bims-proteo Biomed News
on Proteostasis
Issue of 2025–04–20
fifty-two papers selected by
Eric Chevet, INSERM
  1. Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity.
  2. Mechanism of small heat shock protein client sequestration and induced polydispersity.
  3. A nanobody that binds to the backside of the ubiquitin conjugating enzyme Ube2G2 differentially affects interactions with its partner E3 Ligases.
  4. ATG9A controls all stages of autophagosome biogenesis.
  5. Induced proximity to PML protects TDP-43 from aggregation via SUMO-ubiquitin networks.
  6. Targeted Protein Degradation by KLHDC2 Ligands Identified by High Throughput Screening.
  7. Identification of Omaveloxolone as An Endoplasmic Reticulum Associated Degradation Inhibitor That Induces Early Apoptotic Signaling in Multiple Myeloma.
  8. Mechanism of client loading from BiP to Grp94 and its disruption by select inhibitors.
  9. Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.
  10. Deep Visual Proteomics maps proteotoxicity in a genetic liver disease.
  11. The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.
  12. Dynamic RNA binding and unfolding by nonsense-mediated mRNA decay factor UPF2.
  13. Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins.
  14. Integrator loss leads to dsRNA formation that triggers the integrated stress response.
  15. Structural mechanism of FusB-mediated rescue from fusidic acid inhibition of protein synthesis.
  16. The mitochondrial unfolded protein response: acting near and far.
  17. Genetically encoded tool for manipulation of ΔΨm identifies its role as the driver of ISR induced by ATP synthase dysfunction.
  18. PIN1-SUMO2/3 motif suppresses excessive RNF168 chromatin accumulation and ubiquitin signaling to promote IR resistance.
  19. Chemically Induced Nuclear Pore Complex Protein Degradation via TRIM21.
  20. Structural diversity and oligomerization of bacterial ubiquitin-like proteins.
  21. PML mutants from arsenic-resistant patients reveal SUMO1-TOPORS and SUMO2/3-RNF4 degradation pathways.
  22. Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.
  23. Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer.
  24. Disruption of Intracellular Calcium Homeostasis Leads to ERLIN2-Linked Hereditary Spastic Paraplegia in Patient-Derived Stem Cell Models.
  25. The Role of WIPI2, ATG16L1 and ATG12-ATG5 in selective and nonselective autophagy.
  26. Inhibitor-based modulation of huntingtin aggregation mechanisms mitigates fibril-induced cellular stress.
  27. A tripartite cell-free translation system to study mammalian translation.
  28. Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress.
  29. Programmable protein stabilization with language model-derived peptide guides.
  30. Coupling of ribosome biogenesis and translation initiation in human mitochondria.
  31. Usutu virus NS4A induces autophagy and is targeted by the selective autophagy receptor p62/SQSTM1 for degradation.
  32. Proteome-Wide Data Guides the Discovery of Lysine-Targeting Covalent Inhibitors Using DNA-Encoded Chemical Libraries.
  33. A non-syndromic orofacial cleft risk locus links tRNA splicing defects to neural crest cell pathologies.
  34. Thiol isomerase ERp18 enhances platelet activation and arterial thrombosis.
  35. An Msp1-Protease Chimera Captures Transient AAA+ Interactions and Unveils Ost4 Mislocalization Errors.
  36. Integrated analysis of transcriptional and metabolic responses to mitochondrial stress.
  37. RabGAP1L modulates Rab7A and Rab10 to orchestrate cell-autonomous immunity.
  38. Genetic inactivation of FAAP100 causes Fanconi anemia due to disruption of the monoubiquitin ligase core complex.
  39. Tauroursodeoxycholic Acid (TUDCA) Reduces ER Stress and Lessens Disease Activity in Ulcerative Colitis.
  40. The versatile role of YidC in membrane protein biosynthesis and quality control.
  41. A non-canonical cGAS-STING pathway drives cellular and organismal aging.
  42. Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response.
  43. Exploiting the therapeutic vulnerability of IDH-mutant gliomas with zotiraciclib.
  44. Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity.
  45. Structural basis for nucleolin recognition of MYC promoter G-quadruplex.
  46. RNase L Produces tRNA-derived RNAs that Contribute to Translation Inhibition.
  47. Proteomic-based stemness score measures oncogenic dedifferentiation and enables the identification of druggable targets.
  48. RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling.
  49. Emerging roles for microproteins as critical regulators of endoplasmic reticulum function and cellular homeostasis.
  50. RNF149 modulates the type I IFN innate antiviral immune responses through degrading IRF3.
  51. ATF6α inhibits ΔNp63α expression to promote breast cancer metastasis by the GRP78-AKT1-FOXO3a signaling.
  52. Engineered depalmitoylases enable selective manipulation of protein localization and function.
  1. bioRxiv. 2025 Apr 05. pii: 2025.04.04.646800. [Epub ahead of print]
    Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity.
    Gabriel M Kline, Lisa Boinon, Adrian Guerrero, Sergei Kutseikin, Gabrielle Cruz, Marnie P Williams, Ryan J Paxman, William E Balch, Jeffery W Kelly, Tingwei Mu, R Luke Wiseman.
      Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically-diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a compound that promotes adaptive ER proteostasis remodeling through mechanisms including activation of the ATF6 signaling arm of the unfolded protein response (UPR). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated α1-antitrypsin (A1AT) variant and an epilepsy-associated GABA A receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically-diverse protein misfolding disorders.
    DOI:  https://doi.org/10.1101/2025.04.04.646800
  2. Nat Commun. 2025 Apr 16. 16(1): 3635
    Mechanism of small heat shock protein client sequestration and induced polydispersity.
    Adam P Miller, Steve L Reichow.
      Small heat shock proteins (sHSPs) act as first responders during cellular stress, sequestering destabilized proteins (clients) to prevent aggregation and facilitate refolding or degradation. This critical function, conserved across all life, is linked to proteostasis and protein misfolding diseases. However, the extreme molecular plasticity of sHSP/client complexes has limited mechanistic understanding. Here, we present high-resolution cryo-EM structures of Methanocaldococcus jannaschii sHSP (mjHSP16.5) in apo and multiple client-bound states. The ensemble reveals molecular mechanisms of client sequestration, highlighting cooperative chaperone-client interactions. Client engagement polarizes scaffold stability, promoting higher-order assembly and enhanced sequestration. Higher-order states suggest multiple sHSP/client assembly pathways, including subunit insertion at destabilized geometrical features. These findings provide critical insights into sHSP chaperone function and the interplay between polydispersity and client handling under stress.
    DOI:  https://doi.org/10.1038/s41467-025-58964-3
  3. Commun Biol. 2025 Apr 16. 8(1): 614
    A nanobody that binds to the backside of the ubiquitin conjugating enzyme Ube2G2 differentially affects interactions with its partner E3 Ligases.
    Pavana Suresh, Charlotte Wijne, Zhen-Yu J Sun, Nanette Becht, Ishani Sahay, Novalia Pishesha, Hidde Ploegh.
      Ubiquitin conjugating E2 enzymes are a set of ~40 proteins that play a central role in the ubiquitination cascade. They transfer ubiquitin from an E1 enzyme to substrates with the help of an E3 enzyme. The members of the E2 family share structural similarity in their conserved UBC fold. This complicates an assessment of the specificity of E2-E3 interactions. We identified a nanobody that binds to the 'backside' region of Ube2G2, an E2 involved in ER protein quality control. This binding does not affect ubiquitin loading but shows varying degrees of inhibition on E3-mediated ubiquitination, in the order HRD1 > CHIP >> TRC8. A naturally occurring segment that binds Ube2G2's backside, referred to as G2BR (Ube2G2 Binding Region), shows a similar inhibitory effect depending on the identity of the interacting E3. The G2BR in the Ube2G2-cognate E3 Gp78 enhances Ube2G2's activity, but its deletion results in a similar inhibition upon addition of the nanobody. Occupation of a single binding site on an E2 can thus affect its interactions with different E3s.
    DOI:  https://doi.org/10.1038/s42003-025-08038-3
  4. Autophagy. 2025 Apr 16.
    ATG9A controls all stages of autophagosome biogenesis.
    Ruheena Javed, Muriel Mari, Einar Trosdal, Thabata Lopes Alberto Duque, Masroor Paddar, Lee Allers, Prithvi Akepati, Michal H Mudd, Fulvio Reggiori, Vojo Deretic.
      Canonical autophagy is an intracellular pathway that degrades and recycles cellular components. A key step of this pathway is the formation of double-membraned organelles, known as autophagosomes, an emblematic feature of macroautophagy. For convenience, the formation of autophagosomes can be categorized into sequential steps, initiation (X), expansion (Y) and closure (Z). ATG9A is an integral membrane protein known for its role in the X and Y steps. whereby it organizes phagophore membrane assembly and its growth. Here, we report a previously unappreciated function of mammalian ATG9A in directing the last step Z. In particular, ATG9A partners with the key ESCRT-III component CHMP2A through IQGAP1 to facilitate autophagosome closure. Thus, ATG9A orchestrates all stages of autophagosome membrane biogenesis, from phagophore initiation to its closure. This makes ATG9A a unique ATG factor that works as a central hub in autophagosome biogenesis.
    Keywords:  Autophagy; ESCRT; IQGAP; closure; mitophagy; phagophore; tuberculosis
    DOI:  https://doi.org/10.1080/15548627.2025.2494802
  5. Nat Chem Biol. 2025 Apr 17.
    Induced proximity to PML protects TDP-43 from aggregation via SUMO-ubiquitin networks.
    Kristina Wagner, Jan Keiten-Schmitz, Bikash Adhikari, Upayan Patra, Koraljka Husnjak, François McNicoll, Dorothee Dormann, Michaela Müller-McNicoll, Georg Tascher, Elmar Wolf, Stefan Müller.
      The established role of cytosolic and nuclear inclusions of TDP-43 in the pathogenesis of neurodegenerative disorders has multiplied efforts to understand mechanisms that control TDP-43 aggregation and has spurred searches for approaches limiting this process. Formation and clearance of TDP-43 aggregates are controlled by an intricate interplay of cellular proteostasis systems that involve post-translational modifications and frequently rely on spatial control. We demonstrate that attachment of the ubiquitin-like SUMO2 modifier compartmentalizes TDP-43 in promyelocytic leukemia protein (PML) nuclear bodies and limits the aggregation of TDP-43 in response to proteotoxic stress. Exploiting this pathway through proximity-inducing recruitment of TDP-43 to PML triggers a SUMOylation-ubiquitylation cascade protecting TDP-43 from stress-induced insolubility. The protective function of PML is mediated by ubiquitylation in conjunction with the p97 disaggregase. Altogether, we demonstrate that SUMO-ubiquitin networks protect cells from insoluble TDP-43 inclusions and propose the functionalization of PML as a potential future therapeutic avenue countering aggregation.
    DOI:  https://doi.org/10.1038/s41589-025-01886-4
  6. bioRxiv. 2025 Apr 01. pii: 2025.03.31.646306. [Epub ahead of print]
    Targeted Protein Degradation by KLHDC2 Ligands Identified by High Throughput Screening.
    Han Zhou, Tongliang Zhou, Wenli Yu, Liping Liu, Yeonjin Ko, Kristen A Johnson, Ian A Wilson, Peter G Schultz, Michael J Bollong.
      Proteolysis targeting chimeras (PROTACs) enable the selective and sub-stoichiometric elimination of pathological proteins, yet only two E3 ligases are routinely used for this purpose. Here, we expand the repertoire of PROTAC compatible E3 ligases by identifying a novel small molecule scaffold targeting the ubiquitin E3 ligase KLHDC2 using a fluorescence polarization-based high throughput screen. We highlight the utility of this ligand with the synthesis of PROTACs capable of potently degrading BRD4 in cells. This work affords additional chemical matter for targeting KLHDC2 and suggests a practical approach for identifying novel E3 binders by high throughput screening.
    DOI:  https://doi.org/10.1101/2025.03.31.646306
  7. bioRxiv. 2025 Apr 03. pii: 2025.04.02.646787. [Epub ahead of print]
    Identification of Omaveloxolone as An Endoplasmic Reticulum Associated Degradation Inhibitor That Induces Early Apoptotic Signaling in Multiple Myeloma.
    Erin M Kropp, Sho Matono, Olivia Y Wang, Aaron M Robida, Malathi Kandarpa, Jineigh L Grant, Bryndon J Oleson, Andrew Alt, Moshe Talpaz, Matthew Pianko, Qing Li.
      Endoplasmic reticulum-associated degradation (ERAD) is essential for maintaining protein homeostasis, yet its regulatory mechanisms remain poorly understood. A major challenge in studying ERAD is the lack of specific inhibitors targeting the ERAD complex. To address this, we conducted a cell-based high-throughput screen using the FDA-repurposing library and identified omaveloxolone (RTA408) as a potent ERAD inhibitor that selectively impairs the degradation of ER luminal and membrane substrates. Beyond its utility in identifying ERAD substrates, RTA408 exhibits strong cytotoxic effects in multiple myeloma (MM), an incurable plasma cell malignancy. RTA408 inhibits ERAD activity and rapidly induces apoptotic signaling via caspase 8 and the death-inducing signaling complex (DISC). Notably, RTA408 is cytotoxic to malignant plasma cells, including those resistant to proteasome inhibitors, and demonstrates in vivo anti-myeloma activity. Our findings establish ERAD inhibitors as valuable tools for dissecting ERAD regulation while also highlighting their potential as therapeutic agents for MM.
    Highlights: We developed a cell-based screening approach to identify novel modulators of endoplasmic reticulum associated degradation (ERAD), with implications in studying ERAD biology and targeting plasma cell neoplasms.Screening of the FDA-repurposing library identified Omaveloxolone (RTA408) as an inhibitor of luminal and membrane ERAD substrate degradation, which can be leveraged to identify ERAD substrates.maveloxolone treatment rapidly induces the unfolded protein response and apoptosis that is dependent on caspase 8 and death-inducing signaling complex (DISC) in multiple myeloma cells. maveloxolone exhibits cytotoxic effects against multiple myeloma cells in vitro and in vivo and induces apoptosis in primary plasma cells from patients with relapsed/refractory myeloma.
    DOI:  https://doi.org/10.1101/2025.04.02.646787
  8. Nat Commun. 2025 Apr 15. 16(1): 3575
    Mechanism of client loading from BiP to Grp94 and its disruption by select inhibitors.
    Tara P Azam, Luna Han, Erin E Deans, Bin Huang, Reyal Hoxie, Larry J Friedman, Jeff Gelles, Timothy O Street.
      Hsp90 chaperones are a long-standing cancer drug target with numerous ATP-competitive inhibitors in clinical trials. Client proteins are transferred from Hsp70 to Hsp90 in a stepwise process of client delivery, loading, and trapping, but little is known about how inhibitors influence these steps. By examining the ER-resident BiP/Grp94 system (Hsp70/Hsp90 paralogs), we discover that some inhibitors allow BiP to push Grp94 into the client loading conformation, whereas other inhibitors block this conformational change and destabilize a BiP/client/Grp94 ternary complex. We uncover how BiP drives Grp94 into the client loading state and identify a structural explanation for why only a select group of inhibitors disrupt client loading on Grp94. These results show a client loading mechanism with specific shared features between the Hsp70/Hsp90 systems in the ER and cytosol and open a new avenue for rational Hsp90 drug design.
    DOI:  https://doi.org/10.1038/s41467-025-58658-w
  9. Nat Commun. 2025 Apr 17. 16(1): 3401
    Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.
    Koki Nakamura, Saeko Aoyama-Ishiwatari, Takahiro Nagao, Mohammadreza Paaran, Christopher J Obara, Yui Sakurai-Saito, Jake Johnston, Yudan Du, Shogo Suga, Masafumi Tsuboi, Makoto Nakakido, Kouhei Tsumoto, Yusuke Kishi, Yukiko Gotoh, Chulhwan Kwak, Hyun-Woo Rhee, Jeong Kon Seo, Hidetaka Kosako, Clint Potter, Bridget Carragher, Jennifer Lippincott-Schwartz, Franck Polleux, Yusuke Hirabayashi.
      Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.
    DOI:  https://doi.org/10.1038/s41467-025-58538-3
  10. Nature. 2025 Apr 16.
    Deep Visual Proteomics maps proteotoxicity in a genetic liver disease.
    Florian A Rosenberger, Sophia C Mädler, Katrine Holtz Thorhauge, Sophia Steigerwald, Malin Fromme, Mikhail Lebedev, Caroline A M Weiss, Marc Oeller, Maria Wahle, Andreas Metousis, Maximilian Zwiebel, Niklas A Schmacke, Sönke Detlefsen, Peter Boor, Ondřej Fabián, Soňa Fraňková, Aleksander Krag, Pavel Strnad, Matthias Mann.
      Protein misfolding diseases, including α1-antitrypsin deficiency (AATD), pose substantial health challenges, with their cellular progression still poorly understood1-3. We use spatial proteomics by mass spectrometry and machine learning to map AATD in human liver tissue. Combining Deep Visual Proteomics (DVP) with single-cell analysis4,5, we probe intact patient biopsies to resolve molecular events during hepatocyte stress in pseudotime across fibrosis stages. We achieve proteome depth of up to 4,300 proteins from one-third of a single cell in formalin-fixed, paraffin-embedded tissue. This dataset reveals a potentially clinically actionable peroxisomal upregulation that precedes the canonical unfolded protein response. Our single-cell proteomics data show α1-antitrypsin accumulation is largely cell-intrinsic, with minimal stress propagation between hepatocytes. We integrated proteomic data with artificial intelligence-guided image-based phenotyping across several disease stages, revealing a late-stage hepatocyte phenotype characterized by globular protein aggregates and distinct proteomic signatures, notably including elevated TNFSF10 (also known as TRAIL) amounts. This phenotype may represent a critical disease progression stage. Our study offers new insights into AATD pathogenesis and introduces a powerful methodology for high-resolution, in situ proteomic analysis of complex tissues. This approach holds potential to unravel molecular mechanisms in various protein misfolding disorders, setting a new standard for understanding disease progression at the single-cell level in human tissue.
    DOI:  https://doi.org/10.1038/s41586-025-08885-4
  11. bioRxiv. 2025 Apr 03. pii: 2025.04.01.646556. [Epub ahead of print]
    The glycosyltransferase ALG3 is an AKT substrate that regulates protein N-glycosylation.
    Adrija J Navarro-Traxler, Laura Ghisolfi, Evan C Lien, Alex Toker.
      The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.
    DOI:  https://doi.org/10.1101/2025.04.01.646556
  12. RNA. 2025 Apr 17. pii: rna.080300.124. [Epub ahead of print]
    Dynamic RNA binding and unfolding by nonsense-mediated mRNA decay factor UPF2.
    Jenn-Yeu Alvin Szeto, Mirella Vivoli Vega, Justine Mailliot, George Orriss, Lingling Sun, Joshua C Bufton, Kyle T Powers, Sathish K N Yadav, Imre Berger, Christiane Schaffitzel.
      Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance pathway involved in translational control and gene expression regulation. Core NMD factors UPF1, UPF2 and UPF3B are conserved from yeast to humans and essential to target mRNAs with a premature stop codon for decay. UPF2 binding to UPF1 activates UPF1's ATPase and helicase activities, and UPF2 binding to UPF3B is important for its association with the exon-junction complex and efficient NMD. However, UPF2's association with RNA remains largely uncharacterized. Here, we analyze nucleic acid binding, identifying the first and third MIF4G domains of UPF2 as main RNA-/DNA-binding modules. We find that UPF2's MIF4G domain-3 has RNA annealing activity while full-length UPF2 unfolds our reporter hairpin-RNA structure. We show that UPF2 preferentially binds and stabilizes single-stranded RNA (ss-RNA) in a sequence-independent manner. Concomitant to ss-RNA binding, UPF2 undergoes a distinct conformational change in its otherwise highly dynamic structure. UPF2's RNA binding and unfolding activity may support UPF1's helicase and mRNP remodeling activity and, in combination with UPF3B, stabilize UPF1's association with nonsense mRNA.
    Keywords:  Dynamic RNA-UPF Protein Complexes; Nonsense-Mediated mRNA Decay; RNA Unfolding; Up-Frameshift Protein 2
    DOI:  https://doi.org/10.1261/rna.080300.124
  13. Biochem J. 2025 Apr 09. pii: BCJ20240473. [Epub ahead of print]
    Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins.
    Lauren Rice, Nicholas Marzano, Dezerae Cox, Bailey Skewes, Antoine van Oijen, Heath Ecroyd.
      Small heat shock proteins (sHsps) are molecular chaperones that act to prevent the aberrant aggregation of misfolded proteins. Whilst it is suggested that sHsps prevent aggregation by binding to misfolded client proteins, the dynamic and heterogeneous nature of sHsps has hindered attempts to establish the mechanistic details of how sHsp-client protein complexes form. Single-molecule approaches have emerged as a powerful tool to investigate dynamic and heterogeneous interactions such as those that can occur between sHsps and their client proteins. Here, we use total internal reflection fluorescence microscopy to observe and characterise the complexes formed between model aggregation-prone client proteins [firefly luciferase (FLUC), rhodanese, and chloride intracellular channel 1 protein (CLIC)], and the human sHsps αB-crystallin (αB-c; HSPB5) and Hsp27 (HSPB1). We show that small (monomeric or dimeric) forms of both αB-c and Hsp27 bind to misfolded or oligomeric forms of the client proteins at early stages of aggregation, resulting in the formation of soluble sHsp-client complexes. Stoichiometric analysis of these complexes revealed that additional αB-c subunits accumulate onto pre-existing sHsp-client complexes to form larger species - this does not occur to the same extent for Hsp27. Instead, Hsp27-client interactions tend to be more transient than those of αB-c. Elucidating these mechanisms of sHsp function is crucial to our understanding of how these molecular chaperones act to inhibit protein aggregation and maintain cellular proteostasis.
    Keywords:  Hsp27; molecular chaperones; photobleaching; proteostasis; single-molecule fluorescence; αB-crystallin
    DOI:  https://doi.org/10.1042/BCJ20240473
  14. Cell. 2025 Apr 10. pii: S0092-8674(25)00343-5. [Epub ahead of print]
    Integrator loss leads to dsRNA formation that triggers the integrated stress response.
    Apoorva Baluapuri, Nicole ChenCheng Zhao, Ryan J Marina, Kai-Lieh Huang, Anastasia Kuzkina, Maria E Amodeo, Chad B Stein, Lucie Y Ahn, Jordan S Farr, Ashleigh E Schaffer, Vikram Khurana, Eric J Wagner, Karen Adelman.
      Integrator (INT) is a metazoan-specific complex that targets promoter-proximally paused RNA polymerase II (RNAPII) for termination, preventing immature RNAPII from entering gene bodies and functionally attenuating transcription of stress-responsive genes. Mutations in INT subunits are associated with many human diseases, including cancer, ciliopathies, and neurodevelopmental disorders, but how reduced INT activity contributes to disease is unknown. Here, we demonstrate that the loss of INT-mediated termination in human cells triggers the integrated stress response (ISR). INT depletion causes upregulation of short genes such as the ISR transcription factor activating transcription factor 3 (ATF3). Further, immature RNAPII that escapes into genes upon INT depletion is prone to premature termination, generating incomplete pre-mRNAs with retained introns. Retroelements within retained introns form double-stranded RNA (dsRNA) that is recognized by protein kinase R (PKR), which drives ATF4 activation and prolonged ISR. Critically, patient cells with INT mutations exhibit dsRNA accumulation and ISR activation, thereby implicating chronic ISR in diseases caused by INT deficiency.
    Keywords:  IR-Alu; Integrator; RNA polymerase II pausing; double-stranded RNA; gene regulation; integrated stress response; premature cleavage and polyadenylation; premature termination; protein kinase R
    DOI:  https://doi.org/10.1016/j.cell.2025.03.025
  15. Nat Commun. 2025 Apr 18. 16(1): 3693
    Structural mechanism of FusB-mediated rescue from fusidic acid inhibition of protein synthesis.
    Adrián González-López, Xueliang Ge, Daniel S D Larsson, Carina Sihlbom Wallem, Suparna Sanyal, Maria Selmer.
      The antibiotic resistance protein FusB rescues protein synthesis from inhibition by fusidic acid (FA), which locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis. Here, we present time-resolved single-particle cryo-EM structures explaining the mechanism of FusB-mediated rescue. FusB binds to the FA-trapped EF-G on the ribosome, causing large-scale conformational changes of EF-G that break interactions with the ribosome, tRNA, and mRNA. This leads to dissociation of EF-G from the ribosome, followed by FA release. We also observe two independent binding sites of FusB on the classical-state ribosome, overlapping with the binding site of EF-G to each of the ribosomal subunits, yet not inhibiting tRNA delivery. The affinity of FusB to the ribosome and the concentration of FusB in S. aureus during FusB-mediated resistance support that direct binding of FusB to ribosomes could occur in the cell. Our results reveal an intricate resistance mechanism involving specific interactions of FusB with both EF-G and the ribosome, and a non-canonical release pathway of EF-G.
    DOI:  https://doi.org/10.1038/s41467-025-58902-3
  16. Biol Chem. 2025 Apr 17.
    The mitochondrial unfolded protein response: acting near and far.
    Nikolaos Charmpilas, Qiaochu Li, Thorsten Hoppe.
      Mitochondria are central hubs of cellular metabolism and their dysfunction has been implicated in a variety of human pathologies and the onset of aging. To ensure proper mitochondrial function under misfolding stress, a retrograde mitochondrial signaling pathway known as UPRmt is activated. The UPRmt ensures that mitochondrial stress is communicated to the nucleus, where gene expression for several mitochondrial proteases and chaperones is induced, forming a protective mechanism to restore mitochondrial proteostasis and function. Importantly, the UPRmt not only acts within cells, but also exhibits a conserved cell-nonautonomous activation across species, where mitochondrial stress in a defined tissue triggers a systemic response that affects distant organs. Here, we summarize the molecular basis of the UPRmt in the invertebrate model organism Caenorhabditis elegans and in mammals. We also describe recent findings on cell-nonautonomous activation of the UPRmt in worms, flies and mice, and how UPRmt activation in specific tissues affects organismal metabolism and longevity.
    Keywords:  cell-nonautonomous regulation; integrated stress response; mitochondria; mitochondrial unfolded protein response; stress signaling
    DOI:  https://doi.org/10.1515/hsz-2025-0107
  17. Cell Chem Biol. 2025 Apr 17. pii: S2451-9456(25)00097-2. [Epub ahead of print]32(4): 620-630.e6
    Genetically encoded tool for manipulation of ΔΨm identifies its role as the driver of ISR induced by ATP synthase dysfunction.
    Mangyu Choe, Alex E Ekvik, Gretchen Stalnaker, Hijai R Shin, Denis V Titov.
      Mitochondrial membrane potential (ΔΨm) is one of the key parameters controlling cellular bioenergetics. Investigation of the role of ΔΨm in live cells is complicated by a lack of tools for its direct manipulation without off-target effects. Here, we adopted the uncoupling protein UCP1 from brown adipocytes as a genetically encoded tool for direct manipulation of ΔΨm. We validated the ability of exogenously expressed UCP1 to induce uncoupled respiration and lower ΔΨm in mammalian cells. UCP1 expression lowered ΔΨm to the same extent as chemical uncouplers but did not inhibit cell proliferation, suggesting that it manipulates ΔΨm without the off-target effects of chemical uncouplers. Using UCP1, we revealed that elevated ΔΨm is the driver of the integrated stress response induced by ATP synthase inhibition in mammalian cells.
    Keywords:  ATP synthase inhibition; GEMMs; ISR; UCP1; genetically encoded tools for manipulation of metabolism; integrated stress response,; mitochondrial membrane potential; ΔΨm
    DOI:  https://doi.org/10.1016/j.chembiol.2025.03.007
  18. Nat Commun. 2025 Apr 14. 16(1): 3399
    PIN1-SUMO2/3 motif suppresses excessive RNF168 chromatin accumulation and ubiquitin signaling to promote IR resistance.
    Anoop S Chauhan, Matthew J W Mackintosh, Joseph Cassar, Alexander J Lanz, Mohammed Jamshad, Hannah L Mackay, Alexander J Garvin, Alexandra K Walker, Satpal S Jhujh, Teresa Carlomagno, Aneika C Leney, Grant S Stewart, Joanna R Morris.
      RNF168 is an E3 ubiquitin ligase critical to the mammalian DNA double-strand break repair response. The protein is recruited to and amplifies ubiquitin signals at damaged chromatin and, if not properly regulated, can drive an uncontrolled ubiquitin cascade potentially harmful to repair outcomes. Several indirect mechanisms restrict RNF168 positive feedback, and a longstanding question has been whether these alone suppress excessive RNF168 signaling or whether mechanisms to remove RNF168 from damaged chromatin exist. Here, we reveal a cascade of post-translational modifications which act at three adjacent amino acids, threonine-208, proline-209 and lysine-210, to process RNF168 actively. Phosphorylation at threonine-208 by CDK1/2 induces interaction with the peptidyl-prolyl isomerase PIN1. PIN1 promotes RNF168 SUMOylation at lysine-210, resulting in p97/VCP mediated removal. These actions promote RNF168 clearance and limit RNF168 chromatin build-up. Thus, single amino acid substitutions of the regulatory motif (SUMO-PIN1-assisted Chromatin Regulator, SPaCR) that restrict PIN1 interaction or SUMOylation are sufficient to drive supraphysiological accumulation of RNF168, increased ubiquitin signaling, excessive 53BP1 recruitment and radiosensitivity. Our findings define a mechanism of direct RNF168 regulation that is part of the normal damage response, promoting RNF168 dissociation from chromatin and limiting deleterious ubiquitin signaling.
    DOI:  https://doi.org/10.1038/s41467-025-56974-9
  19. ACS Chem Biol. 2025 Apr 18.
    Chemically Induced Nuclear Pore Complex Protein Degradation via TRIM21.
    Xiaomei Li, Qingyang Wang, Anping Guo, Yaping Qiu, Qiuxia Chen, You Li, Lanjun Zhang, Yaxin Guo, Xiaoyun Meng, Shiqian Li, Guizhi Liu, Liyun Zhang, Jian Liu, Xianyang Li, Longying Cai, Xuemin Cheng, Chuan Liu, Xiaotao Wang, Andrew Wood, James Murray, Guansai Liu, Jin Li, Xiaodong Huang, Dengfeng Dou.
      Despite the exciting progress of bifunctional degrader molecules, also known as proteolysis-targeting chimeras (PROTACs), the rapidly expanding field is still significantly hampered by the lack of available E3 ligase ligands. Our research bridges this gap by uncovering a series of small-molecule ligands to the E3 ligase TRIM21 through DNA-Encoded Library (DEL) technology. We confirmed their interaction with TRIM21 using crystallography and demonstrated their antiproliferative effects across various cancer cell types. Furthermore, proteomic studies identified that the mRNA Export Factor GLE1 and the Nuclear Pore Complex Protein NUP155 were significantly downregulated on TRIM21 ligand treatment. This degradation required TRIM21 and was ubiquitin-proteasome-dependent. More specifically, NUP155 was the primary target for the TRIM21 ligands, while GLE1 was considered a passenger target on initial degradation of NUP155. Using immunofluorescence techniques, we further demonstrated that the degradation of GLE1 and NUP155 proteins impaired the integrity of the nuclear envelope, leading to cell death. Highlighted by this research, a novel mode of action has been discovered for the TRIM21 E3 ligase ligand, acting as a monovalent degrader that triggers de novo interaction with functional complex proteins and induces their degradation.
    DOI:  https://doi.org/10.1021/acschembio.4c00833
  20. Structure. 2025 Apr 08. pii: S0969-2126(25)00108-X. [Epub ahead of print]
    Structural diversity and oligomerization of bacterial ubiquitin-like proteins.
    Minheng Gong, Qiaozhen Ye, Yajie Gu, Lydia R Chambers, Andrey A Bobkov, Neal K Arakawa, Mariusz Matyszewski, Kevin D Corbett.
      Bacteria possess a variety of operons with homology to eukaryotic ubiquitination pathways that encode predicted E1, E2, E3, deubiquitinase, and ubiquitin-like proteins. Some of these pathways have recently been shown to function in anti-bacteriophage immunity, but the biological functions of others remain unknown. Here, we show that ubiquitin-like proteins in two bacterial operon families show surprising architectural diversity, possessing one to three β-grasp domains preceded by diverse N-terminal domains. We find that a large group of bacterial ubiquitin-like proteins possess three β-grasp domains and form homodimers and helical filaments mediated by conserved Ca2+ ion binding sites. Our findings highlight a distinctive mode of self-assembly for ubiquitin-like proteins and suggest that Ca2+-mediated ubiquitin-like protein filament assembly and/or disassembly enables cells to sense and respond to stress conditions that alter intracellular metal ion concentration.
    DOI:  https://doi.org/10.1016/j.str.2025.03.011
  21. J Cell Biol. 2025 Jun 02. pii: e202407133. [Epub ahead of print]224(6):
    PML mutants from arsenic-resistant patients reveal SUMO1-TOPORS and SUMO2/3-RNF4 degradation pathways.
    Ellis G Jaffray, Michael H Tatham, Barbara Mojsa, Anna Plechanovová, Alejandro Rojas-Fernandez, Julio C Y Liu, Niels Mailand, Adel F M Ibrahim, Graeme Ball, Iain M Porter, Ronald T Hay.
      Arsenic effectively treats acute promyelocytic leukemia by inducing SUMO and ubiquitin-dependent degradation of the promyelocytic leukemia (PML)-retinoic acid receptor alpha oncogenic fusion protein. However, some patients relapse with arsenic-resistant disease because of missense mutations in PML. To determine the mechanistic basis for arsenic resistance, PML-/- cells were reconstituted with YFP fusions of wild-type PML-V and two common patient mutants: A216T and L217F. Both mutants were resistant to degradation by arsenic but for different biochemical reasons. Arsenic did not trigger SUMOylation of A216T PML, which failed to recruit the SUMO-targeting ubiquitin ligases RNF4 and TOPORS. L217F PML did respond with increased SUMO2/3 conjugation that facilitated RNF4 engagement but failed to reach the threshold of SUMO1 conjugation required to recruit TOPORS. Thus, neither mutant accumulated the appropriate polyubiquitin signal required for p97 binding. These PML mutants have revealed a convergence of SUMO1, SUMO2/3, TOPORS, and RNF4 that facilitates the arsenic-induced degradation of PML.
    DOI:  https://doi.org/10.1083/jcb.202407133
  22. Cell Death Dis. 2025 Apr 12. 16(1): 280
    Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.
    Ylauna Christine Mégane Penalva, Sandra Paschkowsky, Jingyun Yang, Sherilyn Junelle Recinto, Jessica K Cinkornpumin, Marina Ruelas, Bin Xiao, Albert Nitu, Sin Young Kwon, Helen Yee-Li Wu, Hans Markus Munter, Bernadeta Michalski, Margaret Fahnestock, William A Pastor, David A Bennett, Lisa Marie Munter.
      Characteristic cerebral pathological changes of Alzheimer's disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aβ peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBDL4 cleaves APP in HEK293T cells, leading to decreased total APP and Aβ. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4's absence on APP physiology in vivo, we crossed APPtg mice to a RHBDL4 knockout (R4-/-) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and amyloidogenic processing when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNA-seq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active β-catenin due to decreased proteasomal clearance. Decreased β-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4's increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of β-catenin, contributing to cognitive impairment.
    DOI:  https://doi.org/10.1038/s41419-025-07579-z
  23. Nat Commun. 2025 Apr 16. 16(1): 3623
    Josephin Domain Containing 2 (JOSD2) inhibition as Pan-KRAS-mutation-targeting strategy for colorectal cancer.
    Tao Yuan, Yue Liu, Ruilin Wu, Meijia Qian, Weihua Wang, Yonghao Li, Hongdao Zhu, Jia'er Wang, Fujing Ge, Chenming Zeng, Xiaoyang Dai, Ronggui Hu, Tianhua Zhou, Qiaojun He, Hong Zhu, Bo Yang.
      KRAS is the most common mutated oncogenes in colorectal cancer (CRC), yet effective therapeutic strategies for targeting multiple KRAS mutations remained challenging. The prolonged protein stability of KRAS mutants contribute to their robust tumor-promoting effects, but the underlying mechanism is elusive. Herein by screening deubiquitinases (DUBs) siRNA library, we identify Josephin domain containing 2 (JOSD2) functions as a potent DUB that regulates the protein stability of KRAS mutants. Mechanistically, JOSD2 directly interacts with and stabilizes KRAS variants across different mutants, by reverting their proteolytic ubiquitination; while KRAS mutants reciprocally inhibit the catalytic activity of CHIP, a bona fide E3 ubiquitin ligase for JOSD2, thus forming a JOSD2/KRAS positive feedback circuit that significantly accelerates KRAS-mutant CRC growth. Inhibition of JOSD2 by RNA interference or its pharmacological inhibitor promotes the polyubiquitination and proteasomal degradation of KRAS mutants, and preferentially impede the growth of KRAS-mutant CRC including patient-derived cells/xenografts/organoids (PDCs/PDXs/PDOs) over that harboring wild-type KRAS. Collectively, this study not only reveals the crucial roles of JOSD2/KRAS positive feedback circuit in KRAS-mutant CRC, but also provides a rationale to target JOSD2 as the promising pan-KRAS-mutation-targeting strategy for the treatment of a broad population of CRC patients with KRAS variant across different mutant types.
    DOI:  https://doi.org/10.1038/s41467-025-58923-y
  24. Hum Mutat. 2023 ;2023 4834423
    Disruption of Intracellular Calcium Homeostasis Leads to ERLIN2-Linked Hereditary Spastic Paraplegia in Patient-Derived Stem Cell Models.
    Xintong Zhu, Xiaoyin Tan, Junwen Wang, Limeng Dai, Jia Li, Xingying Guan, Ziyi Wang, Mao Zhang, Jun Hu, Yun Bai, Hong Guo.
      Hereditary spastic paraplegia (HSP) is a category of neurodegenerative illnesses with significant clinical and genetic heterogeneity. Homozygous truncated variants of the ERLIN2 gene lead to HSP18 (MIM #611225). However, it is still unclear whether there is an autosomal dominant pathogenic pattern. The specific molecular mechanism needs to be investigated. We generated patient-derived iPSC models to study the mechanism of ERLIN2 heterogeneous variants leading to HSP. We identified a heterozygous missense variant p.Val71Ala of ERLIN2 in an HSP family. Based on IP-mass spectrometry, we found that the ERLIN2 heterozygous missense variant protein recruited the ubiquitin E3 ligase RNF213 to degrade IP3R1. The degradation of IP3R1 leads to the reduction of intracellular free calcium, which triggered endoplasmic reticulum (ER) stress-mediated apoptosis. Calcium homeostasis imbalance inhibited the MAPK signaling pathway that contributed to decreased cell proliferation. In summary, these results suggest that the autosomal dominant inheritance of heterozygous missense variants in ERLIN2 is a novel pathogenic mode of HSP. Furthermore, the disruption of intracellular calcium homeostasis is the pathological mechanism.
    DOI:  https://doi.org/10.1155/2023/4834423
  25. J Mol Biol. 2025 Apr 10. pii: S0022-2836(25)00204-9. [Epub ahead of print] 169138
    The Role of WIPI2, ATG16L1 and ATG12-ATG5 in selective and nonselective autophagy.
    Daniele Campisi, N'Toia Hawkins, Kennedy Bonjour, Thomas Wollert.
      Autophagy is a conserved cellular recycling pathway that delivers damaged or superfluous cytoplasmic material to lysosomes for degradation. In response to cytotoxic stress or starvation, autophagy can also sequester bulk cytoplasm and deliver it to lysosomes to regenerate building blocks. In macroautophagy, a membrane cisterna termed phagophore that encloses autophagic cargo is generated. The formation of the phagophore depends on a conserved machinery of autophagy related proteins. The phosphatidylinositol(3)-phosphate binding protein WIPI2 facilitates the transition from phagophore initiation to phagophore expansion by recruiting the ATG12-ATG5-ATG16L1 complex to phagophores. This complex functions as an E3-ligase to conjugate ubiquitin-like ATG8 proteins to phagophore membranes, which promotes tethering of cargo to phagophore membranes, phagophore expansion, maturation and the fusion of autophagosomes with lysosomes. ATG16L1 also has important functions independently of ATG12-ATG5 in autophagy and beyond. In this review, we will summarize the functions of WIPI2 and ATG16L1 in selective and nonselective autophagy.
    DOI:  https://doi.org/10.1016/j.jmb.2025.169138
  26. Nat Commun. 2025 Apr 15. 16(1): 3588
    Inhibitor-based modulation of huntingtin aggregation mechanisms mitigates fibril-induced cellular stress.
    Greeshma Jain, Marina Trombetta-Lima, Irina Matlahov, Hennrique Taborda Ribas, Tingting Chen, Raffaella Parlato, Giuseppe Portale, Amalia M Dolga, Patrick C A van der Wel.
      Huntington's disease (HD) is a neurodegenerative disorder in which mutated fragments of the huntingtin protein (Htt) undergo misfolding and aggregation. Since aggregated proteins can cause cellular stress and cytotoxicity, there is an interest in the development of small molecule aggregation inhibitors as potential modulators of HD pathogenesis. Here, we study how a polyphenol modulates the aggregation mechanism of huntingtin exon 1 (HttEx1) even at sub-stoichiometric ratios. Sub-stoichiometric amounts of curcumin impacted the primary and/or secondary nucleation events, extending the pre-aggregation lag phase. Remarkably, the disrupted aggregation process changed both the aggregate structure and its cell metabolic properties. When administered to neuronal cells, the 'break-through' protein aggregates induced significantly reduced cellular stress compared to aggregates formed in absence of inhibitors. Structural analysis by electron microscopy, small angle X-ray scattering (SAXS), and solid-state NMR spectroscopy identified changes in the fibril structures, probing the flanking domains in the fuzzy coat and the fibril core. We propose that changes in the latter relate to the presence or absence of polyglutamine (polyQ) β-hairpin structures. Our findings highlight multifaceted consequences of small molecule inhibitors that modulate the protein misfolding landscape, with potential implications for treatment strategies in HD and other amyloid disorders.
    DOI:  https://doi.org/10.1038/s41467-025-58691-9
  27. Nat Protoc. 2025 Apr 16.
    A tripartite cell-free translation system to study mammalian translation.
    Frederic S W Arendrup, Kasper L Andersen, Anders H Lund.
      Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate. The proteome constitutes the ultimate manifestation of gene expression programs with multiple layers of regulation to ensure faithful execution. While current high-throughput techniques to investigate regulation at the level of translation, such as Ribo-Seq and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have rigid composition and often rely on non-human model systems and artificially designed mRNA constructs. Here we detail a tripartite cell-free translation system based on the separation of mRNAs, ribosomes and ribosome-depleted cytoplasmic lysate from human cells, allowing for flexible reconstitution of translation reactions, which can be performed in 1-4 days. In this setup, cellular parts such as the cytoplasmic lysate can be kept constant, while ribosome complexes or mRNA can be varied or subjected to treatments or vice versa. We detail how complete mRNA populations can be used as input with subsequent detection of nascent peptides using autoradiography or mass spectrometry. We utilize this protocol to resolve which aspects of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which have been unresolvable until now.
    DOI:  https://doi.org/10.1038/s41596-025-01155-7
  28. Sci Adv. 2025 Apr 18. 11(16): eads6830
    Spatial analysis of mitochondrial gene expression reveals dynamic translation hubs and remodeling in stress.
    Adam Begeman, John A Smolka, Ahmad Shami, Tejashree Pradip Waingankar, Samantha C Lewis.
      Protein- and RNA-rich bodies contribute to the spatial organization of gene expression in the cell and are also sites of quality control critical to cell fitness. In most eukaryotes, mitochondria harbor their own genome, and all steps of mitochondrial gene expression co-occur within a single compartment-the matrix. Here, we report that processed mitochondrial RNAs are consolidated into micrometer-scale translation hubs distal to mitochondrial DNA transcription and RNA processing sites in human cells. We find that, during stress, mitochondrial messenger and ribosomal RNA are sequestered in mesoscale bodies containing mitoribosome components, concurrent with suppression of active translation. Stress bodies are triggered by proteotoxic stress downstream of double-stranded RNA accumulation in cells lacking unwinding activity of the highly conserved helicase SUPV3L1/SUV3. We propose that the spatial organization of nascent polypeptide synthesis into discrete domains serves to throttle the flow of genetic information to support recovery of mitochondrial quality control.
    DOI:  https://doi.org/10.1126/sciadv.ads6830
  29. Nat Commun. 2025 Apr 15. 16(1): 3555
    Programmable protein stabilization with language model-derived peptide guides.
    Lauren Hong, Tianzheng Ye, Tian Z Wang, Divya Srijay, Howard Liu, Lin Zhao, Rio Watson, Sophia Vincoff, Tianlai Chen, Kseniia Kholina, Shrey Goel, Matthew P DeLisa, Pranam Chatterjee.
      Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. While small molecule-based targeted protein degradation (TPD) and targeted protein stabilization (TPS) platforms can address this dysregulation, they rely on structured and stable binding pockets, which do not exist to classically "undruggable" targets. Here, we expand the TPS target space by engineering "deubiquibodies" (duAbs) via fusion of computationally-designed peptide binders to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. Using protein language models to generate target-binding peptides, we engineer duAbs to conformationally diverse target proteins, including key tumor suppressor proteins p53 and WEE1, and heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. We further encapsulate p53-targeting duAbs as mRNA in lipid nanoparticles and demonstrate effective intracellular delivery, p53 stabilization, and apoptosis activation, motivating further in vivo translation.
    DOI:  https://doi.org/10.1038/s41467-025-58872-6
  30. Nat Commun. 2025 Apr 17. 16(1): 3641
    Coupling of ribosome biogenesis and translation initiation in human mitochondria.
    Marleen Heinrichs, Anna Franziska Finke, Shintaro Aibara, Angelique Krempler, Angela Boshnakovska, Peter Rehling, Hauke S Hillen, Ricarda Richter-Dennerlein.
      Biogenesis of mitoribosomes requires dedicated chaperones, RNA-modifying enzymes, and GTPases, and defects in mitoribosome assembly lead to severe mitochondriopathies in humans. Here, we characterize late-step assembly states of the small mitoribosomal subunit (mtSSU) by combining genetic perturbation and mutagenesis analysis with biochemical and structural approaches. Isolation of native mtSSU biogenesis intermediates via a FLAG-tagged variant of the GTPase MTG3 reveals three distinct assembly states, which show how factors cooperate to mature the 12S rRNA. In addition, we observe four distinct primed initiation mtSSU states with an incompletely matured rRNA, suggesting that biogenesis and translation initiation are not mutually exclusive processes but can occur simultaneously. Together, these results provide insights into mtSSU biogenesis and suggest a functional coupling between ribosome biogenesis and translation initiation in human mitochondria.
    DOI:  https://doi.org/10.1038/s41467-025-58827-x
  31. Virol J. 2025 Apr 17. 22(1): 103
    Usutu virus NS4A induces autophagy and is targeted by the selective autophagy receptor p62/SQSTM1 for degradation.
    Tessa Nelemans, Ali Tas, Nina L de Beijer, George M C Janssen, Peter A van Veelen, Martijn J van Hemert, Marjolein Kikkert.
      Usutu virus (USUV) is an emerging orthoflavivirus, which mainly affects birds but in rare cases can cause severe neuroinvasive disease in humans. The virus relies on a multitude of host cell proteins, molecules and cellular processes for its replication, and must subvert host antiviral responses to establish a successful infection. Studying the complex network of virus-host protein interactions by proteomics approaches can therefore provide new insights in the replication cycle of USUV and its pathogenesis. We have previously shown that the USUV protein NS4A acts as an antagonist of the antiviral interferon response, and here we further map the host interaction partners of USUV NS4A using proximity labeling coupled to mass spectrometry. The resulting NS4A interactome revealed many host proteins involved in the autophagy pathway. We showed that both USUV infection and overexpression of USUV NS4A can indeed induce the autophagy pathway. However, stimulation or inhibition of the autophagy pathway in general did not affect USUV replication. Therefore, we decided to specifically analyze the role of the selective autophagy receptor sequestosome 1 (p62/SQSTM1), since we identified this protein as an important interaction partner of USUV NS4A. We found that p62 is involved in the degradation of USUV NS4A. In agreement with this, the knockdown of p62 enhanced replication of USUV in A549 cells. P62 thus plays an antiviral role during USUV infection, although this antiviral effect might also be related to its functions outside the autophagy pathway, such as modulation of the immune response. In conclusion, this study showed that USUV NS4A induces autophagy and is then targeted by p62 for degradation by the autophagic machinery, uncovering a new role of p62 in the antiviral defense against USUV.
    Keywords:  Autophagy; NS4A; Orthoflavivirus; SQSTM1; Usutu virus; p62
    DOI:  https://doi.org/10.1186/s12985-025-02719-5
  32. Angew Chem Int Ed Engl. 2025 Apr 13. e202505581
    Proteome-Wide Data Guides the Discovery of Lysine-Targeting Covalent Inhibitors Using DNA-Encoded Chemical Libraries.
    Xiaojie Lu, Xinyuan Wu, Shunyao Li, Ting Liang, Qing Yu, Yiwei Zhang, Jiaxiang Liu, Kaige Li, Zijian Liu, Mengqing Cui, Yongchao Zhao, Xin Han, Rui Jin, Minjia Tan, Xiaohua Chen, Yujun Zhao, Mingyue Zheng, Yi Sun, Lu Zhou.
      Broadening the application of covalent inhibitors requires the exploration of nucleophilic residues beyond cysteine. The covalent DNA-encoded chemical library (CoDEL) represents an advanced technology for covalent drug discovery. However, its application in lysine-targeting inhibitors remains uncharted territory. Here, we report the utilization of CoDEL selection guided by proteome-wide data to identify lysine-targeting covalent inhibitors. A comprehensive assessment of activity-based protein profiling (ABPP) data on lysine distribution and ligandability reveals potential targets for selective covalent inhibition, including phosphoglycerate mutase 1 (PGAM1), bromodomain (BRD) family proteins, and ubiquitin-conjugating enzyme E2 N (UBE2N). The 10.7-million-member CoDELs, featuring diverse lysine-reactive warheads, enables the discovery of a series of covalent inhibitors, covering photo-covalent, reversible covalent, and irreversible covalent reaction mechanisms. In-depth characterization of binding sites and modes of action provides structural and functional insights. Notably, irreversible covalent inhibitors unveil a novel mechanism for regulating UBE2N-mediated ubiquitination by modulating the conformation of the protein complex. Our work adopts the ABPP-CoDEL strategy, offering an efficient and versatile selection method for the development of covalent inhibitors targeting functional lysines.
    Keywords:  Covalent DNA-encoded chemical library, activity-based protein profiling data, covalent ligand selection, lysine-targeting covalent inhibitors
    DOI:  https://doi.org/10.1002/anie.202505581
  33. Am J Hum Genet. 2025 Apr 15. pii: S0002-9297(25)00138-7. [Epub ahead of print]
    A non-syndromic orofacial cleft risk locus links tRNA splicing defects to neural crest cell pathologies.
    Michaela Bartusel, Skylar X Kim, Rizwan Rehimi, Alicia M Darnell, Miloš Nikolić, Julia Heggemann, Petros Kolovos, Wilfred F J van Ijcken, Jade Varineau, Giuliano Crispatzu, Elisabeth Mangold, Samantha A Brugmann, Matthew G Vander Heiden, Magdalena Laugsch, Kerstin U Ludwig, Alvaro Rada-Iglesias, Eliezer Calo.
      Orofacial clefts are the most common form of congenital craniofacial malformation worldwide. The etiology of these birth defects is multifactorial, involving genetic and environmental factors. However, in most cases, the underlying causes remain unexplained, precluding a molecular understanding of disease mechanisms. Here, we integrated genome-wide association data, targeted resequencing of case and control cohorts, tissue- and cell-type-specific epigenomic profiling, and genome architecture analyses to molecularly dissect a genomic locus associated with an increased risk of non-syndromic orofacial cleft. We found that common and rare risk variants associated with orofacial cleft intersect with an enhancer (e2p24.2) that is active in human embryonic craniofacial tissue. We mapped e2p24.2 long-range interactions to a topologically associated domain harboring MYCN, DDX1, and CYRIA. We found that MYCN and DDX1, but not CYRIA, are required during craniofacial development in chicken embryos. We investigated the role of DDX1, a key component of the tRNA splicing complex, in cranial neural crest cells (cNCCs). The loss of DDX1 in cNCCs resulted in the accumulation of unspliced tRNA fragments, depletion of mature intron-containing tRNAs, and ribosome stalling at codons decoded by these tRNAs. This was accompanied by defects in both global protein synthesis and cNCC migration. We further showed that the induction of tRNA fragments is sufficient to disrupt craniofacial development. Together, these results uncovered a molecular mechanism in which impaired tRNA splicing affects cNCCs and craniofacial development and positioned MYCN, DDX1, and tRNA processing defects as risk factors in the pathogenesis of orofacial clefts.
    Keywords:  DDX1; MYCN; craniofacial development; epigenetics; neural crest cells; orofacial cleft; tRNA; tRNA fragments
    DOI:  https://doi.org/10.1016/j.ajhg.2025.03.017
  34. Res Pract Thromb Haemost. 2025 Feb;9(2): 102706
    Thiol isomerase ERp18 enhances platelet activation and arterial thrombosis.
    Chao He, Aizhen Yang, Keyu Lv, Yuxin Zhang, Zhenzhen Zhao, Yi Lu, Chao Fang, Yue Han, Depei Wu, Miao Jiang, Jingyu Zhang, Yi Wu.
       Background: Thiol isomerases regulate the thiol-disulfide exchange of functional proteins in cells. Using genetically modified mouse models and inhibitors, we and others demonstrated that 7 thiol isomerases (ERp57, protein diisulfide isomerase, ERp72, ERp46, ERp5, TMX4, and TMX1) participate in thrombosis. There are 21 thiol isomerases in mammals, but whether other enzymes of this family also contribute to thrombosis remains unknown.
    Objectives: Investigate whether and how ERp18 participates in arterial thrombosis.
    Methods: ERp18 knockout mice and arterial thrombosis models were used to determine the role of ERp18 in thrombosis. Platelets from ERp18 knockout mice were used to detect aggregation, activation, spreading, and clot retraction. Finally, flow cytometry and immunoprecipitation were used to detect the binding between ERp18 and αIIbβ3.
    Results: The mice lacking ERp18 exhibited a prolonged tail bleeding time and decreased platelet thrombus formation in FeCl3-induced carotid arterial injury and laser-induced cremaster artery injury models. ERp18 deficiency inhibited platelet aggregation, adenosine triphosphate release, integrin αIIbβ3 activation, P-selectin expression, platelet adhesion, as well as clot retraction. Flow cytometry and coimmunoprecipitation analyses revealed that ERp18 binds to the platelet surface via interaction with integrin αIIbβ3. Moreover, the ERp18 protein promoted the binding of integrin αIIbβ3 to fibrinogen and platelet aggregation. Furthermore, the recombinant ERp18 protein exhibited reductase activity and cleaved integrin αIIbβ3 disulfides.
    Conclusion: ERp18 participates in platelet activation and thrombosis. Its function is, at least in part, through the regulation of integrin αIIbβ3 function. This finding expands our understanding of the role of thiol isomerases in the redox regulation of thrombosis and platelet function.
    Keywords:  ERp18; integrin αIIbβ3; platelets; redox regulation; thrombosis
    DOI:  https://doi.org/10.1016/j.rpth.2025.102706
  35. bioRxiv. 2025 Apr 01. pii: 2025.03.31.646376. [Epub ahead of print]
    An Msp1-Protease Chimera Captures Transient AAA+ Interactions and Unveils Ost4 Mislocalization Errors.
    Deepika Gaur, Brian Acquaviva, Matthew L Wohlever.
      Membrane protein homeostasis (proteostasis) is essential for maintaining the integrity of eukaryotic organelles. Msp1 is a membrane anchored AAA+ (ATPase Associated with cellular Activities) protein that maintains mitochondrial proteostasis by extracting aberrant proteins from the outer mitochondrial membrane. A comprehensive understanding of the physiological roles of Msp1 has been hindered because AAA+ proteins interact with substrates transiently and common strategies to stabilize this interaction lead to undesirable mitochondrial phenotypes. To circumvent these drawbacks, we fused catalytically active Msp1 to the inactivated protease domain of the AAA+ protease Yme1. The resulting chimera sequesters substrates in the catalytically inactive degradation chamber formed by the protease domain. We performed mass spectrometry analysis with the Msp1-protease chimera and identified the signal anchored protein Ost4 as a novel Msp1 substrate. Topology experiments show that Ost4 adopts mixed orientations when mislocalized to mitochondria and that Msp1 extracts mislocalized Ost4 regardless of orientation. Together, this work develops new tools for capturing transient interactions with AAA+ proteins, identifies new Msp1 substrates, and shows a surprising error in targeting of Ost4.
    DOI:  https://doi.org/10.1101/2025.03.31.646376
  36. Cell Rep Methods. 2025 Apr 08. pii: S2667-2375(25)00063-3. [Epub ahead of print] 101027
    Integrated analysis of transcriptional and metabolic responses to mitochondrial stress.
    Liam P Kelley, Song-Hua Hu, Sarah A Boswell, Peter K Sorger, Alison E Ringel, Marcia C Haigis.
      Mitochondrial stress arises from a variety of sources, including mutations to mitochondrial DNA, the generation of reactive oxygen species, and an insufficient supply of oxygen or fuel. Mitochondrial stress induces a range of dedicated responses that repair damage and restore mitochondrial health. However, a systematic characterization of transcriptional and metabolic signatures induced by distinct types of mitochondrial stress is lacking. Here, we defined how primary human fibroblasts respond to a panel of mitochondrial inhibitors to trigger adaptive stress responses. Using metabolomic and transcriptomic analyses, we established integrated signatures of mitochondrial stress. We developed a tool, stress quantification using integrated datasets (SQUID), to deconvolute mitochondrial stress signatures from existing datasets. Using SQUID, we profiled mitochondrial stress in The Cancer Genome Atlas (TCGA) PanCancer Atlas, identifying a signature of pyruvate import deficiency in IDH1-mutant glioma. Thus, this study defines a tool to identify specific mitochondrial stress signatures, which may be applied to a range of systems.
    Keywords:  CP: Metabolism; CP: Systems biology; cancer metabolism; integrated multi-omics; integrated stress response; metabolomics; mitochondria; mitochondrial stress response; mitochondrial unfolded protein response; stress signatures
    DOI:  https://doi.org/10.1016/j.crmeth.2025.101027
  37. Cell Rep. 2025 Apr 16. pii: S2211-1247(25)00370-5. [Epub ahead of print]44(5): 115599
    RabGAP1L modulates Rab7A and Rab10 to orchestrate cell-autonomous immunity.
    Atsuko Minowa-Nozawa, Takashi Nozawa, Kazunori Murase, Ichiro Nakagawa.
      Cell-autonomous immunity protects cells by utilizing membrane trafficking to detect and counteract diverse microbial pathogens, including selective autophagy and extracellular expulsion. However, the mechanisms underlying the mutual regulation among these systems has remained unknown. Here, we demonstrate that Rab GTPase-activating protein 1-like (RabGAP1L) modulates cell-autonomous immune responses via inactivation of two distinct Rab GTPases during group A Streptococcus (GAS) infection. Confocal microscopy analyses revealed that Rab7A positively regulates selective autophagy induction against GAS by facilitating endolysosomal trafficking and that Rab7A and Rab10 negatively regulate GAS expulsion from infected cells by inhibiting Rab11A-positive recycling endosome formation. RabGAP1L suppressed these pathways via inactivation of Rab7A and Rab10. By contrast, ATG7 and ATG5 knockout, resulting in autophagy deficiency, increased RabGAP1L-dependent bacterial expulsion from infected cells via the endocytic recycling pathway. Our findings suggest a regulatory mechanism of cell-autonomous immunity mediated by RabGAP1L, which contributes to the efficient elimination of intracellular pathogens.
    Keywords:  CP: Cell biology; CP: Immunology; Rab10; Rab7A; RabGAP1L; cell-autonomous immunity; extracellular expulsion; group A Streptococcus; selective autophagy
    DOI:  https://doi.org/10.1016/j.celrep.2025.115599
  38. J Clin Invest. 2025 Apr 15. pii: e187323. [Epub ahead of print]
    Genetic inactivation of FAAP100 causes Fanconi anemia due to disruption of the monoubiquitin ligase core complex.
    Julia Kuehl, Yutong Xue, Fenghua Yuan, Ramanagouda Ramanagoudr-Bhojappa, Simone Pickel, Reinhard Kalb, Settara C Chandrasekharappa, Weidong Wang, Yanbin Zhang, Detlev Schindler.
      The Fanconi anemia (FA)/BRCA DNA repair network promotes the removal of DNA interstrand crosslinks (ICLs) to counteract their devastating consequences, including oncogenesis. Network signaling is initiated by the FA core complex, which consists of seven authentic FA proteins and an FA-associated protein, FAAP100, with incompletely characterized roles and unknown disease associations. Upon activation, the FA core complex functions as a multiprotein E3 ubiquitin ligase centered on its catalytic module, the FANCB-FANCL-FAAP100 (BLP100) subcomplex, for FANCD2 and FANCI monoubiquitylation. Here, we identified a homozygous variant in FAAP100, c.1642A>C, predicting p.(T542P), in a fetus with malformations suggestive of FA. The mutation causes sensitivity to ICL-inducing agents in cells from the affected individual and genetically engineered, FAAP100-inactivated human, avian, zebrafish, and mouse cells. All FAAP100-deficient cell types were rescued by ectopic expression of wild-type FAAP100, but not FAAP100T542P. In a confirmatory animal model, customized Faap100-/- mice exhibit embryonic lethality, microsomia, malformations, and gonadal atrophy resembling mice with established FA subtypes. Mechanistically, FAAP100T542P impairs ligase activity by preventing BLP100 subcomplex formation, resulting in defective FAAP100T542P nuclear translocation and chromatin recruitment. FAAP100 dysfunction that disrupts the FA pathway and impairs genomic maintenance, together with FAconsistent human manifestations, recommends FAAP100 as a legitimate FA gene, FANCY.
    Keywords:  Cell biology; DNA repair; Genetic instability; Genetics; Monogenic diseases
    DOI:  https://doi.org/10.1172/JCI187323
  39. medRxiv. 2025 Apr 04. pii: 2025.04.02.25322684. [Epub ahead of print]
    Tauroursodeoxycholic Acid (TUDCA) Reduces ER Stress and Lessens Disease Activity in Ulcerative Colitis.
    Vladimir Lamm, Katherine Huang, Ruishu Deng, Siyan Cao, Miao Wang, Saeed Soleymanjahi, Thanyarat Promlek, Rachel Rodgers, Deanna Davis, Darren Nix, Guadalupe Oliva Escudero, Yan Xie, Chien-Huan Chen, Anas Gremida, Richard P Rood, Ta-Chiang Liu, Megan T Baldridge, Parakkal Deepak, Nicholas O Davidson, Randal J Kaufman, Matthew A Ciorba.
       BACKGROUND AND AIMS: In inflammatory bowel disease, protein misfolding in the endoplasmic reticulum (ER) potentiates epithelial barrier dysfunction and impairs mucosal healing. Tauroursodeoxycholic acid (TUDCA), a naturally occurring bile acid, acts as a chemical chaperone to reduce protein aggregation and colitis severity in preclinical models. We conducted an open label trial evaluating oral TUDCA as therapy in patients with active ulcerative colitis (UC).
    METHODS: Patients with moderate-to-severely active UC (Mayo score ≥6, endoscopic subscore ≥1) received oral TUDCA at 1.75 or 2 g/day for 6 weeks. Exclusion criteria included known hepatic disorders or change in UC therapy within 60 days. Clinical disease activity questionnaires, endoscopy with biopsy, blood, and stool were collected at enrollment and after 6 weeks. The primary outcome measure was change in ER stress markers while safety, tolerability and change in UC disease activity were secondary outcomes.
    RESULTS: Thirteen participants completed the study with eleven evaluable for clinical response. TUDCA was well-tolerated with transient dyspepsia being the most common side effect. Mucosal biopsies revealed significant reductions in ER stress and inflammation as well as an increase in markers of epithelial restitution. Clinical, endoscopic, and histologic disease activity were significantly improved at week 6 (mean total Mayo Score: 9 to 4.5, p<0.001).
    CONCLUSIONS: Six weeks of oral TUDCA treatment was well-tolerated in patients with active ulcerative colitis and promoted mucosal healing, lessened ER stress, and reduced clinical disease activity. A randomized controlled trial of adjunctive TUDCA therapy in patients with UC is warranted.
    TRIAL REGISTRATION: ClinicalTrials.gov ( NCT04114292 ).
    KEY WORDS: chemical chaperones, unfolded protein response, inflammatory bowel disease, protein misfolding.
    DOI:  https://doi.org/10.1101/2025.04.02.25322684
  40. Biochim Biophys Acta Mol Cell Res. 2025 Apr 10. pii: S0167-4889(25)00061-8. [Epub ahead of print] 119956
    The versatile role of YidC in membrane protein biosynthesis and quality control.
    Mehmet Caliseki, Christiane Schaffitzel, Burak Veli Kabasakal.
      Membrane proteins are essential for bacterial survival, facilitating vital processes such as energy production, nutrient transport, and cell wall synthesis. YidC is a key player in membrane protein biogenesis, acting as both an insertase and a chaperone to ensure proper protein folding and integration into the lipid bilayer. Its conserved structure and adaptability enable it to mediate co-translational and post-translational protein insertion into the membrane through both Sec-dependent and Sec-independent pathways. In addition to facilitating protein insertion, YidC collaborates with FtsH in protein quality control, preventing the accumulation of misfolded proteins that could impair cellular function. This important relationship between YidC and FtsH is poorly understood, and there is a need for further investigation into their collaboration. Understanding how YidC and FtsH coordinate their roles could provide valuable insights into the links between bacterial membrane protein biogenesis and quality control pathways. Moreover, given its central functions, YidC represents a potential target for antimicrobial development. Small molecules disrupting its function in protein folding and insertion, hold promise. However, achieving bacterial specificity without impacting eukaryotic homologs remains a challenge. Here, we review our current understanding of YidC's structure, molecular function in membrane protein biogenesis and quality control, known interactions and its therapeutic potential.
    Keywords:  Chaperone; Insertase; Membrane protein biosynthesis; Membrane protein insertion; Membrane protein quality control; YidC
    DOI:  https://doi.org/10.1016/j.bbamcr.2025.119956
  41. bioRxiv. 2025 Apr 04. pii: 2025.04.03.645994. [Epub ahead of print]
    A non-canonical cGAS-STING pathway drives cellular and organismal aging.
    Rafael Cancado de Faria, Lilian Silva, Barbara Teodoro-Castro, Kyle S McCommis, Elena V Shashkova, Susana Gonzalo.
      Accumulation of cytosolic DNA has emerged as a hallmark of aging, inducing sterile inflammation. STING (Stimulator of Interferon Genes) protein translates the sensing of cytosolic DNA by cGAS (cyclic-GMP-AMP synthase) into an inflammatory response. However, the molecular mechanisms whereby cytosolic DNA-induced cGAS-STING pathway leads to aging remain poorly understood. We show that STING does not follow the canonical pathway of activation in human fibroblasts passaged (aging) in culture, senescent fibroblasts, or progeria fibroblasts (from Hutchinson Gilford Progeria Syndrome patients). Despite cytosolic DNA buildup, features of the canonical cGAS-STING pathway like increased cGAMP production, STING phosphorylation, and STING trafficking to perinuclear compartment are not observed in progeria/senescent/aging fibroblasts. Instead, STING localizes at endoplasmic reticulum, nuclear envelope, and chromatin. Despite the non-conventional STING behavior, aging/senescent/progeria cells activate inflammatory programs such as the senescence-associated secretory phenotype (SASP) and the interferon (IFN) response, in a cGAS and STING-dependent manner, revealing a non-canonical pathway in aging. Importantly, progeria/aging/senescent cells are hindered in their ability to activate the canonical cGAS-STING pathway with synthetic DNA, compared to young cells. This deficiency is rescued by activating vitamin D receptor signaling, unveiling new mechanisms regulating the cGAS-STING pathway in aging. Significantly, in HGPS, inhibition of the non-canonical cGAS-STING pathway ameliorates cellular hallmarks of aging, reduces tissue degeneration, and extends the lifespan of progeria mice. Our study reveals that a new feature of aging is the progressively reduced ability to activate the canonical cGAS-STING pathway in response to cytosolic DNA, triggering instead a non-canonical pathway that drives senescence/aging phenotypes.
    Significance Statement: Our study provides novel insights into the mechanisms driving sterile inflammation in aging and progeria. We reveal a previously unrecognized characteristic of aging cells: the progressive loss of ability to activate the canonical response to foreign or self-DNA at the cytoplasm. Instead, aging, senescent, and progeria cells activate inflammatory programs via a non-conventional pathway driven by cGAS and the adaptor protein STING. Importantly, pharmacological inhibition of the non-canonical cGAS-STING pathway ameliorates cellular, tissue and organismal decline in a devastating accelerated aging disease (Hutchinson Gilford Progeria Syndrome), highlighting it as a promising therapeutic target for age-related pathologies.
    DOI:  https://doi.org/10.1101/2025.04.03.645994
  42. Sci Adv. 2025 Apr 18. 11(16): eado7660
    Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response.
    Jingyu Hou, Yunyi Gao, Bing Han, Sujun Yan, Saisai Wei, Xiangwei Gao.
      YTH domain-containing family protein 1 (YTHDF1), a reader of N6-methyladenosine (m6A), has been implicated in regulating RNA metabolism in the cytosol. Here, we report a role of YTHDF1 within the nucleus in response to genotoxic stress. Upon radiation, YTHDF1 is phosphorylated at serine-182 in an ataxia telangiectasia and Rad3-related-dependent manner. This phosphorylation inhibits exportin 1-mediated nuclear export of YTHDF1, resulting in its accumulation within the nucleus. Nuclear YTHDF1 enhances the binding capacity of serine- and arginine-rich splicing factor 2 to a group of m6A-modified exons, leading to increased exon inclusion. Specifically, YTHDF1 promotes splicing and expression of DNA repair genes, such as BRCA1 and TP53BP1, thereby mitigating excessive DNA damage. Depletion of YTHDF1 sensitizes cancer cells to radiation treatment. Together, our study reveals a crucial role of YTHDF1 in m6A-mediated messenger RNA splicing in the DNA damage response, proposing it as a potential target for radiation therapy.
    DOI:  https://doi.org/10.1126/sciadv.ado7660
  43. iScience. 2025 Apr 18. 28(4): 112283
    Exploiting the therapeutic vulnerability of IDH-mutant gliomas with zotiraciclib.
    Ying Pang, Qi Li, Zach Sergi, Guangyang Yu, Olga Kim, Peng Lu, Marina Chan, Xueyu Sang, Herui Wang, Alice Ranjan, Robert W Robey, Ferri Soheilian, Bao Tran, Felipe J Núñez, Meili Zhang, Hua Song, Wei Zhang, Dionne Davis, Mark R Gilbert, Michael M Gottesman, Zhenggang Liu, Craig J Thomas, Maria G Castro, Taranjit S Gujral, Jing Wu.
      Isocitrate dehydrogenase (IDH)-mutant gliomas have distinctive metabolic and biological traits that potentially render them susceptible to targeted treatments. Here, by conducting a high-throughput drug screen, we pinpointed a specific vulnerability of IDH-mutant gliomas to zotiraciclib (ZTR). ZTR exhibited selective growth inhibition across multiple IDH-mutant glioma in vitro and in vivo models. Mechanistically, ZTR at low doses suppressed CDK9 and RNA Pol II phosphorylation in IDH-mutant cells, disrupting mitochondrial function and NAD+ production, resulting in oxidative stress. Integrated biochemical profiling of ZTR kinase targets and transcriptomics unveiled that ZTR-induced bioenergetic failure was linked to the suppression of PIM kinase activity. We posit that the combination of mitochondrial dysfunction and an inability to adapt to oxidative stress resulted in significant cell death upon ZTR treatment, ultimately increasing the therapeutic vulnerability of IDH-mutant gliomas. These findings prompted a clinical trial evaluating ZTR in IDH-mutant gliomas (NCT05588141).
    Keywords:  Cancer; Therapeutics
    DOI:  https://doi.org/10.1016/j.isci.2025.112283
  44. Nat Commun. 2025 Apr 14. 16(1): 3546
    Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity.
    Yun Huang, Wen-Qing Zhong, Xiao-Yu Yang, Jia-Lu Shan, Ling Zhou, Zhi-Ling Li, Yi-Qing Guo, Kai-Ming Zhang, Tian Du, Hai-Liang Zhang, Bing-Xin Hu, Yu-Hong Chen, Dong Yang, Gong-Kan Feng, Jun Tang, Xiao-Feng Zhu, Rong Deng.
      B7H3, an immune checkpoint molecule, is a highly N-glycosylated membrane protein. However, the key glycosylated asparagine residues that mediate the function of the B7H3 protein are still unclear. Here we identify that N-glycans attached to asparagine residues N91/309 and N104/322 are required for proper B7H3 localization on the cell surface membrane. We demonstrate that mutations in these two pairs of N-glycosylation sites induce ER accumulation of B7H3 by blocking its ER-to-Golgi translocation and subsequently promote its degradation via the endoplasmic reticulum-associated protein degradation pathway. Additional evidence suggests that N-glycosylation at N91/309 and N104/322 of B7H3 is essential for its inhibition of T-cell proliferation and activation. More importantly, a monoclonal antibody, Ab-82, preferentially targeting B7H3 glycosylated at N91/309 and N104/322 is developed, which exhibits the ability to elicit cytotoxic T lymphocyte-mediated antitumor immunity via B7H3 internalization. Together, these findings offer a rationale for targeting glycosylated B7H3 as a potential strategy for immunotherapy.
    DOI:  https://doi.org/10.1038/s41467-025-58740-3
  45. Science. 2025 Apr 18. 388(6744): eadr1752
    Structural basis for nucleolin recognition of MYC promoter G-quadruplex.
    Luying Chen, Jonathan Dickerhoff, Ke-Wei Zheng, Satchal Erramilli, Hanqiao Feng, Guanhui Wu, Buket Onel, Yuwei Chen, Kai-Bo Wang, Megan Carver, Clement Lin, Saburo Sakai, Jun Wan, Charles Vinson, Laurence Hurley, Anthony A Kossiakoff, Nanjie Deng, Yawen Bai, Nicholas Noinaj, Danzhou Yang.
      The MYC oncogene promoter G-quadruplex (MycG4) regulates transcription and is a prevalent G4 locus in immortal cells. Nucleolin, a major MycG4-binding protein, exhibits greater affinity for MycG4 than for nucleolin recognition element (NRE) RNA. Nucleolin's four RNA binding domains (RBDs) are essential for high-affinity MycG4 binding. We present the 2.6-angstrom crystal structure of the nucleolin-MycG4 complex, revealing a folded parallel three-tetrad G-quadruplex with two coordinating potassium ions (K+), interacting with RBD1, RBD2, and Linker12 through its 6-nucleotide (nt) central loop and 5' flanking region. RBD3 and RBD4 bind MycG4's 1-nt loops as demonstrated by nuclear magnetic resonance (NMR). Cleavage under targets and tagmentation sequencing confirmed nucleolin's binding to MycG4 in cells. Our results revealed a G4 conformation-based recognition by a regulating protein through multivalent interactions, suggesting that G4s are nucleolin's primary cellular substrates, indicating G4 epigenetic transcriptional regulation and helping G4-targeted drug discovery.
    DOI:  https://doi.org/10.1126/science.adr1752
  46. RNA. 2025 Apr 15. pii: rna.080419.125. [Epub ahead of print]
    RNase L Produces tRNA-derived RNAs that Contribute to Translation Inhibition.
    Yoshika Takenaka, Asuka Yamada, Yoshihisa Tomioka, Yasutoshi Akiyama, Pavel Ivanov.
      Ribonuclease L (RNase L) is an RNase which is activated by viral double-stranded RNAs (dsRNAs). RNase L cleaves not only viral RNAs but also host RNAs including mRNAs and tRNAs, which contributes to innate immune defense against viruses. While it has been reported that RNase L-mediated bulk mRNA cleavage induces rapid translation repression independently of the integrated stress response, the significance of RNase L-mediated tRNA cleavage remains largely unknown. Here we show that RNase L cleaves various tRNA species in the anticodon loops, generating transfer RNA-derived RNAs (tDRs) similar to tRNA-derived stress-induced RNAs (tiRNAs) that are generated by a stress-responsive RNase angiogenin (ANG). Three tRNA species (tRNALeu, tRNASeC and tRNASer) were cleaved within the variable loops as well as in the anticodon loops by RNase L, generating non-canonical tDRs. As RNase L-induced 5'-tDRAla/Cys were similar in length to 5'-tiRNAAla/Cys that possess a translation inhibitory effect, we examined whether RNase L-induced 5'-tDRAla also inhibited translation. In vitro translation analysis showed that RNase L-induced 5'-tDRAla significantly inhibits mRNA translation like 5'-tiRNAAla, suggesting that the production of 5'-tDRAla may be involved in the mechanism of RNase L-mediated stress response during viral infection. Our data shed new light on the potential roles of tDRs in innate immunity against viral infection.
    Keywords:  RNase L; protein synthesis; ribonuclease; tRNA; tRNA-derived RNAs
    DOI:  https://doi.org/10.1261/rna.080419.125
  47. Cell Genom. 2025 Apr 15. pii: S2666-979X(25)00107-7. [Epub ahead of print] 100851
    Proteomic-based stemness score measures oncogenic dedifferentiation and enables the identification of druggable targets.
    Clinical Proteomic Tumor Analysis Consortium
      Cancer progression and therapeutic resistance are closely linked to a stemness phenotype. Here, we introduce a protein-expression-based stemness index (PROTsi) to evaluate oncogenic dedifferentiation in relation to histopathology, molecular features, and clinical outcomes. Utilizing datasets from the Clinical Proteomic Tumor Analysis Consortium across 11 tumor types, we validate PROTsi's effectiveness in accurately quantifying stem-like features. Through integration of PROTsi with multi-omics, including protein post-translational modifications, we identify molecular features associated with stemness and proteins that act as active nodes within transcriptional networks, driving tumor aggressiveness. Proteins highly correlated with stemness were identified as potential drug targets, both shared and tumor specific. These stemness-associated proteins demonstrate predictive value for clinical outcomes, as confirmed by immunohistochemistry in multiple samples. The findings emphasize PROTsi's efficacy as a valuable tool for selecting predictive protein targets, a crucial step in customizing anti-cancer therapy and advancing the clinical development of cures for cancer patients.
    Keywords:  biomarkers; cancer; drug targets; kinase activity; machine learning; mass spectrometry; multiomics; proteomics; stemness; tumor plasticity
    DOI:  https://doi.org/10.1016/j.xgen.2025.100851
  48. Brief Bioinform. 2025 Mar 04. pii: bbaf164. [Epub ahead of print]26(2):
    RIBOSS detects novel translational events by combining long- and short-read transcriptome and translatome profiling.
    Chun Shen Lim, Alexandra K Gibbon, Anh Thu Tran Nguyen, Gabrielle S W Chieng, Chris M Brown.
      Ribosome profiling is a high-throughput sequencing technique that captures the positions of translating ribosomes on RNAs. Recent advancements in ribosome profiling include achieving highly phased ribosome footprints for plant translatomes and more recently for bacterial translatomes. This substantially increases the specificity of detecting open reading frames (ORFs) that can be translated, such as small ORFs located upstream and downstream of the annotated ORFs. However, most genomes (e.g. bacterial genomes) lack the annotations for the transcription start and termination sites. This hinders the systematic discovery of novel ORFs in the 'untranslated' regions in ribosome profiling data. Here, we develop a new computational pipeline called RIBOSS to discover noncanonical ORFs and assess their translational potential against annotated ORFs. The RIBOSS Python modules are versatile, and we use them to analyse both prokaryotic and eukaryotic data. We present a resulting list of noncanonical ORFs with high translational potential in Homo sapiens, Arabidopsis thaliana, and Salmonella enterica. We further illustrate RIBOSS utility when studying organisms with incomplete transcriptome annotations. We leverage long-read and short-read data for reference-guided transcriptome assembly and highly phased ribosome profiling data for detecting novel translational events in the assembled transcriptome for S. enterica. In sum, RIBOSS is the first integrated computational pipeline for noncanonical ORF detection and translational potential assessment that incorporates long- and short-read sequencing technologies to investigate translation. RIBOSS is freely available at https://github.com/lcscs12345/riboss.
    Keywords:  Nanopore long-read direct RNA sequencing; gene annotation; protein synthesis; ribosome profiling analysis method; transcriptome assembly
    DOI:  https://doi.org/10.1093/bib/bbaf164
  49. Semin Cell Dev Biol. 2025 Apr 16. pii: S1084-9521(25)00018-7. [Epub ahead of print]170 103608
    Emerging roles for microproteins as critical regulators of endoplasmic reticulum function and cellular homeostasis.
    Taylor M Coughlin, Catherine A Makarewich.
      The endoplasmic reticulum (ER) is a multifunctional organelle essential for key cellular processes including protein synthesis, calcium homeostasis, and the cellular stress response. It is composed of distinct domains, such as the rough and smooth ER, as well as membrane regions that facilitate direct communication with other organelles, enabling its diverse functions. While many well-characterized ER proteins contribute to these processes, recent studies have revealed a previously underappreciated class of small proteins that play critical regulatory roles. Microproteins, typically under 100 amino acids in length, were historically overlooked due to size-based biases in genome annotation and often misannotated as noncoding RNAs. Advances in ribosome profiling, mass spectrometry, and computational approaches have now enabled the discovery of numerous previously unrecognized microproteins, significantly expanding our understanding of the proteome. While some ER-associated microproteins, such as phospholamban and sarcolipin, were identified decades ago, newly discovered microproteins share similar fundamental characteristics, underscoring the need to refine our understanding of the coding potential of the genome. Molecular studies have demonstrated that ER microproteins play essential roles in calcium regulation, ER stress response, organelle communication, and protein translocation. Moreover, growing evidence suggests that ER microproteins contribute to cellular homeostasis and are implicated in disease processes, including cardiovascular disease and cancer. This review examines the shared and unique functions of ER microproteins, their implications for health and disease, and their potential as therapeutic targets for conditions associated with ER dysfunction.
    Keywords:  Calcium; Cellular stress response; Endoplasmic reticulum; Micropeptide; Microprotein; sORF
    DOI:  https://doi.org/10.1016/j.semcdb.2025.103608
  50. PLoS Pathog. 2025 Apr;21(4): e1013051
    RNF149 modulates the type I IFN innate antiviral immune responses through degrading IRF3.
    Mengyun Wu, Jiamin Cai, Guodong Qiao, Xiaoping Li, Ji Zhou, Fei Xu, Yunfei Ye, Yufeng Wang, Xuena Xu, Jiaoyang Li, Xiaoyu Tian, Yu Shao, Chunsheng Dong, Zhengrong Chen, Chuangli Hao, Yi Yang, Jinping Zhang.
      E3 ubiquitin ligases are key molecules in regulating the innate immune responses against virus. They catalyze the activation or degradation of various signaling proteins involved in the innate immune responses. Herein, we found the regulatory role of RNF149 in the host's innate immune responses against viral infection. Virus infection induced the expression of RNF149. Overexpression of RNF149 was associated with reduced production of IFN-β and enhanced viral replication. Mechanically, RNF149 interacted with IRF3 and downregulated its protein level. As an E3 ubiquitin ligase, RNF149 promoted the K27-linked ubiquitination of IRF3 at K409 and K33-linked ubiquitination at K366 and K409, which promoted IRF3 degradation through the proteasome pathway. Our results revealed the regulatory mechanism of RNF149 during viral infection and provided new insights into host cells responding to viral infection. Downregulating the expression of RNF149 may help enhance the antiviral ability of host cells and inhibit viral replication, thus providing a new strategy for the treatment of viral infection.
    DOI:  https://doi.org/10.1371/journal.ppat.1013051
  51. Cell Death Dis. 2025 Apr 13. 16(1): 289
    ATF6α inhibits ΔNp63α expression to promote breast cancer metastasis by the GRP78-AKT1-FOXO3a signaling.
    Hong Wang, Xin Yang, Liyuan Deng, Xuanyu Zhou, Jin Tao, Zhiqiang Wu, Hu Chen.
      Endoplasmic reticulum (ER) stress is increasingly recognized as a driver of cancer progression; however, the precise molecular mechanisms by which ER stress facilitates tumor metastasis remain incompletely understood. In this study, we demonstrate that ER stress-activated ATF6α promotes breast cancer cell migration and metastasis by downregulating the expression of ΔNp63α, a key metastasis suppressor. Mechanistically, ATF6α reduces ΔNp63α expression through GRP78, which interacts with and activates AKT1. Activated AKT1 subsequently phosphorylates FOXO3a, leading to its degradation. Since FOXO3a directly transactivates ΔNp63α expression, its degradation results in reduced ΔNp63α levels. Furthermore, pharmacological inhibition or genetic knockdown of AKT1 upregulates ΔNp63α in vitro and suppresses tumor metastasis in vivo. Clinical analyses reveal that TP63 and FOXO3a expression are significantly reduced in breast cancer tissues compared to normal tissues, whereas ATF6 and GRP78 expression are elevated. Moreover, low TP63 and high GRP78 expression are associated with a poor prognosis in breast cancer patients. Collectively, these findings elucidate the pivotal role of the ATF6α-GRP78-AKT1-FOXO3a axis in chronic ER stress-mediated downregulation of ΔNp63α, establishing a molecular framework for targeting this pathway as a potential therapeutic strategy against breast cancer metastasis.
    DOI:  https://doi.org/10.1038/s41419-025-07619-8
  52. Nat Commun. 2025 Apr 13. 16(1): 3514
    Engineered depalmitoylases enable selective manipulation of protein localization and function.
    Srinidhi Jayaraman, Audrey Kochiss, Thy-Lan Alcalay, Pedro J Del Rivero Morfin, Manu Ben-Johny.
      S-Palmitoylation is a reversible post-translational modification that tunes the localization, stability, and function of an impressive array of proteins including ion channels, G-proteins, and synaptic proteins. Indeed, altered protein palmitoylation is linked to various human diseases including cancers, neurodevelopmental and neurodegenerative diseases. As such, strategies to selectively manipulate protein palmitoylation with enhanced temporal and subcellular precision are sought after to both delineate physiological functions and as potential therapeutics. Here, we develop chemogenetically and optogenetically inducible engineered depalmitoylases to manipulate the palmitoylation status of target proteins. We demonstrate that this strategy is programmable allowing selective depalmitoylation in specific organelles, triggered by cell-signaling events, and of individual protein complexes. Application of this methodology revealed bidirectional tuning of neuronal excitability by distinct depalmitoylases. Overall, this strategy represents a versatile and powerful method for manipulating protein palmitoylation in live cells, providing insights into their regulation in distinct physiological contexts.
    DOI:  https://doi.org/10.1038/s41467-025-58908-x
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