bims-proteo Biomed News
on Proteostasis
Issue of 2026–01–04
forty-nine papers selected by
Eric Chevet, INSERM



  1. J Biol Chem. 2025 Dec 27. pii: S0021-9258(25)02968-0. [Epub ahead of print] 111116
      Hyper- and hypo-activation of the Integrated Stress Response (ISR) results in impaired regulation of global and mRNA-specific translation in multiple disease contexts. During the ISR, specific stress-sensing kinases modulate translation by regulating the activity of the heterotrimeric eukaryotic translation initiation factor eIF2. Here, we identify the chaperone CDC123, which promotes eIF2 biogenesis, as a novel regulator of the ISR. We find that impaired CDC123 activity reduces eIF2 complex assembly, promoting the translational and cellular outcomes of the ISR through a noncanonical mechanism. Pharmacological or genetic strategies are sufficient to rescue the translational defects associated with impaired CDC123 activity. Additionally, we report functional insights into eIF2 heterotrimer formation and provide the first evidence that CDC123-mediated eIF2 complex assembly may be regulated by ATP hydrolysis. These data emphasize the essential contribution of eIF2 biogenesis in mRNA translation regulation, and highlight CDC123 as a possible therapeutic target in the treatment of ISR-related diseases.
    DOI:  https://doi.org/10.1016/j.jbc.2025.111116
  2. Autophagy. 2025 Dec 31.
      Endoplasmic reticulum (ER) exit sites (ERES) serve as essential hubs for the packaging and export of secretory proteins into the COPII vesicular pathway. Previous studies have shown that ERES are dynamic and capable of adapting to stress, but the molecular details controlling their degradation under nutrient stress conditions were largely unknown. The study by Liao et al. (2024) introduces a new mechanism in which ERES are degraded through lysosome-dependent microautophagy in response to nutrient stress. This process is uniquely facilitated by COPII components, the calcium-binding adaptor ALG2, and the ESCRT machinery. The authors demonstrate that inhibiting MTOR triggers calcium release from lysosomes, which then recruits ALG2, leading to SEC31 ubiquitination and subsequently promoting PDCD6IP/ALIX-ESCRT-dependent lysosomal engulfment of ERES. This research reveals an unexplored pathway for the quality control and recycling of secretory machinery, thereby improving our understanding of ER turnover and establishing a mechanistic link between nutrient sensing, autophagy, and remodeling of the secretory pathway.
    Keywords:  Autophagy; COPII; ESCRT; er exit sites; microautophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2608387
  3. Sci Adv. 2026 Jan 02. 12(1): eaea9302
      (Macro)autophagy is a conserved cellular degradation pathway that delivers substrates to lysosomes via autophagosomes. Among various physiological stimuli, nutrient starvation is the most potent inducer of autophagy. In response to starvation, transcription factor EB (TFEB) is activated and up-regulates a broad set of autophagy-related genes. However, the mechanisms by which TFEB promotes autophagosome biogenesis remain incompletely understood. Here, we demonstrate that TFEB-mediated transcriptional induction of sequestosome 1 (SQSTM1; p62) triggers the formation of SQSTM1-positive bodies that recruit essential autophagy factors, thereby initiating autophagosome biogenesis. Genetic disruption of TFEB-dependent SQSTM1 regulation markedly impairs starvation-induced autophagy, underscoring the critical role of the TFEB-SQSTM1 axis in the autophagic response to nutrient stress. Furthermore, we show that these SQSTM1 bodies contain ubiquitinated ribosomal proteins and that TFEB promotes ribosomal protein ubiquitination by inducing the E3 ubiquitin ligase ZNF598. Collectively, our findings uncover a transcriptionally coordinated mechanism that regulates both autophagosome biogenesis and substrate ubiquitination, facilitating efficient cargo clearance during starvation-induced autophagy.
    DOI:  https://doi.org/10.1126/sciadv.aea9302
  4. Proc Natl Acad Sci U S A. 2026 Jan 06. 123(1): e2524899122
      E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous different E3s. Here, focusing on E3s in the RING-between-RING (RBR) family and their partner UBE2L3 and UBE2D-family E2s, we report an approach to interrogate selected pathways. We screened phage-displayed libraries of structure-based E2 variants (E2Vs) to discover enzymes with enhanced affinity and specificity toward half of all RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, and RNF14). Collectively, these E2Vs allowed distinguishing actions of different cofunctioning E3s, obtaining high-resolution cryogenic Electron Microscopy (cryo-EM) structures of an RBR E3 in the context of a substrate-bound multiprotein complex, and profiling an endogenous RBR E3 response to an extracellular stimulus. Overall, we anticipate that E2V technology will be a generalizable tool to enable in-depth mechanistic and structural analysis of E3 ligase functions, and mapping their activity states and protein partners in cellular signaling cascades.
    Keywords:  E2; E3 ligase; RBR; activity-based probe; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2524899122
  5. Mol Cell. 2025 Dec 30. pii: S1097-2765(25)00982-7. [Epub ahead of print]
      The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acid tails (CAT tails) in an mRNA-free, non-canonical elongation process. The relationship between CAT tail composition (alanine [Ala] and threonine [Thr] in yeast) and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanical forces on the NC regulate CAT tailing. We propose that CAT tailing initially operates in "extrusion mode," which increases NC lysine accessibility for on-ribosome ubiquitylation. Thr in CAT tails prevents the formation of polyalanine, which forms α-helices that lower extrusion efficiency and disrupt termination of CAT tailing. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode," which facilitates NC release and degradation. Failure to switch from extrusion to release mode leads to the accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails.
    Keywords:  CAT tails; RQC; mechanochemistry; protein folding; protein quality control; ribosome; ribosome stalling; ribosome-associated quality control; translation
    DOI:  https://doi.org/10.1016/j.molcel.2025.12.008
  6. J Cell Biol. 2026 Mar 02. pii: e202503087. [Epub ahead of print]225(3):
      Exosomes are multivesicular body-derived extracellular vesicles that are secreted by metazoan cells. Exosomes have utility as disease biomarkers, and exosome-mediated miRNA secretion has been proposed to facilitate tumor growth and metastasis. Previously, we demonstrated that the Lupus La protein (La) mediates the selective incorporation of miR-122 into metastatic breast cancer-derived exosomes; however, the mechanism by which La itself is sorted into exosomes remains unknown. Using unbiased proximity labeling proteomics, biochemical fractionation, superresolution microscopy, and genetic tools, we establish that the selective autophagy receptor p62 sorts La and miR-122 into exosomes. We then performed small RNA sequencing and found that p62 depletion reduces the exosomal secretion of tumor suppressor miRNAs and results in their accumulation within cells. Our data indicate that p62 is a quality control factor that modulates the miRNA composition of exosomes. Cancer cells may exploit p62-dependent exosome cargo sorting to eliminate tumor suppressor miRNAs and thus to promote cell proliferation.
    DOI:  https://doi.org/10.1083/jcb.202503087
  7. Autophagy. 2025 Dec 31. 1-3
      Cells maintain organelle integrity and metabolic balance through tightly coordinated quality control systems. Autophagy plays a central role by recycling damaged and unnecessary cellular components, with selective pathways providing specificity through dedicated receptors. Although OPTN is well-established as a receptor for mitophagy, aggrephagy, and xenophagy, its role in pexophagy, the selective autophagic degradation of peroxisomes, has only recently been recognized. Our recent work identifies the peroxisomal membrane protein PEX14 as a critical docking platform for OPTN, enabling recruitment of autophagic machinery and initiation of pexophagy. How PEX14 engages OPTN, what triggers this interaction, and how it drives the autophagic engulfment of peroxisomes remain unclear. In this punctum, we contextualize our findings and highlight unresolved questions that must be addressed to understand the physiological and pathological relevance of this process.
    Keywords:  Autophagy receptors; TBK1; organelle quality control; peroxisome biogenesis; pexophagy; phosphorylation; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2025.2610452
  8. Biochem Biophys Res Commun. 2025 Dec 26. pii: S0006-291X(25)01931-X. [Epub ahead of print]797 153215
      Oncogenic signaling and stress response pathways interact to drive tumorigenesis and therapy resistance. However, little is known about such interactions for HER3, a member of the HER/ErbB receptor family that is aberrantly expressed in many tumors, including breast cancer. Here, we show that HER3 cooperates with HER2 to enhance induction of ATF4, a central transcription factor of the integrated stress response and the unfolded protein response, during endoplasmic reticulum (ER) stress. ATF4 induction was enhanced by ligand-activated HER3 and conversely reduced by genetic knockdown or pharmacological inhibition of HER2/HER3-mediated signaling in both HER2-overexpressing SKBR3 and non-overexpressing MCF7 breast cancer cells. HER3 knockdown in SKBR3 cells also increased cell death during ER stress. Notably, depletion of HER3, likely occurring through ER stress-associated downregulation mechanisms, was accompanied by attenuation of ATF4 induction during sustained stress. These findings suggest that the HER3-ATF4 axis functions as a dynamically regulated mechanism for tuning the cellular stress response.
    Keywords:  ATF4; Breast cancer; HER2; HER3; Integrated stress response
    DOI:  https://doi.org/10.1016/j.bbrc.2025.153215
  9. FEBS Lett. 2026 Jan 03.
      The maintenance of protein homeostasis is a fundamental premise for the survival of all life. The synthesis, folding, localization, and degradation of thousands of proteins must be organized according to various conditions. To ensure such a stable and functional proteome, the proteostasis network evolved. Dedicated to this, the fourth School on Proteostasis, a co-funded EMBO|FEBS Lecture Course in memory of Susan Lindquist, took place in Espoo, Finland on 16-19 September 2025, with 59 early career researchers (PhD students or postdoctoral fellows), 18 leading scientists, and two editors attending and discussing the current state of the field. From basic principles to the latest therapeutic developments, this meeting provided a comprehensive overview of proteostasis. This report summarizes the lecture course and highlights selected presentations.
    Keywords:  aging and disease; molecular chaperones; protein degradation; protein quality control; proteostasis; stress responses
    DOI:  https://doi.org/10.1002/1873-3468.70261
  10. Biochem Biophys Rep. 2026 Mar;45 102393
       Background: The most common loss-of-function mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) is F508del. The misfolded F508del-CFTR protein is targeted for endoplasmic reticulum associated degradation (ERAD), a pathway in which non-native proteins are ubiquitinated and degraded by the proteasome. Because the identification of ubiquitinated residues would highlight how F508del-CFTR is selected for premature degradation, ubiquitination profiles in CFTR- and F508del-CFTR-expressing cells have been examined. Several ubiquitin ligases modify CFTR, however, the relative CFTR-directed activity of each ligase is unknown.
    Methods: We reconstituted CFTR ubiquitination using purified CFTR and components of the ubiquitination machinery. Since prior work implicated the Carboxyl terminus of Hsp70-Interacting Protein (CHIP) ubiquitin ligase in both ERAD and plasma membrane turnover, CFTR ubiquitination was examined in the presence of CHIP and a companion ubiquitin conjugating enzyme.
    Results: Mass spectrometry identified 16 modified lysines, half of which were previously identified after CFTR was isolated from cells. One lysine, K420, which resides in the regulatory insertion, had been implicated in cyclic nucleotide-dependent activation of CFTR. Here, we find that mutation of K420 increases cell surface levels of CFTR, an effect which in turn increases forskolin-dependent short circuit current.
    Conclusions: We establish a system in which residue-specific modifications of CFTR by any component of the ubiquitin machinery can now be surveyed.
    DOI:  https://doi.org/10.1016/j.bbrep.2025.102393
  11. Plant Cell Physiol. 2025 Dec 29. pii: pcaf173. [Epub ahead of print]
      The AAA+ ATPase CDC48A is a central regulator of proteostasis in plants, functioning through interactions with a diverse set of cofactors. Among these, the plant-specific ubiquitin regulatory X (UBX) domain-containing proteins (PUX) are key adaptors that direct CDC48A to specific substrates and pathways. The molecular basis of PUX-CDC48A interactions remains incompletely understood. Here, we combine structural, biophysical, and computational approaches to dissect the binding modes of representative PUX proteins from different subfamilies in Arabidopsis thaliana. Although all PUX proteins tested exhibit low micromolar affinities for CDC48A, they form unexpectedly stable complexes, suggesting additional mechanisms of interaction. We identify two distinct strategies for complex stabilisation, producing different dynamic features. One relies on combining two weak associations: PUX5 employs a SHP-UBX module that engages the CDC48A N domain at two proximal sites, whereas PUX2 uses a SHP motif and a distant PUB domain to engage the N- and C-termini of CDC48A. In contrast, PUX6, PUX7, and PUX9 allosterically stabilise the association between their UBX domain and the CDC48A N domain. These multi-pronged strategies likely enable durable yet reversible associations, facilitating fine-tuned competitive regulation of CDC48A activity across diverse cellular contexts. Our findings provide a mechanistic framework for understanding how PUX proteins achieve specificity, stability, and regulatory flexibility in directing CDC48A function.
    Keywords:  multivalency; protein-protein interactions; targeted protein degradation; unfolding
    DOI:  https://doi.org/10.1093/pcp/pcaf173
  12. Life Sci Alliance. 2026 Mar;pii: e202503523. [Epub ahead of print]9(3):
      Every cell must adapt to environmental changes. When nutrients decrease, oxygen levels fall, or protein synthesis outpaces resources, cells activate stress pathways to restore balance. Among these, the integrated stress response (ISR) stands out for its capability to integrate diverse stress signals into a unified translational output. By temporarily slowing global protein synthesis while maintaining the selective translation of stress-adaptive factors, the ISR saves energy, redirects metabolism, and promotes either recovery or, if challenges surpass repair capacity, cell death. In many chronic diseases-including cancer, metabolic, inflammatory, and fibrotic disorders-ISR activity persists. Is this persistence merely a prolonged defensive phase, or does it represent a rewired, self-sustaining state with its own control mechanisms actively reshaping cell fate and disease? We argue that chronic ISR cannot be defined by time alone, challenging the monolithic view. It signifies a qualitative shift in regulation-from rhythmic homeostasis to entrenched maladaptation. Understanding this Janus framework is essential for elucidating the origins of pathology and for guiding future fundamental and translational research.
    DOI:  https://doi.org/10.26508/lsa.202503523
  13. Cell Chem Biol. 2025 Dec 31. pii: S2451-9456(25)00396-4. [Epub ahead of print]
      The NCI-60 cancer cell line panel is one of the most extensively characterized and publicly accessible resources in cancer research. Here, we expand this platform by generating a comprehensive proteomic and cysteine reactivity atlas using shotgun proteomics and quantitative chemoproteomics. We quantified over 12,000 proteins and identified more than 36,000 reactive cysteines, including over 10,000 hyperreactive sites, across the panel. Our analyses reveal widespread heterogeneity in cysteine reactivity, while conserved hyperreactive cysteines strongly correlate with functional roles. Tissue-specific cysteine reactivity occurred independently of protein abundance, highlighting context-dependent regulation. To enable exploration and integration with existing datasets, we developed an interactive online database that allows users to visualize protein and cysteine coverage and design custom cell line panels. Together, these data and tools illuminate the landscape of cysteine reactivity in cancer and provide a foundational resource to advance future studies in protein function, redox biology, and covalent drug discovery.
    Keywords:  NCI-60; chemical proteomics; cysteine reactivity; mass spectrometry
    DOI:  https://doi.org/10.1016/j.chembiol.2025.12.003
  14. Bioorg Med Chem Lett. 2025 Dec 27. pii: S0960-894X(25)00437-8. [Epub ahead of print]133 130528
      The proteolysis-targeting chimera (PROTAC) technology utilizes heterobifunctional molecules to induce targeted protein degradation through the ubiquitin-proteasome system. Structurally, PROTAC molecules consist of a target protein ligand and an E3 ligase ligand covalently linked by a suitable linker arm, forming the target protein-PROTAC-E3 ligase stable ternary complex and bringing the target protein in proximity to the E3 ligase for ubiquitination and subsequent proteasomal degradation. However, only a few E3 ligases have been used to generate effective PROTACs with limited small molecule E3 ligase ligands. Therefore, there is an urgent need to discover novel E3 ligase ligands to expand the toolbox for PROTACs. Unlike traditional E3 ligases such as ‌CRBN‌ and ‌VHL‌, GID4 E3 ligase recognizes substrates bearing N-terminal proline or other small residues through the Pro/N-degron pathway, and has already been successfully leveraged in ‌PROTAC technology. Here, we reported the development of a fluorescent probe YG11, with a Kd value of 8.1 ± 0.7 nM for GID4. With this probe, we established a robust fluorescence polarization (FP)-based competition assay for evaluation of GID4 ligands. The assay exhibited a high signal-to-noise ratio of over 20, 2.5 % DMSO tolerance, and a Z'-factor of 0.84, confirming its suitability and robustness for high-throughput screening. Thus, by enabling rapid identification of GID4 ligands, this FP competition assay promises to substantially advance PROTAC development initiatives.
    Keywords:  E3 ligases; Fluorescence polarization assay; GID4; High-throughput screening
    DOI:  https://doi.org/10.1016/j.bmcl.2025.130528
  15. Proc Natl Acad Sci U S A. 2026 Jan 06. 123(1): e2525043123
      Cholesterol homeostasis in mammalian cells relies on the interaction between two endoplasmic reticulum (ER) sterol-sensing membrane proteins, Scap and Insig. Their interaction regulates activation of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control genes for cholesterol biosynthesis and uptake. Previous studies suggested a model where cholesterol sensing by Scap involves communication across the ER membrane between two functional domains, a cholesterol-binding domain on the luminal side and a COPII-binding domain on the cytosolic side. When ER cholesterol is low, Scap binds COPII adapter proteins to facilitate ER-to-Golgi transport and proteolytic activation of SREBPs. When ER cholesterol is above a threshold concentration, this transport is blocked, a process that requires Insigs. However, the precise molecular mechanisms by which cholesterol and Insigs control the conformations of Scap remain unknown. Here, we elucidate the 3.2 Å cryo-EM structure of a Scap/Insig complex in the presence of saturating amounts of cholesterol. Structure-guided mutagenesis of the Scap/Insig interface indicates that Scap's transmembrane helix 7 (TM7) plays a critical role in transducing conformational changes between the luminal and cytosolic sides of the ER membrane to control Scap/SREBP transport from ER to Golgi. An intramembrane cholesterol bound at the Scap/Insig interface competes with the intramolecular interaction of Scap's TM7 at this interface to modulate cholesterol sensing. These results further advance our understanding of how Scap senses cholesterol and provides additional targets for controlling cholesterol and lipid synthesis in the context of metabolic diseases and even some cancers.
    Keywords:  SREBP; Scap; cholesterol; lipid metabolism
    DOI:  https://doi.org/10.1073/pnas.2525043123
  16. J Cell Biol. 2026 Feb 02. pii: e202502112. [Epub ahead of print]225(2):
      Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one leaflet, scramblase activity should be essential for equilibrated membrane growth. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane and participates in the transfer of phosphatidylserine. We show both in vitro and in silico that the ER domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity that is not regulated by Ca2+. In cells, overexpression or deletion of the ER domain of Ist2 affects ER-related processes including COPII-mediated vesicular transport, lipid droplet homeostasis, and general phospholipid transport, with a specific contribution of residues implicated in lipid scrambling. The weak phenotypes can be augmented by the deletion of another putative scramblase, the protein insertase Get1, suggesting that the combined action of different proteins supports lipid scrambling at the ER.
    DOI:  https://doi.org/10.1083/jcb.202502112
  17. Proc Natl Acad Sci U S A. 2026 Jan 06. 123(1): e2517547123
      Nuclear factor erythroid 2-like 1 (NFE2L1/Nrf1), an endoplasmic reticulum (ER)-associated transcription factor, is responsible for the coordinated expression of proteasome subunit genes upon proteasomal dysfunction. N-glycosylated proteins undergo protein sequence editing by peptide:N-glycanase (NGLY1)-mediated conversion of N-glycosylated asparagine residues to aspartic acid. Nrf proteins are the only transcription factors that undergo sequence editing for transcriptional activation. However, the mechanism via which sequence editing regulates the transcriptional activity of Nrf1 has remained unclear. Here, we demonstrated that sequence editing of the ninth N-glycosylation site (Asn574) in human Nrf1 is required for proteasome gene expression in HeLa cells. Editing of Asn574 is essential for interaction with host cell factor C1 and O-GlcNAc transferase, which is required for Nrf1 chromatin binding and sufficient proteasome expression. Furthermore, sequence editing of N-glycosylation sites other than Asn574 is required for the interaction with the coactivator CREBBP/EP300, thereby enhancing Nrf1's transcriptional activity. Unexpectedly, the expression of Nrf1 mutants that mimic proteolytic processing by DNA-damage-inducible 1 homolog 2 and sequence editing by NGLY1 markedly diminished the growth rate in HeLa cells, suggesting that the constitutive activation of Nrf1 exhibits cytotoxicity. Collectively, our study explains the strategy of on-demand Nrf1 activation for survival benefits. Nrf1 is synthesized as a proteasome-targeting protein and is highly glycosylated in the ER. Nrf1 is activated via sequence editing-dependent coactivator complex formation only when the proteasome needs to be compensated for.
    Keywords:  HCFC1; NFE2L1; NGLY1; Nrf1; proteasome
    DOI:  https://doi.org/10.1073/pnas.2517547123
  18. STAR Protoc. 2025 Dec 27. pii: S2666-1667(25)00709-9. [Epub ahead of print]7(1): 104303
      The dynamics of the early steps of protein aggregation remain poorly understood, particularly in the case of α-synuclein (α-syn) aggregation, the hallmark of synucleinopathies. Here, we present a protocol that combines light-inducible protein aggregation (LIPA) with proximity biotinylation using an UltraID construct. We describe the workflow from protein expression to biochemical validation, including the purification of biotinylated proteins prior to liquid chromatography-mass spectrometry (LC-MS) analysis and subsequent validation. This platform provides a powerful strategy to identify proteins interacting with nascent α-syn aggregates. For complete details on the use and execution of this protocol, please refer to Teixeira et al.1.
    Keywords:  Cell biology; Cell culture; Molecular biology; Proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2025.104303
  19. Neural Regen Res. 2025 Dec 30.
      Error-prone translation, resulting in inaccuracies in protein synthesis, is increasingly recognized as a critical contributor to proteostasis disruption and the pathogenesis of age-related neurological disorders. In recent years, numerous studies have elucidated that stochastic errors during mRNA translation may act as a molecular "tipping point" initiating pathogenic protein misfolding. A detailed analysis of how translation errors lead to protein misfolding, aggregation, and subsequent neurotoxicity will facilitate the identification of promising therapeutic targets for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. This article explores the contribution of mistranslation to proteostasis decline, focusing on the unique vulnerabilities of neuronal cells. We review the sources of translation errors, effects of ribosomal ambiguity and error-restrictive mutations, role of proteostatic mechanisms (such as molecular chaperones, ubiquitin-proteasome system, and unfolded protein response), and provide a unified perspective that links age-related translational infidelity to neurodegeneration. By synthesizing the most recent data obtained with genetically modified cellular and animal model studies, we highlight how age-associated decline in translational fidelity exacerbates proteostasis failure and propose potential therapeutic interventions targeting translation accuracy to mitigate neurodegeneration.
    Keywords:  aging; neurological disease; protein aggregation; protein misfolding; protein synthesis; proteostasis; ribosomal mistranslation; translation accuracy
    DOI:  https://doi.org/10.4103/NRR.NRR-D-25-00795
  20. Clin Transl Med. 2026 Jan;16(1): e70577
       BACKGROUND: Colorectal adenocarcinoma (COAD) cells exploit stress-adaptation programs, such as the unfolded protein response (UPR), to survive in hostile tumour microenvironments. However, the role of specific E3 ubiquitin ligases in regulating these survival pathways remains poorly understood. We investigated Ring Finger Protein 39 (RNF39), an E3 ligase previously implicated in immune signalling, as a potential regulator of COAD progression.
    METHODS: We analyzed RNF39 expression using public transcriptomic datasets (TCGA, GEO) and a clinical COAD cohort via immunohistochemistry. Functional roles were assessed in COAD cell lines using shRNA knockdown, CRISPR/Cas9 knockout, and overexpression systems. In vitro assays (proliferation, invasion, colony formation) and in vivo xenograft models were employed. Mechanistic investigations included co-immunoprecipitation, ubiquitination assays, chromatin immunoprecipitation, and luciferase reporter assays to delineate the MEF2D-RNF39-RINT1 axis.
    RESULTS: RNF39 was aberrantly upregulated in COAD tissues, and its high expression correlated with poor patient survival. We identified the transcription factor MEF2D as a direct activator of RNF39. Functionally, RNF39 promoted COAD cell proliferation and invasion in vitro and tumour growth in vivo, dependent on its E3 ligase activity. Mechanistically, RNF39 directly interacted with, polyubiquitinated (K48-linked), and promoted the proteasomal degradation of RAD50-interacting protein 1 (RINT1). Consequently, RNF39 depletion stabilized RINT1, amplified the UPR and CHOP expression, and sensitized cells to ER stress-induced apoptosis. Crucially, the anti-tumour phenotypes of RNF39 loss were partially reversed by simultaneous RINT1 knockdown.
    CONCLUSION: RNF39 acts as a pro-tumorigenic E3 ligase in COAD by driving the degradation of RINT1, thereby suppressing ER stress-induced apoptosis and promoting malignant progression. Our findings delineate a novel MEF2D-RNF39-RINT1 signalling axis that governs tumour cell adaptation to stress. Targeting RNF39 could represent a promising therapeutic strategy to overcome stress resistance in COAD.
    KEY POINTS: RNF39 is identified as an oncogenic E3 ubiquitin ligase that is upregulated in colorectal adenocarcinoma and associated with poor prognosis. Mechanistically, RNF39 targets the tumour suppressor RINT1 for K48-linked polyubiquitination and proteasomal degradation. By degrading RINT1, RNF39 suppresses the unfolded protein response (UPR) and limits endoplasmic reticulum (ER) stress-induced apoptosis, thereby promoting tumour progression. The study reveals a novel MEF2D-RNF39-RINT1 axis governing stress adaptation, positioning RNF39 as a potential prognostic biomarker and therapeutic target in colorectal cancer.
    Keywords:  COAD; ER stress; RINT1; RNF39; ubiquitin‐dependent degradation
    DOI:  https://doi.org/10.1002/ctm2.70577
  21. Sci Adv. 2026 Jan 02. 12(1): eaea7735
      Adaptive immunity depends on major histocompatibility complex class I (MHC I) presentation of peptides, a process orchestrated by the peptide-loading complex (PLC) in the endoplasmic reticulum (ER). The PLC ensures precise peptide selection and loading and is a major target of viral immune evasion, notably by human cytomegalovirus (HCMV). Here, we report the 2.59- to 2.88-Å cryo-electron microscopy structure of native human PLC bound to the HCMV immune evasin US6. US6 inhibits the transporter associated with antigen processing 1/2 (TAP1/2) by laterally attaching its transmembrane helix to TAP2 using a disulfide-rich domain to mimic a translocating peptide. This domain blocks the ER-lumenal exit and locks TAP in an outward-facing conformation with closed nucleotide-binding domains and asymmetric adenosine 5'-triphosphate/adenosine 5'-diphosphate occlusion. The structure also reveals how TAP's amino-terminal transmembrane domains scaffold the MHC I chaperone tapasin. These findings elucidate the mechanism of US6-mediated immune evasion and highlight potential targets for therapeutic modulation of immune presentation in infection and cancer.
    DOI:  https://doi.org/10.1126/sciadv.aea7735
  22. Nat Chem Biol. 2025 Dec 29.
      The E3 ligase substrate adapter cereblon (CRBN), the primary target of clinical agents thalidomide and lenalidomide, recognizes endogenous substrates bearing the C-terminal cyclic imide modification. Although C-terminal cyclic imides can form spontaneously, an enzyme that regulates their formation and thereby promotes a biological pathway connecting substrates to CRBN is unknown. Here we report that protein carboxymethyltransferase (PCMT1) promotes formation of C-terminal cyclic imides on C-terminal asparagine residues of CRBN substrates. PCMT1 and CRBN coregulate the levels of metabolic enzymes including glutamine synthetase and inorganic pyrophosphatase 1 in vitro, in cells and in vivo, and this regulation is associated with the proepileptic phenotype of CRBN knockout mouse models. The discovery of an enzyme that regulates CRBN substrates through the C-terminal cyclic imide reveals a previously unknown biological pathway that is perturbed by thalidomide derivatives and provides a biochemical basis for the connection between multiple biological processes and CRBN.
    DOI:  https://doi.org/10.1038/s41589-025-02106-9
  23. J Biol Chem. 2025 Dec 30. pii: S0021-9258(25)02974-6. [Epub ahead of print] 111122
      UBE2O, an E3-independent E2 ubiquitin-conjugating enzyme, directly engages substrates to mediate Ubiquitin conjugation and ligation. Despite its critical role in ubiquitination of multiple substrates, the catalytic and regulatory mechanisms of Homo sapiens UBE2O (hUBE2O) remain incompletely understood. Here, combining domain truncation, systematic mutagenesis and well-designed biochemical approaches, we demonstrate that hUBE2O mediates both mono- and polyubiquitination through a unique catalytic architecture. Specifically, hUBE2O lacks dedicated catalytic cysteines for E3 ligase activity. Instead, its E3-independent ubiquitination function relies on the coordination of multiple domains: the catalytically essential UBC domain requires the flanking coiled-coil (CC) and C-terminal regulatory (CTR) domains to maintain its full enzymatic competence, whereas the N-terminal region imposes an autoinhibitory constraint on the enzyme's activity. Interestingly, hUBE2O's activity is refractory to its phosphorylation and lysine-mediated self-ubiquitination state. Instead, specific non-cysteine residues (H939, T995, S1042, T1046, S1060 and H1130) in the UBC domain emerge as critical regulators of catalytic optimization. Surprisingly, zinc ions emerge as potent allosteric inhibitors that bind cysteines of hUBE2O, sterically occluding the access of catalytic site C1040 to Ubiquitin. Our findings reveal that hUBE2O-mediated E3-independent ubiquitination is governed by dynamic interdomain cooperation and allosteric modulation, establishing a mechanistic framework for understanding non-canonical ubiquitination and informing the development of targeted hUBE2O modulators.
    Keywords:  UBE2O; ubiquitin-conjugating enzyme; ubiquitination; zinc regulation
    DOI:  https://doi.org/10.1016/j.jbc.2025.111122
  24. J Pharmacol Exp Ther. 2025 Dec;pii: S0022-3565(25)39907-6. [Epub ahead of print]392(12): 103694
      Targeted protein degradation is an emerging strategy for experimental and therapeutic ablation of biologically important proteins. To elicit the degradation of their cellular targets, targeted protein degraders act by co-opting the endogenous cellular degradation machineries through chemically-induced proximity. While targeted protein degradation was serendipitously discovered as the mode of action of approved anticancer drugs including fulvestrant and thalidomide, recent years have witnessed systematic endeavors for the rational design of targeted protein degraders for diverse biological targets. Such endeavors have led to 3 major classes of targeted protein degraders including molecular glue degraders, proteolysis targeting chimeras, and hydrophobic tag-based degraders. Of these, several agents are clinically approved or currently evaluated in clinical trials for use in diseases such as cancer, neurodegenerative disorders, autoimmune and dermatologic conditions. The novel chemical and pharmacologic nature of targeted protein degraders heralds an emerging paradigm of pharmacology, known as event-driven pharmacology, which is different in many aspects from the occupancy-based pharmacology of conventional small-molecule inhibitors. In this review, we discuss the emerging pharmacology of different classes of targeted protein degraders including the molecular basis of their drug action and key pharmacologic properties pertinent to efficacy, selectivity, safety, and dosing considerations. SIGNIFICANCE STATEMENT: Targeted protein degradation is a novel strategy that establishes induced-proximity pharmacology as a promising next-generation therapeutic modality. This review provides insights into the common organizing principles of this emerging approach and the prospects for this rapidly evolving field.
    Keywords:  Autophagy-lysosomal pathway; Molecular glue degrader; Proteolysis targeting chimera; Selective estrogen receptor degrader; Targeted protein degrader; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.jpet.2025.103694
  25. ACS Chem Biol. 2026 Jan 02.
      Protein-based bispecific degraders, known as bioPROTACs, have emerged as powerful tools for targeted protein degradation through the ubiquitin-proteasome system (UPS). However, the relative efficacy of various recruitment domains within these degraders remains poorly understood. To address this knowledge gap, we conducted a comprehensive comparison of recruitment domains in bioPROTACs, utilizing eGFP as a proof-of-principle degradation target and an eGFP-binding DARPin with known structure as an adapter. Our innovative approach combined microinjection and live-cell microscopy, enabling a detailed assessment of directly measured degradation rates as a single-cell kinetic readout, unaffected by uptake or biosynthesis rates of the degrader, and across the different chemical classes. We examined nine degron peptides, three E3 ligase domains or adapters, and two series of small-molecule binders, linked in various geometries. Our results revealed that bioPROTACs based on E3 or adapter protein domains and small molecules generally exhibited the highest degradation rates, while most degron peptides showed comparatively low efficacy. Notably, for VHL-ligand-1 and thalidomide, the placement of the coupling site and linker position significantly influenced performance. This study provides crucial insights into the design and optimization of bioPROTACs, paving the way for the development of more effective degraders for specific applications. Our findings contribute to the growing field of targeted protein degradation and offer valuable guidance for researchers seeking to enhance the efficacy of bioPROTAC-based therapeutic approaches.
    DOI:  https://doi.org/10.1021/acschembio.5c00569
  26. Nat Commun. 2025 Dec 31.
      Sigma Non-Opioid Intracellular Receptor 1 (SigmaR1) is a member of the sigma family of receptors that interacts with a variety of psychotomimetic drugs and is involved in a wide range of cellular and physiological functions. Despite its increasing importance in human physiology and disease, the subcellular localization of SigmaR1 and its molecular function remain poorly defined. Using endogenous tagging and cell fractionation, we show that SigmaR1 is a type II integral ER membrane protein that is specifically enriched at ER sheets. A short region at the N-terminus of SigmaR1 promotes its ER-sheet localization. Importantly, our biochemical studies demonstrate that SigmaR1 directly interacts with components of the translocon complex including TRAPα and Nicalin. In addition, we found that a β-barrel at the C-terminal of SigmaR1 binds phosphatidylcholine (PC), and the binding of PC strengthens the association of SigmaR1 with the translocon complex. SigmaR1 knockout systematically impaired cellular protein and lipid homeostasis, resulting in accumulation of lipid droplets in hepatocytes. Collectively, we propose that SigmaR1 is an auxiliary translocon factor that binds lipids to regulate protein and lipid droplet homeostasis, which may underlie the broad and vital roles of SigmaR1 in physiology and disease.
    DOI:  https://doi.org/10.1038/s41467-025-68157-7
  27. Cell Res. 2026 Jan 02.
      Lysosomes are essential for cellular homeostasis, serving as degradative organelles that recycle nutrients. Whether and how lysosomes maintain membrane integrity under energy stress is poorly understood. Here, we found that the uptake of lipid droplets by lysosomes during glucose starvation provokes disruption of lysosomal membranes. We identified tectonin beta-propeller repeat-containing protein 1 (TECPR1) as a critical mediator of lysosomal repair during glucose starvation or LLOMe-induced lysosomal membrane permeabilization. TECPR1 is recruited to damaged lysosomes via interaction with PI4P on damaged lysosomal membranes. It interacts with KIF1A to facilitate tubule formation from damaged lysosomes, enabling the removal of damaged membrane components and promoting lysosomal repair. Our in vitro reconstituted tubulation process provided further evidence that TECPR1 coordinates with KIF1A to drive tubulation from PI4P-enriched giant unilamellar vesicles. TECPR1-mediated lysosomal repair is essential for maintaining lipid metabolism and cellular survival during an energy crisis, as TECPR1 deficiency exacerbates starvation-induced liver damage in a high-fat diet-induced MAFLD mouse model. Our findings demonstrate a previously unrecognized role of TECPR1 in lysosomal repair, revealing its critical contributions to energy stress adaptation and liver protection. This work provides new insight into mechanisms of lysosomal repair and their implications for metabolic and lysosome-related disorders.
    DOI:  https://doi.org/10.1038/s41422-025-01193-6
  28. Exp Hematol. 2025 Dec 31. pii: S0301-472X(25)00643-5. [Epub ahead of print] 105364
      Hematopoietic stem cells (HSCs) maintain the blood system by balancing self-renewal versus mature blood cell generation. One mechanism contributing to this balance is asymmetric cell division (ACD), which relies on tightly regulated intracellular compartmentalization. In other cell types, endoplasmic reticulum diffusion barriers (ER-DBs) contribute to the targeted distribution of cellular components and cell fate regulators during ACDs. Here, we identify ER-DBs as a feature of a subset of HSC divisions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse confocal microscopy, we observe ER-DBs in around 30% of mitotic HSCs. These ER-DBs are significantly weakened by Fingolimod, a potent inhibitor of sphingosine-1-phosphate receptor and ceramide synthesis, implicating sphingolipid metabolism in their regulation. We find that strong ER-DBs are not required for the asymmetric inheritance of lysosomes during HSC ACD. This demonstrates that ER diffusion barriers are present and regulated during HSC division and are an additional mechanism orchestrating molecular polarization and asymmetric inheritance in HSC divisions, independently of the mechanism regulating lysosomal asymmetry. TEASER ABSTRACT: Endoplasmic diffusion barriers (ER-DBs) control the distribution of cell fate regulators during divisions, also contributing to asymmetric cell division (ACD). Here, we demonstrate that ER-DBs are also present in a subset of HSC divisions and that their strength depends on sphingolipid metabolism. While they are not required for HSC lysosomal asymmetric cell division, these barriers are an additional mechanism orchestrating polarization and asymmetric inheritance in HSC divisions.
    Keywords:  Endoplasmic reticulum; asymmetric cell division; diffusion barrier; expansion culture; hematopoietic stem cell; organelle inheritance; sphingolipids
    DOI:  https://doi.org/10.1016/j.exphem.2025.105364
  29. J Cell Biol. 2026 Feb 02. pii: e202503127. [Epub ahead of print]225(2):
      Insect cuticles with nano-level structures exhibit functional surface properties such as the photonic nanocrystal of the butterfly wing scale with structural color and the corneal nipple arrays of superhydrophobic compound eye lens. Despite the enormous influence the cuticle has had on biomimetic industrial applications, cellular mechanisms of cuticular nanopatterning remain poorly understood. Drosophila gore-tex/Osiris23 (gox) controls the formation of nanopores, with a molecular filtering function, on the olfactory organs. Here we used 3D electron microscopy imaging of entire hair structures to show that nanopore is formed through a novel process of bidirectional interaction of the ER and the plasma membrane trafficking. ER-resident protein Gox stimulates ER-phagy through regulation of Ref(2)P, the fly counterpart of the autophagy protein p62/SQSTM1, and initiates endocytosis. Dynamin on the plasma membrane completes endocytosis and sustains ER-phagy. The repurposing of ER-phagy for plasma membrane remodeling and the fabrication of nanoscale ECM structures sheds light on the nanopatterning mechanism of insect cuticles and their genetic control.
    DOI:  https://doi.org/10.1083/jcb.202503127
  30. Protein Sci. 2026 Jan;35(1): e70390
      Here, we investigate the effects of glycosylation at position N99 on the structural dynamics and lipid scrambling activity of ATG9A, a key autophagy protein, using microsecond all-atom molecular dynamics simulations. ATG9A is an integral membrane protein involved in autophagosome biogenesis, and glycosylation at N99 has previously been implicated in intracellular trafficking, although its precise role remains unclear. The simulations reveal that the hydrophilic central cavity of ATG9A supports lipid reorientation and partial trans-bilayer movements, consistent with experiments on its lipid scrambling activity. We propose that N-glycosylation at N99 enhances cooperative interactions between protomers, facilitating lipid insertion and translocation within the central cavity. These findings suggest a mechanism by which glycosylation may influence lipid redistribution across the phagophore membrane during autophagy. To test this hypothesis, we generate N99 variants (ATG9AN99A and ATG9AN99D) lacking N-glycosylation. These mutants show no significant changes in autophagy flux, suggesting that N99 glycosylation may not be essential for bulk autophagic processing. However, the analysis of autophagosome size indicates that the variants fail to rescue the enlarged vesicle phenotype of ATG9A-KO cells, unlike wild-type ATG9A. Thus, glycosylation might fine-tune ATG9A function, influencing vesicle morphology through conformational dynamics and lipid transport. We also observe asymmetric protomer conformations in ATG9A, in contrast to the symmetric structures obtained from cryo-EM, suggesting that structural heterogeneity could be further explored with experimental methods. Overall, our study highlights the importance of including glycosylation in computational models of membrane proteins and provides mechanistic insight into lipid transport during autophagy, with potential implications for other lipid scramblases and flippases.
    Keywords:  ATG9A; autophagy; glycosylation; membranes; molecular dynamics; post‐translational modifications
    DOI:  https://doi.org/10.1002/pro.70390
  31. J Clin Invest. 2026 Jan 02. pii: e189798. [Epub ahead of print]136(1):
      Calorie restriction (CR) extends maximal lifespan and maintains cellular homeostasis in various animal models. We have previously shown that CR induces a global reduction of protein fractional synthesis rates (FSRs) across the hepatic proteome in mice, but the timing and regulatory mechanisms remain unclear. Nitric oxide (NO), a bioactive molecule upregulated during CR, is a potential regulator of protein synthesis. To explore the role of NO in hepatic proteome fluxes during CR, we used in vivo deuterium labeling from heavy water and liquid chromatography/mass spectrometry-based (LC/MS-based) flux proteomics in WT and NO-deficient (NO-) mice. We observed a transition to reduced global protein FSRs that occurred rapidly between days 25 and 30 of CR. NO deficiency, whether genetic or pharmacological, disrupted the slowing of proteome-wide fluxes and the beneficial effects on body composition and physiology. Administering the NO donor molsidomine restored the reduction in hepatic FSRs in NO- mice. Furthermore, inhibiting NO pharmacologically, whether starting on day 1, day 14, or day 24 of CR, mitigated the reduction in hepatic protein FSRs at day 32, highlighting NO's critical role during the transition period. These results underscore the importance of NO in CR-induced changes in proteostasis and suggest NO as a potential CR-mimetic target, while offering a specific time window for identifying other signals and testing therapeutic interventions.
    Keywords:  Aging; Hepatology; Metabolism; Nitric oxide; Proteomics
    DOI:  https://doi.org/10.1172/JCI189798
  32. J Mol Cell Biol. 2026 Jan 02. pii: mjaf060. [Epub ahead of print]
      The endoplasmic reticulum (ER) lipid raft proteins (Erlins) belong to the stomatin-prohibitin-flotillin-HflC/K (SPFH) family and form highly oligomeric platforms that mediate the degradation of activated inositol 1,4,5-trisphosphate receptors by facilitating their interaction with the E3 ligase RNF170. However, the molecular mechanisms underlying this process remain unclear. Here, we successfully reconstituted the Erlin1-Erlin2 complex and its complex with RNF170 by overexpressing these components in HEK293F cells. We also isolated the Erlin2 oligomer by solely expressing Erlin2 in the cells. Using cryo-EM, we determined the structures of the Erlin1-Erlin2 complex, Erlin1-Erlin2-RNF170 complex, and Erlin2 oligomer at resolutions of 3.29 Å, 3.05 Å, and 2.12 Å, respectively. Both the Erlin1-Erlin2 complex and the Erlin2 oligomer exhibit similar cage-like architectures, with the Erlin1-Erlin2 complex containing 13 pairs of Erlin1 and Erlin2 subunits, whereas the Erlin2 oligomer comprises 26 Erlin2. Although RNF170 was clearly identified during protein purification, it was invisible in the final 3D reconstruction, suggesting a high degree of flexibility between RNF170 and the Erlin complex. Multiple water molecules were identified in the Erlin2 oligomer, underscoring their critical roles in facilitating the high degree of oligomerization of the Erlin2 complex. Taken together, our structural investigation elucidates the molecular basis for the assembly of the Erlin complex and provides a framework for further investigation.
    Keywords:  ERAD; Erlins; SPFH family; cryo-EM
    DOI:  https://doi.org/10.1093/jmcb/mjaf060
  33. J Am Chem Soc. 2026 Jan 02.
      Extracellular targeted protein degradation (eTPD) is an emerging modality to regulate protein levels without genomic interruption. Current strategies co-opt lysosome-targeting receptors (LTRs) that are ubiquitously present in most cells, offering a high success rate of eTPD across cell types and tissues. To circumvent on-target, off-site protein degradation, exploring alternative strategies is becoming a main focus to advance conventional platforms. Until now, many efforts have been focused on developing degraders by repurposing the surface receptors with intrinsically internalizing function. Yet, exploring novel access to eTPD would introduce an arguably nimble molecular design paradigm that opens up new opportunities in many diseases without such receptor availability. Opening up the binding complementarity requirement from LTRs to any overexpressed cell surface receptor offers to endow eTPD platforms with new cellular targeting capabilities. Here, we report polymeric lysosome-targeting chimeras (PolyTACs), a polymer-antibody conjugate based platform for the targeted degradation of membrane-bound and soluble proteins without the need for involving LTRs. Mechanistic investigations suggest a nonclassical uptake pathway caused by the multivalent interactions between the PolyTACs and the overexpressed functionalities on the cell surface. The utility of PolyTACs in eTPD has been demonstrated with four disease-relevant membrane proteins. Additionally, the same design principle has also been leveraged to bind and drag soluble extracellular proteins into the lysosome. The design and fabrication simplicity, nonreliance on LTRs, and tissue-targeting capabilities open up new avenues for eTPD in many disease-specific applications.
    DOI:  https://doi.org/10.1021/jacs.5c17519
  34. Neural Regen Res. 2025 Dec 30.
      The devastating neurodegenerative disorder of Alzheimer's disease hallmarks the presence of protein aggregates known as amyloid-β plaques and neurofibrillary tangles, composed of amyloid-β peptides and aberrantly phosphorylated Tau protein, respectively. The accumulation of these inclusions leads to significant alterations in neuronal homeostasis and overall brain function, resulting in a progressive and rapid cognitive decline. Autophagy, the molecular mechanism of cellular waste removal through the lysosomal pathway, accounts for the degradation of both amyloid-β plaques and neurofibrillary tangles in the brain, conferring therefore protection against the pathology. In addition to general autophagy, several lines of evidence have reported the implication of selective autophagy receptors, including sequestosome1/p62, the neighbor of BRCA1 gene, the nuclear-dot protein 52, and optineurin, in mediating the autophagic clearance of amyloid-β, phosphorylated Tau, or both. Herein, we have highlighted autophagy and selective autophagy as pivotal mechanisms in Alzheimer's disease, underlining selective autophagy receptors as a potential target for treatments in the future.
    Keywords:  Alzheimer's diseases; amyloid-β pathology; amyloid-β plaques; autophagy; neurodegeneration; neurofibrillary tangles; selective autophagy; selective autophagy receptors; tauopathy
    DOI:  https://doi.org/10.4103/NRR.NRR-D-25-00976
  35. Sci Adv. 2026 Jan 02. 12(1): eadz9069
      Intestinal fibrosis (IF), a severe complication of inflammatory bowel disease (IBD), remains a critical unmet clinical need. Although the LL37 peptide and probiotics demonstrate therapeutic potential against IF, their clinical translation is hampered by enzymatic hydrolysis and rapid clearance. Here, inspired by the strategy of bacteriocin transport by bacteria (BTB), we developed an orally administered biotherapeutic platform [BTB-alginate (Alg)] featuring an "all-in-one" architecture that enables spatiotemporal coordination of LL37 and probiotics. The BTB-Alg effectively restored intestinal homeostasis through inflammation resolution, immune modulation, and gut microbiota reconstitution. Notably, integrated multiomics analysis and molecular dynamics simulations revealed that LL37 exerts antifibrotic effects by inducing adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin-mediated autophagy, a mechanism validated in clinical specimens. BTB-Alg exhibited potent therapeutic efficacy in three murine models of acute colitis, IBD-associated IF, and Clostridioides difficile-complicated colitis, highlighting its potential as an IBD treatment paradigm. This study offers a clinically translatable strategy for broad gastrointestinal applications.
    DOI:  https://doi.org/10.1126/sciadv.adz9069
  36. Science. 2026 Jan;391(6780): eadv6127
      Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.
    DOI:  https://doi.org/10.1126/science.adv6127
  37. Neuron. 2025 Dec 30. pii: S0896-6273(25)00890-6. [Epub ahead of print]
      Defective nucleocytoplasmic transport (NCT) has emerged as a contributing factor in the pathogenesis of neurodegenerative diseases and aging. Valosin-containing protein (VCP) is an AAA+ATPase required for disassembly of protein complexes, and mutations in VCP cause neurodegenerative and neuromuscular diseases. We find that VCP is required for quality control of nuclear pore complexes (NPCs) by extracting selected nucleoporins from NPCs for proteasome-mediated degradation. Pathogenic VCP variants cause a reduction in nucleoporins in Drosophila, induced pluripotent stem cell (iPSC)-derived motor neurons, and muscle biopsies from patients, indicating a dominant gain-of-function mechanism. Mechanistically, disease-associated mutations in VCP result in increased recruitment to NPCs through interactions with Ufd1-Npl4, leading to the removal of a subset of nucleoporins from NPCs and disrupting NCT. These findings show that the VCP-Ufd1-Npl4 pathway regulates NPC quality control and that disease-associated variants aberrantly activate the VCP-Ufd1-Npl4 complex to degrade NPCs and disrupt NCT.
    Keywords:  ALS; Drosophila; TDP-43; VCP; muscle disease; neurodegeneration; nuclear pore complex; nucleocytoplasmic transport; nucleoporin; protein quality control
    DOI:  https://doi.org/10.1016/j.neuron.2025.11.017
  38. Cell Death Dis. 2025 Dec 27.
      Vanishing White Matter Disease (VWMD) is a devastating, currently incurable neurodevelopmental disorder primarily affecting white matter. The prevailing view attributes VWMD to the activation of the canonical integrated stress response (c-ISR). However, recent studies have identified a novel, distinct pathway called the split ISR (s-ISR), though its activation has so far only been documented in mouse stem cells harboring a single eIF2B mutation, leaving uncertainty about whether it occurs in human cells, whether other mutations can trigger it, and what role it plays in the disease. Here, we used prime editing (PE) to engineer multiple eIF2B pathogenic mutations into HEK293T and induced pluripotent stem cells (iPSCs), generating human models. We demonstrated PE's effectiveness and safety, marking the first successful application of PE for modeling VWMD. We found that all modeled mutations activate the s-ISR, indicating that this response is a common feature across VWMD mutations, and that it can be further amplified by stress-induced c-ISR and effectively suppressed by ISRIB. Mechanistically, we show that s-ISR hinders mutant iPSCs from achieving the high protein synthesis levels necessary for proper differentiation, expecially into astrocytes. This impairment disrupts their maturation process, directly linking s-ISR activation to the white matter abnormalities of VWMD.
    DOI:  https://doi.org/10.1038/s41419-025-08399-x
  39. Cell Mol Life Sci. 2025 Dec 29.
      UFMylation, similar to ubiquitination, is a unique post-translational modification which is indispensable in hematopoiesis, neurogenesis and chondrogenesis. However, its role in intervertebral disc development remains unclear. In this study, we focused on DDRGK domain containing protein 1 (DDRGK1), a pivotal component involved in UFMylation, and generated Ddrgk1fl/fl; Acan-CreERT2 (Ddrgk1cKO) mice to explore DDRGK1's regulatory function in the nucleus pulposus and cartilage endplate. We found that Ddrgk1 conditional knockout led to severe retardation of spinal growth, disruption of disc cellularity and initiation of disc degeneration during early postnatal phase. Furthermore, Ddrgk1 conditional knockout in late postnatal phase resulted in profound degeneration of mouse discs, mainly characterized by substantially reduced thickness of cartilage endplate. In addition, Ddrgk1cKO mice exhibited exacerbated disc degeneration compared to the WT mice after the lumbar spine instability surgery. RNA sequencing of disc cells from Ddrgk1cKO mice showed upregulation of genes related to apoptosis, matrix metalloproteinase activation, extracellular matrix (ECM) degradation and endoplasmic reticulum (ER) unfolded protein response after Ddrgk1 conditional knockout. Immunohistochemical analysis further verified increased apoptosis, ECM disruption and ER stress in both nucleus pulposus and cartilage endplate after Ddrgk1 conditional knockout. In summary, this study demonstrated that DDRGK1 preserves the normal cellularity and structure of intervertebral discs by regulating the cell fate of nucleus pulposus and cartilage endplate cells, maintaining the ER homeostasis and regulating the metabolic balance of ECM.
    Keywords:  Apoptosis; DDRGK1; ECM; ER stress; Intervertebral disc; UFMylation
    DOI:  https://doi.org/10.1007/s00018-025-06034-8
  40. Aging Cell. 2026 Jan;25(1): e70347
      N6-methyladenosine (m6A) methylation, a dynamic and reversible modification of eukaryotic mRNAs, plays critical roles in diverse cellular processes. Although METTL3-mediated m6A deposition has been implicated in cellular senescence, the mechanisms controlling METTL3 stability and activity during senescence remain poorly defined. Here, we demonstrate that both m6A levels and METTL3 protein abundance are significantly reduced in replication-induced and stress-induced senescence models. METTL3 depletion promotes senescence by inducing telomere dysfunction via diminished expression of shelterin components TRF2 and POT1. Mechanistically, we identify PRKN (Parkin) as a senescence-associated E3 ubiquitin ligase that promotes METTL3 proteasomal degradation through K48-linked polyubiquitination at lysine 164. Genetic PRKN inhibition in pre-senescent cells rescues METTL3 expression, restores TRF2/POT1 levels, reduces telomere dysfunction-induced foci (TIFs), and attenuates senescence-associated β-galactosidase (SA-β-gal) activity. Crucially, PRKN overexpression accelerates telomere dysfunction and senescence in wild-type METTL3-expressing cells but not in cells expressing the ubiquitination-resistant K164R METTL3 mutant. Our findings establish METTL3 ubiquitination as a pivotal regulator of telomere integrity and senescence progression, unveiling a therapeutic target for age-related pathologies.
    Keywords:  METTL3; PRKN; m6A; senescence; telomere
    DOI:  https://doi.org/10.1111/acel.70347
  41. Proc Natl Acad Sci U S A. 2026 Jan 06. 123(1): e2524916123
      A subset of the four-pass transmembrane proteins called C8 tetraspanins (TspanC8) can bind and promote the cell surface localization of ADAM10 (A Disintegrin and Metalloproteinase 10). ADAM10 is a conserved transmembrane metalloprotease essential for metazoan embryonic development and human health. However, the in vivo functional relationships between C8 tetraspanins and ADAM10 are not fully understood. Caenorhabditis elegans has two paralogous tetraspanins, TSP-12 and TSP-14, that resemble the mammalian TspanC8 proteins. We have previously shown that TSP-12/TspanC8 can bind SUP-17/ADAM10 and promote its cell surface localization in early embryos. In this study, we identified the specific step in the secretory pathway where TSP-12-SUP-17 interaction is needed. We found that TSP-12 and SUP-17 share a mutually dependent, yet cell type- and developmental stage-specific relationship in their Golgi trafficking itinerary. In the early embryo, TSP-12 and SUP-17 depend on each other for their transit through the Golgi. However, only SUP-17 is required for proper Golgi trafficking of TSP-12 in the developing oocytes. We further showed that the ER accumulation of SUP-17 in embryos lacking TSP-12 is mediated by the Retention in Endoplasmic Reticulum 1 protein RER-1. These findings, combined with our previous work showing that TSP-12 and TSP-14 function redundantly in endosomes for the recycling of the type II receptor of the BMP signaling pathway, showcase the dynamic and versatile functions of TSP-12 in membrane trafficking in specific cellular contexts. They further highlight the importance of dissecting the functional relationships between TspanC8 proteins and ADAM10 in vivo.
    Keywords:  ADAM10; Golgi; SUP-17; TSP-12; tetraspanin
    DOI:  https://doi.org/10.1073/pnas.2524916123
  42. Sci Adv. 2026 Jan 02. 12(1): eaeb2571
      Hepatocyte polyploidization promotes liver homeostasis by enhancing resistance to cellular stress. Caspase-2, a proapoptotic protease, restricts polyploidization by deleting polyploid and aneuploid cells. While caspase-2 protects against diet-induced hepatic injury, it also acts as a tumor suppressor by controlling genomic instability and oxidative stress. To investigate these roles, we assessed hepatic ploidy dynamics, liver damage, and age-associated tumorigenesis in caspase-2-deficient and catalytically inactive mutant mice. We found that caspase-2 loss promotes early-onset hepatocyte hyperpolyploidy, accompanied by progressive liver inflammation, fibrosis, oxidative liver damage, ferroptosis, and higher incidence of spontaneous hepatocellular carcinoma in aged animals. Proteomic profiling revealed a pathogenic polyploidy-associated signature associated with caspase-2 deficiency and increased predisposition to liver disease and malignancy. These findings establish caspase-2 enzymatic activity as a critical regulator of hepatic genome stability and preventing age-related liver cancer that strongly argue against therapeutic caspase-2 inhibition as a strategy for managing liver injury or cancer risk.
    DOI:  https://doi.org/10.1126/sciadv.aeb2571
  43. Nat Immunol. 2026 Jan 02.
      Type 1 conventional dendritic cells (cDC1s) acquire and cross-present tumor antigens to prime CD8⁺ T cells. Whether this selects for specific neoantigens is unclear. DNGR-1 (CLEC9A), a cDC1 receptor for F-actin exposed on dead cells, promotes cross-presentation of cell-associated antigens. Here we show that DNGR-1-deficient mice develop chemically induced tumors more rapidly and at higher incidence, and these are more frequently rejected on transplantation into wild-type recipients. Whole-exome sequencing reveals enrichment of predicted neoantigens derived from mutated F-actin-binding proteins. Consistent with this observation, tethering model antigens to F-actin enhances DNGR-1-dependent cross-presentation. These results suggest that DNGR-1-mediated recognition of F-actin exposed by dead cancer cells favors priming of CD8⁺ T cells specific for cytoskeletal neoantigens, which can then drive immune escape of cancer cells lacking or reverting those mutations. Thus, neoantigen cross-presentation by cDC1 can determine the immune visibility of the tumor mutational landscape and sculpt cancer evolution by immunoediting.
    DOI:  https://doi.org/10.1038/s41590-025-02354-w
  44. Proc Natl Acad Sci U S A. 2026 Jan 06. 123(1): e2517258123
      The chemical modification N6-methyladenosine (m6A) is catalyzed by the m6A methyltransferase complex (MTC) comprising METTL3 and METTL14 in the nucleus. Structural evidence reveals that METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding scaffold. However, the mechanism directing the complex assembly in vivo remains enigmatic. Here, we demonstrate that MTC is formed by a cotranslational mechanism in which nascent METTL3 interacts with METTL14 polypeptide chain exposed from the ribosome exit tunnel. The methyltransferase domains in the subunits determine the specificity of their cotranslational interaction. In contrast, WTAP, the regulatory subunit of MTC, is recruited to the complex posttranslationally. We further identify CCT4, the key subunit of cytosolic chaperonin TCP-1, as an essential facilitator of the endogenous MTC assembly process. Depletion of CCT4 results in dramatic reduction of METTL3-METTL14 heterodimer formation. Remarkably, we engineer a cell-permeable peptide M14P1, which could disrupt cotranslational assembly of MTC, thereby impairing m6A deposition and significantly attenuating the proliferation of acute myeloid leukemia (AML) cells and promoting apoptosis. Collectively, our findings unravel intrinsic mechanisms governing the in vivo assembly of MTC, and provide a potential therapeutic strategy to disrupt oncogenic m6A pathways and impede AML progression.
    Keywords:  N6-methyladenosine; cotranslational assembly; interfering peptide; methyltransferase complex
    DOI:  https://doi.org/10.1073/pnas.2517258123
  45. J Clin Invest. 2026 Jan 02. pii: e195506. [Epub ahead of print]136(1):
      Neuromyelitis optica (NMO) is an autoimmune disorder characterized by autoantibodies against the astrocyte water channel aquaporin-4 (AQP4) that cause demyelination in the optic nerves and spinal cord. How astrocytopathy leads to myelination deficits remains unclear. Chitinase-3-like protein 1 (CHI3L1, also known as YKL-40) is predominantly secreted by activated astrocytes, serves as a robust NMO biomarker, and plays a role in immune responses, but how it is induced and shapes astrocyte activation in NMO is not well defined. Using ex vivo and in vivo NMO mouse models together with mice with astrocyte-specific CHI3L1 knockout, we demonstrated that CHI3L1 directly contributed to demyelinating lesions elicited by AQP4 autoantibody-activated astrocytes. With complementary in vitro assays and inducible transgenic lines, we uncovered an astrocyte-intrinsic cascade in which AQP4 autoantibody exposure activated STAT3, which in turn drove CHI3L1 expression and secretion. Secreted CHI3L1 then engaged the astrocytic receptor RAGE in an autocrine manner, activating downstream NF-κB signaling that drove proinflammatory gliosis and damaged myelination. Pharmacological blockade of this pathway in NMO models rescued demyelinating pathology and improved motor function. These findings reveal an astrocyte-intrinsic CHI3L1 pathway that contributed to demyelination in NMO and identify actionable therapeutic targets.
    Keywords:  Autoimmunity; Biomarkers; Mouse models; Neuroscience; Therapeutics
    DOI:  https://doi.org/10.1172/JCI195506
  46. Nat Commun. 2025 Dec 30.
      With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.
    DOI:  https://doi.org/10.1038/s41467-025-67697-2
  47. Nat Chem. 2026 Jan 02.
      Despite the crucial biological functions of arginine, its reactivity and ligandability within the human proteome remain largely unexplored. Here we apply activity-based protein profiling (ABPP) with phenylglyoxal-based chemical probes to map arginine reactivity globally. Screening phenylglyoxal derivatives identified a probe with enhanced coverage and selectivity, enabling quantification of 4,606 arginine sites across human cell lines. Among these, critical residues regulate liquid-liquid phase separation. Arginine reactivity was further assessed by on-beads reductive dimethylation proteomics, revealing a subset of hyper-reactive sites. Competitive fragment screening using data-independent acquisition ABPP (DIA-ABPP) generated a ligandability map of arginine residues across 60 dicarbonyl compounds. This dataset revealed ligandable arginines that modulate protein activity, in particular protein-protein interactions, highlighting potential covalent drug targets. Together, this work provides a proteome-wide profile of arginine reactivity and ligandability, offering insights into the functional landscape of arginines and expanding the scope of covalent drug discovery to include arginine-targeting molecules.
    DOI:  https://doi.org/10.1038/s41557-025-02012-6
  48. Metabol Open. 2025 Dec;28 100396
      Lysosomes, the cellular recycling hubs, are indispensable for maintaining homeostasis by degrading misfolded proteins, damaged organelles, and foreign pathogens. Their dysfunction is a hallmark of aging and age-related neurodegenerative diseases, where impaired clearance of toxic protein aggregates drives pathogenesis. Nevertheless, the mechanisms by which lysosomal function can be enhanced to mitigate these detrimental processes remain inadequately understood. A recent study conducted by Li et al. describes a newly identified transcriptional program, the Lysosomal Surveillance Response (LySR), that, when activated, significantly extends healthspan and reduces proteotoxicity in C. elegans. This adaptive transcriptional program, governed by the GATA transcription factor, ELT-2, and modulated by the acetyltransferase CBP-1, operates independently of canonical longevity pathways such as the DAF-2 insulin-like signaling. This work not only unveils a previously unrecognized longevity pathway but also charts a new course for developing therapies targeting aging and neurodegeneration.
    DOI:  https://doi.org/10.1016/j.metop.2025.100396
  49. Aging Cell. 2026 Jan;25(1): e70359
      Testicular aging, a key feature of late-onset hypogonadism (LOH), is closely associated with Sertoli cells dysfunction. Emerging evidence implicates lipid droplet (LD) accumulation as a hallmark of aging in Sertoli cells, but its role in Sertoli cells senescence and the associated molecular mechanisms are unknown. We found that aging and obesity drove progressive LD accumulation in Sertoli cells, accompanied by mitochondrial dysfunction and ROS overproduction. Palmitic Acid (PA)-induced LD overload in vitro replicated these aging phenotypes, triggering ROS overproduction that provoked ribosome collisions and caused decreased protein synthesis globally. Moreover, LD-driven ROS disrupted mRNA translation, particularly at GA-rich sequences encoding aspartate and glutamate. Collided ribosomes activated the ZAKα-p38 axis in Sertoli cells, causing cellular senescence and impairing the blood-testis barrier. ZAKα inhibitor Nilotinib attenuated testicular atrophy, restored testosterone levels, and mitigated Sertoli cells dysfunction in aged mice. Targeting this pathway with ZAKα inhibitor offers a therapeutic strategy for age-related gonadal decline, bridging lipid metabolism dysfunction, and reproductive aging.
    Keywords:  ROS; Sertoli cells; lipid droplet; ribosome collisions; testicular aging
    DOI:  https://doi.org/10.1111/acel.70359