bims-proteo Biomed News
on Proteostasis
Issue of 2025–12–07
sixty-nine papers selected by
Eric Chevet, INSERM
  1. Competition between Der1 and ERAD-M substrates controls Hrd1 complex function.
  2. Dynamic Organellar Mapping in yeast reveals extensive protein localization changes during ER stress.
  3. O-mannosylation of misfolded ER proteins promotes ERAD.
  4. eIF2D promotes 40S ribosomal subunit recycling during intrinsic ribosome destabilization.
  5. Systematic characterization of cancer-associated SPOP mutants reveals novel and reprogrammable degradative activities.
  6. Membralin Assembles a MAN1B1-VCP Complex to Target Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation.
  7. A non-radioactive method to detect and measure 48S initiation complex and 80S ribosome formation in vitro.
  8. Capturing Translation in Action with Protein Synthesis Profiling.
  9. Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants.
  10. E3 ligase AREL1 controls perinuclear localization of lysosomes and supports Purkinje cell survival.
  11. Applications of KinTek Explorer Kinetic Simulations of Targeted Protein Degradation for Evaluation and Design of PROTACs.
  12. Structural insights into the role of eIF3 in translation mediated by the HCV IRES.
  13. FAM134B Controls Collagen I Dynamics in Hepatic Stellate Cell-Driven Fibrosis.
  14. Multiple Quality Control Checkpoints Safeguard Small Nuclear RNA Biogenesis and Prevent Assembly of Aberrant Spliceosomes.
  15. Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress.
  16. Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation.
  17. Proteome-Wide Discovery of Degradable Proteins Using Bifunctional Molecules.
  18. UBA6 specificity for ubiquitin E2 conjugating enzymes reveals a priority mechanism of BIRC6.
  19. 5' UTR Cis-Regulatory Logic Governs Ribosome Engagement on Canonical and Non-Coding RNAs.
  20. Sprint interval exercise disrupts mitochondrial ultrastructure driving a unique mitochondrial stress response and remodelling in men.
  21. Machine learning in targeted protein degradation drug design: a technical review of PROTACs and molecular glues.
  22. Proteome-wide computational analyses reveal links between protein condensate formation and RNA biology.
  23. Rapid estimation of protein folding pathways from sequence alone using AlphaFold2.
  24. Development of a p62 biodegrader for autophagy targeted degradation.
  25. Rbfox2 selectively governs hematopoietic stem cell self-renewal by regulating proteostasis.
  26. Structural mechanism of mRNA decoding by mammalian GTPase GTPBP1.
  27. Atom-level enzyme active site scaffolding using RFdiffusion2.
  28. Tumor microenvironment-targeted PROTAC nanoparticle self-assembly broadly predicted by structural descriptors.
  29. Computational design of metallohydrolases.
  30. Accurate single-domain scaffolding of three nonoverlapping protein epitopes using deep learning.
  31. Vacuolar-type H+-ATPase-mediated extra-organellar buffering resolves mitochondrial dysfunction.
  32. A lunapark for secretory mRNA translation at ER junctions.
  33. The transcription factor SKN-1 drives lysosomal enlargement during aging to maintain function.
  34. Age-related decline of chaperone-mediated autophagy in skeletal muscle leads to progressive myopathy.
  35. Tom70-mediated mitochondria-nuclear envelope contacts regulate nuclear pore complex inheritance during gametogenesis.
  36. Evolution of proteolysis-targeting chimeras (PROTAC) technology to overcome challenges of antimicrobial resistance.
  37. Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway.
  38. Structural insights into cauliflower mitoribosome in translation state and in association with a late assembly factor.
  39. Molecular mechanism of PINK1 regulation by the Hsp90 machinery.
  40. Decoding protein binding plasticity via integrated deep ribosome display and deep learning.
  41. The unfolded protein response in progeria arteries originates from non-endothelial cell types.
  42. Towards a phylogenetically informed approach to solving protein-protein interactions.
  43. Bidirectional crosstalk between ER stress and lipid metabolism: From proteostasis to tumor adaptation.
  44. Selective CDK6 Degradation via the KLHDC2 E3 Ubiquitin Ligase.
  45. Sec61 translocon inhibitor flavitransin blocks selectively dengue virus polyprotein insertion in the ER with pan-orthoflavivirus antiviral potency.
  46. Integrative and accurate annotations enhance current nonsense-mediated mRNA decay rules.
  47. Molecular glue neferine induces BAP31 homodimerisation to disrupt endoplasmic reticulum-mitochondria crosstalk for anti-neuroinflammation in ischaemic stroke.
  48. VAPB is a negative regulator of STING-mediated innate immune signaling.
  49. High-throughput mapping of modular regulatory domains in human RNA-binding proteins.
  50. Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles.
  51. Computational enzyme design by catalytic motif scaffolding.
  52. Reprogramming of cholesterol sensing in epithelial cells supports pancreatic inflammation.
  53. C-COMPASS: a user-friendly neural network tool profiles cell compartments at protein and lipid levels.
  54. NEMO recruitment at single cytokine-receptor complexes shows quantized dynamics independent of ligand affinity.
  55. Non-canonical functions of UGT2B17 promote castrate-resistant prostate cancer progression.
  56. Benchmarking interpretability of deep learning for predictive genomics: Recall, precision, and variability of feature attribution.
  57. The Role of STING-Mediated Activation of the Integrated Stress Response in Inflammation-Induced Depletion of Ovarian Reserve.
  58. An smFRET assay to probe the impact of antibiotics on intersubunit rotation in eukaryotic ribosomes.
  59. LRRK2 integrates Rab and GABARAP interactions to sense and respond to distinct lysosomal stresses.
  60. The ubiquitin system targets translocated EspH to proteasomal degradation.
  61. Innate immune and metabolic signals induce mitochondria-dependent membrane lysis via mitoxyperiosis.
  62. DTBind: A Mechanism-Driven Deep Learning Framework for Accurate Prediction of Drug-Target Molecular Recognition.
  63. Astrocytic PERK Deficiency Drives Prefrontal Circuit Dysfunction and Depressive-Like Behaviors.
  64. The handshake between the continuous remodeling of the endoplasmic reticulum and the store-operated calcium entry.
  65. Attenuation of ATM signaling by ROS delays replicative senescence at physiological oxygen.
  66. Clathrin-mediated endocytosis in budding yeast at a glance: animated.
  67. Microbial metabolites shape mammalian protein translation.
  68. Using evolutionary context to classify difficult protein folds.
  69. Translational activators align mRNAs at the small mitoribosomal subunit for translation initiation.
  1. Proc Natl Acad Sci U S A. 2025 Dec 09. 122(49): e2513595122
    Competition between Der1 and ERAD-M substrates controls Hrd1 complex function.
    Jennifer E Russ, Brian G Peterson, Sophia Taylor, Ryan D Baldridge.
      Endoplasmic reticulum-associated degradation (ERAD) is a quality control process which removes misfolded proteins from the ER. The central component of the most conserved ERAD system is an integral membrane ubiquitin ligase called Hrd1. The Hrd1 ligase functions within a complex to mediate the recognition and ubiquitination of both soluble, lumenal substrates and integral membrane substrates, all of which are ultimately targeted for degradation by the cytosolic proteasome. Here, we used deep mutational scanning to identify Hrd1 residues exclusively involved in the degradation of integral membrane substrates. We report single residue Hrd1 variants that are broadly deficient in the degradation of all integral membrane substrates tested. Using in vivo assays to characterize Hrd1 variant deficiency, we explain how integral membrane substrates compete with other complex components to control Hrd1 function. This work reveals competition for the retrotranslocon cavity between both lumenal and membrane substrate degradation paths and highlights Hrd1 complex assembly as the primary determinant for tuning ERAD function.
    Keywords:  ERAD; deep mutational scanning; endoplasmic reticulum–associated degradation; protein degradation; ubiquitin proteasome system
    DOI:  https://doi.org/10.1073/pnas.2513595122
  2. Nat Commun. 2025 Dec 02. 16(1): 10842
    Dynamic Organellar Mapping in yeast reveals extensive protein localization changes during ER stress.
    Anna Platzek, Klára Odehnalová, Julia P Schessner, Georg H H Borner, Sebastian Schuck.
      Sophisticated techniques are available for systematic studies of yeast cell biology. However, it remains challenging to investigate protein subcellular localization changes on a proteome-wide scale. Here, we apply Dynamic Organellar Mapping by label-free mass spectrometry to detect localization changes of native, untagged proteins during endoplasmic reticulum (ER) stress. We find that hundreds of proteins shift between cellular compartments. For example, we show that numerous secretory pathway proteins accumulate in the ER, thus defining the extent and selectivity of ER retention of misfolded proteins. Furthermore, we identify candidate cargo proteins of the ER reflux pathway, determine constituents of reticulon clusters that segregate from the remainder of the ER and provide evidence for altered nuclear pore complex composition and nuclear import. These findings uncover protein relocalization as a major aspect of cellular reorganization during ER stress and establish Dynamic Organellar Maps as a powerful discovery tool in yeast.
    DOI:  https://doi.org/10.1038/s41467-025-66946-8
  3. EMBO J. 2025 Dec 05.
    O-mannosylation of misfolded ER proteins promotes ERAD.
    Leticia Lemus, Hadar Meyer, Ana I Rodríguez-Rosado, Maya Schuldiner, Veit Goder.
      Protein quality control (PQC) in the secretory pathway, a process critically linked to numerous human diseases, begins in the endoplasmic reticulum (ER) and involves ER-associated degradation (ERAD) of terminally misfolded proteins. In this study, we conducted genome-wide screens in baker's yeast (Saccharomyces cerevisiae) to investigate the degradation of Gas1*, a misfolded version of an O-mannosylated, glycosylphosphatidylinositol (GPI)-anchored protein. In combination with detailed biochemical and genetic analyses, these screens revealed an unexpected bifunctionality of the evolutionarily conserved heteromeric enzyme complex Pbn1-Gpi14: while it has been previously recognized as a GPI-mannosyltransferase, we here find that it catalyzes the O-mannosylation of misfolded proteins, thereby promoting their ERAD. This process is particularly relevant for misfolded proteins that lack N-glycans. Our results suggest that protein O-mannosylation constitutes a distinct type of glycan-dependent mechanism for promoting ERAD.
    Keywords:  ER Quality Control; ERAD; Gpi14; Pbn1; Protein O-Mannosylation
    DOI:  https://doi.org/10.1038/s44318-025-00647-2
  4. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1322. [Epub ahead of print]53(22):
    eIF2D promotes 40S ribosomal subunit recycling during intrinsic ribosome destabilization.
    Kazuya Ichihara, Taichi Shiraishi, Yuhei Chadani, Yuki Kito, Chisa Shiraishi, Mina Hirata, Yuta Takahashi, Akinao Kobo, Atsushi Hatano, Masaki Matsumoto, Kodai Machida, Hiroaki Imataka, Atsushi Toyoda, Emi Mishiro-Sato, Takayuki Nojima, Takuhiro Ito, Hideki Taguchi, Keiichi I Nakayama, Akinobu Matsumoto.
      Although eukaryotic initiation factor 2D (eIF2D) is implicated in translation initiation, reinitiation, and ribosome recycling, its precise role remains unclear. Here, we show that eIF2D promotes 40S ribosome recycling during intrinsic ribosome destabilization (IRD), a process in which ribosomes stochastically destabilize while translating proteins with consecutive acidic amino acids at their NH2-terminus. Unrecycled 40S ribosomes accumulate in eIF2D-deficient cells, leading to 80S ribosome stalling. Selective translation complex profiling (TCP-seq) reveals that eIF2D preferentially associates with IRD-prone regions. The winged helix domain, unique to eIF2D but absent in MCTS1-DENR, enhances its binding to 40S subunits, but likely clashes with ABCE1 during stop-codon-associated recycling. Loss of eIF2D reduces the expression of IRD-inducing proteins, including splicing factors. Together, these findings define a previously unappreciated role for eIF2D in 40S recycling and clarify its mechanistic divergence from the MCTS1-DENR complex.
    DOI:  https://doi.org/10.1093/nar/gkaf1322
  5. bioRxiv. 2025 Nov 23. pii: 2025.11.21.689851. [Epub ahead of print]
    Systematic characterization of cancer-associated SPOP mutants reveals novel and reprogrammable degradative activities.
    Alana G Caldwell, Harshil Parmar, Xiaokang Jin, Chen Zhou, Xiaoyu Zhang.
      Speckle-type POZ protein (SPOP) functions as the substrate adaptor of the Cullin3-RING ligase (CRL3) complex and is recurrently mutated in multiple cancer types. Among these, F102C and F133L are frequent prostate cancer mutations within the substrate-binding domain, yet their biochemical consequences remain incompletely understood. Using quantitative proteomics, we show that SPOP-F133L, unlike SPOP-F102C, retains degradative activity toward the nuclear basket proteins NUP153 and TPR, indicating substrate-dependent loss-of-function. Moreover, SPOP-F133L induces partial down-regulation of p53 through a CRL-dependent, post-translational mechanism, revealing a potential neo-substrate relationship. Finally, we demonstrate that both SPOP-F102C and SPOP-F133L support targeted protein degradation (TPD) in an engineered cellular system. These findings define the degradative capacities of SPOP mutants and highlight opportunities to repurpose these variants as mutant-selective E3 ligases for therapeutic applications.
    DOI:  https://doi.org/10.1101/2025.11.21.689851
  6. Adv Sci (Weinh). 2025 Dec 01. e19256
    Membralin Assembles a MAN1B1-VCP Complex to Target Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation.
    Jing Zhang, Xiaoran Lu, Sunan Li, Tao Wang, Iqbal Ahmad, Yong-Hui Zheng.
      Protein quality control in the endoplasmic reticulum (ER) maintains proteostasis by eliminating aberrant or foreign proteins through ER-associated degradation (ERAD) or ER-to-lysosome-associated degradation (ERLAD). Here, Membralin (TMEM259) is identified as a previously unrecognized ER-phagy receptor that assembles a selective degradation machinery targeting viral class I fusion glycoproteins. Membralin recruits MAN1B1, an α-mannosidase that trims high-mannose N-glycans, through its luminal loop, and VCP/p97 through its cytoplasmic loop, while its cytoplasmic tail contains a functional LC3-interacting region (LIR) essential for autophagic delivery. This Membralin-MAN1B1-VCP axis directs viral glycoproteins such as SARS-CoV-2 spike, Ebola GP, influenza HA, and HIV-1 Env to lysosomes for degradation independently of polyubiquitination or canonical ER-phagy receptors. In contrast, misfolded host glycoproteins are degraded through conventional ERAD or FAM134B-dependent ERLAD pathways. Mechanistically, the Membralin complex selectively recognizes densely glycosylated substrates, likely by sensing clustered N-glycans characteristic of viral envelope proteins. Loss of Membralin or MAN1B1 markedly enhances pseudoviral infectivity, underscoring its antiviral role. These findings reveal a ubiquitin-independent ERLAD pathway that discriminates foreign from host glycoproteins and establish Membralin as a central scaffold coordinating ER quality control and innate antiviral defense.
    Keywords:  ER‐phagy receptor; MAN1B1; TMEM259; membralin; reticulophagy; viral glycoprotein
    DOI:  https://doi.org/10.1002/advs.202519256
  7. Methods Enzymol. 2025 ;pii: S0076-6879(25)00405-7. [Epub ahead of print]725 105-124
    A non-radioactive method to detect and measure 48S initiation complex and 80S ribosome formation in vitro.
    Jose Gabriel R Javellana, Katherine S Olson, Michael G Kearse.
      Canonical eukaryotic translation initiation is cap- and scanning-dependent and is highly regulated by multiple factors, including cis-elements and trans-acting factors within the 5' untranslated region (UTR). Biochemically defining how different sequences and mutations in 5' UTRs affect initiation is critical to understanding the vastly different initiation rates among cellular mRNAs and how disease-linked mutations affect protein synthesis. Here, we provide details for an approach to detect and measure the abundance of 48S initiation complexes and 80S ribosomes on in vitro transcribed mRNAs using a commercially available rabbit reticulocyte lysate, along with the non-hydrolyzable GTP analog, GMPPNP, and the elongation inhibitor, cycloheximide. The outlined method relies on tracking the sedimentation of an experimental mRNA with each complex in sucrose density gradients by RT-qPCR, bypassing the need for radiolabeling mRNAs and scintillation counting. The protocol herein serves as a starting point for assessing translation initiation at distinct biochemical stages in vitro.
    Keywords:  Protein synthesis; Start codon; Translation initiation; Translational control; mRNA
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.013
  8. bioRxiv. 2025 Nov 17. pii: 2025.11.17.688896. [Epub ahead of print]
    Capturing Translation in Action with Protein Synthesis Profiling.
    Cesar Arcasi Matta, Zhi Qi Ten, Simpson Joseph.
      Translation is a central control point of gene expression, linking nucleotide sequences to functional proteins. Dysregulated translation contributes to diverse diseases, underscoring the need for methods that can directly reveal which transcripts are actively translated. Ribosome profiling, the current gold standard, provides nucleotide-resolution maps of ribosome occupancy but requires laborious purification and sacrifices information on mRNA isoforms and mRNA modifications by restricting analysis to short ribosome-protected fragments. Here, we introduce Protein Synthesis Profiling (PSP), a proximity-labeling strategy for transcriptome-wide identification of actively translated mRNAs without ribosome isolation. PSP exploits a fusion of the enzyme APEX2 with the elongation factor eEF2, which transiently associates with ribosomes during elongation, to catalyze selective tagging of mRNAs engaged in translation. Applied in Saccharomyces cerevisiae , PSP captures condition-specific translational programs, recapitulates known stress responses, and expands the detectable repertoire of regulated genes beyond existing methods. By preserving full-length transcript features, PSP is scalable, isoform-aware, and broadly adaptable, providing a versatile platform to dissect translational regulation in health and disease.
    DOI:  https://doi.org/10.1101/2025.11.17.688896
  9. Dev Cell. 2025 Dec 01. pii: S1534-5807(25)00693-8. [Epub ahead of print]
    Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants.
    Víctor Sánchez de Medina Hernández, Marintia M Nava-García, Anita Bianchi, Marion Clavel, Ranjith K Papareddy, Lina Benchalel, Veselin I Andreev, Varsha Mathur, Azadeh Mohseni, Marta García-León, Peng Gao, Juan Carlos de la Concepción, Lorenzo Picchianti, Nenad Grujic, Roksolana Kobylinska, Alibek Abdrakhmanov, Héloïse Duvergé, Gaurav Anand, Nils Leibrock, Juncai Ma, Margot Raffeiner, Timothy Scott Crawford, Luca Argirò, Mateusz Matuszkiewicz, Cheuk-Ling Wun, Jakob Valdbjørn Kanne, Anton Meinhart, Elisabeth Roitinger, Isabel Bäurle, Byung Ho Kang, Morten Petersen, Suayib Üstün, Yogesh Kulathu, Tim Clausen, Silvia Ramundo, Yasin Dagdas.
      Selective autophagy is a fundamental protein quality control pathway that safeguards proteostasis by degrading damaged or surplus cellular components, particularly under stress. This process is orchestrated by selective autophagy receptors (SARs) that recruit specific cargo for degradation. Although significant strides have been made in understanding the molecular framework of selective autophagy, the diversity of SAR repertoires across species remains largely unexplored. Through a comparative interactome analysis across five model organisms, we identified a suite of conserved and lineage-specific SAR candidates. Among these, we validated coupling of ubiquitin to endoplasmic reticulum degradation- and protein rich in the amino acids E, L, K, and S-domain-containing SAR (CESAR) as a conserved SAR critical for proteostasis under heat stress. CESAR specifically facilitates the degradation of ubiquitinated protein aggregates and is indispensable for heat stress tolerance. Altogether, our study establishes a robust pipeline and a rich resource for SAR discovery. It also positions CESAR as a pivotal regulator of proteostasis, with broad implications for improving stress resilience in plants.
    Keywords:  ATG8; aggrephagy; autophagic flux; heat stress; protein aggregate; proteostasis; proteotoxic stress; receptor evolution; selective autophagy; selective autophagy receptor
    DOI:  https://doi.org/10.1016/j.devcel.2025.11.001
  10. EMBO J. 2025 Dec 02.
    E3 ligase AREL1 controls perinuclear localization of lysosomes and supports Purkinje cell survival.
    Luyi Jiang, Jiangfen Tang, Ya-Fen Zhang, Wen-Xuan Zou, Gang Deng, Na Tian, Xiaolu Zhao, Lei Han, Kai Liu, Bao-Liang Song, Jie Luo.
      Localization of lysosomes influences their properties, e.g., perinuclear lysosomes are more acidic but less mobile compared with the peripheral ones. Furthermore, the endoplasmic reticulum (ER) can actively regulate the dynamics and functions of lysosomes via membrane contact sites. In this study, we find that ER-resident apoptosis-resistant E3 ubiquitin protein ligase 1 (AREL1) establishes membrane contacts with lysosomes by directly interacting with the Voa subunit of V-ATPase. AREL1 also catalyzes K33-linked polyubiquitylation of V-ATPase V1B2 subunit, inducing its binding to UBAC2 localized in the perinuclear ER. Depletion of AREL1 or UBAC2 increases the number of peripheral lysosomes that possess partially assembled V-ATPase, elevated luminal pH, and attenuated degradative capacity. Knockdown of ZRANB1, the deubiquitylating enzyme that antagonizes AREL1-mediated V1B2 ubiquitylation, promotes perinuclear clustering of lysosomes and increases lysosomal acidity and degradation. Mice lacking Arel1 exhibit age-dependent Purkinje cell loss, an ataxic phenotype, and motor impairment. Lipofuscin accumulation in the residual Purkinje cells of Arel1-/- mice indicates lysosomal dysfunction. Orchestration of lysosomal positioning and function by the AREL1-UBAC2-V-ATPase axis underscores the physiological significance of ER-regulated perinuclear lysosomal positioning in neurons.
    Keywords:  AREL1; Lysosomal Positioning; Purkinje Neurons; UBAC2; V-ATPase
    DOI:  https://doi.org/10.1038/s44318-025-00654-3
  11. Biochemistry. 2025 Dec 04.
    Applications of KinTek Explorer Kinetic Simulations of Targeted Protein Degradation for Evaluation and Design of PROTACs.
    Yacheng Liu, Jiexin Wu, Danqi Chen, Liang Han.
      Targeted protein degradation (TPD) technology centered on proteolysis-targeting chimeras (PROTACs) has become an increasingly transformative paradigm in drug discovery. PROTACs, by association with a disease-related target protein of interest and an E3 ligase, form a ternary complex in which the target protein undergoes subsequent ubiquitination and proteasomal degradation. This unique event-driven mechanism of action underscores the importance of kinetic simulation in facilitating the understanding of the kinetic parameters in TPD processes within a kinetic context to guide PROTAC design and optimization. Here, we employ KinTek Explorer to develop kinetic models for simulating PROTAC-induced ternary complex formation and the subsequent mechanistic steps leading to TPD. We illustrate the effects of, and interplay between, PROTAC binding specificity, affinity, cooperativity, and mechanism in complex TPD scenarios. Our findings highlight the effectiveness of KinTek Explorer in TPD kinetic simulation to facilitate PROTAC design.
    DOI:  https://doi.org/10.1021/acs.biochem.5c00381
  12. Proc Natl Acad Sci U S A. 2025 Dec 09. 122(49): e2505538122
    Structural insights into the role of eIF3 in translation mediated by the HCV IRES.
    Wakana Iwasaki, Kazuhiro Kashiwagi, Ayako Sakamoto, Madoka Nishimoto, Mari Takahashi, Kodai Machida, Hiroaki Imataka, Akinobu Matsumoto, Yuichi Shichino, Shintaro Iwasaki, Koshi Imami, Takuhiro Ito.
      The genomes of various RNA viruses and a subset of human genes contain structured RNA elements termed internal ribosomal entry sites (IRESs) to initiate translation in a cap-independent manner. The well-studied IRES from Hepatitis C virus (HCV) binds to eukaryotic initiation factor 3 (eIF3), but how the HCV IRES harnesses eIF3 for viral translation remains unclear. Here, we determined multiple cryo-EM structures in which the HCV IRES binds simultaneously to the ribosome and eIF3, covering steps from initiation to elongation. The eIF3 core subunits are displaced from the ribosome by binding more tightly to subdomain IIIb of the HCV IRES. However, cross-linking mass spectrometry suggested that the eIF3 noncore subunits in the HCV-IRES-mediated elongation complex remain in similar positions on the ribosome to those observed in the cap-mediated initiation complex. This currently determined configuration of eIF3 core and noncore subunits reveals the mechanisms through which the HCV IRES overcomes the competition with the host mRNA and promotes viral mRNA translation by utilizing eIF3. Interestingly, cryo-EM structures also revealed that the N-terminal domain of the eIF3 c-subunit (eIF3c-NTD) binds to the large ribosomal subunit (60S) during elongation. These findings suggest that eIF3 contributes to HCV IRES-mediated translation not only during initiation but also elongation and potentially in reinitiation. The interaction between the eIF3c-NTD and the 60S ribosome is likely to occur in general translation processes as well, contributing to 60S joining or eIF3 stabilization on the elongating ribosome.
    Keywords:  IRES; cryo-EM; eIF3; ribosome; virus
    DOI:  https://doi.org/10.1073/pnas.2505538122
  13. Am J Physiol Gastrointest Liver Physiol. 2025 Dec 05.
    FAM134B Controls Collagen I Dynamics in Hepatic Stellate Cell-Driven Fibrosis.
    Jagannath Misra, Zachary Hanquier, Reese Baxter, NIpuni Barupala, Alexander Jackson, Jessica L Maiers.
      Liver fibrosis is driven by the accumulation of scar tissue in response to injury. Activated hepatic stellate cells (HSCs) secrete fibrogenic proteins that deposit into the extracellular matrix, leading to fibrosis. Increased production of fibrogenic proteins by HSCs leads to ER stress, triggering the Unfolded Protein Response (UPR). The UPR is important in regulating HSC activation and fibrogenesis, but mechanisms driving this regulation are unclear. A key process regulated by the UPR is degradation of misfolded proteins through various pathways, including ER-to-Lysosome-Associated Degradation (ERLAD). ERLAD targets proteins for lysosomal degradation and can involve autophagosomes engulfing portions of the ER, termed ER-phagy. ER-phagy is implicated in degradation of misfolded fibrillar collagen, but its role in fibrogenesis is unknown. We show that collagen I levels are post-translationally regulated by autophagy, and this correlates with ER-phagy receptor expression. Furthermore, activation of HSCs induces ER-phagy flux and expression of ER-phagy receptors, including FAM134B, in a process dependent on UPR transducer ATF6α. Loss of FAM134B decreases intracellular collagen I without affecting COL1A1 mRNA. Moreover, FAM134B deletion blocks TGFβ-induced collagen I deposition despite increased secretion. Together, we show that ER-phagy receptor FAM134B is pivotal for collagen I deposition during fibrogenesis.
    Keywords:  Cell-cycle Progression gene 1; ER-phagy; ER-to-Lysosomal Degradation; TGFbeta; Unfolded Protein Response
    DOI:  https://doi.org/10.1152/ajpgi.00170.2025
  14. bioRxiv. 2025 Nov 23. pii: 2025.11.21.689835. [Epub ahead of print]
    Multiple Quality Control Checkpoints Safeguard Small Nuclear RNA Biogenesis and Prevent Assembly of Aberrant Spliceosomes.
    Tiantai Ma, Claire Huntington, Zhuoyi Song, Rea M Lardelli, Eric L Van Nostrand, Jens Lykke-Andersen.
      Defective small nuclear (sn)RNAs are produced from hundreds of human snRNA pseudogenes and mutant snRNA genes associated with human developmental disorders. Machineries that prevent defective snRNAs from disrupting pre-mRNA splicing remain poorly defined. Here, we identify multiple checkpoints in snRNA biogenesis monitored by quality control machineries that subject defective snRNAs to degradation and prevent their assembly into spliceosomes. We show that variant U1 snRNAs produced from human pseudogenes, some at rates approaching the canonical snRNAs, are impaired in 3' cleavage and targeted for degradation by the NEXT-exosome while failures in subsequent protein assembly steps promote NEXT-exosome- or Terminal Uridylyl Transferase 4/7-mediated degradation. These pathways also repress mutant snRNAs associated with human developmental disorders. Impeding snRNA quality control causes formation of aberrant spliceosomes and altered pre-mRNA splicing. These findings define checkpoints in snRNA biogenesis that safeguard pre-mRNA splicing and represent potential therapeutic targets for human disorders associated with snRNA mutations.
    DOI:  https://doi.org/10.1101/2025.11.21.689835
  15. bioRxiv. 2025 Nov 18. pii: 2025.11.17.688722. [Epub ahead of print]
    Differential regulation of p62-ubiquitin conjugates in neurons versus astrocytes during cellular stress.
    David K Sidibe, Erin M Smith, Maeve L Spivey, Maria C Vogel, Sandra Maday.
      Sequestosome 1/p62 (hereafter referred to as p62) is a multifunctional protein that orchestrates various cellular stress response pathways including autophagy, proteasome-mediated degradation, antioxidant defense, nutrient sensing, and inflammatory signaling. Mutations in distinct functional domains of p62 are linked with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), underscoring its importance in neural cells. Neurons and astrocytes perform distinct roles in brain physiology and thus encounter a unique landscape of cellular stress. However, how p62 is regulated in these cell types in response to various stress modalities remains largely unexplored. Several functions for p62 depend on engagement with ubiquitinated substrates. Thus, we investigated how the regulation of p62-ubiquitin conjugates differs between neurons and astrocytes exposed to two stress modalities: lysosomal membrane damage and metabolic stress. Lysosomal damage triggered ubiquitin-dependent assembly of p62 puncta in both neurons and astrocytes. In contrast, nutrient deprivation elicited different responses between neurons and astrocytes. Neurons formed p62-ubiquitin structures more prominently and displayed a greater dependence on ubiquitin for p62 clustering. Together, these findings reveal cell-type-specific and stress-specific regulation of p62-ubiquitin conjugates, indicating that neurons and astrocytes can deploy distinct quality control strategies.
    DOI:  https://doi.org/10.1101/2025.11.17.688722
  16. EMBO J. 2025 Dec 01.
    Ubiquitin pathway blockade reveals endogenous ADP-ribosylation marking PARP7 and AHR for degradation.
    Andrii Gorelik, Nina Đukić, Rebecca Smith, Chatrin Chatrin, Osamu Suyari, Jason Matthews, Ivan Ahel.
      ADP-ribosylation is an important protein post-translational modification catalysed by a family of PARP enzymes in humans and is involved in DNA damage and immunity among other processes. While poly-ADP-ribosylation has been established as a protein degradation signal in several cases, the role of mono-ADP-ribosylation in protein turnover has remained elusive and mostly relies on overexpression systems. Here, we describe a way to visualise high levels of endogenous ADP-ribosylation by inhibiting the ubiquitin pathway. By blocking ubiquitylation/proteasome, we found that ADP-ribosylation by at least three different PARPs (PARP7, PARP1 and TNKS) can be greatly induced. We discovered that specific activation of the aryl hydrocarbon receptor (AHR) pathway in combination with the ubiquitin pathway inhibition promotes quantitative ADP-ribosylation of PARP7 targets, including the mono-ADP-ribosyltransferase PARP7 itself and AHR. We found that DTX2 is the E3 ligase responsible for degrading ADP-ribosylated PARP7, AHR and other PARP7 substrates. This PARP7-DTX2 crosstalk establishes a mechanism to rapidly shut down AHR-mediated transcription by decreasing its protein levels. Taken together, our findings uncover a paradigm where mono-ADP-ribosylation acts as a degradation mark.
    Keywords:  ADP-Ribose; Aryl Hydrocarbon Receptor; PARP7; Protein Degradation; Ubiquitin
    DOI:  https://doi.org/10.1038/s44318-025-00656-1
  17. ACS Cent Sci. 2025 Nov 26. 11(11): 2240-2256
    Proteome-Wide Discovery of Degradable Proteins Using Bifunctional Molecules.
    Ines Forrest, Louis P Conway, Clara Gathmann, Appaso M Jadhav, Tzu-Yuan Chiu, Christian M Chaheine, Michelle Estrada, Anurupa Shrestha, Kathy Sarris, Justin M Reitsma, Scott E Warder, Anil Vasudevan, Shaun M McLoughlin, Christopher G Parker.
      Targeted protein degradation (TPD) is an emergent therapeutic strategy with the potential to circumvent challenges associated with targets unamenable to conventional pharmacological inhibition. Among TPD approaches, proteolysis-targeting chimeras (PROTACs) have shown marked advancement, with numerous candidates in clinical development. Despite their potential, most PROTACs utilize advanced small-molecule inhibitors, inherently limiting the scope of this approach. More generally, the fraction of the proteome tractable to small-molecule-induced degradation strategies is unknown. Here we describe a chemical proteomic strategy for the agnostic discovery of degradable proteins in cells using a new class of bifunctional degrader molecules called "AgnoTACs". Proteome-wide screening of 72 AgnoTACs in human cells uncovered downregulation events spanning >50 functionally and structurally diverse proteins, most of which lack chemical probes. While many events progressed through canonical degradation pathways, we also observed instances of alternative mechanisms, indicating that AgnoTACs can impact protein stability via multiple modes of action. Our findings highlight the potential of function-biased chemical libraries coupled with proteomic profiling to discover degrader starting points as well as furnish a blueprint for expanding our understanding of the chemically degradable proteome.
    DOI:  https://doi.org/10.1021/acscentsci.5c01594
  18. Nat Struct Mol Biol. 2025 Dec 05.
    UBA6 specificity for ubiquitin E2 conjugating enzymes reveals a priority mechanism of BIRC6.
    Carlos Riechmann, Cara J Ellison, Jake W Anderson, Kay Hofmann, Peter Sarkies, Paul R Elliott.
      In mammals, ubiquitylation is orchestrated by the canonical ubiquitin-activating E1 enzyme UBA1 and the orthogonal E1 UBA6. Growing evidence underscores the essentiality of both E1s, which differentiate between 29 active ubiquitin-conjugating enzymes (E2s). The mechanisms governing this distinction have remained unclear. Here we establish a framework for ubiquitin E1-E2 specificity. Focusing on UBA6-controlled ubiquitylation cascades, we reveal that BIRC6, a UBA6-exclusive E2, gains priority over all other UBA6-competent E2s, underpinning the functional importance of defined UBA6-BIRC6 ubiquitylation events in regulating cell death, embryogenesis and autophagy. By capturing BIRC6 receiving ubiquitin from UBA6 in different states, we observe BIRC6 engaging with the UBA6 ubiquitin fold domain, driving an exceptionally high-affinity interaction that is modulated by the UBA6 Cys-Cap loop. Using this interaction as a template, we demonstrate how to confer activity between E2s and their noncognate E1, providing a tool to delineate E1-E2-dependent pathways. Lastly, we explain how BIRC6 priority does not lead to inhibition of UBA6, through a bespoke thioester switch mechanism that disengages BIRC6 upon receiving ubiquitin. Our findings propose a concept of hierarchy of E2 activity with cognate E1s, which may explain how ubiquitin E1s can each function with over a dozen E2s and orchestrate E2-specific cellular functions.
    DOI:  https://doi.org/10.1038/s41594-025-01717-z
  19. RNA. 2025 Dec 02. pii: rna.080625.125. [Epub ahead of print]
    5' UTR Cis-Regulatory Logic Governs Ribosome Engagement on Canonical and Non-Coding RNAs.
    Siang Chen, Dongdong Zhang, Hao Wang, Meng Wang, Haiyan Yue, Runsheng Chen, Jianjun Luo.
      Eukaryotic translation initiation is critically regulated by 5' UTR features, including uORFs, Kozak sequences, and secondary structures, that modulate ribosome dynamics. Although canonical mRNAs dominate protein synthesis, ribosome profiling and peptidomics reveal ribosomes actively engaging putative non-coding RNAs (ncRNAs), translating enigmatic short ORFs (sORFs). We systematically analyzed 5' UTR architectures across canonical mRNAs, ribosome-associated ncRNAs (translationally active), and non-translated ncRNAs using curated human datasets. mRNAs exhibited optimal translational features (short 5' UTRs, few uORFs), while translated ncRNAs showed intermediate features, and non-translated ncRNAs the weakest. Notably, mRNAs with long 5' UTRs maintained high translational efficiency through conserved regulatory elements. Integrating these features into our newly developed random forest model, plusCE, surpassed existing methods in predicting translation efficiency, suggesting their potential relevance to translation mechanisms and providing guidance for rational 5' UTR design to modulate translation. Although some ncRNAs are frequently bound by ribosomes, they show no evidence of stable translation, consistent with their lack of coding-related evolutionary signatures. Our analysis suggests that ribosome-bound ncRNAs may not reflect adaptive evolution toward coding function, but rather represent a reservoir of untranslated transcripts that engage the translation machinery through permissive sequence features. Together, these results demonstrate that ribosome engagement is primarily shaped by 5' UTR sequence features, highlighting the importance of regulatory grammar in translation control and complementing current models of ncRNA evolution.
    Keywords:  5’ untranslated regions; non-coding RNAs; translational efficiency prediction; translational features
    DOI:  https://doi.org/10.1261/rna.080625.125
  20. Nat Commun. 2025 Dec 01.
    Sprint interval exercise disrupts mitochondrial ultrastructure driving a unique mitochondrial stress response and remodelling in men.
    J Botella, E Perri, N J Caruana, S López-Calcerrada, M Brischigliaro, N A Jamnick, V Oorschot, N J Saner, J Díaz-Lara, D F Taylor, A Garnham, E Fernández-Vizarra, C Ugalde, G Ramm, D A Stroud, M Lazarou, D J Bishop.
      Exercise is a key lifestyle intervention for mitochondrial health, yet the molecular mechanisms by which different exercise prescriptions regulate mitochondrial remodeling remain unclear. We conducted an open-label counterbalanced randomized controlled trial (ACTRN12617001105336) and observed that sprint-interval exercise (SIE; n = 14), compared to moderate-intensity continuous exercise (MICE; n = 14), induces a mitochondrial stress signature and unfolded protein response (UPRmt). SIE triggers morphological and structural mitochondrial alterations along with activation of the integrated stress response (ISR) and mitochondrial quality control (MQC) pathways. Following eight weeks of training, moderate-intensity continuous training (MICT) increases mitochondrial content, complex I activity, and displays an enrichment of tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) proteins, while sprint-interval training (SIT) improves respiratory function and upregulates pathways involved in 1-carbon metabolism and protein quality control. We identify COX7A2L accumulating in III2 + IV1 supercomplexes only after SIT. These findings elucidate how exercise intensity shapes mitochondrial remodeling, informing tailored exercise prescriptions.
    DOI:  https://doi.org/10.1038/s41467-025-66625-8
  21. Drug Discov Today. 2025 Nov 27. pii: S1359-6446(25)00276-4. [Epub ahead of print] 104563
    Machine learning in targeted protein degradation drug design: a technical review of PROTACs and molecular glues.
    Chieh-Te Lin, Ya-Ping Shiau, Chu-Chung Lin.
      Targeted protein degradation (TPD) allows catalytic removal of disease-associated proteins by exploiting the ubiquitin-proteasome system (UPS). Proteolysis-targeting chimeras (PROTACs) and molecular glues represent two complementary TPD modalities, yet their rational design remains hindered by challenges in ternary complex formation, ligand discovery, and pharmacokinetic optimization. Recent machine learning (ML) advances address these barriers through predictive modeling, virtual screening, and generative design of degrader candidates. In this review, we summarize how ML is integrated across PROTAC and molecular glue development, including ternary complex prediction, linker and fragment design, degradation efficiency modeling, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) optimization. We also highlight emerging artificial intelligence (AI)-driven strategies for de novo glue discovery. Together, these innovations demonstrate how ML is accelerating degrader design and expanding the landscape of druggable targets.
    Keywords:  PROTAC; deep generative design; machine learning; molecular glue; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.drudis.2025.104563
  22. Sci Adv. 2025 Dec 05. 11(49): eady1420
    Proteome-wide computational analyses reveal links between protein condensate formation and RNA biology.
    Snigdha Maiti, Swarnendu Tripathi, David W Baggett, Aaron H Phillips, Cheon-Gil Park, Jina Wang, Wahiduzzaman, William T Freyaldenhoven, Swati Kinger, Brittany J Pioso, John C Bollinger, Ramiz Somjee, Benjamin Lang, M Madan Babu, Richard W Kriwacki.
      Biomolecular condensates mediate dynamic compartmentalization of cellular processes. The multivalent interactions that underlie biomolecular condensation are often promoted by intrinsically disordered regions (IDRs) within proteins. Although the role of IDRs in biomolecular condensates is well appreciated, predicting whether an IDR forms condensates in cells remains challenging. Here, we developed a machine learning model to accurately predict the condensation behavior of IDRs, analyzing 215 IDRs from fusion oncoproteins in human embryonic kidney (HEK) 293T cells. We identified distinct sequence-derived physicochemical features associated with condensation. Leveraging these data, our model predicts that ~12% of the ~13,000 IDRs in the human proteome are likely to form cellular condensates, establishing a robust framework for proteome-wide analysis of IDR-mediated biomolecular condensation. Notably, proteins with condensate-forming IDRs are significantly enriched in RNA processing and splicing functions and are predominantly localized to membraneless organelles, highlighting a central role of IDR-mediated biomolecular condensation in cellular organization and RNA biology.
    DOI:  https://doi.org/10.1126/sciadv.ady1420
  23. Nat Commun. 2025 Dec 01.
    Rapid estimation of protein folding pathways from sequence alone using AlphaFold2.
    Liwei Chang, Alberto Perez.
      AlphaFold2 (AF2) revolutionized protein structure prediction, yet it is often conflated with the protein folding problem. Structure prediction seeks a static conformation, whereas folding concerns the dynamic process of structure formation. We challenge the current status quo, showing that AF2 has implicitly learned some folding principles. Its learned biophysical energy function, though imperfect, enables rapid discovery of folding pathways within minutes. Operating AF2 without multiple sequence alignments (MSAs) or templates forces sampling across its entire energy landscape, akin to ab initio modeling. Among over 7000 proteins, a fraction folds from sequence alone, highlighting the smoothness of AF2's learned surface. Iterating and recycling predictions uncover intermediate structures consistent with experiments, suggesting a "local-first, global-later" mechanism. For designed proteins with optimized local interactions, AF2's landscape becomes too smooth to reveal intermediates. These findings illuminate what AF2 has learned and open avenues for probing protein folding mechanisms and experimental intermediates.
    DOI:  https://doi.org/10.1038/s41467-025-66870-x
  24. Nat Commun. 2025 Dec 03. 16(1): 10858
    Development of a p62 biodegrader for autophagy targeted degradation.
    Zacharias Thiel, David Marcellin, Carole Manneville, Benedikt Goretzki, Luca Egger, Rob Maher, Noémie Siccardi, Laura Torres, Alexandra Probst, Catrin S Müller, Nathalie George, Markus Vogel, Sabine Sinterhauf, Alexandra Lavoisier, Ji-Young Choi, Laurianne Forcellino, Alexandro Landshammer, Patrick Hauck, Celine Be, Frédéric Villard, Sascha Gutmann, Marc Meyer, Felix Freuler, Alexandra Hinniger, César Fernández, Suzanne Chau, Maude Patoor, Gilles Sansig, Gabriel Mitchell, Beat Nyfeler.
      Autophagy-based targeted degradation offers a powerful complement to proteasomal degradation leveraging the capacity and versatility of lysosomes to degrade complex cargo. However, it remains unclear which components of the autophagy-lysosomal pathway are most effective for targeted degradation. Here, we describe two orthogonal induced-proximity strategies to identify autophagy effectors capable of degrading organelles and soluble targets. Recruitment of autophagy cargo receptors, ATG8-like proteins, or the kinases ULK1 and TBK1 is sufficient to trigger mitophagy, while only autophagy cargo receptors capable of self-oligomerization degrade soluble cytosolic proteins. We further report a single-domain antibody against p62 and its use as a heterobifunctional degrader to clear mitochondria. Fusing the p62 single-domain antibody to PINK1 enables selective targeting of damaged mitochondria. Our study highlights the importance of avidity for targeted autophagy and suggests that autophagy cargo receptors are attractive entry points for the development of heterobifunctional degraders for organelles or protein aggregates.
    DOI:  https://doi.org/10.1038/s41467-025-65868-9
  25. Sci Adv. 2025 Dec 05. 11(49): eaea7451
    Rbfox2 selectively governs hematopoietic stem cell self-renewal by regulating proteostasis.
    Longfei Gao, Desmond Dickson, Huijuan Feng, Chaolin Zhang, Lei Ding.
      Self-renewing hematopoietic stem cells (HSCs) generate all blood cells and give rise to long-term reconstitution of the hematopoietic system after transplantation, but the molecular mechanisms that specifically regulate HSCs remain poorly defined. Here, we found that HSCs displayed a distinct messenger RNA alternative splicing pattern and preferentially expressed Rbfox2, an alternative splicing regulator, compared with multipotent progenitors (MPPs). Deletion of Rbfox2 from the hematopoietic compartment specifically depleted HSCs, but not progenitors in the adult bone marrow. Rbfox1 did not function redundantly with Rbfox2 in HSCs. Mechanistically, Rbfox2 loss led to proteostasis stress, including increased protein synthesis rate and accumulated misfolded/unfolded protein contents, in HSCs, but not in progenitors. Small molecules that restore proteostasis rescued HSC defects in Rbfox2-deficient mice. Our work thus reveals that HSCs, but not progenitors, selectively rely on Rbfox2 for their self-renewal and maintenance.
    DOI:  https://doi.org/10.1126/sciadv.aea7451
  26. Nat Commun. 2025 Dec 05.
    Structural mechanism of mRNA decoding by mammalian GTPase GTPBP1.
    Denis Susorov, Anna Miścicka, Dmitrij Golovenko, Anna B Loveland, Alexandra Zinoviev, Tatyana V Pestova, Andrei A Korostelev.
      GTP-binding protein 1 (GTPBP1) is a widespread translational GTPase closely related to elongation factor eEF1A. The loss of GTPBP1 leads to neurodevelopmental and neurodegenerative disorders in animals. Although linked to translation and quality control mechanisms, GTPBP1 molecular functions remain largely obscure. Similarly to eEF1A, GTPBP1 delivers aminoacyl-tRNA to the ribosome, but the ensuing GTPBP1-mediated elongation is slow. Here, using cryo-EM of mammalian 80S ribosomal complexes bound to GTPBP1 and aa-tRNA with GTP or the non-hydrolysable analog GDPCP, we show that the distinct GTPBP1 architecture and interactions with tRNA underlie slow GTPBP1 dissociation after GTP hydrolysis, resulting in delayed tRNA accommodation. Slow dissociation correlates with an extended proofreading stage and higher accuracy of GTPBP1-mediated decoding, potentially allowing GTPBP1 to elicit its putative quality control functions. GTPBP1 visualization provides the foundation for mapping and elucidating GTPBP1 mutations associated with human diseases.
    DOI:  https://doi.org/10.1038/s41467-025-66833-2
  27. Nat Methods. 2025 Dec 03.
    Atom-level enzyme active site scaffolding using RFdiffusion2.
    Woody Ahern, Jason Yim, Doug Tischer, Saman Salike, Seth M Woodbury, Donghyo Kim, Indrek Kalvet, Yakov Kipnis, Brian Coventry, Han Raut Altae-Tran, Magnus S Bauer, Regina Barzilay, Tommi S Jaakkola, Rohith Krishna, David Baker.
      Designing new enzymes typically begins with idealized arrangements of catalytic functional groups around a reaction transition state, then attempts to generate protein structures that precisely position these groups. Current AI-based methods can create active enzymes but require predefined residue positions and rely on reverse-building residue backbones from side-chain placements, which limits design flexibility. Here we show that a new deep generative model, RoseTTAFold diffusion 2 (RFdiffusion2), overcomes these constraints by designing enzymes directly from functional group geometries without specifying residue order or performing inverse rotamer generation. RFdiffusion2 successfully generates scaffolds for all 41 active sites in a diverse benchmark, compared to 16 using previous methods. We further design enzymes for three distinct catalytic mechanisms and identify active candidates after experimentally testing fewer than 96 sequences in each case. These results highlight the potential of atomic-level generative modeling to create de novo enzymes directly from reaction mechanisms.
    DOI:  https://doi.org/10.1038/s41592-025-02975-x
  28. Sci Adv. 2025 Dec 05. 11(49): eadu2292
    Tumor microenvironment-targeted PROTAC nanoparticle self-assembly broadly predicted by structural descriptors.
    Kristen C Vogt, Magdalini Panagiotakopoulou, Mandana T Manzari Honu, Ana Marie Perea, Xiaoping Hu, Xufen Yu, Raashed Raziuddin, Quincey LaPlant, Stephen Ruiz, G Praveen Raju, Jian Jin, David A Scheinberg, Daniel A Heller.
      Proteolysis-targeting chimeras (PROTACs) are catalytic protein degraders with promising preclinical activity. The clinical translation of PROTACs has been limited by poor pharmacologic properties and toxicities, in part due to their "non-druglike" characteristics, including large molecular weights. We found that the vast majority of PROTACs can self-assemble into nanoparticles, yielding nanoparticle PROTACs (nanoPROTACs) with ultrahigh drug loadings. While PROTAC molecular features can be deleterious to their pharmacokinetic properties, we found that they can drive nanoencapsulation more efficiently than FDA-approved small-molecule drugs. Using structure-based prediction algorithms, we identified spatial autocorrelation molecular descriptors that defined nanoPROTAC formation with 96% sensitivity at 100% specificity. NanoPROTACs, targeted to the tumor microenvironment via P-selectin, led to significantly enhanced tumor drug uptake, target degradation, tumor growth inhibition, and overall survival in solid tumor xenografts. These findings offer a broad strategy to improve the pharmacologic properties and therapeutic index of PROTACs and potentially other non-druglike experimental therapeutics.
    DOI:  https://doi.org/10.1126/sciadv.adu2292
  29. Nature. 2025 Dec 03.
    Computational design of metallohydrolases.
    Donghyo Kim, Seth M Woodbury, Woody Ahern, Doug Tischer, Alex Kang, Emily Joyce, Asim K Bera, Nikita Hanikel, Saman Salike, Rohith Krishna, Jason Yim, Samuel J Pellock, Anna Lauko, Indrek Kalvet, Donald Hilvert, David Baker.
      De novo enzyme design seeks to build proteins containing ideal active sites with catalytic residues surrounding and stabilizing the transition state(s) of the target chemical reaction1-7. The generative artificial intelligence method RFdiffusion8,9 solves this problem, but requires specifying both the sequence position and backbone coordinates for each catalytic residue, limiting sampling. Here we introduce RFdiffusion2, which eliminates these requirements, and use it to design zinc metallohydrolases starting from quantum chemistry-derived active site geometries. From an initial set of 96 designs tested experimentally, the most active has a catalytic efficiency (kcat/KM) of 16,000 M-1 s-1, orders of magnitude higher than previously designed metallohydrolases6,7,10,11. A second round of 96 designs yielded 3 additional highly active enzymes, with kcat/KM values of up to 53,000 M-1 s-1 and a catalytic rate constant (kcat) of up to 1.5 s-1. The design models of the four most active designs differ from known structures and from each other, and the crystal structure of the most active design is very close to the design model, demonstrating the accuracy of the design method. The most active enzymes are predicted by PLACER12 and Chai-1 (ref. 13) to have preorganized active sites that effectively position the substrate for nucleophilic attack by a water molecule activated by the bound metal. The ability to generate highly active enzymes directly from the computer, without experimental optimization, should enable a new generation of potent designer catalysts14,15.
    DOI:  https://doi.org/10.1038/s41586-025-09746-w
  30. Nat Chem Biol. 2025 Dec 05.
    Accurate single-domain scaffolding of three nonoverlapping protein epitopes using deep learning.
    Karla M Castro, Joseph L Watson, Jue Wang, Joshua Southern, Reyhaneh Ayardulabi, Sandrine Georgeon, Stéphane Rosset, David Baker, Bruno E Correia.
      De novo protein design has seen major success in scaffolding single functional motifs; however, in nature, most proteins present multiple functional sites. Here, we describe an approach to simultaneously scaffold multiple functional sites in a single-domain protein using deep learning. We designed small single-domain immunogens, under 130 residues, that present three distinct and irregular motifs from respiratory syncytial virus. These motifs together comprise nearly half of the designed proteins; hence, the overall folds are quite unusual with little global similarity to proteins in the Protein Data Bank. Despite this, X-ray crystal structures confirmed the accuracy of presentation of each of the motifs and the multiepitope design yields improved cross-reactive titers and neutralizing response compared to a single-epitope immunogen. The successful presentation of three distinct binding surfaces in a small single-domain protein highlights the power of generative deep learning methods to solve complex protein design problems.
    DOI:  https://doi.org/10.1038/s41589-025-02083-z
  31. Nat Commun. 2025 Dec 03.
    Vacuolar-type H+-ATPase-mediated extra-organellar buffering resolves mitochondrial dysfunction.
    Geoffray Monteuuis, Ryan Awadhpersad, Daan van der Kolk, Sachin K Singh, Tuula A Nyman, Alina Malyutina, Nicola Zamboni, Kari Moisio, Juhana Juutila, Ville Hietakangas, Sara Seneca, Christopher J Carroll, Christopher B Jackson.
      Mitochondrial dysfunction underlies a wide range of human diseases, including primary mitochondrial disorders, neurodegeneration, cancer, and ageing. To preserve cellular homeostasis, organisms have evolved adaptive mechanisms that coordinate nuclear and mitochondrial gene expression. Here, we use genome-wide CRISPR knockout screening to identify cell fitness pathways that support survival under impaired mitochondrial protein synthesis. The strongest suppressor of aberrant mitochondrial translation defects - besides a compendium of known mitochondrial translation quality control factors - is the loss of the vacuolar-type H+-ATPase (v-ATPase), a key regulator of intracellular acidification, nutrient sensing, and growth signaling. We show that partial v-ATPase loss reciprocally modulates mitochondrial membrane potential (ΔΨm) and cristae structure in both cancer cell lines and mitochondrial disease patient-derived models. Our findings uncover an extra-organellar buffering mechanism whereby partial v-ATPase inhibition mitigates mitochondrial dysfunction by altering pH homeostasis and driving metabolic rewiring as a protective response that promotes cell fitness.
    DOI:  https://doi.org/10.1038/s41467-025-66656-1
  32. Nat Rev Mol Cell Biol. 2025 Dec 04.
    A lunapark for secretory mRNA translation at ER junctions.
    Lisa Heinke.
      
    DOI:  https://doi.org/10.1038/s41580-025-00938-w
  33. PLoS Biol. 2025 Dec 05. 23(12): e3003540
    The transcription factor SKN-1 drives lysosomal enlargement during aging to maintain function.
    Xinyu Wang, Huimin Liu, Xiaoman Wang, Ben Zhou, Haiqing Tang, Shanshan Pang.
      Lysosomes are critical hubs for both cellular degradation and signal transduction, yet their function declines with age. Aging is also associated with significant changes in lysosomal morphology, but the physiological significance of these alterations remains poorly understood. Here, we find that a subset of aged lysosomes undergo enlargement resulting from lysosomal dysfunction in C. elegans. Importantly, this enlargement is not merely a passive consequence of functional decline but represents an active adaptive response to preserve lysosomal degradation capacity. Blocking lysosomal enlargement exacerbates the impaired degradation of dysfunctional lysosomes. Mechanistically, lysosomal enlargement is a transcriptionally regulated process governed by the longevity transcription factor SKN-1, which responds to lysosomal dysfunction by restricting fission and thereby induces lysosomal enlargement. Furthermore, in long-lived germline-deficient animals, SKN-1 activation induces lysosomal enlargement, thereby promoting lysosomal degradation and contributing to longevity. These findings unveil a morphological adaptation that safeguards lysosomal homeostasis, with potential relevance for lysosomal aging and life span.
    DOI:  https://doi.org/10.1371/journal.pbio.3003540
  34. Nat Metab. 2025 Dec 03.
    Age-related decline of chaperone-mediated autophagy in skeletal muscle leads to progressive myopathy.
    Olaya Santiago-Fernández, Luisa Coletto, Inmaculada Tasset, Susmita Kaushik, Axel R Concepcion, Rizwan Qaisar, Adrián Macho-González, Kristen Lindenau, Antonio Diaz, Rabia R Khawaja, Stefano Donega, Nirad Banskota, Ceereena Ubaida-Mohien, Gavin Pharaoh, Bumsoo Ahn, Lisa M Hartnell, Ignacio Ramírez-Pardo, Bhakti Chavda, Aiara Gazteluiturri, Michael Kinter, Luigi Ferrucci, Julie A Reisz, Angelo D'Alessandro, Holly Van Remmen, Pura Muñoz-Cánoves, Stefan Feske, Ana Maria Cuervo.
      Chaperone-mediated autophagy (CMA) contributes to proteostasis maintenance by selectively degrading a subset of proteins in lysosomes. CMA declines with age in most tissues, including skeletal muscle. However, the role of CMA in skeletal muscle and the consequences of its decline remain poorly understood. Here we demonstrate that CMA regulates skeletal muscle function. We show that CMA is upregulated in skeletal muscle in response to starvation, exercise and tissue repair, but declines in ageing and obesity. Using a muscle-specific CMA-deficient mouse model, we show that CMA loss leads to progressive myopathy, including reduced muscle force and degenerative myofibre features. Comparative proteomic analyses reveal CMA-dependent changes in the mitochondrial proteome and identify the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) as a CMA substrate. Impaired SERCA turnover in CMA-deficient skeletal muscle is associated with defective calcium (Ca2+) storage and dysregulated Ca2+ dynamics. We confirm that CMA is also downregulated with age in human skeletal muscle. Remarkably, genetic upregulation of CMA activity in old mice partially ameliorates skeletal muscle ageing phenotypes. Together, our work highlights the contribution of CMA to skeletal muscle homoeostasis and myofibre integrity.
    DOI:  https://doi.org/10.1038/s42255-025-01412-9
  35. bioRxiv. 2025 Nov 21. pii: 2025.11.20.689567. [Epub ahead of print]
    Tom70-mediated mitochondria-nuclear envelope contacts regulate nuclear pore complex inheritance during gametogenesis.
    Cyrus T Ruediger, Benjamin S Styler, Eric M Sawyer, Silvan Spiri, Grant King, Madison E Walsh, Gloria A Brar, Danielle M Jorgens, Elçin Ünal.
      Gametogenesis rejuvenates the cellular lineage and excludes senescence-associated factors from gametes. In Saccharomyces cerevisiae , this involves sequestration of nuclear constituents into the Gametogenesis-Uninherited Nuclear Compartment (GUNC), which is excluded from gametes. Here we identify the conserved mitochondrial import receptor Tom70 as a key regulator of GUNC-mediated exclusion. Loss of TOM70 disrupts the sequestration of nuclear pore complexes, but not senescence-associated aggregates and nucleolar components, into the GUNC. Tom70's role appears independent of its canonical function in mitochondrial import and instead reflects a meiosis-specific requirement for mitochondria-nuclear envelope tethering. During meiosis II, Tom70 concentrates around the GUNC, where it recruits the nuclear envelope tethering protein Cnm1. Loss of CNM1 partially phenocopies tom70Δ , consistent with parallel tethering interactions. These findings uncover a previously unrecognized organelle contact-dependent pathway that remodels the nuclear envelope to support selective nuclear inheritance. More broadly, they highlight how organelle contacts integrate with nuclear quality control to safeguard gamete integrity.
    DOI:  https://doi.org/10.1101/2025.11.20.689567
  36. NPJ Antimicrob Resist. 2025 Dec 04. 3(1): 94
    Evolution of proteolysis-targeting chimeras (PROTAC) technology to overcome challenges of antimicrobial resistance.
    Dhanashree N Sarwan, Pramod B Khedekar, Ritesh P Bhole, Rupesh V Chikhale.
      Targeted protein degradation (TPD) is an innovative therapeutic approach that bypasses traditional drug inhibition methods. Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that harness degradation machinery to remove target proteins. This review examines the evolution of PROTACs and their application in targeting microorganisms that develop drug resistance, covering their development, advancements in linker design, E3 ligase selection, and delivery methods, including nanoparticles and exosomes.
    DOI:  https://doi.org/10.1038/s44259-025-00136-w
  37. J Cell Biol. 2026 Feb 02. pii: e202508155. [Epub ahead of print]225(2):
    Regulation of cell dynamics by rapid integrin transport through the biosynthetic pathway.
    Martina Lerche, Mathilde Mathieu, Hellyeh Hamidi, Megan Chastney, Guillaume Jacquemet, Bart Marlon Herwig Bruininks, Shreyas Kaptan, Lene Malerød, Nina Marie Pedersen, Andreas Brech, Nobuyuki Matoba, Yuichiro Sato, Ilpo Vattulainen, Pere Roca-Cusachs, Franck Perez, Gaelle Boncompain, Stéphanie Miserey, Johanna Ivaska.
      Constitutive integrin endocytosis and recycling control cell movement and morphology. In contrast, the role of newly synthesized integrins delivered via the biosynthetic pathway has been largely overlooked. We used the retention using selective hooks system to monitor the localization of new integrins exiting the endoplasmic reticulum in space and time. We discovered that new integrin delivery to the plasma membrane is polarized and enhances cell protrusion and focal adhesion growth in an extracellular matrix-ligand-dependent manner. Motor-clutch modeling explained the increased adhesion as higher integrin availability driving recruitment of additional receptors. Unexpectedly, live-cell imaging revealed a small subset of fast-emerging integrin vesicles rapidly transported to the cell surface to facilitate localized spreading. This unconventional secretion depended on cell adhesion and correlated with increased surface levels of immature, high-mannose glycosylated integrin, indicating bypass of the canonical Golgi-dependent secretory pathway. Thus, spatial plasma membrane-targeting of new integrins rapidly alters adhesion receptor availability, providing cells with added plasticity to respond to their environment.
    DOI:  https://doi.org/10.1083/jcb.202508155
  38. Nat Commun. 2025 Dec 02. 16(1): 10839
    Structural insights into cauliflower mitoribosome in translation state and in association with a late assembly factor.
    Vasileios Skaltsogiannis, Philippe Wolff, Tan-Trung Nguyen, Nicolas Corre, David Pflieger, Todd Blevins, Yaser Hashem, Philippe Giegé, Florent Waltz.
      Ribosomes are key molecular machines that translate mRNA into proteins. Mitoribosomes are specific ribosomes found in mitochondria, which have been shown to be remarkably diverse across eukaryotic lineages. In plants, they possess unique features, including additional rRNA domains stabilized by plant-specific proteins. However, the structural specificities of plant mitoribosomes in translation state remained unknown. We used cryo-electron microscopy to provide a high-resolution structural characterization of the cauliflower mitoribosome, in translating and maturation states. The structure reveals the mitoribosome bound with a tRNA in the peptidyl site, along with a segment of mRNA and a nascent polypeptide. Moreover, using structural data, nanopore sequencing and mass spectrometry, we identify a set of 19 ribosomal RNA modifications. Additionally, we observe a late assembly intermediate of the small ribosomal subunit, in complex with the RsgA assembly factor. This reveals how a plant-specific extension of RsgA blocks the mRNA channel to prevent premature mRNA association before complete small subunit maturation. Our findings elucidate key aspects of translation in angiosperm plant mitochondria, revealing its distinct features compared to other eukaryotic lineages.
    DOI:  https://doi.org/10.1038/s41467-025-65864-z
  39. Nat Commun. 2025 Dec 01. 16(1): 10801
    Molecular mechanism of PINK1 regulation by the Hsp90 machinery.
    Xuyang Tian, Jiayue Su, Ziyi Wang, Tao Liu, Huifang Xiong, Shan Sun, Kunrong Mei, Sen-Fang Sui.
      Hundreds of human kinases, including PINK1-a protein kinase associated with familial Parkinson's disease-are regulated by Hsp90 and its cochaperones. While previous studies have elucidated the mechanism of kinase loading into the Hsp90 machinery, the subsequent regulation of kinases by Hsp90 and its cochaperones remains poorly understood. In this study, using complexes obtained through PINK1 pulldown, we determine the cryo-EM structures of the human Hsp90-Cdc37-PINK1 complex at 2.84 Å, Hsp90-FKBP51-PINK1 at approximately 6 Å, and Hsp90- PINK1 at 2.98 Å. These structures, along with the bound nucleotide in the Hsp90 dimers of the three complexes, provide insights into the Hsp90 chaperone machinery for kinases and elucidate the molecular mechanisms governing cytosolic PINK1 regulation.
    DOI:  https://doi.org/10.1038/s41467-025-65859-w
  40. Commun Biol. 2025 Dec 02.
    Decoding protein binding plasticity via integrated deep ribosome display and deep learning.
    Mengtong Tang, Jiawei Li, Zhixi Li, Jingsong Cui, Hao Qi.
      The plasticity in protein interaction is central to understanding biological networks and de novo protein design. However, the systematic exploration remains impeded by the astronomic dimensionality of sequence space. Here, we present a platform that synergizes deep experimental screening with deep learning to decode interaction plasticity. By developing a ribosome display stripped of all known ribosome termination and rescue functions, we produce a comprehensive dataset comprising 47.8 million unique peptides spanning a broad spectrum of Streptactin-binding activity. A deep learning architecture, systematically trained on sequence context, enrichment dynamics, and subsequence abundance, achieves high accuracy (Pearson's r = 0.902) on predicting Streptactin-binding activity. Through sequence dimensionality reduction, exhaustive subsequence elucidation, and enriched motif elicitation, we identify 799 strong-binding sequences containing a canonical motif and 219 sequences harboring novel motifs with divergent docking conformations. These findings reveal an unanticipated depth and breadth in protein-binding plasticity. We propose that this integrated experimental-AI framework will facilitate the systematic exploration of protein interactions and enable the data-driven design of synthetic peptides.
    DOI:  https://doi.org/10.1038/s42003-025-09160-y
  41. Life Sci Alliance. 2026 Feb;pii: e202503485. [Epub ahead of print]9(2):
    The unfolded protein response in progeria arteries originates from non-endothelial cell types.
    Raquel A Silva, Fatih Sarigol, G Elif Karagöz, Selma Osmanagic-Myers, Roland Foisner.
      Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease caused by a mutation in LMNA, leading to the expression of a prelamin A variant called progerin. HGPS hallmarks include accelerated cardiovascular disease and atherosclerosis, caused in part by ER stress-induced apoptosis of vascular smooth muscle cells. As a dysregulated unfolded protein response (UPR) can induce endothelial cell (EC) pathology during aging, we investigated whether loss of proteostasis contributes to EC dysfunction in HGPS, using an endothelium-specific HGPS mouse model. Contrary to previous reports in vascular smooth muscle cells and fibroblasts, we found no robust activation of UPR in ECs constitutively expressing progerin, and cells retained the ability to elicit potent UPR when exposed to external ER stress. Unlike aortic tissue derived from mice with endothelium-specific progerin expression, aorta from Lmna G609G/+ mice with ubiquitous progerin expression showed up-regulation of the UPR, suggesting that the UPR in HGPS aorta is primarily rooted in non-ECs. Analysis of scRNA-Seq datasets from aorta in Lmna G609G/G609G mice confirmed this hypothesis. Our data indicate that UPR activation is a cell-type-specific phenomenon in progerin-expressing arteries.
    DOI:  https://doi.org/10.26508/lsa.202503485
  42. Biochem Soc Trans. 2025 Dec 03. pii: BST20253031. [Epub ahead of print]53(6):
    Towards a phylogenetically informed approach to solving protein-protein interactions.
    Chun Shen Lim, Peter Mace, Peter C Fineran, Paul P Gardner.
      Protein-protein interactions (PPIs) are critical to all cellular activities. Despite having a large number of proteins, cells have spatial and temporal control over PPIs to avoid dysregulation in cellular pathways. Considerable research efforts have aimed to find new PPIs, curate PPIs from the literature and build searchable PPI databases. These databases have been widely used by experimental and computational scientists. Here we find that the PPIs captured by these databases are highly heterogeneous and concentrated on a small number of species. These issues hamper researchers from capturing the full landscape of reliable PPIs, affecting the accuracy of machine-learning models and the effectiveness of experimental designs. However, there are opportunities to fill gaps computationally and experimentally. We suggest developing a phylogenetically informed approach to test PPIs experimentally and computationally.
    Keywords:  artificial intelligence; evolutionary conserved protein–protein interactions; machine learning; protein complex prediction
    DOI:  https://doi.org/10.1042/BST20253031
  43. Cell Death Discov. 2025 Dec 05.
    Bidirectional crosstalk between ER stress and lipid metabolism: From proteostasis to tumor adaptation.
    Yueling Wu, Huijuan Luo, Zhiwei Pan, Weiping Chen, Lei Bi.
      Endoplasmic reticulum (ER) stress is a central adaptive response that maintains proteostasis under diverse metabolic and environmental challenges. In cancer, ER stress and lipid metabolism form a tightly coupled, bidirectional regulatory network that integrates protein quality control with lipid remodeling. Through the unfolded protein response (UPR), ER stress reprograms lipid synthesis, oxidation, and storage to sustain energy balance and membrane integrity. Conversely, dysregulated lipid accumulation disrupts ER homeostasis and amplifies stress signaling, creating a feedback loop between metabolic and proteostatic imbalance. Proteostasis systems, including the ubiquitin-proteasome system (UPS) and autophagy, cooperate with UPR signaling to fine-tune this adaptive balance and enhance tumor survival under stress. This review highlights the bidirectional crosstalk between ER stress and lipid metabolism from the perspective of proteostasis-driven tumor adaptation and summarizes emerging therapeutic strategies such as small-molecule modulators, natural products, and combination therapies that target this adaptive network to overcome drug resistance and improve cancer treatment.
    DOI:  https://doi.org/10.1038/s41420-025-02878-y
  44. J Med Chem. 2025 Dec 02.
    Selective CDK6 Degradation via the KLHDC2 E3 Ubiquitin Ligase.
    Eunhye Jeon, Younghoon Kim, Hyunwoo Ahn, Michael J Martinez, Kyubin Hwang, Soyeong Cho, Brendan G Dwyer, Bryan A Romero, Stephen M Hinshaw, Nathanael S Gray, Taebo Sim.
      We discovered novel small molecule ligands of KLHDC2 and leveraged them to generate KLHDC2-mediated CDK6-selective degraders. Degrader 48a exhibited potent and selective CDK6 degradation (DC50 = 0.037 μM) over CDK4 (DC50 > 10 μM) in MOLM-14 cells, leading to pronounced G0/G1 cell-cycle arrest and apoptosis through inhibition of CDK6 downstream signaling. In addition, 48a demonstrated superior growth-inhibitory activity compared to the warhead, palbociclib, in several leukemia cells and displayed favorable microsomal stability. Proteomic profiling confirmed that 48a selectively degrades CDK6 with minimal effects on other CDK family members. Furthermore, 48a reduced tumor burden and CDK6 levels in an in vivo xenograft model. Collectively, these findings highlight the potential of KLHDC2-mediated degraders as a novel strategy for selective CDK6 degradation and underscore the promise of KLHDC2 as an alternative E3 ligase platform for targeted protein degradation.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c02713
  45. Cell Rep. 2025 Dec 04. pii: S2211-1247(25)01414-7. [Epub ahead of print]44(12): 116642
    Sec61 translocon inhibitor flavitransin blocks selectively dengue virus polyprotein insertion in the ER with pan-orthoflavivirus antiviral potency.
    Marijke Verhaegen, Marianne Croonenborghs, Nidhi Sorout, Andrea Sartori, Becky Provinciael, Eef Meyen, Joren Stroobants, Robin Hermans, Maarten Jacquemyn, Dirk Daelemans, Ana Lucia Rosales-Rosas, Leen Delang, Piet Maes, Enno Hartmann, Volkhard Helms, Kurt Vermeire.
      Dengue virus (DENV) infections impose a significant healthcare threat worldwide. Here, we report the antiviral activity of cyclotriazadisulfonamide (CADA) derivative VGD020, renamed as flavitransin (FT), against all four DENV serotypes, Zika, and yellow fever virus in various cell types. FT exerts a post-entry antiviral effect, in line with its anticipated Sec61 inhibition. Cell-free in vitro translation assays demonstrate a direct inhibitory effect of FT on DENV polyprotein translocation across the endoplasmic reticulum (ER) membrane of both human and insect (vector) origin, with high selectivity for the N-terminal transmembrane domain of the capsid subunit of different orthoflaviviruses. Selection and genotyping of FT-resistant HCT116 cells reveal a unique A70V mutation in the Sec61α subunit. Finally, a high barrier to FT resistance development is observed for wild-type DENV. In conclusion, our data demonstrate that FT selectively interferes with the initiation of DENV polyprotein ER translocation and confirm the critical role of the ER in orthoflavivirus replication.
    Keywords:  CP: microbiology; Sec61 translocon; Zika virus; antiviral; co-translational translocation; cyclotriazadisulfonamide; dengue virus
    DOI:  https://doi.org/10.1016/j.celrep.2025.116642
  46. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1306. [Epub ahead of print]53(22):
    Integrative and accurate annotations enhance current nonsense-mediated mRNA decay rules.
    Hiroyuki Iha, Chie Kikutake, Mikita Suyama.
      Variants generating premature termination codons (PTCs) are a major cause of human genetic disease, and it is crucial to accurately assess their impact. Nonsense-mediated mRNA decay (NMD) is a surveillance system that degrades mRNAs containing PTCs to prevent the production of truncated proteins. Prior studies have shown that PTC position-dependent rules are major contributors to NMD efficiency, though they leave a portion of the variability unexplained. To improve the coverage of current NMD escape rules, we used matched human genome and transcriptome data from 1086 individuals and re-evaluated NMD efficiency by considering multi-nucleotide variants (MNVs), translation status, and RNA isoform expression as part of an accurate annotation. Integrated data assessment and accurate annotation resulted in a 12.0% improvement in the explanatory power of NMD efficiency. Furthermore, we found that variants with high allele frequency or occurring in regions of low genomic conservation escape NMD due to the presence of MNVs or the absence of translation by ribosomes. Our results emphasize the importance of accurate annotation in assessing the impact of nonsense variants.
    DOI:  https://doi.org/10.1093/nar/gkaf1306
  47. Br J Pharmacol. 2025 Dec 02.
    Molecular glue neferine induces BAP31 homodimerisation to disrupt endoplasmic reticulum-mitochondria crosstalk for anti-neuroinflammation in ischaemic stroke.
    Ya-Xuan Zhu, Zhuo Yang, Ling Li, Ze-Kun Chen, Fang-Fang Zhuo, Yu-Hui Wang, Zheng-Ping Liu, Bo Han, Wei Yu, Peng-Fei Tu, Tian-Tian Wei, Hua Wang, Ke-Wu Zeng.
       BACKGROUND AND PURPOSE: B-cell receptor-associated protein 31 (BAP31), an endoplasmic reticulum (ER)-resident transmembrane protein, has emerged as a critical regulator of immune cell activation, yet its role in neuroinflammation remains unexplored. Here, we uncovered the natural compound neferine (Nef) as a pharmacological modulator of BAP31 that suppressed microglial activation.
    EXPERIMENTAL APPROACH: Using thermal protein profiling (TPP), we identified BAP31 as the primary target of Nef. Biochemical and structural analyses were employed to characterise Nef-BAP31 interactions. We evaluated ER stress and mitochondrial energy metabolism homeostasis using techniques such as STER super-resolution technology, flow cytometry, western blot, etc. In vivo validation utilised two models: lipopolysaccharide (LPS)-induced endotoxaemia and middle cerebral artery occlusion (MCAO) for ischaemic stroke, combining behavioural tests, cytokine profiling and histopathological assessments.
    KEY RESULTS: Nef functioned as a 'molecular glue' by binding to BAP31's coiled-coil CC2 domain to induce stable dimerisation. We revealed that dimerised BAP31 triggered ER membrane remodelling, which disrupted ER-mitochondria contact sites and preserved mitochondrial energy metabolism homeostasis, thereby blocking inflammatory cytokine release. In vivo, Nef attenuated neuroinflammation in endotoxaemia mice and further conferred neuroprotection against ischaemic stroke in a MCAO model by inhibiting microglia-driven neuronal injury.
    CONCLUSIONS AND IMPLICATIONS: In summary, our work reveals that BAP31 is a master regulator of ER-mitochondria communication during microglial activation and introduces a paradigm-shifting 'molecular glue' strategy for targeting ER-resident proteins. Additionally, these findings redefine the pharmacological landscape for modulating organelle interactions in microglia involved in neuroinflammatory diseases.
    Keywords:  B cell receptor‐associated protein 31 (BAP31); endoplasmic reticulum (ER); ischaemic stroke; microglial polarization; neferine (Nef); neuroinflammation
    DOI:  https://doi.org/10.1111/bph.70277
  48. Sci Adv. 2025 Dec 05. 11(49): eaea3996
    VAPB is a negative regulator of STING-mediated innate immune signaling.
    Wangsheng Ji, Yin Zhang, Lianfei Zhang, Qingqing Liu, Shengbo Niu, Yuqun Feng, Feilong Chen, Xinqi Liu, Xia Li.
      Stimulator of IFN genes (STING) is an endoplasmic reticulum (ER) signaling receptor involved in the type I interferon response to pathogen- or self-derived cytosolic double-stranded DNA. Excessive activation of STING is associated with many diseases, but the regulatory mechanism of STING activation remains to be further elucidated. Here, we identify VAPB as a negative regulator of STING-mediated innate immune response. VAPB deficiency increases the expression of type I interferons under resting conditions or upon stimulation. Mechanistically, VAPB associates and translocates with STING, thereby regulating STING translocation, oligomerization, and recruitment of TBK1. In vivo, deficiency of VAPB enhances the expression of type I interferons and prevents lethality following HSV-1 infection. Furthermore, VAPB P56S, a pathogenic mutation causing amyotrophic lateral sclerosis (ALS), can promote STING-mediated innate immune response under resting conditions, which might contribute to further understanding of the relationship between cGAS-STING pathway and ALS. Our study identifies VAPB as a critical regulating factor in cGAS-STING-mediated innate immune responses.
    DOI:  https://doi.org/10.1126/sciadv.aea3996
  49. Cell Syst. 2025 Dec 01. pii: S2405-4712(25)00283-2. [Epub ahead of print] 101450
    High-throughput mapping of modular regulatory domains in human RNA-binding proteins.
    Abby R Thurm, Yaara Finkel, Cecelia Andrews, Xiangmeng S Cai, Colette Benko, Lacramioara Bintu.
      RNA regulation is central to tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). While many RBPs require their full sequence to function, some act through modular domains that recruit larger regulatory complexes. Mapping these RNA-regulatory effector domains is important for understanding RBP function and designing compact RNA regulators. We developed a high-throughput recruitment assay (HT-RNA-Recruit) to identify RNA-downregulatory effector domains within human RBPs. By recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA, we discovered over 100 RNA-downregulatory effector domains in 86 RBPs. Certain domains-for instance, KRABs-suppress gene expression upon recruitment to both DNA and RNA. We engineered inducible synthetic RNA regulators based on NANOS that can downregulate endogenous RNAs or maintain reporter expression at defined intermediate levels, as predicted by mathematical modeling. This work serves as a resource for understanding RNA regulators and expands the repertoire of RNA control tools. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  RNA degradation; RNA-binding proteins; gene regulation; high-throughput screening; mammalian synthetic biology
    DOI:  https://doi.org/10.1016/j.cels.2025.101450
  50. J Cell Biol. 2026 Jan 05. pii: e202501255. [Epub ahead of print]225(1):
    Loss of ErbB3 redirects Integrin β1 from early endosomal recycling to secretion in extracellular vesicles.
    Dorival Mendes Rodrigues-Junior, Ana Rosa Sáez-Ibáñez, Takeshi Terabayashi, Nina Daubel, Taija Mäkinen, Olof Idevall-Hagren, Aristidis Moustakas, Ingvar Ferby.
      Receptor tyrosine kinases (RTKs) are important cargo in endocytic trafficking, yet their role in endosomal sorting and maturation of multivesicular bodies remains unclear. Here, we show that the ErbB3 (HER3) receptor sorts internalized Integrin β1 and the transferrin receptor, for endocytic recycling, in a manner that does not require ligand-induced ErbB3 signaling in breast epithelial cells. Loss of ErbB3 abrogates recycling of Integrin β1, likely from a Rab4-positive compartment, and redirects it toward lysosomal degradation or secretion as an extracellular vesicle (EV) cargo. ErbB3 depletion impairs the collective migration of breast epithelial cell sheets, coinciding with reduced cell-surface levels of Integrin β1 and increased release of Integrin β1-containing EVs. In contrast, EVs secreted from ErbB3-depleted cells enhance the motility of wild-type cells. Mechanistically, ErbB3 promotes assembly of the Arf6-GGA3-Rabaptin5 endosomal sorting complex to facilitate early recycling and suppress EV release. These findings provoke the notion that pseudo-RTKs play an active role in vesicular trafficking.
    DOI:  https://doi.org/10.1083/jcb.202501255
  51. Nature. 2025 Dec 03.
    Computational enzyme design by catalytic motif scaffolding.
    Markus Braun, Adrian Tripp, Morakot Chakatok, Sigrid Kaltenbrunner, Celina Fischer, David Stoll, Aleksandar Bijelic, Wael Elaily, Massimo G Totaro, Melanie Moser, Shlomo Y Hoch, Horst Lechner, Federico Rossi, Matteo Aleotti, Mélanie Hall, Gustav Oberdorfer.
      Enzymes find broad use as biocatalysts in industry and medicine owing to their exquisite selectivity, efficiency and mild reaction conditions. Custom-designed enzymes can produce tailor-made biocatalysts with potential applications that extend beyond natural reactions. However, current design methods require testing a large number of designs and mostly produce de novo enzymes with low catalytic activities1-3. As a result, they require costly experimental optimization and high-throughput screening to be industrially viable4,5. Here we present rotamer inverted fragment finder-diffusion (Riff-Diff), a hybrid machine learning and atomistic modelling strategy for scaffolding catalytic arrays in de novo proteins. We highlight the general applicability of Riff-Diff by designing enzymes for two mechanistically distinct chemical transformations, the retro-aldol reaction and the Morita-Baylis-Hillman reaction. We show that in both cases, it is possible to generate catalysts that exhibit activities rivalling those optimized by in vitro evolution, along with exquisite stereoselectivity. High-resolution structures of six of the designs revealed near-atomic active site design precision. The design strategy can, in principle, be applied to any catalytically competent amino acid array. These findings lay the basis for practical applicability of de novo protein catalysts in synthesis and describe fundamental principles of protein design and enzyme catalysis.
    DOI:  https://doi.org/10.1038/s41586-025-09747-9
  52. Mol Metab. 2025 Dec 02. pii: S2212-8778(25)00199-1. [Epub ahead of print] 102292
    Reprogramming of cholesterol sensing in epithelial cells supports pancreatic inflammation.
    Giulia Milan, Olga A Mareninova, Marco Fantuz, Martina Spacci, Carlotta Paoli, Jerik A Pineda, Roberta Noè, Beatrice Calciolari, Roberto Zoncu, Anna S Gukovskaya, Alessandro Carrer.
      Pancreatitis is a common cause of hospitalization that necessitates attentive clinical management. Affected individuals are at risk for pancreatic cancer due to aberrant signaling and empowered cell plasticity. Yet, molecular and cellular dynamics that govern epithelial cell behavior in response to inflammation remain largely elusive. Here we found that inflammation induces Endoplasmic Reticulum-Associated Degradation protein (ERAD)-mediated downregulation of Niemann-Pick type C protein 1 (NPC1), which leads to the sequestration of free cholesterol within acinar cells' lysosomes. Reducing intra-pancreatic cholesterol levels through genetic ablation of Acly ameliorates cerulein-induced pancreatitis, while pharmacological targeting of NPC1 exacerbates tissue damage. Mechanistically, the accumulation of lysosomal cholesterol is sensed by the mechanistic Target of Rapamycin Complex 1 (mTORC1) that promotes metaplasia of pancreatic acinar cells, an event commonly associated to pancreatitis and tissue regeneration. Indeed, cholesterol supplementation or NPC1 inhibition facilitate acinar-to-ductal metaplasia (ADM) both ex vivo and in vivo, in an mTORC1-dependent manner. These results identify a metabolic/signaling axis driving the reprogramming of pancreatic epithelial cells in response to inflammation. This hinges on a nutrient sensing paradigm, previously documented exclusively in pathological conditions.
    Keywords:  acinar-to-ductal metaplasia (ADM); cholesterol; lysosome; mTORC1; pancreatitis
    DOI:  https://doi.org/10.1016/j.molmet.2025.102292
  53. Nat Methods. 2025 Dec 04.
    C-COMPASS: a user-friendly neural network tool profiles cell compartments at protein and lipid levels.
    Daniel T Haas, Daniel Weindl, Pamela Kakimoto, Eva-Maria Trautmann, Julia P Schessner, Xia Mao, Mathias J Gerl, Maximilian Gerwien, Timo D Müller, Christian Klose, Xiping Cheng, Jan Hasenauer, Natalie Krahmer.
      Systematic proteomic organelle profiling methods including protein correlation profiling and LOPIT have advanced our understanding of cellular compartmentalization. To manage the complexity of organelle profiling data, we introduce C-COMPASS, a user-friendly open-source software that employs a neural network-based regression model to predict the spatial cellular distribution of proteins. C-COMPASS handles complex multilocalization patterns and integrates protein abundance to model organelle composition changes across conditions. We apply C-COMPASS to mice with humanized livers to elucidate organelle remodeling during metabolic perturbations. Additionally, by training neural networks with co-generated marker protein profiles, C-COMPASS extends spatial profiling to lipids, overcoming the lack of organelle-specific lipid markers, allowing for determination of localization and tracking of lipid species across different compartments. This provides integrated snapshots of organelle lipid and protein compositions. Overall, C-COMPASS offers an accessible tool for multiomic studies of organelle dynamics without needing advanced computational skills, empowering researchers to explore new questions in lipidomics, proteomics and organelle biology.
    DOI:  https://doi.org/10.1038/s41592-025-02880-3
  54. Cell Rep. 2025 Dec 02. pii: S2211-1247(25)01409-3. [Epub ahead of print]44(12): 116637
    NEMO recruitment at single cytokine-receptor complexes shows quantized dynamics independent of ligand affinity.
    A Hyun Kim, Benjamin Krummenacher, Jason Yeung, David R Koes, Robin E C Lee.
      Cells use a limited number of receptors to sense and process molecular information from their environment. In the classical view of signaling, receptor-ligand affinities determine binding kinetics, on timescales of diffusion, where their time-averaged contact duration regulates rapid cytoplasmic signaling events to coordinate cellular responses. For some cytokines, single receptor-ligand binding events can initiate large multiprotein complexes in the cytoplasm that assemble over tens of minutes, raising the question of how cytokine affinity influences the sensitivity and strength of signaling. Here, we leverage naturally occurring variations of interleukin (IL)-1β from multiple species to determine the impact of affinity on human IL-1 receptor signaling. Using experiments and models, we investigate single receptor complexes activated by ligands that vary across multiple orders of magnitude in affinity. Our results show that while the receptor-ligand affinity establishes cytokine response sensitivity, activated IL-1 receptor complexes signal as discrete, quantized packets of signaling flux independent of affinity.
    Keywords:  CP: cell biology; CP: molecular biology; IL-1β; Il-1R1; affinity; cross-species protein interaction; cytokine structure; live-cell imaging; receptor signaling; signal quantization; single particle tracking
    DOI:  https://doi.org/10.1016/j.celrep.2025.116637
  55. J Clin Invest. 2025 Dec 04. pii: e196495. [Epub ahead of print]
    Non-canonical functions of UGT2B17 promote castrate-resistant prostate cancer progression.
    Tingting Feng, Ning Xie, Lin Gao, Qiongqiong Jia, Sonia Kung, Tunc Morova, Yinan Li, Lin Wang, Ladan Fazli, Louis Lacombe, Chantal Guillemette, Eric Lévesque, Nathan A Lack, Jianfei Qi, Bo Han, Xuesen Dong.
      Androgen deprivation therapy is the primary treatment for advanced prostate tumors. While initially effective, tumor progression to the therapy-resistant stage is inevitable. Paradoxically, UDP-glucuronosyltransferase 2B17 (UGT2B17), the key enzyme responsible for androgen catabolism in prostate tumor cells, is upregulated in therapy-resistant tumors, though its role in tumor progression remains unclear. Here, we demonstrate that UGT2B17 possesses multiple oncogenic functions independent of androgen catabolism. It modulates protein-folding pathways, allowing tumor cells to endure therapy-induced stress. UGT2B17 also regulates transcription associated with cell division and the DNA damage response, enabling unchecked cell proliferation. Targeting the newly identified UGT2B17 functions using a combination of inhibitors reduces tumor growth in therapy-resistant tumor models, highlighting a promising therapeutic strategy. Collectively, these findings reveal a mechanism by which prostate tumors exploit UGT2B17 to evade therapy and highlight its potential as a therapeutic target in advanced prostate cancer.
    Keywords:  Cell stress; Clinical Research; Oncology; Prostate cancer; Protein misfolding
    DOI:  https://doi.org/10.1172/JCI196495
  56. PLoS Comput Biol. 2025 Dec;21(12): e1013784
    Benchmarking interpretability of deep learning for predictive genomics: Recall, precision, and variability of feature attribution.
    Justin Reynolds, Chongle Pan.
      Deep neural networks can model the nonlinear architecture of polygenic traits, yet the reliability of attribution methods to identify the genetic variants driving model predictions remains uncertain. We introduce a benchmarking framework that quantifies three aspects of interpretability: attribution recall, attribution precision, and stability, and apply it to deep learning models trained on UK Biobank genotypes for standing height prediction. After quality control, feed-forward neural networks were trained on more than half a million autosomal variants from approximately 300 thousand participants and evaluated using four attribution algorithms (Saliency, Gradient SHAP, DeepLIFT, Integrated Gradients) with and without SmoothGrad noise averaging. Attribution recall was assessed using synthetic spike-in variants with known additive, dominant, recessive, and epistatic effects, enabling direct measurement of sensitivity to diverse genetic architectures. Attribution precision estimated specificity using an equal number of null decoy variants that preserved allele structure while disrupting genotype-phenotype correspondence. Stability was measured by the consistency of variant-level attributions across an ensemble of independently trained models. SmoothGrad increased average recall across effect types by approximately 0.16 at the top 1% of the most highly attributed variants and improved average precision by about 0.06 at the same threshold, while stability remained comparable with median relative standard deviations of 0.4 to 0.5 across methods. Among the evaluated attribution methods, Saliency achieved the highest composite score, indicating that its simple gradient formulation provided the best overall balance of recall, precision, and stability.
    DOI:  https://doi.org/10.1371/journal.pcbi.1013784
  57. FASEB J. 2025 Dec 15. 39(23): e71286
    The Role of STING-Mediated Activation of the Integrated Stress Response in Inflammation-Induced Depletion of Ovarian Reserve.
    Qiumin Wang, You Wu, Xiaotong Dong, Yuxiu Wu, Yu Dai, Yongzhi Cao, Yanbo Du, Hong Lv, Keke Wei, Lianbao Cao, Lei Yan.
      Inflammatory responses within the ovarian microenvironment are increasingly recognized as significant disruptors of ovarian function, yet their specific effects on early follicular development, particularly the activation of primordial follicles, remain poorly understood. In this study, we employ a mouse model of transient lipopolysaccharide (LPS)-induced inflammation to mimic the inflammatory conditions associated with chronic pelvic inflammatory disease (PID) and systemic infections. We demonstrate that LPS stimulation triggers the premature activation of primordial follicles, leading to a depletion of the ovarian reserve. This finding underscores the detrimental impact of inflammation on ovarian health. However, we also identify a protective mechanism mediated by the cGAS-STING pathway, a central regulator of innate immunity and cellular stress responses. Activation of the cGAS-STING pathway effectively inhibits LPS-induced primordial follicle activation, thereby preserving ovarian function. Further mechanistic investigations reveal that the Integrated Stress Response (ISR), a downstream effector of STING signaling, plays a critical role in this protective process. The ISR, orchestrated by kinases such as PERK, modulates cellular homeostasis under stress conditions by phosphorylating eukaryotic initiation factor 2α (eIF2α). Our data show that STING activation induces ISR signaling, which in turn suppresses the overactivation of primordial follicles. To explore the therapeutic potential of this pathway, we utilized STING and ISR agonists, which successfully mitigated LPS-induced primordial follicle activation and preserved ovarian reserve in our experimental model. These findings highlight the dual role of inflammation in ovarian biology: while acute inflammatory stimuli can disrupt follicular quiescence, the cGAS-STING-ISR axis serves as a critical regulatory network to counteract these adverse effects. Our study not only elucidates the molecular mechanisms underlying inflammation-induced ovarian dysfunction but also identifies STING and ISR as promising therapeutic targets for preserving ovarian reserve in women exposed to chronic inflammatory conditions. These insights have significant clinical implications, offering potential strategies to protect ovarian function in patients with inflammatory diseases or those undergoing treatments that compromise ovarian health.
    DOI:  https://doi.org/10.1096/fj.202502370RR
  58. Methods Enzymol. 2025 ;pii: S0076-6879(25)00404-5. [Epub ahead of print]725 77-103
    An smFRET assay to probe the impact of antibiotics on intersubunit rotation in eukaryotic ribosomes.
    Amy K Grove, Ananya Das, Hironao Wakabayashi, Aleksandr V Ivanov, Dmitri N Ermolenko.
      Protein translation involves large-scale structural rearrangements within the ribosome. During translation elongation, the two subunits of the ribosome rotate with respect to each other to allow for tRNA-mRNA translocation. While conformational changes of bacterial ribosomes have been extensively explored using cryo-EM and single-molecule microscopy, structural dynamics of eukaryotic ribosomes are less well studied. Here we describe procedures for fluorescent labeling, assembly of elongation complexes and real-time observation of intersubunit rotation in yeast ribosomes using single-molecule Förster resonance energy transfer. We demonstrate that this assay can be used to interrogate mechanisms by which translation inhibitors perturb protein synthesis and alter ribosome structural dynamics.
    Keywords:  Cycloheximide; Intersubunit rotation; Yeast ribosome; smFRET
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.012
  59. bioRxiv. 2025 Nov 19. pii: 2025.11.19.689251. [Epub ahead of print]
    LRRK2 integrates Rab and GABARAP interactions to sense and respond to distinct lysosomal stresses.
    Devin Clegg, Amanda Bentley-DeSousa, Agnes Roczniak-Ferguson, Shawn M Ferguson.
      Increased activity of leucine-rich repeat kinase 2 (LRRK2) is an important risk factor for Parkinson's disease. LRRK2 localizes to lysosomal membranes, and changes in lysosome physiology are emerging as key regulators of its activation, yet the mechanisms by which distinct perturbations engage this kinase remain unclear. Analysis of osmotic and membrane-integrity challenges revealed that LRRK2 integrates multiple upstream cues through parallel interactions with Rab GTPases and GABARAP. Manipulations that caused lysosome enlargement, including inhibition of PIKfyve, showed that osmotic swelling leads to the accumulation of multiple Rabs on lysosomes and Rab-dependent LRRK2 activation independently of GABARAP. In contrast, under conditions of lysosome deacidification, CASM-dependent lipidation of GABARAP creates a platform that cooperates with Rabs in LRRK2 activation. These findings demonstrate how LRRK2 interprets perturbations of lysosome function through a combination of Rab- and GABARAP-dependent mechanisms, providing a framework for understanding both normal physiological regulation and pathological dysregulation in Parkinson's disease.
    Significance Statement: This study reveals how LRRK2 integrates lysosomal stress signals through coordinated interactions with Rab GTPases and GABARAP. Osmotic swelling drives strong Rab-dependent activation, whereas deacidification requires CASM-mediated GABARAP lipidation as a scaffold for LRRK2 activation at lysosomes. These results define how LRRK2 activation at lysosomes is tuned across physiological and pathogenic contexts.
    DOI:  https://doi.org/10.1101/2025.11.19.689251
  60. Gut Microbes. 2025 Dec 31. 17(1): 2595775
    The ubiquitin system targets translocated EspH to proteasomal degradation.
    Ipsita Nandi, Efrat Zlotkin-Rivkin, Hanan Schoffman, Benjamin Aroeti.
      EspH is an effector protein secreted by the type III secretion system of various pathogenic Escherichia coli strains, including enteropathogenic E. coli (EPEC). The ability of EspH to inhibit host RhoGTPases, disrupt the actin cytoskeleton, and induce host cell cytotoxicity has been well-documented. Mass spectrometry analysis of EspH translocated into EPEC-infected cells revealed that a lysine at position 106 (K106) is modified with ubiquitin. Immunoblotting using the FK2 anti-ubiquitin antibodies has confirmed these results, suggesting that EspH undergoes polyubiquitylation. Prediction algorithms have identified a single ubiquitylation site at K106 and a phosphodegron in EspH. Moreover, we show that wild-type (EspHwt), but not the EspHK106R mutant, is subjected to degradation following translocation in an MG132-sensitive manner, indicating that the proteasome degrades the polyubiquitylated effector following translocation. Finally, we show that translocated EspHK106R induces higher cytotoxicity than translocated EspHwt. EspHwt translocated into MG132-pretreated cells also displayed higher cytotoxicity levels than EspHwt in untreated cells. These data reinforce the idea that EspH is polyubiquitylated and that the host proteasome degrades the translocated effector, possibly limiting its ability to toxicate the host cells. Additional implications of these effects on bacterial-host interactions are discussed.
    Keywords:  Enteropathogenic E. coli (EPEC); EspH; effector polyubiquitylation; effector post-translational modifications; host-pathogen interactions; mass spectrometry; proteasomal degradation; type III secreted effectors
    DOI:  https://doi.org/10.1080/19490976.2025.2595775
  61. Cell. 2025 Nov 28. pii: S0092-8674(25)01251-6. [Epub ahead of print]
    Innate immune and metabolic signals induce mitochondria-dependent membrane lysis via mitoxyperiosis.
    Yaqiu Wang, Jianlin Lu, Alexandre F Carisey, Sangappa B Chadchan, Ha Won Lee, R K Subbarao Malireddi, Bhesh Raj Sharma, Nagakannan Pandian, Rebecca E Tweedell, Gustavo Palacios, Nathalie Becerra Mora, Camenzind G Robinson, Aaron Pitre, Peter Vogel, Taosheng Chen, Michael P Murphy, Thirumala-Devi Kanneganti.
      The combination of innate immune activation and metabolic disruption plays critical roles in many diseases, often leading to mitochondrial dysfunction and oxidative stress that drive pathogenesis. However, mechanistic regulation under these conditions remains poorly defined. Here, we report a distinct lytic cell death mechanism induced by innate immune signaling and metabolic disruption, independent of caspase activity and previously described pyroptosis, PANoptosis, necroptosis, ferroptosis, and oxeiptosis. Instead, mitochondria undergoing BAX/BAK1/BID-dependent oxidative stress maintained prolonged plasma membrane contact, leading to local oxidative damage, a process we termed mitoxyperiosis. This process then caused membrane lysis and cell death, termed mitoxyperilysis. mTORC2 regulated the cell death, and mTOR inhibition restored cytoskeletal activity for lamellipodia to retract and mobilize mitochondria away from the membrane, preserving integrity. Activating this pathway in vivo regressed tumors in an mTORC2-dependent manner. Overall, our results identify a lytic cell death modality in response to the synergism of innate immune signaling and metabolic disruption.
    Keywords:  carbon starvation; cytokine; inflammasome; inflammatory cell death; innate immunity; mTOR; metabolism; mitochondria; oxidative damage; tumor
    DOI:  https://doi.org/10.1016/j.cell.2025.11.002
  62. Research (Wash D C). 2025 ;8 1022
    DTBind: A Mechanism-Driven Deep Learning Framework for Accurate Prediction of Drug-Target Molecular Recognition.
    Qiuyu Li, Zeyu Xu, Yanhao Zhu, Wanyun Zhou, Mingming Guan, Shiqing Zhao, Mengyuan Liu, Bin Liu, Juntao Liu.
      Accurate prediction of drug-target molecular recognition is essential for early-stage drug discovery, spanning binding occurrence, binding site localization, and binding affinity estimation. However, current approaches frequently treat these tasks independently, thereby overlooking the shared mechanistic principles that underlie them. We present DTBind, a unified and mechanism-driven framework that hierarchically adapts to sequence, structure, and complex-level inputs for predicting binding occurrence, site, and affinity, grounded in the shared mechanistic determinants of molecular recognition. Comprehensive benchmarking demonstrates that DTBind achieves superior performance over state-of-the-art methods in terms of accuracy and generalizability. Analysis of hierarchical protein encodings demonstrates that determinant-based protein representations gradually drive the accurate prediction of molecular recognition. Validation based on molecular dynamics simulations shows that DTBind reliably predicts drug-target binding regions for 3 famous proteins that lack experimental structures.
    DOI:  https://doi.org/10.34133/research.1022
  63. Adv Sci (Weinh). 2025 Nov 30. e10780
    Astrocytic PERK Deficiency Drives Prefrontal Circuit Dysfunction and Depressive-Like Behaviors.
    Kai Chen, Riya Gupta, Yosuke M Morizawa, Yu Qin, Xingyu Du, Cynthia Pang, Osama Al-Dalahmah, Maura B Dupont, Guang Yang.
      Major depressive disorder (MDD) is associated with dysfunction in prefrontal cortex (PFC) circuits, yet the glial mechanisms underlying these abnormalities remain unclear. Here, downregulation of the endoplasmic reticulum (ER) stress sensor PERK in PFC astrocytes is identified as a mechanistic contributor to depression-related phenotypes. PERK expression is markedly reduced in PFC astrocytes from individuals with MDD and in two chronic-stress mouse models. Astrocyte-specific PERK deletion in stress-naïve mice is sufficient to induce robust depressive-like behaviors and widespread PFC circuit pathology, including dendritic spine loss, pyramidal neuron hypoactivity, and weakened functional connectivity. Mechanistically, PERK-deficient astrocytes display reduced Nrf2 abundance, dysregulated ER and cytosolic Ca2+ dynamics, and decreased expression of the synaptogenic protein thrombospondin-1 (TSP1). Restoring astrocytic TSP1 via a blood-brain barrier-penetrant adeno-associated virus rescues PFC circuit function and reverses depressive-like behaviors. These findings establish astrocytic PERK deficiency as a sufficient driver of synaptic and network dysfunction underlying depressive phenotypes and highlight astrocyte-directed TSP1 augmentation as a potential therapeutic strategy for MDD.
    Keywords:  Nrf2; PERK; TSP1; astrocyte; depression; gene therapy; prefrontal cortex; synaptic plasticity
    DOI:  https://doi.org/10.1002/advs.202510780
  64. Cell Calcium. 2025 Nov 30. pii: S0143-4160(25)00108-3. [Epub ahead of print]133 103100
    The handshake between the continuous remodeling of the endoplasmic reticulum and the store-operated calcium entry.
    Alicia Sampieri, Alexander Asanov, Aaron Pavel Rodríguez-Hernández, Ileana Tobías-Juárez, Luis Vaca.
      The two main components of store-operated calcium entry (SOCE) are the ion channel Orai1 and the multifunctional protein STIM1. Recently STIM1 has been recognized as a microtubule plus end tracking protein involved in remodeling of the endoplasmic reticulum. However, the relationship between ER remodeling and SOCE are poorly understood. In the present study we have found that activation of Orai1 channels by the expression of the SOAR (STIM1 Orai activating region) from STIM1 induces Orai1 puncta formation and SOCE but does not alter the role of STIM1 in ER remodeling. Furthermore, expression of the STIM1-EB1-D (the STIM1-EB1 association domain) from STIM1 significantly reduces SOCE and prevents the traveling of STIM1 in ER projections. However, ER projections driven by EB1 remained unaffected. These findings suggest that there may be several mechanisms responsible for ER remodeling but only one in which STIM1 participates. This type of ER remodeling mechanism where STIM1 plays an active role participates in the control of SOCE.
    Keywords:  EB1; ER remodeling; Orai1; SOCE; STIM1
    DOI:  https://doi.org/10.1016/j.ceca.2025.103100
  65. Mol Cell. 2025 Dec 01. pii: S1097-2765(25)00902-5. [Epub ahead of print]
    Attenuation of ATM signaling by ROS delays replicative senescence at physiological oxygen.
    Alexander J Stuart, Kaori K Takai, Railia R Gabbasova, Henry Sanford, Ekaterina V Vinogradova, Titia de Lange.
      Replicative senescence is a powerful tumor suppressor pathway that curbs proliferation of human cells when a few critically-short telomeres activate the DNA damage response (DDR). We show that ATM is the sole DDR kinase responsible for the induction and maintenance of replicative senescence and that ATM inhibition can induce normal cell divisions in senescent cells. Compared to non-physiological atmospheric (∼20%) oxygen, primary fibroblast cells grown at physiological (3%) oxygen were more tolerant to critically short telomeres, explaining their extended replicative lifespan. We show that this tolerance is due to attenuation of the ATM response to double-strand breaks (DSBs) and unprotected telomeres. Our data indicate that the reduced ATM response to DSBs at 3% oxygen is due to increased ROS, which induces disulfide crosslinked ATM dimers that do not respond to DSBs. This regulation of cellular lifespan through attenuation of ATM at physiological oxygen has implications for tumor suppression through telomere shortening.
    Keywords:  ATM; DDR; Hayflick; ROS; TRF2; cancer; hypoxia; oxygen; senescence; telomere
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.006
  66. J Cell Sci. 2025 Nov 15. pii: jcs264222. [Epub ahead of print]138(22):
    Clathrin-mediated endocytosis in budding yeast at a glance: animated.
    Leanna Owen, Margot Riggi, David G Drubin, Janet H Iwasa, Yidi Sun.
      Clathrin-mediated endocytosis (CME) is an essential, highly conserved process in eukaryotic cells that facilitates the internalization of plasma membrane components, transmembrane proteins and extracellular nutrients. This complex pathway involves the concerted assembly and disassembly of many different proteins at the plasma membrane. Budding yeast has served as a powerful model for dissecting CME through combined genetic, biochemical, quantitative imaging and mathematical approaches. In this Cell Science at a Glance article, we integrate decades of quantitative data to generate a three-dimensional molecular animation depicting the full progression of CME in budding yeast (Movie 1). The animation and accompanying poster capture the spatial and temporal dynamics of key protein players. In addition, we highlight recent advances in understanding of the condensation of endocytic proteins into distinct sites and the organization of actin networks that generate the forces necessary to deform and internalize the membrane against the high internal turgor pressure of the budding yeast cell.
    Keywords:   Saccharomyces cerevisiae ; 3D molecular animation; Actin; Clathrin-mediated endocytosis; Condensation; Force generation
    DOI:  https://doi.org/10.1242/jcs.264222
  67. Cell Metab. 2025 Dec 02. pii: S1550-4131(25)00487-5. [Epub ahead of print]37(12): 2301-2302
    Microbial metabolites shape mammalian protein translation.
    Francesca Tuorto, Frank Lyko.
      Eukaryotic queuosine tRNA modification depends on the intake of microbial queuine. In a recent issue of Nature Cell Biology, Zhang et al. show that pre-queuosine 1 (preQ1), an intermediate of the bacterial queuine biosynthesis pathway, competes with queuine for the same mammalian tRNA targets to antagonistically modulate protein translation and cell proliferation.
    DOI:  https://doi.org/10.1016/j.cmet.2025.11.003
  68. PLoS Comput Biol. 2025 Dec 01. 21(12): e1013773
    Using evolutionary context to classify difficult protein folds.
    Jimin Pei, R Dustin Schaeffer, Qian Cong, Nick V Grishin.
      Recent advances in protein structure prediction, such as AlphaFold2, have enabled identification of vast numbers of putative novel protein domains across the sequence space, many of which adopt structures dissimilar to known folds. Based on structural segmentation and classification, the Encyclopedia of Domains (TED) project recently cataloged more than 7400 low-symmetry, structure-based domains as candidate novel-fold (CNF) domains. To place these domains in their broader evolutionary and structural context, we applied DPAM (Domain Parser for AlphaFold Models), a complementary method that combines AlphaFold-derived confidence metrics with sensitive sequence and structure similarity searches, to parse domains for the AlphaFold models of proteins containing TED CNF domains. We identified 8044 DPAM domains with significant overlap with TED CNF domains, among which 2490 were confidently assigned to entries in the ECOD (Evolutionary Classification of protein Domains) structural classification hierarchy. Our results suggest that a substantial subset of TED candidate novel-fold domains are distant homologs of existing ECOD domains. Comparison of domain boundaries between TED and DPAM showed varied patterns: more than one-third of cases featured TED CNF domains largely embedded within DPAM domains-often representing insertions or extensions into enzymatic or repeat folds. A smaller fraction (17%) exhibited consistent domain boundaries between TED and DPAM. These consistently defined domains are often characterized by significant structural diversity, including long insertions and duplications. An even smaller subset showed the reverse relationship, with DPAM domains largely embedded within TED CNF domains. In these cases, DPAM effectively separated multiple structural units that TED grouped as single domains. Together, these findings highlight the complementarity of structural and evolutionary approaches for domain annotation and demonstrate the power of integrative methods, such as DPAM, in refining the classification of challenging protein folds and uncovering distant evolutionary relationships.
    DOI:  https://doi.org/10.1371/journal.pcbi.1013773
  69. Nat Struct Mol Biol. 2025 Dec 03.
    Translational activators align mRNAs at the small mitoribosomal subunit for translation initiation.
    Joseph B Bridgers, Andreas Carlström, Dawafuti Sherpa, Mary T Couvillion, Urška Rovšnik, Jingjing Gao, Bowen Wan, Sichen Shao, Martin Ott, L Stirling Churchman.
      Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In Saccharomyces cerevisiae, nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats using selective mitoribosome profiling and cryo-electron microscopy (cryo-EM) structural analysis. These analyses show that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicate that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to mitoribosomes stalled in the post-initiation, pre-elongation state revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' untranslated region of the client mRNA and the mRNA channel exit to align the mRNA in the small subunit during initiation. Our findings provide a mechanistic basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position mitoribosomes at start codons.
    DOI:  https://doi.org/10.1038/s41594-025-01726-y
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