bims-proteo Biomed News
on Proteostasis
Issue of 2025–12–21
fifty-one papers selected by
Eric Chevet, INSERM
  1. 5' UTR-mediated retention of eIF3 on 80S ribosomes promotes co-translational folding of ER membrane proteins.
  2. Protein quality control: from molecular mechanisms to aging and disease - EMBO workshop, May 18-23, 2025, Hersonissos, Greece.
  3. The G3BP stress-granule proteins reinforce the integrated stress response translation programme.
  4. Structural Basis for Substrate Recruitment and Catalytic Ubiquitin Transfer by the E2/E3 Hybrid Enzyme UBE2O.
  5. Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae.
  6. AUTOPHAGIC punctum a self-sensing vacuole/lysosome: V-ATPase dysfunction activates selective autophagy.
  7. Covalent Reprogramming of Kinase Binders to Modulate Protein Homeostasis.
  8. Rif-seq reveals Caulobacter crescentus mRNA decay is globally coordinated with transcription and translation.
  9. Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency.
  10. Stress to exhaustion: Proteotoxicity in T cells.
  11. Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response.
  12. How Tailored Ribo-Seq Methods Probe Unique Translation Events.
  13. TBK1 Induces the Formation of Optineurin Filaments That Condensate with Polyubiquitin and LC3 for Cargo Sequestration.
  14. Mechanism of cotranslational modification of histones H2A and H4 by MetAP1 and NatD.
  15. 1-Deoxysphingolipids Require Very-Long-Chain Ceramide Synthesis to Induce ER Stress and Neurotoxicity.
  16. Pathogenic podocin variants exhibit distinct defects in trafficking, membrane organization, and degradation pathways.
  17. Inhibiting translation elongation by reducing eIF5A activity induces feedback inhibition of initiation, limiting tumour cell proliferation.
  18. A practical method for determining the rate of covalent modification of fragments and leads.
  19. NoxO1 promotes endosome formation and reduces intracellular vesicle processing.
  20. DIS3L2-mediated RNA surveillance and extracellular vesicle packaging prevent innate immune activation by aberrant cellular RNAs.
  21. A matter of selectivity: ER-phagy suppression at the onset of pancreatic cancer.
  22. CD147 promotes NSCLC metastasis by inducing secretory autophagy-dependent exosome secretion via TRIM56-mediated ubiquitination and degradation of GCN2.
  23. Stress-specific NONO interactomes reveal a key role of Hsp70 chaperone activity in regulation of paraspeckle formation.
  24. Ribosome-binding protein 1 maintains peroxisome biogenesis.
  25. Vesicle-mediated mitochondrial clearance presents an actionable metabolic vulnerability in triple-negative breast cancer.
  26. The ribosome derives the energy to translocate and unwind mRNA from EF-G binding.
  27. Cell-surface RNA forms ternary complex with RNA-binding proteins and heparan sulfate to recruit immune receptors.
  28. Subcellular In Vivo Cardiac Proteomics Reveals Sarcomere and Ribosome Interactomes and skNAC's Impact on Cardiac Proteostasis.
  29. Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease.
  30. RHOA regulates mitochondria-ER contact sites through modulation of the VAPB/PTPIP51 tether.
  31. Computational Modeling of PROTAC Ternary Complexes as Ensembles Using SILCS-xTAC.
  32. Mutant CHCHD10 disrupts cytochrome c oxidation and activates mitochondrial retrograde signaling.
  33. Development of Cathepsin B-Responsive GalNAc-PROTACs for Hepatocyte-Targeting Protein Degradation.
  34. VarLand: A pipeline to map the structural landscape of missense variants at the proteome scale.
  35. Lymphodepleting chemotherapy potentiates neoantigen-directed T cell therapy by enhancing antigen presentation.
  36. SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.
  37. ARAP2 regulates responses to interferon-gamma by restricting SOCS1.
  38. Interaction of lncRNA LENT with DHX36 regulates translation and suppresses autophagy in melanoma.
  39. Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay.
  40. A Facile Protocol for C(sp2)-C(sp3) Bond Formation Reactions Toward Functionalized E3 Ligase Ligands.
  41. MLKL/retromer axis controls PD-L1 recycling to compromise antitumor immunity during VCP inhibition-induced necroptosis.
  42. Discovery of Non-Degradative Covalent Molecular Glues for Transcriptional Reprogramming.
  43. Targeting PRDX6-dependent localization and function of GPX4 enhances ferroptosis-mediated tumor suppression.
  44. BoltzGen: Toward Universal Binder Design.
  45. TDP-43 dysfunction compromises UPF1-dependent mRNA metabolism in ALS.
  46. SLAP controls mTORC2 integrity via UBE3C-mediated non-degradative mLST8 ubiquitination to suppress colorectal tumorigenesis.
  47. Homotypic endoplasmic reticulum membrane tethering is critical for flavivirus replication.
  48. PDIA3 Inhibition Facilitates Sensitivity of IKE-Induced Ferroptosis via STAT3/LCN2 Axis to Improve Glioblastoma Therapy.
  49. Degradation factor 1, Def1, regulates mRNA translation and decay through Ccr4-Not-dependent ubiquitylation of the ribosome.
  50. Vesicle-coupled mRNA transport and translation govern intracellular organelle networking.
  51. An open-source screening platform accelerates discovery of drug combinations.
  1. Cell Rep. 2025 Dec 12. pii: S2211-1247(25)01434-2. [Epub ahead of print]44(12): 116662
    5' UTR-mediated retention of eIF3 on 80S ribosomes promotes co-translational folding of ER membrane proteins.
    Baochun Han, Siqiong Zhang, Yurui Zhang, Binbin Yu, Weimin Lin, Yabin Cheng, Haoran Duan, Dieter A Wolf.
      Co-translational folding of nascent polypeptides is essential for protein function and cellular homeostasis. Ribosome-associated chaperones assist in this process, but coordination between their recruitment and translation initiation remains poorly understood. We report here that specific binding sites for eukaryotic translation initiation factor eIF3 within the 5' UTRs of mRNAs promote its retention on 80S ribosomes during the synthesis of select endoplasmic reticulum (ER) membrane proteins. Disruption of these eIF3 binding sites leads to misfolding and sequestration of newly synthesized membrane proteins into ER whorls. Sequestration into ER whorls is exacerbated by HSP70 inhibition but can be rescued by overexpressing HSPA1 and HSPA8. Cross-linking assays reveal that 5' UTR binding sites stabilize eIF3-80S interactions during early elongation, facilitating recruitment of HSPA1 and HSPA8 to ribosomes. These findings indicate that genetic instructions within 5' UTRs direct eIF3-mediated chaperone recruitment, ensuring proper co-translational folding of ER membrane proteins.
    Keywords:  5′ UTR; CP: molecular biology; ER membrane proteins; ER whorls; HSP70 chaperones; co-translational folding; eIF3
    DOI:  https://doi.org/10.1016/j.celrep.2025.116662
  2. Cell Stress Chaperones. 2025 Dec 16. pii: S1355-8145(25)00084-7. [Epub ahead of print] 100139
    Protein quality control: from molecular mechanisms to aging and disease - EMBO workshop, May 18-23, 2025, Hersonissos, Greece.
    Claes Andréasson, Anat Ben-Zvi.
      Cells safeguard the functionality of the proteome using complex pathways of protein quality control. The centerpiece of this proteostasis network is a large set of molecular chaperones and proteases that impact the entire lifespan of proteins by controlling protein folding and degradation. Dysfunction of the proteostasis network is associated with many diseases and age-associated functional decline of neurons, including Alzheimer's and Parkinson's diseases, as well as several motor neuron diseases. The 2025 EMBO workshop "Protein quality control: from molecular mechanisms to aging and disease" gathered the large and interdisciplinary community of researchers that study protein quality control, from its fundamental molecular mechanisms via higher order organization in organisms to its impact on and use in the medical field. Here we summarize the workshop and report research findings.
    Keywords:  Aggregation, Autophagy; Chaperone; Folding, Protein quality control; Proteostasis; Stress ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.1016/j.cstres.2025.100139
  3. Nat Cell Biol. 2025 Dec 19.
    The G3BP stress-granule proteins reinforce the integrated stress response translation programme.
    Jarrett Smith, David P Bartel.
      When mammalian cells are exposed to stress, they co-ordinate the condensation of stress granules (SGs) through the action of proteins G3BP1 and G3BP2 (G3BPs) and, simultaneously, undergo a massive reduction in translation. Although SGs and G3BPs have been linked to this translation response, their overall impact has been unclear. Here we investigate the question of how, and indeed whether, G3BPs and SGs shape the stress translation response. We find that SGs are enriched for mRNAs that are resistant to the stress-induced translation shutdown. Although the accurate recruitment of these stress-resistant mRNAs does require the context of stress, a combination of optogenetic tools and spike-normalized ribosome profiling demonstrates that G3BPs and SGs are necessary and sufficient to both help prioritize the translation of their enriched mRNAs and help suppress cytosolic translation. Together, these results support a model in which G3BPs and SGs reinforce the stress translation programme by prioritizing the translation of their resident mRNAs.
    DOI:  https://doi.org/10.1038/s41556-025-01834-3
  4. J Biol Chem. 2025 Dec 17. pii: S0021-9258(25)02925-4. [Epub ahead of print] 111073
    Structural Basis for Substrate Recruitment and Catalytic Ubiquitin Transfer by the E2/E3 Hybrid Enzyme UBE2O.
    Darja Kordic, Thomas L Williams, Luiza Deszcz, Julian F Ehrmann, Renato Arnese, Alexander Schleiffer, Tim Clausen, Anton Meinhart.
      UBE2O is a promiscuous ubiquitin ligase involved in cellular quality control pathways. Along with BIRC6, UBE2O is one of only two E2 enzymes that can ubiquitinate substrates in an E3 independent manner. The E2/E3 hybrid targets and multi-monoubiquitinates a multitude of orphan proteins, however the mechanisms underlying substrate specificity and ubiquitin transfer remain poorly understood. By combining structural and biochemical approaches, we show that substrate binding by UBE2O occurs through a conserved acidic pocket formed by the N-terminal SH3-like domains and that this platform allows the recruitment of a broad range of proteins. Furthermore, we identified specific residues in the catalytic UBC domain that position ubiquitin in a closed state confirmation, priming it for nucleophilic attack by the incoming substrate. Importantly, the activated E2∼Ub conjugate is protected by a tryptophan residue avoiding premature hydrolysis. By incorporating these findings into the UBC domain of BIRC6 our data provide the molecular basis of how specialized E2/E3 hybrid proteins function as potent ubiquitination enzymes reminiscent of the catalytic principle of RING E3 ligases.
    DOI:  https://doi.org/10.1016/j.jbc.2025.111073
  5. G3 (Bethesda). 2025 Dec 17. pii: jkaf284. [Epub ahead of print]
    Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae.
    Matthew D Berg, Alexis T Chang, Ricard A Rodriguez-Mias, Judit Villén.
      Transfer RNAs (tRNAs) ensure accurate decoding of the genetic code. However, mutations in tRNAs can lead to misincorporation of an amino acid that differs from the genetic message in a process known as mistranslation. As mistranslating tRNAs modify how the genetic message is decoded, they have potential as therapeutic tools for diseases caused by nonsense and missense mutations. Despite this, they also produce proteome-wide mismade proteins, which can disrupt proteostasis. To better understand the impact of mistranslating tRNA variants, we profile the proteome and phosphoproteome of yeast expressing three different mistranslating tRNAs. While the overall impacts were similar, the extent of growth defects and proteome changes varied with the substitution type. Although the global impacts were modest, mistranslation influenced key cellular processes, including proteostasis, cell cycle, and translation. These findings highlight the need to consider cellular consequences when developing mistranslating tRNAs for therapeutic applications.
    Keywords:  mass spectrometry; mistranslation; phosphoproteomics; proteomics; proteostasis; tRNA biology
    DOI:  https://doi.org/10.1093/g3journal/jkaf284
  6. Autophagy. 2025 Dec 18.
    AUTOPHAGIC punctum a self-sensing vacuole/lysosome: V-ATPase dysfunction activates selective autophagy.
    Yuxiang Huang, Daniel J Klionsky.
      Macroautophagy/autophagy has long been viewed as being strictly dependent on vacuolar or lysosomal acidity, with the vacuolar-type H+ -translocating ATPase (V-ATPase) functioning mainly as a proton pump that sustains degradation. Our recent paper overturns this paradigm, revealing that loss of V-ATPase activity paradoxically induces a selective autophagy program in nutrient-replete Saccharomyces cerevisiae. Vacuolar deacidification triggers a signaling cascade through the Gcn2-Gcn4/ATF4 integrated stress response, which drives Atg11-dependent ribophagy even when TORC1 remains active. This "V-ATPase-dependent autophagy" operates as a self-corrective feedback loop: when the vacuole's degradative capacity falters, it signals its own dysfunction to restore homeostasis. Tryptophan and NAD+ metabolism modulate this response, linking metabolic balance to autophagy induction. This discovery reframes the vacuole/lysosome from a passive endpoint to an active sensor of cellular integrity. It also challenges the use of V-ATPase inhibitors such as bafilomycin A1 as neutral autophagy flux blockers, because inhibition itself can stimulate autophagy induction. Collectively, these findings position the V-ATPase as a bidirectional regulator - both gatekeeper and sentinel - governing how cells translate organelle stress into adaptive autophagy.
    Keywords:  ATF4/Gcn4; NAD+ metabolism; V-ATPase; ribosome biogenesis; selective autophagy; tryptophan metabolism
    DOI:  https://doi.org/10.1080/15548627.2025.2604345
  7. bioRxiv. 2025 Dec 01. pii: 2025.11.30.691461. [Epub ahead of print]
    Covalent Reprogramming of Kinase Binders to Modulate Protein Homeostasis.
    Chen Mozes, Xiaokang Jin, Miguel A Campos, Chen Zhou, Xiaoyu Zhang.
      Small molecules that modulate protein abundance through induced proximity have expanded the landscape beyond traditional inhibition. Here, we explore how introducing covalent or latent electrophilic groups into a multi-kinase binder scaffold can reprogram protein homeostasis within the kinase family. Using the broad-spectrum kinase ligand TL13-87 as a template, we synthesized analogs bearing α-chloroacetamide, acrylamide, or terminal amine groups. Quantitative proteomics revealed that while most analogs had minimal global impact, MKI-AA uniquely stabilized the mitotic kinase AURKA, a protein often destabilized by ATP-competitive inhibitors. Mechanistic studies showed that MKI-AA acts post-translationally to suppress AURKA ubiquitination and proteasomal degradation. Proteomic mapping of MKI-AA-induced AURKA interactors revealed changes in protein associations upon treatment, providing mechanistic insights into how MKI-AA influences AURKA stability. Intriguingly, adding a short linker to MKI-AA converted it from a stabilizer into a degrader, highlighting how subtle structural variations can invert functional outcomes. These findings demonstrate that electrophilic ligand design can modulate kinase stability and reveal a previously unrecognized mode of covalent proximity-driven protein stabilization.
    DOI:  https://doi.org/10.1101/2025.11.30.691461
  8. Cell Rep. 2025 Dec 12. pii: S2211-1247(25)01463-9. [Epub ahead of print]44(12): 116691
    Rif-seq reveals Caulobacter crescentus mRNA decay is globally coordinated with transcription and translation.
    Kulathungage H Dilrangi, James R Aretakis, Kavya Vaidya, Nadra Al-Husini, Nisansala S Muthunayake, Sangjin Kim, Jared M Schrader.
      While transcription and translation have been shown to be coordinated with mRNA decay across various single-gene studies, their global coordination remains poorly defined. Therefore, we utilize rifampicin sequencing experiments in Caulobacter crescentus to measure genome-wide mRNA lifetimes and analyze the impact of transcription and translation. Based on the RNA polymerase elongation speed, we identify that approximately 20% of the transcriptome is cotranscriptionally degraded. To investigate translation's impact on mRNA decay, we find that translation efficiency measured by ribosome profiling correlates with mRNA lifetime. We compare the 5' P cleavage sites to ribosome occupancy and find that cleavage sites occur preferentially in regions of low ribosome occupancy. Using the translation initiation inhibitor retapamulin, which traps ribosomes at the start codon, and chloramphenicol, which arrests elongating ribosomes, we show that ribosomes directly protect the occupied mRNA regions. Taken altogether, mRNA decay is globally interconnected with transcription and translation.
    Keywords:  CP: Microbiology; CP: Molecular biology; Caulobacter crescentus; Rif-seq; absolute quantitation; mRNA decay; ribosome occupancy
    DOI:  https://doi.org/10.1016/j.celrep.2025.116691
  9. bioRxiv. 2025 Nov 30. pii: 2025.11.26.690573. [Epub ahead of print]
    Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency.
    Huigang Shi, Xiaorong Wang, Clinton Yu, Haibin Mao, Fenglong Jiao, Merav Braitbard, Ben Shor, Zhongsheng Zhang, Thomas R Hinds, Shiyun Cao, Erkang Fan, Dina Schneidman-Duhovny, Lan Huang, Ning Zheng.
      Orthosteric inhibitors block enzyme active sites and prevent substrates from binding. Enhancing their specificity through substrate dependence seems inherently unlikely, as their mechanism hinges on direct competition rather than selective recognition. Here, we show that a molecular glue mechanism unexpectedly imparts substrate-dependent potency to CSN5i-3, an orthosteric inhibitor of the COP9 signalosome (CSN). We first confirm that CSN5i-3 inhibits CSN, which catalyzes NEDD8 deconjugation from the cullin-RING ubiquitin ligases (CRLs), by occupying the active site of its catalytic subunit, CSN5, and directly competing with the iso-peptide bond substrate. Curiously, the orthosteric inhibitor binds free CSN with only micromolar affinity, yet achieves nanomolar potency in blocking its deneddylase activity. Cryo-EM structures of the enzyme-substrate-inhibitor complex reveal that active site-engaged CSN5i-3 occludes the substrate iso-peptide linkage while simultaneously extending an NEDD8-binding exosite of CSN5, acting as a molecular glue to cement the NEDD8-CSN5 interaction. The cooperativity of this tri-molecular CSN5i-3-NEDD8-CSN5 assembly, in turn, sequesters CSN5i-3 at its binding site, conferring high potency to the orthosteric inhibitor despite its low affinity for the free enzyme. Together, our findings highlight the modest affinity requirements of molecule glues for individual target proteins and establish "orthosteric molecular glue inhibitors" as a new class of substrate-dependent enzyme antagonists.
    DOI:  https://doi.org/10.1101/2025.11.26.690573
  10. Mol Cell. 2025 Dec 18. pii: S1097-2765(25)00933-5. [Epub ahead of print]85(24): 4490-4491
    Stress to exhaustion: Proteotoxicity in T cells.
    Ting Zhang, Chen Dong.
      In a recent Nature paper, Yi et al.1 uncover that a noncanonical proteotoxic stress response (PSR) in exhausted T cells (Tex), termed "Tex-PSR," drives T cell exhaustion. This response is characterized by sustained global protein synthesis, accumulation of protein aggregate, and selective upregulation of chaperone proteins.
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.016
  11. Nat Commun. 2025 Dec 13.
    Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response.
    Siddhi Omkar, Jake T Kline, James H Grissom, Diyun Sun, Richard J Chi, Jared A M Bard, Luca Fornelli, Andrew W Truman.
      Cells must respond rapidly to heat stress by activating multiple signaling pathways that preserve proteostasis. In budding yeast, this includes induction of Hsf1 and Msn2/4-mediated transcription, cell integrity signaling, stress-triggered phase separation of proteins, and inhibition of translation. How these pathways are so rapidly activated and coordinated remains unclear. We show that the mechanosensor Mid2 senses heat-induced membrane stretch and leads to rapid phosphorylation of the cytosolic Hsp70 Ssa1 at a well-conserved threonine (T492). Phosphorylation of T492 leads to epichaperome rearrangement promoting fine-tuning of multiple cellular processes including translational pausing, HSF activity, MAPK signaling and stress granule resolution. Taken together, these results provide a comprehensive, unified theory of the global yeast heat shock response mediated by the Hsp70 chaperone code.
    DOI:  https://doi.org/10.1038/s41467-025-67204-7
  12. RNA. 2025 Dec 15. pii: rna.080654.125. [Epub ahead of print]
    How Tailored Ribo-Seq Methods Probe Unique Translation Events.
    James Marks, Sezen Meydan.
      Ribosome profiling (Ribo-seq) is a next-generation, high-resolution sequencing technique that captures ribosome-protected mRNA fragments to map ribosome positions across the transcriptome. This method serves as a powerful proxy for global translational activity by revealing where ribosomes engage with mRNAs. Recent advances have expanded the utility of Ribo-seq to resolve distinct ribosome populations, including initiating ribosomes, small subunits, collided ribosomes, mitochondrial ribosomes, and those associated with specific translation factors or localized to subcellular compartments. These methodological advances have significantly broadened the scope of Ribo-seq, enabling new insights into the molecular mechanisms that govern translation across diverse eukaryotic systems. In this mini-review, we highlight key innovations in Ribo-seq technology and discuss how they have deepened our understanding of the spatial, temporal, and regulatory dimensions of translational control.
    Keywords:  Ribo-seq; next-generation sequencing; ribosome; ribosome profiling; translation regulation
    DOI:  https://doi.org/10.1261/rna.080654.125
  13. Adv Sci (Weinh). 2025 Dec 17. e09927
    TBK1 Induces the Formation of Optineurin Filaments That Condensate with Polyubiquitin and LC3 for Cargo Sequestration.
    Maria G Herrera, Lena Kühn, Lisa Jungbluth, Verian Bader, Laura J Krause, David Kartte, Elias Adriaenssens, Sascha Martens, Jörg Tatzelt, Carsten Sachse, Konstanze F Winklhofer.
      Optineurin is an autophagy receptor that plays an important role in the selective degradation of mitochondria, protein aggregates, and intracellular pathogens. It recognizes ubiquitylated cargo by its ubiquitin-binding in ABIN and NEMO (UBAN) domain and recruits the autophagic machinery through its LC3-interacting region (LIR) domain. Phosphorylation of Optineurin by TANK-binding kinase 1 (TBK1) increases the binding of Optineurin to both ubiquitin chains and lipidated microtubule-associated protein light chain 3 (LC3). Optineurin has been reported to form foci at ubiquitylated cargo, but the underlying mechanism and how these foci are linked to selective autophagy has remained largely unknown. This study shows that phosphorylation of Optineurin by TBK1 induces the formation of filaments that phase separate upon binding to linear polyubiquitin. LC3 anchored to unilamellar vesicles co-partitions into Optineurin/polyubiquitin condensates, resulting in the local deformation of the vesicle membrane. Thus, the condensation of filamentous Optineurin with ubiquitylated cargo promotes the nucleation of cargo and its subsequent alignment with LC3-positive nascent autophagosomes, suggesting that co-condensation processes ensure directionality in selective autophagy.
    Keywords:  Optineurin; TBK1; autophagy; phase separation; ubiquitin
    DOI:  https://doi.org/10.1002/advs.202509927
  14. Sci Adv. 2025 Dec 19. 11(51): eaeb1017
    Mechanism of cotranslational modification of histones H2A and H4 by MetAP1 and NatD.
    Denis Yudin, Mateusz Jaskolowski, Ziyi Fan, Nicolas Burg, Sowmya Chandrasekar, Alfred M Lentzsch, Alain Scaiola, Adrian Bothe, Elke Deuerling, Martin Gamerdinger, Shu-Ou Shan, Nenad Ban.
      The replication-dependent histones H2A and H4 are among the most highly expressed proteins in eukaryotes during the S phase to ensure packaging of replicated chromosomes. Nearly all newly synthesized H2A and H4 are N-terminally acetylated by N-terminal acetyltransferase D (NatD) following excision of the initiator methionine by methionine aminopeptidases (MetAPs). These modifications influence chromatin function, but how they occur cotranslationally on these exceptionally abundant and small proteins was not understood. Here, we show that the nascent polypeptide-associated complex controls the cotranslational modification of histones H2A and H4 by recruiting NatD and the upstream enzyme MetAP1 to ribosomes. MetAP1 and NatD cooperate on the ribosome to create a confined environment for the efficient sequential modification of the nascent histone chain. Our work provides a mechanistic model for the early steps of histone maturation.
    DOI:  https://doi.org/10.1126/sciadv.aeb1017
  15. bioRxiv. 2025 Dec 08. pii: 2025.12.06.692689. [Epub ahead of print]
    1-Deoxysphingolipids Require Very-Long-Chain Ceramide Synthesis to Induce ER Stress and Neurotoxicity.
    Colleen Byrnes, Benjamin A Clarke, Hongling Zhu, Saurav Majumder, Mari Kono, Richard L Proia.
      Sphingolipids play key roles in cellular systems both as membrane components and as signaling molecules. Their biosynthesis, which occurs in the endoplasmic reticulum (ER), begins with the condensation of an amino acid, typically serine, and a fatty acyl-CoA. Under certain pathological conditions, alanine can be substituted for serine in the condensation reaction, producing 1-deoxysphingolipids, which lack the 1-hydroxyl group on the sphingoid base. Unlike typical sphingolipids, 1-deoxysphingolipids are unable to accept a head group modification, which alters their metabolic processing and prevents their canonical degradation. The accumulation of these "headless" 1-deoxysphingolipids causes neurotoxicity in various neurological and metabolic disorders. Here, we conducted a genome-wide CRISPR-Cas9 screen to identify pathways leading to 1-deoxysphinganine-induced toxicity in SH-SY5Y cells, a model used to study neurotoxic responses. Our top genetic hits highlighted the pathway involved in synthesizing ceramides with very-long-chain fatty acids (C22-C26). Using CRISPR-Cas9-modified SH-SY5Y cells with loss-of-function (LOF) mutations in the TECR or CERS2 genes-both critical for producing very-long-chain ceramides-we validated that this pathway was essential for 1-deoxysphinganine-mediated toxicity. Furthermore, we demonstrated that the ceramide synthesis pathway is required for 1-deoxysphinganine to trigger ER stress, as evidenced by significantly increased expression of the unfolded protein response in WT, but not TECR or CERS2 LOF mutant, SH-SY5Y cells exposed to 1-deoxysphinganine. Collectively, these findings identify a specific metabolic pathway for 1-deoxysphinganine leading to very-long-chain 1-deoxyceramide production that culminates in ER stress and toxicity. The findings highlight potential therapeutic targets for neuropathological diseases caused by 1-deoxysphingolipid accumulation.
    DOI:  https://doi.org/10.64898/2025.12.06.692689
  16. Eur J Cell Biol. 2025 Dec 16. pii: S0171-9335(25)00051-2. [Epub ahead of print]105(1): 151526
    Pathogenic podocin variants exhibit distinct defects in trafficking, membrane organization, and degradation pathways.
    Pei-Chen Lu, Ruth Rollason, Chia-An Chou, Valeryia Kuzmuk, Kate J Heesom, Moin A Saleem, Gavin I Welsh.
      Nephrotic syndrome is frequently associated with pathogenic variants in NPHS2 (podocin), including the common and severe R138Q substitution. Using conditionally immortalized human podocytes expressing Myc-tagged podocin variants (G92C, V180M, R138Q, R238S, and R291W), we systematically compared variant-specific defects in plasma-membrane trafficking, detergent-resistant microdomain (DRM) localization, and protein stability. All variants displayed reduced plasma membrane abundance and altered DRM distribution. Among them, R138Q-podocin showed uniquely reduced protein stability. Consistent with previous reports, quantitative proteomics revealed a strong enrichment of endoplasmic reticulum (ER) quality-control and ubiquitin-proteasome components in the R138Q interactome, confirming its identity as an ER-associated degradation substrate. Proteasome inhibition with MG132 stabilized R138Q-podocin and restored its trafficking to both the plasma membrane and DRMs, indicating that impaired stability-rather than an intrinsic trafficking defect-restricts its surface localization. Proteomic profiling further identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings expand the podocin interaction network and suggest potential roles in adhesion-associated membrane organization. Collectively, these results demonstrate that pathogenic podocin variants disrupt podocyte function through distinct mechanisms involving degradation, trafficking, and membrane microdomain association, providing insight into variant-specific disease pathways in nephrotic syndrome. SYNOPSIS: This study examined the trafficking, membrane localization, and stability of disease-associated podocin variants. All variants showed reduced plasma-membrane abundance and altered detergent-resistant microdomain distribution, whereas only R138Q-podocin exhibited marked proteasomal degradation. Quantitative proteomics confirmed the endoplasmic reticulum-associated degradation signature of R138Q and identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings reveal variant-specific mechanisms governing podocin stability and membrane organization.
    Keywords:  Gene mutation; Genetic disease; Kidney; Nephrotic syndrome; Podocin
    DOI:  https://doi.org/10.1016/j.ejcb.2025.151526
  17. Nat Commun. 2025 Dec 13.
    Inhibiting translation elongation by reducing eIF5A activity induces feedback inhibition of initiation, limiting tumour cell proliferation.
    Aristeidis P Sfakianos, Rebecca M Raven, Tom Smith, Xiao-Ming Sun, Thomas E Mulroney, Mariavittoria Pizzinga, Veronica Dezi, Angela Rubio Tenor, Mark Stoneley, Cameron H Cole, Karam Al-Doori, Hashim Ahmed Nur, Rachel L Pennie, Ian Powley, Leah Officer-Jones, Ritwick Sawarkar, Martin Turner, Marion MacFarlane, Owen J Sansom, Martin Bushell, John Le Quesne, Robert F Harvey, Anne E Willis.
      Cancer development is associated with dysregulation of the translatome, and targeting canonical eukaryotic initiation and elongation factors can offer treatment avenues for various neoplasms. Emerging evidence indicates that dysregulated mRNA elongation, involving alterations in eEF2 activity and eIF5A expression, also contributes to tumour cell growth. In this study, we investigate whether targeting eIF5A with the inhibitor GC7 is a viable strategy to curtail aberrant cell growth. Our findings demonstrate that inhibiting elongation by reducing eIF5A activity induces feedback inhibition of initiation through eIF2α phosphorylation, decreasing ternary complex formation and shutting down bulk protein synthesis. Employing dynamic SILAC, we identify proteins impacted by reduced eIF5A activity, and show their decreased translation results from feedback inhibition to initiation or other processes downstream of eIF5A. Decreased eIF5A activity impairs mitochondrial function, which activates signalling through HRI to eIF2α phosphorylation, reducing cancer cell proliferation. These effects are reversed by treatment with the integrated stress response inhibitor, implying that the impact of GC7 on cancer cell proliferation is mediated via translation initiation rather than elongation inhibition. These data suggest that eIF5A inhibition could be used to target cancer cells that depend on mitochondrial function for their proliferation and survival.
    DOI:  https://doi.org/10.1038/s41467-025-66531-z
  18. Nat Commun. 2025 Dec 18. 16(1): 11234
    A practical method for determining the rate of covalent modification of fragments and leads.
    Janice Jeon, Svetlana A Kholodar, Brian H Tran, Kimberly E Mallinger, Daniel A Erlanson, Robert A Everley.
      The clinical success of covalent drugs such as sotorasib has renewed interest in covalency for rational drug design. The most rigorous potency metric for covalent modifiers is the second-order rate constant kinact/KI. However, existing methods for measuring kinact/KI are resource-intensive and involve complex data interpretation. We describe the diagonal dose-response time-course (dDRTC), an efficient mass spectrometry-based method for determining kinact/KI, enabling routine kinact/KI quantification earlier in programs and accelerating SAR interpretation for lead discovery. We apply dDRTC to a dozen covalent fragment and lead-like modifiers for three targets, KRASG12C and two E3 ligase complexes. Kinetic simulations comparing a range of kinact and KI values establish recommended parameters for dDRTC and reveal that the approach is particularly suited for covalent fragments and leads. Our results demonstrate accurate determination of kinact/KI values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis.
    DOI:  https://doi.org/10.1038/s41467-025-66924-0
  19. Redox Biol. 2025 Dec 12. pii: S2213-2317(25)00486-0. [Epub ahead of print]89 103973
    NoxO1 promotes endosome formation and reduces intracellular vesicle processing.
    Maureen Hebchen, Falk Herwig, Tim Schader, Manuela Spaeth, Niklas Müller, Katrin Schröder.
      NADPH oxidase organizer 1 (NoxO1) is known as a scaffold cytoplasmic subunit of the reactive oxygen species (ROS) forming Nox1 complex. We previously identified an interaction between NoxO1 and Erbin, a cytosolic scaffold protein that associates with Epidermal Growth Factor Receptor (EGFR), but its ROS-independent roles remain poorly understood. Here, we demonstrate that NoxO1 overexpression remodels the endolysosomal system by expanding early endosomes and lysosomes. A calibrated six-compartment ordinary differential equation model of EGFR trafficking predicts a slowed down intracellular trafficking: NoxO1 overexpression increased internalization rates by 14 % while reducing degradative sorting by 48 %, lysosomal transfer by 24 %, and final degradation by 41 %. Using fluorescent cargo (EGF and BSA), we confirmed enhanced internalization and cargo accumulation in lysosomes, supporting the idea of prolonged lysosomal retention in NoxO1 overexpressing cells. Mechanistically, NoxO1 activated transcription factor EB (TFEB), the master regulator of lysosomal biogenesis, in an Erbin-dependent but ROS independent manner. Proximity ligation assays revealed spatial association of NoxO1, Erbin, EGFR, and TFEB, suggesting a multi-protein regulatory complex. Genetic ablation of Erbin abolished NoxO1-induced increases in early endosome (EEA1) and lysosome (LAMP1) markers, confirming Erbin's essential role. In conclusion, via its interaction with Erbin NoxO1 promotes activation of TFEB, contributes to lysosome formation while delaying cargo degradation.
    Keywords:  EGFR; Endosomal trafficking; Erbin; Lysosome biogenesis; NoxO1; TFEB
    DOI:  https://doi.org/10.1016/j.redox.2025.103973
  20. Proc Natl Acad Sci U S A. 2025 Dec 23. 122(51): e2526318122
    DIS3L2-mediated RNA surveillance and extracellular vesicle packaging prevent innate immune activation by aberrant cellular RNAs.
    Sandra G Williams, Soyeong Sim, Wilfried M Guiblet, Jubin George, Jennifer C Jones, Sandra L Wolin.
      Although most studies of the RNAs within extracellular vesicles (EVs) have focused on messenger RNA (mRNA) and microRNAs (miRNAs), recent analyses have revealed that transfer RNAs (tRNAs) and other noncoding RNAs (ncRNAs) are far more abundant. However, the extent to which EV ncRNAs resemble overall cellular RNAs and the benefits to host cells of packaging them into EVs remain unknown. Here, we purified EVs from the culture media of mouse and human cells and characterized their RNA components using high-throughput sequencing and Northern blotting. We report that EVs are enriched for numerous aberrant ncRNAs, including ncRNA fragments, ncRNAs that have failed to mature, and short structured introns. Many RNAs contain oligouridine tails, a modification that can promote degradation by DIS3L2, an exoribonuclease that degrades defective structured ncRNAs. Both the numbers of EVs released and the fractions of tailed RNAs in EVs increase on DIS3L2 depletion, indicating that EV packaging of aberrant ncRNAs occurs in competition with DIS3L2 decay. Multiple type I interferon-stimulated genes (ISGs) are upregulated on DIS3L2 depletion, and cells treated with a neutral sphingomyelinase inhibitor that reduces exosome biogenesis show moderate ISG upregulation, an effect that is enhanced in DIS3L2-depleted cells and accompanied by accumulation of aberrant ncRNAs in cellular RNA. Thus, DIS3L2 degradation and packaging of aberrant RNAs into vesicles may prevent these RNAs from activating innate immune sensors and triggering an interferon response.
    Keywords:  DIS3L2; RNA surveillance; exosomes; extracellular vesicles; interferon
    DOI:  https://doi.org/10.1073/pnas.2526318122
  21. Dev Cell. 2025 Dec 15. pii: S1534-5807(25)00715-4. [Epub ahead of print]60(24): 3359-3360
    A matter of selectivity: ER-phagy suppression at the onset of pancreatic cancer.
    Carmine Settembre.
      KRAS mutations drive pancreatic ductal adenocarcinoma (PDAC). In this issue of Developmental Cell, Salomó Coll et al.1 reveal that KRAS suppresses endoplasmic reticulum (ER)-phagy in pancreatic acinar cells by inhibiting CCPG1 transcription. Impaired ER-phagy triggers protein aggregation, inflammation, and acinar-to-ductal metaplasia, promoting tumorigenesis. These findings highlight selective autophagy's role in cancer, with possible therapeutic implications.
    DOI:  https://doi.org/10.1016/j.devcel.2025.11.005
  22. Cell Death Differ. 2025 Dec 18.
    CD147 promotes NSCLC metastasis by inducing secretory autophagy-dependent exosome secretion via TRIM56-mediated ubiquitination and degradation of GCN2.
    Jie Yang, Chenggong Liao, Xiaohua Liang, Yuan Ke, Ying Sun, Minmin Huang, Meirui Qian, Xu Yang, Hongyong Cui, Huijie Bian, Zhinan Chen, Lingmin Kong.
      Tumor-derived exosome secretion dynamically correlates with malignant progression, although the mechanisms by which tumor-associated antigens regulate exosome production remain unclear. Here, we found that the number of plasma exosomes increased significantly with the progression of non-small-cell lung cancer (NSCLC) patients and identified that CD147 as a crucial mediator of exosome secretion using mass spectrometry. CD147 exhibited a positive correlation with exosomes release in NSCLC patients and various cell lines and it drove the release of exosome to promote tumor metastasis in vitro and in vivo. Transcriptomic profiling of transgenic CD147 models identified differential gene expression patterns enriched in autophagy-related pathways. Intriguingly, CD147 was found to specifically enhance autophagosome and amphisome biogenesis to promote exosomes release by using transmission electron microscopy, high-sensitivity structured light microscope, RFP-GFP-LC3 adenovirus reporters and immunofluorescence, which indicated the role of CD147 in mediating non-canonical autophagy processes. Mechanistically, CD147 activated the GCN2/EIF2α/ATG12 signaling axis to drive autophagosome assembly but blocked autolysosome maturation by inhibiting VAMP8/STX17/SNAP29-dependent fusion, leading to amphisome accumulation. Proteomics identified TRIM56 as a novel E3 ligase mediating K619 ubiquitination-dependent GCN2 proteasomal degradation. Subsequently, we found that CD147 suppresses TRIM56 expression, thereby stabilizing GCN2 to activate the GCN2/EIF2α/ATG12 axis. Meanwhile, CD147-induced IP3R3-mediated calcium overload facilitated the fusion of autophagosomes with multivesicular bodies to form amphisomes, thus enhancing exosome release. Collectively, our findings reveal a novel mechanism whereby CD147 promotes crinophagy-mediated exosome secretion through dual regulation of GCN2 stability and calcium homeostasis, thereby accelerating NSCLC progression. Our work establishes a new molecular link between autophagy modulation and cancer progression.
    DOI:  https://doi.org/10.1038/s41418-025-01636-y
  23. J Cell Sci. 2025 Dec 19. pii: jcs.264115. [Epub ahead of print]
    Stress-specific NONO interactomes reveal a key role of Hsp70 chaperone activity in regulation of paraspeckle formation.
    Isaac Odonkor, Birendra Kumar Shrestha, Stephanie Rose Nielsen, Athanasios Kournoutis, Ida Emilie Bjørlo, Saikat Das Sajib, Annica Hedberg, Toril Anne Grønset, Kenneth Bowitz Larsen, Jack-Ansgar Brun, Erik Knutsen, Seyed Mohammad Lellahi, Maria Perander.
      Paraspeckles are stress-induced nuclear RNA-protein condensates that assemble on the long non-coding RNA NEAT1. Their increased formation under certain cellular circumstances has gained growing interest due to their association with serious human diseases such as neurodegenerative disorders and cancer. The biological functions of paraspeckles still appear obscure, but increasing evidence suggests that they contribute to regulation of gene expression by recruiting specific proteins and RNA molecules. Here, we have characterized and compared two stress-enriched interactomes of the essential paraspeckle protein NONO in both wild type and paraspeckle-deficient NEAT1 knockout cells. We identified Hsp70 as part of stress-enriched NONO complexes in wild type, but not in NEAT1-depleted cells. We show that proteotoxic stress-induced paraspeckle formation and NEAT1 expression are strictly dependent on Hsp70 chaperone activity. Our data demonstrate that both NONO and Hsp70 transiently translocate to the nucleolus during heat shock and that paraspeckle formation during recovery follows Hsp70-dependent relocation of NONO from the nucleolus to the nucleoplasm. Taken together, we demonstrate an important role of Hsp70 in paraspeckle assembly and identify a possible link between the nuclear protein quality control system and paraspeckles.
    Keywords:   NEAT1 ; Cellular stress responses; Heat shock response; Hsp70; NONO; Paraspeckles; Proteostasis; TurboID
    DOI:  https://doi.org/10.1242/jcs.264115
  24. J Cell Sci. 2024 Dec 15. pii: jcs264075. [Epub ahead of print]138(24):
    Ribosome-binding protein 1 maintains peroxisome biogenesis.
    Kaneez Fatima, Helena Vihinen, Ani Akpinar, Tamara Somborac, Anja Paatero, Eija Jokitalo, Ville Paavilainen, Pekka Katajisto, Svetlana Konovalova.
      Peroxisomes are single-membrane-bound organelles essential for human health, yet the mechanisms of peroxisome biogenesis are not fully understood. Here using a systematic double screening approach, we identified ribosome-binding protein 1 (RRBP1) as a novel peroxisome biogenesis factor in human cells. Deletion of RRBP1 in HEK293T cells led to a reduction in both peroxisome number and peroxisomal protein levels as well as in defects in processing of peroxisomal matrix proteins, such as ACOX1 and thiolase. However, cell proliferation and protein translation were not altered in cells lacking RRBP1. RRBP1 depletion did not affect peroxisome-endoplasmic reticulum (ER) contact sites, and pexophagy did not contribute to the reduction of peroxisomes in RRBP1 knockout cells. Instead, in the absence of RRBP1, peroxisomal proteins were processed by proteasomal degradation, suggesting that RRBP1 plays a role in the insertion of these proteins into ER membranes and their stabilization. Altogether, our results show that RRBP1 promotes peroxisome biogenesis in human cells, highlighting the power of systematic approaches in discovering novel factors of organellar biogenesis.
    Keywords:  CRISPR/Cas9; Peroxisome; Peroxisome biogenesis; RRBP1
    DOI:  https://doi.org/10.1242/jcs.264075
  25. Cell Rep Med. 2025 Dec 16. pii: S2666-3791(25)00551-8. [Epub ahead of print]6(12): 102478
    Vesicle-mediated mitochondrial clearance presents an actionable metabolic vulnerability in triple-negative breast cancer.
    Jody Vykoukal, Yihui Chen, Mingxin Zuo, Riccardo Ballarò, Monica J Hong, Hansini Krishna, Daniela B Rodriquez-Perera, Hiroyuki Katayama, Ehsan Irajizad, Ranran Wu, Ricardo A León-Letelier, Jennifer B Dennison, Angelica M Gutierrez, Adriana Paulucci-Holthauzen, Timothy C Thompson, Leona Rusling, Yining Cai, Fu Chung Hsiao, Soyoung Park, Banu Arun, Samir Hanash, Johannes F Fahrmann.
      Selective autophagy of mitochondria is known to promote cancer cell survival and progression, including in triple-negative breast cancer (TNBC). Here, we apply an integrated multi-omics approach together with functional experimental analyses to investigate metabolic adaptations that support mitochondrial quality control in TNBC. We detail a mitochondrial quality control mechanism, complementary to mitophagy, that is enabled by a program of heightened extracellular sphingomyelin salvaging in TNBC coupled with extracellular vesicle-mediated intracellular clearance of mitochondrial damage. Targeting of this onco-metabolic pathway via repurposing of eliglustat, a selective small molecule inhibitor of glucosylceramide synthase, results in ceramide-mediated compensatory mitophagy and cancer cell death in vitro and attenuates tumor growth and prolongs overall survival at clinically achievable doses in orthotopic syngeneic mouse models of TNBC as well as in human cell line-derived xenograft models. Our study defines an unexplored mechanism of aberrant sphingolipid metabolism that underlies an actionable metabolic vulnerability for anti-cancer treatment.
    Keywords:  autophagy; eliglustat; extracellular vesicles; glucosylceramide synthase; mitochondria; sphingolipids; triple-negative breast cancer
    DOI:  https://doi.org/10.1016/j.xcrm.2025.102478
  26. Nat Commun. 2025 Dec 16.
    The ribosome derives the energy to translocate and unwind mRNA from EF-G binding.
    Hossein Amiri, William J Van Patten, Gillian Rexroad, Varsha P Desai, Benjamen A Sterwerf, Laura Lancaster, Harry F Noller, Carlos Bustamante.
      The GTPase EF-G catalyzes translocation of mRNA and tRNAs relative to the ribosome and helps maintain the reading frame during protein synthesis. Which events directly require EF-G-mediated GTP hydrolysis during translocation are still debated. Using high-resolution optical tweezers endowed with single-molecule fluorescence detection, we simultaneously monitored binding of fluorescently-labeled EF-G to ribosomes and either mRNA unwinding or mRNA translocation relative to the body domain of the small ribosomal subunit. Using EF-G mutants and GTP analogs, we find that neither mRNA unwinding nor translocation require GTP hydrolysis and that these are independent events that may or may not temporally coincide. We propose that "tight binding" of EF-G to the ribosome triggers mRNA unwinding and translocation of mRNA relative to the 30S body domain and that while GTP hydrolysis kinetically accelerates translocation, it is thermodynamically required only to liberate the tightly bound EF-G from the ribosome.
    DOI:  https://doi.org/10.1038/s41467-025-66812-7
  27. Mol Cell. 2025 Dec 18. pii: S1097-2765(25)00937-2. [Epub ahead of print]85(24): 4633-4650.e11
    Cell-surface RNA forms ternary complex with RNA-binding proteins and heparan sulfate to recruit immune receptors.
    Zeshi Li, Bhagyashree S Joshi, Hongbo Yin, Ruud H Wijdeven, Azen Koç, Dick W Zijlmans, Irene Santos-Barriopedro, Hailiang Mei, Wei Wu, Milad Shademan, Filip M Zawisza, Eric Bos, Pradeep Chopra, Marvin E Tanenbaum, Thomas H Sharp, Michiel Vermeulen, Vered Raz, Chirlmin Joo.
      Recent discoveries have shown the presence of ribonucleic acid (RNA) on the cell surface, defying the view that RNA only functions intracellularly. However, how RNA is presented on the cell surface and what its biological relevance is are poorly understood. We established Toll-like receptor 7 (TLR7) as a cell-surface RNA (csRNA) probe. Employing it in a genome-wide knockout screening, we identified heparan sulfate (HS) as a crucial factor for csRNA presentation. Cell-surface proximity labeling revealed that HS-associated csRNAs (hepRNAs) are in the vicinity of RNA-binding proteins (RBPs). These observations led us to a model wherein cell-surface HS, RNA, and RBP form ternary complexes, validated by our spatio-selective RNA-protein crosslinking technology in a TLR7-orthogonal manner. We further revealed the identities of hepRNA and found that they can recruit the immune receptor killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), potentially enhancing receptor-ligand interactions. Employing human cell lines, our findings lay the groundwork for investigating how cell-surface ribonucleoproteins contribute to immune modulation.
    Keywords:  RNA-binding protein; RNA-protein crosslinking; Toll-like receptor 7; cell-surface RNA; genome-wide screening; heparan sulfate; immune receptor; proximity labeling; quantitative proteomics; ribonucleoprotein
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.020
  28. Circ Res. 2025 Dec 15.
    Subcellular In Vivo Cardiac Proteomics Reveals Sarcomere and Ribosome Interactomes and skNAC's Impact on Cardiac Proteostasis.
    Rami Haddad, Omer Sadeh, Tamar Ziv, Nardeen Shehdeh, Nadav Keren, Izhak Kehat.
       BACKGROUND: Proteostasis and the regulation of protein folding and sorting play a critical role in maintaining cellular homeostasis. The failure of proteostasis contributes to heart failure and aging, but, despite its importance, the mechanisms and factors regulating proteostasis in cardiomyocytes remain poorly characterized.
    METHODS: Subcellular proteomes of cardiomyocytes were analyzed in vivo using biotin proximity labeling in mouse hearts. We employed a novel homology-independent targeting integration strategy for genetic tagging and for substitution of the muscle-specific skNAC (skeletal nascent polypeptide-associated complex alpha isoform) isoform with the ubiquitous short isoform in cardiomyocytes.
    RESULTS: We identified hundreds of proteins localized to the Z- and M-lines of sarcomeres, the ribosomes, and the desmosomes, including multiple chaperones. A universal homology-independent targeted integration strategy allowed us to genetically tag endogenous genes in the mouse heart and confirm protein localization. We identified the large muscle-specific isoform of the nascent polypeptide-associated complex protein skNAC as a Z-line and ribosome-associated protein. Replacement of skNAC with a ubiquitous isoform induced dilated cardiomyopathy, accompanied by altered ribosome positioning and markedly reduced mitochondrial protein levels.
    CONCLUSIONS: We unraveled the cardiomyocyte subcellular proteome and show that skNAC, an isoform downregulated in disease, is a key ribosome and Z-line-associated protein responsible for cardiomyocyte proteostasis.
    Keywords:  biotin; desmosomes; heart failure; mitochondria; proteostasis
    DOI:  https://doi.org/10.1161/CIRCRESAHA.125.326929
  29. JACC Basic Transl Sci. 2025 Dec 15. pii: S2452-302X(25)00386-9. [Epub ahead of print]11(1): 101433
    Pharmacological Enhancement of Integrated Stress Response Confers Protection in Calcific Aortic Valve Disease.
    Libo Wang, Xulei Duan, Huibing Liu, Fei Lin, Chaoyuan Zhou, Katrin Schröder, Ajay M Shah, Guoan Zhao, Min Zhang.
      Previous studies have shown that Nox4 activates eIF2α/ATF4 signaling during the integrated stress response (ISR) and protects heart injury. However, their roles in calcific aortic valve disease (CAVD) remain unclear. Here, we show that both ATF4 and Nox4 are up-regulated in porcine aortic valve interstitial cells (AVIC) and in human aortic valves with CAVD. Nox4 knockdown promotes while Nox4 overexpression suppresses CAVD by modulating ISR. Importantly, ISR activators Guanabenz and Sephin1 effectively attenuate AVIC osteoblastic-like differentiation and mitigate CAVD in rabbits and mice, respectively. These findings highlight that pharmacological enhancement of the ISR is a promising therapeutic strategy for CAVD.
    Keywords:  ATF4; Nox4; aortic valve; apoptosis; calcification; reactive oxygen species
    DOI:  https://doi.org/10.1016/j.jacbts.2025.101433
  30. Nat Commun. 2025 Dec 14. 16(1): 11260
    RHOA regulates mitochondria-ER contact sites through modulation of the VAPB/PTPIP51 tether.
    Zheng Yang, Shintaro Nakajima, Hsiuchen Chen, Yogaditya Chakrabarty, Huibao Feng, David C Chan.
      The mitochondria-endoplasmic reticulum contact site (MERCS) is critical for calcium exchange, phospholipid transfer, and bioenergetics. Impairment of MERCS is implicated in numerous pathological conditions, including cancer and neurodegenerative diseases. Remodeling of MERCS can affect calcium signaling or metabolism, but the mechanisms involved in dynamic MERCS remodeling are unknown. Employing a genome-wide CRISPRi screen, we uncover the ability of the small GTPase RHOA to tune the cellular MERCS level. RHOA knockdown, or increasing its degradation by CUL3 overexpression, reduces the MERCS level; conversely, upregulation of RHOA increases the MERCS level. RHOA binds to the ER protein VAPB and regulates complex formation between VAPB and mitochondrial PTPIP51, which form a tethering complex at the interface between ER and mitochondria. Furthermore, this regulatory mechanism is perturbed by disease alleles of RHOA, CUL3, and VAPB involved in cancer, hyperkalemia, and neurodegeneration, suggesting that MERCS may be affected in a range of pathological conditions. This study identifies RHOA as a regulator of mitochondria-ER communication, providing mechanistic insights into the dynamic remodeling of MERCS and potential therapeutic strategies for diseases linked to MERCS dysfunction.
    DOI:  https://doi.org/10.1038/s41467-025-66138-4
  31. J Chem Inf Model. 2025 Dec 19.
    Computational Modeling of PROTAC Ternary Complexes as Ensembles Using SILCS-xTAC.
    Erik B Nordquist, Mingtian Zhao, Wenbo Yu, Alexander D MacKerell.
      Proteolysis targeting chimeras, or PROTACs, are an emerging class of drugs that offer the potential to develop therapeutics targeting "undruggable" proteins by stabilizing protein-protein interactions (PPI). This involves leveraging the physiological protein degradation mechanism based on ubiquitination through stabilization of target protein-E3 ubiquitin ligase PPI mediated by the PROTAC. Existing computational methods for ligand design are not typically designed for the ternary complex problem and may have limited accuracy or efficiency due to the use of either rigid docking or full molecular dynamics (MD) simulations. Here we present a method which uses SILCS (site identification by ligand competitive saturation) to address the challenge of designing ligands which stabilize PPI by using precomputed ensembles of (1) functional group affinity patterns, termed FragMaps, for efficient and accurate ligand docking and of (2) a collection of putative PPI dimer 3D structures as docking targets. SILCS simulations involving aqueous, multisolute grand canonical Monte Carlo (GCMC)/MD calculations generate the FragMaps for both the target and ligase proteins. An ensemble of PPI dimer conformations is generated using the FragMaps and then dimer FragMaps are generated by merging the two sets of FragMaps. PROTAC molecules are docked into the ensemble of dimer FragMaps, and the final scoring metrics are extracted from the most favorable ternary complex. The scoring metrics, including energetics, binding site geometry, and physicochemical terms, are weighted together to construct an activity score. The method is benchmarked on a diverse set of ternary crystal structures of different proteins and PROTACs, and the derived activity score shows modest correlation with DC50 values in cells for a wide variety of systems. The SILCS-xTAC method is a powerful tool to facilitate PROTAC optimization by predicting the binding geometries and energetics of ternary complexes.
    DOI:  https://doi.org/10.1021/acs.jcim.5c02045
  32. EMBO Mol Med. 2025 Dec 19.
    Mutant CHCHD10 disrupts cytochrome c oxidation and activates mitochondrial retrograde signaling.
    Márcio Augusto Campos-Ribeiro, Erminia Donnarumma, Hendrik Nolte, Paul Cobine, Elodie Vimont, Dusanka Milenkovic, Juan Diego Hernandez-Camacho, Francina Langa-Vives, Etienne Kornobis, Esthel Pénard, Sonny Yde, Thomas Langer, Véronique Paquis-Flucklinger, Timothy Wai.
      Mutations in CHCHD10, a mitochondrial intermembrane space (IMS) protein implicated in proteostasis and cristae maintenance, cause mitochondrial disease. Knock-in mice modeling the human CHCHD10S59L variant associated with ALS-FTD develop a mitochondrial cardiomyopathy driven by CHCHD10 aggregation and activation of the mitochondrial integrated stress response (mtISR). We show that cardiac dysfunction is associated with dual defects originating at the onset of disease: (1) bioenergetic failure linked to impaired mitochondrial copper homeostasis and cytochrome c oxidation, and (2) maladaptive mtISR signaling via the OMA1-DELE1-HRI axis. Using protease-inactive Oma1E324Q/E324Q knock-in mice, we show that blunting mtISR in Chchd10S55L/+ mice delays cardiomyopathy onset without rescuing CHCHD10 insolubility, cristae defects or OXPHOS impairment. Proteomic profiling of insoluble mitochondrial proteins in Chchd10S55L/+ mice reveals widespread disruptions of mitochondrial proteostasis, including IMS proteins involved in cytochrome c biogenesis. Defective respiration in mutant mitochondria is rescued by the addition of cytochrome c, pinpointing IMS proteostasis disruption as a key pathogenic mechanism. Thus, mutant CHCHD10 insolubility compromises metabolic resilience by impairing bioenergetics and stress adaptation, offering new perspectives for the development of therapeutic targets.
    Keywords:  CHCHD10; Cardiomyopathy; Cytochrome c; Mitochondrial Disease; OMA1
    DOI:  https://doi.org/10.1038/s44321-025-00358-5
  33. J Med Chem. 2025 Dec 16.
    Development of Cathepsin B-Responsive GalNAc-PROTACs for Hepatocyte-Targeting Protein Degradation.
    Yunhua Peng, Tao Li, Donghua Liu, Wenxin Li, Yimeng Zhang, Ying Zhao, Xuehan Jiang, Yaqi Liang, Pengxiao Chen, Bohan Ma, Jing Liu, He Chen.
      Targeted protein degradation (TPD) has arisen as a therapeutic revolution for eliminating disease-relevant proteins, but its tissue-specific delivery remains a critical challenge. Here, we developed an asialoglycoprotein receptor (ASGPR)-based platform for the selective degradation of target proteins in hepatocytes. By conjugating the ASGPR ligand triantennary N-acetylgalactosamine (tri-GalNAc) with a BRD4-targeted proteolysis targeting chimera (PROTAC) via a cathepsin B (CTSB)-cleavable Val-Cit-PABC linker, we generated a prototype GalNAc-PROTAC conjugate, TMU454. TMU454 selectively degraded BRD4 in ASGPR-positive hepatocellular carcinoma cells while sparing ASGPR-negative cancer cells and normal cells. Mechanistic investigations confirmed that TMU454-mediated BRD4 degradation is dependent on the ASGPR-mediated endocytosis, CTSB-mediated linker cleavage, and ubiquitin-proteasome system (UPS). Furthermore, a fluorescein-labeled analogue, TMU670, revealed preferential liver accumulation. Importantly, TMU454 significantly inhibited tumor growth in a Huh7-derived liver cancer xenograft model without apparent systemic toxicity. Collectively, this study establishes a versatile approach for tissue-selective protein degradation and advances targeted therapies for liver cancer.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c02908
  34. J Biol Chem. 2025 Dec 17. pii: S0021-9258(25)02923-0. [Epub ahead of print] 111071
    VarLand: A pipeline to map the structural landscape of missense variants at the proteome scale.
    Francisco J Guzmán-Vega, Kelly J Cardona-Londoño, Ana C González-Álvarez, Karla A Peña-Guerra, Azza Althagafi, Tanisha Khan, Robert Hoehndorf, Stefan T Arold.
      Missense variant pathogenicity often arises from disruptions to protein structural features. The integration of large-scale genetic sequencing into clinical workflows, and the availability of accurate AI-based protein structure predictions present an opportunity to assess the structure-function relationship of missense variants at a population scale. To harness this potential, we developed VarLand, a computational pipeline that extracts 29 structural and biophysical features from AlphaFold-predicted protein models and nine complementary annotation tools. We applied VarLand to pathogenic missense variants from ClinVar and a population-specific dataset of rare Middle Eastern variants, comparing their feature profiles to high-frequency benign variants from the Genome Aggregation Database (gnomAD). Our analysis confirms that pathogenic variants are significantly enriched in ordered regions, buried residues, and sites with high intramolecular contact density, whereas benign variants preferentially occur in disordered, solvent-exposed regions. However, VarLand also uncovered feature landscape variations across protein functional classes and disease categories, suggesting differences in underlying disease mechanisms. Furthermore, variants from the AI-based AlphaMissense database showed a stronger association between structural order and pathogenicity than clinical datasets, indicating residual bias from structure-centric training. These findings demonstrate the effectiveness of multidimensional structural profiling by VarLand to uncover not only broad structure-pathogenicity relationships but also dataset-specific and class-specific deviations, offering deeper insight into disease mechanisms.
    DOI:  https://doi.org/10.1016/j.jbc.2025.111071
  35. Cell Rep Med. 2025 Dec 16. pii: S2666-3791(25)00579-8. [Epub ahead of print]6(12): 102506
    Lymphodepleting chemotherapy potentiates neoantigen-directed T cell therapy by enhancing antigen presentation.
    Shira Sagie, Tomer Babu, Chen Weller, Claire Tabachnik, Ido Livneh, Nele P Quast, Nofar Gumpert, Alina Shomuradova, Matthew I Raybould, Ronen Levy, Dmitry Malko, Adi Alfia, Talal Ben Lulu, Michal Alon, Franco Herrera, Mikhail Kutuzov, Mirie Zerbib, Polina Greenberg, Talya Wasserman-Bartov, Gil Benedek, Yishai Levin, Chani Stossel, Iris Kamer, Talia Golan, Roni Oren, Merav Shmueli, Osnat Bartok, Jair Bar, Jonathan Cohen, Omer Dushek, Charlotte M Deane, Yardena Samuels.
      Adoptive cell therapy (ACT) targeting tumor-specific antigens holds promise for solid tumors, but limited neoantigen presentation remains a key barrier to efficacy. Here, we identify and characterize a T cell receptor (TCR), T104, for the KRAS.G12V mutation, a prevalent neoantigen in colorectal, lung, and pancreatic cancers. TCR-T104 selectively recognizes and kills KRAS.G12V-expressing tumor cells. Combining T cell therapy with lymphodepleting chemotherapy significantly enhances tumor cell killing, particularly by TCR-T cells, tumor-infiltrating lymphocytes (TILs), and T cell engager antibodies across multiple cancer types and target antigens. Mechanistically, chemotherapy upregulates immunoproteasome activity and human leukocyte antigen (HLA)-I surface expression. HLA-immunopeptidome analyses reveal that chemotherapy remodels the antigenic landscape across tumor cell lines and in vivo models, increasing peptide abundance and hydrophobicity while altering proteasomal cleavage preferences. These findings establish a synergistic role for chemotherapy in enhancing neoantigen presentation and T cell-mediated tumor recognition and suggest that fine-tuning these regimens could improve ACT efficacy, particularly in tumors with low-abundance neoantigens.
    Keywords:  Adoptive Cell therapy; KRAS G12V; TCR-T therapy; colon cancer; immunopeptidomics; immunoproteasome; lung cancer; lymphodepleting chemotherapy; neoantigens; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.xcrm.2025.102506
  36. Nat Commun. 2025 Dec 19.
    SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.
    Qianqian Ying, Yongchen Chen, Luochen Shen, Yang Xu, Ruilin Tian.
      RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.
    DOI:  https://doi.org/10.1038/s41467-025-67633-4
  37. Cell Rep. 2025 Dec 15. pii: S2211-1247(25)01439-1. [Epub ahead of print]44(12): 116667
    ARAP2 regulates responses to interferon-gamma by restricting SOCS1.
    Narelle Keating, Karen Doggett, Grace M Bidgood, Lizeth G Meza Guzman, Laura F Dagley, Kunlun Li, Bailey E Williams, Anna Gabrielyan, Carolina Alvarado, Benjamin J Broomfield, Brigette C Duckworth, Colin Hockings, Jumana Yousef, Evelyn Leong, Rhiannon Morris, Andrew Kueh, Alexandra L Garnham, Göknur Giner, Jean-Laurent Casanova, Stephanie Boisson-Dupuis, Jeffrey J Babon, Edmond M Linossi, Michelle D Tate, Joanna R Groom, Sandra E Nicholson.
      Interferon-gamma (IFNγ) is critical for immunity against intra-macrophagic pathogens, signaling through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to induce a tyrosine-phosphorylation cascade that ensures a potent immune response. Excessive JAK-STAT signaling can drive hyperinflammation and autoimmunity; thus, signaling is tightly and selectively regulated by the IFNγ-inducible protein, suppressor of cytokine signaling 1 (SOCS1). SOCS1 inhibits signaling by directly blocking JAK kinase activity. Here, we identified a SOCS1-interacting partner, ankyrin repeat and pleckstrin homology domain 2 (ARAP2), that fine-tunes SOCS1 function. We report that tyrosine 415 in ARAP2 binds the SOCS1-Src homology 2 (SH2) domain and limits the ability of SOCS1 to inhibit IFNγ signaling. Our findings show that ARAP2 promotes the IFNγ response through a phosphorylation-dependent interaction with the negative regulator SOCS1, and this exacerbates inflammation in a mouse model of influenza virus infection.
    Keywords:  ARAP2; CP: immunology; IFNγ; JAK; SH2 domain; SOCS1; STAT; influenza; interferon; tyrosine
    DOI:  https://doi.org/10.1016/j.celrep.2025.116667
  38. Cell Death Dis. 2025 Dec 19.
    Interaction of lncRNA LENT with DHX36 regulates translation and suppresses autophagy in melanoma.
    Alexandre Haller, Giovanni Gambi, Mattia D'Agostino, Guillaume Davidson, Antonin Lallement, Gabrielle Mengus, Chadia Nahy, Nadia Messaddeq, Guillaume Bec, Angelita Simonetti, Eric Ennifar, Irwin Davidson.
      The melanocyte lineage-determining Microphthalmia-associated transcription factor (MITF) drives proliferation and survival of melanocytic melanoma cells through regulation of both coding genes and long non-coding RNAs (LncRNAs). Here we characterize LINC00520 (hereafter called LncRNA ENhancer of Translation, LENT) regulated by MITF and strongly expressed in melanocytic melanoma cells. LENT is essential for the proliferation and survival of cultured melanocytic melanoma cells and xenograft tumors. LENT interacts with the G4 quadruplex resolvase DHX36, and both associate with the ribosome in the 80S and light polysome fractions. LENT modulates DHX36 association with a collection of mRNAs regulating their engagement with polysomes and fine-tuning their subsequent translation. These mRNAs encode proteins involved in endoplasmic reticulum (ER) and mitochondrial homeostasis as well as autophagy. Consequently, LENT silencing leads to extensive autophagy and mitophagy, compromised oxidative metabolic capacity, accompanied by an accumulation and mis-localization of mitochondrial proteins leading to proteotoxic stress and apoptosis. The LENT-DHX36 axis therefore fine-tunes translation of proteins involved in ER and mitochondrial homeostasis, suppressing autophagy and promoting survival and proliferation of melanoma cells.
    DOI:  https://doi.org/10.1038/s41419-025-08296-3
  39. EMBO Mol Med. 2025 Dec 19.
    Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay.
    Søren H Hough, Satpal S Jhujh, Samah W Awwad, Oliver E Lewis, Simon Lam, John C Thomas, Thorsten Mosler, Aldo Bader, Lauren Bartik, Shane McKee, Shivarajan Amudhavalli, Estelle Colin, Nadirah Damseh, Emma Clement, Pilar Cacheiro, Anirban Majumdar, Damian Smedley, Joël Fluss, Rosalinda Giannini, Isabelle Thiffault, Guido Zagnoli Vieira, Rimma Belotserkovskaya, Stephen J Smerdon, Petra Beli, Yaron Galanty, Christopher J Carnie, Grant S Stewart, Stephen P Jackson.
      Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. We performed a CRISPR-Cas9 knockout screen focused on ubiquitin E3 ligases and related proteins with the DNA topoisomerase I inhibitor camptothecin. This led us to establish that MAEA, a core subunit of the CTLH E3 ligase complex, is a critical regulator of homologous recombination and the replication stress response. In tandem, we identified eight patients with variants in MAEA who present with a neurodevelopmental disorder that we term DIADEM (Developmental delay and Intellectual disability Associated with DEfects in MAEA). Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart and protection as a likely cause of disease. Mechanistically, we find that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which we propose may contribute to the presentation of DIADEM by compromising genome integrity and cell division during development.
    Keywords:  DNA Repair; DNA Replication; Neurodevelopmental Disorder; Ubiquitin
    DOI:  https://doi.org/10.1038/s44321-025-00352-x
  40. ChemMedChem. 2025 Dec 16. e202500929
    A Facile Protocol for C(sp2)-C(sp3) Bond Formation Reactions Toward Functionalized E3 Ligase Ligands.
    Anita Maksutova, Thomas M Geiger, Lorenzo Cianni, Dominika E Pieńkowska, Jan Gerhartz, Lina Read, Aleša Bricelj, Alexander Herrmann, Maurice Leon Nelles, Yuen Lam Dora Ng, Marcus D Hartmann, Jan Krönke, Izidor Sosič, Radosław P Nowak, Michael Gütschow, Christian Steinebach.
      A straightforward method for creating C(sp2)-C(sp3) bonds is employed to develop novel cereblon (CRBN) E3 ligase ligands, essential for targeted protein degradation (TPD) applications. While most prior studies focus on biological activities, this work explores how the linker attachment and bond types affect physicochemical stability, binding affinity, and degrading performance. Utilizing N-hydroxyphthalimide (NHP) esters and aryl bromides, a resilient decarboxylative cross-coupling technique that broadens the available chemical space beyond traditional C(sp2)-N connections is developed. Several well-established and underexplored CRBN binders and their derivatives are synthesized and studied. Binding affinity, aqueous solubility, stability in microsomes, and degradation of typical CRBN ligand off-targets are then investigated. Selected compounds are transformed into GSPT1-targeting molecular glue degraders or BRD4-targeting proteolysis-targeting chimeras (PROTACs). Benzamide-based degraders obtained using the new method have a very high ability to break down BRD4. This research shows that C(sp2)-C(sp3) connections open up new ways to fine-tune PROTAC characteristics, which unlock degrader chemotypes that were not accessible before. The results demonstrate the importance of synthetic innovation in developing ligands for TPD applications.
    Keywords:  C(sp2)–C(sp3) bond formation; E3 ligands; cereblon; medicinal chemistry; proteolysis‐targeting chimeras
    DOI:  https://doi.org/10.1002/cmdc.202500929
  41. Proc Natl Acad Sci U S A. 2025 Dec 23. 122(51): e2518675122
    MLKL/retromer axis controls PD-L1 recycling to compromise antitumor immunity during VCP inhibition-induced necroptosis.
    Yang Wang, Zi-Jia Huang, Nan-Nan Yu, Jun-Ze Liang, Yong-Hui Zeng, Qian Zhao, Yi-Ping Zhou, Yu-Jie Zeng, Zheng-Hua Sun, Shuixing Zhang, Qing-Yu He, Jing Zhang.
      The activation of the vesicular trafficking system, including endosomal recycling and extracellular vesicle (EV) release, represents an adaptive mechanism in cancer cells to counteract cellular stress or necroptotic death signals, which hampers cancer therapy. However, the underlying mechanisms remain poorly understood. Indeed, our study demonstrate that inhibition of valosin-containing protein (VCPi) induces EV release and necroptosis in colorectal cancer (CRC), but does not achieve a synergistic effect with immunotherapy. Mechanistically, VCPi delays the degradation of mixed lineage kinase domain-like protein (MLKL), a critical regulator of necroptosis and endosomal trafficking. Knockdown of MLKL reduces VCPi-mediated necroptosis, EV release, and programmed death ligand 1 (PD-L1) recycling. VCPi leads to MLKL accumulation, which recruits the retromer complex and GTPase-activating protein TBC1D5 to inactivate Rab7A. This redirects PD-L1-loaded vesicles to the cell surface via the retromer complex, thereby resulting in immunosuppression. Notably, targeting the retromer complex enhances the therapeutic efficacy of combined VCP inhibitors and anti-PD-L1 therapy in CRC, offering a promising immunotherapeutic strategy. Our study elucidates the role of the retromer complex in mediating PD-L1 recycling during VCPi-induced necroptosis and reveals that dual inhibition of VCP and the retromer complex potentiates immunotherapy efficacy in CRC.
    Keywords:  PD-L1; mixed lineage kinase domain-like protein; necroptosis; retromer complex; valosin-containing protein
    DOI:  https://doi.org/10.1073/pnas.2518675122
  42. bioRxiv. 2025 Dec 14. pii: 2025.12.12.694031. [Epub ahead of print]
    Discovery of Non-Degradative Covalent Molecular Glues for Transcriptional Reprogramming.
    Tuong Nghi Duong, Edward Pandji, Qian Shao, Daniel K Nomura.
      Transcriptional reprogramming through induced proximity has emerged as a powerful strategy for modulating the expression of oncogenic and tumor-suppressive genes. Inspired by transcriptional reprogramming approaches such as transcriptional/epigenetic chemical inducers of proximity (TCIPs) that link BCL6 inhibitors to transcriptional regulators, we sought to develop covalent ligands that rewire BCL6 proximity to selectively suppress MYC transcriptional output while derepressing BCL6 target loci. Through a chemistry-driven and chemoproteomics-enabled design strategy, we generated a panel of BCL6-based electrophile-bearing hybrid ligands and identified a nondegradative molecular glue, ZD-1-186, that potently suppresses MYC and robustly induces CDKN1A (p21) in diffuse large B-cell lymphoma cells. ZD-1-186 downregulates MYC more effectively than BCL6 inhibitors or degraders, while strongly derepressing canonical BCL6 targets, including p21. Through BCL6 pulldown proteomics, ZD-1-186 induced a selective recruitment of the noncanonical BAF complex subunit BRD9 to BCL6 and covalently modified BRD9 at C288. Pharmacologic inhibition or genetic knockdown of BRD9 attenuated ZD-1-186-mediated MYC suppression and blunted p21 induction. Transcriptomic profiling of ZD-1-186 showed simultaneous derepression of BCL6-repressive loci and suppression of MYC transcriptional programs. These findings demonstrated that ZD-1-186 acted as a transcriptional rewiring glue, recruiting BRD9 to BCL6-repressive loci to activate tumor-suppressive transcription, while also potentially redirecting BCL6 to BRD9-bound oncogenic loci. Overall, our work provides a blueprint for the rational discovery and design of electrophile-enabled, nondegradative molecular glues for targeted transcriptional rewiring.
    DOI:  https://doi.org/10.64898/2025.12.12.694031
  43. Mol Cell. 2025 Dec 18. pii: S1097-2765(25)00940-2. [Epub ahead of print]85(24): 4602-4620.e9
    Targeting PRDX6-dependent localization and function of GPX4 enhances ferroptosis-mediated tumor suppression.
    Yameng Hu, Ziwen Li, Man Li, Xingui Wu, Shuxia Zhang, Miaoling Tang, Ruyuan Yu, Meisongzhu Yang, Xin Chen, Libing Song, Guido Kroemer, Valerian E Kagan, Hülya Bayir, Rui Kang, Jinbao Liu, Daolin Tang, Jun Li.
      Inducing lipid peroxidation-dependent ferroptosis is a promising anticancer strategy; however, the development of resistance poses a considerable challenge. This study identifies peroxiredoxin 6 (PRDX6) as a crucial modulator of glutathione peroxidase 4 (GPX4), affecting its localization and functional roles, thus contributing to ferroptosis resistance. PRDX6, endowed with phospholipase A2 activity, catalyzes the conversion of peroxy-phospholipids to lysophospholipids and oxidized fatty acids. Through targeted structural mutations and biochemical analyses, we demonstrate that PRDX6 binds to GPX4 via a C47 disulfide bond, facilitating GPX4's membrane translocation and enhanced production of hydroxy fatty acids. Combining the inhibition of PRDX6 with ferroptosis inducers increases lipid peroxidation, effectively suppressing tumor growth in liver and ovarian cancer mouse models, including patient-derived models. Furthermore, high PRDX6 expression correlates with shorter progression-free survival across multiple human cancer types. Collectively, our findings delineate a PRDX6-dependent mechanism in ferroptosis defense, offering new perspectives for targeted cancer therapy.
    Keywords:  PRDX6; cancer therapy; lysophospholipids; membrane translocation of GPX4
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.023
  44. bioRxiv. 2025 Nov 24. pii: 2025.11.20.689494. [Epub ahead of print]
    BoltzGen: Toward Universal Binder Design.
    Hannes Stark, Felix Faltings, MinGyu Choi, Yuxin Xie, Eunsu Hur, Timothy O'Donnell, Anton Bushuiev, Talip Uçar, Saro Passaro, Weian Mao, Mateo Reveiz, Roman Bushuiev, Tomáš Pluskal, Josef Sivic, Karsten Kreis, Arash Vahdat, Shamayeeta Ray, Jonathan T Goldstein, Andrew Savinov, Jacob A Hambalek, Anshika Gupta, Diego A Taquiri-Diaz, Yaotian Zhang, A Katherine Hatstat, Angelika Arada, Nam Hyeong Kim, Ethel Tackie-Yarboi, Dylan Boselli, Lee Schnaider, Chang C Liu, Gene-Wei Li, Denes Hnisz, David M Sabatini, William F DeGrado, Jeremy Wohlwend, Gabriele Corso, Regina Barzilay, Tommi Jaakkola.
      We introduce BoltzGen , an all-atom generative model for designing proteins and peptides across all modalities to bind a wide range of biomolecular targets. BoltzGen builds strong structural reasoning capabilities about target-binder interactions into its generative design process. This is achieved by unifying design and structure prediction, resulting in a single model that also reaches state-of-the-art folding performance. BoltzGen's generation process can be controlled with a flexible design specification language over covalent bonds, structure constraints, binding sites, and more. We experimentally validate these capabilities in a total of eight diverse wetlab design campaigns with functional and affinity readouts across 26 targets. The experiments span binder modalities from nanobodies to disulfide-bonded peptides and include targets ranging from disordered proteins to small molecules. For instance, we test 15 nanobody and protein binder designs against each of nine novel targets with low similarity to any protein with a known bound structure. For both binder modalities, this yields nanomolar binders for 66% of targets. We release model weights, data, and both inference and training code at: https://github.com/HannesStark/boltzgen .
    DOI:  https://doi.org/10.1101/2025.11.20.689494
  45. Neuron. 2025 Dec 12. pii: S0896-6273(25)00848-7. [Epub ahead of print]
    TDP-43 dysfunction compromises UPF1-dependent mRNA metabolism in ALS.
    Francesco Alessandrini, Matthew Wright, Tatsuaki Kurosaki, Olga S Perloff, Marilyn Ngo, Julia K Kofler, Christopher J Donnelly, Lynne E Maquat, Evangelos Kiskinis.
      Up-frameshift protein 1 (UPF1)-mediated mRNA decay maintains transcriptome integrity and cellular homeostasis. However, its role in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by TAR DNA-binding protein 43 (TDP-43) pathology and disrupted mRNA metabolism in motor neurons (MNs), remains unresolved. Here, we integrated RNA sequencing (RNA-seq) after UPF1 knockdown with RNA immunoprecipitation (RIP)-seq of phosphorylated UPF1 to delineate direct UPF1 targets in induced pluripotent stem cell (iPSC)-derived MNs. These transcripts are enriched for autophagy and structurally characterized by GC-rich, long 3' untranslated regions (3' UTRs). UPF1 activity, measured by this transcript signature, is diminished in TDP-43-depleted and ALS patient MNs. Mechanistically, TDP-43 depletion impairs UPF1 phosphorylation; the two proteins interact in an RNA-dependent manner and co-aggregate in pathological inclusions in ALS tissue. Transcriptomic analyses reveal convergent regulation of alternative polyadenylation and 3' UTR length by UPF1 and TDP-43, processes disrupted in ALS models and patient neurons. Our study defines the mRNA surveillance network of UPF1 in MNs and uncovers a link between RNA decay, TDP-43 dysfunction, and ALS neurodegeneration.
    Keywords:  3′ UTR; ALS; APA; NMD; TDP-43; UPF1; alternative polyadenylation; amyotrophic lateral sclerosis; iPSC-derived motor neurons; nonsense-mediated mRNA decay
    DOI:  https://doi.org/10.1016/j.neuron.2025.11.001
  46. Cell Death Differ. 2025 Dec 15.
    SLAP controls mTORC2 integrity via UBE3C-mediated non-degradative mLST8 ubiquitination to suppress colorectal tumorigenesis.
    Rudy Mevizou, Dana Naim, Florent Cauchois, Cécile Naudin, Georgia Greaves, Kevin Espie, Bastien Felipe, Valérie Simon, Yvan Boublik, Julie Nguyen, Serge Urbach, Serge Roche, Audrey Sirvent.
      The mechanistic target of rapamycin complex 2 (mTORC2) signaling pathway, which regulates cell growth and migration, exhibits oncogenic function in colorectal cancer (CRC). mTORC2 signaling is primarily activated by a complex assembly of mTOR, RICTOR, SIN1, and mLST8; however, the mechanisms by which dysregulation of this pathway contributes to its oncogenic function remain elusive. Here, we show that the Src-Like Adaptor Protein (SLAP), a negative regulator of tyrosine kinase signaling receptors, controls mTORC2 integrity to mediate its tumor-suppressive function in CRC. Mechanistically, SLAP interacts with mLST8 and facilitates its non-degradative ubiquitination at lysines 86 and 215, thereby reducing the integrity of mTORC2 and mTORC2-AKT signaling. The E3 ubiquitin ligase UBE3C was identified as a novel SLAP interactor involved in this ubiquitination process. Functionally, SLAP inhibition of CRC cell growth and invasion was dependent upon mTORC2 signaling inhibition. In immunodeficient mice CRC xenografts, SLAP depletion enhanced mTORC2 activity and sensitized CRC cells to mTOR catalytic inhibitors. Together, our findings reveal a previously unrecognized SLAP-UBE3C-mLST8 axis that regulates mTORC2 integrity and suggest a potential therapeutic avenue for targeting mTORC2 in CRC.
    DOI:  https://doi.org/10.1038/s41418-025-01633-1
  47. bioRxiv. 2025 Nov 27. pii: 2025.11.26.690702. [Epub ahead of print]
    Homotypic endoplasmic reticulum membrane tethering is critical for flavivirus replication.
    Jonathan Einterz Owen, Cheyanne Lee Bemis, Qingyi Wang, Ambarish C Varadan, Jacob W Vander Velden, Laura Andačić, Olus Uyar, Mansi Gupta, Christopher D Scharer, Laurent Chatel-Chaix, Pietro Scaturro, Mehul S Suthar, Christopher J Neufeldt.
      Flaviviruses (genus Orthoflavivirus ) are arthropod-borne viruses which cause approximately 400 million annual global infections in humans. Flavivirus infection requires cellular machinery to facilitate replication and spread. All known flaviviruses replicate in association with the host endoplasmic reticulum (ER), where genome replication is confined within virus-induced ER invaginations called viral replication organelles (vROs). Despite the central role of these structures during flavivirus infection, the mechanisms underlying vRO biogenesis remain undefined - particularly the membrane rearrangements required for their formation. In this work, we report a conserved role for a cellular ER remodeling protein, atlastin-2 (ATL2), in the organization of vROs within infected cells. Using confocal and electron microscopy, we show that ATL2 depletion leads to a reduction in vRO spatial distribution in flavivirus-infected cells. Changes in vRO distribution corresponded with a decrease in virus production and robust induction of innate immune responses. We also demonstrate that ATL2 accumulates in areas of vRO formation during flavivirus infection. Critically, mutational analysis showed that a tethering-competent but fusion-defective ATL2 mutant was sufficient to rescue DENV and ZIKV replication in ATL2-knockout cells. Finally, inhibition of ATL2 activity using synthetic peptides significantly reduced DENV replication in both immortalized and human primary cells, suggesting a possible avenue for targeting host ER functions to limit flavivirus replication. Taken together, these results show that membrane tethering plays a critical and conserved role in flavivirus infection, functioning to organize membranes for vRO biogenesis and limit cellular immune activation. Importantly, we provide evidence that ATL2-mediated membrane organization can be targeted to inhibit viral replication.
    DOI:  https://doi.org/10.1101/2025.11.26.690702
  48. Adv Sci (Weinh). 2025 Dec 14. e14191
    PDIA3 Inhibition Facilitates Sensitivity of IKE-Induced Ferroptosis via STAT3/LCN2 Axis to Improve Glioblastoma Therapy.
    Jie Zhang, Wei Wang, Xin Liu, Peifen Lu, Xinjing Liu, Qiucheng Nie, Siyuan Xin, Hong Li, Donglin Yu, Xinyue Zhang, Kailong Li, Xiaomin Han, Shuping Zhang, Wei Chong, Lili Sun, Wei Li, Tao Xin, Jin Jiao, Qiang Ma, Yiju Wei.
      Ferroptosis, an iron-dependent form of programmed cell death, has emerged as a promising therapeutic approach in glioblastoma (GBM). Nonetheless, the role and mechanism governing vulnerability to ferroptosis in GBM have remained unknown. In this study, we identify protein disulfide isomerase A3 (PDIA3) as a crucial factor mediating the vulnerability of glioma cells to ferroptosis and demonstrate that inhibition or depletion of PDIA3 enhances IKE-induced ferroptosis in GBM cells. Mechanistically, NEDD4L functions as an E3 ubiquitin ligase to promote ferroptosis by facilitating K29-linked ubiquitination of PDIA3 via its C-terminal HECT domain. Furthermore, NEDD4L-mediated ubiquitination of PDIA3 enhances ferroptosis by downregulating the expression of LCN2 through its interaction with STAT3 independently of ATF4. Here, a drug delivery system is presented using a tetrahedral DNA nanostructure (TDN) encapsulating IKE (TDN-IKE) to penetrate the blood-brain barrier. The combined use of TDN-IKE and PDIA3 inhibitors exhibits a synergistic antitumor effect against GBM therapy in vivo, providing a potential therapeutic approach for ferroptosis-based therapy in GBM. Overall, these findings demonstrate a novel mechanism by which PDIA3 regulates ferroptosis, indicating that a promising therapeutic strategy for GBM is through inhibiting of SLC7A11 and PDIA3.
    Keywords:  LCN2; NEDD4L; PDIA3; ferroptosis; glioblastoma
    DOI:  https://doi.org/10.1002/advs.202514191
  49. bioRxiv. 2025 Dec 12. pii: 2025.12.10.693450. [Epub ahead of print]
    Degradation factor 1, Def1, regulates mRNA translation and decay through Ccr4-Not-dependent ubiquitylation of the ribosome.
    O T Akinniyi, A Sebastian, S Kulkarni, I Albert, J C Reese.
      Yeast Def1 is well known for its role in regulating RNA polymerase II elongation and degrading the large subunit of polymerase during transcriptional stress. It is an abundant cytoplasmic protein that undergoes stress-induced processing and is then transported to the nucleus. Previous research from our lab has shown that Def1 interacts with various proteins involved in mRNA decay and translation control, and that it regulates mRNA half-lives, suggesting an important role in the cytoplasm. In this study, we report that Def1 binds polyribosomes and that its null mutant strain exhibits phenotypes indicating a role in translation. Ribo-seq analysis revealed that deleting DEF1 altered ribosome footprints on mRNAs and increased the dwell time of ribosomes at non-optimal codons in the A-site. Additionally, results from a codon-optimality reporter assay suggest that Def1 facilitates the degradation of mRNAs containing non-optimal codons. The Ccr4-Not complex links codon optimality to mRNA decay, and Def1's binding to ribosomes depends on its ubiquitin-binding domain, as well as the ubiquitylation of eS7a in the small ribosomal subunit by the Ccr4-Not complex. Moreover, the polyglutamine-rich, unstructured C-terminus of Def1 is crucial for its interaction with RNA decay and translation factors. This indicates that Def1 functions as a ubiquitin-dependent scaffold, connecting translation status to mRNA decay. In summary, we have identified a cytoplasmic function for Def1 in translation and established it as a regulator of gene expression, spanning both transcription and translation processes.
    DOI:  https://doi.org/10.64898/2025.12.10.693450
  50. EMBO Rep. 2025 Dec 15.
    Vesicle-coupled mRNA transport and translation govern intracellular organelle networking.
    Melissa Vázquez-Carrada, Sainath Shanmugasundaram, Sander H J Smits, Lasse van Wijlick, Michael Feldbrügge.
      Eukaryotic cells are highly compartmentalized, enabling sophisticated division of labour. For example, genetic information is stored in the nucleus while energy is produced in mitochondria. Despite this clear specialisation, compartments depend on intensive communication, including the exchange of metabolites and macromolecules. This is achieved through intracellular trafficking with membranous carriers such as endosomes, which constitute versatile transport vehicles. Key cargos include mRNAs and ribosomes that hitchhike on endosomes, linking RNA and membrane biology. In this review, we summarize recent advances showing how mRNAs are mechanistically attached to membranes of endosomes and lysosomal vesicles and how cargos are identified for transport. The encoded proteins illuminate the biological processes that rely on such spatiotemporal control. This is particularly true for the regulation of subcellular mitochondrial homeostasis, disclosing intensive multi-organelle networking. As a general concept, the underlying protein/protein and protein/RNA interactions exhibit significant redundancy yet are organized in a strict hierarchy with distinct core and accessory functions. This ensures both the robustness and specificity of mRNA hitchhiking.
    Keywords:  Endosomes; Local Translation; Mitochondria; RNA Transport, SLiMs
    DOI:  https://doi.org/10.1038/s44319-025-00666-4
  51. Nat Commun. 2025 Dec 15. 16(1): 11005
    An open-source screening platform accelerates discovery of drug combinations.
    William C Wright, Min Pan, Gregory A Phelps, Jonathan Low, Duane Currier, Ankita Sanjali, Marlon Trotter, Jihye Hwang, Richard H Chapple, Xueying Liu, Declan Bennett, Yinwen Zhang, Richard E Lee, Taosheng Chen, Paul Geeleher.
      Drug combinations are essential to modern medicine, but their discovery remains slow and inefficient as experimental complexity expands rapidly with each additional drug tested. Although modern liquid handling systems enable complex and highly customizable experimental designs, a lack of strategies integrating these technologies with combination-specific analytical methods has limited throughput. Here we introduce Combocat, an open-source and streamlined framework that combines acoustic liquid handling protocols with machine learning-based inference to achieve ultrahigh-throughput drug combination screening. Using Combocat, we generate a reference dataset of over 800 unique combinations in a dense 10 × 10 matrix format across multiple cell types, and use this to train a predictive model that accurately infers drug combination effects from sparse data, drastically reducing the number of experimental measurements required. As proof of concept, we screened 9,045 combinations in a neuroblastoma cell line-the largest number of combinations tested in a single cell line to date-achieved using minimal resources. By integrating advanced drug dispensing technologies with predictive computational modeling, Combocat provides a scalable solution to accelerate the discovery of novel drug combinations.
    DOI:  https://doi.org/10.1038/s41467-025-66223-8
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