bims-proteo Biomed News
on Proteostasis
Issue of 2025–09–28
sixty-four papers selected by
Eric Chevet, INSERM
  1. Autophagy in Proteostasis AND AGING in C. ELEGANS.
  2. Quantitative insights into protein turnover and ubiquitination with HiBiT and NanoBRET.
  3. Harnessing indole scaffolds to identify small-molecule IRE1α inhibitors modulating XBP1 mRNA splicing.
  4. Identification of the Ac/N-degron recognition domain in the MARCHF6 E3 ubiquitin ligase.
  5. Decoding the Impact of Lipid Saturation on ER Signaling Networks, ERSU, UPR, and ERAD.
  6. DEGRONOPEDIA: A practical guide to identifying and targeting protein degrons.
  7. PKR-driven ISR signaling controls synaptic translation and structural plasticity in an age-dependent manner.
  8. Depurination of sarcin/ricin loop 25S rRNA is signaled through the small ribosomal subunit during translation.
  9. A Leishmania virulence factor harnesses an allosteric kinase switch to regulate its ubiquitin ligase activity.
  10. GCN2 enhances host survival and drives eIF2α phosphorylation during mouse adenovirus type 1 infection.
  11. Profiling of terminating ribosomes reveals translational control at stop codons.
  12. Structural basis for L-isoaspartyl-containing protein recognition by the human PCMTD1 cullin-RING E3 ubiquitin ligase.
  13. Ubiquitination-activated TAB-TAK1-IKK-NF-κB axis modulates gene expression for cell survival in the lysosomal damage response.
  14. PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules independent of its kinase activity.
  15. The shutdown of food digestion due to endoplasmic reticulum homeostasis disruption acts as a protective mechanism in C. elegans.
  16. Increased expression of ATase1/NAT8B or ATase2/NAT8 in the mouse results in an autistic-like phenotype with altered dendritic branching and spine formation.
  17. Ageing-related decline of translation as a consequence of transcription dysregulation.
  18. Endoplasmic reticulum stress in lung cancer.
  19. Predicting protein-protein interactions in the human proteome.
  20. V-ATPase-dependent induction of selective autophagy.
  21. Hsp70 and Hsp90 post-translational modifications and translating the chaperone code.
  22. TRIM52 ubiquitin ligase acts as a key recognition component of the mammalian fMet/N-degron pathway.
  23. Steric repulsion counteracts ER-to-lipid droplet protein movement.
  24. "Design principles of a membrane-spanning ubiquitin ligase".
  25. IRE1/Xbp1 promotes the clearance of poly(GR) dipeptide repeats in Amyotrophic Lateral Sclerosis.
  26. Cell-Selective Targeting Chimeras (SelecTACB) for Membrane Protein Degradation on B Cells.
  27. Proteome analysis of puromycin-labeled nascent polypeptides.
  28. USP13 stabilizes NLRP3 to facilitate inflammasome activation by preventing TRIM31-mediated NLRP3 ubiquitination and degradation.
  29. Principles of protein abundance regulation across single cells in a mammalian tissue.
  30. Intrinsically disordered cytoplasmic tail in netrin receptor DCC binds the large ribosomal subunit to inhibit translation.
  31. Unfolding the potential-chemical chaperones in Alport syndrome.
  32. Rapid remodeling of NTP levels enables immediate translational adaptation to energy stress in yeast.
  33. The ubiquitin ligase Nedd4-2 promotes localization of DNMBP/Tuba to P-bodies under hyperosmotic stress.
  34. The SARS-CoV-2 spike protein interacts with HAX1 to modulate cellular stress responses through activation of the UPR.
  35. Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae.
  36. Indolequinone-Based Hypoxia-Activated Proteolysis Targeting Chimeras Selectively Degrade BRD4 in Hypoxic Cancer Cells.
  37. DHX36 modulates stress granule assembly independent of recruitment of mRNAs with G-quadruplex sequence motifs.
  38. HSP90 Mediates Targeted Degradation of Nonclient Protein PARP1 for Breast Cancer Treatment.
  39. Spatiotemporal dynamics and selectivity of mRNA translation during mouse pre-implantation development.
  40. Intra-tumoral hypoxia promotes CD8+ T cell dysfunction via chronic activation of integrated stress response transcription factor ATF4.
  41. Organelle-specific signaling of cGAS-STING.
  42. Charcot-Marie-Tooth type 2A variants of mitofusin 2 sensitize cells to apoptotic cell death.
  43. An Enigma of N-termini dependent protein degradation.
  44. Disease-specific tau polymorphs define unique protein interaction networks across proteinopathies.
  45. Ubiquitin precursor with C-terminal extension promotes proteostasis and longevity.
  46. Ultrasmall Chemogenetic Tags with Group-Transfer Ligands.
  47. Characterization of the autophagic N-degron pathway and monitoring its chemical modulation for therapeutic development.
  48. GCN2-Mediated Integrated Stress Response Attenuates Periodontitis.
  49. JIP4 and RILPL1 utilize opposing motor force to dynamically regulate lysosomal tubulation.
  50. ribofootPrinter: A precision python toolbox for analysis of ribosome profiling data.
  51. Collective homeostasis of condensation-prone proteins via their mRNAs.
  52. Repeatability of protein structural evolution following convergent gene fusions.
  53. Design of facilitated dissociation enables timing of cytokine signalling.
  54. Protocol to set up the CellMinerCDB pharmacogenomics analysis web application with custom cell line data.
  55. Parental S-adenosylmethionine diet defines offspring immune response via histone H3K4me3 complex and Endoplasmic Reticulum UPR.
  56. Systematic Mapping of Protein Interactions Underlying IL-2 Secretion in Human T Cells.
  57. CRISPR screens identify the ATPase VCP as a druggable therapeutic vulnerability in cholangiocarcinoma.
  58. A diverse family of bacterial deubiquitinases is defined by the Coxiella burnetii effector EmcB.
  59. Collagen Prolyl Hydroxylases Regulate HIF-α Levels Independently of pVHL in ccRCC.
  60. UBE2O-mediated ubiquitylation directs cytoplasmic CTNNA1 to promote cell-to-ECM adhesions.
  61. A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality.
  62. TRPML1 signaling at lysosomes-mitochondria nexus drives triple-negative breast cancer mitophagy, metabolic reprogramming and chemoresistance.
  63. Hsp90 and associates shaping parasite biology.
  64. Diverse Conformational Ensembles Define the Shared Folding-Allosteric Landscapes of Protein Kinases.
  1. Cell Stress Chaperones. 2025 Sep 18. pii: S1355-8145(25)00060-4. [Epub ahead of print] 100115
    Autophagy in Proteostasis AND AGING in C. ELEGANS.
    Caitlin M Lange, Ryo Higuchi-Sanabria, Caroline Kumsta.
      Proteostasis (protein homeostasis), the balance of protein synthesis, folding, and degradation, is critical for cellular function and organismal health. Its disruption leads to the accumulation of misfolded and aggregated proteins, hallmarks of aging and age-related diseases including neurodegeneration. Autophagy, a conserved lysosome-mediated degradation pathway, is central to proteostasis by clearing toxic proteins and damaged organelles. In Caenorhabditis elegans, studies across conserved longevity paradigms and models of neurodegenerative diseases have defined key mechanisms by which autophagy maintains proteostasis during aging and stress. Beyond its degradative functions, autophagy contributes to spatial quality control by promoting the formation of potentially protective protein inclusions and coordinating with the ubiquitin-proteasome system. Emerging evidence also points to noncanonical autophagy pathways, such as unconventional secretion and inter-tissue communication, that broaden its role in systemic proteostasis. Together, these advances underscore autophagy's multifaceted contribution to protein quality control, with wide-ranging implications for aging, stress resistance and neurodegenerative disease.
    Keywords:  Aggresome; Aging; Autophagy; C. elegans; Inclusion body; Inter-tissue signaling; Longevity; Neurodegeneration; Protein Aggregation; Proteostasis; Secretion; Stress Response
    DOI:  https://doi.org/10.1016/j.cstres.2025.100115
  2. Methods Enzymol. 2025 ;pii: S0076-6879(25)00234-4. [Epub ahead of print]719 95-120
    Quantitative insights into protein turnover and ubiquitination with HiBiT and NanoBRET.
    Małgorzata Piechota, Wojciech Pokrzywa.
      Protein turnover and ubiquitination are fundamental processes regulating cellular homeostasis, proteostasis, and targeted protein degradation. Here, we present an optimized methodology for studying protein stability, ubiquitination dynamics, and degron function using HiBiT (11-amino-acid peptide tag) and NanoBRET (bioluminescence resonance energy transfer BRET) assays, specifically tailored for investigating Cullin-RING E3 ligase receptors, including von Hippel-Lindau (VHL) and FBXL15. HiBiT enables real-time quantification of protein abundance and degradation kinetics, making it ideal for CHX chase experiments and degron validation, while NanoBRET allows live-cell monitoring of ubiquitination events, facilitating the assessment of substrate ubiquitination efficiency and E3 ligase interactions. Given the relevance of protein termini in degradation, we leveraged HiBiT tagging to mask terminal degrons, enabling controlled analysis of N- and C-terminal degron function in protein stability. Using FBXL15 as a model, we demonstrate that degron accessibility significantly impacts turnover rates. Additionally, we applied NanoBRET to explore non-canonical ubiquitination mechanisms, using VHL as a case study to assess ubiquitination dynamics in a live-cell-compatible system. This study establishes HiBiT and NanoBRET as versatile tools for investigating Cullin-RING receptor stability and terminal degron function, providing a valuable methodological resource for dissecting protein turnover, ubiquitination pathways, and post-translational modifications in a quantitative and physiologically relevant manner.
    Keywords:  Cullin-RING ligases live-cell degradation assays; Degrons; HiBiT stability analysis; NanoBRET; Protein turnover; Ubiquitination dynamics
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.026
  3. Nat Commun. 2025 Sep 26. 16(1): 8531
    Harnessing indole scaffolds to identify small-molecule IRE1α inhibitors modulating XBP1 mRNA splicing.
    Yang Liu, Amrutha K Avathan Veettil, Raphael Gasper, Mao Jiang, Leon Wagner, Oguz Hastürk, Peng Wu.
      The inositol-requiring enzyme 1 alpha (IRE1α) is an important sensor protein with dual kinase and ribonuclease function. It induces X-box binding protein 1 (XBP1) mRNA splicing and mediates endoplasmic reticulum (ER) stress-triggered downstream unfolded protein response signaling pathways. The dysregulation of IRE1α has been associated with multiple human diseases, and thus IRE1α-targeting small molecules harbor great therapeutic potential. We herein report a series of substituted indoles as IRE1α inhibitors (such as IA107) of excellent potency and selectivity. We also report a resolved co-crystal structure that reveals a unique inhibition mode of IA107 that allosterically inhibits IRE1α RNase activity via binding to the IRE1α kinase domain but without inhibiting the IRE1α dimerization. The following cellular evaluation results demonstrate that IA107 concentration-dependently inhibits the cellular ER stress-induced XBP1 mRNA splicing, and the ester-containing prodrug exhibits a ~ 50-fold increase in cellular activity. Collectively, our results establish the indoles as a potent and selective IRE1α-inhibiting chemotype that modulates RNA splicing and expands the biological application potential associated with IRE1α targeting via small molecules.
    DOI:  https://doi.org/10.1038/s41467-025-64291-4
  4. Methods Enzymol. 2025 ;pii: S0076-6879(25)00235-6. [Epub ahead of print]719 237-257
    Identification of the Ac/N-degron recognition domain in the MARCHF6 E3 ubiquitin ligase.
    Jihye Yang, Cheol-Sang Hwang.
      Nα-terminal (Nt-) acetylation is a prevalent post-translational modification that regulates protein stability and turnover. The Ac/N-degron pathway, a branch of the N-degron pathways, recognizes Nt-acetyl groups as degradation signals (Ac/N-degrons), mediating proteolysis. MARCHF6, an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase, acts as a principal Ac/N-recognin, targeting Ac/N-degron-bearing substrates for polyubiquitylation and subsequent proteasomal degradation. However, the molecular mechanisms underlying Ac/N-degron recognition by MARCHF6 remain elusive. Here, we utilized a comprehensive alanine-stretch mutational screen combined with split-ubiquitin (Split-Ub) assays to define the Ac/N-degron recognition domain (Ac/N-domain) within MARCHF6. Sequence alignment with its yeast ortholog, Doa10, revealed conserved cytosolic residues essential for substrate recognition. Biochemical approaches, including chemical crosslinking and co-immunoprecipitation, identified key residues critical for Ac/N-degron recognition, while truncation and Split-Ub assays delineated the specific Ac/N-domain necessary for binding. These findings establish a mechanistic framework for Ac/N-degron recognition by MARCHF6, deepening our understanding of Nt-acetylation-mediated proteostasis and its therapeutical implications for diseases linked to dysregulation of Nt-acetylation or MARCHF6, including cancer, birth defects, and metabolic and neurological disorders.
    Keywords:  Ac/N-degron; Ac/N-domain; Endoplasmic reticulum; Ferroptosis; MARCHF6; Nα-terminal acetylation; Transmembrane protein
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.027
  5. bioRxiv. 2025 Sep 17. pii: 2025.09.15.676312. [Epub ahead of print]
    Decoding the Impact of Lipid Saturation on ER Signaling Networks, ERSU, UPR, and ERAD.
    Xia Li, Maho Niwa.
      The faithful inheritance of a functional endoplasmic reticulum (ER) in Saccharomyces cerevisiae is safeguarded by the ER Stress Surveillance (ERSU) checkpoint, which delays cytokinesis when ER homeostasis is perturbed. Under stress, ER transmission to the daughter cell is halted, while in parallel-but through independent pathways-the Unfolded Protein Response (UPR) restores ER function and ER-associated degradation (ERAD) eliminates misfolded proteins, ultimately allowing cell cycle re-entry. ER stress also transiently stimulates sphingolipid biosynthesis, with the intermediate phytosphingosine (PHS) acting as a key activator of ERSU. Yet, how broader lipid parameters-such as membrane composition, saturation, and fluidity-reshape ER quality control and, in particular, govern ER inheritance during division remains poorly understood. To address this, we employed a tightly controlled experimental system to selectively alter lipid saturation and phospholipid composition while monitoring ER inheritance within the framework of ER homeostasis maintained by UPR and ERAD. Strikingly, we found that perturbations in lipid balance exerted specific effects on ER inheritance that were distinct from their impact on UPR and ERAD. These findings reveal lipid homeostasis as a critical integrator of ER functional regulation, linking ERSU, UPR, and ERAD into a unified adaptive network that ensures robust ER transmission and cellular resilience under stress.
    DOI:  https://doi.org/10.1101/2025.09.15.676312
  6. Methods Enzymol. 2025 ;pii: S0076-6879(25)00222-8. [Epub ahead of print]719 67-94
    DEGRONOPEDIA: A practical guide to identifying and targeting protein degrons.
    Natalia A Szulc, Wojciech Pokrzywa.
      Degrons are recognition motifs mediating substrate binding to E3 ubiquitin ligases within the ubiquitin-proteasome system, driving protein ubiquitination and degradation. These motifs, located at protein N- and C-termini or within internal regions, are essential for maintaining proteostasis. Effective degradation relies on a tripartite architecture: a degron motif, a ubiquitination site, and a proteasomal unwinding seed. This chapter introduces DEGRONOPEDIA, a web server for identifying and predicting degrons across eukaryotic proteomes. It integrates machine learning, solvent accessibility modeling, and proteolysis simulations to analyze degrons in sequential and structural contexts. We provide detailed guidance on its workflow and applications, highlighting its role in studying terminal and internal degrons.
    Keywords:  Degron; E3 ligase; Protein degradation; Protein stability; Protein turnover; SLIMs; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.014
  7. Neurobiol Dis. 2025 Sep 19. pii: S0969-9961(25)00330-4. [Epub ahead of print] 107113
    PKR-driven ISR signaling controls synaptic translation and structural plasticity in an age-dependent manner.
    Nicolás W Martínez, Felipe Gómez, Ariel Tapia-Godoy, Juan Francisco Roa, Yuwei Liu, Claudia Jara, Cheril Tapia-Rojas, Iván Alfaro, Mauro Costa-Mattioli, Soledad Matus.
      The integrated stress response (ISR) modulates protein homeostasis in response to both intracellular and extracellular signals. The four kinases involved in the ISR all phosphorylate the same target, the alpha subunit of eukaryotic initiation factor 2 (eIF2a), to integrate various stress signals, thereby regulating cell fate. The activation of the ISR reprograms the proteome by inhibiting general protein synthesis while increasing the translation of specific mRNAs. In the brain, the ISR regulates the type of synaptic plasticity necessary for forming long-term memory. More importantly, the activation of the ISR has emerged as a causal mechanism underlying cognitive decline associated with a wide range of neurological disorders, prompting several pharmaceutical companies to target the ISR to promote brain health. However, whether the ISR acts at specific localities within neurons, including synapses, remains unclear. Here, we examined the presence, activity, and spatial arrangement of the ISR branch driven by the double-stranded RNA-dependent protein kinase (PKR) (PKR-eIF2a axis) in synapses and assessed the role of PKR in maintaining synaptic proteostasis over time. Our findings demonstrate that both PKR and eIF2a are localized at synapses, where a dynamic PKR-eIF2a axis regulates synaptic size and the abundance of synaptic proteins in an age-dependent manner. Moreover, PKR deficiency leads to an increase in protein synthesis in synapse-enriched fractions. Thus, the PKR branch of the ISR serves as a new regulator of synaptic structural plasticity.
    Keywords:  Neurons; PKR; PSD95; Proteostasis; Synapsin; eIF2a
    DOI:  https://doi.org/10.1016/j.nbd.2025.107113
  8. RNA. 2025 Sep 23. pii: rna.080559.125. [Epub ahead of print]
    Depurination of sarcin/ricin loop 25S rRNA is signaled through the small ribosomal subunit during translation.
    Tanya Prashar, Katalin A Hudak.
      In addition to their function in protein synthesis, translating ribosomes serve as sensors that communicate the presence of aberrant messenger RNAs (mRNAs); however, how they recognize damage to their ribosomal RNA (rRNA) remains poorly understood. The conserved sarcin/ricin loop (SRL) of the 25S rRNA is a component of the GTPase centre essential for ribosome movement during translation. In this study, we expressed an RNA N-glycosylase called pokeweed antiviral protein (PAP) in yeast Saccharomyces cerevisiae to specifically damage rRNA by hydrolysis of a purine base from the SRL. 25S rRNA depurination inhibited translation elongation, as shown by reduced incorporation of a methionine analog and binding of eukaryotic elongation factor 2 (eEF2) to ribosomes. PAP expression altered sucrose gradient profiles, increasing free subunits and 80S peaks and reducing polysomes without causing ribosome collisions. We discovered depurinated rRNA associated with 80S monosomes and polysomes, suggesting that cells would detect damage to rRNA during active translation. These ribosomes were ubiquitinated by E3 ligases Mag2 and Hel2, elements of the 18S non-functional rRNA decay (NRD) pathway involved in recognizing slow-moving ribosomes. Furthermore, mass spectrometry analysis revealed ubiquitination of ribosomal protein uS3, characteristic of 18S NRD. Even though the SRL is a component of the large ribosomal subunit, its depurination is signaled by ubiquitin ligases that recognize damage to the small subunit. We suggest that slow translation elongation is the factor that communicates SRL depurination to E3 ubiquitin ligases, which extends our understanding of how rRNA integrity is surveilled in yeast.
    Keywords:  Sarcin/ricin loop; nonfunctional ribosome decay; pokeweed antiviral protein; rRNA depurination; ribosome quality control
    DOI:  https://doi.org/10.1261/rna.080559.125
  9. Mol Cell. 2025 Sep 24. pii: S1097-2765(25)00743-9. [Epub ahead of print]
    A Leishmania virulence factor harnesses an allosteric kinase switch to regulate its ubiquitin ligase activity.
    Thornton J Fokkens, Elisa T Rauh, Madita Wolter, Heidi Sebald, Melina Mitnacht, Sofia Ainatzi, Sebastian Sprick, Lars Teschke, Nicole Eisenhuth, Jon M Huibregtse, Falk Butter, Henning Urlaub, Anna Karnkowska, Wieland Steinchen, Ulrike Schleicher, Christian J Janzen, Sonja Lorenz.
      Stringent control of ubiquitylation is a central requirement of signaling specificity in eukaryotes. Here, we discover a domain module integrating protein kinase and ubiquitin ligase domains within a single protein. This module is widespread across unicellular eukaryotic lineages and particularly conserved in Leishmania, the causative agents of major neglected tropical diseases with a strong therapeutic need. We reveal that a gene encoding the module, tetratricopeptide repeat (TPR)-kinase-ubiquitin ligase (TKUL), is essential for L. mexicana to sustain macrophage infections and that TKUL can cooperate with parasite heat shock protein 70 (HSP70) to modify unfolded proteins with degradative ubiquitin chains. Intriguingly, the homologous to E6AP C-terminus (HECT)-type ubiquitin ligase activity of TKUL requires its atypical kinase domain, with kinase autophosphorylation triggering activating conformational changes across the catalytic module. Consistent with the ligase domain harnessing the kinase domain for regulation, TKUL-driven ubiquitylation can allosterically be suppressed by small-molecule kinase inhibitors. Together, this work establishes an unprecedented allosteric coupling mechanism in the realms of phosphorylation and ubiquitylation.
    Keywords:  HDX-MS; HECT E3; HSP70; Ser/Thr kinase; TPR domain; X-ray crystallography; allostery; enzyme regulation; heat shock protein 70; hydrogen-deuterium exchange mass spectrometry; kinase inhibitor; phosphorylation; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.molcel.2025.09.002
  10. J Virol. 2025 Sep 24. e0128825
    GCN2 enhances host survival and drives eIF2α phosphorylation during mouse adenovirus type 1 infection.
    Luiza A Castro Jorge, Daniel F Edwards, Rosario Labastida, Danielle E Goodman, Estela A Pereira, Oded Foreman, Katherine R Spindler.
      The integrated stress response (ISR) is a cellular signaling pathway that reduces protein synthesis in the face of cellular stress, including viral infection. Two eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and general control nonderepressible 2 (GCN2), are commonly activated during viral infections. Mouse adenovirus type 1 (MAV-1) infection leads to a steep reduction of PKR levels by proteasomal degradation. We assayed whether GCN2, a sensor of amino acid starvation and UV damage, plays a role in the ISR to MAV-1 infection. There was more phosphorylated GCN2 in MAV-1-infected cells, and its activation was dependent on virus replication since UV-inactivated virus was not able to increase the phosphorylation of GCN2. Infected Eif2ak4tm1.2Dron mice (designated here Gcn2-/- mice) had lower survival than wild-type (WT) mice, but results indicated that this was not due to increased viral replication. Both Gcn2-/- and WT mice developed multifocal brain parenchymal microhemorrhages during infection. While Gcn2-/- animals had more lesions, their higher mortality is likely not due to the microhemorrhages alone. Cytokine RNA and protein assays of WT and Gcn2-/- mice only showed a difference for IL- β levels, which were higher in Gcn2-/- mice. Our results also indicate that of the two eIF2α kinases, PKR and GCN2, GCN2 is the primary inducer of phosphorylated-eIF2α during MAV-1 infection. GCN2 is thus antiviral and contributes to the host response to MAV-1 infection.IMPORTANCECells often respond to viral infection by activation of the host protein kinase R (PKR), part of the integrated stress response (ISR). We show that a second host protein kinase, general control nonderepressible 2 (GCN2), is activated by phosphorylation in response to mouse adenovirus type 1 (MAV-1) infection. Our results indicate GCN2 is antiviral: without it, the mortality in MAV-1-infected mouse is higher. Furthermore, the data show that GCN2, rather than PKR, is the main inducer of eIf2α phosphorylation (and thus the ISR) upon MAV-1 infection. This is consistent with PKR exerting antiviral effects in MAV-1 infections through a pathway independent of eIf2α phosphorylation.
    Keywords:  PKR; general control nonderepressible 2; integrated stress response; protein kinase R
    DOI:  https://doi.org/10.1128/jvi.01288-25
  11. bioRxiv. 2025 Sep 18. pii: 2025.09.16.676599. [Epub ahead of print]
    Profiling of terminating ribosomes reveals translational control at stop codons.
    Longfei Jia, Yuanhui Mao, Saori Uematsu, Xinyi Ashley Liu, Leiming Dong, Leonardo Henrique França de Lima, Shu-Bing Qian.
      Accurate termination of protein synthesis is paramount for the integrity of cellular proteome, but our understanding of the dynamics and fidelity of terminating ribosomes is far from complete. Here we establish profiling of terminating ribosomes in mammalian cells and report a wide range of ribosome pausing at individual stop codons. We identify a sequence motif upstream of the stop codon that contributes to termination pausing, which was confirmed by massively paralleled reporter assays. Unexpectedly, lack of termination pausing increases the chance of stop codon slippage, generating proteins with mixed C-terminal extensions. We demonstrate that the sequence-dependent termination pausing is a result of post-decoding mRNA scanning by the 3' end of 18S rRNA. We further observe tissue-specific termination pausing that correlates with the stoichiometry of Rps26, which constrains mRNA:rRNA interaction. Thus, termination pausing represents a translational signature associated with mRNA sequence contexts, ribosome heterogeneity, and cell type-specific translational control.
    DOI:  https://doi.org/10.1101/2025.09.16.676599
  12. J Biol Chem. 2025 Sep 18. pii: S0021-9258(25)02587-6. [Epub ahead of print] 110735
    Structural basis for L-isoaspartyl-containing protein recognition by the human PCMTD1 cullin-RING E3 ubiquitin ligase.
    Eric Z Pang, Boyu Zhao, Cameron Flowers, Elizabeth Oroudjeva, Jasmine B Winter, Vijaya Pandey, Michael R Sawaya, James Wohlschlegel, Joseph A Loo, Jose A Rodriguez, Steven G Clarke.
      A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of human PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.
    Keywords:  Aging; E3 ubiquitin ligase; S-adenosylmethionine (SAM); cryo-electron microscopy; mass spectrometry; protein complex; protein methylation; protein turnover
    DOI:  https://doi.org/10.1016/j.jbc.2025.110735
  13. Elife. 2025 Sep 24. pii: RP106901. [Epub ahead of print]14
    Ubiquitination-activated TAB-TAK1-IKK-NF-κB axis modulates gene expression for cell survival in the lysosomal damage response.
    Akinori Endo, Chikage Takahashi, Naoko Ishibashi, Yasumasa Nishito, Koji Yamano, Keiji Tanaka, Yukiko Yoshida.
      The lysosomal damage response is important for the maintenance of cellular homeostasis in human cells. Although the mechanisms underlying the repair and autophagic elimination of damaged lysosomes have been elucidated, the early signal transduction pathways and genes induced in response to lysosomal damage remain elusive. We performed transcriptome and proteome analyses and found that the TAB-TAK1-IKK-NF-κB axis is activated by K63-linked ubiquitin chains that accumulate on damaged lysosomes. This activates the expression of various transcription factors and cytokines that promote anti-apoptosis and intercellular signaling. The findings highlight the crucial role of ubiquitin-regulated signal transduction and gene expression in cell survival and cell-cell communication in response to lysosomal damage. The results suggest that the ubiquitin system is not only involved in the removal of damaged lysosomes by lysophagy, but also functions in the activation of cellular signaling for cell survival.
    Keywords:  NF-κB; TAB; TAK1; cell biology; human; lysosomal damage response; ubiquitin
    DOI:  https://doi.org/10.7554/eLife.106901
  14. Mol Biol Cell. 2025 Sep 24. mbcE25060277
    PERK protein kinase facilitates keratinocyte collective cell migration by engagement with cell adhesion molecules independent of its kinase activity.
    Miguel Barriera Diaz, Kirk A Staschke, Anthony J Baucum, Dan F Spandau, Ronald C Wek.
      Successful cutaneous wound healing requires re-epithelialization by keratinocytes using a coordinated migratory process called keratinocyte collective cell migration (KCCM). Environmental stresses such as wounding induce the Integrated Stress Response (ISR) initiated by protein kinases that phosphorylate the α subunit of eIF2 and mitigate translational control to alleviate stress damage. We previously reported that the ISR protein kinase GCN2 (EIF2AK4) facilitates KCCM via sustained phosphorylation of eIF2α and coordinated production of reactive oxygen species and amino acid transport. In this study, we show that a second ISR protein kinase PERK (EIF2AK3) also contributes to KCCM. PERK promotes KCCM by protein-protein interactions requiring the cytoplasmic portion of PERK but independent of its catalytic functions. To discern these PERK interactions, we used BioID proximity labeling, immunoprecipitation analyses, and immunofluorescence microscopy to show that PERK interacts with multiple cell adhesion and cytoskeletal complexes important for KCCM. PERK engages with the hemidesmosome proteins ITGA6, ITGB4, COLXVII, and the desmosome proteins JUP, DSG2, and DSG3. Loss of PERK disrupts expression and localization of these cell adhesion proteins, which alters keratinocyte morphology and increases cell-substrate and intercellular adhesions. Our results define an underappreciated scaffolding function for PERK involving cell adhesions that are critical for KCCM.
    DOI:  https://doi.org/10.1091/mbc.E25-06-0277
  15. Nat Commun. 2025 Sep 25. 16(1): 8417
    The shutdown of food digestion due to endoplasmic reticulum homeostasis disruption acts as a protective mechanism in C. elegans.
    YongJuan He, Yunqing Zhang, Huijuan Xie, Zhao Shan, Zongliu Hou, Bin Qi.
      Food digestion is essential for nutrient absorption, supporting protein synthesis and maintaining endoplasmic reticulum (ER) homeostasis. However, whether animals can sense ER stress and suppress digestion to reduce ER overload remains unclear. Here, we show that Caenorhabditis elegans can sense ER stress and shut down digestion as a protective response. Food intake activates the unfolded protein response in the ER, and loss of its central regulator, XBP-1, impairs digestion, highlighting the importance of ER homeostasis in food digestion. We identify FDR-1, a food-induced protein, as a key factor that promotes digestion by preserving ER balance through its interaction with DPY-11. Disruption of FDR-1 triggers the innate immune p38/PMK-1 pathway, leading to a protective shutdown of digestion to mitigate ER stress. These findings reveal an adaptive mechanism by which animals limit digestion under ER stress and suggest that modulating nutrient intake may offer therapeutic strategies for diseases related to ER dysfunction.
    DOI:  https://doi.org/10.1038/s41467-025-63712-8
  16. Mol Psychiatry. 2025 Sep 24.
    Increased expression of ATase1/NAT8B or ATase2/NAT8 in the mouse results in an autistic-like phenotype with altered dendritic branching and spine formation.
    Balagangadharan Kalimuthu, Haiyan Lu, Angelique Steenhagen, Qiping Dong, Mitchell Gray, Michael J Rigby, Andreas Endresen, Qiang Chang, Lingjun Li, Luigi Puglielli.
      Neurons heavily depend on the ability of the secretory pathway to deliver correctly folded polypeptides to the periphery of the cell for the assembly, maintenance, and normal functioning of synapses. The endoplasmic reticulum (ER) acetylation machinery has emerged as a novel branch of the more general ER quality control machinery. It regulates the positive selection of correctly folded nascent glycoproteins, thus ensuring the efficiency of the conventional secretory pathway. ER acetylation requires the activity of two ER-luminal acetylCoA:lysine acetyltransferases, ATase1/NAT8B and ATase2/NAT8. Both acetyltransferases depend on the influx of acetyl-CoA into the ER from the cytosol, which is ensured by the coordinated action of the citrate transporters, SLC25A1 and SLC13A5, and the ER acetyl-CoA transporter, AT-1. Gene duplication events affecting ATase1 and ATase2 are associated with rare disease phenotypes that include autism and intellectual disability with dysmorphism. Here, we generated mice with neuron-specific overexpression of human ATase1 or ATase2. The animals display autistic-like behaviors with altered synaptic plasticity, altered neuronal morphology, and altered synaptic structure and function. Mechanistic assessment demonstrates that widespread proteomic changes and altered dynamics of the secretory pathway underly the synaptic defects. The phenotype of ATase1 and ATase2 overexpressing mice is reminiscent of SLC25A1, SLC13A5 and AT-1 overexpressing models. Therefore, when taken together, our results support the argument that the intracellular citrate/acetyl-CoA pathway, with the ATases acting as the last output, is immediately connected to the pathogenesis of certain rare forms of autism spectrum disorder.
    DOI:  https://doi.org/10.1038/s41380-025-03228-1
  17. J R Soc Interface. 2025 Sep;22(230): 20250323
    Ageing-related decline of translation as a consequence of transcription dysregulation.
    Sebastian Pechmann.
      The ageing-related decline of translational fidelity disrupts cellular protein homeostasis, thus contributing to the onset of cancer and neurodegeneration. However, it remains unclear what alters speed and accuracy of translation at advanced age. Here, I show that the shift in translation kinetics upon ageing is systematic and a direct consequence of transcription deregulation. Computational modelling of ageing yeast and worm Riboseq data demonstrates that the loss of translational fidelity is independent of codon identity, tRNA abundances or the specificities of anticodon-codon interactions at the ribosome. Instead, large-scale transcriptional changes during ageing perturb the codon usage of the transcriptome, which at the systems level induces a dramatic remodelling and increase in ribosome collisions and stalling. Ribosome collisions in turn reduce control over translation elongation and effect an assimilation of codon translation rates. The presented results thus explain the ageing-related decline of translational fidelity, and provide important insights towards a systems-level understanding of ageing-related human diseases linked to mistranslation and protein homeostasis failure that are especially prevalent in the brain.
    Keywords:  ageing; codon usage; computational biology; stochastic modelling; translational fidelity
    DOI:  https://doi.org/10.1098/rsif.2025.0323
  18. Front Oncol. 2025 ;15 1550075
    Endoplasmic reticulum stress in lung cancer.
    Donghuan Zhang, Lanlan Lin, Hui Jin, Huajun Mao, Luying Wang, Wenwen Ma, Zhenghong Lao.
      Endoplasmic reticulum is the primary site of eukaryotic cells involved in biosynthesis, lipid metabolism, glucose metabolism, protein folding and secretion. Multiple factors in the tumor microenvironment may induce the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum and trigger endoplasmic reticulum (ER) stress. Adaptive mechanisms including unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) are activated in response to ER stress. Previous studies have revealed that ER stress may participate in epithelial mesenchymal transformation, apoptosis, metabolic regulation and drug resistance of lung cancer cells. Herein, we summarized the potential effects and regulatory mechanisms of ER stress on the biological process of lung cancer, which may provide scientific significance and clinical value for elucidating the adaptability of lung cancer cells under stress and developing novel targeted therapies.
    Keywords:  endoplasmic reticulum stress; lung cancer; targeted therapy; tumor microenvironment; unfolded protein response
    DOI:  https://doi.org/10.3389/fonc.2025.1550075
  19. Science. 2025 Sep 25. eadt1630
    Predicting protein-protein interactions in the human proteome.
    Jing Zhang, Ian R Humphreys, Jimin Pei, Jinuk Kim, Chulwon Choi, Rongqing Yuan, Jesse Durham, Siqi Liu, Hee-Jung Choi, Minkyung Baek, David Baker, Qian Cong.
      Protein-protein interactions (PPI) are essential for biological function. Coevolutionary analysis and deep learning (DL) based protein structure prediction have enabled comprehensive PPI identification in bacteria and yeast, but these approaches have had limited success for the more complex human proteome. We overcame this challenge by enhancing the coevolutionary signals with 7-fold deeper multiple sequence alignments harvested from 30 petabytes of unassembled genomic data and developing a new DL network trained on augmented datasets of domain-domain interactions from 200 million predicted protein structures. We systematically screened 200 million human protein pairs and predicted 17,849 interactions with an expected precision of 90%, of which 3,631 interactions were not identified in previous experimental screens. Three-dimensional models of these predicted interactions provide numerous hypotheses about protein function and mechanisms of human diseases.
    DOI:  https://doi.org/10.1126/science.adt1630
  20. Nat Commun. 2025 Sep 26. 16(1): 8508
    V-ATPase-dependent induction of selective autophagy.
    Yuxiang Huang, Dimitra Dialynaki, Yuchen Lei, Zhihai Zhang, Charles R Evans, Daniel J Klionsky.
      The general consensus is that the vacuolar-type H+-translocating ATPase (V-ATPase) is critical for macroautophagy/autophagy. However, there is a fundamental conundrum because follicular lymphoma-associated mutations in the V-ATPase result in lysosomal/vacuolar deacidification but elevated autophagy activity under nutrient-replete conditions and the underlying mechanisms remain unclear. Here, working in yeast, we show that V-ATPase dysfunction activates a selective autophagy flux termed "V-ATPase-dependent autophagy ". By combining transcriptomic and proteomic profiling, along with genome-wide suppressor screening approaches, we found that V-ATPase-dependent autophagy is regulated through a unique mechanism distinct from classical nitrogen starvation-induced autophagy. Tryptophan metabolism negatively regulates V-ATPase-dependent autophagy via two parallel effectors. On the one hand, it activates ribosome biogenesis, thus repressing the translation of the transcription factor Gcn4/ATF4. On the other hand, tryptophan fuels NAD+ de novo biosynthesis to inhibit autophagy. These results provide an explanation for the mutational activation of autophagy seen in follicular lymphoma patients.
    DOI:  https://doi.org/10.1038/s41467-025-63472-5
  21. Cell Stress Chaperones. 2025 Sep 19. pii: S1355-8145(25)00063-X. [Epub ahead of print] 100118
    Hsp70 and Hsp90 post-translational modifications and translating the chaperone code.
    Sarah J Backe, Jennifer A Heritz, Mehdi Mollapour.
      Molecular chaperones maintain proteostasis by assisting protein folding, stability, and activity. Heat shock protein 70 (Hsp70) and Hsp90 are ATP-dependent chaperones essential for protein quality control, signaling, and stress adaptation. Their activities are controlled not only by co-chaperones but also by dynamic post-translational modifications (PTMs). The review dissects phosphorylation, acetylation, methylation, ubiquitination, glycosylation, and other PTMs of Hsp70 and Hsp90 across systems. These PTMs regulate the ATPase activity, localization, and interactions of the molecular chaperones with major implications in health and disease. The term 'chaperone code' describes the PTMs landscape that fine tunes the chaperone function. This code governs client fate, drug sensitivity, and stress responses. Importantly, combinatorial PTMs introduce regulatory complexity and flexibility, especially in cancer, neurodegeneration, and inflammation. The crosstalk between various PTMs and feedback loops add new regulatory layers to chaperone function. Additionally, these PTMs impact the function of the clients that are central in regulating a specific cellular processes or pathways such as transcription, autophagy, metabolism, and immune regulation. These pathways are usually affected in different maladies such cancer, neurodegenerative, infectious and chronic diseases. Unlocking the chaperone code is essential for directing chaperone activity toward therapeutic benefit. This can be achieved by targeting enzymes that write, erase, or read the chaperone code therefore offering new therapeutic strategies.
    Keywords:  Molecular chaperone; O-GlcNAcylation; acetylation; cancer; chaperone code; co-chaperone; heat shock protein 70 (Hsp70) post-translational modification (PTM); heat shock protein 90 (Hsp90); phosphorylation
    DOI:  https://doi.org/10.1016/j.cstres.2025.100118
  22. J Mol Biol. 2025 Sep 19. pii: S0022-2836(25)00519-4. [Epub ahead of print] 169453
    TRIM52 ubiquitin ligase acts as a key recognition component of the mammalian fMet/N-degron pathway.
    Dasom Kim, Juhee Park, Sang-Yoon Kim, Jaehwan Sim, Tae Su Choi, Cheol-Sang Hwang.
      Eukaryotic translation can initiate with formylmethionine (fMet), generating N-terminally formylated proteins in mitochondria and, unexpectedly, in the cytosol. However, the specific mechanism for eliminating cytosolic fMet-bearing proteins has remained elusive. Here, we identify the E3 ubiquitin ligase TRIM52 as the key recognition component of the mammalian fMet/N-degron pathway. TRIM52 targets N-terminally formylated proteins, including TPD54 and SPTAN1, for proteasomal degradation. It recognizes the N-terminal fMet through an evolutionarily conserved acidic loop embedded between its bipartite RING domain. Structural modeling and mutagenesis identify Tyr148 in the acidic loop as critical for fMet recognition without intervening E3 ligase activity. TRIM52 depletion stabilizes fMet-bearing proteins, disrupts proteostasis, and induces caspase-3-dependent apoptosis-phenotypes rescued by enhancing deformylation. These findings establish TRIM52 as a dedicated sensor and effector of the mammalian fMet/N-degron pathway, linking N-terminal formylation to proteostasis and cell survival.
    Keywords:  Apoptosis; Formylmethionine; N-degron pathway; Proteolysis; TRIM52; Ubiquitin
    DOI:  https://doi.org/10.1016/j.jmb.2025.169453
  23. Sci Adv. 2025 Sep 26. 11(39): eadu6998
    Steric repulsion counteracts ER-to-lipid droplet protein movement.
    Alicia Damm, Mohyeddine Omrane, Ozren Stojanović, Bianca M Esch, Mehdi Zouiouich, Maxime Carpentier, Robin Klemm, Florian Fröhlich, Lionel Forêt, Abdou Rachid Thiam.
      Lipid droplets (LDs) are organelles with a neutral lipid core surrounded by a phospholipid monolayer, which is continuous with the cytoplasmic leaflet of the endoplasmic reticulum (ER). LD function depends on a highly dynamic LD surface proteome. Key proteins continuously exchange between the ER and LDs; however, the mechanisms governing the interorganelle movement and accumulation on the LD surface remain poorly understood. Here, we developed an ex cellulo tool introducing a classification of ER-derived proteins based on their different affinity for LDs. We find that proteins with higher LD affinity can effectively displace those with lower affinity from the LD surface, identifying steric hindrance as a key mechanism in regulating ER-to-LD protein transfer. Consistent with this model, we show that, during adipocyte differentiation Plin1-an adipocyte-specific high-affinity LD protein-reduces the recruitment of ER proteins with lower affinity by displacing them from the LD surface. These findings highlight lateral protein-protein exclusion as a fundamental mechanism in shaping the LD proteome.
    DOI:  https://doi.org/10.1126/sciadv.adu6998
  24. bioRxiv. 2025 Sep 16. pii: 2025.09.11.675358. [Epub ahead of print]
    "Design principles of a membrane-spanning ubiquitin ligase".
    Carys Williams, Laura M Nocka, George Hedger, Pragya Parashara, Els Pardon, Naomi R Latorraca, Ganesh V Pusapati, Dorothy Lartey, Lei Gao, Ljiljana Milenkovic, Rod Chalk, Jan Steyaert, Susan Marqusee, Loïc Carrique, J Fernando Bazan, Sarah L Rouse, Jennifer H Kong, Christian Siebold, Rajat Rohatgi.
      Receptor-type E3 ubiquitin ligases are membrane-spanning assemblies that enable extracellular signals to directly control ubiquitylation in the cytoplasm. Despite playing widespread roles in tissue patterning and homeostasis, metabolism, and immunity, their structures and mechanisms remain poorly understood. Using cryo-electron microscopy, integrated with biophysical and functional studies, we visualized an E3 complex composed of two transmembrane proteins, MEGF8 and MOSMO, and the intracellular RING-family protein MGRN1. This MEGF8-MOSMO-MGRN1 (MMM) complex regulates left-right patterning of the body axis and the development of multiple organs, partly by attenuating signaling through the Hedgehog pathway. We find that the MMM complex functions like a fishing pole: a long, flexible helix attached to a membrane platform suspends an activated and precisely oriented RING domain-like a fishhook-to ubiquitylate the cytoplasmic surfaces of target receptors. Our structure explains how mutations in MEGF8 cause multi-organ birth defects in humans and defines a paradigm for receptor regulation by ubiquitylation.
    DOI:  https://doi.org/10.1101/2025.09.11.675358
  25. J Biol Chem. 2025 Sep 24. pii: S0021-9258(25)02616-X. [Epub ahead of print] 110764
    IRE1/Xbp1 promotes the clearance of poly(GR) dipeptide repeats in Amyotrophic Lateral Sclerosis.
    Yu Li, Dongyue Liu, Shuangxi Li.
      Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders characterized by the expansion of GGGGCC (G4C2) repeats in the C9orf72 gene and progressive motor neuron degeneration. A key pathological hallmark of these diseases is the accumulation and cytoplasmic mislocalization of dipeptide repeat (DPR) proteins, particularly poly(GR), which are neurotoxic. Enhancing the clearance of poly(GR) represents a promising therapeutic strategy; however, the molecular mechanisms regulating poly(GR) turnover are not fully understood. Our previous work demonstrated that translationally stalled poly(GR) is targeted by the ribosome-associated quality control (RQC) pathway. In the present study, we identify the IRE1/Xbp1s signaling axis as an essential regulator of poly(GR) degradation. Ectopic expression of IRE1 or its downstream effector Xbp1s, as well as pharmacological activation of IRE1 using IXA4, significantly reduces poly(GR) protein levels in a Drosophila disease model, mammalian cell lines, fibroblasts derived from C9orf72-ALS patients, and a C9orf72 transgenic mouse model. Mechanistically, RNA-sequencing analysis reveals that IRE1/Xbp1s signaling upregulates heat shock protein Hsp70Ba, which plays a critical role in maintaining poly(GR) proteostasis. Additionally, we show that the Rictor/AKT/VCP pathway contributes to the translational regulation and turnover of poly(GR). Importantly, activation of IRE1, either through ectopic expression or IXA4 treatment, mitigates motor neuron loss in the C9orf72 mouse model. Collectively, our findings highlight the IRE1/Xbp1s axis as a key modulator of poly(GR) clearance and suggest its therapeutic potential in ALS/FTD.
    Keywords:  Drosophila melanogaster; IRE1; Xbp1; poly(GR)
    DOI:  https://doi.org/10.1016/j.jbc.2025.110764
  26. J Am Chem Soc. 2025 Sep 26.
    Cell-Selective Targeting Chimeras (SelecTACB) for Membrane Protein Degradation on B Cells.
    Zhenlin Yang, Jianfei Jiang, Yuhui Cao, Haoming Fang, Xianrui Zhang, Yuxuan Zhang, Heng Zhang, Peng R Chen.
      Membrane protein degradation chimeras (MPDs) have expanded the application space of current targeted protein degradation technologies (TPDs), yet cell-selective MPDs are still lacking, particularly for surface proteins on immune cells. Precise degradation of a target protein on a specific cell type is challenging when it is expressed across multiple immune cell types. Here, we introduce SelecTACB as a targeted membrane protein degradation chimera that is selective on B cells. By leveraging CD22 as the B cell specific lysosome-targeting receptor to induce endocytosis and lysosome degradation, SelecTACB allowed membrane protein degradation in a targeted and cell-selective manner. We show that SelecTACB can effectively degrade indicated proteins such as CD40 and ICOSL on B cells from human peripheral blood mononuclear cells (PBMCs) without affecting protein expression in other immune cell types. The degradation of key proteins involved in T cell stimulation pathways led to sustained inhibition of B cell functions. Together, our work established a cell-type selective degradation platform that offers a general strategy for targeted membrane protein degradation on immune cells.
    DOI:  https://doi.org/10.1021/jacs.5c09630
  27. Methods Enzymol. 2025 ;pii: S0076-6879(25)00226-5. [Epub ahead of print]719 1-23
    Proteome analysis of puromycin-labeled nascent polypeptides.
    Koshi Imami.
      Emerging nascent polypeptides from ribosomes and their protein N-termini have significant impact on protein stability, folding, interaction and subcellular targeting. To profile elongating nascent polypeptides on a proteome-wide scale, here we present a streamlined protocol that combined a biochemical enrichment of puromycin-labeled nascent polypeptides and mass spectrometry-based quantitative proteomics. This chapter includes the detailed protocol for metabolic pulse labeling with puromycin and SILAC amino acids, immunoprecipitation for nascent polypeptides, fractionation and enrichment of N-terminal acetylated peptides as well as MS and data analysis. These protocols are illustrated using HeLa cells treated with cycloheximide, a protein synthesis inhibitor, but can be broadly applied to any cell culture systems, including primary cultures, or any treatments (e.g., drugs).
    Keywords:  Mass spectrometry; Nascent polypeptide; Protein N-terminal acetylation; Puromycin; Ribosome
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.018
  28. Sci Adv. 2025 Sep 26. 11(39): eadx3827
    USP13 stabilizes NLRP3 to facilitate inflammasome activation by preventing TRIM31-mediated NLRP3 ubiquitination and degradation.
    Ya-Ting Li, Ke-Ying Li, Shou-Song Tao, Ting Wang, Ying Lu, Hui Chen, Yi-Qun Zhan, Ke Zhao, Shen-Si Xiang, Jing-Jing Li, Hui-Ying Gao, Miao Yu, Chang-Yan Li, Lin Wang, Xiao-Ming Yang, Guang-Ming Ren, Rong-Hua Yin.
      NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) has a fundamental role in host defense and is involved in diverse inflammatory diseases. NLRP3 protein expression is tightly controlled by the ubiquitin system. In particular, NLRP3 protein degradation has been extensively studied. In contrast, the mechanisms to stabilize NLRP3 protein are much less known. Here, we demonstrated the critical role of ubiquitin-specific protease 13 (USP13) in regulating NLRP3 protein stability and inflammasome activation independently of its deubiquitinating enzyme activity. USP13 competes with E3 ubiquitin ligase TRIM31 to interact with NLRP3 and prevents TRIM31-mediated NLRP3 ubiquitination at K192 and K496 sites, thereby inhibiting proteasomal degradation of NLRP3. USP13 deficiency reduces NLRP3 protein expression in both human and mouse macrophages, which consequently inhibits NLRP3 inflammasome assembly and activation. Accordingly, deficiency of USP13 attenuates monosodium urate crystal-induced mouse peritonitis. Overall, our findings reveal a previously unrecognized regulatory mechanism of NLRP3 stability by USP13 and provide a potential therapeutic target for NLRP3-driven diseases.
    DOI:  https://doi.org/10.1126/sciadv.adx3827
  29. bioRxiv. 2025 Sep 20. pii: 2025.09.17.676955. [Epub ahead of print]
    Principles of protein abundance regulation across single cells in a mammalian tissue.
    Andrew Leduc, Gergana Shipkovenska, Yanxin Xu, Alexander Franks, Nikolai Slavov.
      Protein synthesis and clearance are major regulatory steps of gene expression, but their in vivo regulatory roles across the cells comprising complex tissues remains unexplored. Here, we systematically quantify protein synthesis and clearance across over 4,200 cells from a primary tissue. Through integration with single-cell transcriptomics, we report the first quantitative analysis of how individual cell types regulate their proteomes across the continuum of gene expression. Our analysis quantifies the relative contributions of RNA abundance, translation, and protein clearance to the abundance variation of thousands of proteins. These results reveal an putative organizing principle: The contributions of both translation and protein clearance are linearly dependent on the cell growth rate. Further, we find that some proteins are primarily regulated by one mechanism (RNA abundance, translation, or clearance) across all cell types while the abundances of other proteins is dominated by different regulatory mechanisms across cell types. Our reliable multimodal measurements enabled quantifying and functionally interpreting molecular variation across single cells from the same cell type. The protein-protein correlations are substantially stronger than the mRNA-mRNA ones, which is mediated by protein clearance regulation. The protein-protein correlations are stronger not only for directly interacting proteins but also between functional sets of proteins. Further, these protein correlations allow identifying cell-type specific functional clusters. These clusters vary across cell types, revealing differences in metabolic processes coordination, partially mediated by protein clearance regulation. Our approach provides a scalable multiplexed framework for quantifying the regulatory processes shaping mammalian tissues and reveals organizing principles determining the relative contributions of translation and protein clearance to the proteomes of primary mammalian cells.
    DOI:  https://doi.org/10.1101/2025.09.17.676955
  30. J Biol Chem. 2025 Sep 18. pii: S0021-9258(25)02593-1. [Epub ahead of print] 110741
    Intrinsically disordered cytoplasmic tail in netrin receptor DCC binds the large ribosomal subunit to inhibit translation.
    Natasia Paukovich, Elizabeth A Spear, Andrea MacFadden, Nathan N Nowling, Morkos A Henen, Beat Vögeli, Megan E Filbin.
      Transmembrane receptors in neurons act as transducers of extrinsic growth cues by regulating local protein synthesis of specific mRNAs to facilitate axon guidance. In the absence of cues, receptors tether and silence translation at the membrane, but little is known about this receptor-ribosome interaction. Here, we show the direct and specific interaction between the transmembrane receptor deleted in colorectal cancer, DCC, and the 60S subunit that leads to translation inhibition in the absence of DCC's growth cue, netrin-1. We combined translation assays, equilibrium binding, and NMR spectroscopic approaches and identified the plasma membrane-proximal portion of DCC's cytoplasmic tail, specifically residues 1123-1158, bind the 60S subunit. We show that this region is unstructured, providing evidence for how the subunit is tethered at the membrane. Pinpointing the electrostatic interaction between DCC and the 60S subunit protein eL5/uL18 that leads to translational silencing, we propose a two-part binding interaction that facilitates this function. Our findings reveal how DCC directly regulates local translation, shedding light on the role of transmembrane receptors in controlling protein synthesis during axon guidance.
    Keywords:  intrinsically disordered protein; neurodevelopment; neuron; nuclear magnetic resonance (NMR); receptor structure-function; ribosome; ribosome function; translation; translation control
    DOI:  https://doi.org/10.1016/j.jbc.2025.110741
  31. Kidney Int. 2025 Oct;pii: S0085-2538(25)00569-1. [Epub ahead of print]108(4): 527-529
    Unfolding the potential-chemical chaperones in Alport syndrome.
    Gema Bolas, Rachel Lennon.
      Chemical chaperones are small molecules that prevent protein aggregation by improving folding, relieving endoplasmic reticulum stress, and restoring secretion of misfolded proteins. Variants in COL4A3, COL4A4, and COL4A5 cause Alport syndrome, which is characterized by abnormal assembly, secretion, and incorporation of type IV collagen into basement membranes. Ioannou et al. discovered that the chaperone 4-phenylbutyric acid improved the basement membrane and kidney function in Alport mice, raising prospects for clinical translation of chaperone therapy in Alport syndrome.
    DOI:  https://doi.org/10.1016/j.kint.2025.07.006
  32. Mol Cell. 2025 Sep 19. pii: S1097-2765(25)00736-1. [Epub ahead of print]
    Rapid remodeling of NTP levels enables immediate translational adaptation to energy stress in yeast.
    Katherine Bexley, Michaela Ristová, Sushma Sharma, Christos Spanos, Andrei Chabes, David Tollervey.
      In Saccharomyces cerevisiae, glucose depletion induces metabolic reprogramming through widespread transcriptional and translational reorganization. We report that initial, very rapid translational silencing is driven by a specialized metabolic mechanism. Following glucose withdrawal, intracellular NTP levels drop drastically over 30 s before stabilizing at a regulated, post-stress set point. Programmed translational control results from the differential NTP affinities of key enzymes; ATP falls below the (high) binding constants for DEAD-box helicase initiation factors, including eIF4A, driving mRNA release and blocking 80S assembly. Contrastingly, guanosine triphosphate (GTP) levels always greatly exceed the (low) binding constants for elongation factors, allowing ribosome run-off and orderly translation shutdown. Translation initiation is immediately lost on all pre-existing mRNAs before being preferentially re-established on newly synthesized, upregulated stress-response transcripts. We conclude that enzymatic constants are tuned for metabolic remodeling. This response counters energy depletion rather than being glucose specific, allowing hierarchical inhibition of energy-consuming processes on very rapid timescales.
    Keywords:  RNA-protein interaction; gene expression; metabolomics; stress; translation regulation; yeast
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.031
  33. J Biol Chem. 2025 Sep 18. pii: S0021-9258(25)02590-6. [Epub ahead of print] 110738
    The ubiquitin ligase Nedd4-2 promotes localization of DNMBP/Tuba to P-bodies under hyperosmotic stress.
    Zetao Liu, Chong Jiang, Faith Yeung, Brian Raught, Daniela Rotin.
      The ubiquitin ligase Nedd4-2/NEDD4L, comprised of C2-WW(x4)-HECT domains, is known to regulate several ion transporters and channels. We recently showed that elevated intracellular [Na+] and osmolarity enhances Nedd4-2 enzymatic activity. To globally identify its interactome and substrates in cells under hyperosmotic stress, we performed a BioID screen using miniTurbo with Nedd4-2 as a bait under hyperosmotic (vs. isosmotic) conditions. One of the top hits identified that preferentially binds Nedd4-2 under hyperosmolarity was Dynamin-Binding-Protein (DNMBP)/Tuba, a known GEF for Cdc42. We then showed that DNMBP is a substrate for Nedd4-2, and that active Nedd4-2 targets DNMBP to P-body condensates under hyperosmotic stress. Moreover, DNMBP itself promotes P-body formation under hyperosmolarity. Both Nedd4-2 and DNMBP are required for the activation of Cdc42 following hyperosmotic treatment, and accordingly, knockout of DNMBP results in suppression of Cdc42 and its downstream effector p38-MAPK. We thus propose that Nedd4-2 - mediated targeting of DNMBP to P-bodies under hyperosmotic stress facilitates the activation of Cdc42 by this GEF.
    Keywords:  NEDD4L; condensates; dynamin binding protein; osmolarity
    DOI:  https://doi.org/10.1016/j.jbc.2025.110738
  34. FEBS J. 2025 Sep 21.
    The SARS-CoV-2 spike protein interacts with HAX1 to modulate cellular stress responses through activation of the UPR.
    Tony Avril, Elodie Lafont.
      During cell infection, viruses maintain the lifespan of host cells by preserving key functions of cellular organelles such as the endoplasmic reticulum (ER) and mitochondria to guarantee protein secretion and energy production. The host secretory pathway is rapidly hijacked to produce viral proteins and reconstitute viral particles for further viral dissemination. However, secreted protein synthesis and proper folding are tightly regulated in the host ER to maintain homeostasis, otherwise this organelle is subjected to ER stress that triggers an adaptive response named the unfolded protein response (UPR). The UPR first aims at restoring ER function by producing enzymes to correct or eliminate misfolded proteins. If ER stress remains unresolved, the UPR triggers cell death. In the work published by Zhu et al. in this issue of The FEBS Journal, the authors explore a previously undescribed molecular hijacking function of SARS-CoV-2 to limit host cell death. Indeed, the viral spike protein directly interacts with the host HAX1 molecule to promote UPR activation, limiting the production of deleterious reactive oxygen species and mitochondrial dysfunction to maintain host cell survival.
    Keywords:  ER stress; HAX1; ROS production; SARS‐CoV‐2; cell death; spike
    DOI:  https://doi.org/10.1111/febs.70259
  35. bioRxiv. 2025 Sep 18. pii: 2025.09.15.676438. [Epub ahead of print]
    Mistranslating tRNA variants impact the proteome and phosphoproteome of Saccharomyces cerevisiae.
    Matthew D Berg, Alexis T Chang, Ricard A Rodriguez-Mias, Judit Villén.
      Transfer RNAs (tRNAs) ensure accurate decoding of the genetic code. However, mutations in tRNAs can lead to mis-incorporation of an amino acid that differs from the genetic message in a process known as mistranslation. As mistranslating tRNAs modify how the genetic message is decoded, they have potential as therapeutic tools for diseases caused by nonsense and missense mutations. Despite this, they also produce proteome-wide mis-made proteins which can disrupt proteostasis. To better understand the impact of mistranslating tRNA variants, we profile the proteome and phosphoproteome of yeast expressing three different mistranslating tRNAs. While the overall impacts were similar, the extent of growth defects and proteome changes varied with the substitution type. Although the global impacts were modest, mistranslation influenced key cellular processes, including proteostasis, cell cycle and translation. These findings highlight the need to consider cellular consequences when developing mistranslating tRNAs for therapeutic applications.
    DOI:  https://doi.org/10.1101/2025.09.15.676438
  36. J Am Chem Soc. 2025 Sep 24.
    Indolequinone-Based Hypoxia-Activated Proteolysis Targeting Chimeras Selectively Degrade BRD4 in Hypoxic Cancer Cells.
    Marta Serafini, Sophie A Twigger, George Delfas, Maxim Mallerman, Elliot P Bailey, Ewen D D Calder, Ester M Hammond, Stuart J Conway.
      Proteolysis targeting chimeras (PROTACs) have helped to establish proximity induction as an exciting strategy in drug discovery, and there are multiple clinical trials focused on this modality. However, degradation of a full protein in a physiological setting might lead to dose-limiting toxicities, giving rise to the need for PROTACs that are activated in a context-dependent nature. Here, we report the development of hypoxia-activated PROTACs (HAP-TACs) which are selectively activated in conditions of low oxygen (hypoxia) such as those found in solid tumors. To develop HAP-TACs, we have attached an indolequinone bioreductive group to an essential functional group of either the VHL- or cereblon-recruiting component of the PROTAC, reducing affinity for its cognate E3 ligase and preventing degradation of the protein of interest. Using BRD4, we have conducted proof-of-concept studies which demonstrate that the indolequinone group is bioreduced under hypoxic conditions, releasing the active PROTAC, resulting in selective degradation of BRD4 in hypoxia. As the bioreductive group is attached to the VHL or cereblon ligand, this approach is potentially applicable to all PROTACs that recruit these commonly employed E3 ligases.
    DOI:  https://doi.org/10.1021/jacs.5c10240
  37. Nucleic Acids Res. 2025 Sep 23. pii: gkaf938. [Epub ahead of print]53(18):
    DHX36 modulates stress granule assembly independent of recruitment of mRNAs with G-quadruplex sequence motifs.
    Li Yi Cheng, Nina Ripin, Thomas R Cech, Roy Parker.
      Stress granules are RNA-protein condensates that form in response to an increase in untranslating mRNPs (messenger ribonucleoproteins). Stress granules form by the condensation of mRNPs through a combination of protein-protein, protein-RNA, and RNA-RNA interactions. Several reports have suggested that G-rich RNA sequences capable of forming G-quadruplexes (rG4s) promote stress granule formation. Here, we provide three observations arguing that G-tracts do not promote messenger RNA (mRNA) accumulation in stress granules in human osteosarcoma cells. First, we observed no difference in the accumulation in stress granules of reporter mRNAs with and without G-tracts in their 3' UTRs. Second, in U-2 OS cell lines with reduced expression of DHX36, which is thought to unwind G-quadruplexes, the accumulation of endogenous mRNAs was independent of their predicted rG4-forming potential. Third, while mRNAs in stress granules initially appeared to have more rG4 motifs than bulk mRNAs, this effect disappeared when rG4 motif abundance was normalized to mRNA length. However, we observed that in a G3BP1/2 double knockout cell line, which strongly inhibits stress granule formation, reducing DHX36 expression rescued stress granule-like foci formation. This indicates that DHX36 can limit stress granule formation, potentially by unwinding trans-rG4s or limiting other intermolecular RNA-RNA interactions that promote stress granule formation.
    DOI:  https://doi.org/10.1093/nar/gkaf938
  38. J Med Chem. 2025 Sep 26.
    HSP90 Mediates Targeted Degradation of Nonclient Protein PARP1 for Breast Cancer Treatment.
    Wei Liu, Jianfeng Liu, Huangliang Shu, Lixiao Zhang, Xingyu Yin, Xiaoli Xu, Qidong You, Lei Wang.
      Heat shock protein 90 (HSP90) has been developed as an effector in mediating targeted protein degradation (TPD), representing a novel strategy in TPD drug design. The majority of reported cases of HSP90-mediated degradation targeted HSP90 client proteins, including BRD4-CHAMPs, CDK4/6-HEMTACs, and GPX4-HIM-PROTACs. However, HSP90 ATPase inhibitor was used to design the above molecules, which might cause nonspecific degradation of other client proteins. In this study, we sought to broaden the scope of HSP90-mediated proteolysis-targeting chimeras (HSPTACs) from client protein degradation to include nonclient protein degradation. Herein, we induced unnatural interactions between poly(ADP-ribose) polymerase-1 (PARP1), a nonclient protein of HSP90, and HSP90 by bridging them with a small molecule (DDO3602). DDO3602 effectively induced PARP1 degradation through a multi-E3 ubiquitin ligase-mediated degradation pathway. In general, this study demonstrates that DDO3602 can degrade the HSP90 nonclient protein PARP1 through the ubiquitin-proteasome pathway and exhibits tumor-selective pharmacokinetics.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c00409
  39. Nucleic Acids Res. 2025 Sep 23. pii: gkaf956. [Epub ahead of print]53(18):
    Spatiotemporal dynamics and selectivity of mRNA translation during mouse pre-implantation development.
    Hao Ming, Rajan Iyyappan, Kianoush Kakavand, Michal Dvoran, Andrej Susor, Zongliang Jiang.
      Translational regulation plays a pivotal role during pre-implantation development. However, the mechanisms by which messenger RNAs (mRNAs) are selectively regulated over time, along with their dynamic utilization and fate during this period, remain largely unknown. Here, we performed fraction-resolved polysome profiling and characterized translational dynamics across oocytes and early embryo development. This approach allowed us to examine the changes in translation during pre-implantation development in high resolution and uncover previously unrecognized modes of translational selectivity. We observed a stage-specific delay in translation, characterized by the postponed recruitment of stored mRNAs-either unbound or associated with light ribosomal fractions-into actively translating polysomes (heavy fraction). Comparative analysis of translatome with proteomics, RNA N6-methyladenosine modifications, and mRNA features further revealed both coordinated and distinct regulatory mechanisms during pre-implantation development. Furthermore, we identified a eukaryotic initiation factor 1A domain containing 3, Eif1ad3, which is exclusively translated at the two-cell stage and is essential for embryonic development by regulating ribosome biogenesis and protein synthesis. Collectively, our study provides a valuable resource of spatiotemporal translational regulation in mammalian pre-implantation development and highlights a previously uncharacterized translation initiation factor critical for early embryos.
    DOI:  https://doi.org/10.1093/nar/gkaf956
  40. Immunity. 2025 Sep 25. pii: S1074-7613(25)00414-5. [Epub ahead of print]
    Intra-tumoral hypoxia promotes CD8+ T cell dysfunction via chronic activation of integrated stress response transcription factor ATF4.
    Coral Del Mar Alicea Pauneto, Brian P Riesenberg, Evelyn J Gandy, Andrew S Kennedy, Genevieve T Clutton, Jessica W Hem, Katie E Hurst, Elizabeth G Hunt, Jarred M Green, Brian C Miller, Steven P Angus, Gary L Johnson, Robert J Esther, Jennifer L Guerriero, Peng Gao, David R Soto-Pantoja, Robert L Ferris, Jennifer L Modliszewski, Michael F Coleman, H Kay Chung, J Justin Milner, Stergios J Moschos, R Luke Wiseman, Jessica E Thaxton.
      Metabolic stress in the tumor microenvironment (TME) promotes T cell dysfunction and immune checkpoint inhibitor (ICI) resistance. We examined the contribution of activating transcription factor 4 (ATF4), the central node of the integrated stress response (ISR), to T cell dysfunction in tumors. CD8+ tumor-infiltrating lymphocytes (TILs) in patient samples exhibited chronic ATF4 activity, which was reflected across various tumor models. Hypoxia in the TME imposed chronic ATF4 activity via the ISR kinases. ATF4 overexpression in CD8+ T cells induced metabolic polarity, mitochondrial oxidative stress, and cell death, impairing antitumor immunity. Chronic ATF4 transcriptional activity replicated the terminal exhaustion CD8+ T cell state independent of T cell receptor (TCR) stimulation. Genetic or pharmacologic attenuation of ATF4 reduced mitochondrial oxidative stress and promoted CD8+ TIL viability, enabling response to programmed cell death protein-1 (PD-1) inhibitor therapy and conferring protection from re-emergent disease. Thus, the ISR converges on chronic ATF4 activity in CD8+ TILs as a barrier to ICI response, positioning ISR therapeutics as candidates for immunotherapy.
    Keywords:  ATF4; T cell; hypoxia; immunotherapy; integrated stress response; metabolism; mitochondria; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.immuni.2025.09.003
  41. Trends Cell Biol. 2025 Sep 19. pii: S0962-8924(25)00198-9. [Epub ahead of print]
    Organelle-specific signaling of cGAS-STING.
    Shengduo Liu, Ailian Wang, Chen Chen, Pinglong Xu.
      Innate immune sensing through cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) surveils cytosolic DNA from invading pathogens or damaged organelles and initiates a spectrum of immune responses. It is well established that upon 2'3'-cyclic GMP-AMP (cGAMP) binding, STING exits the endoplasmic reticulum (ER), traverses the Golgi to trigger interferon programs, and finally reaches lysosomes for signal resolution through degradation, revealing a tightly choreographed itinerary for cytokine-driven immunity. However, emerging studies reveal additional layers of spatiotemporal complexity: ER-resident STING tunes in messenger RNA translation and Ca2+ efflux, Golgi-localized STING functions as a proton channel that initiates H+-dependent autophagy and transcription factor EB-directed programs for organelle homeostasis, and various mechanisms for metabolic remodeling and cell fate determination. This review synthesizes emerging organelle-specific mechanisms of cGAS-STING, delineates their roles in physiology and disease, and discusses how an organelle-centric perspective may inform selective, context-sensitive immunotherapies.
    Keywords:  cGAS–STING; cellular function; innate immunity; organelle; signaling mechanism; trafficking
    DOI:  https://doi.org/10.1016/j.tcb.2025.08.007
  42. J Cell Sci. 2025 Sep 15. pii: jcs263691. [Epub ahead of print]138(18):
    Charcot-Marie-Tooth type 2A variants of mitofusin 2 sensitize cells to apoptotic cell death.
    Mariana Joaquim, Maria-Bianca Bulimaga, Marie A Mohn, Solenn Plouzennec, Leon Osinski, Selver Altin, Esther Mahabir, Arnaud Chevrollier, Mafalda Escobar-Henriques.
      The neuropathy Charcot-Marie-Tooth (CMT) is an incurable disease with a lack of genotype-phenotype correlation. Variants of the mitochondrial protein mitofusin 2 (MFN2), a large GTPase that mediates mitochondrial fusion, are responsible for the subtype CMT type 2A (CMT2A). Interestingly, beyond membrane remodelling, additional roles of MFN2 have been identified, expanding the possibilities to explore its involvement in disease. Here, we investigated how cellular functions of MFN2 are associated with variants present in individuals with CMT2A. Using human cellular models, we observed that cells expressing CMT2A variants display increased endoplasmic reticulum (ER) stress and apoptotic cell death. Increased cleavage of PARP1, caspase 9, caspase 7 and caspase 3, alongside BAX translocation to mitochondria, pointed towards effects on intrinsic apoptosis. Moreover, although disruption of fusion and fission dynamics per se did not correlate with cell death markers, expression of MFN1 or MFN2 alleviated the apoptosis markers of CMT2A variant cell lines. In sum, our results highlight excessive cell death by intrinsic apoptosis as a potential target in CMT2A disease.
    Keywords:  Apoptosis; CMT2A; Cell death; Charcot–Marie–Tooth; Fusion; MFN2; Mitochondria
    DOI:  https://doi.org/10.1242/jcs.263691
  43. Biochem Biophys Rep. 2025 Dec;44 102247
    An Enigma of N-termini dependent protein degradation.
    Lina Alhourani, Alaa M Elgohary, Richard P Fahlman.
      The N-Degron rules that target proteins for degradation via their N-terminal sequences has expanded to encompass most N-terminal sequences. These destabilizing N-termini include many sequence combinations with the initiator methionine intact, including the recently reported N-terminal methionine followed by a basic residue. Despite the diverse sequences reported for N-Degron recognition and degradation system wide proteomic analysis has currently not observed these rules with endogenous proteins. Here we report on these apparent inconsistencies by validating the degradation of reporter proteins with N-terminal MK- and MR-sequences and also investigating global endogenous protein turnover by proteomics. In addition to verifying the reported degradation of proteins with MK- and MR- N-termini we have also identified an additional sequence dependency where an acidic residue following the basic residue inhibits protein degradation. Global protein degradation analysis using a metabolic labelling approach with azidohomoalanine failed to observe trends in cytoplasmic protein stability that correlates to the N-terminal sequence. Together we have been able to reproduce the apparent contradictory results reported using different methodologies. This included the use of different recombinant protein reporters to investigate MK- and MR- N-termini dependent protein degradation and an alternative proteomic method to quantify global protein degradation. This highlights the need into further investigations to complete our understanding of the mechanisms and roles of N-Degron pathways.
    Keywords:  N-Degron; N-End rule; Protein degradation
    DOI:  https://doi.org/10.1016/j.bbrep.2025.102247
  44. bioRxiv. 2025 Sep 15. pii: 2025.09.09.675096. [Epub ahead of print]
    Disease-specific tau polymorphs define unique protein interaction networks across proteinopathies.
    Nicha Puangmalai, Nemil Bhatt, Nopparat Suthprasertporn, Ernesto G Miranda-Morales, Nikita Shchankin, Madison Samples, Ahmet E Balci, Jia Yi Liew, Mauro Montalbano, Yingxin Zhao, Rakez Kayed.
      Tau protein aggregates exhibit distinct conformations across tauopathies, but their disease-specific protein interactions remain poorly understood. Here, we demonstrate that disease-specific tau conformations determine unique protein interaction landscapes across Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Through comprehensive interactome profiling of misfolded tau aggregates from PBS- and sarkosyl-soluble fractions. We identified 493 high-confidence proteins with remarkable disease specificity-notably, no common interactors overlapping across all three tauopathies. Machine learning classification achieved compelling discrimination between diseases using as few as 4-6 proteins features, demonstrating robust molecular signatures underlying clinical heterogeneity. AD derived tau aggregates uniquely engaged cellular metabolism machinery, including key glycolytic enzymes and TCA cycle proteins, alongside glutamate/GABA neurotransmitter cycling components, with the astrocytic glutamate transporter SLC1A2 showing 27-fold enrichment over other tauopathies. In contrast, PSP tau displayed the most distinctive profile, with extensive protein depletion (52/57 significant proteins) and selective enrichment of proteasome components, particularly PSMB7 showing >3000-fold abundance. DLB tau is associated with neurogenesis modulators while depleting neuroinflammatory mediators. These interaction patterns were validated through proximity ligation assays and correlated with distinct post-translational modification profiles, with PSP tau exhibiting globally elevated ubiquitination, AD showing mixed modification patterns, and DLB displaying minimal ubiquitination. Critically, sarkosyl-soluble fractions revealed reduced interactome complexity across diseases, except for PSP tau which maintained robust interactions with GPCR-ERK signaling and kinetochore proteins, suggesting unique aggregation mechanisms. Our findings establish that conformationally distinct tau strains dictate disease-specific protein interaction networks, providing molecular insight into tauopathy diversity and identifying novel therapeutic targets for precision medicine approaches in neurodegeneration.
    DOI:  https://doi.org/10.1101/2025.09.09.675096
  45. Mol Cell. 2025 Sep 23. pii: S1097-2765(25)00737-3. [Epub ahead of print]
    Ubiquitin precursor with C-terminal extension promotes proteostasis and longevity.
    Selver Altin, Tânia Simões, Christina Behrendt, Vincent Anton, Dennis Domke, Kai Mayor Völtzke, Rajesh Kumar, Hendrik Nolte, Thomas Hermanns, Nahal Brocke-Ahmadinejad, Katja Bendrin, Marcel Zimmermann, Reinhard Büttner, Natascia Ventura, Marcus Krüger, R Jürgen Dohmen, Kay Hofmann, Thorsten Hoppe, Andreas S Reichert, Mafalda Escobar-Henriques.
      Ubiquitin is a conserved modifier regulating the stability and function of numerous target proteins. In all eukaryotes, polyubiquitin precursors are generated and processed into ubiquitin monomers. The final ubiquitin unit always contains a C-terminal extension, but its physiological significance is unknown. Here, we show that C-terminally extended ubiquitin, termed CxUb, is essential for stress resistance, mitophagy, and longevity in Saccharomyces cerevisiae and Caenorhabditis elegans. CxUb forms ubiquitin chains and binds to a previously undescribed region within the ubiquitin chain-elongating E4 enzyme Ufd2, which also functions during stress and aging. Ufd2 recognizes CxUb and conjugates it to substrate proteins, triggering their degradation. By contrast, CxUb is not required for basal housekeeping functions of the ubiquitin-proteasome system. These data suggest that the CxUb encodes a functionally unique ubiquitin form, specialized for proteostasis defects, expanding the code of post-translational modification processes.
    Keywords:  CxUb; E4; Ufd2; aging; mitochondria; mitofusin; mitophagy; proteostasis; stress; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.032
  46. Angew Chem Int Ed Engl. 2025 Sep 27. e202506997
    Ultrasmall Chemogenetic Tags with Group-Transfer Ligands.
    Vedagopuram Sreekanth, Shaimaa H Sindi, Santosh K Chaudhary, Rajaiah Pergu, Prashant Singh, Endri Karaj, Surached Siriwongsup, Jeffrey E Fung, Arghya Deb, Stephan J DeCarlo, Jaron A M Mercer, Kei Yamada, Diego Rodriguez, David R Liu, Amit Choudhary.
      Chemogenetic tags facilitate exploration of activities of a protein-of-interest (POI) that lacks small-molecule ligands; however, most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest. These tags exhibit transferase-type reactivity with their ligands, allowing the attachment of any moiety-of-interest to the tag. cTag utilizes an engineered C1 domain-bearing cysteine that undergoes a group-transfer reaction with its ligand. Likewise, mgTag utilizes an engineered zinc-finger domain-bearing cysteine that undergoes a group-transfer reaction with its molecular glue ligand in the presence of cereblon (CRBN). While the fusion of HaloTag (297 aa) or SNAPTag (182 aa) to the KRASG12D (188 aa) disrupted its growth-signaling pathway, fusion of mgTag or cTag did not, pointing to the importance of tag size. Group-transfer of BRD4 binder to tags appended to Abelson kinase (ABL) induced proximity between ABL and BRD4, resulting in the latter's phosphorylation. Deletion of the transferase-type reactivity reduced phosphorylation levels, suggesting that proximity-inducing chimeras with group-transfer design may be more efficacious. We envision these ultrasmall tags to have wide-ranging applications, including in basic science, biotechnology, and medicine.
    Keywords:  C1 domain; Chemogenetic tags; Induced proximity; KRAS signaling; Molecular glue
    DOI:  https://doi.org/10.1002/anie.202506997
  47. Methods Enzymol. 2025 ;pii: S0076-6879(25)00217-4. [Epub ahead of print]719 173-210
    Characterization of the autophagic N-degron pathway and monitoring its chemical modulation for therapeutic development.
    Jihoon Lee, Gee Eun Lee, Chan Hoon Jung, Yoon Jee Lee, Yong Tae Kwon.
      Cellular homeostasis relies on the regulated degradation of unnecessary or harmful biomaterials including proteins. The N-degron pathway plays a central role in quality control at the crossroad of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy-lysosome system (ALS). In this pathway, single N-terminal (Nt) residues of substrate proteins act as degradation determinants, called N-degrons. In the UPS, N-degrons are recognized and bound by their cognate N-recognins such as a family of E3 Ub ligases carrying the UBR box to induce substrate ubiquitination for proteasomal degradation. Our earlier studies identified an autophagic version of the N-degron pathway that works cooperatively with the UPS. In this process, N-degrons bind the N-recognin p62/SQSTM1 and modulate its activity as an autophagy receptor, leading to lysosomal destruction of UPS-resistant cargoes. We also developed chemical mimetics of N-degrons that exert efficacy to accelerate the degradation of excessive or harmful biomaterials such as cellular wastes and subcellular organelles. Here, we describe biochemical assays to characterize p62 as an autophagic N-recognin. We also delve into methodologies to assess therapeutic efficacy of chemical N-degrons in the degradation of lipid droplets (LDs) through lipophagy and the clearance of invading bacteria through xenophagy. These protocols can be used to identify new ZZ-type N-recognins in mammals and other species as well as under various pathophysiological conditions in both the UPS and ALS.
    Keywords:  ATE1 R-transferase; Lipophagy; N-recognin; N-terminal arginylation; P62/SQSTSM-1; The N-degron pathway; The autophagy-lysosome system; Xenophagy
    DOI:  https://doi.org/10.1016/bs.mie.2025.06.009
  48. J Dent Res. 2025 Sep 22. 220345251363525
    GCN2-Mediated Integrated Stress Response Attenuates Periodontitis.
    D Huang, Q Li, S Peng, Y Li, Q Yin, X Yang, X Gan, Y Wang, X Li, Y Zhao, Y Guo, W Lin, Y Li, N Feng, Q Yuan.
      The integrated stress response (ISR), regulated by general control nonderepressible 2 (GCN2), is essential for maintaining tissue homeostasis, yet its role in periodontitis remains poorly understood. Here, through transcriptomic analysis and immunohistochemistry of gingival biopsies from patients and a ligature-induced mouse periodontitis model, we demonstrate that GCN2-mediated ISR is activated in both human and mouse periodontitis and mainly functioned in macrophages. Using Gcn2-/- mice, we show that Gcn2 deletion exacerbates gingival inflammation and bone loss in experimental periodontitis. Mechanistically, bulk RNA-seq and in vitro assays revealed that the loss of GCN2 impairs autophagy and leads to overactivation of the NLRP3/CASPASE1 inflammasome pathway. Notably, local administration of halofuginone, a GCN2 activator, mitigates oral inflammation and tissue destruction in a GCN2-dependent manner. In summary, our work highlights the protective role of the GCN2-mediated ISR in oral mucosa and indicates GCN2 as a promising therapeutic target for periodontitis.
    Keywords:  immunomodulation; inflammasomes; inflammation; macrophages; periodontal diseases; protein kinases
    DOI:  https://doi.org/10.1177/00220345251363525
  49. J Cell Biol. 2025 Nov 03. pii: e202404018. [Epub ahead of print]224(11):
    JIP4 and RILPL1 utilize opposing motor force to dynamically regulate lysosomal tubulation.
    Luis Bonet-Ponce, Tsion Tegicho, Nuria Fernandez-Martinez, Irene A Rozenberg, Mia Ashriem, Alexandra Beilina, Jillian H Kluss, Yan Li, Mark R Cookson.
      Lysosomes are dynamic organelles that remodel their membrane in response to stimuli. We previously uncovered a process we term LYsosomal Tubulation/sorting driven by LRRK2 (LYTL), wherein damaged lysosomes generate tubules sorted into vesicles. LYTL is orchestrated by the Parkinson's disease kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes. JIP4 enhances LYTL tubule extension toward the plus-end of microtubules. To identify new players involved in LYTL, we mapped the lysosomal proteome after LRRK2 kinase inhibition. We found that RILPL1 is recruited to dysfunctional lysosomes in an LRRK2 kinase activity-dependent manner, facilitated by pRAB proteins. Unlike JIP4, RILPL1 induces retraction of LYTL tubules by binding to p150Glued, thereby moving lysosomal tubules toward the minus-end of microtubules. Our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, acting as opposing motor adaptor proteins. These opposing forces create a metastable lysosomal membrane deformation, enabling dynamic tubulation events.
    DOI:  https://doi.org/10.1083/jcb.202404018
  50. bioRxiv. 2025 Sep 15. pii: 2021.07.04.451082. [Epub ahead of print]
    ribofootPrinter: A precision python toolbox for analysis of ribosome profiling data.
    Kyra Kerkhofs, Nicholas R Guydosh.
      Ribosome profiling is a valuable methodology for measuring changes in a cell's translational program. The approach can report how efficiently mRNA coding sequences are translated and pinpoint positions along mRNAs where ribosomes slow down or arrest. It can also reveal when translation takes place outside coding regions, often with important regulatory consequences. While many useful software tools have emerged to facilitate analysis of these data, packages can become complex and challenging to adapt to specialized needs. We therefore introduce ribofootPrinter, a suite of Python tools designed to offer an accessible and modifiable set of code for analysis of data from ribosome profiling and related types of small RNA sequencing experiments. Alignments are made to a simplified transcriptome to keep the code intuitive and multiple normalization options help facilitate interpretation of meta analysis, particularly outside coding regions. We demonstrate how mapping of short reads to the transcriptome increases the frequency of matches to multiple sites and we provide multimapper identifier files to highlight these regions. Overall, this tool has the capability to carry out sophisticated analysis while maintaining enough simplicity to make it readily understandable and adaptable.
    DOI:  https://doi.org/10.1101/2021.07.04.451082
  51. Nature. 2025 Sep 24.
    Collective homeostasis of condensation-prone proteins via their mRNAs.
    Rupert Faraway, Neve Costello Heaven, Holly Digby, Klara Kuret Hodnik, Jure Rebselj, Oscar G Wilkins, Anob M Chakrabarti, Ira A Iosub, Neža Vadnjal, Rhys Dore, Lea Knez, Stefan L Ameres, Clemens Plaschka, Jernej Ule.
      The concentration of proteins containing intrinsically disordered regions must be tightly controlled to maintain cellular homeostasis1,2. However, mechanisms for collective control of these proteins, which tend to localize to membraneless condensates, are less understood than pathways mediated by membrane-bound organelles3,4. Here we report 'interstasis', a homeostatic mechanism in which increased concentration of proteins within RNA-protein condensates induces the sequestration of their own mRNAs. The selectivity of interstatic mRNA capture relies on the structure of the genetic code and conserved codon biases, which ensure that similar multivalent RNA regions encode similar low-complexity domains. For example, arginine-enriched mixed charge domains (R-MCDs) tend to be encoded by repetitive purine-rich sequences in mRNAs. Accumulation of proteins containing R-MCDs increases the cohesion of nuclear speckles, which induces selective capture of purine-rich multivalent mRNAs. The multivalent regions are bound by specific RNA-binding proteins, including TRA2 proteins, which relocalize to speckles upon interstasis to promote selective mRNA capture. CLK-mediated phosphorylation of TRA2 proteins counters their localization to speckles, thereby modulating interstasis. Thus, the condensation properties of nuclear speckles act as a sensor for interstasis, a collective negative-feedback loop that co-regulates mRNAs of highly dosage-sensitive genes, which primarily encode nuclear condensation-prone proteins.
    DOI:  https://doi.org/10.1038/s41586-025-09568-w
  52. Nat Commun. 2025 Sep 22. 16(1): 8278
    Repeatability of protein structural evolution following convergent gene fusions.
    Naoki Konno, Keita Miyake, Satoshi Nishino, Kimiho Omae, Haruaki Yanagisawa, Saburo Tsuru, Yuki Nishimura, Masahide Kikkawa, Chikara Furusawa, Wataru Iwasaki.
      Convergent evolution of proteins provides insights into repeatability of genetic adaptation. While local convergence of proteins at residue or domain level has been characterized, global structural convergence by inter-domain/molecular interactions remains largely unknown. Here we present structural convergent evolution on fusion enzymes of aldehyde dehydrogenases (ALDHs) and alcohol dehydrogenases (ADHs). We discover BdhE (bifunctional dehydrogenase E), an enzyme clade that emerged independently from the previously known AdhE family through distinct gene fusion events. AdhE and BdhE show shared enzymatic activities and non-overlapping phylogenetic distribution, suggesting common functions in different species. Cryo-electron microscopy reveals BdhEs form donut-like homotetramers, contrasting AdhE's helical homopolymers. Intriguingly, despite distinct quaternary structures and < 30% amino acid sequence identity, both enzymes forms resemble dimeric structure units by ALDH-ADH interactions via convergently elongated loop structures. These findings suggest convergent gene fusions recurrently led to substrate channeling evolution to enhance two-step reaction efficiency. Our study unveils structural convergence at inter-domain/molecular level, expanding our knowledges on patterns behind molecular evolution exploring protein structural universe.
    DOI:  https://doi.org/10.1038/s41467-025-63898-x
  53. Nature. 2025 Sep 24.
    Design of facilitated dissociation enables timing of cytokine signalling.
    Adam J Broerman, Christoph Pollmann, Yang Zhao, Mauriz A Lichtenstein, Mark D Jackson, Maxx H Tessmer, Won Hee Ryu, Masato Ogishi, Mohamad H Abedi, Danny D Sahtoe, Aza Allen, Alex Kang, Joshmyn De La Cruz, Evans Brackenbrough, Banumathi Sankaran, Asim K Bera, Daniel M Zuckerman, Stefan Stoll, K Christopher Garcia, Florian Praetorius, Jacob Piehler, David Baker.
      Protein design has focused on the design of ground states, ensuring that they are sufficiently low energy to be highly populated1. Designing the kinetics and dynamics of a system requires, in addition, the design of excited states that are traversed in transitions from one low-lying state to another2,3. This is a challenging task because such states must be sufficiently strained to be poorly populated, but not so strained that they are not populated at all, and because protein design methods have focused on generating near-ideal structures4-7. Here we describe a general approach for designing systems that use an induced-fit power stroke8 to generate a structurally frustrated9 and strained excited state, allosterically driving protein complex dissociation. X-ray crystallography, double electron-electron resonance spectroscopy and kinetic binding measurements show that incorporating excited states enables the design of effector-induced increases in dissociation rates as high as 5,700-fold. We highlight the power of this approach by designing rapid biosensors, kinetically controlled circuits and cytokine mimics that can be dissociated from their receptors within seconds, enabling dissection of the temporal dynamics of interleukin-2 signalling.
    DOI:  https://doi.org/10.1038/s41586-025-09549-z
  54. STAR Protoc. 2025 Sep 24. pii: S2666-1667(25)00482-4. [Epub ahead of print]6(4): 104076
    Protocol to set up the CellMinerCDB pharmacogenomics analysis web application with custom cell line data.
    Fathi Elloumi, Camille Tlemsani, Christine M Heske, Lorinc Pongor, Prashant Khandagale, Sudhir Varma, Paul S Meltzer, Javed Khan, William C Reinhold, Yves Pommier, Augustin Luna.
      CellMiner Cross-Database (CellMinerCDB) is an interactive web application for integrating and analyzing molecular and pharmacological data across human cancer cell lines. Here, we detail the setup process, including installing necessary software, preparing compatible datasets, and customizing configuration files; we use sarcoma data as an example. The protocol involves data loading, software configuration, and deployment to enable univariate and multivariate analyses. For complete details on the use and execution of this protocol, please refer to Luna et al.1 and Tlemsani et al.2.
    Keywords:  Bioinformatics; Cancer; Genomics; Health Sciences
    DOI:  https://doi.org/10.1016/j.xpro.2025.104076
  55. Cell Commun Signal. 2025 Sep 24. 23(1): 397
    Parental S-adenosylmethionine diet defines offspring immune response via histone H3K4me3 complex and Endoplasmic Reticulum UPR.
    Fang Liu, Qingyao Wang, Jun Xiong, Mengqi Wang, Hanlin Zhou, Yi Xiao.
      S-adenosylmethionine (SAM), is a ubiquitous cofactor necessary for methyltransferase reactions. Deficiency in SAM results in dysregulation of crucial methylation and cellular dysfunction. SAM promotes innate immunity via histone H3K4me3 complex, raising the question of whether SAM supplementation in the parental generation could be reprogrammed histone modifications in offspring and thereby affect the innate immunity of descendants. In this study, we fed Caenorhabditis elegans with SAM, which led to enhance innate immunity. Furthermore, this enhancement is capable of transmitting the phenotype to subsequent generations. Transcriptome sequencing and GO functional enrichment analysis revealed that SAM induced the expression of genes involved in immune responses and IRE-1-mediated endoplasmic reticulum unfolded protein response (UPRER), revealing those genes were required for transgenerational innate immunity enhancement. Additionally, histone H3K4me3 marked immune response genes and IRE-1-mediated UPRER genes and promoted their transcription response to multigenerational innate immunity enhancement effects. Our findings indicate that the endoplasmic reticulum unfolded protein response (UPRER) in parental somatic cells mediates the establishment of epigenetic memory, which is preserved through the histone H3K4me3 complex in the germline across generations. Surprisingly, the transgenerational epigenetic inheritance (TEI) of the immune response induced by a SAM diet occurs independently of small RNAs. These findings offer valuable insights into the mechanisms driving multigenerational innate immunity reprogramming and clarify the effects of SAM supplementation.
    Keywords:   Caenorhabditis elegans ; Endoplasmic reticulum UPR; Histone H3K4me3 complex; Innate immunity; S-adenosylmethionine
    DOI:  https://doi.org/10.1186/s12964-025-02386-7
  56. bioRxiv. 2025 Sep 15. pii: 2025.09.09.675165. [Epub ahead of print]
    Systematic Mapping of Protein Interactions Underlying IL-2 Secretion in Human T Cells.
    Seunghyeon Shin, Frances Rocamora, Nathan E Lewis.
      Protein secretion plays a crucial role in maintaining immune homeostasis, yet the molecular interactions governing this process remain incompletely understood. While transcriptional and post-transcriptional regulation of protein expression is well characterized, the subcellular interactions between secreted proteins and trafficking machinery are less explored. To address this, we systematically mapped protein-protein interactions (PPIs) involved in the secretion of interleukin-2 (IL-2) from human T cells using proximity-based labeling coupled with mass spectrometry. Our analysis revealed significant enrichment of proteins associated with conventional secretory pathways, including ER-to-Golgi transport, protein folding, and vesicle-mediated trafficking. Functional validation confirmed that several of these proteins are critical for IL-2 secretion, underscoring their direct roles in cytokine processing. In addition, time-resolved profiling of PPIs and transcriptomic changes following T cell stimulation revealed dynamic remodeling of the cytokine secretion machinery, reflecting multilayered regulation at both the protein and gene expression levels. These findings offer a systems-level understanding of IL-2 secretion and identify new molecular components that can be targeted to modulate immune responses. This work provides a framework for dissecting complex secretory processes and has broad implications for therapeutic strategies in immune-related diseases.
    DOI:  https://doi.org/10.1101/2025.09.09.675165
  57. Proc Natl Acad Sci U S A. 2025 Sep 30. 122(39): e2519568122
    CRISPR screens identify the ATPase VCP as a druggable therapeutic vulnerability in cholangiocarcinoma.
    Wu Yang, Siying Wang, Shuyi Ji, Jian Wang, Shuo Lian, Zhe Li, Robin A Jansen, Wei Wu, Kongyan Niu, Zhen Sun, Qi Jia, Jiaojiao Zheng, Huijue Zhu, Xuan Deng, Liqin Wang, Zhoulong Fan, Yaoping Shi, Cor Lieftink, Ming Guan, Roderick L Beijersbergen, Wenxin Qin, Qiang Gao, René Bernards, Haojie Jin.
      Cholangiocarcinoma (CCA) remains a lethal malignancy with limited therapeutic options. Through genome-wide CRISPR-Cas9 screening, we identified the adenosine triphosphatase (ATPase) valosin-containing protein (VCP) as a critical dependency in CCA. Compound screens revealed that the VCP inhibitor CB-5339 potently suppresses CCA proliferation in a panel of patient-derived organoids by inducing cellular senescence. It is known that senescent cells persist, and this can contribute to therapy resistance. To address this, we combined CB-5339 with senolytic agents (ABT-263 and conatumumab), which selectively eliminate senescent CCA cells, resulting in enhanced tumor suppression both in vitro and in vivo. Clinical analysis showed that VCP overexpression in CCA patients correlates with poor prognosis. Our study unveils a "one-two punch" strategy, targeting VCP-mediated senescence followed by senolytic clearance, offering a promising therapeutic approach for CCA.
    Keywords:  CRISPR-Cas9 screening; VCP; cholangiocarcinoma; senescence
    DOI:  https://doi.org/10.1073/pnas.2519568122
  58. Proc Natl Acad Sci U S A. 2025 Sep 30. 122(39): e2513714122
    A diverse family of bacterial deubiquitinases is defined by the Coxiella burnetii effector EmcB.
    Jeffrey K Duncan-Lowey, Craig R Roy.
      The obligate intracellular pathogen Coxiella burnetii encodes a deubiquitinase called EmcB that prevents signaling by the host immune sensor Retinoic acid Inducible Gene I (RIG-I). The evolutionary relationship between EmcB and other deubiquitinases is currently unknown. Here, we show that EmcB defines a broad family of bacterial deubiquitinases divergently evolved from the eukaryotic Ovarian Tumor (OTU) family of deubiquitinases. Our data indicate that the emcB gene has an internal gene rearrangement that resulted in a circular permutation of the OTU fold. Proteins with a region homologous to the deubiquitinase domain of EmcB were identified in numerous members of the bacterial order Legionellales. Proteins with an EmcB-related deubiquitinase domain demonstrated cysteine protease activity that cleaved ubiquitin and ubiquitin-related modifiers. Most of the bacteria encoding EmcB-related proteins had components of the Dot/Icm Type IVB secretion system that delivers EmcB into host cells. Indeed, many of these EmcB-related proteins were translocated into eukaryotic cells by the Dot/Icm system of Legionella pneumophila, consistent with their predicted roles as bacterial effector proteins that target host pathways. Comparison of EmcB family deubiquitinases revealed a two-domain architecture of EmcB comprising a deubiquitinase domain and a ubiquitin binding module that confers enzyme specificity for certain polyubiquitin chains. This ubiquitin binding region in EmcB was found to be necessary for efficient inhibition of RIG-I signaling. Thus, these EmcB-related proteins represent a large family of deubiquitinating enzymes that arose by divergent evolution in Legionellales, likely enabling these bacteria to infect different host cells by targeting signaling pathways regulated by ubiquitin.
    Keywords:  bacterial effectors; bacterial pathogenesis; deubiquitinases; intracellular pathogens; type IV secretion
    DOI:  https://doi.org/10.1073/pnas.2513714122
  59. bioRxiv. 2025 Sep 17. pii: 2025.09.14.676157. [Epub ahead of print]
    Collagen Prolyl Hydroxylases Regulate HIF-α Levels Independently of pVHL in ccRCC.
    Yangsook Song Green, Karen Acuña-Pilarte, Marin Jones, Lily Halberg, Thai Huu Ho, Mei Yee Koh.
      The hypoxia-inducible factors, HIF-1α and HIF-2α, are master regulators of the hypoxia response. Under ambient conditions, both are hydroxylated by the HIF prolyl hydroxylases (HIF PHDs) resulting in ubiquitination by the pVHL E3 ligase complex, leading to subsequent proteasomal degradation. During hypoxia, the HIF PHDs are inhibited, resulting in HIF-α stabilization and transcriptional activation of genes involved in the adaptation to hypoxia. Previous studies have shown that the collagen PHD, P4HA1, which promotes proline hydroxylation of collagen, inhibits the HIF PHDs by modulating levels of α-ketoglutarate and succinate, thus enhancing HIF-1α stability by preventing pVHL-mediate degradation. Here, we investigate the role of collagen PHDs in the regulation of HIF-1/2α in the setting of pVHL deficiency in ccRCC. We show that the collagen PHDs P4HA1 and P4HA2 are required for HIF-1α translation and HIF-2α transcription and translation independently of pVHL function, through a mechanism regulated in part by P4HA1/2-driven collagen production. Thus, we reveal a novel pVHL-independent mechanism of HIF-1/2α regulation driven by P4HA1/2 in ccRCC. Since the HIFs have been strongly implicated in ccRCC initiation and progression, our data suggests that inhibition of P4HA1/2 may be a promising therapeutic strategy in ccRCC.
    DOI:  https://doi.org/10.1101/2025.09.14.676157
  60. EMBO Rep. 2025 Sep 22.
    UBE2O-mediated ubiquitylation directs cytoplasmic CTNNA1 to promote cell-to-ECM adhesions.
    Dan Xiang, Wenfeng Wu, Ruona Shi, Xiaoxiao Tang, Xiaofei Zhang.
      CTNNA1, a multifunctional protein that localizes at both the plasma membrane and the cytosol, plays crucial roles in actin dynamics regulation, cell-to-cell and cell-to-the extracellular matrix (ECM) adhesions and tumor suppression. Despite its diverse functions, the regulatory mechanisms by which cells coordinate CTNNA1's roles remain poorly understood. In this study, we identified UBE2O, a unique hybrid E2/E3 enzyme, as a key regulator that selectively interacts with and ubiquitylates cytosolic CTNNA1 in a phosphorylation-independent manner. Through comprehensive mass spectrometry-based interactome analysis of ubiquitylated CTNNA1, we reveal that the ubiquitylation of CTNNA1 diminishes its interaction with β-catenin while allowing its interaction with vinculin. This switch of molecular interactions promotes focal adhesions maturation, facilitates cell extension and matrix adhesion during the initial phases of cell spreading. Importantly, our findings demonstrate that ubiquitylation serves as a molecular switch that directs the regulatory roles of CTNNA1 to cell-to-ECM adhesions. This study advances our understanding of how ubiquitylation fine-tunes protein function in cell adhesion dynamics.
    Keywords:  CTNNA1; Cell Adhesions; Proteomics; UBE2O; Ubiquitylation
    DOI:  https://doi.org/10.1038/s44319-025-00585-4
  61. bioRxiv. 2025 Sep 19. pii: 2025.09.18.677077. [Epub ahead of print]
    A widespread protein misfolding mechanism is differentially rescued by chaperones based on gene essentiality.
    Ian Sitarik, Quyen Vu, Justin Petucci, Paulina Frutos, Hyebin Song, Edward P O'Brien.
      Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism explains why, in the mass spectrometry data, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest widespread NCLE misfolding, that such misfolded states in non-essential proteins can bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.
    DOI:  https://doi.org/10.1101/2025.09.18.677077
  62. bioRxiv. 2025 Sep 16. pii: 2025.09.11.675705. [Epub ahead of print]
    TRPML1 signaling at lysosomes-mitochondria nexus drives triple-negative breast cancer mitophagy, metabolic reprogramming and chemoresistance.
    Alia K Syeda, Shekoufeh Almasi, J Cory Benson, Barry E Kennedy, Ryan M Yoast, Scott M Emrich, Logan Slade, Shanmugasundaram Pakkiriswami, Vishnu V Vijayan, Shashi Gujar, Thomas Pulinilkunnil, Mohamed Trebak, Yassine El Hiani.
      Inter-organelle signaling mechanisms, particularly those at the lysosomes-mitochondria interface, are critical for cancer cell metabolism, mitophagy and survival. However, the incomplete understanding of these mechanisms has limited the development of effective therapies, especially for triple-negative breast cancers (TNBC). Here, we demonstrate the lysosomal Ca²⁺-release channel TRPML1 as a master regulator of mitochondrial bioenergetics in TNBC cells. TRPML1 knockdown (ML1-KD) in TNBC cells selectively compromises mitochondrial respiration, reprograms cell metabolism, and induces mitochondrial fragmentation without impacting non-cancerous cells. Mitochondria of ML1-KD TNBC cells sequester around the endoplasmic reticulum (ER), increasing mitochondria-ER contact sites at the expense of mitochondria-lysosomes contacts. Mechanistically, ML1-KD reduces lysosomal acidification, thus hindering autophagic flux and completion of autophagy. ML1-KD inhibits TFEB-mediated mitophagy and oxidative defense mechanisms while causing mitochondrial Ca 2+ overload, further impairing mitochondrial function. These alterations render ML1-KD TNBC cells highly sensitive to doxorubicin and paclitaxel at low doses that are typically ineffective on their own. Together, our findings establish TRPML1 as a critical inter-organelle regulator and highlight its potential as a therapeutic target to exploit the metabolic vulnerabilities of TNBC cells.
    DOI:  https://doi.org/10.1101/2025.09.11.675705
  63. mSphere. 2025 Sep 24. e0032925
    Hsp90 and associates shaping parasite biology.
    Abhilasha Gahlawat, Sunanda Bhattacharyya, Mrinal Kanti Bhattacharyya.
      Hsp90 is considered to be the master regulator of chaperone activity within the cellular context. In addition to aiding client maturation and maintaining protein homeostasis, Hsp90 serves various non-canonical functions in model eukaryotes: ranging from protein-trafficking into the nucleus to transcriptional regulation, from chromatin remodeling to assembly and disassembly of protein complexes during DNA repair and telomere maintenance. In performing all these trades, Hsp90 collaborates with its co-chaperones in a client-specific or function-specific manner. Hsp90 undergoes various conformational changes during its chaperone cycle, which is regulated via several post-translational modifications (PTM). Different combinations of such PTMs, known as the chaperone code, also play key regulatory roles for Hsp90 functions. Here, we examine various cellular functions of Hsp90 in protozoan parasites, particularly those that shuttle between insect host and human host, adapting to a temperature difference of at least 10°C. Our analyses reveal that most of the prominent co-chaperones are present in all these parasites, except for one that is essential in model eukaryotes. We reviewed the biochemical correlates of Hsp90 and its co-chaperone interactions and realized that the physiological significance of such interplay is largely unknown in the protozoan parasites. One striking observation is the lack of sequence conservation of the parasitic co-chaperones with their human counterparts, making them attractive drug targets. Our analyses revealed that in spite of the identification of few PTMs of parasitic Hsp90 proteins, the chaperone codes remain largely elusive.IMPORTANCEHsp90 is a pivotal molecular chaperone involved in maintaining proteostasis and facilitating the maturation of diverse client proteins. Beyond its canonical folding functions, Hsp90 plays non-canonical roles in nuclear trafficking, transcriptional regulation, chromatin remodeling, and DNA repair. These activities are tightly regulated through interactions with specific co-chaperones and through post-translational modifications, collectively forming the "chaperone code." This study examines Hsp90's role in thermal adaptation of protozoan parasites when shuttling between the insect and human hosts. Here, we summarize the canonical and diverse non-canonical functions of Hsp90 in three protozoan parasites: Plasmodium, Leishmania, and Trypanosoma. We highlight all the Hsp90 isoforms found in these three parasites and also illustrate all the co-chaperones and post-translational modifications of Hsp90 found to be present in these protozoan parasites. Importantly, the divergence in co-chaperone sequences from human homologs in these parasites presents a promising avenue for targeted antiparasitic drug discovery and development.
    Keywords:  Hsp90; Leishmania; Plasmodium; Trypanosoma; chaperone code; co-chaperone
    DOI:  https://doi.org/10.1128/msphere.00329-25
  64. Biophys J. 2025 Sep 24. pii: S0006-3495(25)00615-0. [Epub ahead of print]
    Diverse Conformational Ensembles Define the Shared Folding-Allosteric Landscapes of Protein Kinases.
    Dhruv Kumar Chaurasiya, Athi N Naganathan.
      Sequence variation across members of an enzyme family contributes to diverse ensemble behaviors, which subtly influence substrate affinity, selectivity and regulation. A classic example is the family of eukaryotic protein kinases (EPKs), which regulate numerous cellular processes and serve as important drug targets. Here, we dissect the consequences of sequence variation on the folding-conformational landscapes by performing a meta-analysis of 274 EPKs through a structure-based statistical mechanical framework. We find that EPKs populate several partially structured states in their native ensemble with a hierarchy of structural order in the N-terminal lobe that is critical for catalysis and activation. Despite this, the (un)folding mechanism is uniquely conserved across the majority of kinases, with the N-terminal lobe unfolding first. Kinase activation modulates the local stability and thermodynamic connectivity in a non-conserved manner and across the entire structure, due to the strong coupling between the active site residues to distant sites, including the established allosteric pockets. We further show how activation drives the Abl kinase ensemble towards a more folded and thermodynamically coupled system in a graded manner. Our work uncovers the thermodynamic design principles of kinases with insights into allostery, while shedding light on the extents to which ensemble behaviors are impacted by sequence variations in paralogs.
    Keywords:  Activity; Conformational landscape; Folding mechanism; Function; Mutations; Thermodynamic coupling
    DOI:  https://doi.org/10.1016/j.bpj.2025.09.035
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