bims-proteo 2025-01-26 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–01–26
38 papers selected by
Eric Chevet, INSERM

The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy.
The deubiquitinase USP5 prevents accumulation of protein aggregates in cardiomyocytes.
The Kinesin Kar3 is Required for Endoplasmic Reticulum-Associated Degradation.
Protein quality control is a master modulator of molecular evolution in bacteria.
Leveraging Structural and Computational Biology for Molecular Glue Discovery.
Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.
Preventing inappropriate signals pre- and post-ligand perception by a toggle switch mechanism of ERECTA.
Mitochondrial protein import stress.
19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation.
Autophagy in Plant Health and Disease.
Induction of lysosome biogenesis is a novel function of the CGAS-STING1 pathway.
Epstein-Barr virus BALF0/1 subverts the Caveolin and ERAD pathways to target B cell receptor complexes for degradation.
De novo design of peptide binders to conformationally diverse targets with contrastive language modeling.
Engineered initiator tRNAs can effectively start translation at non-AUG start codons and diversify N-terminal amino acids for mRNA Display.
Multi-modal investigation reveals pathogenic features of diverse DDX3X missense mutations.
Tango1L but not Tango1S, Tali and cTAGE5 is required for export of type II collagen in medaka fish.
The Cul3 ubiquitin ligase engages Insomniac as an adaptor to impact sleep and synaptic homeostasis.
Diphthamide synthesis is linked to the eEF2-client chaperone machinery.
Redox proteomics reveal a role for peroxiredoxinylation in stress protection.
Structures of aberrant spliceosome intermediates on their way to disassembly.
Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation.
Endoplasmic Reticulum-Targeted Polymer-Manganese Nanocomplexes for Tumor Immunotherapy.
Ubiquitin-A structural perspective.
FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis.
UFMylation promotes orthoflavivirus infectious particle production.
Molecular determinants of condensate composition.
Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin.
Inhibition of vascular smooth muscle cell PERK/ATF4 ER stress signaling protects against abdominal aortic aneurysms.
Dynamic allostery in the peptide/MHC complex enables TCR neoantigen selectivity.
Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.
Reduced UPF1 levels in senescence impair nonsense-mediated mRNA decay.
Design and Discovery of Preclinical Candidate LYG-409 as a Highly Potent and Selective GSPT1 Molecular Glue Degraders.
Lipidic folding pathway of α-Synuclein via a toxic oligomer.
A computational tool to infer enzyme activity using post-translational modification profiling data.
AggNet: Advancing protein aggregation analysis through deep learning and protein language model.
DDX1 is required for non-spliceosomal splicing of tRNAs but not of XBP1 mRNA.
The FIRE biosensor illuminates iron regulatory protein activity and cellular iron homeostasis.
Metagenome-informed metaproteomics of the human gut microbiome, host, and dietary exposome uncovers signatures of health and inflammatory bowel disease.

Autophagy. 2025 Jan 22. 1-23

The Epstein-Barr virus deubiquitinase BPLF1 regulates stress-induced ribosome UFMylation and reticulophagy.

Jiangnan Liu, Noemi Nagy, Carlos Ayala-Torres, Solenne Bleuse, Francisco Aguilar-Alonso, Ola Larsson, Maria G Masucci.

  The synthesis of membrane and secreted proteins is safeguarded by an endoplasmic reticulum-associated ribosome quality control (ER-RQC) that promotes the disposal of defective translation products by the proteasome or via a lysosome-dependent pathway involving the degradation of portions of the ER by macroautophagy (reticulophagy). The UFMylation of RPL26 on ER-stalled ribosomes is essential for activating the ER-RQC and reticulophagy. Here, we report that the viral deubiquitinase (vDUB) encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein BPLF1 hinders the UFMylation of RPL26 on ribosomes that stall at the ER, promotes the stabilization of ER-RQC substrates, and inhibits reticulophagy. The vDUB did not act as a de-UFMylase or interfere with the UFMylation of the ER membrane protein CYB5R3 by the UFL1 ligase. Instead, it copurified with ribosomes in sucrose gradients and abrogated a ZNF598- and LTN1-independent ubiquitination event required for RPL26 UFMylation. Physiological levels of BPLF1 impaired the UFMylation of RPL26 in productively EBV-infected cells, pointing to an important role of the enzyme in regulating the translation quality control that allows the efficient synthesis of viral proteins and the production of infectious virus.Abbreviation: BPLF1, BamH1 P fragment left open readingframe-1; CDK5RAP3, CDK5regulatory subunit associated protein 3; ChFP, mCherry fluorescent protein; DDRGK1, DDRGKdomain containing 1; EBV, Epstein-Barr virus; eGFP, enhancedGFP; ER-RQC, endoplasmicreticulum-associated ribosome quality control; LCL, EBV-carryinglymphoblastoid cell line; GFP, green fluorescent protein; RQC, ribosome quality control; SRP, signal recognition particle; UFM1, ubiquitin fold modifier 1; UFL1, UFM1 specific ligase 1.

Keywords:  EBV; Macroautophagy; Ribosome; UFM1; viral DUB

DOI:  https://doi.org/10.1080/15548627.2024.2440846
Sci Adv. 2025 Jan 24. 11(4): eado3852

The deubiquitinase USP5 prevents accumulation of protein aggregates in cardiomyocytes.

Yvonne Eibach, Silke Kreher, Mareike S Poetsch, Ay Lin Kho, Ulrich Gaertner, Christoph S Clemen, Rolf Schröder, Kai Guo, Hendrik Milting, Benjamin Meder, Michael Potente, Manfred Richter, Andre Schneider, Silke Meiners, Mathias Gaute, Thomas Braun.

Protein homeostasis is crucial for maintaining cardiomyocyte (CM) function. Disruption of proteostasis results in accumulation of protein aggregates causing cardiac pathologies such as hypertrophy, dilated cardiomyopathy (DCM), and heart failure. Here, we identify ubiquitin-specific peptidase 5 (USP5) as a critical determinant of protein quality control (PQC) in CM. CM-specific loss of mUsp5 leads to the accumulation of polyubiquitin chains and protein aggregates, cardiac remodeling, and eventually DCM. USP5 interacts with key components of the proteostasis machinery, including PSMD14, and the absence of USP5 increases activity of the ubiquitin-proteasome system and autophagic flux in CMs. Cardiac-specific hUSP5 overexpression reduces pathological remodeling in pressure-overloaded mouse hearts and attenuates protein aggregate formation in titinopathy and desminopathy models. Since CMs from humans with end-stage DCM show lower USP5 levels and display accumulation of ubiquitinated protein aggregates, we hypothesize that therapeutically increased USP5 activity may reduce protein aggregates during DCM. Our findings demonstrate that USP5 is essential for ubiquitin turnover and proteostasis in mature CMs.

DOI: https://doi.org/10.1126/sciadv.ado3852
Mol Biol Cell. 2025 Jan 22. mbcE24100437

The Kinesin Kar3 is Required for Endoplasmic Reticulum-Associated Degradation.

Emmanuel Akoto, Ellen M Doss, Kieran P Claypool, Sophia L Owutey, Kyle A Richards, Katie M Lehman, Mahmoud M Daraghmi, Samantha M Turk, Christopher J Indovina, James A Avaala, Melissa D Evans, Abigail R Scott, Hayden O Schneider, Evan M Rogers, Jason D True, Philip J Smaldino, Eric M Rubenstein.

Degradation of aberrant, excess, and regulatory proteins at the endoplasmic reticulum (ER) is a conserved feature of eukaryotic cells, disruption of which contributes to disease. While remarkable progress has been made in recent years, mechanisms and genetic requirements for ER-Associated Degradation (ERAD) remain incompletely understood. We recently conducted a screen for genes required for turnover of a model ER translocon-associated substrate of the Hrd1 ubiquitin ligase in Saccharomyces cerevisiae. This screen revealed loss of Kar3 impedes degradation of Deg1*-Sec62, which persistently and aberrantly engages the translocon. Kar3 is a microtubule-associated kinesin 14 family member that impacts multiple aspects of microtubule dynamics during cell division and karyogamy. We investigated involvement of Kar3 and its cofactors in ERAD. Loss of Kar3 hindered ERAD mediated by three ubiquitin ligases but did not impair turnover of a soluble nuclear protein. Further, KAR3 deletion caused hypersensitivity to conditions associated with proteotoxic stress. Kar3's cytoplasmic cofactor Vik1 was also required for efficient degradation of Deg1*-Sec62. Our results reveal a profound and underappreciated role for microtubule-associated proteins in ERAD.

DOI: https://doi.org/10.1091/mbc.E24-10-0437
Genome Biol Evol. 2025 Jan 22. pii: evaf010. [Epub ahead of print]

Protein quality control is a master modulator of molecular evolution in bacteria.

Carolina Diaz Arenas, Maristella Alvarez, Robert H Wilson, Eugene I Shakhnovich, C Brandon Ogbunugafor.

  The bacterial protein quality control (PQC) network comprises a set of genes that promote proteostasis (proteome homeostasis) through proper protein folding and function via chaperones, proteases, and a protein translational machinery. It participates in vital cellular processes and influences organismal development and evolution. In this review, we examine the mechanistic bases for how the bacterial PQC network influences molecular evolution. We discuss the relevance of PQC components to contemporary issues in evolutionary biology including epistasis, evolvability, and the navigability of protein space. We examine other areas where proteostasis affects aspects of evolution and physiology, including host-parasite interactions. More generally, we demonstrate that the study of bacterial systems can aid in broader efforts to understand the relationship between genotype and phenotype across the biosphere.

Keywords:  chaperone; evolvability; molecular evolution; protease; protein evolution; protein quality control; proteostasis

DOI:  https://doi.org/10.1093/gbe/evaf010
J Med Chem. 2025 Jan 24.

Leveraging Structural and Computational Biology for Molecular Glue Discovery.

Congbao Kang, Weijun Xu.

The discovery of molecular glues has made significant strides, unlocking new avenues for targeted protein degradation as a therapeutic strategy, thereby expanding the scope of drug discovery into territories previously considered undruggable. Pioneering molecules like thalidomide and its derivatives have paved the way for the development of small molecules that can induce specific protein degradation by hijacking the cellular ubiquitin-proteasome system. Recent advancements have focused on expanding the range of E3 ligases and target proteins that can be modulated by molecular glues. Structural elucidation of E3 ligase in complex with molecular glue and the target of interest, combined with computational modeling, facilitates the understanding of the underlying mechanisms of how molecular glues induce targeted degradation. By leveraging these tools, the next generation of molecular glues are expected to offer unprecedented opportunities for combating a wide range of diseases, including cancer, autoimmune disorders, and neurodegenerative conditions.

DOI: https://doi.org/10.1021/acs.jmedchem.5c00076
Sci Adv. 2025 Jan 24. 11(4): eadu5787

Global cellular proteo-lipidomic profiling of diverse lysosomal storage disease mutants using nMOST.

Felix Kraus, Yuchen He, Sharan Swarup, Katherine A Overmyer, Yizhi Jiang, Johann Brenner, Cristina Capitanio, Anna Bieber, Annie Jen, Nicole M Nightingale, Benton J Anderson, Chan Lee, Joao A Paulo, Ian R Smith, Jürgen M Plitzko, Steven P Gygi, Brenda A Schulman, Florian Wilfling, Joshua J Coon, J Wade Harper.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

DOI: https://doi.org/10.1126/sciadv.adu5787
Proc Natl Acad Sci U S A. 2025 Jan 28. 122(4): e2420196122

Preventing inappropriate signals pre- and post-ligand perception by a toggle switch mechanism of ERECTA.

Liangliang Chen, Michal Maes, Alicia M Cochran, Julian R Avila, Paul Derbyshire, Jan Sklenar, Kelsey M Haas, Judit Villén, Frank L H Menke, Keiko U Torii.

  Dynamic control of signaling events requires swift regulation of receptors at an active state. By focusing on the Arabidopsis ERECTA (ER) receptor kinase, which perceives peptide ligands to control multiple developmental processes, we report a mechanism preventing inappropriate receptor activity. The ER C-terminal tail (ER_CT) functions as an autoinhibitory domain: Its removal confers higher kinase activity and hyperactivity during inflorescence and stomatal development. ER_CT is required for the binding of a receptor kinase inhibitor, BKI1, and two U-box E3 ligases, PUB30 and PUB31, that trigger activated ER to degradation through ubiquitination. We further identify ER_CT as a phosphodomain transphosphorylated by the coreceptor BAK1. The phosphorylation impacts the tail structure, likely releasing ER from autoinhibition. The phosphonull version enhances BKI1 association, whereas the phosphomimetic version promotes PUB30/31 association. Thus, ER_CT acts as an off-on-off toggle switch, facilitating the release of BKI1 inhibition, enabling signal activation, and swiftly turning over the receptors afterward. Our results elucidate a mechanism that fine-tunes receptor signaling via a phosphoswitch module, maintaining the receptor at a low basal state while ensuring robust yet transient activation upon ligand perception.

Keywords:  inflorescence growth; peptide hormone; phosphoswitch; receptor kinase; stomatal development

DOI:  https://doi.org/10.1073/pnas.2420196122
Nat Cell Biol. 2025 Jan 22.

Mitochondrial protein import stress.

Nikolaus Pfanner, Fabian den Brave, Thomas Becker.

Mitochondria have to import a large number of precursor proteins from the cytosol. Chaperones keep these proteins in a largely unfolded state and guide them to the mitochondrial import sites. Premature folding, mitochondrial stress and import defects can cause clogging of import sites and accumulation of non-imported precursors, representing a critical burden for cellular proteostasis. Here we discuss how cells respond to mitochondrial protein import stress by regenerating clogged import sites and inducing stress responses. The mitochondrial protein import machinery has a dual role by serving as sensor for detecting mitochondrial dysfunction and inducing stress-response pathways. The production of chaperones that fold or sequester precursor proteins in deposits is induced and the proteasomal activity is increased to remove the excess precursor proteins. Together, these pathways reveal how mitochondria are tightly integrated into a cellular proteostasis and stress response network to maintain cell viability.

DOI: https://doi.org/10.1038/s41556-024-01590-w
bioRxiv. 2025 Jan 09. pii: 2025.01.08.632038. [Epub ahead of print]

19S proteasome loss causes monopolar spindles through ubiquitin-independent KIF11 degradation.

Océane Marescal, Iain M Cheeseman.

To direct regulated protein degradation, the 26S proteasome recognizes ubiquitinated substrates through its 19S particle and then degrades them in the 20S enzymatic core. Despite this close interdependency between proteasome subunits, we demonstrate that knockouts from different proteasome subcomplexes result in distinct highly cellular phenotypes. In particular, depletion of 19S PSMD lid proteins, but not that of other proteasome subunits, prevents bipolar spindle assembly during mitosis, resulting in a mitotic arrest. We find that the monopolar spindle phenotype is caused by ubiquitin-independent proteasomal degradation of the motor protein KIF11 upon loss of 19S proteins. Thus, negative regulation of 20S-mediated proteasome degradation is essential for mitotic progression and 19S and 20S proteasome components can function independently outside of the canonical 26S structure. This work reveals a role for the proteasome in spindle formation and identifies the effects of ubiquitin-independent degradation on cell cycle control.

DOI: https://doi.org/10.1101/2025.01.08.632038
Annu Rev Plant Biol. 2025 Jan 22.

Autophagy in Plant Health and Disease.

Angelina S Gross, Margot Raffeiner, Yonglun Zeng, Suayib Üstün, Yasin Dagdas.

Autophagy has emerged as an essential quality control pathway in plants that selectively and rapidly removes damaged or unwanted cellular components to maintain cellular homeostasis. It can recycle a broad range of cargoes, including entire organelles, protein aggregates, and even invading microbes. It involves the de novo biogenesis of a new cellular compartment, making it intimately linked to endomembrane trafficking pathways. Autophagy is induced by a wide range of biotic and abiotic stress factors, and autophagy mutant plants are highly sensitive to stress, making it an attractive target for improving plant stress resilience. Here, we critically discuss recent discoveries related to plant autophagy and highlight open questions and future research areas.

DOI: https://doi.org/10.1146/annurev-arplant-060324-094912
Autophagy. 2025 Jan 21.

Induction of lysosome biogenesis is a novel function of the CGAS-STING1 pathway.

Yinfeng Xu, Wei Wan.

  Induction of macroautophagy/autophagy has been established as an important function elicited by the CGAS-STING1 pathway during pathogen infection. However, it remains unknown whether lysosomal activity within the cell in these settings is concurrently enhanced to cope with the increased autophagic flux. Recently, we discovered that the CGAS-STING1 pathway elevates the degradative capacity of the cell by activating lysosome biogenesis. Intriguingly, we found that STING1-induced GABARAP lipidation, rather than TBK1 activation, serves as the key mediator triggering the nuclear translocation of transcription factor TFEB and enhances the expression of lysosome-related genes. Mechanistically, we demonstrated that lipidated GABARAP on single membranes, regulated by the V-ATPase-ATG16L1 axis, sequesters the FLCN-FNIP complex to abolish its function toward RRAGC-RRAGD, leading to a specific impairment of MTORC1-dependent phosphorylation of TFEB and resulting in its subsequent nuclear translocation. Functionally, we showed that STING1-induced lysosome biogenesis is essential for the clearance of cytoplasmic DNA and the elimination of invading pathogens. Collectively, our findings underscore the induction of lysosome biogenesis as a novel function of the CGAS-STING1 pathway.China; Yinfeng Xu; Email: yinfengxu@hnfnu.edu.cn; Hunan First Normal University, 1015 Feng-Lin-San Road, Changsha, Hunan 410,205, China.

Keywords:  Autophagy; CGAS; GABARAP; STING1; TFEB; lysosome

DOI:  https://doi.org/10.1080/15548627.2025.2456064
Proc Natl Acad Sci U S A. 2025 Jan 28. 122(4): e2400167122

Epstein-Barr virus BALF0/1 subverts the Caveolin and ERAD pathways to target B cell receptor complexes for degradation.

Stephanie Pei Tung Yiu, Yifei Liao, Jinjie Yan, Michael P Weekes, Benjamin E Gewurz.

  Epstein-Barr virus (EBV) establishes persistent infection, causes infectious mononucleosis, is a major trigger for multiple sclerosis and contributes to multiple cancers. Yet, knowledge remains incomplete about how the virus remodels host B cells to support lytic replication. We previously identified that EBV lytic replication results in selective depletion of plasma membrane (PM) B cell receptor (BCR) complexes, composed of immunoglobulin and the CD79A and CD79B signaling chains. Here, we used proteomic and biochemical approaches to identify that the EBV early lytic protein BALF0/1 is responsible for EBV lytic cycle BCR degradation. Mechanistically, an immunoglobulin heavy chain (HC) cytoplasmic tail KVK motif was required for ubiquitin-mediated BCR degradation, while CD79A and CD79B were dispensable. BALF0/1 subverted caveolin-mediated endocytosis to internalize PM BCR complexes and to deliver them to the endoplasmic reticulum. BALF0/1 stimulated immunoglobulin HC cytoplasmic tail ubiquitination, which together with the ATPase valosin-containing protein/p97 drove ER-associated degradation of BCR complexes by cytoplasmic proteasomes. BALF0/1 knockout reduced the viral load of secreted EBV particles from B cells that expressed a monoclonal antibody against EBV glycoprotein 350 but not a control anti-influenza hemagglutinin antibody and increased viral particle immunoglobulin incorporation. Consistent with downmodulation of PM BCR, BALF0/1 overexpression reduced viability of a diffuse large B cell lymphoma cell line whose survival is dependent upon BCR signaling. Collectively, our results suggest that EBV BALF0/1 downmodulates immunoglobulin upon lytic reactivation to block BCR signaling and support virion release, but await the development of suitable models to test its roles in EBV reactivation in vivo.

Keywords:  ER-associated degradation (ERAD); caveolae; gamma-herpesvirus; host–virus response; humoral immunity

DOI:  https://doi.org/10.1073/pnas.2400167122
Sci Adv. 2025 Jan 24. 11(4): eadr8638

De novo design of peptide binders to conformationally diverse targets with contrastive language modeling.

Suhaas Bhat, Kalyan Palepu, Lauren Hong, Joey Mao, Tianzheng Ye, Rema Iyer, Lin Zhao, Tianlai Chen, Sophia Vincoff, Rio Watson, Tian Z Wang, Divya Srijay, Venkata Srikar Kavirayuni, Kseniia Kholina, Shrey Goel, Pranay Vure, Aniruddha J Deshpande, Scott H Soderling, Matthew P DeLisa, Pranam Chatterjee.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery. In this work, we provide an algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model and subsequently screen these novel sequences for target-selective interaction activity via a contrastive language-image pretraining (CLIP)-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains. PepPrCLIP-derived constructs demonstrate functionally potent binding and degradation of conformationally diverse, disease-driving targets in vitro. In total, PepPrCLIP empowers the modulation of previously inaccessible proteins without reliance on stable and ordered tertiary structures.

DOI: https://doi.org/10.1126/sciadv.adr8638
Nucleic Acids Res. 2025 Jan 11. pii: gkaf003. [Epub ahead of print]53(2):

Engineered initiator tRNAs can effectively start translation at non-AUG start codons and diversify N-terminal amino acids for mRNA Display.

Christina Helmling, Alix I Chan, Christian N Cunningham.

mRNA display is an effective tool to identify high-affinity macrocyclic binders for challenging protein targets. The success of an mRNA display selection is dependent on generating highly diverse libraries with trillions of peptides. While translation elongation can canonically accommodate the 61 proteinogenic triplet codons, translation initiation is restricted to the native start codon AUG. Here, we investigate the ability of the Escherichia coli ribosome to initiate translation for 31 initiator tRNA (tRNAini) anticodon mutants at their respective cognate start codon using a NanoBiT translation assay. We show that the ability of those anticodon mutant tRNAsini to initiate translation is highly variable and is, in part, inhibited by tRNA misfolding induced by the anticodon mutations. We demonstrate based on two distinct misfolding patterns that translation efficiency can be effectively restored by introducing additional mutations that restore the active tRNA fold. We then used 10 of the engineered tRNAsini in a mutational analysis experiment for three reported macrocyclic peptides binding to Ubiquitin Specific Protease 7 (USP7). The observed enrichment of peptides correlates strongly with dissociation constants measured by surface plasmon resonance, and provides insights into the structure-activity relationship of the N-terminal amino acid without the requirement for peptide synthesis.

DOI: https://doi.org/10.1093/nar/gkaf003
PLoS Genet. 2025 Jan 21. 21(1): e1011555

Multi-modal investigation reveals pathogenic features of diverse DDX3X missense mutations.

Federica Mosti, Mariah L Hoye, Carla F Escobar-Tomlienovich, Debra L Silver.

De novo mutations in the RNA binding protein DDX3X cause neurodevelopmental disorders including DDX3X syndrome and autism spectrum disorder. Amongst ~200 mutations identified to date, half are missense. While DDX3X loss of function is known to impair neural cell fate, how the landscape of missense mutations impacts neurodevelopment is almost entirely unknown. Here, we integrate transcriptomics, proteomics, and live imaging to demonstrate clinically diverse DDX3X missense mutations perturb neural development via distinct cellular and molecular mechanisms. Using mouse primary neural progenitors, we investigate four recurrently mutated DDX3X missense variants, spanning clinically severe (2) to mild (2). While clinically severe mutations impair neurogenesis, mild mutations have only a modest impact on cell fate. Moreover, expression of severe mutations leads to profound neuronal death. Using a proximity labeling screen in neural progenitors, we discover DDX3X missense variants have unique protein interactors. We observe notable overlap amongst severe mutations, suggesting common mechanisms underlying altered cell fate and survival. Transcriptomic analysis and subsequent cellular investigation highlights new pathways associated with DDX3X missense variants, including upregulated DNA Damage Response. Notably, clinically severe mutations exhibit excessive DNA damage in neurons, associated with increased cytoplasmic DNA:RNA hybrids and formation of stress granules. These findings highlight aberrant RNA metabolism and DNA damage in DDX3X-mediated neuronal cell death. In sum our findings reveal new mechanisms by which clinically distinct DDX3X missense mutations differentially impair neurodevelopment.

DOI: https://doi.org/10.1371/journal.pgen.1011555
Cell Struct Funct. 2025 Jan 23.

Tango1L but not Tango1S, Tali and cTAGE5 is required for export of type II collagen in medaka fish.

Yusuke Yasuda, Tomoka Yoshida, Mahiro Oue, Masaya Sengiku, Tokiro Ishikawa, Shunsuke Saito, Byungseok Jin, Kazutoshi Mori.

  Newly synthesized proteins destined for the secretory pathway are folded and assembled in the endoplasmic reticulum (ER) and then transported to the Golgi apparatus via COPII vesicles, which are normally 60-90 nm. COPII vesicles must accordingly be enlarged to accommodate proteins larger than 90 nm, such as long-chain collagen. Key molecules involved in this enlargement are Tango1 and Tango1-like (Tali), which are transmembrane proteins in the ER encoded by the MIA3 and MIA2 genes, respectively. Interestingly, two splicing variants are expressed from each of these two genes: Tango1L and Tango1S from the MIA3 gene, and Tali and cTAGE5 from the MIA2 gene. Here, we constructed Tango1L-knockout (KO), Tango1S-KO, Tali-KO, and cTAGE5-KO separately in medaka fish, a vertebrate model organism, and characterized them. Results showed that only Tango1L-KO conferred a lethal phenotype to medaka fish. Only Tango1L-KO medaka fish exhibited a shorter tail than wild-type (WT) fish and showed the defects in the export of type II collagen from the ER, contrary to the previous reports analyzing Tango1-KO or Tali-KO mice and the results of knockdown experiments in human cultured cells. Medaka fish may employ a simpler system than mammals for the export of large molecules from the ER.Key words: intracellular transport, COPII vesicles, enlargement, endoplasmic reticulum, Golgi apparatus.

Keywords:  COPII vesicles; Golgi apparatus; endoplasmic reticulum; enlargement; intracellular transport

DOI:  https://doi.org/10.1247/csf.25001
PLoS Genet. 2025 Jan 22. 21(1): e1011574

The Cul3 ubiquitin ligase engages Insomniac as an adaptor to impact sleep and synaptic homeostasis.

Qiuling Li, Kayla Y Lim, Raad Altawell, Faith Verderose, Xiling Li, Wanying Dong, Joshua Martinez, Dion Dickman, Nicholas Stavropoulos.

Mutations of the Cullin-3 (Cul3) E3 ubiquitin ligase are associated with autism and schizophrenia, neurological disorders characterized by sleep disturbances and altered synaptic function. Cul3 engages dozens of adaptor proteins to recruit hundreds of substrates for ubiquitination, but the adaptors that impact sleep and synapses remain ill-defined. Here we implicate Insomniac (Inc), a conserved protein required for normal sleep and synaptic homeostasis in Drosophila, as a Cul3 adaptor. Inc binds Cul3 in vivo, and mutations within the N-terminal BTB domain of Inc that weaken Inc-Cul3 associations impair Inc activity, suggesting that Inc function requires binding to the Cul3 complex. Deletion of the conserved C-terminus of Inc does not alter Cul3 binding but abolishes Inc activity in the context of sleep and synaptic homeostasis, indicating that the Inc C-terminus has the properties of a substrate recruitment domain. Mutation of a conserved, disease-associated arginine in the Inc C-terminus also abolishes Inc function, suggesting that this residue is vital for recruiting Inc targets. Inc levels are negatively regulated by Cul3 in neurons, consistent with Inc degradation by autocatalytic ubiquitination, a hallmark of Cullin adaptors. These findings link Inc and Cul3 in vivo and support the notion that Inc-Cul3 complexes are essential for normal sleep and synaptic function. Furthermore, these results indicate that dysregulation of conserved substrates of Inc-Cul3 complexes may contribute to altered sleep and synaptic function in autism and schizophrenia associated with Cul3 mutations.

DOI: https://doi.org/10.1371/journal.pgen.1011574
FEBS Lett. 2025 Jan 17.

Diphthamide synthesis is linked to the eEF2-client chaperone machinery.

Lars Kaduhr, Klaus Mayer, Raffael Schaffrath, Johannes Buchner, Ulrich Brinkmann.

  The diphthamide modification of eukaryotic translation elongation factor (eEF2) is important for accurate protein synthesis. While the enzymes for diphthamide synthesis are known, coordination of eEF2 synthesis with the diphthamide modification to maintain only modified eEF2 is unknown. Physical and genetic interactions extracted from BioGRID show a connection between diphthamide synthesis enzymes and chaperones in yeast. This includes the Hsp90 co-chaperones Hgh1 and Cpr7. The respective co-chaperone deletion strains contained eEF2 without diphthamide. Notably, strains deficient in other co-chaperones showed no defect in the eEF2-diphthamide modification. Our results demonstrate that diphthamide synthesis involves not only Dph enzymes but also the eEF2-interacting co-chaperones Hgh1 and Cpr7 and may thus require a conformational state of eEF2 which is maintained by specific (co-)chaperones.

Keywords:  Cpr7; Hgh1; J‐protein; chaperone; diphthamide; elongation factor 2 (eEF2)

DOI:  https://doi.org/10.1002/1873-3468.15095
Cell Rep. 2025 Jan 21. pii: S2211-1247(24)01575-4. [Epub ahead of print]44(2): 115224

Redox proteomics reveal a role for peroxiredoxinylation in stress protection.

Gerhard Seisenbacher, Zrinka Raguz Nakic, Eva Borràs, Eduard Sabidó, Uwe Sauer, Eulalia de Nadal, Francesc Posas.

  The redox state of proteins is essential for their function and guarantees cell fitness. Peroxiredoxins protect cells against oxidative stress, maintain redox homeostasis, act as chaperones, and transmit hydrogen peroxide signals to redox regulators. Despite the profound structural and functional knowledge of peroxiredoxins action, information on how the different functions are concerted is still scarce. Using global proteomic analyses, we show here that the yeast peroxiredoxin Tsa1 interacts with many proteins of essential biological processes, including protein turnover and carbohydrate metabolism. Several of these interactions are of a covalent nature, and we show that failure of peroxiredoxinylation of Gnd1 affects its phosphogluconate dehydrogenase activity and impairs recovery upon stress. Thioredoxins directly remove TSA1-formed mixed disulfide intermediates, thus expanding the role of the thioredoxin-peroxiredoxin redox cycle pair to buffer the redox state of proteins.

Keywords:  CP: Molecular biology; Tsa1; oxidative stress; peroxiredoxins; redox biology; stress

DOI:  https://doi.org/10.1016/j.celrep.2024.115224
Nat Struct Mol Biol. 2025 Jan 20.

Structures of aberrant spliceosome intermediates on their way to disassembly.

Komal Soni, Attila Horvath, Olexandr Dybkov, Merlin Schwan, Sasanan Trakansuebkul, Dirk Flemming, Klemens Wild, Henning Urlaub, Tamás Fischer, Irmgard Sinning.

Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-electron microscopy structures of post-Bact spliceosome intermediates from Schizosaccharomyces pombe primed for disassembly. We identify the DEAH-box helicase-G-patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an overstabilization of the U5 loop I interaction with the 5' exon leading to a single-nucleotide insertion at the 5' splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.

DOI: https://doi.org/10.1038/s41594-024-01480-7
bioRxiv. 2025 Jan 09. pii: 2025.01.09.632186. [Epub ahead of print]

Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation.

Yannan Tian, Qingrong Li, Shirin Fatma, Junyi Jiang, Hong Jin, Fuxing Zeng, Raven H Huang.

In bacteria, spontaneous mRNAs degradation and ribotoxin-induced RNA damage are two main biological events that lead to the stall of protein translation. The ubiquitous trans-translation system as well as several alternative rescue factors (Arfs) are responsible for rescuing the stalled ribosomes caused by truncated mRNAs that lack the stop codons. To date, protein release factor homolog (PrfH) is the only factor known to rescue the stalled ribosome damaged by ribotoxins. Here we show that a subfamily of PrfH, exemplified by PrfH from Capnocytophaga gingivalis ( Cg PrfH), rescues both types of stalled ribosomes described above. Our in vitro biochemical assays demonstrate that Cg PrfH hydrolyzes the peptides attached to P-site tRNAs when in complex with both the damaged and intact ribosomes. Two cryo-EM structures of Cg PrfH in complex with the damaged and intact 70S ribosomes revealed that Cg PrfH employs two different regions of the protein to recognize two different stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization described here, we have uncovered a family of ribosome rescue factors that possesses dual activities to resolve two distinct stalled protein translation in bacteria.

DOI: https://doi.org/10.1101/2025.01.09.632186
ACS Nano. 2025 Jan 24.

Endoplasmic Reticulum-Targeted Polymer-Manganese Nanocomplexes for Tumor Immunotherapy.

Haoru Zhu, Chang Xu, Yu Geng, Youqing Shen, Nasha Qiu.

  Manganese ions (Mn2+) are an immune activator that enhances the activation of both cGAS and STING proteins. The STING signaling activation and subsequential immune responses are predominantly associated with endoplasmic reticulum (ER). Therefore, ER targeting of Mn2+ in the subcellular compartments would promote the activation of STING signaling pathways. Herein, we report the design of ER-targeted manganese-based nanocomplexes (NCs) by complexation of Mn2+ with a zwitterionic polymer, poly[2-(N-oxide-N,N-dimethylamino) ethyl methacrylate] (OPDMA). The Mn/OPDMA nanocomplexes (Mn/OPDMA NCs) keep a long blood circulation for tumor accumulation and trigger adsorption-mediated transcytosis for extravasation and deep tumor penetration. Notably, in the tumor-associated macrophages, the Mn/OPDMA NCs can preferentially translocate to their ERs, significantly enhancing cGAS-STING pathway activation for tumor-associated macrophage polarization and IFN-β secretion. In mouse colon and hepatocellular cancer models, the intravenously administrated Mn/OPDMA NCs efficiently remodel tumor immune microenvironment, greatly retard tumor growths by 2.4- to 5-fold, and prolong the mouse survivals compared to free Mn2+-treated mice. This study provides the ER-targeted delivery of Mn2+ that achieves robust STING activation and, thus, potent systemic tumor inhibition without the toxicity of free Mn2+.

Keywords:  cGAS/STING activation; cancer immune therapy; endoplasmic reticulum-targeted nanocomplexes; manganese ions; metalloimmunology

DOI:  https://doi.org/10.1021/acsnano.4c17279
Mol Cell. 2025 Jan 16. pii: S1097-2765(24)01010-4. [Epub ahead of print]85(2): 323-346

Ubiquitin-A structural perspective.

Rashmi Agrata, David Komander.

  The modification of proteins and other biomolecules with the small protein ubiquitin has enthralled scientists from many disciplines for decades, creating a broad research field. Ubiquitin research is particularly rich in molecular and mechanistic understanding due to a plethora of (poly)ubiquitin structures alone and in complex with ubiquitin machineries. Furthermore, due to its favorable properties, ubiquitin serves as a model system for many biophysical and computational techniques. Here, we review the current knowledge of ubiquitin signals through a ubiquitin-centric, structural biology lens. We amalgamate the information from 240 structures in the Protein Data Bank (PDB), combined with single-molecule, molecular dynamics, and nuclear magnetic resonance (NMR) studies, to provide a comprehensive picture of ubiquitin and polyubiquitin structures and dynamics. We close with a discussion of the latest frontiers in ubiquitin research, namely the modification of ubiquitin by other post-translational modifications (PTMs) and the notion that ubiquitin is attached to biomolecules beyond proteins.

Keywords:  linkage specificity; non-proteinaceous; phospho-ubiquitin; post-translational modification; ubiquitin; ubiquitin chain

DOI:  https://doi.org/10.1016/j.molcel.2024.12.015
bioRxiv. 2025 Jan 06. pii: 2025.01.06.631537. [Epub ahead of print]

FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis.

Mike Lange, Michele Wölk, Cody E Doubravsky, Joseph M Hendricks, Shunji Kato, Yurika Otoki, Benjamin Styler, Kiyotaka Nakagawa, Maria Fedorova, James A Olzmann.

Lipid droplets (LDs) are organelles that store and supply lipids based on cellular needs. While mechanisms preventing oxidative damage to membrane phospholipids are established, the vulnerability of LD neutral lipids to peroxidation and protective mechanisms are unknown. Here, we identify LD-localized Ferroptosis Suppressor Protein 1 (FSP1) as a critical regulator that prevents neutral lipid peroxidation by recycling coenzyme Q10 (CoQ10) to its lipophilic antioxidant form. Lipidomics reveal that FSP1 loss leads to the accumulation of oxidized triacylglycerols and cholesteryl esters, and biochemical reconstitution of FSP1 with CoQ10 and NADH suppresses triacylglycerol peroxidation in vitro. Notably, polyunsaturated fatty acid (PUFA)-rich triacylglycerols enhance cancer cell sensitivity to FSP1 loss and inducing PUFA-rich LDs triggers triacylglycerol peroxidation and LD-initiated ferroptosis when FSP1 activity is impaired. These findings uncover the first LD lipid quality control pathway, wherein LD-localized FSP1 maintains neutral lipid integrity to prevent the buildup of oxidized lipids and induction of ferroptosis.

DOI: https://doi.org/10.1101/2025.01.06.631537
bioRxiv. 2025 Jan 09. pii: 2025.01.09.632082. [Epub ahead of print]

UFMylation promotes orthoflavivirus infectious particle production.

Hannah M Schmidt, Grace C Sorensen, Matthew R Lanahan, Jenna Grabowski, Moonhee Park, Stacy M Horner.

Post-translational modifications play crucial roles in viral infections, yet many potential modifications remain unexplored in orthoflavivirus biology. Here we demonstrate that the UFMylation system, a post-translational modification system that catalyzes the transfer of UFM1 onto proteins, promotes infection by multiple orthoflaviviruses including dengue virus, Zika virus, West Nile virus, and yellow fever virus. We found that depletion of the UFMylation E3 ligase complex proteins UFL1 and UFBP1, as well as other UFMylation machinery components (UBA5, UFC1, and UFM1), significantly reduces infectious virion production for orthoflaviviruses but not the hepacivirus, hepatitis C. Mechanistically, UFMylation does not regulate viral RNA translation or RNA replication but instead affects a later stage of the viral lifecycle. We identified novel interactions between UFL1, and several viral proteins involved in orthoflavivirus virion assembly, including NS2A, NS2B-NS3, and Capsid. These findings establish UFMylation as a previously unrecognized post-translational modification system that promotes orthoflavivirus infection, likely through modulation of viral assembly. This work expands our understanding of the post-translational modifications that control orthoflavivirus infection and identifies new potential therapeutic targets.

DOI: https://doi.org/10.1101/2025.01.09.632082
Mol Cell. 2025 Jan 16. pii: S1097-2765(24)01039-6. [Epub ahead of print]85(2): 290-308

Molecular determinants of condensate composition.

Alex S Holehouse, Simon Alberti.

  Cells use membraneless compartments to organize their interiors, and recent research has begun to uncover the molecular principles underlying their assembly. Here, we explore how site-specific and chemically specific interactions shape the properties and functions of condensates. Site-specific recruitment involves precise interactions at specific sites driven by partially or fully structured interfaces. In contrast, chemically specific recruitment is driven by complementary chemical interactions without the requirement for a persistent bound-state structure. We propose that site-specific and chemically specific interactions work together to determine the composition of condensates, facilitate biochemical reactions, and regulate enzymatic activities linked to metabolism, signaling, and gene expression. Characterizing the composition of condensates requires novel experimental and computational tools to identify and manipulate the molecular determinants guiding condensate recruitment. Advancing this research will deepen our understanding of how condensates regulate cellular functions, providing valuable insights into cellular physiology and organization.

Keywords:  binding; biomolecular condensates; cellular organization; chemical specificity; disordered protein regions; phase separation; specificity

DOI:  https://doi.org/10.1016/j.molcel.2024.12.021
Nat Commun. 2025 Jan 17. 16(1): 737

Engineering a membrane protein chaperone to ameliorate the proteotoxicity of mutant huntingtin.

Jeonghyun Oh, Christy Catherine, Eun Seon Kim, Kwang Wook Min, Hae Chan Jeong, Hyojin Kim, Mijin Kim, Seung Hae Ahn, Nataliia Lukianenko, Min Gu Jo, Hyeon Seok Bak, Sungsu Lim, Yun Kyung Kim, Ho Min Kim, Sung Bae Lee, Hyunju Cho.

Toxic protein aggregates are associated with various neurodegenerative diseases, including Huntington's disease (HD). Since no current treatment delays the progression of HD, we develop a mechanistic approach to prevent mutant huntingtin (mHttex1) aggregation. Here, we engineer the ATP-independent cytosolic chaperone PEX19, which targets peroxisomal membrane proteins to peroxisomes, to remove mHttex1 aggregates. Using yeast toxicity-based screening with a random mutant library, we identify two yeast PEX19 variants and engineer equivalent mutations into human PEX19 (hsPEX19). These variants effectively delay mHttex1 aggregation in vitro and in cellular HD models. The mutated hydrophobic residue in the α4 helix of hsPEX19 variants binds to the N17 domain of mHttex1, thereby inhibiting the initial aggregation process. Overexpression of the hsPEX19-FV variant rescues HD-associated phenotypes in primary striatal neurons and in Drosophila. Overall, our data reveal that engineering ATP-independent membrane protein chaperones is a promising therapeutic approach for rational targeting of mHttex1 aggregation in HD.

DOI: https://doi.org/10.1038/s41467-025-56030-6
JCI Insight. 2025 Jan 23. pii: e183959. [Epub ahead of print]10(2):

Inhibition of vascular smooth muscle cell PERK/ATF4 ER stress signaling protects against abdominal aortic aneurysms.

Brennan Callow, Xiaobing He, Nicholas Juriga, Kevin D Mangum, Amrita Joshi, Xianying Xing, Andrea Obi, Abhijnan Chattopadhyay, Dianna M Milewicz, Mary X O'Riordan, Johann Gudjonsson, Katherine Gallagher, Frank M Davis.

  Abdominal aortic aneurysms (AAA) are a life-threatening cardiovascular disease for which there is a lack of effective therapy preventing aortic rupture. During AAA formation, pathological vascular remodeling is driven by vascular smooth muscle cell (VSMC) dysfunction and apoptosis, for which the mechanisms regulating loss of VSMCs within the aortic wall remain poorly defined. Using single-cell RNA-Seq of human AAA tissues, we identified increased activation of the endoplasmic reticulum stress response pathway, PERK/eIF2α/ATF4, in aortic VSMCs resulting in upregulation of an apoptotic cellular response. Mechanistically, we reported that aberrant TNF-α activity within the aortic wall induces VSMC ATF4 activation through the PERK endoplasmic reticulum stress response, resulting in progressive apoptosis. In vivo targeted inhibition of the PERK pathway, with VSMC-specific genetic depletion (Eif2ak3fl/fl Myh11-CreERT2) or pharmacological inhibition in the elastase and angiotensin II-induced AAA model preserved VSMC function, decreased elastin fragmentation, attenuated VSMC apoptosis, and markedly reduced AAA expansion. Together, our findings suggest that cell-specific pharmacologic therapy targeting the PERK/eIF2α/ATF4 pathway in VSMCs may be an effective intervention to prevent AAA expansion.

Keywords:  Cardiovascular disease; Cell stress; Surgery; Vascular biology

DOI:  https://doi.org/10.1172/jci.insight.183959
Nat Commun. 2025 Jan 20. 16(1): 849

Dynamic allostery in the peptide/MHC complex enables TCR neoantigen selectivity.

Jiaqi Ma, Cory M Ayres, Chad A Brambley, Smita S Chandran, Tatiana J Rosales, W W J Gihan Perera, Bassant Eldaly, William T Murray, Steven A Corcelli, Evgenii L Kovrigin, Christopher A Klebanoff, Brian M Baker.

The inherent antigen cross-reactivity of the T cell receptor (TCR) is balanced by high specificity. Surprisingly, TCR specificity often manifests in ways not easily interpreted from static structures. Here we show that TCR discrimination between an HLA-A*03:01 (HLA-A3)-restricted public neoantigen and its wild-type (WT) counterpart emerges from distinct motions within the HLA-A3 peptide binding groove that vary with the identity of the peptide's first primary anchor. These motions create a dynamic gate that, in the presence of the WT peptide, impedes a large conformational change required for TCR binding. The neoantigen is insusceptible to this limiting dynamic, and, with the gate open, upon TCR binding the central tryptophan can transit underneath the peptide backbone to the opposing side of the HLA-A3 peptide binding groove. Our findings thus reveal a novel mechanism driving TCR specificity for a cancer neoantigen that is rooted in the dynamic and allosteric nature of peptide/MHC-I binding grooves, with implications for resolving long-standing and often confounding questions about T cell specificity.

DOI: https://doi.org/10.1038/s41467-025-56004-8
Nat Commun. 2025 Jan 22. 16(1): 957

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.

Griffin J Davis, Anthony O Omole, Yejin Jung, Wioletta Rut, Ronald Holewinski, Kiall F Suazo, Hong-Rae Kim, Mo Yang, Thorkell Andresson, Marcin Drag, Euna Yoo.

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling the selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) such as USP5 and USP14. Combined with a ubiquitin-based DUB ABP, the USP18 ABP is employed in a chemoproteomics screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

DOI: https://doi.org/10.1038/s41467-025-56336-5
Commun Biol. 2025 Jan 18. 8(1): 83

Reduced UPF1 levels in senescence impair nonsense-mediated mRNA decay.

Dahyeon Koh, Yebin Lee, Kyuchan Kim, Hyeong Bin Jeon, Chaehwan Oh, Sangik Hwang, Minjung Lim, Kwang-Pyo Lee, Yeonkyoung Park, Yong Ryoul Yang, Yoon Ki Kim, Donghwan Shim, Myriam Gorospe, Ji Heon Noh, Kyoung Mi Kim.

Cells regulate gene expression through various RNA regulatory mechanisms, and this regulation often becomes less efficient with age, contributing to accelerated aging and various age-related diseases. Nonsense-mediated mRNA decay (NMD), a well-characterized RNA surveillance mechanism, degrades aberrant mRNAs with premature termination codons (PTCs) to prevent the synthesis of truncated proteins. While the role of NMD in cancer and developmental and genetic diseases is well documented, its implications in human aging remain largely unexplored. This study reveals a significant decline in the levels of the protein UPF1, a key player in NMD, during cellular senescence. Additionally, NMD substrates accumulate in senescent cells, along with decreased levels of cap-binding protein 80/20 (CBP80/20)-dependent translation (CT) factors and reduced binding to active polysomes, indicating reduced efficiency of NMD. Moreover, knockdown of UPF1 in proliferating WI-38 cells induces senescence, as evidenced by increased senescence-associated β-galactosidase activity, alterations in senescence-associated molecular markers, increased endogenous γ-H2AX levels, and reduced cell proliferation. These findings suggest that the decline in UPF1 levels during cellular senescence accelerates the senescent phenotype by impairing NMD activity and the consequent accumulation of abnormal mRNA.

DOI: https://doi.org/10.1038/s42003-025-07502-4
J Med Chem. 2025 Jan 24.

Design and Discovery of Preclinical Candidate LYG-409 as a Highly Potent and Selective GSPT1 Molecular Glue Degraders.

Yanqing Zhang, Wenjing Liu, Chao Tong, Xinhong Wang, Xiujin Chang, Fangui Qu, Zhiming Zhang, Zhongpen Fan, Monong Zhao, Cheng Tang, Beichen Song, Ming Ding, Zhixia Qiu, Jubo Wang, Jinlei Bian, Zhiyu Li, Hongxi Wu, Xi Xu.

Molecular glue degraders induce "undruggable" protein degradation by a proximity-induced effect. Inspired by the clinical success of immunomodulatory drugs, we aimed to design novel molecular glue degraders targeting GSPT1. Here, we report the design of a series of GSPT1 molecular glue degraders. LYG-409, a 2H-chromene derivative, was identified as a potent, selective, and orally bioavailable GSPT1 degrader with excellent antitumor activity in vivo (anti-Acute Myeloid Leukemia MV4-11 xenograft model: TGI = 94.34% at 30 mg/kg; prostate cancer 22Rv1 xenograft model: TGI = 104.49% at 60 mg/kg) and in vitro (KG-1 cells: IC50 = 9.50 ± 0.71 nM, DC50 = 7.87 nM) mediated by the degradation of GSPT1. In conclusion, LYG-409 exhibits potent GSPT1 degradation activity, demonstrating promising therapeutic efficacy and favorable safety profile. However, its potential drug resistance profile needs to be thoroughly evaluated in comparison with existing treatments. We hope LYG-409 can provide a valuable direction for the development of GSPT1 degraders.

DOI: https://doi.org/10.1021/acs.jmedchem.4c01787
Nat Commun. 2025 Jan 17. 16(1): 760

Lipidic folding pathway of α-Synuclein via a toxic oligomer.

Vrinda Sant, Dirk Matthes, Hisham Mazal, Leif Antonschmidt, Franz Wieser, Kumar T Movellan, Kai Xue, Evgeny Nimerovsky, Marianna Stampolaki, Magdeline Nathan, Dietmar Riedel, Stefan Becker, Vahid Sandoghdar, Bert L de Groot, Christian Griesinger, Loren B Andreas.

Aggregation intermediates play a pivotal role in the assembly of amyloid fibrils, which are central to the pathogenesis of neurodegenerative diseases. The structures of filamentous intermediates and mature fibrils are now efficiently determined by single-particle cryo-electron microscopy. By contrast, smaller pre-fibrillar α-Synuclein (αS) oligomers, crucial for initiating amyloidogenesis, remain largely uncharacterized. We report an atomic-resolution structural characterization of a toxic pre-fibrillar aggregation intermediate (I1) on pathway to the formation of lipidic fibrils, which incorporate lipid molecules on protofilament surfaces during fibril growth on membranes. Super-resolution microscopy reveals a tetrameric state, providing insights into the early oligomeric assembly. Time resolved nuclear magnetic resonance (NMR) measurements uncover a structural reorganization essential for the transition of I1 to mature lipidic L2 fibrils. The reorganization involves the transformation of anti-parallel β-strands during the pre-fibrillar I1 state into a β-arc characteristic of amyloid fibrils. This structural reconfiguration occurs in a conserved structural kernel shared by a vast number of αS-fibril polymorphs including extracted fibrils from Parkinson's and Lewy Body Dementia patients. Consistent with reports of anti-parallel β-strands being a defining feature of toxic αS pre-fibrillar intermediates, I1 impacts viability of neuroblasts and disrupts cell membranes, resulting in an increased calcium influx. Our results integrate the occurrence of anti-parallel β-strands as salient features of toxic oligomers with their significant role in the amyloid fibril assembly pathway. These structural insights have implications for the development of therapies and biomarkers.

DOI: https://doi.org/10.1038/s41467-025-55849-3
Commun Biol. 2025 Jan 21. 8(1): 103

A computational tool to infer enzyme activity using post-translational modification profiling data.

Dehui Kong, Aijun Zhang, Ling Li, Zuo-Fei Yuan, Yingxue Fu, Long Wu, Ashutosh Mishra, Anthony A High, Junmin Peng, Xusheng Wang.

Enzymes play a pivotal role in orchestrating complex cellular responses to external stimuli and environmental changes through signal transduction pathways. Despite their crucial roles, measuring enzyme activities is typically indirect and performed on a smaller scale, unlike protein abundance measured by high-throughput proteomics. Moreover, it is challenging to derive the activity of enzymes from proteome-wide post-translational modification (PTM) profiling data. To address this challenge, we introduce enzyme activity inference with structural equation modeling under the JUMP umbrella (JUMPsem), a novel computational tool designed to infer enzyme activity using PTM profiling data. We demonstrate that the JUMPsem program enables estimating kinase activities using phosphoproteome data, ubiquitin E3 ligase activities from the ubiquitinome, and histone acetyltransferase (HAT) activities based on the acetylome. In addition, JUMPsem is capable of establishing novel enzyme-substrate relationships through searching motif sequences. JUMPsem outperforms widely used kinase activity tools, such as IKAP and KSEA, in terms of the number of kinases and the computational speed. The JUMPsem program is scalable and publicly available as an open-source R package and user-friendly web-based R/Shiny app. Collectively, JUMPsem provides an improved tool for inferring protein enzyme activities, potentially facilitating targeted drug development.

DOI: https://doi.org/10.1038/s42003-025-07548-4
Protein Sci. 2025 Feb;34(2): e70031

AggNet: Advancing protein aggregation analysis through deep learning and protein language model.

Wenjia He, Xiaopeng Xu, Haoyang Li, Juexiao Zhou, Xin Gao.

  Protein aggregation is critical to various biological and pathological processes. Besides, it is also an important property in biotherapeutic development. However, experimental methods to profile protein aggregation are costly and labor-intensive, driving the need for more efficient computational alternatives. In this study, we introduce "AggNet," a novel deep learning framework based on the protein language model ESM2 and AlphaFold2, which utilizes physicochemical, evolutionary, and structural information to discriminate amyloid and non-amyloid peptides and identify aggregation-prone regions (APRs) in diverse proteins. Benchmark comparisons show that AggNet outperforms existing methods and achieves state-of-the-art performance on protein aggregation prediction. Also, the predictive ability of AggNet is stable across proteins with different secondary structures. Feature analysis and visualizations prove that the model effectively captures peptides' physicochemical properties effectively, thereby offering enhanced interpretability. Further validation through a case study on MEDI1912 confirms AggNet's practical utility in analyzing protein aggregation and guiding mutation for aggregation mitigation. This study enhances computational tools for predicting protein aggregation and highlights the potential of AggNet in protein engineering. Finally, to improve the accessibility of AggNet, the source code can be accessed at: https://github.com/Hill-Wenka/AggNet.

Keywords:  APR; amyloid; computational biology; machine learning; protein aggregation; protein engineering

DOI:  https://doi.org/10.1002/pro.70031
Commun Biol. 2025 Jan 20. 8(1): 92

DDX1 is required for non-spliceosomal splicing of tRNAs but not of XBP1 mRNA.

Teruhiko Suzuki, Satoko Takagi, Junta Funada, Yuka Egawa, Mana Yamakawa, Takahiko Hara.

RNA helicase DEAD-box helicase 1 (DDX1) forms a complex with the RNA ligase 2´,3´-cyclic phosphate and 5´-OH ligase (RTCB), which plays a vital role in non-spliceosomal splicing of tRNA and X-box binding protein 1 (XBP1) mRNA. However, the importance of DDX1 in non-spliceosomal splicing has not been clarified. To analyze the functions of DDX1 in mammalian cells, we generated DDX1 cKO cells from the polyploid human U2OS cell line and found that splicing of intron-containing tRNAs was significantly disturbed in DDX1-deficient cells, whereas endoplasmic reticulum (ER) stress-induced splicing of XBP1 mRNA was unaffected. Additionally, the enforced expression of DDX1, but not of its helicase-inactive mutant, rescued the splicing defects of tRNAs in DDX1-deficient cells. These results indicate that RTCB is required for the splicing of both tRNA and XBP1 mRNA, whereas the DDX1 enzymatic activity is specifically required for tRNA splicing in vivo.

DOI: https://doi.org/10.1038/s42003-025-07523-z
Cell Rep Methods. 2025 Jan 11. pii: S2667-2375(24)00350-3. [Epub ahead of print] 100960

The FIRE biosensor illuminates iron regulatory protein activity and cellular iron homeostasis.

Carolyn Sangokoya.

  On Earth, iron is abundant, bioavailable, and crucial for initiating the first catalytic reactions of life from prokaryotes to plants to mammals. Iron-complexed proteins are critical to biological pathways and essential cellular functions. While it is well known that the regulation of iron is necessary for mammalian development, little is known about the timeline of how specific transcripts network and interact in response to cellular iron regulation to shape cell fate, function, and plasticity in the developing embryo and beyond. Here, we present a ratiometric genetically encoded dual biosensor called FIRE (Fe-IRE [iron-responsive element]) to evaluate iron regulatory protein (IRP)-binding activity and cellular iron status in live cells, allowing for the study and dissection of dynamic changes in cellular iron and IRP activity over developmental time. FIRE reveals a previously unrecognized foundational timeline of IRP activity and cellular iron homeostasis during stem cell pluripotency transition and early differentiation.

Keywords:  CP: Metabolism; CP: Molecular biology; IRPs; biosensor; embryoid; embryonic stem cells; iron; iron homeostasis; iron regulatory protein; pluripotent stem cells; post-transcriptional regulation; stem cell biology

DOI:  https://doi.org/10.1016/j.crmeth.2024.100960
Cell. 2025 Jan 15. pii: S0092-8674(24)01429-6. [Epub ahead of print]

Metagenome-informed metaproteomics of the human gut microbiome, host, and dietary exposome uncovers signatures of health and inflammatory bowel disease.

Rafael Valdés-Mas, Avner Leshem, Danping Zheng, Yotam Cohen, Lara Kern, Niv Zmora, Yiming He, Corine Katina, Shimrit Eliyahu-Miller, Tal Yosef-Hevroni, Liron Richman, Barbara Raykhel, Shira Allswang, Reut Better, Merav Shmueli, Aurelia Saftien, Nyssa Cullin, Fernando Slamovitz, Dragos Ciocan, Kyanna S Ouyang, Uria Mor, Mally Dori-Bachash, Shahar Molina, Yishai Levin, Koji Atarashi, Ghil Jona, Jens Puschhof, Alon Harmelin, Noa Stettner, Minhu Chen, Jotham Suez, Kenya Honda, Wolfgang Lieb, Corinna Bang, Michal Kori, Nitsan Maharshak, Yifat Merbl, Oren Shibolet, Zamir Halpern, Dror S Shouval, Raanan Shamir, Andre Franke, Suhaib K Abdeen, Hagit Shapiro, Alon Savidor, Eran Elinav.

  Host-microbiome-dietary interactions play crucial roles in regulating human health, yet their direct functional assessment remains challenging. We adopted metagenome-informed metaproteomics (MIM), in mice and humans, to non-invasively explore species-level microbiome-host interactions during commensal and pathogen colonization, nutritional modification, and antibiotic-induced perturbation. Simultaneously, fecal MIM accurately characterized the nutritional exposure landscape in multiple clinical and dietary contexts. Implementation of MIM in murine auto-inflammation and in human inflammatory bowel disease (IBD) characterized a "compositional dysbiosis" and a concomitant species-specific "functional dysbiosis" driven by suppressed commensal responses to inflammatory host signals. Microbiome transfers unraveled early-onset kinetics of these host-commensal cross-responsive patterns, while predictive analyses identified candidate fecal host-microbiome IBD biomarker protein pairs outperforming S100A8/S100A9 (calprotectin). Importantly, a simultaneous fecal nutritional MIM assessment enabled the determination of IBD-related consumption patterns, dietary treatment compliance, and small intestinal digestive aberrations. Collectively, a parallelized dietary-bacterial-host MIM assessment functionally uncovers trans-kingdom interactomes shaping gastrointestinal ecology while offering personalized diagnostic and therapeutic insights into microbiome-associated disease.

Keywords:  biomarker; diet; inflammatory bowel disease; metagenome-informed metaproteomics; microbiome

DOI:  https://doi.org/10.1016/j.cell.2024.12.016