaz Tirosh, Ronald K Castellano, Brian K Law.
      New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity. TRAIL analogs or agonistic antibodies targeting these receptors are available but have not yet received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may activate TRAIL receptors as a monotherapy or potentiate the efficacy of TRAIL analogs and agonistic antibodies. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy and as research tools. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands. Implications: Extreme endoplasmic reticulum stress triggers triage of transmembrane receptor production, whereby mitogenic receptors are downregulated and death receptors are simultaneously elevated.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-24-0756
  13. Cell Rep. 2025 Mar 18. pii: S2211-1247(25)00220-7. [Epub ahead of print]44(4): 115449
    The E3 ligase HRD1 enhances plant antiviral immunity by targeting viral movement proteins.
    Zhi-Hong Guo, Xin-Yu Qin, Hong-Fang Guo, Chuan Zheng, Zong-Ying Zhang, Qian Chen, Xian-Bing Wang, Cheng-Gui Han, Ying Wang.
      The ubiquitin-26S proteasome system (UPS) is a conserved protein degradation process involved in plant growth and immunity. However, whether some UPS E3 ligases directly target plant viruses in the endoplasmic reticulum (ER) remains less understood. Here, we identify an E3 ubiquitin ligase Hmg-CoA reductase degradation 1 of Nicotiana benthamiana (NbHRD1) interacting with the triple gene block (TGB) movement proteins of beet necrotic yellow vein virus (BNYVV) in the ER. The TGB proteins are ubiquitinated by NbHRD1 and then degraded by the UPS. Consequently, overexpression of NbHRD1a significantly inhibits BNYVV infection, whereas silencing of NbHRD1 promotes BNYVV infection in N. benthamiana. Moreover, NbHRD1a mainly impairs BNYVV cell-to-cell movement, rather than virus replication. Interestingly, NbHRD1 also targets the TGB proteins of potato virus X for ubiquitination and virus inhibition. Collectively, our results demonstrate that NbHRD1 is an important antiviral component targeting plant viruses with TGB movement proteins.
    Keywords:  CP: Plants; E3 ligase activity; HRD1; antiviral; beet necrotic yellow vein virus; movement protein; potato virus X; ubiquitination; viral infection
    DOI:  https://doi.org/10.1016/j.celrep.2025.115449
  14. Curr Opin Cell Biol. 2025 Mar 20. pii: S0955-0674(25)00039-0. [Epub ahead of print]94 102501
    License to drive: Receptor-mediated ER exit of proteins and lipids.
    Xiao Wang, Tiantian Li, Yusong Guo, Xiao-Wei Chen.
      The secretory pathway, which begins at the endoplasmic reticulum (ER) through the COPII complex, is responsible for transporting proteins and lipid carriers to various destined cellular compartments or extracellular space. The fundamental mechanism by which the COPII operates is evolutionarily conserved. Nevertheless, the vast diversity of mammalian cargos poses significant challenges to the secretory pathway, especially considering the intricate physiology in vivo. Particularly, certain physiologically essential cargos, including procollagen and lipoproteins, appear to be oversized for these canonical carriers, implying the need for additional sophisticated regulation at the onset step so-called ER exit. Emerging evidence highlights the critical role of cargo receptors in selective sorting for ER export, illuminating the complex biology of the trafficking dynamics, which holds broad implications for human health and diseases.
    DOI:  https://doi.org/10.1016/j.ceb.2025.102501
  15. Nat Commun. 2025 Mar 18. 16(1): 2684
    A phenopushing platform to identify compounds that alleviate acute hypoxic stress by fast-tracking cellular adaptation.
    Li Li, Heinz Hammerlindl, Susan Q Shen, Feng Bao, Sabrina Hammerlindl, Steven J Altschuler, Lani F Wu.
      Severe acute hypoxic stress is a major contributor to the pathology of human diseases, including ischemic disorders. Current treatments focus on managing consequences of hypoxia, with few addressing cellular adaptation to low-oxygen environments. Here, we investigate whether accelerating hypoxia adaptation could provide a strategy to alleviate acute hypoxic stress. We develop a high-content phenotypic screening platform to identify compounds that fast-track adaptation to hypoxic stress. Our platform captures a high-dimensional phenotypic hypoxia response trajectory consisting of normoxic, acutely stressed, and chronically adapted cell states. Leveraging this trajectory, we identify compounds that phenotypically shift cells from the acutely stressed state towards the adapted state, revealing mTOR/PI3K or BET inhibition as strategies to induce this phenotypic shift. Importantly, our compound hits promote the survival of liver cells exposed to ischemia-like stress, and rescue cardiomyocytes from hypoxic stress. Our "phenopushing" platform offers a general, target-agnostic approach to identify compounds and targets that accelerate cellular adaptation, applicable across various stress conditions.
    DOI:  https://doi.org/10.1038/s41467-025-57754-1
  16. Nat Commun. 2025 Mar 14. 16(1): 2535
    ER O-glycosylation in synovial fibroblasts drives cartilage degradation.
    Le Son Tran, Joanne Chia, Xavier Le Guezennec, Keit Min Tham, Anh Tuan Nguyen, Virginie Sandrin, Way Cherng Chen, Tan Tong Leng, Sreedharan Sechachalam, Khai Pang Leong, Frederic A Bard.
      How arthritic synovial fibroblasts (SFs) activate cartilage ECM degradation remains unclear. GALNT enzymes initiate O-glycosylation in the Golgi; when relocated to the ER, their activity stimulates ECM degradation. Here, we show that in human rheumatoid and osteoarthritic synovial SFs, GALNTs are relocated to the ER. In an RA mouse model, GALNTs relocation occurs shortly before arthritis symptoms and abates as the animal recovers. An ER GALNTs inhibitor prevents cartilage ECM degradation in vitro and expression of this chimeric protein in SFs results in the protection of cartilage. One of the ER targets of GALNTs is the resident protein Calnexin, which is exported to the cell surface of arthritic SFs. Calnexin participates in matrix degradation by reducing ECM disulfide bonds. Anti-Calnexin antibodies block ECM degradation and protect animals from RA. In sum, ER O-glycosylation is a key switch in arthritic SFs and glycosylated surface Calnexin could be a therapeutic target.
    DOI:  https://doi.org/10.1038/s41467-025-57401-9
  17. ACS Med Chem Lett. 2025 Mar 13. 16(3): 420-427
    Quantum Mechanics-Based Ranking of Predicted Proteolysis Targeting Chimeras-Mediated Ternary Complexes.
    Stefania Monteleone, Inaki Morao, Dmitri G Fedorov, Tahsin F Kellici.
      Targeted protein degradation has become the most pursued alternative modality to small-molecule inhibition over the past decade. The traditional strategy of blocking protein activity by tightly binding to a functional substrate pocket has progressed toward proteolysis-targeting chimeras (PROTACs), bivalent molecules that induce the knockdown of targeted proteins. Herein, a combined protocol is described for modeling ternary complexes via well-established approaches. We performed local protein-protein docking using Rosetta protocol and sampled the conformational landscape of a specific PROTAC molecule that was compatible with the generated protein-protein docking poses, followed by double and independent single-linkage/nearest-neighbor clustering for representative selection. Subsequently, we combined the fragment molecular orbital and density functional tight-binding methods to facilitate fast quantum mechanics-based energy calculations of the clustered ternary complexes. Finally, the computed energy values were utilized to score and select the best ternary poses, achieving good agreement with available crystallographic data.
    DOI:  https://doi.org/10.1021/acsmedchemlett.4c00534
  18. Biomed Pharmacother. 2025 Mar 19. pii: S0753-3322(25)00186-6. [Epub ahead of print]186 117992
    Proteostasis regulation of GABAA receptors in neuronal function and disease.
    Xi Chen, Ya-Juan Wang, Ting-Wei Mu.
      The γ-aminobutyric acid type A receptors (GABAARs) are ligand-gated anion channels that mediate fast inhibitory neurotransmission in the mammalian central nervous system. GABAARs form heteropentameric assemblies comprising two α1, two β2, and one γ2 subunits as the most common subtype in mammalian brains. Proteostasis regulation of GABAARs involves subunit folding within the endoplasmic reticulum, assembling into heteropentamers, receptor trafficking to the cell surface, and degradation of terminally misfolded subunits. As GABAARs are surface proteins, their trafficking to the plasma membrane is critical for proper receptor function. Thus, variants in the genes encoding GABAARs that disrupt proteostasis result in various neurodevelopmental disorders, ranging from intellectual disability to idiopathic generalized epilepsy. This review summarizes recent progress about how the proteostasis network regulates protein folding, assembly, degradation, trafficking, and synaptic clustering of GABAARs. Additionally, emerging pharmacological approaches that restore proteostasis of pathogenic GABAAR variants are presented, providing a promising strategy to treat related neurological diseases.
    Keywords:  4-phenylbutyric acid (PubChem CID: 4775); AA147 (PubChem CID: 882909); AA263 (PubChem CID: 135509553); BIX (PubChem CID: 16656807); Dihydroergocristine (PubChem CID: 107715); Dinoprost (PubChem CID: 5280363); Epilepsy; GABA(A) receptors; Hispidulin (PubChem CID: 5281628); Pharmacological Chaperones; Proteostasis; Proteostasis regulators; SAHA (PubChem CID: 5311); TP003 (PubChem CID: 10001434); Variants; Verapamil (PubChem CID: 2520)
    DOI:  https://doi.org/10.1016/j.biopha.2025.117992
  19. Nephron. 2025 Mar 20. 1-17
    Lysosomal storage-independent Fabry disease variants with α-galactosidase A misprocessing-induced ER stress and the unfolded protein response.
    Martina Živná, Malte Lenders, Stanislav Kmoch.
       BACKGROUND: Clinical findings in Fabry disease have classically been attributed to loss-of- function variants in the GLA gene that result in α-galactosidase A deficiency, intracellular accumulation of globotriaosylceramides and clinical manifestations. However, over time, increasing number of patients have been identified with GLA variants causing either non-classic Fabry disease or having unclear clinical effects.
    SUMMARY: Searching for additional etiologic and lysosomal storage-independent factors, investigators have recently identified that certain missense GLA variants not only affect enzymatic activity, but also encode for misfolded α-galactosidase A that itself induces chronic endoplasmic reticulum stress and the unfolded protein response. Thus, Fabry disease pathogenesis may be caused by decreased enzymatic activity as well as cellular toxicity from accumulation of the misfolded α-galactosidase A protein, with the contribution of each factor determined by the type of the genetic variant and host factors.
    KEY MESSAGES: Defective proteostasis and misfolding of certain missense α-galactosidase A variants induce chronic endoplasmic reticulum stress and the unfolded protein response that may contribute to intra-familial and inter-familial variation in disease penetrance and clinical expressivity. Pharmacologic modulation of defective proteostasis may have therapeutic implications in Fabry disease.
    DOI:  https://doi.org/10.1159/000545388
  20. FEBS Open Bio. 2025 Mar 18.
    METTL3 knockout accelerates hepatocarcinogenesis via inhibiting endoplasmic reticulum stress response.
    Bo Cui, Silin Tu, Haibo Li, Zhancheng Zeng, Ruiqi Xiao, Jing Guo, Xiaoqi Liang, Chang Liu, Lijie Pan, Wenjie Chen, Mian Ge, Xiaofen Zhong, Linsen Ye, Huaxin Chen, Qi Zhang, Yan Xu.
      Hepatocellular carcinoma (HCC) is among the most common causes of cancer-related deaths worldwide. Previous studies showed that N6-methyladenosine (m6A), the most abundant chemical modification in eukaryotic RNAs, is implicated in HCC progression. Using liver-specific conditional knockout mice, we found that the loss of METTL3, the core catalytic subunit of m6A methyltransferase, significantly promoted hepatic tumor initiation under various oncogenic challenges, contrary to the previously reported oncogenic role of METTL3 in liver cancer cell lines or xenograft models. Mechanistically, we hypothesized that METTL3 deficiency accelerated HCC initiation by inhibiting m6A deposition on MANF transcripts, impairing nuclear export and thus MANF protein levels, which led to insufficient endoplasmic reticulum (ER) stress response pathway activation. Our findings suggest a tumor-suppressive role for METTL3 in the early stages of HCC, emphasizing the importance of understanding the dynamic role of epigenetic regulation in tumorigenesis and targeted therapy.
    Keywords:  ER stress response; METTL3; hepatocarcinogenesis; hepatocellular carcinoma; m6A
    DOI:  https://doi.org/10.1002/2211-5463.70023
  21. EMBO Rep. 2025 Mar 20.
    Lack of AtMC1 catalytic activity triggers autoimmunity dependent on NLR stability.
    Jose Salguero-Linares, Laia Armengot, Joel Ayet, Nerea Ruiz-Solaní, Svenja C Saile, Marta Salas-Gómez, Esperanza Fernandez, Lode Denolf, Fernando Navarrete, Jenna Krumbach, Markus Kaiser, Simon Stael, Frank Van Breusegem, Kris Gevaert, Farnusch Kaschani, Morten Petersen, Farid El Kasmi, Marc Valls, Núria S Coll.
      Plants utilize cell surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to detect non-self and elicit robust immune responses. Fine-tuning the homeostasis of these receptors is critical to prevent their hyperactivation. Here, we show that Arabidopsis plants lacking metacaspase 1 (AtMC1) display autoimmunity dependent on immune signalling components downstream of NLR and PRR activation. Overexpression of a catalytically inactive AtMC1 in an atmc1 background triggers severe autoimmunity partially dependent on the same immune signalling components. Overexpression of the E3 ligase SNIPER1, a master regulator of NLR homeostasis, fully reverts the AtMC1-dependent autoimmunity phenotype, inferring that a broad defect in NLR turnover may underlie the severe phenotype observed. Catalytically inactive AtMC1 localizes to punctate structures that are degraded through autophagy. Considering also previous evidence on the proteostatic functions of AtMC1, we speculate that Wt AtMC1 may either directly or indirectly control NLR protein levels, thereby preventing autoimmunity.
    Keywords:  Autoimmunity; Autophagy; Condensates; Metacaspases; Proteostasis
    DOI:  https://doi.org/10.1038/s44319-025-00426-4
  22. Cell Rep. 2025 Mar 18. pii: S2211-1247(25)00212-8. [Epub ahead of print]44(4): 115441
    Lactylation orchestrates ubiquitin-independent degradation of cGAS and promotes tumor growth.
    Keqiang Rao, Xinchao Zhang, Yi Luo, Qiang Xia, Yuting Jin, Jing He.
      Lactate extensively associates with metabolic reprogramming, signal transduction, and immune modulation. Nevertheless, the regulatory role of lactate in immune sensing of cytosolic DNA remains uncertain. Here, we report that lactate serves as an initiator to facilitate proteasomal degradation of cyclic GMP-AMP synthase (cGAS) independent of ubiquitin, thus repressing the production of interferon and contributing to tumor growth. Mechanistically, lactylation of K21 stimulates cGAS translocation from the nucleus to the proteasome for degradation, which is compromised by phosphorylation of PSMA4 S188 via disrupting its association with cGAS. Concurrently, lactylation of K415 rewires PIK3CB activity and impairs ULK1-driven phosphorylation of PSMA4 S188. Physiologically, lactylation of cGAS sustains tumor growth. Expression of cGAS correlates with the antitumor effect of the LDHA inhibitor FX11. Finally, the lactate-cGAS axis indicates a prognostic outcome of lung adenocarcinoma. Collectively, these findings not only put forth a mechanism of cGAS degradation but also unravel the clinical relevance of cGAS lactylation.
    Keywords:  CP: Cancer; PIK3CB; PSMA4; ULK1; cGAS; innate immune system; lactylation; tumor growth; ubiquitin-independent degradation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115441
  23. Nat Commun. 2025 Mar 20. 16(1): 2751
    Structurally heterogeneous ribosomes cooperate in protein synthesis in bacterial cells.
    Karla Helena-Bueno, Sophie Kopetschke, Sebastian Filbeck, Lewis I Chan, Sonia Birsan, Arnaud Baslé, Maisie Hudson, Stefan Pfeffer, Chris H Hill, Sergey V Melnikov.
      Ribosome heterogeneity is a paradigm in biology, pertaining to the existence of structurally distinct populations of ribosomes within a single organism or cell. This concept suggests that structurally distinct pools of ribosomes have different functional properties and may be used to translate specific mRNAs. However, it is unknown to what extent structural heterogeneity reflects genuine functional specialization rather than stochastic variations in ribosome assembly. Here, we address this question by combining cryo-electron microscopy and tomography to observe individual structurally heterogeneous ribosomes in bacterial cells. We show that 70% of ribosomes in Psychrobacter urativorans contain a second copy of the ribosomal protein bS20 at a previously unknown binding site on the large ribosomal subunit. We then determine that this second bS20 copy appears to be functionally neutral. This demonstrates that ribosome heterogeneity does not necessarily lead to functional specialization, even when it involves significant variations such as the presence or absence of a ribosomal protein. Instead, we show that heterogeneous ribosomes can cooperate in general protein synthesis rather than specialize in translating discrete populations of mRNA.
    DOI:  https://doi.org/10.1038/s41467-025-57955-8
  24. Autophagy. 2025 Mar 20.
    TBK1 is a signaling hub in coordinating stress-adaptive mechanisms in head and neck cancer progression.
    Hyo Jeong Kim, Haeng-Jun Kim, Sun-Yong Kim, Jin Roh, Ju Hyun Yun, Chul-Ho Kim.
      Tumorigenesis is closely linked to the ability of cancer cells to activate stress-adaptive mechanisms in response to various cellular stressors. Stress granules (SGs) play a crucial role in promoting cancer cell survival, invasion, and treatment resistance, and influence tumor immune escape by protecting essential mRNAs involved in cell metabolism, signaling, and stress responses. TBK1 (TANK binding kinase 1) functions in antiviral innate immunity, cell survival, and proliferation in both the tumor microenvironment and tumor cells. Here, we report that MUL1 loss results in the hyperactivation of TBK1 in both HNC cells and tissues. Mechanistically, under proteotoxic stress induced by proteasomal inhibition, HSP90 inhibition, or Ub+ stress, MUL1 promotes the degradation of active TBK1 through K48-linked ubiquitination at lysine 584. Furthermore, TBK1 facilitates autophagosome-lysosome fusion and phosphorylates SQSTM1, regulating selective macroautophagic/autophagic clearance in HNC cells. TBK1 is required for SG formation and cellular protection. Moreover, we found that MAP1LC3B is partially localized within SGs. TBK1 depletion enhances the sensitivity of HNC cells to cisplatin-induced cell death. GSK8612, a novel TBK1 inhibitor, significantly inhibits HNC tumorigenesis in xenografts. In summary, our study reveals that TBK1 facilitates the rapid removal of ubiquitinated proteins within the cell through protective autophagy under stress conditions and assists SG formation through the use of the autophagy machinery. These findings highlight the potential of TBK1 as a therapeutic target in HNC treatment.
    Keywords:  Autophagic flux; GSK8612; MUL1; TBK1; head and neck cancer; stress granule formation
    DOI:  https://doi.org/10.1080/15548627.2025.2481661
  25. Nat Commun. 2025 Mar 19. 16(1): 2723
    A cysteine-less and ultra-fast split intein rationally engineered from being aggregation-prone to highly efficient in protein trans-splicing.
    Christoph Humberg, Zahide Yilmaz, Katharina Fitzian, Wolfgang Dörner, Daniel Kümmel, Henning D Mootz.
      Split inteins catalyze protein trans-splicing by ligating their extein sequences while undergoing self-excision, enabling diverse protein modification applications. However, many purified split intein precursors exhibit partial or no splicing activity for unknown reasons. The Aes123 PolB1 intein, a representative of the rare cysteine-less split inteins, is of particular interest due to its resistance to oxidative conditions and orthogonality to thiol chemistries. In this work, we identify β-sheet-dominated aggregation of its N-terminal intein fragment as the origin of its low (~30%) splicing efficiency. Using computational, biochemical, and biophysical analyses, we characterize the fully active monomeric fraction and pinpoint aggregation-prone regions. Supported by a crystal structure, we design stably monomeric mutants with nearly complete splicing activity. The optimized CLm intein (Cysteine-Less and monomeric) retains the wild-type's ultra-fast reaction rate and serves as an efficient, thiol-independent protein modification tool. We find that other benchmark split inteins show similar precursor aggregation, suggesting that this general phenomenon arises from the intrinsic challenge to maintain the precursor in a partially disordered state while promoting stable folding upon fragment association.
    DOI:  https://doi.org/10.1038/s41467-025-57596-x
  26. Sci Adv. 2025 Mar 21. 11(12): eads2923
    Transfer RNA acetylation regulates in vivo mammalian stress signaling.
    Supuni Thalalla Gamage, Roxane Khoogar, Shereen Howpay Manage, Judey T DaRos, McKenna C Crawford, Joe Georgeson, Bogdan V Polevoda, Chelsea Sanders, Kendall A Lee, Kellie D Nance, Vinithra Iyer, Anatoly Kustanovich, Minervo Perez, Chu T Thu, Sam R Nance, Ruhul Amin, Christine N Miller, Ronald J Holewinski, Sudipto Das, Thomas J Meyer, Vishal Koparde, Acong Yang, Parthav Jailwala, Joe T Nguyen, Thorkell Andresson, Kent Hunter, Shuo Gu, Beverly A Mock, Elijah F Edmondson, Simone Difilippantonio, Raj Chari, Schraga Schwartz, Mitchell R O'Connell, Colin Chih-Chien Wu, Jordan L Meier.
      Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here, we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification catalyzed by the essential acetyltransferase Nat10. By targeting Thumpd1, a nonessential adapter protein required for Nat10-catalyzed tRNA acetylation, we determine that loss of tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality in mice, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translational control and therapeutic interventions.
    DOI:  https://doi.org/10.1126/sciadv.ads2923
  27. Autophagy. 2025 Mar 20.
    Chaperone-mediated autophagy degrades SERPINA1E342K/α1-antitrypsin Z variant and alleviates cell stress.
    Jiayu Lin, Xinyue Wei, Yan Dai, Haorui Lu, Yajian Song, Jiansong Ju, Rihan Wu, Qichen Cao, Hao Yang, Lang Rao.
      Chaperone-mediated autophagy (CMA) is a specific form of autophagy that selectively targets proteins containing a KFERQ-like motif and relies on the chaperone protein HSPA8/HSC70 for substrate recognition. In SERPINA1/a1-antitrypsin deficiency (AATD), a disease characterized by the hepatic buildup of the SERPINA1E342K/ATZ, CMA's role had been unclear. This work demonstrates the critical role that CMA plays in preventing SERPINA1E342K/ATZ accumulation; suppressing CMA worsens SERPINA1E342K/ATZ accumulation while activating it through chemical stimulation or LAMP2A overexpression promotes SERPINA1E342K/ATZ breakdown. Specifically, SERPINA1E342K/ATZ's 121QELLR125 motif is critical for HSPA8/HSC70 recognition and LAMP2A's charged C-terminal cytoplasmic tail is vital for substrate binding, facilitating CMA-mediated degradation of SERPINA1E342K/ATZ. This selective activation of CMA operates independently from other autophagy pathways and alleviates SERPINA1E342K/ATZ aggregate-induced cellular stress. In vivo administration of AR7 promotes hepatic SERPINA1E342K/ATZ elimination and mitigates hepatic SERPINA1E342K/ATZ aggregation pathology. These findings highlight CMA's critical function in cellular protein quality control of SERPINA1E342K/ATZ and place it as a novel target for AATD treatment approaches.
    Keywords:  Cellular stress; HSPA8/HSC70; LAMP2A; chaperone-mediated autophagy; protein degradation
    DOI:  https://doi.org/10.1080/15548627.2025.2480037
  28. Nat Commun. 2025 Mar 17. 16(1): 2614
    MyoD1 localization at the nuclear periphery is mediated by association of WFS1 with active enhancers.
    Konstantina Georgiou, Fatih Sarigol, Tobias Nimpf, Christian Knapp, Daria Filipczak, Roland Foisner, Nana Naetar.
      Spatial organization of the mammalian genome influences gene expression and cell identity. While association of genes with the nuclear periphery is commonly linked to transcriptional repression, also active, expressed genes can localize at the nuclear periphery. The transcriptionally active MyoD1 gene, a master regulator of myogenesis, exhibits peripheral localization in proliferating myoblasts, yet the underlying mechanisms remain elusive. Here, we generate a reporter cell line to demonstrate that peripheral association of the MyoD1 locus is independent of mechanisms involved in heterochromatin anchoring. Instead, we identify the nuclear envelope transmembrane protein WFS1 that tethers MyoD1 to the nuclear periphery. WFS1 primarily associates with active distal enhancer elements upstream of MyoD1, and with a subset of enhancers genome-wide, which are enriched in active histone marks and linked to expressed myogenic genes. Overall, our data identify a mechanism involved in tethering regulatory elements of active genes to the nuclear periphery.
    DOI:  https://doi.org/10.1038/s41467-025-57758-x
  29. Sci Adv. 2025 Mar 21. 11(12): eadn8402
    AMPK-dependent Parkin activation suppresses macrophage antigen presentation to promote tumor progression.
    Xinyu Wang, Yiyi Li, Yan Li, Xiumei Wang, Hongrui Song, Yingzhao Wang, Chunliu Huang, Chengzhou Mao, Lixiang Wang, Cheng Zhong, Di Yu, Zijin Xia, Yongyi Feng, Jingjing Duan, Yujia Liu, Juanjuan Ou, Congzhou Luo, Wenhao Mai, Hai Hong, Weibin Cai, Limin Zheng, Jean-François Trempe, Edward A Fon, Jing Liao, Wei Yi, Jun Chen.
      The constrained cross-talk between myeloid cells and T cells in the tumor immune microenvironment (TIME) restricts cancer immunotherapy efficacy, whereas the underlying mechanism remains elusive. Parkin, an E3 ubiquitin ligase renowned for mitochondrial quality control, has emerged as a regulator of immune response. Here, we show that both systemic and macrophage-specific ablations of Parkin in mice lead to attenuated tumor progression and prolonged mouse survival. By single-cell RNA-seq and flow cytometry, we demonstrate that Parkin deficiency reshapes the TIME through activating both innate and adaptive immunities to control tumor progression and recurrence. Mechanistically, Parkin activation by AMP-activated protein kinase rather than PTEN-induced kinase 1 mediated major histocompatibility complex I down-regulation on macrophages via Autophagy related 5-dependent autophagy. Furthermore, Parkin deletion synergizes with immune checkpoint blockade treatment and Park2-/- signature aids in predicting the prognosis of patients with solid tumor. Our findings uncover Parkin's involvement in suppressing macrophage antigen presentation for coordinating the cross-talk between macrophages and T cells.
    DOI:  https://doi.org/10.1126/sciadv.adn8402
  30. Sci Adv. 2025 Mar 21. 11(12): eadq9111
    USP8 and Hsp70 regulate endoreplication by synergistically promoting Fzr deubiquitination and stabilization.
    Wenliang Qian, Xing Zhang, Dongqin Yuan, Yuting Wu, Hao Li, Ling Wei, Zheng Li, Zongcai Dai, Pei Song, Qiaoling Sun, Zizhang Zhou, Qingyou Xia, Daojun Cheng.
      Endoreplication is characterized by multiple rounds of DNA replication without cell division and determines the growth and final size of endoreplicating cells and tissues in eukaryotes. The cyclic ubiquitination and degradation of several cell cycle regulators are required for endoreplication progression. However, the deubiquitinase that deubiquitinates and stabilizes key factors to modulate endoreplication remains unknown. Here, we found in the endoreplicating Drosophila salivary gland and Bombyx silk gland that the depletion of ubiquitin-specific peptidase 8 (USP8) led to endoreplication arrest and a decrease in gland size. Mechanistically, we showed that USP8 interacted with the Fizzy-related (Fzr) protein, a conserved master regulator of endoreplication, thereby deubiquitinating and stabilizing Fzr to modulate endoreplication. Moreover, the molecular chaperone heat shock protein 70 (Hsp70) mediated proper folding of Fzr and increased the interaction between Fzr and USP8, thereby promoting the deubiquitination and stabilization of Fzr. Together, our study demonstrates that USP8 and Hsp70 regulate endoreplication by synergistically maintaining Fzr stability though deubiquitination.
    DOI:  https://doi.org/10.1126/sciadv.adq9111
  31. J Cell Biol. 2025 May 05. pii: e202408050. [Epub ahead of print]224(5):
    TMBIM-2 orchestrates systemic mitochondrial stress response via facilitating Ca2+ oscillations.
    Jiasheng Li, Jimeng Cui, Xinyu Li, Di Zhu, Zhenhua Chen, Xiahe Huang, Yingchun Wang, Qingfeng Wu, Ye Tian.
      Neuronal mitochondrial function is critical for orchestrating inter-tissue communication essential for overall fitness. Despite its significance, the molecular mechanism underlying the impact of prolonged mitochondrial stresses on neuronal activity and how they orchestrate metabolism and aging remains elusive. Here, we identified the evolutionarily conserved transmembrane protein XBX-6/TMBIM-2 as a key mediator in the neuronal-to-intestinal mitochondrial unfolded protein response (UPRmt). Our investigations reveal that intrinsic neuronal mitochondrial stress triggers spatiotemporal Ca2+ oscillations in a TMBIM-2-dependent manner through the Ca2+ efflux pump MCA-3. Notably, persistent Ca2+ oscillations at synapses of ADF neurons are critical for facilitating serotonin release and the subsequent activation of the neuronal-to-intestinal UPRmt. TMBIM2 expression diminishes with age; however, its overexpression counteracts the age-related decline in aversive learning behavior and extends the lifespan of Caenorhabditis elegans. These findings underscore the intricate integration of chronic neuronal mitochondrial stress into neurotransmission processes via TMBIM-2-dependent Ca2+ equilibrium, driving metabolic adaptation and behavioral changes for the regulation of aging.
    DOI:  https://doi.org/10.1083/jcb.202408050
  32. Blood. 2025 Mar 16. pii: blood.2024024956. [Epub ahead of print]
    Proteostasis Disruption in Inherited Bone Marrow Failure Syndromes.
    Helena Yu, Robert Signer.
      Inherited bone marrow failure syndromes (IBMFS) are genetic disorders of impaired hematopoiesis that manifest in childhood with both cytopenias and extra-hematologic findings. While several IBMFS are categorized as ribosomopathies due to shared underlying ribosomal dysfunction, there is a broader disruption of the protein homeostasis (proteostasis) network across both classic and emerging IBMFS. Precise regulation of the proteostasis network, including mechanisms of protein synthesis, folding, trafficking, and degradation as well as associated stress response pathways, has emerged as essential for maintaining hematopoietic stem cell (HSC) function, providing new potential mechanistic insights into IBMFS pathogenesis. Furthermore, the varied clinical trajectories of patients with IBMFS with possible divergent outcomes of malignancy and spontaneous remission may reflect developmental and temporal changes in proteostasis activity and be driven by strong selective pressures to restore proteostasis. These new insights are spurring fresh therapeutic approaches to target proteostasis. Thus, further evaluation of proteostasis regulation and the consequences of proteostasis disruption in IBMFS could aid in developing new biomarkers, therapeutic agents, and preventative approaches for patients.
    DOI:  https://doi.org/10.1182/blood.2024024956
  33. Bioanalysis. 2025 Mar 20. 1-16
    Conquering PROTAC molecular design and drugability.
    Ritesh P Bhole, Sonali Labhade, Shilendra S Gurav.
      PROTACs are reshaping drug discovery by enabling targeted protein degradation, overcoming the limitations of traditional inhibitors, and addressing previously "undruggable" proteins. The present perspective explores advancements in PROTAC molecular design, focusing on ligand discovery, E3 ligase recruitment, and ternary complex optimization. Integrating AI-driven modeling, FBDD, and SBDD accelerates PROTAC development. In contrast, emerging innovations, such as PHOTACs, hypoxia-responsive systems, and Ab-PROTACs, enhance precision and reduce systemic toxicity. Clinical successes, including ARV-110 for castration-resistant prostate cancer and ARV-471 for breast cancer, exemplify their ability to overcome resistance and provide durable effects. PROTACs are expanding into neurodegenerative diseases and rare conditions, highlighting their versatility. By addressing challenges in pharmacokinetics, safety, and scalability, PROTACs are poised to revolutionize precision medicine. This article presents a forward-looking perspective on conquering the molecular design and drugability of PROTACs, paving the path for transformative therapies.
    Keywords:  AI and machine learning; E3 ligase recruitment; Precision medicine; immunotherapy; ligand discovery; targeted and combination therapy; targeted protein degradation; therapeutic innovation
    DOI:  https://doi.org/10.1080/17576180.2025.2481021
  34. Nat Commun. 2025 Mar 20. 16(1): 2755
    Structural recognition and stabilization of tyrosine hydroxylase by the J-domain protein DNAJC12.
    Mary Dayne S Tai, Lissette Ochoa, Marte I Flydal, Lorea Velasco-Carneros, Jimena Muntaner, César Santiago, Gloria Gamiz-Arco, Fernando Moro, Kunwar Jung-Kc, David Gil-Cantero, Miguel Marcilla, Juha P Kallio, Arturo Muga, José María Valpuesta, Jorge Cuéllar, Aurora Martinez.
      Pathogenic variants of the J-domain protein DNAJC12 cause parkinsonism, which is associated with a defective interaction of DNAJC12 with tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis. In this work, we characterize the formation of the TH:DNAJC12 complex, showing that DNAJC12 binding stabilizes both TH and the variant TH-p.R202H, associated with TH deficiency. This binding delays their time-dependent aggregation in an Hsp70-independent manner, while preserving TH activity and feedback regulatory inhibition by dopamine. DNAJC12 alone barely activates Hsc70 but synergistically stimulates Hsc70 ATPase activity when complexed with TH. Cryo-electron microscopy supported by crosslinking-mass spectroscopy reveals two DNAJC12 monomers bound per TH tetramer, each embracing one of the two regulatory domain dimers, leaving the active sites available for substrate, cofactor and inhibitory dopamine interaction. Our results also reveal the key role of the C-terminal region of DNAJC12 in TH binding, explaining the pathogenic mechanism of the DNAJC12 disease variant p.W175Ter.
    DOI:  https://doi.org/10.1038/s41467-025-57733-6
  35. Nat Commun. 2025 Mar 17. 16(1): 2617
    Deep indel mutagenesis reveals the impact of amino acid insertions and deletions on protein stability and function.
    Magdalena Topolska, Antoni Beltran, Ben Lehner.
      Amino acid insertions and deletions (indels) are an abundant class of genetic variants. However, compared to substitutions, the effects of indels on protein stability are not well understood. To better understand indels here we analyse new and existing large-scale deep indel mutagenesis (DIM) of structurally diverse proteins. The effects of indels on protein stability vary extensively among and within proteins and are not well predicted by existing computational methods. To address this shortcoming we present INDELi, a series of models that combine experimental or predicted substitution effects and secondary structure information to provide good prediction of the effects of indels on both protein stability and pathogenicity. Moreover, quantifying the effects of indels on protein-protein interactions suggests that insertions can be an important class of gain-of-function variants. Our results provide an overview of the impact of indels on proteins and a method to predict their effects genome-wide.
    DOI:  https://doi.org/10.1038/s41467-025-57510-5
  36. Nat Commun. 2025 Mar 17. 16(1): 2631
    Transcriptional heterogeneity shapes stress-adaptive responses in yeast.
    Mariona Nadal-Ribelles, Guillaume Lieb, Carme Solé, Yaima Matas, Ugo Szachnowski, Sara Andjus, Maria Quintana, Mònica Romo, Aitor Gonzalez Herrero, Antonin Morillon, Serge Pelet, Eulàlia de Nadal, Francesc Posas.
      In response to stress, cells activate signaling pathways that coordinate broad changes in gene expression to enhance cell survival. Remarkably, complex variations in gene expression occur even in isogenic populations and in response to similar signaling inputs. However, the molecular mechanisms underlying this variability and their influence on adaptive cell fate decisions are not fully understood. Here, we use scRNA-seq to longitudinally assess transcriptional dynamics during osmoadaptation in yeast. Our findings reveal highly heterogeneous expression of the osmoresponsive program, which organizes into combinatorial patterns that generate distinct cellular programs. The induction of these programs is favored by global transcriptome repression upon stress. Cells displaying basal expression of the osmoresponsive program are hyper-responsive and resistant to stress. Through a transcription-focused analysis of more than 300 RNA-barcoded deletion mutants, we identify genetic factors that shape the heterogeneity of the osmostress-induced transcriptome, define regulators of stress-related subpopulations and find a link between transcriptional heterogeneity and increased cell fitness. Our findings provide a regulatory map of the complex transcriptional phenotypes underlying osmoadaptation in yeast and highlight the importance of transcriptional heterogeneity in generating distinct adaptive strategies.
    DOI:  https://doi.org/10.1038/s41467-025-57911-6
  37. RNA. 2025 Mar 21. pii: rna.080450.125. [Epub ahead of print]
    Natural human tRNAAla anticodon variants mistranslate the genetic code.
    Rasangi Tennakoon, Teija M I Bily, Farah Hasan, Kyle S Hoffman, Patrick O'Donoghue.
      Transfer RNAs (tRNAs) play an essential role in protein synthesis by linking the nucleic acid sequences of gene products to the amino acid sequences of proteins. There are > 400 functional tRNA genes in humans, and adding to this diversity, there are many single nucleotide polymorphisms in tRNAs across our population, including anticodon variants that mistranslate the genetic code. In human genomes, we identified three rare alanine tRNA (tRNAAla) variants with non-synonymous anticodon mutations: tRNAAlaCGC G35T, tRNAAlaUGC G35A, and tRNAAlaAGC C36T. Since alanyl-tRNA synthetase (AlaRS) does not recognize the anticodon, we hypothesized that these tRNAAla variants will mis-incorporate Ala at glutamate (Glu), valine (Val), and threonine (Thr) codons, respectively. We found that expressing the naturally occurring tRNAAla variants in human cells led to defects in protein production without a substantial impact on cell growth. Using mass spectrometry, we confirmed and estimated Ala mis-incorporation levels at Glu (0.7%), Val (5%) and Thr (0.1%) codons. Although Ala mis-incorporation was higher at Val codons, cells mis-incorporating Ala at Glu codons had the most severe defect in protein production. The data demonstrate the ability of natural human tRNAAla variants to generate mistranslation leading to defects in protein production that depend on the nature of the amino acid replacement.
    Keywords:  alanine; anticodon; missense suppression; mistranslation; tRNA
    DOI:  https://doi.org/10.1261/rna.080450.125
  38. Biochim Biophys Acta Mol Cell Res. 2025 Mar 18. pii: S0167-4889(25)00037-0. [Epub ahead of print] 119932
    Defective gating of the Sec61 translocon results in reorganisation of the actin cytoskeleton and perturbed formation of the actomyosin contractile ring.
    Lee V Thomasson, Christopher M Witham, Robert F L Steuart, Danielle E Dye, Carl J Mousley.
      The Sec61 complex sits between the distinct environments of the cytosol and the ER lumen and it's appropriate gating is essential to prevent the deleterious flux of molecules and ions between them. Using the sss1-7 mutant we show that actin dynamics is grossly perturbed when translocon gating is defective. Importantly, normal actin morphology is restored when sss1-7 translocon gating defects are suppressed or when these cells are treated with cell-permeable Ca2+ chelators. Our findings underscore the importance of translocon gating, particularly in regulating Ca2+ homeostasis, in the overall regulation and functional distribution of the actin cytoskeleton.
    Keywords:  Actin; Sec61 complex; Sss1p; Translocon gating
    DOI:  https://doi.org/10.1016/j.bbamcr.2025.119932
  39. Nat Commun. 2025 Mar 19. 16(1): 2702
    Deciphering the folding code of collagens.
    Jean-Daniel Malcor, Noelia Ferruz, Sergio Romero-Romero, Surbhi Dhingra, Vamika Sagar, Abhishek A Jalan.
      Collagen proteins contain a characteristic structural motif called a triple helix. During the self-assembly of this motif, three polypeptides form a folding nucleus at the C-termini and then propagate towards the N-termini like a zip-chain. While polypeptides from human collagens contain up to a 1000 amino acids, those found in bacteria can contain up to 6000 amino acids. Additionally, the collagen polypeptides are also frequently interrupted by non-helical sequences that disrupt folding and reduce stability. Given the length of polypeptides and the disruptive interruptions, compensating mechanisms that stabilize against local unfolding during propagation and offset the entropic cost of folding are not fully understood. Here, we show that the information for the correct folding of collagen triple helices is encoded in their sequence as interchain electrostatic interactions, which likely act as molecular clamps that prevent local unfolding. In the case of humans, disrupting these electrostatic interactions is associated with severe to lethal diseases.
    DOI:  https://doi.org/10.1038/s41467-024-54046-y
  40. bioRxiv. 2025 Mar 06. pii: 2025.03.06.641798. [Epub ahead of print]
    Murine Leukemia Virus GlycoGag Antagonizes SERINC5 via ER-phagy Receptor RETREG1.
    Iqbal Ahmad, Jing Zhang, Rongrong Li, Wenqiang Su, Weiqi Liu, You Wu, Ilyas Khan, Xiaomeng Liu, Lian-Feng Li, Sunan Li, Yong-Hui Zheng.
      Serine incorporator 5 (SERINC5) is a host restriction factor that targets certain enveloped viruses, including human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). It integrates into the viral envelope from the cell surface, inhibiting viral entry. SERINC5 is transported to the cell surface via polyubiquitination, while a single K130R mutation retains it in the cytoplasm. Both HIV-1 Nef and MLV glycoGag proteins antagonize SERINC5 by reducing its expression in producer cells. Here, we report that MLV glycoGag employs selective autophagy to downregulate SERINC5, demonstrating a more potent mechanism for decreasing its cell surface expression. Although glycoGag is a type II integral membrane protein, it primarily localizes to the cytoplasm and undergoes rapid proteasomal degradation. Employing the K130R mutant, we show that Nef, primarily associated with the plasma membrane, downregulates SERINC5 only after it has trafficked to the cell surface, whereas glycoGag can reduce its expression before reaching the plasma membrane while still in the cytoplasm. Nonetheless, an interaction with SERINC5 stabilizes and recruits glycoGag to the plasma membrane, enabling it to downregulate SERINC5 from the cell surface. Through affinity-purified mass spectrometry analysis combined with CRISPR/Cas9 knockouts, we find that glycoGag's activity depends on reticulophagy regulator 1 (RETREG1), an ER-phagy receptor. Further knockout experiments of critical autophagy genes demonstrate that glycoGag downregulates cytoplasmic SERINC5 via micro-ER-phagy. These findings provide crucial new insights into the ongoing arms race between retroviruses and SERINC5 during infection.
    AUTHOR SUMMARY: HIV-1 Nef and MLV glycoGag are unrelated viral proteins, yet both counteract the same host restriction factor, SERINC5, to facilitate productive infection. In this study, we report a novel pathway through which glycoGag downregulates SERINC5. We demonstrate that while Nef downregulates SERINC5 only after it has trafficked to the cell surface, glycoGag can directly downregulate SERINC5 in the cytoplasm before it reaches the plasma membrane. Furthermore, we show that this pathway is mediated by the ER-phagy receptor RETREG1, which targets SERINC5 for degradation via micro-ER-phagy. This mechanism provides a more effective means of blocking SERINC5 antiviral activity. These findings reveal that retroviruses have evolved different strategies to antagonize SERINC5, highlighting the critical role of SERINC5 in restricting retroviral infections.
    DOI:  https://doi.org/10.1101/2025.03.06.641798
  41. Cell. 2025 Mar 14. pii: S0092-8674(25)00209-0. [Epub ahead of print]
    Turnover atlas of proteome and phosphoproteome across mouse tissues and brain regions.
    Wenxue Li, Abhijit Dasgupta, Ka Yang, Shisheng Wang, Nisha Hemandhar-Kumar, Surendhar R Chepyala, Jay M Yarbro, Zhenyi Hu, Barbora Salovska, Eugenio F Fornasiero, Junmin Peng, Yansheng Liu.
      Understanding how proteins in different mammalian tissues are regulated is central to biology. Protein abundance, turnover, and post-translational modifications such as phosphorylation are key factors that determine tissue-specific proteome properties. However, these properties are challenging to study across tissues and remain poorly understood. Here, we present Turnover-PPT, a comprehensive resource mapping the abundance and lifetime of 11,000 proteins and 40,000 phosphosites in eight mouse tissues and various brain regions using advanced proteomics and stable isotope labeling. We reveal tissue-specific short- and long-lived proteins, strong correlations between interacting protein lifetimes, and distinct impacts of phosphorylation on protein turnover. Notably, we discover a remarkable pattern of turnover changes for peroxisome proteins in specific tissues and that phosphorylation regulates the stability of neurodegeneration-related proteins, such as Tau and α-synuclein. Thus, Turnover-PPT provides fundamental insights into protein stability, tissue dynamic proteotypes, and functional protein phosphorylation and is accessible via an interactive web-based portal at https://yslproteomics.shinyapps.io/tissuePPT.
    Keywords:  DIA-MS; TMT; brain regions; mouse tissues; protein lifetime; protein phosphorylation; protein turnover; proteomics; pulse SILAC
    DOI:  https://doi.org/10.1016/j.cell.2025.02.021
  42. Commun Biol. 2025 Mar 18. 8(1): 455
    POT, an optogenetics-based endogenous protein degradation system.
    Yunyue Chen, Siyifei Wang, Luhao Zhang, Dandan Peng, Ke Huang, Baohua Ji, Junfen Fu, Yingke Xu.
      Precise regulation of protein abundance is critical for cellular homeostasis, whose dysfunction may directly lead to human diseases. Optogenetics allows rapid and reversible control of precisely defined cellular processes, which has the potential to be utilized for regulation of protein dynamics at various scales. Here, we developed a novel optogenetics-based protein degradation system, namely Peptide-mediated OptoTrim-Away (POT) which employs expressed small peptides to effectively target endogenous and unmodified proteins. By engineering the light-induced oligomerization of the E3 ligase TRIM21, POT can rapidly trigger protein degradation via the proteasomal pathway. Our results showed that the developed POT-PI3K and POT-GPX4 modules, which used the iSH2 and FUNDC1 domains to specifically target phosphoinositide 3-kinase (PI3K) and glutathione peroxidase 4 (GPX4) respectively, were able to potently induce the degradation of these endogenous proteins by light. Both live-cell imaging and biochemical experiments validated the potency of these tools in downregulating cancer cell migration, proliferation, and even promotion of cell apoptosis. Therefore, we believe the POT offers an alternative and practical solution for rapid manipulation of endogenous protein levels, and it could potentially be employed to dissect complex signaling pathways in cell and for targeted cellular therapies.
    DOI:  https://doi.org/10.1038/s42003-025-07919-x
  43. Nat Commun. 2025 Mar 18. 16(1): 2541
    GraphBAN: An inductive graph-based approach for enhanced prediction of compound-protein interactions.
    Hamid Hadipour, Yan Yi Li, Yan Sun, Chutong Deng, Leann Lac, Rebecca Davis, Silvia T Cardona, Pingzhao Hu.
      Understanding compound-protein interactions is crucial for early drug discovery, offering insights into molecular mechanisms and potential therapeutic effects of compounds. Here, we introduce GraphBAN, a graph-based framework that inductively predicts these interactions using compound and protein feature information. GraphBAN effectively handles inductive link predictions for unseen nodes, providing a robust solution for predicting interactions between entirely unseen compounds and proteins. This capability enables GraphBAN to transcend the constraints of traditional methods that are typically limited to known contexts. GraphBAN employs a knowledge distillation architecture through a teacher-student learning model. The teacher block leverages network structure information, while the student block focuses on node attributes, enhancing learning and prediction accuracy. Additionally, GraphBAN incorporates a domain adaptation module, increasing its effectiveness across different dataset domains. Empirical tests on five benchmark datasets demonstrate that GraphBAN outperforms ten baseline models, while a case study analysis with the Pin1 protein further supports the model's effectiveness in real world scenarios, making it as a promising tool for early drug discovery.
    DOI:  https://doi.org/10.1038/s41467-025-57536-9
  44. Nucleic Acids Res. 2025 Feb 27. pii: gkaf176. [Epub ahead of print]53(5):
    Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles.
    Jessica Corsi, Pouriya Sharbatian Semnani, Daniele Peroni, Romina Belli, Alessia Morelli, Michelangelo Lassandro, Viktoryia Sidarovich, Valentina Adami, Chiara Valentini, Paolo Cavallerio, Julian Grosskreutz, Fabrizio Fabbiano, Dajana Grossmann, Andreas Hermann, Gianluca Tell, Manuela Basso, Vito G D'Agostino.
      Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and noncoding RNAs can be detected as EV cargo, and RNA-binding proteins (RBPs), such as hnRNPA2B1, have been circumstantially implicated in EV-RNA sorting mechanisms. However, the contribution of competitive RBP-RNA interactions responsible for RNA-sorting outcomes is still unclear, especially for predicting the EV-RNA content. We designed a reverse proteomic analysis exploiting the EV-RNA to identify intracellular protein binders in vitro. Using cells expressing a recombinant hnRNPA2B1 to normalize competitive interactions, we prioritized a network of heterogeneous nuclear ribonucleoproteins and purine-rich RNA sequences subsequently validated in secreted EV-RNA through short fluorescent RNA oligos. Then, we designed a GGGAG-enriched RNA probe that efficiently interacted with a full-length human hnRNPA2B1 protein. We exploited the interaction to conduct a pharmacological screening and identify inhibitors of the protein-RNA binding. Small molecules were orthogonally validated through biochemical and cell-based approaches. Selected drugs remarkably impacted secreted EV-RNAs and reduced an RNA-dependent, EV-mediated paracrine activation of NF-kB in recipient cells. These results demonstrate the relevance of post-transcriptional mechanisms for EV-RNA sorting and the possibility of predicting the EV-RNA quality for developing innovative strategies targeting discrete paracrine functions.
    DOI:  https://doi.org/10.1093/nar/gkaf176
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