bims-proteo Biomed News
on Proteostasis
Issue of 2025–06–08
forty-six papers selected by
Eric Chevet, INSERM
  1. Clearance of protein aggregates during cell division.
  2. Arkadia and Ark2C promote substrate ubiquitylation with multiple E2 enzymes.
  3. Coiled-coil-domain-mediated TRIM E3 protein condensation modulates enzymatic activity and functional specificity.
  4. 1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway.
  5. Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies.
  6. Visible-Light-Controlled Lysine-Selective Crosslinking Decodes Protein Complexes and Dynamic Interactomes in Live Cells.
  7. The highly conserved intron of tyrosine tRNA is critical for m1A58 modification and controls the integrated stress response.
  8. Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase.
  9. Proteome-wide Ligand and Target Discovery by Using β-Nitrostyrene Electrophiles: Supporting Targeted Protein Degradation.
  10. ATG9A and ARFIP2 cooperate to control PI4P levels for lysosomal repair.
  11. Structures of Fab-stabilized CHIP reveal a conformational switch important in E3 ligase and chaperone functions.
  12. Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans.
  13. A Protein Disulfide Isomerase Coordinates Redox Homeostasis and ER Calcium Regulation for Optimal Lytic Cycle Progression in Toxoplasma gondii.
  14. Mechanism by which the molecular glue-like verteporfin induces IRE1α dimerization and activation to synergize with AKT inhibition in breast cancer.
  15. p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic.
  16. The endoplasmic reticulum promotes microtubule organization and region-specific disassembly to execute Compartmentalized Cell Elimination.
  17. A complete map of human cytosolic degrons and their relevance for disease.
  18. Concerted transport and phosphorylation of diacylglycerol at ER-PM contact sites regulate phospholipid dynamics during stress.
  19. Perturbations in L-serine metabolism regulates protein quality control through sensor of retrograde response pathway Rtg2 in S.cerevisae.
  20. Biochemical analysis of PD-L1 ubiquitination by CRL3SPOP, ARIH1, and NEDD4 family ubiquitin ligases.
  21. The IRE1-bZIP60 branch of Unfolded Protein Response is required for Arabidopsis immune response to Botrytis cinerea.
  22. Landscape and regulation of mRNA translation in the early C. elegans embryo.
  23. Autophagy hub-protein p62 orchestrates oxidative, endoplasmic reticulum stress, and inflammatory responses post-ischemia, exacerbating stroke outcome.
  24. Type II kinase inhibitors that target Parkinson's disease-associated LRRK2.
  25. Inhibition of virally induced TFEB proteasomal degradation as a host-centric therapeutic approach for coronaviral infection.
  26. A low-complexity linker as a driver of intra- and intermolecular interactions in DNAJB chaperones.
  27. Wild-type EGFR and the oncogenic mutant EGFR (L858R) employ distinct mechanisms for export from the endoplasmic reticulum, a process critical for their activation.
  28. Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress.
  29. Biophysical Approaches for Investigating the Dynamics and Cooperativity of Ternary Complexes in Targeted Protein Degradation.
  30. UFMylation promotes orthoflavivirus infectious particle production.
  31. β-propeller protein-associated neurodegeneration protein WDR45 regulates stress granule disassembly via phase separation with Caprin-1.
  32. Parkin-dependent ubiquitination of TAX1BP1 directs efficient autophagic removal of defective mitochondria.
  33. Explainable Machine Learning Identifies Factors for Dosage Compensation in Aneuploid Human Cancer Cells.
  34. A synthetic chlorophagy receptor promotes plant fitness by mediating chloroplast microautophagy.
  35. Mechanism of ATP hydrolysis in the Hsp70 BiP nucleotide-binding domain.
  36. Chaperone-mediated heterotypic phase separation regulates liquid-to-solid phase transitions of tau into amyloid fibrils.
  37. MedSAGE: Bridging Generative AI and Medicinal Chemistry for Structure-Based Design of Small Molecule Drugs.
  38. HOPS-dependent vesicle tethering lock inhibits endolysosomal fusions and autophagosome secretion upon the loss of Syntaxin17.
  39. A family of E3 ligases extend K11 polyubiquitin on sites of MARUbylation.
  40. Of condensates and coats - reciprocal regulation of clathrin assembly and the growth of protein networks.
  41. Enhancement of MAVS UFMylation and inhibition of lysosomal degradation of MAVS aggregates: New findings on broad-spectrum antiviral mechanism of Anemoside B4.
  42. De novo design of protein condensation inhibitors by targeting an allosteric site of cGAS.
  43. Translational buffering tunes gene expression in mouse and human.
  44. Lysosomal TMEM165 controls cellular ion homeostasis and survival by mediating lysosomal Ca2+ import and H+ efflux.
  45. Endolysosomal processing of neuron-derived signaling lipids regulates autophagy and lipid droplet degradation in astrocytes.
  46. Extracellular exosomal RNAs are glyco-modified.
  1. Elife. 2025 Jun 06. pii: RP96675. [Epub ahead of print]13
    Clearance of protein aggregates during cell division.
    Shoukang Du, Yuhan Wang, Bowen Chen, Shuangshuang Xie, Kuan Yoow Chan, David C Hay, Ting Gang Chew.
      Protein aggregates are spatially organized and regulated in cells to prevent the deleterious effects of proteostatic stress. Misfolding of proteins in the endoplasmic reticulum (ER) results in aggregate formation, but how the aggregates are processed, especially during cell division is not well understood. Here, we induced proteostatic stress and protein aggregation using a proteostasis reporter, which is prone to misfolding and aggregation in the ER. Unexpectedly, we detected solid-like protein aggregates deposited mainly in the nucleus and surrounded by the ER membrane. The membrane-bound aggregates were then cleared as cells progressed through mitosis and cytokinesis. Aggregate clearance depended on Hsp70 family chaperones in the ER, particularly BiP, and proteasomal activity. The clearance culminated at mitotic exit and required cyclin-dependent kinase 1 (Cdk1) inactivation but was independent of the anaphase-promoting complex (APC/C). The ER reorganization that is active during mitosis and cytokinesis was required for the aggregate clearance. Thus, dividing cells reorganize the ER networks to allow BiP to clear the protein aggregates to maintain proteostasis in the newly divided cells.
    Keywords:  ER reorganization; aggregates; cell biology; chaperone; human; mitosis; proteostasis
    DOI:  https://doi.org/10.7554/eLife.96675
  2. J Mol Biol. 2025 May 30. pii: S0022-2836(25)00325-0. [Epub ahead of print] 169259
    Arkadia and Ark2C promote substrate ubiquitylation with multiple E2 enzymes.
    Claudia Rossig, Andrej Paluda, Rebecca Chen, Adam J Middleton, Catherine L Day.
      Ubiquitylation is a critical post-translational modification that is required for cell vitality. Attachment of ubiquitin is complex, with the fate of ubiquitylated proteins determined by the length of the attached ubiquitin chains and the nature of the linkage used to build the chains. Many E3 ligases attach ubiquitin chains of different types to substrate proteins in a context specific manner, but the molecular details of how E3 ligases specify chains of different types is poorly understood. Arkadia/RNF111 is a large RING E3 ligase that modifies some substrates with degradative ubiquitin chains, while other substrates are modified with non-degradative ubiquitin chains. Here, using Arkadia and the related E3 ligase, Ark2C, we characterize the RING-E2 complexes that promote assembly of ubiquitin chains of distinct linkages. Our structural studies highlight the conserved nature of the RING-E2 interface, while our binding and activity assays reveal several different E2 enzymes that functionally interact with Ark2C and Arkadia. Using Arkadia, substrate ubiquitylation assays reveal differences in the ability for substrates to be modified, with the E2 enzymes Ubc13 and Ube2K requiring addition of a 'priming' ubiquitin molecule before subsequent modification can occur. We also show that substrates that bind Arkadia tightly are more extensively modified, and that prior substrate ubiquitylation enhances subsequent modification. While further studies will be required to understand how RING-E2 pairing is modulated in cells, this study advances our understanding of E2 recruitment and chain assembly by Arkadia and provides tools that may help dissect cellular function.
    Keywords:  E2 specificity; E3 ligase; substrate ubiquitylation
    DOI:  https://doi.org/10.1016/j.jmb.2025.169259
  3. Cell Rep. 2025 May 30. pii: S2211-1247(25)00537-6. [Epub ahead of print]44(6): 115766
    Coiled-coil-domain-mediated TRIM E3 protein condensation modulates enzymatic activity and functional specificity.
    Qi Chen, Peiguo Yang.
      Tripartite motif (TRIM) proteins are E3 ligases with modular structures involved in diverse cellular functions. Whether biomolecular condensation broadly regulates TRIM proteins remains unclear. Here, we systematically examine 75 TRIMs in mammalian cells and find that the majority can form condensates, either individually or together with other TRIMs. The coiled-coil (CC) domain is essential for TRIM condensation, and disease-related single-nucleotide polymorphisms (SNPs) in this domain impair condensate formation. We show that condensation modulates E3 ligase activity of several TRIMs in a context-dependent manner using cellular auto-ubiquitination and biotinylated-ubiquitination assays. Proteomic analysis reveals that proteins within 21 TRIM condensates are linked to key cellular pathways and diseases. Functional screening uncovers that certain TRIM condensates regulate centriolar satellite organization, cilia assembly, and microtubule stabilization. Our work provides foundational insights into the condensation and functions of TRIM family proteins and establishes a framework for studying co-condensation specificity among highly homologous proteins.
    Keywords:  CP: Molecular biology; TRIM E3 ligase; biomolecular condensates; centriolar satellite; cilia; coiled-coil domain; microtubule stability; phase separation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115766
  4. bioRxiv. 2025 May 17. pii: 2025.05.13.652513. [Epub ahead of print]
    1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway.
    Yi-Ting Tsai, Nicolas-Frédéric Lipp, Olivia Seidel, Riya Varma, Aurelie Laguerre, Kristina Solorio-Kirpichyan, Adrian Wong, Roberto J Brea, Grace H McGregor, Thekla Cordes, Neal K Devaraj, Lars Kuerschner, Sonya Neal, Christian M Metallo, Itay Budin.
      1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, but their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We first applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged 1-deoxysphingolipids in RPE-1 cells, observing that they are retained in the endoplasmic reticulum (ER). We found that 1-deoxysphingolipids can be transported by the non-vesicular transporter CERT in vitro but are retained at ER exit sites (ERES) in cells, suggesting that they do not efficiently sort into vesicular carriers. Cells expressing disease-associated variants of serine palmitoyl-CoA transferase (SPT) accumulated long-chain 1-deoxysphingolipids, which reduced ER membrane fluidity and enlarged ERES. We observed that the rates of membrane protein release from the ER were altered in response to mutant SPT expression, in a manner that was dependent on the cargo affinity for ordered or disordered membranes. We propose that dysregulation of sphingolipid metabolism alters secretory membrane properties, which can then modulate protein trafficking.
    DOI:  https://doi.org/10.1101/2025.05.13.652513
  5. bioRxiv. 2025 May 22. pii: 2025.05.22.655499. [Epub ahead of print]
    Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies.
    Jasleen Singh, Justin Krish Williams, Quinn Elliott, Rohit Jhawar, Lucas Ferguson, Kathleen Collins, Randy Schekman.
      All eukaryotic cells secrete exosomes, a type of extracellular vesicles (EVs) derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs (scaRNAs), small-nucleolar RNAs (snoRNAs) and small-nuclear RNAs (snRNAs) traffic to MVBs. We show that this RNA-trafficking requires the function of ESCRT machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes (RNPs), such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway.
    Significance Statement: Endosomes are cytoplasmic, membrane-bound subcellular organelles that are sites for biogenesis of exosomes, a class of extracellular vesicles, thought to mediate intercellular communication via their packaged cargo such as RNA. Previous studies have focused on the transcriptome of exosomes however very little is known about the identity of RNAs and mechanisms by which they are sorted into endosomes. Here we report a comprehensive endosome transcriptome and provide evidence that several nuclear RNA-protein complexes (RNPs) sort into endosomes, a previously unappreciated phenomenon. We show that this process requires the activity of endosomal sorting complexes and phospholipids characteristic of cellular endocytic compartments. Our study provides a mechanism for recycling and disposal of unwanted nuclear RNPs by the cytoplasmic endolysosomal pathway.
    DOI:  https://doi.org/10.1101/2025.05.22.655499
  6. Angew Chem Int Ed Engl. 2025 Jun 04. e202507254
    Visible-Light-Controlled Lysine-Selective Crosslinking Decodes Protein Complexes and Dynamic Interactomes in Live Cells.
    Yali Xu, Hao Hu, Yu Ran, Wensi Zhao, An-Di Guo, Hui-Jun Nie, Linhui Zhai, Guang-Liang Yin, Jintao Cheng, Shengna Tao, Bing Yang, Minjia Tan, Xiao-Hua Chen.
      Crosslinking strategies have emerged as an attractive technology for deciphering protein complexes and protein-protein interactions (PPIs). However, commonly used crosslinking strategies present significant challenges for the precise analysis of protein complexes and dynamic PPIs in native biological environments. Here, we report the development of the first visible-light-inducible lysine-specific homobifunctional photo-crosslinkers and introduce Visible-light-controlled Lysine-selective crosslinking (VL-XL) strategy for in-depth analysis of protein complexes and profiling dynamic interactomes in live cells. By synergistically integrating the advantages of temporal control, high biocompatibility, and lysine selectivity, the VL-XL strategy not only provides an effective solution for protein complexes studies-achieving residue-specific crosslinked peptides, delivering high-confidence data and streamlined MS data analysis-but also reveals dynamic interactomes in various scenarios. The VL-XL strategy successfully profiles the time-resolved, EGF-stimulated EGFR interactome, providing valuable insights into regulatory mechanisms of EGFR signaling. More importantly, the VL-XL strategy effectively unveils molecular glue degrader-induced E3 ligase interactome, leading to discovery of neo-substrates such as SESN2 and opening an innovative avenue for identifying novel targets for degradation. Overall, the VL-XL strategy offers a robust chemical tool for decoding protein complexes and dynamic interactomes, inspiring innovative solutions to address unresolved questions in proteomics, systems biology and drug discovery.
    Keywords:  Chemoproteomics; Molecular glue degraders; Photo-crosslinking; Protein-protein interactions; Targeted protein degradation
    DOI:  https://doi.org/10.1002/anie.202507254
  7. Proc Natl Acad Sci U S A. 2025 Jun 10. 122(23): e2502364122
    The highly conserved intron of tyrosine tRNA is critical for m1A58 modification and controls the integrated stress response.
    Catherine J Stuart, Jennifer E Hurtig, Talia Tzadikario, Niki K Thomas, Miten Jain, Ambro van Hoof.
      tRNA introns are a universally conserved feature of eukaryotic genomes, but the reason for their conservation has remained obscure. We have previously shown that a defect in the essential tRNA splicing endonuclease of yeast results in transcriptome remodeling, resembling that of the integrated stress response (ISR). In this study, we show that ISR activation in this mutant requires the canonical ISR components, including the collided ribosome sensor Gcn1. We further show that splicing of tyrosine tRNA, but not splicing of any of the other intron-containing tRNAs, controls the ISR. Using nanopore direct RNA sequencing, we show that the intron of tyrosine tRNA affects m1A58 modification in the T arm loop of the mature tRNA. In support of these results, we show that deletion of either subunit of the enzyme that adds the m1A58 modification also controls the ISR. Unlike the few intron-dependent modifications previously described, the intron-dependent m1A58 modification site is distal from the intron and has a clear physiological impact. Finally, we survey the occurrence of tRNA introns in eukaryotic genomes and show that the tyrosine tRNA intron is more prevalent than any other tRNA intron. These data suggest that tRNA splicing is conserved in eukaryotes because hypomodified tyrosine tRNAs lead to collided ribosomes, resulting in stress to cell physiology.
    Keywords:  intron conservation; m1A; tRNA intron; tRNA modification; tRNA splicing
    DOI:  https://doi.org/10.1073/pnas.2502364122
  8. bioRxiv. 2025 May 21. pii: 2025.05.21.654933. [Epub ahead of print]
    Structural basis for L-isoaspartyl-containing protein recognition by the PCMTD1 cullin-RING E3 ubiquitin ligase.
    Eric Z Pang, Boyu Zhao, Cameron Flowers, Elizabeth Oroudjeva, Jasmine B Winter, Vijaya Pandey, Michael R Sawaya, James Wohlschlegel, Joseph A Loo, Jose A Rodriguez, Steven G Clarke.
      A major type of spontaneous protein damage that accumulates with age is the formation of kinked polypeptide chains with L-isoaspartyl residues. Mitigating this damage is necessary for maintaining proteome stability and prolonging organismal survival. While repair through methylation by PCMT1 has been previously shown to suppress L-isoaspartyl accumulation, we provide an additional mechanism for L-isoaspartyl maintenance through PCMTD1, a cullin-RING ligase (CRL). We combined cryo-EM, native mass spectrometry, and biochemical assays to provide insight on how the assembly and architecture of PCMTD1 in the context of a CRL complex fulfils this alternative mechanism. We show that the PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.
    eTOC Blurb: Limiting the accrual of L-isoaspartyl damaged proteins is essential during aging. While this is thought to be mediated solely by the repair activity of the protein, PCMT1, Pang et al. now demonstrate that a related protein, PCMTD1, functions as a cullin-RING ligase to selectively target L-isoaspartyl-damaged substrates for potential regulation by the ubiquitylation-proteosomal system.
    Highlights: Atomic cryo-EM structure of CRL5-PCMTD1 determinedArchitecture of PCMTD1 when complexed as a CRL supports ubiquitylation activityPCMTD1 recognizes L-isoaspartyl residues as a recruitment motif for potential CRL activityRecognition of L-isoaspartyl residues is dependent on cofactor engagement.
    DOI:  https://doi.org/10.1101/2025.05.21.654933
  9. Angew Chem Int Ed Engl. 2025 Jun 04. e202504813
    Proteome-wide Ligand and Target Discovery by Using β-Nitrostyrene Electrophiles: Supporting Targeted Protein Degradation.
    Xinyao Ouyang, Xiaomeng Chai, Lei Huang, Yubin Chen, Shengrong Li, Weizhen Huang, Yifang Li, Ke Ding, Tongzhen Liu, Yi Tan, Zhengqiu Li.
      Bioconjugation chemistry has been a powerful avenue in expanding the repertoire of druggable proteome, as well as in identifying new E3 ligases to support targeted protein degradation (TPD). However, a large fraction of proteome remains inaccessible with existing covalent probes. Herein, we incorporated various electron-withdrawing groups into styrene derivatives and identified β-nitrostyrene as a novel cysteine-targeting warhead for target discovery. Through phenotypic screening and chemoproteomics platforms, we identified new ligandable sites such as C96 of SND1, C110 of PTGES2, modulating cell proliferation in an acute myeloid leukemia cell line. Moreover, incorporation of this warhead into the BRD4 inhibitor (+)-JQ1 demonstrated that the covalent handle engages the novel E3 ligase tripartite motif-containing 28 (TRIM28) at Cys232 residue, thereby promoting the targeted degradation. Notably, when transplanted into other protein-targeting ligands, the β-nitrostyrene warhead effectively induced protein degradation of EGFRL858R/T790M/C797S, PDE5, BTK, LRRK2 and BCR-ABL/c-ABL without eliciting a hook effect. Importantly, the degraders demonstrate significantly enhanced anticancer effects compared to corresponding inhibitors. To our knowledge, this is the first report of small-molecular degraders engaging TRIM28 to support targeted protein degradation, and provides a rational pathway for design and development of potent monovalent degraders.
    Keywords:  Chemical proteomics; E3 ligases; target discovery; β-nitrostyrene Electrophiles, covalent probes
    DOI:  https://doi.org/10.1002/anie.202504813
  10. Dev Cell. 2025 May 27. pii: S1534-5807(25)00318-1. [Epub ahead of print]
    ATG9A and ARFIP2 cooperate to control PI4P levels for lysosomal repair.
    Stefano De Tito, Eugenia Almacellas, Daniel Dai Yu, Emily Millard, Wenxin Zhang, Cecilia de Heus, Christophe Queval, Javier H Hervás, Enrica Pellegrino, Ioanna Panagi, Ditte Fogde, Teresa L M Thurston, Judith Klumperman, Maximiliano Gutierrez, Sharon A Tooze.
      Lysosome damage activates multiple pathways to prevent lysosome-dependent cell death, including a repair mechanism involving endoplasmic reticulum (ER)-lysosome membrane contact sites, phosphatidylinositol 4-kinase-2a (PI4K2A), phosphatidylinositol-4 phosphate (PI4P), and oxysterol-binding protein-like proteins (OSBPLs) lipid transfer proteins. PI4K2A localizes to the trans-Golgi network and endosomes, yet how it is delivered to damaged lysosomes remains unknown. During acute sterile damage and damage caused by intracellular bacteria, we show that ATG9A-containing vesicles perform a critical role in delivering PI4K2A to damaged lysosomes. ADP ribosylation factor interacting protein 2 (ARFIP2), a component of ATG9A vesicles, binds and sequesters PI4P on lysosomes, balancing OSBPL-dependent lipid transfer and promoting the retrieval of ATG9A vesicles through the recruitment of the adaptor protein complex-3 (AP-3). Our results identify a role for mobilized ATG9A vesicles and ARFIP2 in lysosome homeostasis after damage and bacterial infection.
    Keywords:  AP-3; ARFIP2; ATG9A; PI4K2A; PI4P; autophagy; lysosomal damage; lysosome; membrane trafficking
    DOI:  https://doi.org/10.1016/j.devcel.2025.05.007
  11. bioRxiv. 2025 May 15. pii: 2025.05.15.654159. [Epub ahead of print]
    Structures of Fab-stabilized CHIP reveal a conformational switch important in E3 ligase and chaperone functions.
    Aparna Unnikrishnan, Dong Hee Chung, Emily J Connelly, Shih-Wei Chuo, Shweta Devi, Aye C Thwin, Cory M Nadel, Eric Tse, Jason E Gestwicki, Charles S Craik, Daniel R Southworth.
      Carboxyl terminus of Hsc70-interacting protein (CHIP/STUB1) is a U-box E3 ligase essential for protein quality control, targeting misfolded or damaged proteins for clearance and conducting chaperone-like functions by suppressing aggregation of proteins, including tau. The previous structure of full-length CHIP identified an asymmetric homodimer in which one U-box is occluded from E2 binding, indicating an unusual half-of-sites activity. However, the flexibility of CHIP has complicated efforts to further characterize its structure and function. Here we leverage two CHIP-targeting fragment antigen-binding (Fab) antibodies to solve structures by cryo-EM. We identify one Fab binds to the CHIP U-box via interactions mimicking E2 contacts and stabilizes three distinct CHIP dimer states, revealing an asymmetric-to-symmetric conformational switch that would enable both U-box domains to be accessible for E2 binding. Conversely, the second Fab targets CHIP's coiled-coil domains, stabilizing the asymmetric dimer with a single accessible U-box. Remarkably, the Fabs exhibit opposing effects on CHIP's inhibition of tau aggregation, wherein binding to coiled-coil domains abolishes inhibition of aggregation, while binding to the U-box greatly potentiates this activity. Together, this work reveals how CHIP conformational states and binding interfaces may regulate ubiquitination cycles and chaperone-like functions.
    DOI:  https://doi.org/10.1101/2025.05.15.654159
  12. bioRxiv. 2025 May 13. pii: 2025.05.07.652686. [Epub ahead of print]
    Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans.
    Qiuxia Zhao, Blythe Bolton, Reed Rothe, Reiko Tachibana, Can Cenik, Elif Sarinay Cenik.
      Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways-including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)-as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.
    DOI:  https://doi.org/10.1101/2025.05.07.652686
  13. bioRxiv. 2025 May 15. pii: 2025.05.15.654346. [Epub ahead of print]
    A Protein Disulfide Isomerase Coordinates Redox Homeostasis and ER Calcium Regulation for Optimal Lytic Cycle Progression in Toxoplasma gondii.
    Katherine E Moen, Silvia N J Moreno.
      The endoplasmic reticulum (ER) maintains an oxidative environment that facilitates disulfide bond formation, a critical process for proper protein folding. Protein disulfide isomerases (PDIs) are ER resident enzymes that facilitate the formation, breakage, and rearrangement of disulfide bonds between cysteine residues, thereby stabilizing protein structures. Although PDIs are functionally diverse, they all contain at least 1 thioredoxin-like domain and mediate disulfide exchange through their conserved CXXC motifs. The Apicomplexan parasite, Toxoplasma gondii , infects approximately one third of the world population, posing a significant risk to immunosuppressed individuals and unborn fetuses. The fast-replicating tachyzoite form engages in a lytic cycle, causing host tissue damage and contributing to pathogenesis. While approximately 26 PDIs are predicted to be present in T. gondii , their specific roles remain largely unexplored. In this study, we investigate TgPDIA3, a T. gondii PDI localized to the ER, along with several of its interacting protein substrates. We explore its role in ER redox activity and calcium sequestration and assess how these functions contribute to the parasite's lytic cycle.
    Importance: The lytic cycle of Toxoplasma gondii is essential to the pathogenesis of toxoplasmosis, with calcium signaling playing a crucial role in driving this process. Cytosolic calcium is tightly regulated through either sequestration into intracellular stores or extrusion from the cell. The ER, likely the largest calcium store in T. gondii , remains poorly characterized. In this study, we identify a link between ER redox regulation and calcium homeostasis and signaling in T. gondii . These findings suggest that redox-controlled calcium homeostasis and flux in the ER is a key driver of the parasite's lytic cycle progression.
    DOI:  https://doi.org/10.1101/2025.05.15.654346
  14. Cell Chem Biol. 2025 Jun 03. pii: S2451-9456(25)00165-5. [Epub ahead of print]
    Mechanism by which the molecular glue-like verteporfin induces IRE1α dimerization and activation to synergize with AKT inhibition in breast cancer.
    Yongliang Liu, Hui Hua, Yalan Cao, Minjing Li, Hongying Zhang, Shan Du, Jieya Liu, Ting Luo, Yangfu Jiang.
      Inositol-requiring enzyme 1α (IRE1α) signaling is one of three arms of the unfolded protein response, playing a vital role in maintaining endoplasmic reticulum homeostasis. Pharmacological modulation of this pathway offers potential therapeutic strategies for various diseases. Molecular glues may regulate protein stability and activity by inducing protein-protein interaction. Here, we find that verteporfin functions as a molecular glue, promoting IRE1α dimerization and activation. Specifically, verteporfin binds to IRE1α, facilitating its dimerization, which relies on the His692 residue. This activation of IRE1α triggers XBP1 splicing and miR-153-mediated downregulation of PTEN, along with AKT phosphorylation. Additionally, we identify the pro-metastasis gene BACH1 as a novel target of miR-153, which is downregulated by IRE1α and verteporfin. While verteporfin inhibits breast cancer cell viability and invasion, its combination with an AKT inhibitor synergistically suppresses breast cancer progression. Our findings establish a mechanistic link between IRE1α and PI3K/AKT signaling, highlighting a possibility for therapeutic intervention.
    Keywords:  AKT; BACH1; IRE1α; PTEN; cancer; inositol-requiring enzyme 1α; miR-153; molecular glue; unfolded protein response; verteporfin
    DOI:  https://doi.org/10.1016/j.chembiol.2025.05.004
  15. bioRxiv. 2025 May 13. pii: 2025.05.07.652703. [Epub ahead of print]
    p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic.
    Kimberly R Long, Gazal Singh, Marie Villemeur, Nathalie Brouwers, Vivek Malhotra, Ishier Raote, Meir Aridor.
      COPII-coat proteins play a crucial role in generating small vesicles at endoplasmic reticulum (ER) exit sites (ERES). However, they also assemble at the necks of tunnels and tubules connecting the ER exit site (donor) to the ERGIC/cis-Golgi cisterna (acceptor). How can COPII support these two traffic mechanisms? Through cell-free reconstitutions, we have found that the apposition of donor and acceptor membranes is important for the assembly of the outer layer of the COPII coat (Sec13/31) but had minimal impact on Sar1-induced COPII inner layer (Sec23/24) recruitment. The expression of the adaptor protein p125A, which binds to phosphatidylinositol 4-phosphate (PI4P), Sec31 and Sec23, stabilized the contact between the donor and acceptor membranes and promoted COPII outer layer assembly. A p125A-chimera expressing a Golgi-targeted PI4P-binding domain could also support outer layer assembly. Notably, the C-terminal helical domain of Sec31A, which interacts with p125A, was essential for its assembly at ERES. In cells lacking p125A, the assembly of the COPII outer layer was selectively destabilized. Transcriptome and secretome analyses reveal selective adjustments to extracellular matrix remodeling and collagen secretion, which corresponded with selective inhibition of fibrillar collagen traffic from the ER. Thus, p125A connects the outer COPII layer at ERES with PI4P-rich ERGIC/cis-Golgi membranes, coordinating COPII assembly with tunnel-driven collagen traffic.
    DOI:  https://doi.org/10.1101/2025.05.07.652703
  16. bioRxiv. 2025 May 13. pii: 2025.05.08.652974. [Epub ahead of print]
    The endoplasmic reticulum promotes microtubule organization and region-specific disassembly to execute Compartmentalized Cell Elimination.
    Karen Juanez, Hannah Selvarathinam, Madison Jones, Piya Ghose.
      Specialized cells, such as neurons, die during development and disease. How subcellular organization and interactions across diverse compartments direct death is not well-understood. We examine this by studying the Compartmentalized Cell Elimination (CCE) developmental death program of the C. elegans tail-scaffolding cell (TSC). We find endoplasmic reticulum (ER) shape genes and the microtubule (MT) severase SPAS-1/Spastin, all linked to neurodegeneration, cooperate to promote CCE. Super-resolution imaging reveals profound spatiotemporal dynamics of MTs and the ER across CCE, including enrichment in a stereotyped degenerative node. We observe an ER-dependent non-centrosomal microtubule organizing center (ncMTOC) in the degenerative node, where the ER locally promotes both MT organization and SPAS-1/Spastin-mediated MT severing. The ER is spatially confined, such that SPAS-1/Spastin also has an ER-independent role. Our study expands our understanding of the cell biology of specialized cell death during development and presents molecular links to pathological neurodegeneration, paving the way to neurodegenerative therapies.
    DOI:  https://doi.org/10.1101/2025.05.08.652974
  17. bioRxiv. 2025 May 15. pii: 2025.05.10.653233. [Epub ahead of print]
    A complete map of human cytosolic degrons and their relevance for disease.
    Vasileios Voutsinos, Kristoffer E Johansson, Fia B Larsen, Martin Grønbæk-Thygesen, Nicolas Jonsson, Emma Holm-Olesen, Giulio Tesei, Amelie Stein, Douglas M Fowler, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen.
      Degrons are short protein segments that target proteins for degradation via the ubiquitin-proteasome system and thus ensure timely removal of signaling proteins and clearance of misfolded proteins from the intracellular space. Here, we describe a systematic screen for degrons in the human cytosol. We determine degron potency of >200,000 different 30-residue tiles from more than 5,000 cytosolic human proteins with 99.7% coverage. In total, 19.1% of the tiles function as strong degrons, 30.4% as intermediate degrons, while 50.5% did not display degron properties. The vast majority of the degrons are dependent on the E1 ubiquitin-activating enzyme and the proteasome but independent of autophagy. The results reveal both known and novel degron motifs, both internal as well as at the C-terminus. Mapping the degrons onto protein structures, predicted by AlphaFold2, revealed that most of the degrons are located in buried regions, indicating that they only become active upon unfolding or misfolding. Training of a machine learning model allowed us to probe the degron properties further and predict the cellular abundance of missense variants that operate by forming degrons in exposed and disordered protein regions, thus providing a mechanism of pathogenicity for germline coding variants at such positions.
    DOI:  https://doi.org/10.1101/2025.05.10.653233
  18. Proc Natl Acad Sci U S A. 2025 Jun 10. 122(23): e2421334122
    Concerted transport and phosphorylation of diacylglycerol at ER-PM contact sites regulate phospholipid dynamics during stress.
    Selene Garcia-Hernandez, Jorge Morello-López, Richard Haslam, Vitor Amorim-Silva, José Moya-Cuevas, Rafael Catalá, Louise Michaelson, Jessica Pérez-Sancho, Vedrana Marković, Julio Salinas, Johnathan Napier, Yvon Jaillais, Noemí Ruiz-Lopez, Miguel A Botella.
      A universal response of plants to environmental stresses is the activation of plasma membrane (PM) phospholipase C, which hydrolyzes phosphoinositides to produce soluble inositol phosphate and diacylglycerol (DAG). Because of their conical shape, DAG amounts have to be tightly regulated or they can destabilize membranes. We previously showed that upon stress, Synaptotagmin1 (SYT1) transports DAG from the PM to the endoplasmic reticulum (ER) at ER-PM Contact Sites (CS). Here, we addressed the fate of the incoming DAG in the ER. We show that diacylglycerol kinases (DGKs) DGK1 and DGK2 form a module with SYT1 functionally coupling DAG transport and phosphorylation at ER-PM CS. Although SYT1 and DGK1/DGK2 do not show exclusive ER-PM CS localization, their interaction occurs specifically at ER-PM CS and the removal of ER-PM CS abolishes the interaction. Lipidomic analysis of a dgk1dgk2 double mutant supports that DGK1 and DGK2 phosphorylate DAG at the ER and transcriptomic and phenotypic analyses indicate that SYT1 and DGK1/DGK2 are functionally related. Taken together, our results highlight a mechanism at ER-PM CS that coordinates the transfer of DAG from the PM to the ER by SYT1 upon stress and the concomitant phosphorylation of DAG by DGK1 and DGK2 at the ER. These findings underscore the critical role of spatial coordination in lipid metabolism during stress-induced membrane remodeling.
    Keywords:  DAG; PI cycle; abiotic stress; contact sites; signaling
    DOI:  https://doi.org/10.1073/pnas.2421334122
  19. J Biol Chem. 2025 May 31. pii: S0021-9258(25)02179-9. [Epub ahead of print] 110329
    Perturbations in L-serine metabolism regulates protein quality control through sensor of retrograde response pathway Rtg2 in S.cerevisae.
    Kanika Saxena, Rebecca Andersson, Per O Widlund, Sakda Khoomrung, Sarah Hanzén, Jens Nielsen, Navinder Kumar, Mikael Molin, Thomas Nyström.
      Cellular protein homeostasis relies on a complex network of protein synthesis, folding, sub-cellular localization, and degradation to sustain a functional proteome. Since, most of these processes are energy driven, proteostasis is inescapably afflicted by cellular metabolism. Proteostasis collapse and metabolic imbalance are both linked to aging and age-associated disorders, yet they have traditionally been studied as a separate phenomenon in the context of aging. In this study, we indicate that reduced proteostasis capacity is a result of a metabolic imbalance associated with age. We observed increased accumulation of L-serine and L-threonine in replicative old cells of S. cerevisiae, indicating an imbalance in amino acid metabolism with replicative aging. Replicating this metabolic imbalance in young cells through deletion of serine dependent transcriptional activator, CHA4, resulted in increased aggregation of endogenous proteins along with misfolding prone proteins Guk1-7ts-GFP and Luciferase-GFP in both young and old cells. Aggregate formation in the cha4Δ strain required a functional sensor of mitochondrial dysfunction and an activator of the retrograde signalling gene, RTG2. CHA4 and RTG2 exhibited genetic interaction and together regulated mitochondrial metabolism, replicative lifespan, and aggregate formation in young cells, connecting metabolic regulation with proteostasis and aging. Constitutive activation of retrograde signalling through overexpression of RTG2 or deletion of MKS-1, negative regulator of Rtg1-Rtg3 nuclear translocation, resulted in faster resolution of aggregates upon heat shock through RTG3 and was found to be independent of molecular chaperone upregulation.
    Keywords:  Aggregation; aging; mitochondria; proteostasis; replicative aging; retrograde response; serine
    DOI:  https://doi.org/10.1016/j.jbc.2025.110329
  20. Structure. 2025 May 24. pii: S0969-2126(25)00183-2. [Epub ahead of print]
    Biochemical analysis of PD-L1 ubiquitination by CRL3SPOP, ARIH1, and NEDD4 family ubiquitin ligases.
    Guojiao Xie, Lin Gao, Renee Lu, Linxia Tian, Tiantian Zheng, Xinning Li, Yongjun Dang, Philip A Cole, Xian Yu, Hanjie Jiang, Zan Chen.
      As a key immune checkpoint ligand, PD-L1 is a critical target in cancer immunotherapy. While multiple E3 ubiquitin ligases including CRL3SPOP, ARIH1, and NEDD4 have been implicated in PD-L1 degradation, the precise enzymatic mechanisms remain unclear. In this study, we systematically compared the enzymatic activities of CRL3SPOP, ARIH1, and NEDD4 ligases toward the cytoplasmic domain of PD-L1 through in vitro reconstitution with purified components. ARIH1, rather than CRL3SPOP, independently ubiquitinates PD-L1. We reveal a mechanism where ARIH1 acts as a substrate receptor and cooperates with CRLs to catalyze PD-L1 ubiquitination. We also biochemically validated the E3 ligase activity of the NEDD4 family E3s toward PD-L1. By using liposomes in the enzymatic assays, we show that phosphorylation enhances PD-L1 ubiquitination through disrupting its membrane association. Our study provides further biochemical insights into PD-L1 ubiquitination, which advances our understanding of the molecular details of PD-L1 regulation.
    Keywords:  ARIH1; Cullin RING ubiquitin ligase; NEDD4 family ubiquitin ligases; PD-L1; membrane association; phosphorylation; ubiquitination
    DOI:  https://doi.org/10.1016/j.str.2025.05.005
  21. J Exp Bot. 2025 Jun 04. pii: eraf244. [Epub ahead of print]
    The IRE1-bZIP60 branch of Unfolded Protein Response is required for Arabidopsis immune response to Botrytis cinerea.
    Cécile Blanchard, Sébastien Aimé, Amélie Ducloy, Siham Hichami, Mariannes Azzopardi, Jean-Luc Cacas, Olivier Lamotte.
      The Unfolded Protein Response (UPR) is a signalling pathway activated when endoplasmic reticulum (ER) proteostasis is disturbed. We have investigated the contribution of UPR in Arabidopsis thaliana response to two necrotrophic fungi Botrytis cinerea and Alternaria brassicicola. We found out that the IRE1-bZIP60 branch of UPR was specifically activated upon infection with both pathogenic fungi, as evidenced by the production of the active bZIP60 transcription factor forms and the increased expression of UPR-responsive genes. We also demonstrated using reverse genetics that IRE1-bzIP60 axis was necessary to restrict foliar necrotic symptoms induced by both fungi. Furthermore, mutants deficient for two ER-QC components were also more susceptible to infection by B. cinerea. By contrast, investigating the involvement of CDC48, an AAA+-ATPAse that assist ER-Associated Degradation (ERAD) pathway, we showed that a series of mutants and transgenics are more resistant to B. cinerea. To gain molecular insights into how the ER shapes Arabidopsis immune response to B. cinerea, we quantified defence gene and cell death marker expression in bzip60 single and ire1 double mutants. None of those genes were misregulated in mutant backgrounds, indicating that IRE1-bZIP60 branch of UPR modulates the Arabidopsis response to B. cinerea by a yet-to-be-identified mechanism. Interestingly, we identified NAC053 as a potential actor of this unknown mechanism.
    Keywords:   Alternaria brassicicola ; Botrytis cinerea ; Arabidopsis thaliana; CDC48; ER stress; ERAD; ERQC; UPR; plant necrotrophic pathogens; proteostasis
    DOI:  https://doi.org/10.1093/jxb/eraf244
  22. Cell Rep. 2025 May 30. pii: S2211-1247(25)00549-2. [Epub ahead of print]44(6): 115778
    Landscape and regulation of mRNA translation in the early C. elegans embryo.
    Yash Shukla, Vighnesh Ghatpande, Cindy F Hu, Daniel J Dickinson, Can Cenik.
      Animal embryos rely on regulated translation of maternally deposited mRNAs to drive early development. Using low-input ribosome profiling combined with RNA sequencing on precisely staged embryos, we measure mRNA translation during the first four cell cycles of C. elegans development. We uncover stage-specific patterns of developmentally coordinated translational regulation. Our results confirm that mRNA localization correlates with translational efficiency, though initial translational repression in germline precursors occurs before P-granule association. Our analysis suggests that the RNA-binding protein OMA-1 represses the translation of its target mRNAs in a stage-specific manner while indirectly promoting the translational efficiency of other transcripts. These findings illuminate how post-transcriptional mechanisms shape the embryonic proteome to direct cell differentiation, with implications for understanding similar regulation across species where maternal factors guide early development.
    Keywords:  C. elegans; CP: Developmental biology; CP: Molecular biology; OMA-1; P granules; Ribo-ITP; cell fate determination; embryogenesis; mRNA translation; maternal transcripts; post-transcriptional control; ribosome profiling; translational regulation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115778
  23. Redox Biol. 2025 May 27. pii: S2213-2317(25)00213-7. [Epub ahead of print]84 103700
    Autophagy hub-protein p62 orchestrates oxidative, endoplasmic reticulum stress, and inflammatory responses post-ischemia, exacerbating stroke outcome.
    Xingyun Quan, Yukun Yang, Xiaolong Liu, Britta Kaltwasser, Matthias Pillath-Eilers, Bernd Walkenfort, Sylvia Voortmann, Ayan Mohamud Yusuf, Nina Hagemann, Chen Wang, Mike Hasenberg, Dirk M Hermann, Ulf Brockmeier.
      Autophagy has crucial roles for ischemia/reperfusion (I/R) injury. To define the role of the autophagy hub protein p62/SQSTM1 in I/R injury, we conducted gain-of-function and loss-of-function experiments in a set of cell types, including two neuron-like cell lines, primary neurons, brain endothelial and astroglial-like cells, which we combined with mouse ischemic stroke studies. p62 levels post-I/R increased alongside intracellular ROS changes. p62 overexpression increased and p62 knockdown or pharmacological deactivation reduced I/R injury. Autophagic flux was p62-dependent, but oxygen-independent. Using p62 domain deletion mutants we identified p62's ZZ domain as key factor mediating autophagy and cell death. Death-promoting effects of p62 involved elevated ROS burden. At the same time, p62 activated a broad network of cytoprotective responses, which included NRF2-associated antioxidant signaling and inhibition of the pro-inflammatory NFκB pathway, which were bidirectionally linked with p62, and downregulation of the ER stress sensor BiP/GRP78 with consecutive activation of the UPR PERK branch. Our study establishes p62 as a master regulator of I/R injury, which offers itself as target for stroke therapies.
    Keywords:  Apoptosis; Middle cerebral artery occlusion; Necroptosis; Nuclear factor erythroid-2-related factor-2; Nuclear factor-kB; Oxygen-glucose deprivation; Protein kinase RNA-Like endoplasmic reticulum kinase; Reactive oxygen species; Reoxygenation; Stress signaling
    DOI:  https://doi.org/10.1016/j.redox.2025.103700
  24. Sci Adv. 2025 Jun 06. 11(23): eadt2050
    Type II kinase inhibitors that target Parkinson's disease-associated LRRK2.
    Nicolai D Raig, Katherine J Surridge, Marta Sanz-Murillo, Verena Dederer, Andreas Krämer, Martin P Schwalm, Nicholas M Lattal, Lewis Elson, Deep Chatterjee, Sebastian Mathea, Thomas Hanke, Andres E Leschziner, Samara L Reck-Peterson, Stefan Knapp.
      Increased kinase activity of leucine-rich repeat kinase 2 (LRRK2) is associated with Parkinson's disease (PD). Numerous LRRK2-selective type I kinase inhibitors have been developed, and some have entered clinical trials. Here, to our knowledge, we present the first type II kinase inhibitors that target LRRK2. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type I inhibitors. We designed these inhibitors with a combinatorial chemistry approach fusing selective LRRK2 type I and promiscuous type II inhibitors using iterative cycles of synthesis supported by structural biology and activity testing. Our lead compounds are selective and potent toward both LRRK2 and LRRK1, a close relative of LRRK2. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive LRRK2 kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2's function and regulation and expand the potential therapeutic options for PD.
    DOI:  https://doi.org/10.1126/sciadv.adt2050
  25. Sci Adv. 2025 Jun 06. 11(23): eadv4033
    Inhibition of virally induced TFEB proteasomal degradation as a host-centric therapeutic approach for coronaviral infection.
    Travis B Lear, Mads B Larsen, Bo Lin, Benjamin R Treat, Qing Cao, Áine N Boudreau, Karina C Lockwood, Irene Alfaras, Jason R Kennerdell, Laura Salminen, Daniel P Camarco, Yao Tong, Jing Ma, Jie Liu, Jay X Tan, Ferhan Tuncer, John J Villandre, Lucas Hertzel, Michael M Myerburg, Yanwen Chen, Claudette St Croix, Yusuke Sekine, John W Evankovich, Simon M Barratt-Boyes, Toren Finkel, Bill B Chen, Yuan Liu.
      The endolysosomal pathway plays an evolutionarily conserved role in pathogen clearance, and viruses have evolved complex mechanisms to evade this host defense system. Here, we describe a previously unidentified aspect of coronaviral infection, whereby the master transcriptional activator of lysosomal homeostasis-TFEB-is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased small interfering RNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. We derive a series of novel small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit virus-induced TFEB degradation and demonstrate broad antiviral activities including attenuating severe acute respiratory syndrome coronavirus 2 infection in two animal models. Together, these results delineate a virally triggered pathway that impairs lysosomal homeostasis in the host. Small molecule E3 ubiquitin ligase DCAF7 inhibitors that restore lysosomal function represent a novel class of host-directed, antiviral therapies useful for current and potentially future coronaviral variants.
    DOI:  https://doi.org/10.1126/sciadv.adv4033
  26. Nat Commun. 2025 May 31. 16(1): 5070
    A low-complexity linker as a driver of intra- and intermolecular interactions in DNAJB chaperones.
    Billy Hobbs, Noor Limmer, Felipe Ossa, Ella Knüpling, Samuel Lenton, Vito Foderà, Arnout P Kalverda, Theodoros K Karamanos.
      J-domain proteins ( JDPs) act as major regulators of the proteostasis network by driving the specificity of the Hsp70 machine. Their important functions are mediated by a low-complexity glycine-/phenylalanine-rich region (GF-linker) that links the folded J-domain with the substrate binding domain. Recently, we and others have shown that in an autoinhibited JDP state, an α-helix formed within the GF-linker blocks the Hsp70 binding site on the J-domain. However, the role of the disordered GF-linker in autoinhibition and how the latter is released, are still not understood. Here, using autoinhibited DNAJB1 and DNAJB6 constructs, we show that in combination with the J-domain, the GF-linker creates a hydrophobic, partially collapsed cluster that shows a remarkable degree of long-range structural communication, disruption of which can lead to destabilisation of autoinhibition. Apart from this crucial intramolecular role, we reveal that the GF-linker can also be recognised by the substrate-binding domain of Hsp70 and dictate the lifetime of the entire JDP-Hsp70 complex. Strikingly, the GF-linkers of DNAJB1 and DNAJB6 display distinct structural properties that lead to different Hsp70 binding kinetics, showing that the behaviour of the GF-linker can vary dramatically even within the same class of JDPs.
    DOI:  https://doi.org/10.1038/s41467-025-60063-2
  27. J Biol Chem. 2025 May 29. pii: S0021-9258(25)02162-3. [Epub ahead of print] 110312
    Wild-type EGFR and the oncogenic mutant EGFR (L858R) employ distinct mechanisms for export from the endoplasmic reticulum, a process critical for their activation.
    Mo Wang, Pik Ki Lau, Yusong Guo.
      The epidermal growth factor receptor (EGFR) is critical for cell differentiation, growth, proliferation, and migration. To function, newly synthesized EGFR must be transported from the endoplasmic reticulum (ER) to the cell surface, yet the specific molecular mechanisms mediating this trafficking are not fully understood. We found that the ER export of EGFR is facilitated by a conserved poly-arginine (polyR) motif located in the juxtamembrane region of the EGFR cytosolic domain. Mechanistic studies show that this motif interacts directly with the D198 residue on SAR1A, regulating the incorporation of EGFR into COPII vesicles for delivery to the Golgi. A depletion of the polyR motif impairs EGF-induced EGFR activation. Interestingly, we found that the ER export of the oncogenic mutant EGFR (L858R) is critical for its activation but does not depend on the D198 residue of SAR1A or the polyR motif of EGFR. Our study elucidates a mechanism regulating ER export of wild-type EGFR and indicates that the ER exports of the EGFRL858R mutant and wild-type EGFR are mediated by distinct molecular machineries, essential for their activation.
    DOI:  https://doi.org/10.1016/j.jbc.2025.110312
  28. Nat Neurosci. 2025 Jun 02.
    Neuronal aging causes mislocalization of splicing proteins and unchecked cellular stress.
    Kevin Rhine, Rachel Li, Hema M Kopalle, Katherine Rothamel, Xuezhen Ge, Elle Epstein, Orel Mizrahi, Assael A Madrigal, Hsuan-Lin Her, Trent A Gomberg, Anita Hermann, Joshua L Schwartz, Amanda J Daniels, Uri Manor, John Ravits, Robert A J Signer, Eric J Bennett, Gene W Yeo.
      Aging is one of the most prominent risk factors for neurodegeneration, yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging, we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons, which retained their aging hallmarks, and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins, especially spliceosome components. Intriguingly, splicing proteins-like the dementia- and ALS-associated protein TDP-43-mislocalize to the cytoplasm in aged neurons, which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules, but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery, poor HSP90α chaperone activity and the failure to respond to new stress events. Together, our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons.
    DOI:  https://doi.org/10.1038/s41593-025-01952-z
  29. J Med Chem. 2025 Jun 05.
    Biophysical Approaches for Investigating the Dynamics and Cooperativity of Ternary Complexes in Targeted Protein Degradation.
    Alexander A Shcherbakov, Leszek Poppe, Amit Vaish.
      Heterobifunctional small molecules (hSMs) are a rapidly emerging therapeutic modality that induces spatial proximity between biomolecules, such as proteins, by forming a ternary complex. In this study, we introduce biophysical methodologies to study ternary complexes involving hydrodynamic profiling by nuclear magnetic resonance spectroscopy (HAP-NMR) and isothermal titration calorimetry (ITC). These approaches allowed us to evaluate the hydrodynamic and thermodynamic factors that influence molecular dynamics and ternary complex formation in solution. We addressed challenges like the limited solubility of hSMs, particularly in ITC experiments, and the near-ideal equimolar stoichiometry (1:1:1) of the ternary complex in solution, which is necessary for accurate hydrodynamic parameter evaluation. Our methodology facilitated the characterization of a known degrader of SMARCA proteins and revealed subtle higher-level structural differences. This approach provided insights into the overall behavior and function of the SMARCA2 and SMARCA4 protein ternary complexes.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c00652
  30. J Virol. 2025 Jun 03. e0065425
    UFMylation promotes orthoflavivirus infectious particle production.
    Hannah M Schmidt, Grace C Sorensen, Matthew R Lanahan, Katherine M Bland, Caroline J Aufgebauer, Moonhee Park, Jenna Grabowski, Stacy M Horner.
      Post-translational modifications play crucial roles in regulating viral infections, yet roles for many modifications remain unexplored in orthoflavivirus biology. Here, we demonstrate that the UFMylation system, a post-translational modification pathway that catalyzes the transfer of UFM1 onto proteins and promotes infection by multiple orthoflaviviruses, including dengue virus (DENV), Zika virus (ZIKV), 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 orthoflavivirus infectious virion production. This regulation was specific to orthoflaviviruses as the hepacivirus and member of the broader Flaviviridae family, hepatitis C virus, was not regulated by UFL1. Mechanistically, UFMylation did not regulate viral RNA translation, RNA replication, or virion egress but instead affected the assembly of infectious virions. Furthermore, 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 pathway that promotes orthoflavivirus infection 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.IMPORTANCEOrthoflaviviruses depend on host-mediated post-translational modifications to successfully complete their life cycle, yet many of these critical interactions remain undefined. Here, we describe a role for a post-translational modification pathway, UFMylation, in promoting infectious particle production of ZIKV and DENV. We show that UFMylation is dispensable for initial RNA translation and RNA replication but promotes the assembly of infectious virions. Additionally, we find that regulation of infection by UFMylation extends to other orthoflaviviruses, including West Nile virus and yellow fever virus, but not to the broader Flaviviridae family. Finally, we demonstrate that UFMylation machinery directly interacts with specific DENV and ZIKV proteins during infection. These studies reveal a previously unrecognized role for UFMylation in regulating orthoflavivirus infection.
    Keywords:  PTM; UFM1; UFMylation; flavivirus; orthoflavivirus; post-translational modification; ubiquitination
    DOI:  https://doi.org/10.1128/jvi.00654-25
  31. Nat Commun. 2025 Jun 05. 16(1): 5227
    β-propeller protein-associated neurodegeneration protein WDR45 regulates stress granule disassembly via phase separation with Caprin-1.
    Yin Li, Jie Fang, Yuqi Ding, Xilong Zhang, Ying Liu, Wanting Qiu, He Xu, Yunzhe Kang, Jiayu Chen, Yanyan Gao, Yan G Zhao, Peiguo Yang, Bo Wang, Wenmin Tian, Yang Chen, Wenjian Bi, Peipei Zhang.
      β-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked neurodegenerative disorder caused by mutations in the WDR45 gene, yet its molecular mechanisms remain poorly understood. Here, we identify a role for WDR45 in stress granule (SG) disassembly, mediated through its phase separation with Caprin-1. We demonstrate that WDR45 forms gel-like condensates via its WD5 domain, which competitively displaces G3BP1 from Caprin-1 to promote SG disassembly. BPAN-associated WDR45 mutations impair condensate formation and Caprin-1 interaction, leading to delayed SG disassembly, which correlates with earlier disease onset. WDR45 depletion also exacerbates amyotrophic lateral sclerosis-associated pathological SGs, highlighting its broader relevance to neurodegenerative diseases. Using iPSC-derived midbrain neurons from a BPAN patient, we demonstrate delayed SG recovery, directly linking WDR45 dysfunction to neurodegeneration. These findings establish WDR45 as a critical regulator of SG dynamics, uncover a potential molecular basis of BPAN pathogenesis, and identify therapeutic targets for neurodegenerative diseases associated with SG dysregulation.
    DOI:  https://doi.org/10.1038/s41467-025-60583-x
  32. bioRxiv. 2025 May 19. pii: 2025.05.16.654474. [Epub ahead of print]
    Parkin-dependent ubiquitination of TAX1BP1 directs efficient autophagic removal of defective mitochondria.
    Anna Lechado-Terradas, Bianca Lemke, Katharina I Zittlau, Boris Macek, Philipp J Kahle.
      In stressed cells, the recessive Parkinson disease (PD) associated gene products PINK1 and parkin mediate the autophagic removal of damaged mitochondria (mitophagy). Upon mitochondrial membrane potential disruption, PINK1 phosphorylation activates the ubiquitin ligase parkin which ubiquitinates various mitochondrial protein substrates. These feed-forward modifications on the mitochondria surface attract ubiquitin-binding autophagy receptors that target ubiquitinated mitochondria to autophagosomes and indirectly contribute to phagophore elongation. Investigating post-translational protein modifications during this process, we detected transient ubiquitination of K549 within the third coiled-coil domain (CC3) of TAX1BP1 in HeLa cells expressing WT but not catalytically inactive parkin. Parkin-dependent ubiquitination did not target TAX1BP1 to proteasomal degradation but was rather indicative of a regulatory modification. In cells with the full complement of autophagy receptors, TAX1BP1 plays only a minor role in mitophagy. However, when expressed as a sole autophagy receptor, both WT and ubiquitination deficient TAX1BP1 were capable of promoting mitophagy, albeit mitochondria degradation was slightly delayed under mutant conditions. Use of the lysosomal inhibitor bafilomycin A indicated classical autophagolysosomal targeting of damaged mitochondria mediated by WT TAX1BP1. However, for the ubiquitination-deficient TAX1BP1, we observed an increased prevalence of enlarged endolysosomal vesicles carrying accumulated TAX1BP1-positive autophagosomes filled with mitochondrial material. Thus, while ubiquitination of the CC3 domain of TAX1BP1 is not essential for complete mitophagy, the lack of CC3 in TAX1BP1 reroutes the degradation flux to a less efficient endolysosmal degradative pathway. Interestingly, the PD gene product VPS35, becomes prominently engaged in this alternative mitophagy pathway.
    DOI:  https://doi.org/10.1101/2025.05.16.654474
  33. bioRxiv. 2025 May 13. pii: 2025.05.12.653427. [Epub ahead of print]
    Explainable Machine Learning Identifies Factors for Dosage Compensation in Aneuploid Human Cancer Cells.
    Erik Marcel Heller, Karen Barthel, Markus Räschle, Klaske M Schukken, Jason M Sheltzer, Zuzana Storchová.
      Aneuploidy, a hallmark of cancer, leads to widespread changes in chromosome copy number, altering the abundance of hundreds or thousands of proteins. How-ever, evidence suggests that levels of proteins encoded on affected chromosomes are often buffered toward their abundances observed in diploid cells. Despite its preval-ence, the molecular mechanisms driving this protein dosage compensation remain largely unknown. It is unclear whether all proteins are buffered to a similar degree, what factors determine buffering, and whether dosage compensation varies across different cell lines or tumor types. Moreover, its potential adaptive advantage and therapeutic relevance remain unexplored. Here, we established a novel approach to quantify protein dosage buffering in a gene copy number-dependent manner, show-ing that dosage compensation is widespread but variable in cancer cell lines and in vivo tumor samples. By developing multifactorial machine learning models, we identify mean gene dependency, protein complex participation, haploinsufficiency, and mRNA decay as key predictors of buffering. We also show that dosage com-pensation can affect oncogenic potential and that higher buffering correlates with reduced proteotoxic stress and increased drug resistance. These findings highlight protein dosage compensation as a crucial regulatory mechanism and a potential therapeutic target in aneuploid cancers.
    DOI:  https://doi.org/10.1101/2025.05.12.653427
  34. Cell Rep. 2025 May 30. pii: S2211-1247(25)00530-3. [Epub ahead of print]44(6): 115759
    A synthetic chlorophagy receptor promotes plant fitness by mediating chloroplast microautophagy.
    Rui Liu, Xun Weng, Xinjing Li, Yongheng Cao, Qiyun Li, Lin Luan, Danqing Tong, Zhaosheng Kong, Hao Wang, Taotao Wang, Qingqiu Gong.
      Chloroplasts are photosynthetic organelles and one of the major protein-containing organelles in green plants and algae. Although chloroplast contents or entire chloroplasts can be cleared by various vesicular pathways and autophagy, canonical chlorophagy receptors remain unidentified. Also, whether chlorophagy can be enhanced to benefit plants remains unknown. Here, we report the design and validation of a synthetic chlorophagy receptor that promotes plant fitness. The receptor LIR-SNT-BFP contains a fragment spanning the LIR/AIM of NBR1 and the N-terminal amphipathic helix of SFR2. The synthetic receptor localizes to chloroplasts and recruits ATG8a in planta. Induced expression of the synthetic receptor promotes microautophagy of entire chloroplasts, independent of ATG5 or ATG7. Meanwhile, it induces chloroplast fission. Notably, moderate induction of chlorophagy promotes rosette growth, whereas excessive chlorophagy appears detrimental. Induced chlorophagy also partially suppresses herbicide-induced leaf chlorosis. Our study provides proof of concept for controlling chloroplast degradation using a synthetic chlorophagy receptor.
    Keywords:  ATG8; CP: Cell biology; CP: Plants; autophagy; biomass; chloroplast; chloroplast fission; herbicide; microautophagy; receptor; vacuole
    DOI:  https://doi.org/10.1016/j.celrep.2025.115759
  35. Nat Commun. 2025 Jun 01. 16(1): 5086
    Mechanism of ATP hydrolysis in the Hsp70 BiP nucleotide-binding domain.
    Guillaume Mas, Sebastian Hiller.
      The 70 kDa heat shock protein (Hsp70) family of molecular chaperones ensures protein biogenesis and homeostasis, driven by ATP hydrolysis. Here, we introduce in-cyclo NMR, an experimental setup that combines high-resolution NMR spectroscopy with an ATP recovery and a phosphate removal system. In-cyclo NMR simultaneously resolves kinetic rates and structural information along functional cycles of ATP-driven molecular machines. We benchmark the method on the nucleotide binding domain (NBD) of the human Hsp70 chaperone BiP. The protein cycles through ATP binding, hydrolysis, and two parallel pathways of product release. We determine the kinetic rates of all eleven underlying elementary reactions and show these to match independent measurements. The two product release pathways regulate the cycle duration dependent on the products concentration. Under physiological conditions, they are both used. The in-cyclo NMR method will serve as a platform for studies of ATP-driven functional cycles at a remarkable level of detail.
    DOI:  https://doi.org/10.1038/s41467-025-60343-x
  36. Sci Adv. 2025 Jun 06. 11(23): eads1241
    Chaperone-mediated heterotypic phase separation regulates liquid-to-solid phase transitions of tau into amyloid fibrils.
    Sandeep K Rai, Roopali Khanna, Anusha Sarbahi, Ashish Joshi, Samrat Mukhopadhyay.
      Biomolecular condensates formed via phase separation of proteins, and nucleic acids regulate crucial cellular processes. However, such liquid-like membraneless bodies can undergo aberrant liquid-to-solid transitions into amyloid-like pathological species, which necessitates their efficient clearance by the cellular protein quality control machinery comprising molecular chaperones. We present a unique case to demonstrate that a heat shock protein 40 (Ydj1) promotes the heterotypic phase separation of intrinsically disordered tau via a multitude of interactions. Using multicolor imaging, time-resolved fluorescence anisotropy, vibrational Raman spectroscopy, and single-molecule Förster resonance energy transfer, we unmask the crucial molecular events associated with heterotypic phase separation of tau. We show that the presence of Ydj1 within condensates abolishes phase transitions into amyloids, unlike tau-only droplets that spontaneously mature into amyloid fibrils. We identify the amyloidogenic hexapeptide motifs located in the hydrophobic microtubule-binding region of tau that interacts with the peptide-binding regions of Ydj1 promoting tau-Ydj1 condensate formation. Our results provide mechanistic underpinnings of condensate-mediated protein homeostasis.
    DOI:  https://doi.org/10.1126/sciadv.ads1241
  37. bioRxiv. 2025 May 15. pii: 2025.05.10.653107. [Epub ahead of print]
    MedSAGE: Bridging Generative AI and Medicinal Chemistry for Structure-Based Design of Small Molecule Drugs.
    Alexander S Powers, Tianyu Lu, Rohan V Koodli, Minkai Xu, Siyi Gu, Masha Karelina, Ron O Dror.
      While generative AI is transforming the de novo design of proteins, its effectiveness for structure-based design of small molecules remains limited. Current methods, including diffusion models, often produce small molecules with difficult-to-synthesize structures, poor medicinal chemistry properties, and limited target selectivity. To address these limitations, we introduce MedSAGE, a novel generative AI framework that adapts diffusion models specifically for de novo small-molecule design. Rather than using atoms or strings, we develop a novel representation for generating molecules using fragments relevant for medicinal chemistry. Chemical and geometric information characterizing these fragments is embedded in a smooth and interpretable latent space. We also develop an algorithm to optimize the connectivity between generated fragments while preserving chemical validity and synthesizability. In a benchmark of multiple methods across 25 therapeutically relevant protein targets, MedSAGE achieved state-of-the-art performance, producing synthesizable, drug-like molecules with predicted affinity and selectivity closely matching known drugs and drug candidates. Compared to large-scale virtual screening, MedSAGE produced molecules with high predicted affinity over 100 times more efficiently. Our results demonstrate that MedSAGE is already practically useful and paves the way for next-generation tools in structure-guided drug design.
    DOI:  https://doi.org/10.1101/2025.05.10.653107
  38. Sci Adv. 2025 Jun 06. 11(23): eadu9605
    HOPS-dependent vesicle tethering lock inhibits endolysosomal fusions and autophagosome secretion upon the loss of Syntaxin17.
    Dávid Hargitai, Anikó Nagy, Iván Bodor, Győző Szenci, Hajnalka Laczkó-Dobos, Arindam Bhattacharjee, Natali Neuhauser, Szabolcs Takáts, Gábor Juhász, Péter Lőrincz.
      The autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein) Syntaxin17 (Syx17) plays a pivotal role in autophagosome-lysosome fusion, yet the broader impact of its loss remains elusive. Our investigation of Syx17 function in Drosophila nephrocytes and salivary gland cells revealed unexpected effects. We find that Syx17 loss induces the formation of autophagosome-lysosome clusters in a HOPS (homotypic fusion and vacuole protein sorting)-dependent manner, entrapping this tether, autophagosomes, and lysosomes. While locked in clusters, these organelles cannot participate in other vesicle fusions, impeding endosomal progression and autophagosome secretion. Therefore, the absence of Syx17 not only inhibits autophagosome-lysosome fusion but also prevents HOPS release from autophagosome-lysosome tethering sites causing a "tethering lock." Preventing autophagosome formation or removing the HOPS adaptor Plekhm1 (pleckstrin homology domain-containing family M member 1) leads to release of HOPS and lysosomes from these clusters, thus rescuing secondary effects of Syx17 loss. Our findings show that a tethering lock can disrupt multiple vesicle trafficking routes.
    DOI:  https://doi.org/10.1126/sciadv.adu9605
  39. bioRxiv. 2025 May 15. pii: 2025.05.11.653360. [Epub ahead of print]
    A family of E3 ligases extend K11 polyubiquitin on sites of MARUbylation.
    Rachel E Lacoursiere, Kapil Upadhyaya, Jasleen Kaur Sidhu, Daniel S Bejan, Ivan Rodriguez Siordia, Michael S Cohen, Jonathan N Pruneda.
      Ubiquitin (Ub) cooperation with other post-translational modifications provides a tiered opportunity for protein regulation. Small modifications to Ub such as phosphorylation, acetylation, or ADP-ribosylation have varying impacts on signaling. The Deltex family of E3 ligases was previously implicated in the ubiquitylation of ADP-ribose (ADPr) and ADPr-containing macromolecules. Our previous work found ester-linked mono-ADPr ubiquitylation (MARUbylation) on PARP7 and PARP10 in cells and that this mark is extended with K11 polyUb. We previously screened for E3 ligases that interact with PARP7 through three different approaches and identified six candidates, including the Deltex family member DTX2. One of these hits, RNF114, interacts with various other PARPs, leading us to hypothesize that RNF114 binds to sites of MARUbylation and extends K11 polyUb. Here, we show that DTX2 generates the initial MARUbe on PARP7 in cells, which depends on PARP7 catalytic activity. The MARUbe on PARP7 is extended with K11 polyUb by RNF114. To investigate the mechanism of RNF114 reader/writer function, we developed a click chemistry-inspired chemoenzymatic approach to create a novel fluorescent Ub-ADPr probe for studying its interaction with RNF114. Strikingly, we found that RNF114 has a weak affinity for ADPr and Ub separately but explicitly recognizes the linkage between Ub and ADPr present in MARUbylated species. We used AlphaFold3 modeling to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identified a tandem Di19-UIM module in RNF114 as a M AR U be- b inding d omain (MUBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we described a small family of MUBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call M ARUbe- T argeted L igases (MUTLs).
    DOI:  https://doi.org/10.1101/2025.05.11.653360
  40. bioRxiv. 2025 May 14. pii: 2025.05.13.653742. [Epub ahead of print]
    Of condensates and coats - reciprocal regulation of clathrin assembly and the growth of protein networks.
    Brandon T Malady, Andromachi Papagiannoula, Advika Kamatar, Susovan Sarkar, Gavin T Lebrun, Liping Wang, Carl C Hayden, Eileen M Lafer, David J Owen, Sigrid Milles, Jeanne C Stachowiak.
      Clathrin-mediated endocytosis is essential for membrane traffic, impacting a diverse range of cellular processes including cell signaling homeostasis, cell adhesion, and receptor recycling. During endocytosis, invagination of the plasma membrane is coordinated by a network of proteins that recruit and assemble the clathrin coat. Recent work demonstrated that clathrin accessory proteins which arrive early at endocytic sites, such as Eps15 and Fcho2, form phase-separated condensates that recruit downstream machinery, promoting assembly and maturation of clathrin-coated vesicles. However, the mechanisms by which protein condensates regulate - and are regulated by - clathrin assembly remain unclear. Using in vitro reconstitution and nuclear magnetic resonance spectroscopy, we demonstrate that protein condensates provide a platform for recruitment and assembly of clathrin triskelia. This condensate driven assembly is enhanced in the presence of the accessory protein, AP2, which is readily incorporated within condensates. In turn, clathrin assembly restricted the growth of condensates, exhibiting surfactant-like behavior that stabilized protein-protein interactions while imposing the preferred curvature of the clathrin lattice. This mutual regulation promotes efficient assembly of clathrin-coated vesicles while preventing uncontrolled expansion of protein condensates. More broadly, reciprocal regulation of protein condensates and clathrin coats may provide a framework for understanding how intrinsically disordered and structured protein assemblies can work together to build complex cellular architectures.
    DOI:  https://doi.org/10.1101/2025.05.13.653742
  41. Biochem Pharmacol. 2025 Jun 02. pii: S0006-2952(25)00285-0. [Epub ahead of print] 117020
    Enhancement of MAVS UFMylation and inhibition of lysosomal degradation of MAVS aggregates: New findings on broad-spectrum antiviral mechanism of Anemoside B4.
    Nai-Xin Kang, Yun-Meng Luo, Jian-Ping Zhao, Hong-Lv Jiang, Guo-Qiang Xu, Ning-Xun Cui, Ke-Jun Deng, Li Zhang, Ikhlas A Khan, Shi-Lin Yang, Qiong-Ming Xu, Yan-Li Liu.
      RNA viruses have evolved various strategies to bypass the mitochondrial antiviral-signaling protein (MAVS) pathway, effectively sidestepping the host's innate immunity. Therefore, searching for MAVS signalosome regulators is crucial for the development of antiviral drugs. Here, we found RNA viral-drived MAVS lysosomal trafficking was inhibited by Anemoside B4 (B4), a triterpenoid saponin from the herbal medicine Pulsatilla chinensis (Bunge) Regel., has significant antiviral activity. We performed biotin-B4 probe tandem proteomic profiling and identified ubiquitin fold modifier 1 (UFM1), one of the newly discovered Ub-like proteins (UBLs), as a key target of B4. Using cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) analysis, and molecular docking analysis we showed that B4 directly bound to UFM1 through Lys34 and Ile57 sites and promoted UFMylation of target proteins. Furthermore, we found that MAVS UFMylation could promote polyubiquitination, K48- and K63-linked ubiquitination, yet preventing K27-linked ubiquitination. MAVS aggregation and innate immunity response were suppressed in virus infected UFM1 KO cells even in the presence of B4. Moreover, the decrease in K27-ubiquitin (Ub) binding on MAVS and lysosomal degradation induced by B4 was also significantly prevented by UFM1 deficiency. Importantly, to verify that MAVS UFMylation is the actual target for B4, cytoprotective and viral titer analysis were executed in cells lacking UFM1 challenged with Enterovirus 71 (EV71) and Influenza A virus (IAV). The findings uncover the new finding broad-spectrum antiviral mechanism of B4, suggesting that UFMylation of MAVS could be an advantageous approach for countering RNA viral infections.
    Keywords:  Anemoside B4; Innate immunity; Mitochondrial antiviral-signaling protein; RNA virus; Ubiquitin fold modifier 1; Ubiquitination
    DOI:  https://doi.org/10.1016/j.bcp.2025.117020
  42. Nat Commun. 2025 Jun 03. 16(1): 5140
    De novo design of protein condensation inhibitors by targeting an allosteric site of cGAS.
    Wenfeng Zhao, Guofeng Chen, Jian He, Xiaofang Shen, Muya Xiong, Liwei Xiong, Zhihao Qi, Hang Xie, Wanchen Li, Jiameng Li, Huixia Dou, Hangchen Hu, Haixia Su, Qiang Shao, Minjun Li, Hongbin Sun, Yechun Xu.
      Cyclic GMP-AMP synthase (cGAS), a key mediator of the cGAS-STING DNA sensing pathway that triggers type-I interferon responses, plays a crucial role in innate immunity and has been implicated in the pathogenesis of various disease. Despite advances in the development of cGAS inhibitors, none have reached the market and there remains an unmet need for divergent chemical scaffolds with high selectivity, potency across species, and target-adaptive mechanisms of action to explore cGAS's potential as a therapeutic target. Here we report the structural, biochemical, cellular, and mechanistic characterization of the XL series of allosteric inhibitors, designed to engage an innovative allosteric site near the activation loop of cGAS. Among them, XL-3156 and XL-3158 emerge as potent, selective, cross-species cGAS inhibitors that simultaneously occupy allosteric and orthosteric sites, stabilizing the activation loop in a closed, inactive conformation and thereby attenuating the cGAS-DNA interactions. Moreover, these allosteric inhibitors, also known as protein condensation inhibitors (PCIs), significantly suppress cGAS-DNA condensate formation, triggering a morphological transition from liquid-solid phase separation (LSPS) to liquid-liquid phase separation (LLPS) at the molecular level while eliminating LLPS in cells. The distinct mechanism of action enables PCIs to achieve synergistic effects in combination with orthosteric inhibitors. These results establish a mechanism-driven pharmacological strategy to inhibit cGAS through PCIs that modulate phase separation primarily by engagement of the allosteric site.
    DOI:  https://doi.org/10.1038/s41467-025-60297-0
  43. bioRxiv. 2025 May 21. pii: 2025.05.16.654561. [Epub ahead of print]
    Translational buffering tunes gene expression in mouse and human.
    Shilpa Rao, Aden Y Le, Logan Persyn, Can Cenik.
      Translational buffering refers to the regulation of ribosome occupancy to offset the effects of transcriptional variation. While previous work systematically analyzed translational buffering in yeast, it remains unknown whether this is an intrinsic property of genes in mouse and human across cell types. To identify this phenomenon on a global scale and across different experimental conditions, we uniformly analyzed 1515 matched ribosome profiling and RNA-seq datasets from human and mouse tissues or cell lines. This resource enabled us to assess the potential of genes to exhibit translational buffering by comparative analysis of variation at ribosome occupancy and the RNA levels across cell types as well as by examining the relationship between mRNA abundance and translation efficiency. We demonstrate that translational buffering is a conserved property of genes using homologous gene pairs from humans and mice. Although the identified buffered genes show association with some intrinsic sequence features, our modeling results suggest that these alone are insufficient to predict translational buffering, highlighting the importance of cellular context in determining buffering. Further, genes exhibiting translational buffering have lower variation in protein abundance in cancer cell lines, primary human tissues and mouse samples. We also observed that translationally buffered genes are more likely to be haploinsufficient and triplosensitive suggesting a demand for stringent dosage limits in these genes. We hypothesize two models of translational buffering, namely "differential accessibility model" and "change in translation initiation rate model". Our experiment suggests that some transcripts conform to the former and others align with the alternate model. Overall, our work broadens the catalog of genes subjected to translational buffering, underscores the characteristics of genes that demonstrate this phenomenon and additionally provides an insight into the rationale driving this effect.
    DOI:  https://doi.org/10.1101/2025.05.16.654561
  44. Nat Commun. 2025 Jun 05. 16(1): 5209
    Lysosomal TMEM165 controls cellular ion homeostasis and survival by mediating lysosomal Ca2+ import and H+ efflux.
    Ran Chen, Bin Liu, Dawid Jaślan, Lucija Kucej, Veronika Kudrina, Belinda Warnke, Yvonne E Klingl, Arnas Petrauskas, Sandra Prat Castro, Kenji Maeda, Christian Grimm, Marja Jäättelä.
      The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how lysosomes replenish their calcium stores in placental mammals has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the cytosol. These data indicate that in addition to its previously identified role in the glycosylation of proteins and lipids in the Golgi, a fraction of TMEM165 localizes on the lysosomal limiting membrane, where its putative calcium/proton antiporter activity plays an essential role in the regulation of intracellular ion homeostasis and cell survival.
    DOI:  https://doi.org/10.1038/s41467-025-60349-5
  45. Nat Commun. 2025 May 31. 16(1): 5073
    Endolysosomal processing of neuron-derived signaling lipids regulates autophagy and lipid droplet degradation in astrocytes.
    Jagannatham Naidu Bhupana, Angelid Pabon, Ho Hang Leung, Mohamed Asik Rajmohamed, Sang Hoon Kim, Yan Tong, Mi-Hyeon Jang, Ching-On Wong.
      Dynamic regulation of metabolic activities in astrocytes is critical to meeting the demands of other brain cells. During neuronal stress, lipids are transferred from neurons to astrocytes, where they are stored in lipid droplets (LDs). However, it is not clear whether and how neuron-derived lipids trigger metabolic adaptation in astrocytes. Here, we uncover an endolysosomal function that mediates neuron-astrocyte transcellular lipid signaling. We identify Tweety homolog 1 (TTYH1) as an astrocyte-enriched endolysosomal protein that facilitates autophagic flux and LD degradation. Astrocyte-specific deletion of mouse Ttyh1 and loss of its Drosophila ortholog lead to brain accumulation of neutral lipids. Computational and experimental evidence suggests that TTYH1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P), a sphingolipid that dampens autophagic flux and LD breakdown in mouse and human astrocytes. Furthermore, neuronal C1P secretion induced by inflammatory cytokine interleukin-1β causes TTYH1-dependent autophagic flux and LD adaptations in astrocytes. These findings reveal a neuron-initiated signaling paradigm that culminates in the regulation of catabolic activities in astrocytes.
    DOI:  https://doi.org/10.1038/s41467-025-60402-3
  46. Nat Cell Biol. 2025 Jun 04.
    Extracellular exosomal RNAs are glyco-modified.
    Sunny Sharma, Xinfu Jiao, Jun Yang, Kelvin Y Kwan, Megerditch Kiledjian.
      Epitranscriptomic modifications play pivotal roles in regulating RNA stability, localization and function. Recently, glycosylation has also emerged as an RNA modification, though its functional implications remain unclear. Here we report that metabolic labelling with a N-azidoacetylgalactosamine-tetraacylated bioorthogonal probe in mammalian cells reveals small, non-coding, glyco-modified RNAs (glycoRNAs) that exhibit unusual stability imparted by their resistance to RNases. These glycoRNAs are primarily found within exosome vesicles as intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Importantly, exosomal glycoRNAs can be transferred to naive cells, highlighting a role in intercellular RNA communication. The inhibition of exosome biogenesis leads to intracellular glycoRNA accumulation, while blocking glycan transfer to proteins reduces glycoRNA sorting into exosomes. These findings suggest a regulatory link between protein and RNA glycosylation in exosome cargo selection. Our studies support a functional role for glycosylation in targeting RNA into exosomes and uncover potential avenues for exosome-based diagnostics and RNA therapeutic applications.
    DOI:  https://doi.org/10.1038/s41556-025-01682-1
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