bims-proteo Biomed News
on Proteostasis
Issue of 2025–08–03
57 papers selected by
Eric Chevet, INSERM
  1. Membrane Contact Sites in Proteostasis and ER Stress Response.
  2. ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains.
  3. Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery.
  4. Trigger factor accelerates nascent chain compaction and folding.
  5. Disturbances of endoplasmic reticulum proteostasis in neurodevelopmental disorders.
  6. Proteome solubility is differentially reshaped by thermal stress and regulators of ubiquitination.
  7. Single-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes.
  8. The p97 ATPase and its adaptor UBXD8 maintain peroxisome pools by preventing pexophagy.
  9. Mitochondrial protein aggregates recruit key chaperones and are sequestered in a fission/fusion-dependent process.
  10. Diffusing protein binders to intrinsically disordered proteins.
  11. Sequence-based virtual screening using transformers.
  12. Translational control by general control nonderepressible 2 kinase regulates methylglyoxal-induced pain in mice.
  13. Identification of an on-pathway intermediate illuminates the kinetic competition between protein folding and misfolding.
  14. 40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion.
  15. The E3 ubiquitin ligase adaptor KLHL8 targets ZAR1 to regulate maternal mRNA degradation in oocytes.
  16. Stress granules promote quiescence by enhancing p21 levels and reducing phospho-Rb.
  17. Mapping of endosomal proximity proteomes reveals Retromer as a hub for RAB GTPase regulation.
  18. AI-directed voxel extraction and volume EM identify intrusions as sites of mitochondrial contact.
  19. DCAF16-Based Covalent Degradative Handles for the Modular Design of Degraders.
  20. PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex.
  21. Bond-centric modular design of protein assemblies.
  22. Structure of CFTR bound to (R)-BPO-27 unveils a pore-blockage mechanism.
  23. Rapid depletion and super-resolution microscopy reveal dual roles of SRSF5 in coordinating nuclear speckle-paraspeckle crosstalk during cellular stress.
  24. eIF2A regulates cell migration in a translation-independent manner.
  25. Neuronal processes contain the essential components for the late steps of ribosome biogenesis.
  26. cTAGE5/MEA6 Regulates LBR Localization to Maintain Nuclear Envelope Integrity and Safeguard Against Aging.
  27. USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that can temporarily enrich for monoubiquitinated PCNA.
  28. Elevated ubiquitin phosphorylation by PINK1 contributes to proteasomal impairment and promotes neurodegeneration.
  29. Pathway regulation mechanism by cotranslational protein folding.
  30. DNAJC13 localization to endosomes is opposed by its J domain and its disordered C-terminus.
  31. The unfolded protein response influences therapy outcome and disease progression in chronic lymphocytic leukaemia.
  32. eIF1 and eIF5 dynamically control translation start site fidelity.
  33. Endoplasmic reticulum-mediated organelle crosstalk in kidney disease.
  34. Directed evolution of LaccID for cell surface proximity labeling and electron microscopy.
  35. DNA damage response pathway regulates Nrf2 in response to oxidative stress.
  36. Altered translation elongation contributes to key hallmarks of aging in the killifish brain.
  37. PEX39 facilitates the peroxisomal import of PTS2-containing proteins.
  38. Time-dependent catalytic activity in aging condensates.
  39. Reductive stress induces unresolved ER stress and proteotoxic cardiomyopathy.
  40. Germin-like protein 1 interacts with proteasome regulator 1 to regulate auxin signaling by controlling Aux/IAA homeostasis.
  41. Translation efficiency covariation identifies conserved coordination patterns across cell types.
  42. Ubiquitin-Specific Protease 7-Mediated Stabilization of Discoidin Domain Receptor 1 Drives Progression of TP53-Mutant Cancers.
  43. Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation.
  44. Advances in KEAP1-based PROTACs as emerging therapeutic modalities: Structural basis and progress.
  45. Defining STING-sterol interactions with chemoproteomics.
  46. Different roles of ACSL3 and ACSL4 in autophagosome formation.
  47. A dynamic structural framework for the allosteric regulation of Hsp70 chaperones.
  48. Ufmylation Suppresses Unfolded Protein Response to Prevent Peripartum Cardiomyopathy.
  49. A resource of RNA-binding protein motifs across eukaryotes reveals evolutionary dynamics and gene-regulatory function.
  50. Predicting the translation efficiency of messenger RNA in mammalian cells.
  51. NGLY1 deficiency - clinical features and therapeutic strategy.
  52. Mitochondrial glutathione import enables breast cancer metastasis via integrated stress response signaling.
  53. Surface delivery quantification reveals distinct trafficking efficiencies among clustered protocadherin isoforms.
  54. Integrating deep learning for post-translational modifications crosstalk on Hsp90 and drug binding.
  55. Dysfunctional autophagy triggers STING1 activation to exacerbate cartilage degeneration in obesity-associated osteoarthritis.
  56. March5-mediated Trim28 degradation preserves islet β-cell function in mice.
  57. An ethanol-induced loss of the lipid droplet-associated segregase VCP/p97 leads to hepatic steatosis.
  1. Contact (Thousand Oaks). 2025 Jan-Dec;8:8 25152564251363050
    Membrane Contact Sites in Proteostasis and ER Stress Response.
    Febe Vermue, Aysegul Sapmaz, Ilana Berlin.
      Execution of all cellular functions depends on a healthy proteome, whose maintenance requires multimodal oversight. Roughly a third of human proteins reside in membranes and thus present unique topological challenges with respect to biogenesis and degradation. To meet these challenges, eukaryotes have evolved organellar pathways of protein folding and quality control. Most transmembrane proteins originate in the endoplasmic reticulum (ER), where they are subject to surveillance and, if necessary, removal through either ER-associated proteasomal degradation (cytosolic pathway) or selective autophagy (ER-phagy; organellar pathway). In the latter case, ER cargoes are shuttled to (endo)lysosomes - the same organelles that degrade cell surface molecules via endocytosis. Here, we provide an overview of dynamic coordination between the ER and endolysosomes, with a focus on their engagement in specialized physical interfaces termed membrane contact sites (MCSs). We cover how cross-compartmental integration through MCSs allows biosynthetic and proteolytic organelles to fine-tune each other's membrane composition, organization, and dynamics and facilitates recovery from proteotoxic stress. Along the way, we highlight recent developments and open questions at the crossroads between organelle biology and protein quality control and cast them against the backdrop of factor-specific diseases associated with perturbed membrane homeostasis.
    Keywords:  endolysosome; endoplasmic reticulum; membrane contact sites; proteostasis; proteotoxic stress
    DOI:  https://doi.org/10.1177/25152564251363050
  2. EMBO J. 2025 Jul 29.
    ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains.
    Maike Reinders, Bojana Kravic, Pinki Gahlot, Sandra Koska, Johannes van den Boom, Nina Schulze, Sophie Levantovsky, Stefan Kleine, Markus Kaiser, Yogesh Kulathu, Christian Behrends, Hemmo Meyer.
      The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is unclear. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 is required for lysophagic flux after lysosomal damage but is not involved in the initial phagophore formation on terminally damaged lysosomes. Instead, ATXN3 is recruited to a distinct subset of lysosomes that are decorated with phosphatidylinositol-(4,5)-bisphosphate and that are not yet fully reacidified. There, ATXN3, along with its partner VCP/p97, targets and turns over K48-K63-branched ubiquitin conjugates. ATXN3 thus facilitates degradation of a fraction of LAMP2 via microautophagy to regenerate the lysosomal membrane and to thereby reestablish degradative capacity needed also for completion of lysophagy. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.
    Keywords:  Autophagy; Lysosome; Membrane; Stress Response; Ubiquitin
    DOI:  https://doi.org/10.1038/s44318-025-00517-x
  3. Nat Cell Biol. 2025 Jul 25.
    Reconstitution of BNIP3/NIX-mitophagy initiation reveals hierarchical flexibility of the autophagy machinery.
    Elias Adriaenssens, Stefan Schaar, Annan S I Cook, Jan F M Stuke, Justyna Sawa-Makarska, Thanh Ngoc Nguyen, Xuefeng Ren, Martina Schuschnig, Julia Romanov, Grace Khuu, Louise Uoselis, Michael Lazarou, Gerhard Hummer, James H Hurley, Sascha Martens.
      Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. Although the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here we demonstrate that the human transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, whereas FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with important implications for therapeutic interventions.
    DOI:  https://doi.org/10.1038/s41556-025-01712-y
  4. Proc Natl Acad Sci U S A. 2025 Jul 29. 122(30): e2422678122
    Trigger factor accelerates nascent chain compaction and folding.
    Katharina Till, Anne-Bart Seinen, Florian Wruck, Vanda Sunderlikova, Carla V Galmozzi, Alexandros Katranidis, Bernd Bukau, Günter Kramer, Sander J Tans.
      Conformational control of nascent chains is poorly understood. Chaperones are known to stabilize, unfold, and disaggregate polypeptides away from the ribosome. In comparison, much less is known about the elementary conformational control mechanisms at the ribosome. Yet, proteins encounter major folding and aggregation challenges during translation. Here, using selective ribosome profiling and optical tweezers with correlated single-molecule fluorescence, with dihydrofolate reductase (DHFR) as a model system, we show that the Escherichia coli chaperone trigger factor (TF) accelerates nascent chain folding. TF scans nascent chains by transient binding events, and then locks into a stable binding mode as the chain collapses and folds. This interplay is reciprocal: TF binding collapses nascent chains and stabilizes partial folds, while nascent chain compaction prolongs TF binding. Ongoing translation controls these cooperative effects, with TF-accelerated folding depending on the emergence of a peptide segment that is central to the core DHFR beta-sheet. The folding acceleration we report here impacts processes that depend on folding occurring cotranslationally, including cotranslational protein assembly, protein aggregation, and translational pausing, and may be relevant to other domains of life.
    Keywords:  chaperones; optical tweezers; protein folding; ribosomes
    DOI:  https://doi.org/10.1073/pnas.2422678122
  5. Biochem Soc Trans. 2025 Jul 28. pii: BST20253035. [Epub ahead of print]
    Disturbances of endoplasmic reticulum proteostasis in neurodevelopmental disorders.
    Danilo B Medinas, Nayrob Pereira, Rodolfo Pereira, Giovanna R da Silva, Victor H S Santos.
      The endoplasmic reticulum (ER) is a vital organelle involved in the biogenesis of membrane and secreted proteins. Proteostasis (protein homeostasis) in the ER relies on finely co-ordinated mechanisms for translocation of polypeptides from the cytosol to the organelle lumen and membrane, introduction of co- and post-translational modifications, protein folding and quality control, exportation of mature proteins and disposal of unfolded or aggregated species, besides the regulation of gene expression to adjust the proteostasis network to the cellular demands for protein biogenesis. Neurodevelopmental processes involving neurogenesis, neuronal migration and differentiation, neural circuit wiring, synaptogenesis, among others, require extensive proteome diversification and remodeling, with high fluxes through the secretory pathways constantly challenging ER proteostasis. Genetic defects affecting the different nodes of the ER proteostasis network can severely disturb neurodevelopment. Here, we compile evidence illustrating how perturbations to the different steps of protein biogenesis in the ER can lead to neurological disorders and present major questions to guide research in the field.
    Keywords:  endoplasmic reticulum; genetics; nervous system; neurodevelopmental disorders; proteostasis
    DOI:  https://doi.org/10.1042/BST20253035
  6. J Biol Chem. 2025 Jul 24. pii: S0021-9258(25)02368-3. [Epub ahead of print] 110517
    Proteome solubility is differentially reshaped by thermal stress and regulators of ubiquitination.
    Liam C Hunt, Anna Stephan, Suresh Poudel, Kaiwen Yu, Kanisha Kavdia, Vishwajeeth R Pagala, Wei Wang, Yingxue Fu, Yong-Dong Wang, Xusheng Wang, Flavia A Graca, Daniel Alford, John Grime, Anthony A High, Junmin Peng, Fabio Demontis.
      The ubiquitin-proteasome system maintains proteostasis by degrading proteins that unfold and become insoluble upon stress. Some proteins have high insolubility in normal conditions because of their structure, subcellular localization, and interactions but it remains incompletely understood how the ubiquitin-proteasome system regulates them. Here, we utilized mass spectrometry to profile heat-induced solubility changes (insolubilome) and associated post-translational modifications in human cells (http://thermal-stress-insolubilome.stjude.org). We find that the solubility of several protein categories is oppositely modulated by thermal stress. Some proteins become more soluble upon heat shock, whereas others, including several ubiquitin-conjugating enzymes, become more insoluble. By analyzing the changes in protein abundance induced by RNAi for E2 ubiquitin-conjugating enzymes, we identify E2-specific biases in targeting proteins with higher-than-average insolubility. Analysis of the E3 ubiquitin ligase HUWE1, which was previously found to detect proteins with exposed hydrophobic residues, indicates that siHUWE1-downregulated proteins have higher-than-average insolubility, suggesting that HUWE1 stabilizes subsets of insoluble proteins. Altogether, this study identifies components of the ubiquitination cascade that control and remodel the solubility of the human proteome.
    Keywords:  E2 ubiquitin-conjugating enzymes; HUWE1; insolubilome; insoluble proteins; protein-quality control; proteome solubility; thermal stress
    DOI:  https://doi.org/10.1016/j.jbc.2025.110517
  7. EMBO J. 2025 Aug 01.
    Single-protein/RNA imaging reveals ZNF598 as a limiting factor in resolving collided ribosomes.
    Ana C De La Cruz, Garrett Tisdale, Emily Nakayama, Zhiyuan Huang, Niladri K Sinha, Rachel Green, Bin Wu.
      Ribosome-associated protein quality control (RQC) is a surveillance system that identifies and processes aberrant mRNAs with collided ribosomes. ZNF598 plays a key role by ubiquitinating the 40S subunit of collided ribosomes. However, how ZNF598 distinguishes stalled from transient ribosome collisions remains unclear. To address this, we developed a method to visualize the binding of a single protein to a specific mRNA while simultaneously determining its translation status. By endogenously tagging ZNF598 with HaloTag, we observed its strong interaction with RQC reporter mRNAs. We discovered that multiple ZNF598s engage with a single RQC mRNA, suggesting that ZNF598 recognizes more than just the leading collided ribosome in a queue. Overexpressing ZNF598 increased the ribosomal clearance rate, indicating that it is a rate-limiting factor for RQC. Interestingly, a subset of supposedly "normal" mRNAs may be damaged and targeted by ZNF598, underscoring the importance of RQC to maintain the proteome quality even in unstressed conditions. Under global UV-induced RNA damage, ZNF598 recruitment to the reporter RQC mRNA diminished, highlighting its role as a limiting factor in managing widespread ribosome collisions.
    Keywords:  Protein–RNA Interactions; RNA; Ribosome-associated Quality Control; Single Molecule; Translation
    DOI:  https://doi.org/10.1038/s44318-025-00523-z
  8. J Cell Biol. 2025 Sep 01. pii: e202409024. [Epub ahead of print]224(9):
    The p97 ATPase and its adaptor UBXD8 maintain peroxisome pools by preventing pexophagy.
    Iris D Montes, Suganthan Amirthagunanathan, Rakesh Ganji, Joao A Paulo, Brittany A Ahlstedt, Ly Nguyen, Amit S Joshi, Malavika Raman.
      Peroxisomes perform key metabolic functions in eukaryotic cells. Loss of peroxisome function causes peroxisome biogenesis disorders and severe childhood diseases with disrupted lipid metabolism. One mechanism regulating peroxisome abundance is degradation via selective autophagy (pexophagy). However, the mechanisms regulating pexophagy remain poorly understood in mammalian cells. Here, we find that the evolutionarily conserved AAA-ATPase p97/VCP and its adaptor UBXD8/FAF2 are essential for maintaining peroxisome abundance. From quantitative proteomics studies, we show that loss of UBXD8 affects the abundance of many peroxisomal proteins and that the depletion of UBXD8 results in a loss of peroxisomes. Loss of p97-UBXD8 and inhibition of p97 catalytic activity increase peroxisomal turnover through autophagy and can be rescued by depleting key autophagy proteins and E3 ligases or overexpressing the deubiquitylase USP30. We find increased ubiquitylation of PMP70 and PEX5 in cells lacking UBXD8 or p97. Our findings identify a new role of the p97-UBXD8 in regulating peroxisome abundance by removing ubiquitylated peroxisome membrane proteins to prevent pexophagy.
    DOI:  https://doi.org/10.1083/jcb.202409024
  9. J Cell Sci. 2025 Jul 30. pii: jcs.263680. [Epub ahead of print]
    Mitochondrial protein aggregates recruit key chaperones and are sequestered in a fission/fusion-dependent process.
    Laura Ruland, Marc Sylvester, Lydia Maus, Hannes Beckert, Wolfgang Voos.
      The potential proteotoxicity of mitochondrial aggregates in yeast cells is reduced by a sequestration of affected polypeptides into a mitochondrial protein quality control compartment (IMiQ). Based on the expression of an aggregation-prone protein in the mitochondrial matrix, we determined the effect of organelle dynamics on aggregate sequestration. Fusion deficient cells were unable to accumulate the aggregates in the IMiQ, resulting in a stress-sensitive phenotype. In contrast, fission deficient cells could not separate the aggregate from the mitochondrial network. In these mitochondria, the aggregates were neutralized by the formation of a shell formed by mitochondrial chaperones. We also performed quantitative mass spectrometry to analyse the mitochondrial proteome and the extent of co-aggregation of mitochondrial proteins. While only minor changes of the total proteome were detected in response to aggregate accumulation, we found a recruitment of proteins of the respiratory chain complexes and of the protein quality control system (PQC). In particular members of the Hsp70 chaperone family were prominently associated with the aggregate. We conclude that this chaperone-dependent neutralization prevents a major co-aggregation of endogenous mitochondrial proteins.
    Keywords:  Cell biology; Chaperone; Hsp70; Mitochondria; Protein aggregation; Proteostasis; Yeast
    DOI:  https://doi.org/10.1242/jcs.263680
  10. Nature. 2025 Jul 30.
    Diffusing protein binders to intrinsically disordered proteins.
    Caixuan Liu, Kejia Wu, Hojun Choi, Hannah L Han, Xueli Zhang, Joseph L Watson, Green Ahn, Jason Z Zhang, Sara Shijo, Lydia L Good, Charlotte M Fischer, Asim K Bera, Alex Kang, Evans Brackenbrough, Brian Coventry, Derrick R Hick, Seema Qamar, Xinting Li, Justin Decarreau, Stacey R Gerben, Wei Yang, Inna Goreshnik, Dionne Vafeados, Xinru Wang, Mila Lamb, Analisa Murray, Sebastian Kenny, Magnus S Bauer, Andrew N Hoofnagle, Ping Zhu, Tuomas P J Knowles, David Baker.
      Proteins that bind to intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could be useful for therapeutic and diagnostic applications1-4. However, a general methodology for targeting IDPs or IDRs has yet to be developed. Here we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformations, RFdiffusion5 can generate binders to IDPs and IDRs in a wide range of conformations. We used this approach to generate binders to the IDPs amylin, C-peptide, VP48 and BRCA1_ARATH in diverse conformations with a dissociation constant (Kd) ranging from 3 to 100 nM. For the IDRs G3BP1, common cytokine receptor γ-chain (IL-2RG) and prion protein, we diffused binders to β-strand conformations of the targets, obtaining Kd between 10 and 100 nM. Fluorescence imaging experiments show that the binders bind to their respective targets in cells. The G3BP1 binder disrupts stress granule formation in cells, and the amylin binder inhibits amyloid fibril formation and dissociates existing fibres, enables targeting of both monomeric and fibrillar amylin to lysosomes, and increases the sensitivity of mass spectrometry-based amylin detection. Our approach should be useful for creating binders to flexible IDPs or IDRs spanning a wide range of intrinsic conformational preferences.
    DOI:  https://doi.org/10.1038/s41586-025-09248-9
  11. Nat Commun. 2025 Jul 28. 16(1): 6925
    Sequence-based virtual screening using transformers.
    Shengyu Zhang, Donghui Huo, Robert I Horne, Yumeng Qi, Sebastian Pujalte Ojeda, Aixia Yan, Michele Vendruscolo.
      Protein-ligand interactions play central roles in myriad biological processes and are of key importance in drug design. Deep learning approaches are becoming cost-effective alternatives to high-throughput experimental methods for ligand identification. Here, to predict the binding affinity between proteins and small molecules, we introduce Ligand-Transformer, a deep learning method based on the transformer architecture. Ligand-Transformer implements a sequence-based approach, where the inputs are the amino acid sequence of the target protein and the topology of the small molecule to enable the prediction of the conformational space explored by the complex between the two. We apply Ligand-Transformer to screen and validate experimentally inhibitors targeting the mutant EGFRLTC kinase, identifying compounds with low nanomolar potency. We then use this approach to predict the conformational population shifts induced by known ABL kinase inhibitors, showing that sequence-based predictions enable the characterisation of the population shift upon binding. Overall, our results illustrate the potential of Ligand-Transformer to accurately predict the interactions of small molecules with proteins, including the binding affinity and the changes in the free energy landscapes upon binding, thus uncovering molecular mechanisms and facilitating the initial steps in drug design.
    DOI:  https://doi.org/10.1038/s41467-025-61833-8
  12. Pain. 2025 Jul 30.
    Translational control by general control nonderepressible 2 kinase regulates methylglyoxal-induced pain in mice.
    Angela R Meyer, Guadalupe García, Alexander R Mikesell, Sydney O'Flanagan, Cheryl L Stucky, Zachary T Campbell.
       ABSTRACT: Neuropathic pain is pervasive among people with diabetes. The integrated stress response (ISR) is a key mechanism of translational regulation implicated in diabetic pain. In this study, we demonstrate that a reactive glycolytic metabolite, methylglyoxal (MGO), which is strongly associated with painful diabetic neuropathy, activates the ISR through the kinase general control nonderepressible 2 (GCN2). Methylglyoxal disrupts elongating ribosomes, triggering the recruitment of ribosome quality control factors and collision sensors. GCN2 activation by MGO requires the ribosomal P-stalk, a critical sensor for elongation factors. Moreover, neuronal sensitization and mechanical allodynia produced by MGO are GCN2-dependent. Overall, this study links ribosomal elongation dysfunction to metabolic pain and identifies GCN2 as a novel analgesic target for diabetic neuropathy.
    Keywords:  DPN; GCN2; ISR; MGO; eIF2
    DOI:  https://doi.org/10.1097/j.pain.0000000000003761
  13. Proc Natl Acad Sci U S A. 2025 Aug 05. 122(31): e2425999122
    Identification of an on-pathway intermediate illuminates the kinetic competition between protein folding and misfolding.
    Qing Luan, Patricia L Clark.
      Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. However, as protein length increases so does the competition between off-pathway misfolding and on-pathway folding, creating a more complex energy landscape ("folding funnel"). Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa β-helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). Existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump "denaturant challenge" assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a C-to-N-terminal model for P.69T folding, with folding initiated in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.
    Keywords:  autotransporter; energy landscape; folding funnel; parallel β-helix
    DOI:  https://doi.org/10.1073/pnas.2425999122
  14. RNA. 2025 Jul 31. pii: rna.080493.125. [Epub ahead of print]
    40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion.
    Hironao Wakabayashi, Mingyi Zhu, Elizabeth J Grayhack, David H Mathews, Dmitri N Ermolenko.
      During eukaryotic translation initiation, the small (40S) ribosomal subunit is recruited to the 5' cap and subsequently scans the 5' untranslated region (5' UTR) of mRNA in search of the start codon. The molecular mechanism of mRNA scanning remains unclear, particularly the requirement for and identity of a translocase. Here, using GFP reporters in Saccharomyces cerevisiae, we show that order-of-magnitude variations in the length of unstructured 5' UTRs have only modest effects on protein synthesis, while structured 5' UTRs strongly inhibit translation. Thus, when not hindered by secondary structure, mRNA scanning is not rate limiting. Loss-of-function mutations in eIF4A, Ded1 and Slh1 reveal that these translational helicases are dispensable for mRNA scanning. Our data suggest that one-dimensional diffusion predominately enables 40S movement along the 5' UTR during mRNA scanning.
    Keywords:  mRNA scanning; one-dimensional diffusion; ribosome; translation initiation
    DOI:  https://doi.org/10.1261/rna.080493.125
  15. EMBO Rep. 2025 Jul 28.
    The E3 ubiquitin ligase adaptor KLHL8 targets ZAR1 to regulate maternal mRNA degradation in oocytes.
    Huizhen Fan, Ruyi Liu, Ran Yu, Biaobang Chen, Qiaoli Li, Jian Mu, Weijie Wang, Tianyu Wu, Lin He, Lei Wang, Qing Sang, Zhihua Zhang.
      Maternal protein homeostasis and timely degradation of maternal mRNAs are essential for meiotic cell-cycle progression and subsequent embryonic development, but the mechanisms of maternal protein degradation are poorly understood. Here, we show that KLHL8, a substrate adaptor of Cullin-RING E3 ubiquitin ligases, is highly expressed in mouse oocytes and co-localizes with mitochondria. Oocyte-specific deletion of Klhl8 causes oocyte maturation defects and female infertility. ZAR1, an RNA binding protein that is required for mitochondria-associated ribonucleoprotein domain (MARDO) dissolution, is specifically recognized and degraded by KLHL8-mediated ubiquitination. In Klhl8-deficient oocytes, ZAR1 accumulation causes abnormal MARDO and mitochondria clustering, correlating with impaired maternal mRNA decay. Supplementation with exogenous Klhl8 mRNA rescues the degradation of ZAR1 and the dissolution of the MARDO in Klhl8oo-/- oocytes. Taken together, our study shows that KLHL8 mediates the ubiquitination and degradation of ZAR1, thus regulating maternal mRNA clearance during oocyte maturation. These findings provide new insights into the roles of the ubiquitin proteasome system during oocyte maturation and establish an interaction network between ubiquitination modification, RNA binding proteins, and maternal mRNA.
    Keywords:  KLHL8; Maternal mRNA Decay; Oocyte Maturation; Ubiquitination; ZAR1
    DOI:  https://doi.org/10.1038/s44319-025-00537-y
  16. RNA. 2025 Jul 30. pii: rna.080635.125. [Epub ahead of print]
    Stress granules promote quiescence by enhancing p21 levels and reducing phospho-Rb.
    Anthony Khong, Nina Ripin, Luisa Macedo de Vasconcelos, Victor Passanisi, Sabrina Spencer, Roy Parker.
      During the integrated stress response (ISR), most mRNAs exit translation and some condense into stress granules. Stress granules that form during chemotherapy can promote cancer cell survival and chemoresistance by an unknown mechanism. Cells can also spontaneously trigger the ISR at low levels, which promotes cellular quiescence where cells exit the cell cycle and are resistant to therapeutic agents. We hypothesized that the ability of cells to form stress granules might be a critical signal to drive cells into quiescence. Herein, we provide several observations that suggest stress granules enhance cell survival and chemoresistance by promoting cellular quiescence. The mechanism by which stress granules promote quiescence is by stimulating p21 expression, leading to inhibition of Rb phosphorylation. These results demonstrate that stress granule formation is sufficient to trigger cellular quiescence and argue that inhibitors of stress granules may be effective in combination with chemotherapy to limit the development of chemoresistance in treating human tumors.
    Keywords:  RNA granules; cancer; quiescence; stress granules
    DOI:  https://doi.org/10.1261/rna.080635.125
  17. Nat Commun. 2025 Jul 30. 16(1): 6990
    Mapping of endosomal proximity proteomes reveals Retromer as a hub for RAB GTPase regulation.
    Carlos Antón-Plágaro, Kai-En Chen, Qian Guo, Meihan Liu, Ashley J Evans, Philip A Lewis, Kate J Heesom, Kevin A Wilkinson, Brett M Collins, Peter J Cullen.
      Endosomal retrieval and recycling of integral cargo proteins is essential for cell and organism development and homeostasis and is orchestrated through a specialised endosomal nanodomain, the retrieval sub-domain. Sub-domain dysfunction is associated with human disease, but our mechanistic understanding of its function remains poorly described. Here, using proximity proteomics of retrieval sub-domain components Retromer and Retriever we identify mechanistic details of retrieval sub-domain composition and organization, including an unrecognised complexity in the interface with RAB GTPase switching. Combining X-ray crystallography and in silico predictions with biochemical and cellular analysis, we reveal that Retromer directly associates and recruits the RAB10 regulators DENND4A, DENND4C, TBC1D1, and TBC1D4, and the RAB35 regulator TBC1D13 to regulate retrieval sub-domain function. The retrieval sub-domain therefore constitutes a hub for integrating cargo recycling with the regulated switching of selected RAB GTPases. We propose this constitutes a major component of the neuroprotective role of the retrieval sub-domain.
    DOI:  https://doi.org/10.1038/s41467-025-61802-1
  18. J Cell Biol. 2025 Oct 06. pii: e202411138. [Epub ahead of print]224(10):
    AI-directed voxel extraction and volume EM identify intrusions as sites of mitochondrial contact.
    Benjamin S Padman, Runa S J Lindblom, Michael Lazarou.
      Membrane contact sites (MCSs) establish organelle interactomes in cells to enable communication and exchange of materials. Volume EM (vEM) is ideally suited for MCS analyses, but semantic segmentation of large vEM datasets remains challenging. Recent adoption of artificial intelligence (AI) for segmentation has greatly enhanced our analysis capabilities. However, we show that organelle boundaries, which are important for defining MCS, are the least confident predictions made by AI. We outline a segmentation strategy termed AI-directed voxel extraction (AIVE), which refines segmentation results and boundary predictions derived from any AI-based method by combining those results with electron signal values. We demonstrate the precision conferred by AIVE by applying it to the quantitative analysis of organelle interactomes from multiple FIB-SEM datasets. Through AIVE, we discover a previously unknown category of mitochondrial contact that we term the mitochondrial intrusion. We hypothesize that intrusions serve as anchors that stabilize MCS and promote organelle communication.
    DOI:  https://doi.org/10.1083/jcb.202411138
  19. ACS Cent Sci. 2025 Jul 23. 11(7): 1207-1217
    DCAF16-Based Covalent Degradative Handles for the Modular Design of Degraders.
    Lauren M Orr, Sydney J Tomlinson, Hannah R Grupe, Melissa Lim, Emily Ho, Halime Yilmaz, Grace Zhou, Barbara Leon, James A Olzmann, Daniel K Nomura.
      While targeted protein degradation is a powerful strategy for eliminating disease-causing proteins, the rational design of monovalent or molecular glue degraders remains challenging. In this study, we generated a library of BET-domain inhibitor JQ1 analogs bearing elaborated electrophilic handles to identify permissive covalent degradative handles and E3 ligase pairs. We identified an elaborated fumaramide handle that, when appended onto JQ1, led to the proteasome-dependent degradation of BRD4. We revealed that the E3 ubiquitin ligase CUL4DCAF16a common E3 ligase target of electrophilic degraderswas responsible for BRD4 loss by covalently targeting C173 on DCAF16. While this original fumaramide handle was not permissive to the degradation of other neo-substrates, a truncated version of this handle attached to JQ1 was still capable of degrading BRD4, now through targeting both C173 and C178. This truncated fumaramide handle, when appended to various protein targeting ligands, was also more permissive in degrading other neo-substrates, including CDK4/6, SMARCA2/4, the androgen receptor (AR), as well as the undruggable AR truncation variant AR-V7. We have identified a unique DCAF16-targeting covalent degradative handle that can be transplanted across several protein-targeting ligands to induce the degradation of their respective targets for the modular design of monovalent or bifunctional degraders.
    DOI:  https://doi.org/10.1021/acscentsci.5c00959
  20. Sci Adv. 2025 Jul 25. 11(30): eadw4603
    PELP1 coordinates the modular assembly and enzymatic activity of the rixosome complex.
    Jacob Gordon, Andrea M Kaminski, Saisamhita R Bommu, Aleksandra Skrajna, Robert M Petrovich, Lars C Pedersen, Robert K McGinty, Alan J Warren, Robin E Stanley.
      The rixosome is a large multisubunit complex that initiates RNA decay during critical nuclear transactions including ribosome assembly and heterochromatin maintenance. The overall architecture of the complex remains undefined because several subunits contain intrinsically disordered regions (IDRs). Here, we combined structural and functional approaches to establish PELP1 as the central scaffold of the rixosome upon which the enzymatic subunits modularly assemble. The C-terminal half of PELP1 is composed of a proline-rich IDR that mediates association with the AAA-ATPase MDN1, histones, and the SUMO-specific protease SENP3. The PELP1 IDR contains a glutamic acid-rich region that we establish can chaperone the histone octamer in vitro. Last, the x-ray structure of a small linear motif (SLiM) from the PELP IDR bound to SENP3 reveals how PELP1 allosterically activates SUMO protease activity. This work provides an integrated structural model for understanding the rixosome's dynamic architecture and how it modularly coordinates several cellular functions.
    DOI:  https://doi.org/10.1126/sciadv.adw4603
  21. Nat Mater. 2025 Jul 31.
    Bond-centric modular design of protein assemblies.
    Shunzhi Wang, Andrew Favor, Ryan D Kibler, Joshua M Lubner, Andrew J Borst, Nicolas Coudray, Rachel L Redler, Huat Thart Chiang, William Sheffler, Yang Hsia, Neville P Bethel, Zhe Li, Damian C Ekiert, Gira Bhabha, Lilo D Pozzo, David Baker.
      Directional interactions that generate regular coordination geometries are a powerful means of guiding molecular and colloidal self-assembly, but implementing such high-level interactions with proteins remains challenging due to their complex shapes and intricate interface properties. Here we describe a modular approach to protein nanomaterial design inspired by the rich chemical diversity that can be generated from the small number of atomic valencies. We design protein building blocks using deep learning-based generative tools, incorporating regular coordination geometries and tailorable bonding interactions that enable the assembly of diverse closed and open architectures guided by simple geometric principles. Experimental characterization confirms the successful formation of more than 20 multicomponent polyhedral protein cages, two-dimensional arrays and three-dimensional protein lattices, with a high (10%-50%) success rate and electron microscopy data closely matching the corresponding design models. Due to modularity, individual building blocks can assemble with different partners to generate distinct regular assemblies, resulting in an economy of parts and enabling the construction of reconfigurable networks for designer nanomaterials.
    DOI:  https://doi.org/10.1038/s41563-025-02297-5
  22. Nat Commun. 2025 Aug 01. 16(1): 7059
    Structure of CFTR bound to (R)-BPO-27 unveils a pore-blockage mechanism.
    Paul G Young, Karol Fiedorczuk, Jue Chen.
      Hyperactivation of the cystic fibrosis transmembrane conductance regulator (CFTR) contributes to secretory diarrhea, a major cause of pediatric mortality worldwide, and autosomal dominant polycystic kidney disease (ADPKD), the most common inherited cause of end-stage renal disease. Selective CFTR inhibition is a potential therapeutic strategy, with (R)-BPO-27 emerging as a promising candidate. Here, we present a cryo-EM structure of CFTR bound to (R)-BPO-27 at an overall resolution of 2.1 Å. Contrary to the previous hypothesis that it inhibits CFTR current by competition with ATP, we demonstrate that (R)-BPO-27 instead directly occludes the chloride-conducting pore while permitting ATP hydrolysis, thus uncoupling the two activities. Furthermore, we find that inhibitor binding requires some degree of NBD separation, as the inhibition rate inversely correlates with the probability NBD dimerization. These findings clarify the compound's mechanism and provide a molecular basis for optimizing its clinical potential.
    DOI:  https://doi.org/10.1038/s41467-025-62199-7
  23. Nucleic Acids Res. 2025 Jul 19. pii: gkaf713. [Epub ahead of print]53(14):
    Rapid depletion and super-resolution microscopy reveal dual roles of SRSF5 in coordinating nuclear speckle-paraspeckle crosstalk during cellular stress.
    Ellen Kazumi Okuda, Laurell Fridolin Kessler, Benjamin Arnold, Ricarda J Riegger, Maria Clara Hernández Cañás, Ewelina Zebrowska, Cem Bakisoglu, Mara Rudigier, Christine Krost, Helder Y Nagasse, Jan Keiten-Schmitz, Stefan Müller, David Stanek, Dorothee Dormann, Kathi Zarnack, Mike Heilemann, Michaela Müller-McNicoll.
      Nuclear speckles (NS) and paraspeckles (PS) are adjacent yet distinct nuclear condensates that undergo stress-induced reorganization. Here, we identify a dual role for the splicing factor SRSF5 in coordinating the crosstalk between both condensates. Super-resolution imaging shows that SRSF5, while enriched in NS, also overlaps with the shell of a subset of PS. SRSF5 binds purine-rich sequences at the 5' end of NEAT1_2 promoting its alignment to PS shells and the formation of large PS cluster during stress. We propose that SRSF5 binding occurs transiently during PS maturation and must later be removed from NEAT1_2 by nuclear helicases. Inhibition of this remodeling by rocaglamide A, which locks helicases onto purine-rich RNA leads to the aberrant fusion of PS and NS, which can be partially rescued by acute SRSF5 depletion. Surprisingly, while short-term SRSF5 loss impairs PS formation, prolonged depletion activates a feedback loop involving intron retention and premature polyadenylation of TARDBP, reduction of TDP-43 levels and NEAT1_2 isoform switching, ultimately restoring PS clusters. Our findings reveal that SRSF5 serves both architectural and regulatory roles in PS biogenesis and that helicase-mediated remodeling is essential to maintain PS identity and function under stress. These insights uncover fundamental principles of nuclear body dynamics.
    DOI:  https://doi.org/10.1093/nar/gkaf713
  24. Sci Adv. 2025 Aug;11(31): eadu5668
    eIF2A regulates cell migration in a translation-independent manner.
    Jennifer Jungfleisch, Neus Mestre-Farràs, Raúl Gómez-Riera, Oriane Pourcelot, Edouard Bertrand, Nadia Halidi, Fátima Gebauer.
      The RNA-binding protein eukaryotic translation initiation factor 2A (eIF2A) is an alternative translation initiation factor shown to drive tumor formation by facilitating translation from near-cognate initiation codons. Here, we uncover a function for eIF2A in regulating cell migration in a manner independent of overt control of translation. Using a melanoma cell model consisting of nontumoral melanocytic Mel-ST cells and their metastatic counterpart obtained by H-Ras transformation, we unexpectedly find minimal effects of eIF2A depletion on translation. Interactome studies identified centrosomal proteins as major binding partners of eIF2A. We found that eIF2A colocalizes with the centrosome, enhances centrosome composition, and promotes centrosome orientation during cell migration. Migration requires the C-terminal disordered region of eIF2A, involved in mRNA binding. Interaction with mRNA, however, does not require ongoing translation. These findings reveal a role for eIF2A in centrosome dynamics beyond its traditional function in translation.
    DOI:  https://doi.org/10.1126/sciadv.adu5668
  25. Proc Natl Acad Sci U S A. 2025 Aug 05. 122(31): e2502424122
    Neuronal processes contain the essential components for the late steps of ribosome biogenesis.
    Claudia M Fusco, Anja Staab, Ashley M Bourke, Georgi Tushev, Kristina Desch, Erico Moreto Lins, Elena Ciirdaeva, Susanne Tom Dieck, Nina Kaltenschnee, Alexander Heckel, Julian D Langer, Erin M Schuman.
      Neurons rely on spatial and temporal control of protein synthesis to respond rapidly and locally to external stimuli, a process facilitated by the dynamic localization and modification of ribosomes. While previous research has shown that neuronal activity can regulate ribosome localization and modify translation rates, little is known about ribosomal assembly within neuronal processes. Here, we investigated the potential for local ribosome maturation in rat neurons using proteomics, RNA sequencing, and imaging methods. We detected an abundance of ribosome biogenesis factors in distal neuronal compartments, particularly those associated with the late stages of ribosome assembly. Moreover, we detected cytosolic pre-ribosomal RNA species in dendrites, as well as the enzymes necessary for their processing, suggesting that local ribosome maturation can occur far from the nucleus. These findings challenge conventional models that confine ribosome biogenesis to nuclear and perinuclear regions and suggest that neurons may fine-tune local protein synthesis by regulating ribosome assembly near synaptic sites. This mechanism may enable rapid modulation of the translational capacity in response to physiological changes, regulating synaptic plasticity and local protein synthesis in neurons.
    Keywords:  local translation; protein synthesis; ribosome; synapses
    DOI:  https://doi.org/10.1073/pnas.2502424122
  26. Aging Cell. 2025 Jul 30. e70185
    cTAGE5/MEA6 Regulates LBR Localization to Maintain Nuclear Envelope Integrity and Safeguard Against Aging.
    Yaqing Wang, Pengyu Sun, Fuqiang Yang, Tiantian Ma, Junwan Fan, Lei Shi, Nan Li, Fei Gao, Kangmin He, Zhiheng Xu.
      cTAGE5/MEA6 plays a pivotal role in COPII complex assembly, ER-to-Golgi trafficking, and secretion. However, whether cTAGE5/MEA6 is involved in other cellular functions remains unclear. Here, we show that conditional cTAGE5 knockout results in embryonic lethality during development and premature aging in adult mice. cTAGE5 deficiency leads to abnormal nuclear structure and disturbed cell proliferation in MEF cells. Further mechanistic studies reveal that cTAGE5 localizes not only to the ER exit sites but also to other ER structures, where it interacts with the lamin B receptor (LBR). Loss of cTAGE5 disrupts LBR's localization to the inner nuclear membrane, leading to its retention in the ER and instability. This results in abnormal nuclear (envelope) morphology and cellular senescence, likely driven by activation of the P53/P21 senescence pathway. Thus, our study uncovers cTAGE5's role in maintaining nuclear envelope integrity and highlights its function and potential mechanism in preventing cellular senescence and animal aging.
    Keywords:  LBR; aging; cTAGE5/MEA6; nuclear envelope; senescence
    DOI:  https://doi.org/10.1111/acel.70185
  27. Nat Commun. 2025 Jul 30. 16(1): 6991
    USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that can temporarily enrich for monoubiquitinated PCNA.
    Niels Keijzer, Jan Sakoltchik, Kaustav Majumder, Nina van Lil, Farid El Oualid, Alexander Fish, Titia K Sixma.
      DNA damage tolerance (DDT) is an important pathway that allows cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA, where monoubiquitinated PCNA (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as a substrate for further ubiquitination, forming K63-polyubiquitinated PCNA that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that formation of K48-linked chains also occurs on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about removal of ubiquitin chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis suggests that this mechanistic preference is maintained in evolution. This unusual mechanism can cause temporal enrichment of monoubiquitinated PCNA during polyubiquitination. It will be interesting to see how this affects DDT pathway balance.
    DOI:  https://doi.org/10.1038/s41467-025-61768-0
  28. Elife. 2025 Jul 31. pii: RP103945. [Epub ahead of print]14
    Elevated ubiquitin phosphorylation by PINK1 contributes to proteasomal impairment and promotes neurodegeneration.
    Cong Chen, Tong-Yao Gao, Hua-Wei Yi, Yi Zhang, Tong Wang, Zhi-Ling Lou, Tao-Feng Wei, Yun-Bi Lu, Tingting Li, Chun Tang, Wei-Ping Zhang.
      Ubiquitin (Ub), a central regulator of protein turnover, can be phosphorylated by PINK1 (PTEN-induced putative kinase 1) to generate S65-phosphorylated ubiquitin (pUb). Elevated pUb levels have been observed in aged human brains and in Parkinson's disease, but the mechanistic link between pUb elevation and neurodegeneration remains unclear. Here, we demonstrate that pUb elevation is a common feature under neurodegenerative conditions, including Alzheimer's disease, aging, and ischemic injury. We show that impaired proteasomal activity leads to the accumulation of sPINK1, the cytosolic form of PINK1 that is normally proteasome-degraded rapidly. This accumulation increases ubiquitin phosphorylation, which then inhibits ubiquitin-dependent proteasomal activity by interfering with both ubiquitin chain elongation and proteasome-substrate interactions. Specific expression of sPINK1 in mouse hippocampal neurons induced progressive pUb accumulation, accompanied by protein aggregation, proteostasis disruption, neuronal injury, neuroinflammation, and cognitive decline. Conversely, Pink1 knockout mitigated protein aggregation in both mouse brains and HEK293 cells. Furthermore, the detrimental effects of sPINK1 could be counteracted by co-expressing Ub/S65A phospho-null mutant but exacerbated by over-expressing Ub/S65E phospho-mimic mutant. Together, these findings reveal that pUb elevation, triggered by reduced proteasomal activity, inhibits proteasomal activity and forms a feedforward loop that drives progressive neurodegeneration.
    Keywords:  PINK1; biochemistry; chemical biology; mouse; neurodegeneration; phosphorylation; proteasome; ubiquitin
    DOI:  https://doi.org/10.7554/eLife.103945
  29. Commun Chem. 2025 Aug 01. 8(1): 226
    Pathway regulation mechanism by cotranslational protein folding.
    Peng Tao, Yi Xiao.
      Existing experimental results indicate potential disparities between cotranslational protein folding in vivo and free folding in vitro, yet the microscopic mechanisms responsible for these differences remain elusive. In this study, we devised a general protein cotranslational folding (GPCTF) simulations framework by modeling the ribosomal exit tunnel and translation process. Utilizing the GPCTF framework, we conducted extensive molecular dynamics simulations on three proteins of varying topologies, generating over 8 milliseconds of total trajectories. When compared to free folding, cotranslational folding enables the nascent peptide to adopt a more helix-rich structure with less nonnative interactions upon expulsion from the ribosomal exit tunnel. Notably, subsequent folding of this structure adheres to the same pathway as free folding, but with different ratios of folding pathways, modulated by the translation speed. This investigation illuminates the pathway regulation mechanism inherent to cotranslational folding and successfully reconciles discrepancies in pre-existing experimental results, offering significant insights into the protein folding process in vivo.
    DOI:  https://doi.org/10.1038/s42004-025-01636-6
  30. Mol Biol Cell. 2025 Jul 30. mbcE24120575
    DNAJC13 localization to endosomes is opposed by its J domain and its disordered C-terminus.
    Hayden Adoff, Brandon Novy, Emily Holland, Braden T Lobingier.
      Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling and degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Pleckstrin Homology (PH)-like domain, which binds endosomal phosphatidylinositol-3-phosphate (PI(3)P). However, little is known about how DNAJC13 localization is regulated. Here, we show that two regions within DNAJC13, its J domain and disordered C-terminus, act as negative regulators of its PH-like domain. Using a structure-function approach, we map these control points to a conserved YLT motif in the disordered C-terminus as well as the catalytic HPD triad in its J domain. Mutation of either motif enhances DNAJC13 endosomal localization in cells and increases binding to PI(3)P in vitro, and overexpression of these mutants causes multiple defects in endosome function including endosomal clustering and loss of recycling of a membrane protein cargo. Mechanistically, the enhanced endosomal localization of DNAJC13 requires its N-terminal PH-like domain, and we show that the PH-like domain of DNAJC13 binds PI(3)P weakly in isolation and requires oligomerization for efficient PI(3)P binding and endosomal localization. Together, these results demonstrate that interaction between DNAJC13 and PI(3)P serves as a molecular control point for regulating DNAJC13 localization to endosomes.
    DOI:  https://doi.org/10.1091/mbc.E24-12-0575
  31. Sci Rep. 2025 Jul 28. 15(1): 27496
    The unfolded protein response influences therapy outcome and disease progression in chronic lymphocytic leukaemia.
    Umair Tahir Khan, Kim Clarke, Gina Eagle, Melanie Oates, Peter Hillmen, Sandrine Jayne, Martin J S Dyer, Alex Phipps, Francesco Falciani, Rosalind E Jenkins, Andrew R Pettitt.
      Since genomics, epigenomics and transcriptomics have provided only a partial explanation of chronic lymphocytic leukaemia (CLL) heterogeneity, and since concordance between mRNA and protein expression is incomplete, we related the CLL proteome to clinical outcome. CLL samples from patients who received fludarabine-containing chemoimmunotherapy were analysed by mass spectrometry (SWATH-MS). One dataset compared pre-treatment samples associated with an optimal versus suboptimal response, while another compared paired samples collected before treatment and at disease progression. eIF2 signalling (pivotal to the unfolded protein response (UPR)), was identified as the most enriched pathway in both datasets (respective z-scores: - 6.245 and 3.317; p < 0.0001), as well as in a fludarabine-resistant CLL cell line established from HG3 cells (z-score: - 2.121; p < 0.0001). Western blotting revealed that fludarabine-resistant HG3 cells expressed higher levels of PERK, which phosphorylates the regulatory eIF2α subunit, and lower levels of BiP, an HSP70 molecular chaperone that inactivates PERK but preferentially binds to misfolded proteins during ER stress. The PERK inhibitor, GSK2606414, sensitised resistant, but not sensitive, HG-3 cells to fludarabine without affecting background cell viability or cytotoxicity induced by the BCL-2 inhibitor venetoclax. These findings identify the UPR as a novel determinant of therapy outcome and disease progression in CLL.
    Keywords:  CLL cells; Drug resistance and disease progression; PERK linked to resistant phenotype; Proteomics; eIF2 signalling
    DOI:  https://doi.org/10.1038/s41598-025-13495-1
  32. Nat Struct Mol Biol. 2025 Jul 28.
    eIF1 and eIF5 dynamically control translation start site fidelity.
    Rosslyn Grosely, Carlos Alvarado, Ivaylo P Ivanov, Oliver B Nicholson, Joseph D Puglisi, Thomas E Dever, Christopher P Lapointe.
      Human translation initiation requires accurate recognition of translation start sites. While AUG codons are canonical start sites, non-AUG codons are also used, typically with lower efficiency. The initiator tRNA and initiation factors eIF1 and eIF5 control recognition. How they distinguish different start sites yet allow flexible recognition remains unclear. Here we used real-time single-molecule assays and an in vitro reconstituted human system to reveal how eIF1 and eIF5 direct start site selection. eIF1 binds initiation complexes in two modes: stable binding during scanning, followed by transient, concentration-dependent rebinding after start site recognition. Termination of eIF1 rebinding requires transient and concentration-dependent binding by eIF5, which allows the formation of translation competent ribosomes. Non-AUG start sites differentially stabilize eIF1 and destabilize eIF5 binding, blocking initiation at multiple points. We confirmed these opposing effects in human cells. Collectively, our findings uncover that eIF1 and eIF5 directly compete to bind initiation complexes and illuminate how their dynamic interplay tunes the fidelity of start site recognition, which has broad connections to health and disease.
    DOI:  https://doi.org/10.1038/s41594-025-01629-y
  33. Nat Rev Nephrol. 2025 Jul 31.
    Endoplasmic reticulum-mediated organelle crosstalk in kidney disease.
    Yu Ah Hong, Reiko Inagi.
      The endoplasmic reticulum (ER) is a key organelle involved in a wide range of intracellular biological processes, including Ca2+ homeostasis; lipid metabolism; proteostasis through protein synthesis, folding and processing of secretory and transmembrane proteins; and signal transduction. The ER forms extensive physical interactions with various intracellular organelles through the membrane contact sites, enabling direct exchange of ions and lipids without vesicular transport. At mitochondria-associated membranes, ER-mitochondria communication governs calcium transfer, lipid synthesis, mitochondrial dynamics, the unfolded protein response and inflammation, all of which are essential for maintaining cellular homeostasis. The ER also interacts with the Golgi apparatus, endosomes and plasma membrane to facilitate transfer of calcium and lipids. Disruption of ER-organelle communication contributes to the development and progression of various kidney diseases, including diabetic kidney disease, acute kidney injury and polycystic kidney disease. Accordingly, ER-organelle communication has emerged as a promising therapeutic target. Pharmacological agents such as SGLT2 inhibitors, AMPK activators, mTOR inhibitors and RAAS blockers have been shown to restore ER-mitochondria communication and alleviate kidney injury in experimental models. Advancing our understanding of ER-organelle crosstalk may offer new mechanistic insights and contribute to the optimization of current treatment strategies for kidney disease.
    DOI:  https://doi.org/10.1038/s41581-025-00989-4
  34. Nat Chem Biol. 2025 Aug 01.
    Directed evolution of LaccID for cell surface proximity labeling and electron microscopy.
    Song-Yi Lee, Heegwang Roh, David Gonzalez-Perez, Mason R Mackey, Daniel Hoces, Colleen N McLaughlin, Chang Lin, Stephen R Adams, Khanh Nguyen, Keun-Young Kim, David J Luginbuhl, Liqun Luo, Namrata D Udeshi, Steven A Carr, Rogelio A Hernández-López, Mark H Ellisman, Miguel Alcalde, Alice Y Ting.
      Enzymes that oxidize aromatic substrates have been harnessed for cell-based technologies including proximity labeling and electron microscopy; however, they are associated with drawbacks such as the need for toxic H2O2. Here, we explore multicopper oxidases (laccases) as a new enzyme class for proximity labeling and electron microscopy in mammalian cells. LaccID was generated through 11 rounds of directed evolution from an ancestral fungal laccase and catalyzes one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2. Surprisingly, we found that LaccID is selectively active at the surface plasma membrane of both living and fixed cells. We use LaccID proximity labeling and mass spectrometry to map the changing surface proteome of T cells that engage with tumor cells through antigen-specific T cell receptors. In addition, we use LaccID as a genetically encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.
    DOI:  https://doi.org/10.1038/s41589-025-01973-6
  35. Sci Adv. 2025 Jul 25. 11(30): eadu9555
    DNA damage response pathway regulates Nrf2 in response to oxidative stress.
    Xiao-You Jiang, Qi-Qiang Guo, Shan-Shan Wang, Ran Guo, Yu Zou, Jing-Wei Liu, Yan-Ling Feng, Yang Guo, Yu-Han Li, Xi-Yan Liu, Xin-Yue Zhang, Shuang Hao, Xiao-Xu Wu, Meng-Han Li, Ao Liu, Chun-Lu Li, Wen-Dong Guo, Hong-De Xu, Xiao-Yu Song, Toren Finkel, Liu Cao.
      Nrf2 acts as a transcriptional master regulator to orchestrate antioxidant responses and maintain redox balance. However, the cellular pathway for translating oxidative stress signals into Nrf2-dependent antioxidant responses remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules in modulating Nrf2's stability and transcriptional activity by activating the DNA damage response (DDR) signaling pathway. When activated, CHK2 phosphorylates the autophagy adaptor protein p62 at serine-349, promoting its interaction with Keap1 and disrupting the Keap1-Nrf2 interaction, thereby inhibiting Nrf2 ubiquitination-dependent degradation. In addition, CHK2 directly phosphorylates Nrf2 at serine-566/serine-577, enhancing its transcriptional activity and antioxidant capacity. Consistent with these effects, Chk2-/- mice show impaired expression of Nrf2 and its downstream antioxidant target genes, along with more severe renal tissue damage in an ROS-dependent model of renal ischemia/reperfusion injury. Our study reveals a direct mechanism linking the DDR signaling pathway to ROS-triggered Nrf2-dependent antioxidant responses, providing critical insight into cellular protection against oxidative stress-induced damage.
    DOI:  https://doi.org/10.1126/sciadv.adu9555
  36. Science. 2025 Jul 31. 389(6759): eadk3079
    Altered translation elongation contributes to key hallmarks of aging in the killifish brain.
    Domenico Di Fraia, Antonio Marino, Jae Ho Lee, Erika Kelmer Sacramento, Mario Baumgart, Sara Bagnoli, Till Balla, Felix Schalk, Stephan Kamrad, Rui Guan, Cinzia Caterino, Chiara Giannuzzi, Pedro Tomaz da Silva, Amit Kumar Sahu, Hanna Gut, Giacomo Siano, Max Tiessen, Eva Terzibasi-Tozzini, Eugenio F Fornasiero, Julien Gagneur, Christoph Englert, Kiran R Patil, Clara Correia-Melo, Danny D Nedialkova, Judith Frydman, Alessandro Cellerino, Alessandro Ori.
      Aging is a major risk factor for neurodegeneration and is characterized by diverse cellular and molecular hallmarks. To understand the origin of these hallmarks, we studied the effects of aging on the transcriptome, translatome, and proteome in the brain of short-lived killifish. We identified a cascade of events in which aberrant translation pausing led to altered abundance of proteins independently of transcriptional regulation. In particular, aging caused increased ribosome stalling and widespread depletion of proteins enriched in basic amino acids. These findings uncover a potential vulnerable point in the aging brain's biology-the biogenesis of basic DNA and RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity, proteostasis, and the biosynthesis of macromolecules.
    DOI:  https://doi.org/10.1126/science.adk3079
  37. Nat Cell Biol. 2025 Jul 30.
    PEX39 facilitates the peroxisomal import of PTS2-containing proteins.
    Walter W Chen, Tony A Rodrigues, Daniel Wendscheck, Ana G Pedrosa, Chendong Yang, Tânia Francisco, Till Möcklinghoff, Alexandros Zografakis, Bernardo Nunes-Silva, Reut E Avraham, Ana R Silva, Maria J Ferreira, Hirak Das, Janet Koster, Simone Neuwirth, Julian Bender, Silke Oeljeklaus, Varun Sondhi, Christos Gatsogiannis, Maya Schuldiner, Einat Zalckvar, Kay Hofmann, Hans R Waterham, Ralph J DeBerardinis, Jorge E Azevedo, Bettina Warscheid.
      Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (also known as peroxins (PEXs)) cause devastating disease. PEX7 binds proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this process remain enigmatic. Utilizing in vitro assays, yeast and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates the import of PTS2-containing proteins by binding PEX7 and stabilizing its interaction with cargo proteins containing a PTS2. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess an (R/K)PWE motif necessary for PEX7 binding. Handover of PEX7 from PEX39 to PEX13 via these motifs provides a new paradigm for peroxisomal protein import and biogenesis. Collectively, this work reveals how PEX39 and (R/K)PWE motifs facilitate the import of PTS2-containing proteins and advances our understanding of peroxisomal disease.
    DOI:  https://doi.org/10.1038/s41556-025-01711-z
  38. Nat Commun. 2025 Jul 29. 16(1): 6959
    Time-dependent catalytic activity in aging condensates.
    Wei Kang, Zhiyue Wu, Xinzhi Huang, Hongbin Qi, Jiaxuan Wu, Jiahui Wang, Jing Li, Sijin Wu, Byung-Ho Kang, Bo Li, Juncai Ma, Chuang Xue.
      Biomolecular condensates are dynamic cellular compartments that concentrate proteins and enzymes to regulate biochemical reactions in time and space. While these condensates can enhance enzyme activity, how this function changes as condensates age remains poorly understood. Here, we design synthetic catalytic condensates that selectively recruit enzymes to investigate this temporal evolution. We show that catalytic condensates exhibit time-dependent activity: they initially accelerate enzymatic reactions but gradually lose efficiency due to the transition from liquid-like to solid-like states. This aging process, characterized by protein aggregation and loss of selective barriers, impairs enzyme function both in vitro and living cells. We further demonstrate that small molecules which influence aging dynamics can modulate catalytic efficiency of condensates. Our findings show that condensate aging as a key regulator of enzymatic activity and provide crucial insights for designing functional synthetic condensates.
    DOI:  https://doi.org/10.1038/s41467-025-62074-5
  39. Redox Biol. 2025 Jun 09. pii: S2213-2317(25)00226-5. [Epub ahead of print]86 103713
    Reductive stress induces unresolved ER stress and proteotoxic cardiomyopathy.
    Sini Sunny, Rajesh Kumar Radhakrishnan, Asokan Devarajan, Juan Xavier Masjoan Juncos, Mohit Bansal, Xiaosen Ouyang, Jianhua Zhang, Min Xie, Silvio H Litovsky, Joel D Trinity, Matthew Might, Steven M Pogwizd, Maria I Kontaridis, Namakkal S Rajasekaran.
      Chronic reductive stress (cRS), induced by constitutive activation of Nrf2 in transgenic (TG) mouse hearts leads to pathological cardiac remodeling and diastolic dysfunction. Transcriptomic analysis revealed that both pro-reductive (PR) and reductive stress (RS) conditions disrupt ER-associated gene expression in a dose-dependent manner, with pronounced dysregulation in high-expressing TG (TGH) mice. These shifts were associated with persistent activation of the unfolded protein response (UPR), impaired ER function, and redox imbalance marked by elevated glutathione and reduced ROS levels. Proteostasis disruption under cRS led to protein misfolding, ER dilation, and aggregation of mis/unfolded proteins. TGH mice showed increased ubiquitination and accumulation of aggregated proteins, alongside inadequate proteasome activity, indicating inadequate protein quality control (PQC) mechanisms. RNA-seq data revealed transcriptional upregulation of ubiquitin-proteasome genes and downregulation of key chaperones, suggesting a failed compensatory response. Speckle-tracking echocardiography (STE) detected myocardial dyssynchrony and progressive strain abnormalities in TGH mice, correlating with increased proteotoxic burden and impaired redox homeostasis. Elevated TEI index values confirmed systolic and diastolic dysfunction. Time- and dose-dependent upregulation of Nogo/Reticulon4 transcripts and proteins further supported maladaptive cardiac remodeling. Collectively, these findings highlight that chronic RS disrupts ER homeostasis, induces proteotoxicity, and impairs cardiac structure and function, particularly in high transgene-expressing hearts.
    Keywords:  Cardiac hypertrophy; Diastolic dysfunction; ER stress; Nrf2 signaling; Proteotoxicity; Reductive stress
    DOI:  https://doi.org/10.1016/j.redox.2025.103713
  40. Cell Rep. 2025 Jul 24. pii: S2211-1247(25)00827-7. [Epub ahead of print]44(8): 116056
    Germin-like protein 1 interacts with proteasome regulator 1 to regulate auxin signaling by controlling Aux/IAA homeostasis.
    Faqing Xu, Yongqiang Yu, Bin Guan, Tongda Xu, Zhihong Xu, Hongwei Xue.
      Auxin regulates various aspects of plant growth and development by modulating the transcription of target genes through the degradation of auxin/indole-3-acetic acid (Aux/IAA) repressors via the 26S proteasome. Proteasome regulator 1 (PTRE1), a positive regulator of proteasome activity, has been implicated in auxin-mediated proteasome suppression; however, the mechanism by which auxin modulates PTRE1 function remains unclear. Here, we demonstrate that auxin promotes the interaction between germin-like protein 1 (GLP1) and PTRE1, facilitating PTRE1 retention at the plasma membrane. The relocation of PTRE1 results in reduced nuclear 26S proteasome activity, and thus the attenuated Aux/IAA degradation and altered Aux/IAA homeostasis, ultimately resulting in suppressed auxin-mediated transcriptional regulation. Our findings uncover a previously uncharacterized regulatory axis in auxin signaling that controls Aux/IAA protein stability, functioning alongside the TIR1- and TRANSMEMBRANE KINASE 1 (TMK1)-mediated pathways, and highlight the coordination of auxin signaling from the cell surface to the nucleus via auxin-induced PTRE1 relocation, which fine-tunes Aux/IAA protein homeostasis and auxin responses.
    Keywords:  Aux/IAAs; CP: Plants; GLP1; PTRE1; auxin; germin-like protein 1; proteasome; proteasome regulator 1; protein interaction
    DOI:  https://doi.org/10.1016/j.celrep.2025.116056
  41. Nat Biotechnol. 2025 Jul 25.
    Translation efficiency covariation identifies conserved coordination patterns across cell types.
    Yue Liu, Shilpa Rao, Ian Hoskins, Michael Geng, Qiuxia Zhao, Jonathan Chacko, Vighnesh Ghatpande, Kangsheng Qi, Logan Persyn, Jun Wang, Dinghai Zheng, Yochen Zhong, Dayea Park, Elif Sarinay Cenik, Vikram Agarwal, Hakan Ozadam, Can Cenik.
      Characterizing shared patterns of RNA expression between genes across conditions has led to the discovery of regulatory networks and biological functions. However, it is unclear if such coordination extends to translation. In this study, we uniformly analyze 3,819 ribosome profiling datasets from 117 human and 94 mouse tissues and cell lines. We introduce the concept of translation efficiency covariation (TEC), identifying coordinated translation patterns across cell types. We nominate candidate mechanisms driving shared patterns of translation regulation. TEC is conserved across human and mouse cells and uncovers gene functions that are not evident from RNA or protein co-expression. Moreover, our observations indicate that proteins that physically interact are highly enriched for positive covariation at both translational and transcriptional levels. Our findings establish TEC as a conserved organizing principle of mammalian transcriptomes. TEC has potential as a predictive marker for gene function and may offer a framework for designing gene expression systems in synthetic biology and biotechnological applications.
    DOI:  https://doi.org/10.1038/s41587-025-02718-5
  42. J Biol Chem. 2025 Jul 24. pii: S0021-9258(25)02366-X. [Epub ahead of print] 110515
    Ubiquitin-Specific Protease 7-Mediated Stabilization of Discoidin Domain Receptor 1 Drives Progression of TP53-Mutant Cancers.
    Yiying Xue, Bing Xiu, Muye Yang, Yanwei Zhang, Changpeng Yang, Lele Zhang, Yilv Yan, Xiu Luo, Yushan Huang, Husheng Mei, Guiming Li, Lixin Zhou, Yisa Chen, Wenjun Zhang, Changlin Qian, Peng Zhang, Juan Liu, Aibin Liang, Yu Zeng, Jing Yang.
      Receptor tyrosine kinase DDR1 (Discoidin Domain Receptor 1) interacts with the extracellular matrix (ECM) to promote tumor cell proliferation through its intracellular kinase activity, while its extracellular non-enzymatic domain creates a physical barrier for immune evasion. Although DDR1 inhibitors and antibodies have been developed, targeting DDR1 kinase activity alone cannot fully block the biological effects mediated by its scaffold function. Therefore, developing DDR1 degraders presents a potentially more effective therapeutic strategy. Through screening a proprietary small-molecule ubiquitination library, we identified NSC632839, which significantly induces DDR1 protein degradation. Mechanistically, chemical proteomics and genetic studies demonstrated that NSC632839 functions by inhibiting USP7, which interacts with, stabilizes, and deubiquitinates DDR1, preventing its proteasomal degradation. Importantly, we observed that TP53 loss or mutation in tumor cells and clinical samples markedly upregulates DDR1 expression, thereby enhancing its interaction with USP7. Inhibition of USP7 with NSC632839 or other selective inhibitors restores TP53 expression, resulting in a significant reduction in DDR1 levels. In various preclinical models, targeting USP7 with NSC632839 effectively eliminates tumor cells, offering a promising therapeutic strategy to overcome tumor relapse driven by TP53 mutations, both in vitro and in vivo. This study highlights the potential of DDR1 degradation via USP7 inhibition as a novel approach to treat TP53 mutation-enriched tumors.
    Keywords:  Discoidin Domain Receptor 1; Malignancy; Protein degradation; TP53 mutation; Ubiquitin specific peptidase 7; Ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2025.110515
  43. STAR Protoc. 2025 Jul 26. pii: S2666-1667(25)00393-4. [Epub ahead of print]6(3): 103987
    Protocol to isolate stress granules in HeLa cells using fluorescence-activated non-membrane condensate isolation.
    Yilong Zhou, Maria Shvedunova, Asifa Akhtar.
      Various stress stimuli induce cytosolic stress granules (SGs) in mammalian cells, which are composed of RNA and RNA-binding proteins and have important physiological functions. Here, we present a protocol for isolating SGs using fluorescence-activated non-membrane condensate isolation (FANCI). We describe steps for seeding and stressing G3BP1-mCherry HeLa cells. We then detail procedures for purifying SGs by FANCI with flow cytometry. This protocol has potential application in studying SGs from other cells in the future. For complete details on the use and execution of this protocol, please refer to Zhou et al.1.
    Keywords:  Cell Biology; Cell culture; Cell isolation; Flow Cytometry; Genetics; Molecular Biology
    DOI:  https://doi.org/10.1016/j.xpro.2025.103987
  44. Redox Biol. 2025 Jul 21. pii: S2213-2317(25)00294-0. [Epub ahead of print]85 103781
    Advances in KEAP1-based PROTACs as emerging therapeutic modalities: Structural basis and progress.
    Jing Chen, Disheng Feng, Rui Zhu, Hua Li, Lixia Chen.
      Kelch-like ECH-associated protein 1 (KEAP1) functions as a substrate adaptor for the Cullin 3-RING E3 ligase complex, mediating the ubiquitination and subsequent proteasomal degradation of nuclear factor erythroid 2-related factor 2 (NRF2). This regulatory mechanism maintains cellular redox homeostasis by preventing NRF2 overactivation. Proteolysis-targeting chimeras (PROTACs) have emerged as a novel therapeutic strategy that harnesses the ubiquitin-proteasome system for targeted protein degradation. Recent advancements have expanded the repertoire of E3 ligases exploitable for PROTAC design, with KEAP1 identified as a promising candidate. This review provides a comprehensive overview of the structural and functional characteristics of KEAP1, detailing its interactions with NRF2 and Cullin 3. The development of KEAP1- recruiting PROTACs utilizing ligands derived from different classes of known KEAP1 inhibitors-including short peptides, covalent small molecules (e.g., CDDO derivatives), and non-covalent inhibitors (e.g., KI696)-is discussed, highlighting the potential to diversify the available E3 ligase recruiters. Recent progress in developing KEAP1-based PROTACs targeting BRD4, CDK9, FAK, Tau and KEAP1 itself is highlighted, with particular emphasis on ligand optimization strategies employed to enhance degradation efficacy and specificity. Elucidating the structural basis of KEAP1 interactions provides crucial insights for advancing PROTAC applications. However, current challenges in KEAP1-based targeted protein degradation warrant further investigation to fully realize its therapeutic potential. Future research should focus on optimizing KEAP1 ligand properties and exploring novel protein targets amenable to degradation via KEAP1 recruitment to further advance this innovative therapeutic modality.
    Keywords:  CUL3; Inhibitors; KEAP1; NRF2; PROTACs; Ubiquitination
    DOI:  https://doi.org/10.1016/j.redox.2025.103781
  45. RSC Chem Biol. 2025 Jul 21.
    Defining STING-sterol interactions with chemoproteomics.
    Ian Ford, Miranda Villanueva, Min Sub Lee, Quan D Zhou, Constance Yuen, Robert Damoiseaux, Steven J Bensinger, Keriann M Backus.
      Stimulator of interferon genes (STING) is an intracellular pattern recognition receptor that plays a key role in responding to cytosolic DNA and cyclic dinucleotides. STING activity is tightly regulated to avoid aberrant STING activity, excessive type I IFN responses, and resultant autoinflammatory disease. As such understanding the molecular events regulating STING activity is critical. Recent work has revealed cellular cholesterol metabolism also functions to modulate STING activity, although the molecular events linking cholesterol homeostasis with STING remain incompletely understood. Here we pair genetic and chemoproteomic approaches to inform the mechanisms governing cholesterol modulation of STING activity. Using gain- and loss-of-function systems, we find that markedly increasing SCAP-SREBP2 processing and resultant cholesterol synthesis has little impact on STING activity. In contrast, we find that genetic deletion of Srebf2 increased basal and ligand inducible type I IFN responses. Thus, STING can function in the absence of the SCAP-SREBP2 protein apparatus. Through activity-based protein profiling with three distinct sterol-mimetic probes, we provide direct evidence for STING-sterol binding. We also find that the mitochondrial protein VDAC1 co-purifies with STING and binds to sterol-mimetic probes. We also show that STING's subcellular localization is responsive to modulation of cellular sterol content. Our findings support a model where sterol synthesis in the ER regulates STING activity, aligning with recent studies indicating that cholesterol-mediated retention of STING in the endoplasmic reticulum occurs through cholesterol recognition amino acid consensus (CARC) motifs in STING.
    DOI:  https://doi.org/10.1039/d5cb00171d
  46. J Cell Sci. 2025 Jul 15. pii: jcs263677. [Epub ahead of print]138(14):
    Different roles of ACSL3 and ACSL4 in autophagosome formation.
    Shun Kato, Naoki Okada, Toshiki Ohata, Takefumi Uemura, Satoshi Waguri, Yuichi Wakana, Hiroki Inoue, Kohei Arasaki, Mitsuo Tagaya.
      Acyl-CoA synthetases (ACSLs) are a family of enzymes that convert intracellular fatty acids into acyl-CoA. A previous study has demonstrated that the yeast ACSL Faa1 (a homolog of mammalian ACSL4) is involved in autophagosome membrane elongation. In the present study, we investigated the involvement of ACSL3, a key enzyme responsible for lipid droplet formation, in autophagosome formation and compared its role with that of ACSL4. Knockdown of ACSL3 impaired starvation-induced autophagy concomitant with the formation of enlarged autophagosome-like structures negative for WIPI2, whereas its overexpression resulted in the formation of WIPI2-positive, but LC3-negative dots, under normal nutrition conditions, likely in an enzymatic activity-independent manner. In contrast, ACSL4 knockdown inhibited starvation-induced autophagosome formation, whereas its overexpression caused autophagosome formation under normal nutrition conditions. Inhibition of autophagosome formation in ACSL4-depleted cells could be rescued by ethanolamine, suggesting a deficit of phosphatidylethanolamine in ACSL4-depleted cells. These results suggest that ACSL3 and ACSL4 are involved in different stages of autophagosome formation - ACSL3 in the formation of fusion-competent autophagosomal membranes and ACSL4 in the formation of autophagosomes.
    Keywords:  ACSL3; ACSL4; Autophagy; Lipid droplet; Lipid metabolism
    DOI:  https://doi.org/10.1242/jcs.263677
  47. J Biol Chem. 2025 Jul 24. pii: S0021-9258(25)02367-1. [Epub ahead of print] 110516
    A dynamic structural framework for the allosteric regulation of Hsp70 chaperones.
    Lukas Rohland, Roman Kityk, Luka Smalinskaitė-Wolf, Veronika Lashkul, Matthias P Mayer.
      Central to allosteric signaling mechanisms are three processes: (I) initial sensing of the allosterically active ligand, (II) allosteric signal relaying from the ligand binding pocket to the allosterically affected parts of the protein, and (III) stabilization of the allosterically activated state. Hsp70 proteins are ATP-driven molecular chaperones that are essential in many organisms across the tree of life and exert their action using an intricate allosteric mechanism. Although pathways of this allosteric signaling mechanism have been outlined, its spatiotemporal organization is poorly understood. Here, we present the structural and dynamic basis of these allosteric signaling pathways for the prokaryotic model Hsp70 DnaK that rationalizes the action of ATP, several of its analogues, and clients on the allosteric signal transmission. Our data reveal that during the initial sensing of ATP, DnaK tolerates chemical alterations in the α-phosphate group of ATP but not in the γ-phosphate group. Amino acid replacements that interfere with the allosteric regulation of DnaK disrupt the ATP-induced relay of allosteric signal transmission at specific steps by altering the mechanics of allostery or the stability of reaction intermediates, or both. Client binding to a DnaK variant that is unreceptive to the client-sent signals that stimulate DnaK's ATPase activity, also shows diminished client-induced conformational remodeling of ATP-bound DnaK, suggesting that client-induced conformational changes in DnaK are needed to trigger ATP hydrolysis. Based on these observations, we formulate a dynamic structural framework for the allosteric regulation of Hsp70 chaperones that links the molecular mechanics of Hsp70s to their biochemical properties.
    Keywords:  Hsp70; Molecular chaperones; allosteric signal transmission; allostery; conformational dynamics; protein folding
    DOI:  https://doi.org/10.1016/j.jbc.2025.110516
  48. JACC Basic Transl Sci. 2025 Jun 04. pii: S2452-302X(25)00181-0. [Epub ahead of print] 101293
    Ufmylation Suppresses Unfolded Protein Response to Prevent Peripartum Cardiomyopathy.
    Varsha Tandra, Lingxian Zhang, Chang Min Lee, Yongbing Wu, Guihua Yue, Honglin Li, Huabo Su, Jie Li.
      Ufmylation is a novel ubiquitin-like protein modification that plays a critical role in maintaining the homeostasis of different tissues, but its role in the heart remains poorly understood. Here, we showed that mice lacking UFM1 ligase 1 (UFL1), an enzyme essential for ufmylation, in the heart developed peripartum cardiomyopathy. Loss of UFL1 reversed pregnancy-induced adaptive cardiac transcriptome alterations. Moreover, loss of UFL1 triggered excessive endoplasmic reticulum stress, inhibited mitochondrial oxidative metabolism, and caused augmented mTOR signaling, leading to pronounced pathological remodeling and heart failure. These results demonstrate that ufmylation is essential for physiological cardiac remodeling and that disruption of ufmylation predisposes the heart to peripartum cardiomyopathy.
    Keywords:  heart failure; peripartum cardiomyopathy; ufmylation; unfolded protein response
    DOI:  https://doi.org/10.1016/j.jacbts.2025.04.007
  49. Nat Biotechnol. 2025 Jul 25.
    A resource of RNA-binding protein motifs across eukaryotes reveals evolutionary dynamics and gene-regulatory function.
    Alexander Sasse, Debashish Ray, Kaitlin U Laverty, Cyrus L Tam, Mihai Albu, Hong Zheng, Yevgen Levdansky, Olga Lyudovyk, Taykhoom Dalal, Kate Nie, Cedrik Magis, Cedric Notredame, Eugene Valkov, Matthew T Weirauch, Timothy R Hughes, Quaid Morris.
      RNA-binding proteins (RBPs) are key regulators of gene expression; however, their RNA-binding specificities, that is, motifs, have not been comprehensively determined. Here we introduce Eukaryotic Protein-RNA Interactions (EuPRI), a freely available resource of RNA motifs for 34,746 RBPs from 690 eukaryotes. EuPRI includes in vitro binding data for 504 RBPs, including newly collected RNAcompete data for 174 RBPs, along with thousands of predicted motifs. We predict these motifs with an algorithm, Joint Protein-Ligand Embedding, which can detect distant homology relationships and map specificity-determining peptides. EuPRI quadruples the number of available RBP motifs, expanding the motif repertoire across all major eukaryotic clades and assigning motifs to the majority of human RBPs. We demonstrate the utility of EuPRI for inferring post-transcriptional function and evolutionary relationships by identifying rapid, recent evolution of post-transcriptional regulatory networks in worms and plants, in contrast to the vertebrate RNA motif set, which has remained relatively stable after a large expansion between the metazoan and vertebrate ancestors.
    DOI:  https://doi.org/10.1038/s41587-025-02733-6
  50. Nat Biotechnol. 2025 Jul 25.
    Predicting the translation efficiency of messenger RNA in mammalian cells.
    Dinghai Zheng, Logan Persyn, Jun Wang, Yue Liu, Fernando Ulloa-Montoya, Can Cenik, Vikram Agarwal.
      The mechanisms by which mRNA sequences specify translational control remain poorly understood in mammalian cells. Here we generate a transcriptome-wide atlas of translation efficiency (TE) measurements encompassing more than 140 human and mouse cell types from 3,819 ribosomal profiling datasets. We develop RiboNN, a state-of-the-art multitask deep convolutional neural network, and classic machine learning models to predict TEs in hundreds of cell types from sequence-encoded mRNA features. While most earlier models solely considered the 5' untranslated region (UTR) sequence, RiboNN integrates how the spatial positioning of low-level dinucleotide and trinucleotide features (that is, including codons) influences TE, capturing mechanistic principles such as how ribosomal processivity and tRNA abundance control translational output. RiboNN predicts the translational behavior of base-modified therapeutic RNA and explains evolutionary selection pressures in human 5' UTRs. Finally, it detects a common language governing mRNA regulatory control and highlights the interconnectedness of mRNA translation, stability and localization in mammalian organisms.
    DOI:  https://doi.org/10.1038/s41587-025-02712-x
  51. J Hum Genet. 2025 Jul 29.
    NGLY1 deficiency - clinical features and therapeutic strategy.
    Haruhiko Fujihria, Hiroto Hirayama, Tadashi Suzuki.
      NGLY1 deficiency is a rare autosomal recessive genetic disorder caused by biallelic mutations of the human NGLY1 gene. NGLY1 encodes the cytosolic peptide:N-glycanase (PNGase; NGLY1 in mammals), which plays essential roles in cytosolic glycan degradation (non-lysosomal glycan degradation), the endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded proteins, and the complete activation of the transcription factor nuclear factor erythroid 2-like 1 (NEF2L1). NFE2L1 contributes to the regulation of the expression of proteasome subunits and oxidative stress responses. Patients with NGLY1 deficiency exhibit multisystemic clinical features, including global developmental delay, peripheral neuropathy, hypolacrima or alacrima, and the transient elevation of liver transaminases. To date, more than 100 individuals with NGLY1 deficiency and over 70 distinct pathogenic mutations in the NGLY1 gene have been reported. There is currently no approved therapy for this disorder. Moreover, the underlying pathogenic mechanism, including the correlation between patients' symptoms and mutant alleles, remains poorly understood. In this review, we summarize the most frequently reported NGLY1 mutations and their associated clinical features. We also present an overview of the current therapeutic strategy for NGLY1 deficiency.
    DOI:  https://doi.org/10.1038/s10038-025-01376-4
  52. Cancer Discov. 2025 Jul 31.
    Mitochondrial glutathione import enables breast cancer metastasis via integrated stress response signaling.
    Hsi-Wen Yeh, Nicole Lauren DelGaudio, Beste Uygur, Alon Millet, Artem Khan, Gokhan Unlu, Michael Xiao, Rebecca C Timson, Caifan Li, Kerem Ozcan, Karl W Smith, Luiza Martins Nascentes Melo, Gabriele Allies, Olca Basturk, Albert Sickmann, Erol C Bayraktar, Richard Possemato, Alpaslan Tasdogan, Kivanc Birsoy.
      Cancer cells require substantial metabolic adaptations to metastasize to distant organs, but the metabolites essential for successful colonization remain poorly defined. Here, we used a mitochondrial metabolomics approach to compare primary and metastatic breast cancer cells. This analysis revealed accumulation of mitochondrial glutathione (GSH) during lung metastasis, driven by elevated expression of SLC25A39, a mitochondrial GSH transporter. Loss of SLC25A39 impairs metastatic colonization in genetic screens, cell line models, and patient-derived xenografts, without affecting primary tumor growth. Mitochondrial GSH import is specifically required during early colonization and functions independently of its canonical antioxidant role. CRISPR activation screens identified ATF4, a stress-induced transcription factor, as a bypass mechanism that restores metastatic potential in SLC25A39-deficient cells. Mechanistically, SLC25A39 is required for optimal ATF4 activation during metastasis and under hypoxia, linking mitochondrial GSH availability to integrated stress response signaling. These findings identify mitochondrial GSH as a necessary and limiting metabolite for metastatic progression.
    DOI:  https://doi.org/10.1158/2159-8290.CD-24-1556
  53. Proc Natl Acad Sci U S A. 2025 Aug 05. 122(31): e2514178122
    Surface delivery quantification reveals distinct trafficking efficiencies among clustered protocadherin isoforms.
    Elizabeth J May, Rachelle Gaudet.
      Proteins that transmit molecules and signals across the plasma membrane are crucial in cell biology because they enable cells to sense and respond to their surroundings. A major challenge for studying cell surface proteins is that often they do not fold or traffic properly to the plasma membrane when produced in heterologous cells. We developed a strategy for quantifying surface localization from fluorescence microscopy images of surface-stained cells. Using clustered protocadherins, a protein family important for cell-cell recognition during neuronal development, we found that surface delivery levels vary among clustered protocadherin isoforms and between wild-type and engineered variants. Quantifying these differences provides evidence that cis dimerization is not tightly coupled to surface delivery for clustered protocadherins. This work establishes a generalizable framework for screening proteins and variants of interest for proper cell surface localization.
    Keywords:  cell surface trafficking; clustered protocadherins; plasma membrane proteins
    DOI:  https://doi.org/10.1073/pnas.2514178122
  54. J Biol Chem. 2025 Jul 25. pii: S0021-9258(25)02370-1. [Epub ahead of print] 110519
    Integrating deep learning for post-translational modifications crosstalk on Hsp90 and drug binding.
    Jennifer A Heritz, Katherine A Meluni, Sarah J Backe, Sara J Cayaban, Laura A Wengert, Meik Kunz, Mark R Woodford, Dimitra Bourboulia, Mehdi Mollapour.
      Post-translational modification (PTM) of proteins regulates cellular proteostasis by expanding protein functional diversity. This naturally leads to increased proteome complexity as the result of PTM crosstalk. Here, we used the molecular chaperone protein, Heat shock protein-90 (Hsp90), which is subject to a plethora of PTMs, to investigate this concept. Hsp90 is at the hub of proteostasis and cellular signaling networks in cancer and is, therefore, an attractive therapeutic target in cancer. We demonstrated that deletion of histone deacetylase 3 (HDAC3) and histone deacetylase 8 (HDAC8) in human cells led to increased binding of Hsp90 to both ATP and its ATP-competitive inhibitor, Ganetespib. When bound this inhibitor, Hsp90 from both HDAC3 and HDAC8 knock out human cells exhibited similar PTMs, mainly phosphorylation and acetylation, and created a common proteomic network signature. We used both a deep-learning artificial intelligence (AI) prediction model and data based on mass-spectrometry analysis of Hsp90 isolated from the mammalian cells bound to its drugs to decipher PTM crosstalk. The alignment of data from both methods demonstrates that the deep-learning prediction model offers a highly efficient and rapid approach for deciphering PTM crosstalk on complex proteins such as Hsp90.
    Keywords:  Hsp90; acetylation; artificial intelligence; chaperone; cochaperone; deep learning; histone deacetylase; phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110519
  55. Autophagy. 2025 Jul 29.
    Dysfunctional autophagy triggers STING1 activation to exacerbate cartilage degeneration in obesity-associated osteoarthritis.
    Xiaomin Kang, Wenjuan Liu, Xiao Ma, Dongxu Feng, Hongzhi Sun, Shufang Wu.
      Obesity, a major risk factor for osteoarthritis (OA), is related to increased circulating levels of free fatty acids (FFAs). However, the molecular mechanism underlying this metabolic OA phenotype remains unknown. We found that mice fed a high-fat diet (HFD) became obese and developed OA in their knee joints. Macroautophagy/autophagy activity was significantly reduced in articular cartilage of mice fed an HFD or in chondrocytes exposed to FFAs. Using conditional knockout (cKO) mice with cartilage-specific deletion of Atg7 to inhibit autophagy in vivo and shAtg7-lentiviral-transduced chondrocytes in vitro, we showed that autophagy deficiency aggravated HFD-induced OA progression and chondrocyte extracellular matrix (ECM) degradation. Mechanistically, STING1 was degraded in an autophagy-dependent manner. Autophagy deficiency increased STING1 levels, in turn activating the STING1-TBK1-IRF3 and MAP2K3/MKK3-MAPK/p38 signaling pathways, thereby triggering cartilage ECM degradation. These findings suggested that the HFD-autophagy-STING1 axis played a pivotal role in OA development, providing a potential therapeutic strategy for obesity-associated OA.
    Keywords:  ATG7; STING1; autophagy; high-fat diet; obesity; osteoarthritis
    DOI:  https://doi.org/10.1080/15548627.2025.2541388
  56. Nat Commun. 2025 Aug 01. 16(1): 7073
    March5-mediated Trim28 degradation preserves islet β-cell function in mice.
    Yangshan Chen, Wei Pang, Guixing Ma, Yongcong Yan, Zhiyu Xiao, Yong Chen, Zhen Ding, Litong Chen, Xiaoting Hou, Huiling Cao.
      Insulin deficiency from β-cell dysfunction underpins both type 1 and type 2 diabetes. However, the regulatory pathways underlying β-cell function remain incompletely understood. Here, we identify that March5 and Trim28 as key modulators of β-cell function. March5 is downregulated and Trim28 upregulated in islets from human or mouse with impaired glucose tolerance. Loss of March5 in β-cells impairs insulin production and glucose tolerance, while its overexpression improves both. Mechanistically, March5 inhibits Trim28 by targeting it for ubiquitination, thereby preventing Trim28-mediated Kindlin-2 degradation, which elevates MafA and insulin expression in male mice. Trim28 deletion in β-cells rescues glucose intolerance in March5-deficient male mice, highlighting their joint regulatory pathway. Furthermore, March5 and Kindlin-2 double haploinsufficiency significantly impair insulin production and glucose tolerance, underscoring their shared pathway. Importantly, islet transplantation with March5-overexpressing or Trim28-deficient β-cells effectively ameliorates glucose intolerance in streptozotocin-induced diabetic male mice. In conclusion, our results suggest that targeting the March5/Trim28/Kindlin-2/MafA pathway may offer a promising therapeutic strategy to restore β-cell function in diabetes.
    DOI:  https://doi.org/10.1038/s41467-025-62587-z
  57. J Cell Biol. 2025 Aug 04. pii: e202408205. [Epub ahead of print]224(8):
    An ethanol-induced loss of the lipid droplet-associated segregase VCP/p97 leads to hepatic steatosis.
    Sandhya Sen, Shaun Weller, Ryan J Schulze, Donglin Ding, Carol A Casey, Conrad Weihl, Mark A McNiven.
      The liver stores substantial numbers of neutral lipid organelles termed lipid droplets (LDs) that accumulate within hepatocytes in response to chronic ethanol (EtOH) consumption leading to hepatic steatosis. Mass spectrometry analysis of LDs isolated from EtOH-damaged rat livers revealed a substantial reduction in the valosin-containing protein ATPase (VCP/p97) that acts to remove targeted proteins from cellular membranes for degradation. Experimental disruption of VCP function resulted in an increase in LD content in hepatocytes and mouse livers along with a marked increase in LD-associated hydroxysteroid dehydrogenase (HSD17β13) known to contribute to hepatic steatosis. Surprisingly, treatment of hepatocytes with the proteasome inhibitor MG132 had no effect on HSD17β13 levels, while a disruption of lysosome function and chaperone-mediated autophagy increased cellular HSD17β13 levels substantially. These findings provide new insights into the cellular mechanisms by which the liver regulates its lipid stores and how this is disrupted by chronic EtOH exposure.
    DOI:  https://doi.org/10.1083/jcb.202408205
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