bims-proteo Biomed News
on Proteostasis
Issue of 2025–08–17
35 papers selected by
Eric Chevet, INSERM
  1. A multichaperone condensate enhances protein folding in the endoplasmic reticulum.
  2. Principles of cotranslational mitochondrial protein import.
  3. Substrate Structure Determines p97- and RAD23A/B-Mediated Proteasomal Degradation in Human Cells.
  4. Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy.
  5. Co-translational ribosome pairing enables native assembly of misfolding-prone subunits.
  6. ER-phagy and proteostasis defects prime pancreatic epithelial state changes in KRAS-mediated oncogenesis.
  7. A method for the detection and enrichment of endogenous cereblon substrates.
  8. Global profiling of N-terminal cysteine-dependent degradation mechanisms.
  9. Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling.
  10. Endosome maturation during ER stress relies on the ubiquitin binding domain of Histone Deacetylase 6.
  11. Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation.
  12. RNA triggers chronic stress during neuronal aging.
  13. The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.
  14. Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates.
  15. GCN2 regulates paclitaxel-induced neuropathic pain.
  16. Defective Ribosome Recycling: A Bridge Between Translation Fidelity, Organelle Dysfunction, and Diseases: Exploring How Flawed Ribosome Recycling Factors Impact Ribosome Metabolism and Organelle Homeostasis, Leading to Human Diseases.
  17. Protein language models reveal evolutionary constraints on synonymous codon choice.
  18. Subtle variations in a client protein determine bacterial Hsp90 dependence.
  19. The topological properties of the protein universe.
  20. Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains.
  21. Ubiquitination of the histone variant mH2A1.2 prevents toxic RAD18 accumulation at a subset of genomic loci upon replication stress.
  22. DEK Interacts with IRE1α to Modulate Endoplasmic Reticulum Stress in Deoxynivalenol-Induced Intestinal Inflammation.
  23. Manganese suppresses tumor growth through hyper-activating IRE1α.
  24. F-actin disassembly by the oxidoreductase MICAL1 promotes mechano-dependent VWF-GPIbα interaction in platelets.
  25. Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.
  26. Client-scaffold interactions suppress aggregation of a client protein in model condensates.
  27. Localisation of Organelle Proteins using Data-Independent Acquisition (DIA-LOP).
  28. Small molecule dysregulation of ClpP activity via bidirectional allosteric pathways.
  29. Functional Proximity across an mRNA.
  30. Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis.
  31. SCOPE: Revealing Hidden Mechanisms in Phenotypic Screens Through Target and Pathway Enrichment.
  32. Cargo-selective regulation of clathrin-mediated endocytosis by AMP-activated protein kinase.
  33. A common structural mechanism for RNA recognition by the SF3B complex in mRNA splicing and export.
  34. RER1 regulates lipid metabolism in monocytes and macrophages.
  35. UBAP2L-driven stress granule formation links oxaliplatin resistance to gastric cancer.
  1. Nat Cell Biol. 2025 Aug 11.
    A multichaperone condensate enhances protein folding in the endoplasmic reticulum.
    Anna Leder, Guillaume Mas, Viktória Szentgyörgyi, Roman P Jakob, Timm Maier, Anne Spang, Sebastian Hiller.
      Protein folding in the endoplasmic reticulum (ER) relies on a network of molecular chaperones that facilitates the folding and maturation of client proteins. How the ER chaperones organize in a supramolecular manner to exert their cooperativity has, however, remained unclear. Here we report the discovery of a multichaperone condensate in the ER lumen, which is formed around the chaperone PDIA6 during protein folding homeostasis. The condensates form in a Ca2+-dependent manner and we resolve the underlying mechanism at the atomic and cellular levels. The PDIA6 condensates recruit further chaperones-Hsp70 BiP, J-domain protein ERdj3, disulfide isomerase PDIA1 and Hsp90 Grp94-which constitute some of the essential components of the early folding machinery. The chaperone condensates enhance folding of proteins, such as proinsulin, and prevent protein misfolding in the ER lumen. The PDIA6-scaffolded chaperone condensates hence provide the functional basis for spatial and temporal coordination of the dynamic ER chaperone network.
    DOI:  https://doi.org/10.1038/s41556-025-01730-w
  2. Cell. 2025 Aug 07. pii: S0092-8674(25)00811-6. [Epub ahead of print]
    Principles of cotranslational mitochondrial protein import.
    Zikun Zhu, Saurav Mallik, Taylor A Stevens, Riming Huang, Emmanuel D Levy, Shu-Ou Shan.
      Nearly all mitochondrial proteins are translated on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Using selective ribosome profiling, we show that nearly 20% of mitochondrial proteins can be imported cotranslationally in human cells. Cotranslational import requires an N-terminal presequence on the nascent protein and contributes to localized translation at the mitochondrial surface. This pathway does not favor membrane proteins but instead prioritizes large, multi-domain, topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to the early onset of cotranslational protein targeting to the endoplasmic reticulum (ER), the presequence on mitochondrial proteins is inhibited from initiating targeting early during translation until a large globular domain emerges from the ribosome. Our findings reveal a multi-layered protein sorting strategy that controls the timing and specificity of mitochondrial protein targeting.
    Keywords:  NAC; TOM complex; cotranslational protein import; localized translation; mitochondria; mitochondrial targeting sequence; nascent polypeptide-associated complex; protein folding; protein targeting; ribosome profiling
    DOI:  https://doi.org/10.1016/j.cell.2025.07.021
  3. J Biochem. 2025 Aug 11. pii: mvaf046. [Epub ahead of print]
    Substrate Structure Determines p97- and RAD23A/B-Mediated Proteasomal Degradation in Human Cells.
    Yi Ding, Takuya Tomita, Hikaru Tsuchiya, Yasushi Saeki.
      Proteasomal degradation of ubiquitinated proteins involves various accessory factors, including p97 and shuttle factors, but their requirements and relationship with substrate structural properties are not fully understood, especially in human cells. Here, we demonstrate that substrate structure dictates the dependency on p97 and RAD23A/B for proteasomal degradation in human cells, using two ubiquitin-fusion model substrates, Ub-GFP (well-folded) and Ub-GFP-tail (with an unstructured tail). Both substrates exhibited similar ubiquitin chain composition, primarily mediated by the UBR4-KCMF1 E3 ligase. Interactome analyses revealed that Ub-GFP preferentially interacts with p97 and RAD23B, while Ub-GFP-tail binds more strongly with the proteasome. The degradation of Ub-GFP depends on p97 and RAD23A/B, whereas that of Ub-GFP-tail bypasses these accessory factors. RAD23A/B knockdown resulted in a reduction in the apparent lengths of ubiquitin chains on both substrates, yet only affected Ub-GFP degradation, suggesting that even a lower level of ubiquitination is sufficient to support proteasomal degradation of substrates with an unstructured tail. Overall, our findings highlight substrate structure as a key determinant of accessory factor requirement, offering valuable insights for the development of targeted protein degradation.
    Keywords:  RAD23A/B; p97; substrate structure; ubiquitin fusion degradation; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1093/jb/mvaf046
  4. Nat Commun. 2025 Aug 09. 16(1): 7367
    Mitochondrial damage triggers the concerted degradation of negative regulators of neuronal autophagy.
    Bishal Basak, Erika L F Holzbaur.
      Mutations that disrupt the clearance of damaged mitochondria via mitophagy are causative for neurological disorders including Parkinson's. Here, we identify a Mitophagic Stress Response (MitoSR) activated by mitochondrial damage in neurons and operating in parallel to canonical Pink1/Parkin-dependent mitophagy. Increasing levels of mitochondrial stress trigger a graded response that induces the concerted degradation of negative regulators of autophagy including Myotubularin-related phosphatase (MTMR)5, MTMR2 and Rubicon via the ubiquitin-proteasome pathway and selective proteolysis. MTMR5/MTMR2 inhibit autophagosome biogenesis; consistent with this, mitochondrial engulfment by autophagosomes is enhanced upon MTMR2 depletion. Rubicon inhibits lysosomal function, blocking later steps of neuronal autophagy; Rubicon depletion relieves this inhibition. Targeted depletion of both MTMR2 and Rubicon is sufficient to enhance mitophagy, promoting autophagosome biogenesis and facilitating mitophagosome-lysosome fusion. Together, these findings suggest that therapeutic activation of MitoSR to induce the selective degradation of negative regulators of autophagy may enhance mitochondrial quality control in stressed neurons.
    DOI:  https://doi.org/10.1038/s41467-025-62379-5
  5. Nat Commun. 2025 Aug 15. 16(1): 7626
    Co-translational ribosome pairing enables native assembly of misfolding-prone subunits.
    Florian Wruck, Jaro Schmitt, Katharina Till, Kai Fenzl, Matilde Bertolini, Frank Tippmann, Alexandros Katranidis, Bernd Bukau, Günter Kramer, Sander J Tans.
      Protein complexes are pivotal to most cellular processes. Emerging evidence indicating dimer assembly by pairs of ribosomes suggests yet unknown folding mechanisms involving two nascent chains. Here, we show that co-translational ribosome pairing allows their nascent chains to 'chaperone each other', thus enabling the formation of coiled-coil homodimers from subunits that misfold individually. We developed an integrated single-molecule fluorescence and force spectroscopy approach to probe the folding and assembly of two nascent chains extending from nearby ribosomes, using the intermediate filament lamin as a model system. Ribosome proximity during early translation stages is found to be critical: when interactions between nascent chains are inhibited or delayed, they become trapped in stable misfolded states that are no longer assembly-competent. Conversely, early interactions allow the two nascent chains to nucleate native-like quaternary structures that grow in size and stability as translation advances. We conjecture that protein folding mechanisms enabled by ribosome cooperation are more broadly relevant to intermediate filaments and other protein classes.
    DOI:  https://doi.org/10.1038/s41467-025-61500-y
  6. Dev Cell. 2025 Aug 13. pii: S1534-5807(25)00473-3. [Epub ahead of print]
    ER-phagy and proteostasis defects prime pancreatic epithelial state changes in KRAS-mediated oncogenesis.
    Carla Salomó Coll, Marisa Di Monaco, Jocelyn Holkham, Matthew Smith, Morwenna Muir, Philippe Gautier, Hywel Dunn-Davies, Xiaozhong Zheng, Roopesh Krishnankutty, Alain J Kemp, Katie Winnington-Ingram, Alex von Kriegsheim, Jennifer P Morton, Natalia Jimenez-Moreno, Damian Mole, Simon Wilkinson.
      Pre-malignant transformation of pancreatic acinar cells by oncogenic Kras is dependent upon stochastic emergence of metaplastic cell states. Here, we reveal that an early, transcriptionally mediated effect of Kras is sporadic failure of proteostatic endoplasmic reticulum (ER)-phagy. Genetically altered mice deficient in ER-phagy demonstrate that this event cooperates with Kras to drive acinar-ductal metaplasia (ADM) and subsequent cancer. Mechanistically, proteomics and high-resolution imaging uncover pathologic aggregation of a subset of ER proteins, including the injury marker REG3B, resulting from failure to physically interact with the ER-phagy receptor CCPG1. Spatial transcriptomics demonstrate that the appearance of sporadic intracellular aggregates upon Kras activation marks rare acinar cells existing in an injured, ADM-primed state. Importantly, engineered mutants of REG3B establish that aggregate formation is sufficient to directly engender this epithelial cell state. Pancreatic cancer can thus arise from stochastic pathologic protein aggregates that are influenced by, and cooperate with, an oncogene.
    Keywords:  ADM; CANCER; CCPG1; ER-phagy; KRAS; autophagy; inflammation; metaplasia; pancreas; proteostasis
    DOI:  https://doi.org/10.1016/j.devcel.2025.07.016
  7. Cell Chem Biol. 2025 Aug 05. pii: S2451-9456(25)00225-9. [Epub ahead of print]
    A method for the detection and enrichment of endogenous cereblon substrates.
    Hannah C Lloyd, Yuli Li, N Connor Payne, Zhenguang Zhao, Wenqing Xu, Alena Kroupova, David Zollman, Tengfang Long, Farah Kabir, Mei Chen, Rebecca Freeman, Ethan Yang Feng, Sarah Y Xi, Ya-Chieh Hsu, Alessio Ciulli, Ralph Mazitschek, Christina M Woo.
      C-terminal cyclic imides are posttranslational modifications (PTMs) on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here, we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by western blot and enrichment from whole-cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.
    Keywords:  E3 ligase; cereblon; cyclic imide; degron; posttranslational modification; protein degradation; protein engineering
    DOI:  https://doi.org/10.1016/j.chembiol.2025.07.002
  8. Proc Natl Acad Sci U S A. 2025 Aug 19. 122(33): e2501681122
    Global profiling of N-terminal cysteine-dependent degradation mechanisms.
    Aizat Bekturova, Yaara Makaros, Shahar Ben-David, Itay Koren.
      Hypoxia, a condition characterized by insufficient oxygen supply, challenges cellular homeostasis and energy production, triggering adaptive responses to promote survival under these stressful conditions. One key strategy involves enzymatic oxidation of N-terminal cysteine residues coupled with proteolysis through the Cys-Arg/N-degron pathway. Despite hundreds of human proteins possessing N-terminal cysteine, very few have been identified as substrates of this pathway, and its substrate selectivity remains unclear. Moreover, the biological role of this pathway in the cellular response to hypoxia is not well defined. Here, by systematically screening protein stability using an N-terminome library, we reveal a broad set of cysteine-initiating proteins regulated by this pathway. Mutagenesis experiments further revealed the specificity of Cys-Arg/N-degron pathway, showing a preference for hydrophobic and positively charged residues following cysteine. Additionally, we uncovered full-length substrates that are regulated by this pathway during hypoxia, including IP6K1. Loss of IP6K1 impaired glucose uptake, glycolytic ATP production, and overall mitochondrial function. Consequently, IP6K1-deficient cells exhibited disrupted metabolic adaptation under hypoxic conditions and reduced survival under stress. These findings underscore the importance of the Cys-Arg/N-degron pathway in regulating metabolic responses and highlight its potential importance in hypoxia-related disorders.
    Keywords:  E3 ligases; N-degron; cysteine; hypoxia; protein degradation
    DOI:  https://doi.org/10.1073/pnas.2501681122
  9. Nat Commun. 2025 Aug 13. 16(1): 7523
    Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling.
    Cristiana C Santos, Nadine Schweizer, Fátima Cairrão, Juanma Ramirez, Nerea Osinalde, Ming Yang, Catarina J Gaspar, Vanya I Rasheva, Miguel L Trigo, Zach Hensel, Colin Adrain, Tiago N Cordeiro, Franka Voigt, Paulo A Gameiro, Ugo Mayor, Pedro M Domingos.
      The Unfolded Protein Response (UPR) is activated by the accumulation of misfolded proteins in the Endoplasmic Reticulum (ER), a condition known as ER stress. Prolonged ER stress and UPR activation cause cell death, by mechanisms that remain poorly understood. Here, we report that regulation of Ataxin-2 by Fbxo42 is a crucial step during UPR-induced cell death. From a genetic screen in Drosophila, we identify loss of function mutations in Fbxo42 that suppress cell death and retinal degeneration induced by the overexpression of Xbp1spliced, an important mediator of the UPR. We identify the RNA binding protein Ataxin-2 as a substrate of Fbxo42, which, as part of a Skp-A/Cullin-1 complex, promotes the ubiquitylation and degradation of Ataxin-2. Upon ER-stress, the mRNA of Xbp1 is sequestered and stabilized in Ataxin-2 granules, where it remains untranslated. Fbxo42 recruitment to these granules promotes the degradation of Ataxin-2, allowing for the translation of Xbp1 mRNA and triggering cell death during the terminal stages of UPR activation.
    DOI:  https://doi.org/10.1038/s41467-025-62417-2
  10. Mol Biol Cell. 2025 Aug 13. mbcE25010024
    Endosome maturation during ER stress relies on the ubiquitin binding domain of Histone Deacetylase 6.
    Katherine M Piscopo, Brooke Larson, Anna M Christiansen, Jason M Perry, Julie Hollien.
      Histone deacetylase 6 (HDAC6) helps cells manage misfolded proteins by transporting ubiquitin-associated structures toward the microtubule organizing center, where they can be sequestered and degraded by lysosomes. Here we show that when cells are subjected to acute protein folding stress in the endoplasmic reticulum (ER), HDAC6 depletion results in the appearance of enlarged endosomes that are highly decorated with ubiquitin and colocalize with both early and late endosome markers. The C-terminal ubiquitin-binding domain and adjacent disordered regions of HDAC6 are necessary and sufficient to rescue this endosomal phenotype in cells lacking endogenous HDAC6. HDAC6 deficiency does not appear to prevent the recruitment of endosomal sorting complexes required for transport (ESCRTs), which coordinate endosome maturation. However, overexpression of HDAC6 can reverse endosome phenotypes associated with the depletion of the early ESCRT factor HRS. We speculate that HDAC6 facilitates the packaging and processing of endosomal cargo when the endomembrane system is under stress.
    DOI:  https://doi.org/10.1091/mbc.E25-01-0024
  11. bioRxiv. 2025 Aug 07. pii: 2025.08.05.666879. [Epub ahead of print]
    Secreted Protein Production is Improved by Controlling Endoplasmic Reticulum Stress Associated Protein Degradation.
    Ryan Chauncey Splichal, Christina Chan, S Patrick Walton.
      Therapeutic proteins are produced frequently by mammalian cells in large-scale bioreactors. As a result, producer cells are exposed to a chemically (nutrients, gas exchange, target protein overexpression) and physically (shear due to mixing) stressful environment, which can lead to loss of proteostasis and endoplasmic reticulum (ER) stress. In response, cells activate the unfolded protein response (UPR). The UPR includes activation of autophagy and proteasomes, both of which target unfolded/misfolded proteins for degradation. To investigate the impacts of autophagy and proteasome activity on secreted protein production in ER-stressed cells, we used HeLa and MDA-MB-231 cells transfected to express Gaussia luciferase (as a model for therapeutic protein production) and exposed to tunicamycin (TM) (to activate ER stress). As expected, TM exposure decreased protein production and secretion. Inhibiting autophagy improved secretion in stressed cells as expected. However, counterintuitively, increasing proteasomal degradation improved secretion while inhibiting proteasomal activity decreased secretion, that is proteasomal activity was directly correlated to secretion. Taken together, our results demonstrate that protein secretion can be improved through control of autophagy and proteasomal activity, providing insight into strategies for improving yield from protein production bioprocesses.
    DOI:  https://doi.org/10.1101/2025.08.05.666879
  12. bioRxiv. 2025 Aug 05. pii: 2025.08.04.668575. [Epub ahead of print]
    RNA triggers chronic stress during neuronal aging.
    Kevin Rhine, Elle Epstein, Natasha M Carlson, Xuezhen Ge, Orel Mizrahi, Anika Kamat, Anita Hermann, William R Brothers, John Ravits, Eric J Bennett, Gülçin Pekkurnaz, Gene W Yeo.
      Neurodegenerative diseases are linked with dysregulation of the integrated stress response (ISR), which coordinates cellular homeostasis during and after stress events. Cellular stress can arise from several sources, but there is significant disagreement about which stress might contribute to aging and neurodegeneration. Here, we leverage directed transdifferentiation of human fibroblasts into aged neurons to determine the source of ISR activation. We demonstrate that increased accumulation of cytoplasmic double-stranded RNA (dsRNA) activates the eIF2α kinase PKR, which in turn triggers the ISR in aged neurons and leads to sequestration of dsRNA in stress granules. Aged neurons accumulate endogenous mitochondria-derived dsRNA that directly binds to PKR. This mitochondrial dsRNA leaks through damaged mitochondrial membranes and forms cytoplasmic foci in aged neurons. Finally, we demonstrate that PKR inhibition leads to the cessation of stress, resumption of cellular translation, and restoration of RNA-binding protein expression. Together, our results identify a source of RNA stress that destabilizes aged neurons and may contribute to neurodegeneration.
    DOI:  https://doi.org/10.1101/2025.08.04.668575
  13. J Biol Chem. 2025 Aug 09. pii: S0021-9258(25)02433-0. [Epub ahead of print] 110582
    The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.
    Adrija J Navarro-Traxler, Laura Ghisolfi, Evan C Lien, Alex Toker.
      The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT-hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.
    Keywords:  AKT; ALG3; N-glycosylation; PI3-kinase; glycans; phosphorylation; signaling
    DOI:  https://doi.org/10.1016/j.jbc.2025.110582
  14. Nat Commun. 2025 Aug 14. 16(1): 7575
    Targeted degradation of cell surface proteins through endocytosis triggered by cell-penetrating peptide-small molecule conjugates.
    Wanyi He, Congli Chen, Jiwei Zheng, Yanyan Li, Huaihuai Shi, Yimin Zhou, Meiqing Li, Ping Gong, Ke Liu, Ximing Shao, Xiaojun Yao, Hongchang Li, Liang Chen, Lijing Fang.
      Targeted degradation of membrane-associated proteins, which constitute a crucial class of drug targets implicated in diverse disease pathologies, has garnered considerable attention in chemical biology and drug discovery recently. Taking advantage of the endosomal entrapment of cell-penetrating peptides (CPPs) in delivering bioactive macromolecules, we successfully construct a CPP-based platform for specific degradation of cell surface proteins by conjugation of target protein-binding small molecules (SMs) with different CPPs, resulting in the formation of CPP-mediated lysosome-targeting chimeras (CPPTACs). Through the endo-lysosomal pathway, CPPTACs exhibit a remarkable ability to degrade clinically significant plasma membrane proteins, including PD-L1, CAIX, and CB2R. In contrast to LYTACs and similar technologies, CPPTACs drive the degradation of targets in a manner independent of specific lysosome-shuttling receptors, thus providing a widely applicable strategy for plasma membrane protein degradation, regardless of the cell types. Additionally, simpler structural design and broader therapeutic window for CPPTACs are expected since CPPs-mediated endocytosis and lysosomal degradation do not necessitate the three-component binding model typically required by other heterobifunctional degraders. Overall, consisting of small molecules and biocompatible cell-penetrating peptides, CPPTACs developed in this study represent a simple, adaptable, and effective approach for selectively degrading cell surface proteins in various cellular contexts with potential for application in both biological research and therapeutic interventions.
    DOI:  https://doi.org/10.1038/s41467-025-62776-w
  15. Br J Pharmacol. 2025 Aug 13.
    GCN2 regulates paclitaxel-induced neuropathic pain.
    Alexander R Mikesell, Angela R Meyer, Guadalupe García, Luke R Frietze, Cheryl L Stucky, Tao Pan, Zachary T Campbell.
       BACKGROUND AND PURPOSE: Neuropathic pain is debilitating and pervasive. Chemotherapeutic agents commonly induce chronic neuropathic pain. Paclitaxel is a prototypical example, causing painful peripheral neuropathy in a majority of patients. Paclitaxel triggers persistent changes in the excitability of sensory neurons resulting in hypersensitivity to sensory cues. The molecular mechanisms underlying paclitaxel-induced maladaptive plasticity are unclear. Here, we demonstrate a role for the Integrated Stress Response (ISR)-a key translational control mechanism-and its activating kinase, general control non-derepressible 2 kinase (GCN2), in paclitaxel-induced neuropathic pain (PINP).
    EXPERIMENTAL APPROACH: We used genetic and pharmacological techniques, including sensory neuron-specific GCN2 conditional knockout mice and the selective GCN2 inhibitor GCN2-IN-7. Behavioural assays assessed mechanical and cold hypersensitivity, while primary DRG neuron cultures were used to evaluate neuronal excitability via calcium imaging and protein translation by puromycin incorporation (surface sensing of translation, SUnSET). tRNA charging and abundance were measured using MSR-seq.
    KEY RESULTS: Paclitaxel robustly activated the ISR via GCN2 in mouse DRG sensory neurons, shown by increased eIF2α phosphorylation, elevated ATF4 levels and reduced global translation rates. Genetic deletion or pharmacological inhibition of GCN2 blocked paclitaxel-induced sensory neuron sensitisation and significantly attenuated mechanical and cold hypersensitivity in vivo. Mechanistically, paclitaxel reduced global tRNA charging and abundance in DRGs, providing a molecular basis for GCN2 activation.
    CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that GCN2-dependent ISR activation is critical for PINP. Targeting GCN2 may represent a promising therapeutic strategy for preventing or alleviating chemotherapy-induced peripheral neuropathy, potentially improving patient quality of life and chemotherapy tolerance.
    Keywords:  GCN2; ISR; PINP; eIF2
    DOI:  https://doi.org/10.1111/bph.70154
  16. Bioessays. 2025 Aug 11. e70054
    Defective Ribosome Recycling: A Bridge Between Translation Fidelity, Organelle Dysfunction, and Diseases: Exploring How Flawed Ribosome Recycling Factors Impact Ribosome Metabolism and Organelle Homeostasis, Leading to Human Diseases.
    Foozhan Tahmasebinia, Zhihao Wu.
      Ribosome recycling is a fundamental biological process crucial for cellular health. Defective recycling disrupts ribosome biogenesis and organelle function, particularly in mitochondria, contributing to ribosomopathies, neurodegenerative diseases, and cancer. While not directly linked to human diseases via known genetic mutations, emerging evidence suggests a critical interplay between ribosome recycling and organelle quality control. Impaired ribosome recycling leads to aberrant ribosome production, compromised translational quality control, protein misfolding, and subsequent organelle dysfunction and cellular stress. These cascading defects underscore the critical need for effective ribosome reutilization, especially under stress, as disruptions can cause translational arrest and heightened stress signaling, perturbing cellular homeostasis. Our analyses establish an indirect but significant link between ribosome recycling and human disease, offering new perspectives on how translational fidelity and organelle maintenance converge to support cellular well-being.
    Keywords:  40S subunit recycling; CAT‐tailing; cancer; mitochondria; neurodegenerative diseases; ribosome recycling; ribosome‐associated quality control (RQC)
    DOI:  https://doi.org/10.1002/bies.70054
  17. bioRxiv. 2025 Aug 05. pii: 2025.08.05.668603. [Epub ahead of print]
    Protein language models reveal evolutionary constraints on synonymous codon choice.
    Helen Sakharova, Liana F Lareau.
      Evolution has shaped the genetic code, with subtle pressures leading to preferences for some synonymous codons over others. Codons are translated at different speeds by the ribosome, imposing constraints on codon choice related to the process of translation. The structure and function of a protein may impose pressure to translate the associated mRNA at a particular speed in order to enable proper protein production, but the molecular basis and scope of these evolutionary constraints have remained elusive. Here, we show that information about codon constraints can be extracted from protein sequence alone. We leverage a protein language model to predict codon choice from amino acid sequence, combining implicit information about position and protein structure to learn subtle but generalizable constraints on codon choice in yeast. In parallel, we conduct a genome-wide screen of thousands of synonymous codon substitutions in endogenous loci in yeast, reliably identifying a small set of several hundred synonymous variants that increase or decrease fitness while showing that most positions have no measurable effect on growth. Our results suggest that cotranslational localization and translational accuracy, more than cotranslational protein folding, are major drivers of selective pressure on codon choice in eukaryotes. By considering both the small but wide-reaching effects of codon choice that can be learned from evolution and the strong but highly specific effects determined via experiment, we expose unappreciated biological constraints on codon choice.
    DOI:  https://doi.org/10.1101/2025.08.05.668603
  18. J Mol Biol. 2025 Aug 07. pii: S0022-2836(25)00444-9. [Epub ahead of print] 169378
    Subtle variations in a client protein determine bacterial Hsp90 dependence.
    Marie Corteggiani, Amine Ali-Chaouche, Miha Bahun, Flora A Honoré, Deborah Byrne, Sébastien Dementin, Mathieu E Rebeaud, Olivier Genest.
      Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilSSo) in the bacterium Shewanella oneidensis. Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilSSo from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli, suggesting that E. coli TilS (TilSEc) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify determinants of Hsp90 dependence. We found that in contrast to TilSSo, TilSEc was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilSEc is not a client of Hsp90. Chimeras between TilSSo and TilSEc as well as directed mutagenesis revealed a region of TilSSo that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilSEc was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilSSo instead of TilSEc. Altogether, our work reveals that protein-specific features, such as stability and degradation sensitivity, can determine whether orthologous proteins require the bacterial Hsp90 chaperone in vivo.
    Keywords:  Bacteria; Chaperone; Client specificity; HslU HslV; Hsp90; HtpG; Protease
    DOI:  https://doi.org/10.1016/j.jmb.2025.169378
  19. Nat Commun. 2025 Aug 13. 16(1): 7503
    The topological properties of the protein universe.
    Christian D Madsen, Agnese Barbensi, Stephen Y Zhang, Lucy Ham, Alessia David, Douglas E V Pires, Michael P H Stumpf.
      Deep learning methods have revolutionised our ability to predict protein structures, allowing us a glimpse into the entire protein universe. As a result, our understanding of how protein structure drives function is now lagging behind our ability to determine and predict protein structure. Here, we describe how topology, the branch of mathematics concerned with qualitative properties of spatial structures, provides a lens through which we can identify fundamental organising features across the known protein universe. We identify topological determinants that capture global features of the protein universe, such as domain architecture and binding sites. Additionally, our analysis identifies highly specific properties, so-called topological generators, that can be used to provide deeper insights into protein structure-function and evolutionary relationships. We present a practical methodology for mapping the topology of the known protein universe at scale. We then use our approach to determine structural, functional and disease consequences of mutations. Our approach reveals and helps to explain differences in properties of proteins in mesophiles and thermophiles, and the likely structural and functional consequences of polymorphisms in a protein. For eukaryotes we find striking differences between protein topologies in multi-cellular and single-celled organisms.
    DOI:  https://doi.org/10.1038/s41467-025-61108-2
  20. Nat Chem Biol. 2025 Aug 15.
    Structural basis for E4 enzyme Ufd2-catalyzed K48/K29 branched ubiquitin chains.
    Zebin Tong, Xiangwei Wu, Hongyi Cai, Shidian Wu, Tianyi Zhang, Zhiheng Deng, Ziyu Xu, Rujing Yuan, Huasong Ai, Lei Liu, Man Pan.
      E4 enzymes amplify and remodel ubiquitin chain signals beyond the conventional E1-E2-E3 cascade. The first identified E4 enzyme Ufd2 preferentially catalyzes K48/K29 branched ubiquitin chains, yet the structural mechanism remains unknown. Here, we combined chemical biology and cryo-electron microscopy to visualize stable intermediates in Ufd2 loading ubiquitin at K48 of proximal ubiquitin on K29-linked di- and triubiquitin. Our data reveal that the core region of Ufd2 functions as an unprecedented K29 diubiquitin binding domain, interacting extensively with proximal and distal ubiquitin, which orients the K48 site of proximal ubiquitin toward the active site of Ubc4, facilitating K48/K29 branched ubiquitin chain formation. We also identified a unique dimeric conformation where dimerized Ufd2 and Ubc4 stabilize each other's distal ubiquitin during branching on K29 triubiquitin. Our findings provide mechanistic insights into the assembly of K48/K29 branched ubiquitin chains by the E4 enzyme Ufd2 and highlight the spatial cooperation among multiple pairs of ubiquitin-related enzymes on longer ubiquitin chains.
    DOI:  https://doi.org/10.1038/s41589-025-01985-2
  21. Mol Cell. 2025 Aug 11. pii: S1097-2765(25)00614-8. [Epub ahead of print]
    Ubiquitination of the histone variant mH2A1.2 prevents toxic RAD18 accumulation at a subset of genomic loci upon replication stress.
    Maxime Galloy, Andréanne Blondeau, Élodie Vion, Daein Kim, Collin A Bakker, Vincent Gaggioli, Mélissa Thomas, Élise G Lavoie, Isabelle Marois, Alberto David Delgado Monterroso, Jean-Yves Masson, Nitika Taneja, Kyle M Miller, Alexandre Maréchal, Amélie Fradet-Turcotte.
      Spatiotemporal recruitment of DNA repair factors is crucial to coordinate repair across DNA lesions. MacroH2A1.2 (mH2A1.2), which accumulates in subgenomic regions that are difficult to replicate (e.g., common fragile sites, telomeres, and repeated sequences), selectively promotes homology-driven DNA repair; however, the mechanisms remain elusive. We report an unexpected role for RNF168-mediated mH2A1.2 ubiquitination in preventing aberrant RAD18 recruitment to subgenomic loci experiencing prolonged replication arrest in human cancer cells. Biochemical reconstitution revealed that RAD18 cannot bind ubiquitinated mH2A1.2-containing nucleosomes. In cells, loss of mH2A1.2 ubiquitination increases RAD18 and γH2AX levels at collapsed forks and sensitizes cells to replication stress. Depletion of RAD18, fork remodelers, or the endonuclease MUS81 rescues these phenotypes, indicating that mH2A1.2-ubiquitination prevents toxic RAD18 engagement at MUS81-dependent double-strand breaks (DSBs), which arise at collapsed forks in difficult-to-replicate sites experiencing prolonged arrest. Our findings highlight the detrimental consequences of inappropriate DNA processing by MUS81 at these loci.
    Keywords:  53BP1; E3 ubiquitin ligase RNF168; MUS81; RAD18; RNF169; chromatin; histone H2A variant; histone mH2A1.2; replication stress; single-ended double-strand break
    DOI:  https://doi.org/10.1016/j.molcel.2025.07.013
  22. J Agric Food Chem. 2025 Aug 13.
    DEK Interacts with IRE1α to Modulate Endoplasmic Reticulum Stress in Deoxynivalenol-Induced Intestinal Inflammation.
    Lingjie Zeng, Danni Yu, Jintao Xu, Ziwan Qu, Siting Wu, Chongwen Guo, Kaiyu Wang, ZiDa Lin, Ruqin Lin, Jikai Wen, Yiqun Deng.
      Deoxynivalenol (DON), a mycotoxin prevalent in grains and feeds, poses a serious threat to humans and animal health. The intestine is the primary target organ of DON toxicity. However, the molecular mechanisms linking DON exposure to intestinal injury remain poorly understood. Here, we identify DEK, a multifunctional nuclear protein, is a critical target protein of DON-induced intestinal damage through endoplasmic reticulum (ER) stress activation. We demonstrate that DON directly binds to DEK via key residues (Phe56, Lys68, and Leu106) and disrupts its interaction with IRE1α, a central regulator of the ER stress. Structural analyses revealed that Lys68 of DEK is essential for its binding to IRE1α. DON interferes with the interaction between DEK and IRE1α by binding to DEK and reducing its expression. DEK overexpression attenuated ER stress and inflammatory cytokine production (IL-1β, IL-6, TNF-α) in intestinal cells. Conversely, DEK knockdown exacerbates DON-triggered ER stress by activating the IRE1α/PERK/eIF2α pathways, thereby amplifying intestinal inflammation via MAPK signaling. In vivo, DON-treated mice and piglets exhibited intestinal villus atrophy, crypt hyperplasia, and DEK-dependent ER stress activation. Collectively, our findings reveal a novel DEK-IRE1α axis in DON toxicity, underscoring that DEK may serve as a potential therapeutic target for mitigating DON-induced intestinal injury.
    Keywords:  DEK protein; deoxynivalenol; endoplasmic reticulum stress; intestinal toxicity; ire1 α
    DOI:  https://doi.org/10.1021/acs.jafc.5c07460
  23. iScience. 2025 Aug 15. 28(8): 113121
    Manganese suppresses tumor growth through hyper-activating IRE1α.
    Ruoxi Shi, Ming Wang, Si Chen, Xinwei Hu, Xudong Zhao, Shixin Zhou, Congli Fan, Zihan Zhou, Likun Wang.
      IRE1α and its downstream XBP1 signal is the most conserved unfolded protein response pathway that cells utilize to combat endoplasmic reticulum stress, also known to be utilized by tumor cells to adapt to harsh environment, leading to tumor progression. Several inhibitors against IRE1α have been developed, some of which show promising effect in clinical trial for cancer therapy, but none of them have been used in practice. Considering that hyper-activation of IRE1α induces cell death, we hypothesize that activation of IRE1α could be an alternative way for tumor suppression. Here, we identified divalent manganese ion as a potent activator to IRE1α, which interacts with the cytosolic part of IRE1α directly, augmenting the downstream pro-apoptotic pathway but not the pro-survival outcome. Mn2+ limits tumor growth in xenograft model in an IRE1α-dependent way. Our finding suggests pharmacological activation of IRE1α as an underestimated but promising way in cancer therapy.
    Keywords:  Biological sciences; cancer; cancer systems biology; molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2025.113121
  24. Nat Commun. 2025 Aug 10. 16(1): 7375
    F-actin disassembly by the oxidoreductase MICAL1 promotes mechano-dependent VWF-GPIbα interaction in platelets.
    Jean Solarz, Christelle Soukaseum, Stéphane Frémont, Sébastien Eymieux, Camilia Nabli, Christelle Repérant, Elisa Rossi, Jean-Claude Bordet, Cécile V Denis, Pierre Mangin, Yacine Boulaftali, R Jeroen Pasterkamp, Hana Raslova, Dominique Baruch, Frédéric Adam, Arnaud Echard, Alexandre Kauskot.
      Mechano-dependent interactions are key to thrombus formation and hemostasis, enabling stable platelet adhesion to injured vessels. The interaction between von Willebrand factor (VWF) and the platelet receptor GPIb-IX-V is central to this process. While GPIbα connects to the actin cytoskeleton, whether actin dynamics are important for GPIbα function under hemodynamic, high shear conditions remains largely unknown. Here, we show that actin disassembly is critical for proper VWF-GPIbα binding under shear. Mechanistically, we identify the oxidoreductase MICAL1 as a shear-activated regulator that promotes local F-actin disassembly around the GPIb-IX-V complex. This enables its translocation to lipid rafts and reinforces VWF binding. MICAL1-deficient platelets display impaired adhesion, increased deformability under shear, and defective thrombus formation in vivo. Thus, MICAL1 drives shear-dependent actin remodeling that supports GPIb-IX-V mechanotransduction and platelet function. These findings uncover a role for actin oxidation in platelet adhesion, providing a connection between cytoskeletal redox control and platelet function during thrombus formation.
    DOI:  https://doi.org/10.1038/s41467-025-62487-2
  25. Nat Commun. 2025 Aug 14. 16(1): 7566
    Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.
    Yuan-Hung Lo, Hudson T Horn, Mo-Fan Huang, Wei-Chieh Yu, Chia-Mei Young, Qing Liu, Madeline Tomaske, Martina Towers, Antonia Dominguez, Michael C Bassik, Dung-Fang Lee, Lei S Qi, Jonathan S Weissman, Jin Chen, Calvin J Kuo.
      Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.
    DOI:  https://doi.org/10.1038/s41467-025-62818-3
  26. Proc Natl Acad Sci U S A. 2025 Aug 19. 122(33): e2508403122
    Client-scaffold interactions suppress aggregation of a client protein in model condensates.
    Rashik Ahmed, Rhea P Hudson, Julie D Forman-Kay, Lewis E Kay.
      Many studies have shown that sequestration of client proteins into condensates locally increases their concentrations and/or modulates their conformational landscapes to promote aberrant aggregation. Far fewer examples have emerged where the proteinaceous condensed phase environment protects clients from aggregation. Here, we show that a condensate scaffolded by the C-terminal disordered region of Cell Cycle Associated Protein 1 (CAPRIN1) suppresses aggregation of the Fused in Sarcoma (FUS) RNA Recognition Motif (RRM) client, both components of stress granules. Although FUS RRM aggregation is mediated through the unfolded ensemble, comparative NMR studies of the FUS RRM outside and within the condensate establish that CAPRIN1 condensates attenuate FUS RRM aggregation despite locally increasing its concentration by twofold and significantly unfolding the domain. Regions of transient intermolecular contacts between unfolded FUS RRM protomers that could drive aggregation have been identified, including the hydrophobic segments spanning I287-I308 and G335-A369. Intermolecular NOE experiments recorded on the FUS RRM:CAPRIN1 condensate indicate that CAPRIN1 interacts with much of the unfolded FUS RRM, with regions of stronger contacts including the RRM sequences 287IFVQ290, 296VTIES300, 322INLY325, and 351IDWFDG356. These interactions collectively outcompete the homotypic contacts between unfolded FUS RRM clients driving aggregation. Our results demonstrate that condensate scaffold molecules can, in some cases, shield client interprotomer interactions, delaying or completely suppressing their aggregation.
    Keywords:  CAPRIN1; FUS RRM; NMR spectroscopy; phase separation; protein free energy landscape
    DOI:  https://doi.org/10.1073/pnas.2508403122
  27. Mol Cell Proteomics. 2025 Aug 07. pii: S1535-9476(25)00146-X. [Epub ahead of print] 101047
    Localisation of Organelle Proteins using Data-Independent Acquisition (DIA-LOP).
    K J A McCaskie, C Hutchings, R Feret, Y Kim, L M Breckels, M J Deery, K S Lilley.
      Subcellular localisation within the proteome fundamentally influences cellular processes, however the development of high-throughput techniques to allow proteome-wide mapping of the cell has proven difficult. Here we present DIA-LOP, an approach capable of high-throughput spatial proteome mapping with in-depth subcellular resolution. This unified framework integrates differential-ultracentrifugation (DC) with ion-mobility-based data-independent acquisition mass spectrometry, alongside data processing using DIA-NN and spatial analysis within the pRoloc bioinformatics pipeline. We obtain the largest DIA-based subcellular proteomics map, with 8242 protein identifications across 13 organellar compartments in U-2 OS cells. Within the same experimental pipeline, we compare DC fractionation with an alternate detergent-based protocol using either DIA or data-dependent acquisition (DDA) mass spectrometry approaches, highlighting the increased subcellular resolution of the DC approach and the increased proteome coverage when DIA is applied. We demonstrate the ability of DIA-LOP to inform clinical studies by identifying and mapping disease related proteins within our osteosarcoma cell model. With impressive coverage and resolution, DIA-LOP provides a straightforward, high-throughput tool for biochemical discovery. This study thus informs potential users of subcellular proteomics strategies that employ biochemical fractionation of the optimal workflows to achieve high proteome coverage and subcellular resolution.
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101047
  28. Structure. 2025 Aug 05. pii: S0969-2126(25)00261-8. [Epub ahead of print]
    Small molecule dysregulation of ClpP activity via bidirectional allosteric pathways.
    Marim M Barghash, Mark F Mabanglo, Samuel E Hoff, Dmytro Brozdnychenko, Keith S Wong, Gursonika Binepal, Philbert Ip, Jiarui Shen, Tomohiro Furukawa, Hidekazu Katayama, Vincent Trudel, Joanne Tan, Andrei K Yudin, Scott D Gray-Owen, Shohei Sakuda, Robert A Batey, Siavash Vahidi, Massimiliano Bonomi, Walid A Houry.
      The bacterial ClpP protease is essential for the virulence and infectivity of many human pathogens and has emerged as a novel antibacterial drug target. Several classes of small molecules dysregulate or activate ClpP, leading to uncontrolled protein degradation and cell death. Here, we investigate the mechanism of ClpP activation by these compounds using an integrative approach combining structural, biochemical, and computational tools. We identified small molecules that activate ClpP through binding at internal catalytic sites where peptide bond hydrolysis occurs. Combined with knowledge of ClpP activation by small molecules that bind to external hydrophobic sites, this work sheds light on the mechanisms governing ClpP allostery and identifies a common molecular pathway utilized by site-specific effectors to achieve allosteric activation. We propose a consensus, bidirectional ClpP activation mechanism causing protease dysregulation.
    Keywords:  ClpP; Escherichia coli; Neisseria meningitidis; X-ray crystallography; activators of self-compartmentalizing proteases; boronic acids; dioctatin; drug design; hydrogen-deuterium exchange mass spectrometry; molecular dynamics simulations
    DOI:  https://doi.org/10.1016/j.str.2025.07.013
  29. Biochemistry. 2025 Aug 12.
    Functional Proximity across an mRNA.
    Breanne M Hatfield, Chase A Weidmann, Christina A McCutchin, Patrick S Irving, Irma M Nelson, Simon Felder, Kevin M Weeks.
      Extensive RNA-protein interactions occur throughout the lifecycle of an mRNA that up- and down-regulate mRNA translation and degradation. Modulating interactions between regulatory proteins and mRNAs can have large effects on gene expression and might be useful for creating therapeutic manipulations, especially for difficult-to-drug proteins. Here, we directed three degradation-inducing proteins that normally bind the 5' cap, 3'-untranslated region (UTR), or 3' poly(A) tail to unconventional sites spanning coding and noncoding regions across a reporter mRNA. DCP2, the 5' decapping enzyme, reduced expression only when targeted to the 5'-UTR. ZFP36L2, a 3'-UTR adapter protein, reduced expression when directed to either the 3'-UTR or the 3' half of the coding sequence, the latter region outside its conventional site. CNOT7, a catalytic subunit within the CCR4-NOT deadenylase complex, reduced expression when directed anywhere in the mRNA, most strongly at both "ends" in the 5'- and 3'-UTRs. mRNAs can therefore be degraded by directing proteins to positions far from their conventionally understood regulatory sites. Our study reveals extensive through-space functional proximity across an mRNA. These observations have broad implications for understanding large-scale RNA structure and for emerging applications in therapeutically targeted RNA degradation.
    DOI:  https://doi.org/10.1021/acs.biochem.5c00340
  30. Nat Commun. 2025 Aug 09. 16(1): 7360
    Opposing regulation of the K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis.
    Chi Hyun Hwang, Minhong Lee, Ju Won Kim, Young Woo Nam, Gyuho Hwang, Hyun Sung Ryu, Jinho Seo, Eun-Woo Lee, Hyuk Wan Ko, Jaewhan Song.
      Receptor-interacting protein kinase 3 (RIPK3), a key regulator of necroptosis, is modulated by ubiquitination through various E3 ligases and deubiquitinases. However, the effects of different polyubiquitination processes on RIPK3 and necroptosis remain unclear. Using a proteomic approach, we identify SMAD Ubiquitination Regulatory Factor 1 (SMURF1) and Ubiquitin-specific peptidase 5 (USP5) as crucial regulators of RIPK3 within the necrosome during necroptosis. SMURF1 facilitates K63 polyubiquitination of RIPK3 at lysine 55 and 363, inhibiting necrosome formation and necroptosis. SMURF1 depletion accelerates necroptosis, while the reintroduction of functional SMURF1 reverses this. Conversely, USP5 acts as a deubiquitinase, removing K63 ubiquitin chains and promoting necroptosis. Reducing SMURF1, using a RIPK3 mutant defective in SMURF1-mediated ubiquitination, or overexpressing USP5 enhances necroptosis in leukaemia cells, leading to reduced tumour growth in xenograft models treated with birinapant and emricasan. These findings highlight the opposing regulation of K63-linked polyubiquitination of RIPK3 by SMURF1 and USP5 in necroptosis.
    DOI:  https://doi.org/10.1038/s41467-025-62723-9
  31. bioRxiv. 2025 Jul 14. pii: 2025.07.11.664427. [Epub ahead of print]
    SCOPE: Revealing Hidden Mechanisms in Phenotypic Screens Through Target and Pathway Enrichment.
    Abhijeet Kapoor, Keith Kelleher, Suzanne Underhill, Sankalp Jain, Brandon K Harvey, Mark J Henderson.
      Phenotypic screening enables discovery of small molecules without requiring predefined targets, but mechanistic interpretation remains challenging due to polypharmacology and pathway complexity. We developed SCOPE (Screening Compound Ontology for Pathway Enrichment), a KNIME-based computational framework that resolves the molecular drivers of phenotypic activity by linking compound-level screening data to annotated targets and pathways. SCOPE integrates multi-source target annotations and performs statistical enrichment to identify shared mechanisms of action. Applied to a high-throughput screen for modulators of ER-stress induced secretion of endoplasmic reticulum (ER) resident proteins, a process known as exodosis, SCOPE identified calcium signaling as the most enriched KEGG pathway without prior biological context. Target enrichment revealed G protein-coupled receptors (GPCRs) involved in inositol 1,4,5-trisphosphate receptors (IP3Rs)-mediated signaling, with widespread antagonism among hit compounds implicating this pathway in the regulation of exodosis. Notably, SCOPE uncovered a novel role for the histamine receptor HRH1, which was validated by RNAi knockdown and pharmacological inhibition, implicating HRH1 as a potential therapeutic target in ER stress-related disorders. These results highlight SCOPE's potential to deconvolute phenotypic screens and uncover actionable mechanisms in complex cellular systems.
    DOI:  https://doi.org/10.1101/2025.07.11.664427
  32. iScience. 2025 Aug 15. 28(8): 113131
    Cargo-selective regulation of clathrin-mediated endocytosis by AMP-activated protein kinase.
    Laura A Orofiamma, Ralph Christian Delos Santos, Ayshin Mehrabi, Nikol Leshchyshyn, Geoffrey G Hesketh, Sadia Rahmani, Aesha Patel, Farnaz Fekri, Rehman Ata, Colin D H Ratcliffe, Mathieu J F Crupi, Lois Mulligan, Morag Park, Anne-Claude Gingras, Costin N Antonescu.
      The cell surface abundance of many proteins is controlled by clathrin-mediated endocytosis (CME). CME is driven by the assembly of clathrin and other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). Regulation of CCP dynamics allows for control of the function of specific cell surface proteins, impacting a range of cellular outcomes. AMP-activated protein kinase (AMPK) becomes activated upon metabolic insufficiency and facilitates cellular adaptation to nutrient stress. Here, we examined how AMPK regulates CME and the cell surface membrane traffic of β1-integrin. We find that AMPK controls CCP dynamics and regulates the abundance of the endocytic adaptor protein Dab2 within CCPs in a manner that requires the GTPase Arf6, thus selectively promoting the CCP recruitment and internalization of β1-integrin. This study reveals a signaling pathway for cargo-selective metabolic regulation of CME by AMPK that impacts the function of cell surface proteins such as integrins.
    Keywords:  Biochemistry; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2025.113131
  33. Nucleic Acids Res. 2025 Aug 11. pii: gkaf759. [Epub ahead of print]53(15):
    A common structural mechanism for RNA recognition by the SF3B complex in mRNA splicing and export.
    Yuzhu Zhang, Changping Yin, Yimin Wang, Kunming Yan, Bo Zhao, Xin Hu, Youzhong Wan, Hong Cheng, Jing Huang.
      The SF3B complex plays a critical role in branch point adenosine recognition during pre-mRNA splicing. Its largest subunit SF3B1 is frequently mutated in cancers, leading to aberrant alternative splicing. Besides its function in pre-mRNA splicing, the SF3B complex also binds mature or intronless mRNAs to facilitate their nuclear export. Notably, the RNA motifs recognized by the SF3B complex exhibit no apparent sequence similarities, raising the question of how the SF3B complex recognizes diverse mRNA sequences for various cellular activities. Here we report the cryo-EM structures of the human SF3B complex associated with either intronless histone mRNAs or intron-U2 snRNA. These structures unveil that both mRNA molecules adopt a similar conformation featuring a bulged adenosine and bind the SF3B complex in a remarkably resembling manner, suggesting that SF3B recognizes the specific shape rather than the sequence of its RNA targets. Further cryo-EM and molecular dynamics analyses of the hotspot-mutant SF3B complexes bound to intron-U2 snRNA demonstrate that the SF3B1K700E and SF3B1R625H mutations similarly repel the attachment of the intronic polypyrimidine tract around the mutation sites, leading to reduced RNA-binding affinity. Altogether, our study provides structural insights into the RNA-recognition mechanism of the SF3B complex and suggests that the cancer-associated SF3B1 mutations could potentially affect multiple cellular processes including mRNA splicing and export, which advances our understanding of the pathogenic mechanisms of the SF3B1 mutations.
    DOI:  https://doi.org/10.1093/nar/gkaf759
  34. Cell Mol Life Sci. 2025 Aug 13. 82(1): 313
    RER1 regulates lipid metabolism in monocytes and macrophages.
    Yanxia Liu, Sandra Theil, Mohamed H Yaghmour, Anja Kerksiek, Peng Chen, Ingo G H Schmidt-Wolf, Rebecca Barker, Eva Bartok, Dieter Lütjohann, Christoph Thiele, Jochen Walter.
      Retention in endoplasmic reticulum sorting receptor 1 (RER1) mediates the retention and retrieval of select cargo proteins, and thereby regulates protein transport in the secretory pathway and assembly of distinct protein complexes. Recently, RER1 was implicated in the assembly and subcellular transport of the TREM2-DAP12 immune receptor complex, and its function in intracellular signaling and phagocytosis. However, the role of RER1 in the regulation of immune cell metabolism remained unknown. Here, we demonstrate an important role of RER1 in the lipid metabolism of monocytic and macrophage-like differentiated THP-1 cells. The deficiency of RER1 resulted in the accumulation of lipid droplets (LDs) in both monocytes and macrophage-like cells. Comprehensive mass spectrometry analyses revealed complex changes in the cellular lipid metabolism and the composition of LDs. RNA sequencing revealed an important role of RER1 in the regulation of genes related to lipid metabolism. Further, western immunoblotting confirmed an important role of RER1 in the expression of select key proteins involved in cellular lipid metabolism. The combined data indicate that RER1 plays an essential role in lipid metabolism in monocytes and macrophages.
    DOI:  https://doi.org/10.1007/s00018-025-05817-3
  35. Commun Biol. 2025 Aug 13. 8(1): 1208
    UBAP2L-driven stress granule formation links oxaliplatin resistance to gastric cancer.
    Chaorui Wu, Yu Yan, Qichen Chen, Zhiying Lian, Jiayong He, Ruoyu Ling, Xuetao Lei, Yanmei Peng, Boyang Zheng, Qingbin Yang, Gengtai Ye, Wenhui Ma, Guoxin Li.
      Stress granules (SGs), which are phase-separating organelles that serve as protective cellular mechanisms in response to various harmful stimuli, have an unclear role in oxaliplatin resistance. Here, we establish a causal link between SG formation and oxaliplatin resistance in GC. Notably, we identify a key SG nucleator, namely, ubiquitin-associated protein 2-like (UBAP2L), as a previously unrecognized critical factor in mediating oxaliplatin resistance. UBAP2L-nucleated SG-mediated inhibition of apoptosis is associated with the recruitment of receptor of activated protein C kinase 1 (RACK1), a known promoter of apoptosis, to these entities. Transcriptional upregulation of UBAP2L is enhanced by oxaliplatin-induced phosphorylation and activation of heat shock factor protein 1 (HSF1) via AKT. Inhibiting either SG or HSF1 significantly overcomes oxaliplatin resistance in vivo. These findings demonstrate that UBAP2L-nucleated SGs play a vital role in mediating oxaliplatin resistance, with elevated SG levels emerging as a promising therapeutic target for overcoming this resistance.
    DOI:  https://doi.org/10.1038/s42003-025-08584-w
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