bims-proteo Biomed News
on Proteostasis
Issue of 2025–03–09
forty-four papers selected by
Eric Chevet, INSERM
  1. RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation.
  2. VIPER-TACs leverage viral E3 ligases for disease-specific targeted protein degradation.
  3. To degrade or not to degrade: how phase separation modulates selective autophagy.
  4. Crucial roles of Grr1 in splicing and translation of HAC1 mRNA upon unfolded stress response.
  5. An ISR-independent role of GCN2 prevents excessive ribosome biogenesis and mRNA translation.
  6. Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering.
  7. When Proteins Go Berserk: The Unfolded Protein Response and ER stress.
  8. Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma.
  9. Structural basis for the allosteric activation of Lon by the heat shock protein LarA.
  10. A subcellular map of translational machinery composition and regulation at the single-molecule level.
  11. Leveraging efficiency metrics for the optimization of CELMoDs™ as cereblon-based molecular glue degraders.
  12. Requirements for nuclear GRP78 transcriptional regulatory activities and interaction with nuclear GRP94.
  13. Polysome profiling is an extensible tool for the analysis of bulk protein synthesis, ribosome biogenesis, and the specific steps in translation.
  14. Small-Molecule FICD Inhibitors Suppress Endogenous and Pathologic FICD-Mediated Protein AMPylation.
  15. CHIP protects lysosomes from CLN4 mutant-induced membrane damages.
  16. Mechanistic insights into protein biogenesis and maturation on the ribosome.
  17. MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks.
  18. Mesoscale landscaping of the TRIM protein family reveals a novel human condensatopathy.
  19. Coordinated Role of Autophagy and ERAD in Maintaining Neuroendocrine Function by Preventing Prohormone Aggregation.
  20. High-throughput screening identifies a novel small-molecule modulator of Hsp70 that selectively enhances ubiquitination and degradation of misfolded neuronal NO synthase.
  21. A long-lived pool of PINK1 imparts a molecular memory of depolarization-induced activity.
  22. Plant-specific tail-anchored coiled-coil protein MAG3 stabilizes Golgi-associated ERESs to facilitate protein exit from the ER.
  23. The pathways of secretory cargo export at the endoplasmic reticulum.
  24. A family of bacterial Josephin-like deubiquitinases with an irreversible cleavage mode.
  25. The integrated stress response pathway controls cytokine production in tissue-resident memory CD4+ T cells.
  26. Molecular Glue-Design-Evaluator (MOLDE): An Advanced Method for In-Silico Molecular Glue Design.
  27. A BiP-centric view of endoplasmic reticulum functions and of my career.
  28. Substrate Recognition and Cleavage-Site Preferences of Lon Protease.
  29. Virus targeting as a dominant driver of interfacial evolution in the structurally resolved human-virus protein-protein interaction network.
  30. How pore formation in complex biological membranes is governed by lipid composition, mechanics, and lateral sorting.
  31. RNA-binding proteins and glycoRNAs form domains on the cell surface for cell-penetrating peptide entry.
  32. Custom affinity probes reveal DNA-damage-induced, ssDNA-independent chromatin SUMOylation in budding yeast.
  33. IDR-driven TOLLIP condensates antagonize the innate antiviral immunity by promoting the deSUMOylation of MAVS.
  34. Alterations in ether phospholipids metabolism activate the conserved UPR-Xbp1- PDIA3/ERp60 signaling to maintain intestinal homeostasis.
  35. Induced proximity pushes beyond protein degraders, as first RIPTAC moves into the clinic.
  36. Macroevolutionary divergence of gene expression driven by selection on protein abundance.
  37. Differential regulation of BAX and BAK apoptotic activity revealed by small molecules.
  38. Cytoplasmic ribosomes on mitochondria alter the local membrane environment for protein import.
  39. Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity in breast cancer.
  40. Protocol for multi-component reconstitution of stress granules in vitro.
  41. Mapping the nanoscale organization of the human cell surface proteome reveals new functional associations and surface antigen clusters.
  42. TBK1 and IKKε prevent premature cell death by limiting the activity of both RIPK1 and NLRP3 death pathways.
  43. AFsample2 predicts multiple conformations and ensembles with AlphaFold2.
  44. Cell-autonomous innate immunity by proteasome-derived defence peptides.
  1. Cell Rep. 2025 Feb 28. pii: S2211-1247(25)00142-1. [Epub ahead of print]44(3): 115371
    RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation.
    Pierce W Ford, Danielle M Garshott, Mythreyi Narasimhan, Xuezhen Ge, Eric M Jordahl, Shubha Subramanya, Eric J Bennett.
      The initiation-specific ribosome-associated quality control pathway (iRQC) is activated when translation initiation complexes fail to transition to elongation-competent 80S ribosomes. Upon iRQC activation, RNF10 ubiquitylates the 40S proteins uS3 and uS5, which leads to 40S decay. How iRQC is activated in the absence of pharmacological translation inhibitors and what mechanisms govern iRQC capacity and activity remain unanswered questions. Here, we demonstrate that altering 60S:40S stoichiometry by disrupting 60S biogenesis triggers iRQC activation and 40S decay. Depleting the critical scanning helicase eIF4A1 impairs 40S ubiquitylation and degradation, indicating mRNA engagement is required for iRQC. We show that amino acid starvation conditions also stimulate iRQC-dependent 40S decay. We identify RIOK3 as a crucial iRQC factor that interacts with ubiquitylated 40S subunits to mediate degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC pathway activation, establishing a feedforward mechanism that regulates iRQC capacity and subsequent 40S decay.
    Keywords:  40S degradation; 60S biogenesis; CP: Molecular biology; RIOK3; RNF10; amino acid starvation; iRQC; ribosomal subunit imbalance; ribosomal ubiquitylation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115371
  2. Cell Chem Biol. 2025 Mar 04. pii: S2451-9456(25)00034-0. [Epub ahead of print]
    VIPER-TACs leverage viral E3 ligases for disease-specific targeted protein degradation.
    Kyle Mangano, Robert G Guenette, Spencer Hill, Shiqian Li, Jeffrey J Liu, Cory M Nadel, Suresh Archunan, Arghya Sadhukhan, Rajiv Kapoor, Seung Wook Yang, Kate S Ashton, Patrick Ryan Potts.
      In targeted protein degradation (TPD) a protein of interest is degraded by chemically induced proximity to an E3 ubiquitin ligase. One limitation of using TPD therapeutically is that most E3 ligases have broad tissue expression, which can contribute to toxicity via target degradation in healthy cells. Many pathogenic and oncogenic viruses encode E3 ligases (vE3s), which de facto have strictly limited expression to diseased cells. Here, we provide proof-of-concept for viral E3 pan-essential removing targeting chimeras (VIPER-TACs) that are bi-functional molecules that utilize viral E3 ubiquitin ligases to selectively degrade pan-essential proteins and eliminate diseased cells. We find that the human papillomavirus (HPV) ligase E6 can degrade the SARS1 pan-essential target protein in a model of HPV-positive cervical cancer to selectively kill E6 expressing cancer cells. Thus, VIPER-TACs have the capacity to dramatically increase the therapeutic window, alleviate toxicity concerns, and ultimately expand the potential target space for TPD.
    Keywords:  HPV positive cancer; VIPER-TAC; antiviral; biotechnology; chemical biology; induced proximity; targeted protein degradation; viral E3 ligase
    DOI:  https://doi.org/10.1016/j.chembiol.2025.02.002
  3. Autophagy. 2025 Mar 07.
    To degrade or not to degrade: how phase separation modulates selective autophagy.
    Mariya Licheva, Riccardo Babic, Jeremy Pflaum, Hector Mancilla, Florian Wilfling, Claudine Kraft.
      Selective macroautophagy/autophagy relies on newly formed double-membrane compartments, known as phagophores, to sequester and recycle diverse cellular components, including organelles, biomolecular condensates and protein aggregates, maturing into autophagosomes that fuse with the vacuole/lysosome. Autophagosomes originate at the cargo-vacuole/ER interface, where autophagy factors assemble into the phagophore assembly site (PAS). However, how autophagy proteins organize on the surface of structurally and biophysically different cargoes, and achieve spatial confinement at the PAS to support autophagosome formation remains unclear. Mechanisms governing cargo selection are also poorly understood. In this study, we demonstrate that receptor mobility, driven by low affinity cargo-receptor interactions, is crucial for rendering cellular structures degradable by autophagy. We show that cargo surface mobility, combined with the phase separation of scaffold proteins, drives the formation of early PAS precursors, termed "initiation hubs". These hubs dynamically rearrange at the cargo-vacuole/ER interface to promote autophagosome biogenesis, providing new insights into selective autophagy initiation.
    Keywords:  Aggrephagy; Atg11/RB1CC1; autophagy; cargo receptor; initiation hub, phase separation
    DOI:  https://doi.org/10.1080/15548627.2025.2476025
  4. Nat Commun. 2025 Mar 04. 16(1): 2172
    Crucial roles of Grr1 in splicing and translation of HAC1 mRNA upon unfolded stress response.
    Nichika Sato, Yu Nakano, Yasuko Matsuki, Shota Tomomatsu, Sihan Li, Yoshitaka Matsuo, Toshifumi Inada.
      In the process of the unfolded protein response (UPR), the Hac1p protein is induced through a complex regulation of the HAC1 mRNA. This includes the mRNA localization on the endoplasmic reticulum (ER) membrane and stress-triggered splicing. In yeast, a specific ribosome ubiquitination process, the monoubiquitination of eS7A by the E3 ligase Not4, facilitates the translation of HAC1i, a spliced form of the HAC1 mRNA. Upon UPR, the mono-ubiquitination of eS7A increases due to the downregulation of Ubp3, a deubiquitinating enzyme of eS7A. However, the exact mechanisms behind these regulations have remained unknown. In this study, an E3 ligase, Grr1, an F-box protein component of the SCF ubiquitin ligase complex, which is responsible for Ubp3 degradation, has been identified. Grr1-mediated Ubp3 degradation is required to maintain the level of eS7A monoubiquitination that facilitates Hac1p translation depending on the ORF of HAC1i. Grr1 also facilitates the splicing of HAC1u mRNA independently of Ubp3 and eS7A ubiquitination. Finally, we propose distinct roles of Grr1 upon UPR, HAC1u splicing, and HAC1i mRNA translation. Grr1-mediated Ubp3 degradation is crucial for HAC1i mRNA translation, highlighting the crucial role of ribosome ubiquitination in translational during UPR.
    DOI:  https://doi.org/10.1038/s41467-025-57360-1
  5. Life Sci Alliance. 2025 May;pii: e202403014. [Epub ahead of print]8(5):
    An ISR-independent role of GCN2 prevents excessive ribosome biogenesis and mRNA translation.
    Mónica Román-Trufero, Istvan T Kleijn, Kevin Blighe, Jinglin Zhou, Paula Saavedra-García, Abigail Gaffar, Marilena Christoforou, Axel Bellotti, Joel Abrahams, Abdelmadjid Atrih, Douglas Lamont, Marek Gierlinski, Pooja Jayaprakash, Audrey M Michel, Eric O Aboagye, Mariia Yuneva, Glenn R Masson, Vahid Shahrezaei, Holger W Auner.
      The integrated stress response (ISR) is a corrective physiological programme to restore cellular homeostasis that is based on the attenuation of global protein synthesis and a resource-enhancing transcriptional programme. GCN2 is the oldest of four kinases that are activated by diverse cellular stresses to trigger the ISR and acts as the primary responder to amino acid shortage and ribosome collisions. Here, using a broad multi-omics approach, we uncover an ISR-independent role of GCN2. GCN2 inhibition or depletion in the absence of discernible stress causes excessive protein synthesis and ribosome biogenesis, perturbs the cellular translatome, and results in a dynamic and broad loss of metabolic homeostasis. Cancer cells that rely on GCN2 to keep protein synthesis in check under conditions of full nutrient availability depend on GCN2 for survival and unrestricted tumour growth. Our observations describe an ISR-independent role of GCN2 in regulating the cellular proteome and translatome and suggest new avenues for cancer therapies based on unleashing excessive mRNA translation.
    DOI:  https://doi.org/10.26508/lsa.202403014
  6. bioRxiv. 2025 Feb 17. pii: 2025.02.17.637997. [Epub ahead of print]
    Phosphorylation-State Modulated Binding of HSP70: Structural Insights and Compensatory Protein Engineering.
    Mariah Stewart, Chathura Paththamperuma, Colleen McCann, Kelsey Cottingim, Huaqun Zhang, Rian DelVecchio, Ivy Peng, Erica Fennimore, Jay C Nix, Morcos N Saeed, Kathleen George, Katherine Makaroff, Meagan Colie, Ethan Paulakonis, Michael F Almeida, Adeleye J Afolayan, Nicholas G Brown, Richard C Page, Jonathan C Schisler.
      Protein quality control is crucial for cellular homeostasis, involving the heat shock response, the ubiquitin-proteasome system, and the autophagy-lysosome pathway. Central to these systems are the chaperone homologs heat shock protein 70 (HSP70) and heat shock cognate 70 (HSC70), which manage protein folding and degradation. This study investigated the impact of the C-terminal phosphorylation of HSP70 on its interaction with the co-chaperone CHIP (C-terminus of HSC70 interacting protein), an E3 ligase that ubiquitinates protein substrates for degradation. Using both cell-free and cell-based approaches, including X-ray crystallography, biolayer interferometry, and live cell biocomplementation assays, we demonstrate that phosphorylation at HSP70 T636 reduces CHIP's binding affinity, shifting the preference toward other co-chaperones like HOP. Structural analysis reveals that phosphorylation disrupts key hydrogen bonds, altering binding dynamics. We engineered a CHIP variant (CHIP-G132N) to restore binding affinity to phosphorylated HSP70. While CHIP-G132N effectively restored binding without additional functional domains, its effectiveness was diminished in full-length phosphomimetic constructs in cell-free and in-cell assays, suggesting that additional interactions may influence binding. Functional assays indicate that phosphorylation of HSP70 affects its stability and degradation, with implications for diseases such as cancer and neurodegeneration. Our findings highlight the complexity of chaperone-co-chaperone interactions and underscore the importance of post-translational modifications in regulating protein quality control mechanisms. By elucidating the molecular details of HSP70 and CHIP interactions, our study provides a foundation for developing therapeutic interventions for diseases characterized by proteostasis imbalance.
    DOI:  https://doi.org/10.1101/2025.02.17.637997
  7. Nephron. 2025 Mar 04. 1-14
    When Proteins Go Berserk: The Unfolded Protein Response and ER stress.
    Doria Meiseles, Narkis Arbeli, Moran Dvela-Levitt.
      Background The cellular proteostasis machinery is essential for maintaining protein homeostasis by employing quality control systems that identify, sequester, and eliminate damaged or misfolded proteins. However, the accumulation of misfolded proteins can overwhelm these protective mechanisms, disrupting proteostasis. This phenomenon is a hallmark of numerous pathologies, including a variety of genetic disorders. In the secretory pathway, the buildup of misfolded proteins triggers endoplasmic reticulum (ER) stress, which activates the unfolded protein response (UPR). The UPR serves as an adaptive mechanism, aiming to alleviate stress and restore cellular homeostasis. However, if ER stress is prolonged or severe, the UPR may fail to restore balance and apoptosis is induced. Summary This review introduces the intricate signaling pathways activated by the three UPR transmembrane sensors: protein-kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6). We briefly present the roles of the distinct transcriptional programs activated by each sensor in modulating the cellular response to protein stress and in determining cell fate. We discuss how genetic variants and environmental factors contribute to the heterogeneity observed in protein misfolding diseases. Finally, we critically evaluate select therapeutic strategies, specifically protein stabilization, trafficking modulation, and UPR sensor targeting approaches. Key message This review introduces the potential consequences of protein misfolding, which may not only impair protein function, but can also lead to toxic protein accumulation and stress induction. Using Fabry disease as a compelling example, we suggest that future therapeutic intervention may require nuanced, combination approaches that address both loss and gain of protein function.
    DOI:  https://doi.org/10.1159/000544971
  8. bioRxiv. 2025 Feb 23. pii: 2025.02.22.639686. [Epub ahead of print]
    Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma.
    Christopher Nardone, Jingjing Gao, Hyuk-Soo Seo, Julian Mintseris, Lucy Ort, Matthew C J Yip, Milen Negasi, Anna K Besschetnova, Nolan Kamitaki, Steven P Gygi, Sirano Dhe-Paganon, Nikhil Munshi, Mariateresa Fulciniti, Michael E Greenberg, Sichen Shao, Stephen J Elledge, Xin Gu.
      The midnolin-proteasome pathway degrades many nuclear proteins without ubiquitination, but how it operates mechanistically remains unclear. Here, we present structures of the midnolin-proteasome complex, revealing how established proteasomal components are repurposed to enable a unique form of proteolysis. While the proteasomal subunit PSMD2/Rpn1 binds to ubiquitinated or ubiquitin-like proteins, we discover that it also interacts with the midnolin nuclear localization sequence, elucidating how midnolin's activity is confined to the nucleus. Likewise, PSMD14/Rpn11, an enzyme that normally cleaves ubiquitin chains, surprisingly functions non-enzymatically as a receptor for the midnolin ubiquitin-like (Ubl) domain, positioning the substrate-binding Catch domain directly above the proteasomal entry site to guide substrates into the proteasome. Moreover, we demonstrate that midnolin downregulation is critical for the survival of myeloma cells by promoting the expression of its transcription factor substrate IRF4. Our findings uncover the mechanisms underlying the midnolin-proteasome pathway and midnolin downregulation as a driver of multiple myeloma.
    DOI:  https://doi.org/10.1101/2025.02.22.639686
  9. Nat Commun. 2025 Mar 05. 16(1): 2212
    Structural basis for the allosteric activation of Lon by the heat shock protein LarA.
    Hsiu-Jung Wang, Yun-Erh Kuan, Meng-Ru Ho, Chung-I Chang.
      Lon is a conserved AAA+ (ATPases associated with diverse cellular activities) proteolytic machine that plays a key regulatory role in cells under proteotoxic stress. Lon-mediated proteolysis can be stimulated by either the unfolded or specific protein substrates accumulated under stress conditions. However, the molecular basis for this substrate-controlled proteolysis remains unclear. Here, we have found that the heat shock protein LarA, a recently discovered Lon substrate and allosteric activator, binds to the N-terminal domain (NTD) of Lon. The crystal structure of the LarA-NTD complex shows that LarA binds to a highly conserved groove in the NTD through the terminal aromatic residue of its C-terminal degron. Crystallographic and biochemical evidence further reveals that this binding exposes the hydrophobic core of LarA, which can bind a leucine residue and promote local protein unfolding. These results define the mechanistic role of the NTD in regulating Lon-mediated proteolysis in response to varying cellular conditions.
    DOI:  https://doi.org/10.1038/s41467-025-57482-6
  10. Science. 2025 Mar 07. 387(6738): eadn2623
    A subcellular map of translational machinery composition and regulation at the single-molecule level.
    Zijian Zhang, Adele Xu, Yunhao Bai, Yuxiang Chen, Kitra Cates, Craig Kerr, Abel Bermudez, Teodorus Theo Susanto, Kelsie Wysong, Fernando J García Marqués, Garry P Nolan, Sharon Pitteri, Maria Barna.
      Millions of ribosomes are packed within mammalian cells, yet we lack tools to visualize them in toto and characterize their subcellular composition. In this study, we present ribosome expansion microscopy (RiboExM) to visualize individual ribosomes and an optogenetic proximity-labeling technique (ALIBi) to probe their composition. We generated a super-resolution ribosomal map, revealing subcellular translational hotspots and enrichment of 60S subunits near polysomes at the endoplasmic reticulum (ER). We found that Lsg1 tethers 60S to the ER and regulates translation of select proteins. Additionally, we discovered ribosome heterogeneity at mitochondria guiding translation of metabolism-related transcripts. Lastly, we visualized ribosomes in neurons, revealing a dynamic switch between monosomes and polysomes in neuronal translation. Together, these approaches enable exploration of ribosomal localization and composition at unprecedented resolution.
    DOI:  https://doi.org/10.1126/science.adn2623
  11. RSC Med Chem. 2025 Mar 03.
    Leveraging efficiency metrics for the optimization of CELMoDs™ as cereblon-based molecular glue degraders.
    Lei Jia, Jennifer R Riggs, Dahlia R Weiss, Brian L Claus, Veerabahu Shanmugasundaram, Stephen R Johnson, Christoph W Zapf.
      Efficiency metrics are useful in medicinal chemistry to track small molecule progress in lead optimization (LO). Molecular glue degraders are small molecules that mediate targeted protein degradation by chemically inducing proximity between an E3 ligase and a protein target. The potency and depth of protein degradation are important factors in identifying molecular glue drug candidates. We developed degradation efficiency metrics based on both potency and depth of degradation to track lead optimization objectives. We applied these efficiency metrics retrospectively to track optimization of a clinical molecular glue degrader series, resulting in the identification of Golcadomide (CC-99282). This work illustrates that efficiency metrics are beneficial for the identification of molecular glue drug candidates.
    DOI:  https://doi.org/10.1039/d4md00870g
  12. J Biol Chem. 2025 Feb 28. pii: S0021-9258(25)00218-2. [Epub ahead of print] 108369
    Requirements for nuclear GRP78 transcriptional regulatory activities and interaction with nuclear GRP94.
    Ze Liu, Dat P Ha, Liangguang Leo Lin, Ling Qi, Amy S Lee.
      GRP78, a molecular chaperone primarily located in the endoplasmic reticulum (ER), has recently been discovered to translocate into the nucleus of stressed and cancer cells where it assumes a new function reprogramming the transcriptome. This study explores the requirements of GRP78 nuclear translocation and its transcriptional activity and investigates the role of ER-associated degradation (ERAD) in the process. We show that the ER-processed, mature form of GRP78 is the major form of nuclear GRP78 and is the form with transcriptional regulatory activity. In contrast, exogenously expressed GRP78 designed to lack its ER signal peptide, thus preventing it from entering the ER or undergoing any ER-related processing/modification, while able to enter the nucleus, lacks transcriptional regulatory activity towards E-Box containing target genes. Additionally, the ATP-binding and substrate-binding activities of GRP78 are critical for this transcriptional regulatory function. We further discover that GRP94, an ER chaperone that acts in concert with GRP78 on protein folding, can translocate to the nucleus and co-localize with nuclear GRP78 upon ER stress. These findings suggest that some form of ER processing of GRP78, in addition to cleavage of the ER signal peptide, is critical for its nuclear activity and that in stressed cells, ER chaperones may assume new functions in the nucleus yet to be explored.
    Keywords:  ER stress; ERAD; GRP78; GRP94; ID2; nuclear translocation; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108369
  13. Mol Biol Cell. 2025 Mar 05. mbcE24080341
    Polysome profiling is an extensible tool for the analysis of bulk protein synthesis, ribosome biogenesis, and the specific steps in translation.
    Ambar Rodriguez-Martinez, Sara K Young-Baird.
      Protein synthesis is an essential and highly regulated cellular process. Here, we demonstrate the versatility of polysome profiling - a methodology traditionally used to assess levels of protein synthesis - to monitor ribosomal integrity and modulation of specific steps in mRNA translation. Using expanded polysome profiling methodologies, we systematically illustrate defects in ribosome biogenesis, translation initiation, and translational elongation in different cellular conditions. We additionally provide instruction for how a modified polysome profiling protocol can be leveraged to identify and characterize the function of factors that regulate protein synthesis. These methodologies are broadly applicable to a range of physiological conditions and human diseases in which ribosome biogenesis or the phases of protein synthesis are distinctly regulated or dysregulated.
    DOI:  https://doi.org/10.1091/mbc.E24-08-0341
  14. ACS Chem Biol. 2025 Mar 04.
    Small-Molecule FICD Inhibitors Suppress Endogenous and Pathologic FICD-Mediated Protein AMPylation.
    Bhaskar K Chatterjee, Mohammad Maroof Alam, Arghya Chakravorty, Shannon M Lacy, William Giblin, Jason Rech, Charles L Brooks, Peter Arvan, Matthias C Truttmann.
      The AMP transferase, FICD, is an emerging drug target fine-tuning stress signaling in the endoplasmic reticulum (ER). FICD is a bifunctional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER-resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules that inhibit pathogenic FICD variants are lacking. Using an in vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also inhibit pathogenic FICD variants and improve proinsulin processing in β cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.
    DOI:  https://doi.org/10.1021/acschembio.4c00847
  15. bioRxiv. 2025 Feb 19. pii: 2025.02.18.638932. [Epub ahead of print]
    CHIP protects lysosomes from CLN4 mutant-induced membrane damages.
    Juhyung Lee, Jizhong Zou, Wan Nur Atiqah Binti Mazli, Natalie Chin, Michal Jarnik, Layla Saidi, Yue Xu, John Replogle, Michael Ward, Juan Bonifacino, Wei Zheng, Ling Hao, Yihong Ye.
      Understanding how cells mitigate lysosomal damage is critical for unraveling pathogenic mechanisms of lysosome-related diseases. Here we use organelle-specific proteomics in iPSC-derived neurons (i3Neuron) and an in vitro lysosome-damaging assay to demonstrate that lysosome damage, caused by the aggregation of Ceroid Lipofuscinosis Neuronal 4 (CLN4)-linked DNAJC5 mutants on lysosomal membranes, serves as a critical pathogenic linchpin in CLN4-associated neurodegeneration. Intriguingly, in non-neuronal cells, a ubiquitin-dependent microautophagy mechanism downregulates CLN4 aggregates to counteract CLN4-associated lysotoxicity. Genome-wide CRISPR screens identify the ubiquitin ligase CHIP as a central microautophagy regulator that confers ubiquitin-dependent lysosome protection. Importantly, CHIP's lysosome protection function is transferrable, as ectopic CHIP improves lysosomal function in CLN4 i3Neurons, and effectively alleviates lipofuscin accumulation and neurodegeneration in a Drosophila CLN4 disease model. Our study establishes CHIP-mediated microautophagy as a key organelle damage guardian that preserves lysosome integrity, offering new insights into therapeutic development for CLN4 and other lysosome-related neurodegenerative diseases.
    Keywords:  CHIP/STUB1; Ceroid Lipofuscinosis Neuronal/CLN4; DNAJC5/CSPα; Drosophila disease model; autophagy/microautophagy; lysosome membrane damage; lysosome storage disease/LSD; neurodegenerative disease; ubiquitin
    DOI:  https://doi.org/10.1101/2025.02.18.638932
  16. J Mol Biol. 2025 Feb 28. pii: S0022-2836(25)00122-6. [Epub ahead of print] 169056
    Mechanistic insights into protein biogenesis and maturation on the ribosome.
    Alfred Lentzsch, Jae Ho Lee, Shu-Ou Shan.
      The ribosome is a major cellular machine that converts genetic information into biological function. Emerging data show that the ribosome is not only a protein synthesis machine, but also participates in the maturation of the nascent protein into properly folded and active molecules. The ribosome surface near the opening of the polypeptide exit tunnel can interact directly with the newly synthesized protein and, more importantly, provides a platform where numerous protein biogenesis factors assemble, gain access to the nascent chain, and direct them into diverse biogenesis pathways. In this article, we review the current understanding of cotranslational protein maturation pathways, with an emphasis on systems in which biochemical studies provided a high-resolution molecular understanding and yielded generalizable mechanistic principles.
    Keywords:  membrane protein folding; molecular chaperone; protein folding; protein modification and processing; protein targeting; ribosome
    DOI:  https://doi.org/10.1016/j.jmb.2025.169056
  17. Life Sci Alliance. 2025 May;pii: e202403074. [Epub ahead of print]8(5):
    MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks.
    Mònica Torres Esteban, Matthew J Stewart, Eilis Bragginton, Alice Meroni, Annica Pellizzari, Alain Jeanrenaud, Stephen J Smerdon, Manuel Stucki.
      Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino 1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.
    DOI:  https://doi.org/10.26508/lsa.202403074
  18. bioRxiv. 2025 Jan 02. pii: 2025.01.02.630836. [Epub ahead of print]
    Mesoscale landscaping of the TRIM protein family reveals a novel human condensatopathy.
    Hari R Singh, Vineeta Sharma, Jik Nijssen, Andrei Pozniakovski, Alexander Rubin, Lorrin Liang, David Ball, Sunwoo Hong, Victoria Gauntner, Guanghao Yu, Arathi Ranga, David Salant, Friedhelm Hildebrandt, Anthony A Hyman, Amar J Majmundar.
      The mesoscale organization of cells is central to cellular physiology and pathology. Cellular condensates often form via biomolecular phase separation, mediated by intrinsically disordered regions (IDRs) and represent a key mechanism for mesoscale organization. The TRI-partite Motif (TRIM) family of ubiquitin ligases is implicated in diverse cellular functions and disease, yet the role of biomolecular condensation in TRIM family organization remains understudied. Here, we systematically investigate the mesoscale localization of 72 TRIM proteins, revealing that a majority form condensates in distinct cellular compartments. IDR content correlates with dynamic condensate formation, suggesting a critical role in mesoscale organization. Focusing on TRIM8, associated with a neuro-renal disorder, we demonstrate that disease-causing truncations of the TRIM8 C-terminal IDR result in a condensatopathy , characterized by disrupted condensation, proteasomal regulation, and TAK1/NFκB signaling. Functional assays in cellular and animal models link these disruptions to podocyte dysfunction and impaired response to injury. Our findings establish a framework for understanding condensatopathies and the mesoscale principles governing TRIM family organization and function.
    DOI:  https://doi.org/10.1101/2025.01.02.630836
  19. Adv Sci (Weinh). 2025 Mar 07. e2411662
    Coordinated Role of Autophagy and ERAD in Maintaining Neuroendocrine Function by Preventing Prohormone Aggregation.
    Xuya Pan, Xing He, Su Wu, Na Xiong, Xinyu Hou, Heting Wang, Diane Somlo, Martin Spiess, Haiyang Wang, Jifeng Yang, Chunliang Li, Shasha Li, Wenbin Ma, Yanming Chen, Jun Cui, Ling Qi, Guojun Shi.
      Proteotoxicity induced by misfolded or aggregated proteins causes progressive neuronal damage. The endoplasmic reticulum (ER) protein quality control (ERQC) pathways are responsible for mitigating the accumulation of these misfolded or aggregated proteins, thus reducing proteotoxicity. Enhancing ERQC pathways is a promising strategy for treating neurodegenerative diseases. However, the mechanisms governing the initiation and degradation of misfolded or aggregated proteins in neurons remain largely unknown in vivo. In studying the maturation of proAVP in mouse AVP neurons, this study discovers that autophagy and ER-associated degradation (ERAD) ERQC pathways collaborate to maintain proAVP maturation and protect AVP neuron survival against proteotoxicity. Autophagy deficiency in mouse AVP neurons leads to the late-onset of diabetes insipidus. Mechanistically, autophagy selectively degrades mutant proAVP aggregates and endogenous HRD1 of the SEL1L-HRD1 ERAD complex through FAM134B mediated ER-phagy. HRD1 induction is responsible for reducing proAVP aggregation and maintaining AVP neuron function and survival under autophagy deficiency. Thus, autophagy and ERAD form a dual-protection system that orchestrates prohormone maturation and endocrine neuron survival, providing new insights in the complexity of neuroendocrinology and the intrinsic mechanism of neurodegenerative diseases, with therapeutic potential in protein folding diseases.
    Keywords:  ERAD; autophagy; diabetes insipidus; endocrine neurons; protein aggregates
    DOI:  https://doi.org/10.1002/advs.202411662
  20. Mol Pharmacol. 2025 Feb;pii: S0026-895X(24)23014-5. [Epub ahead of print]107(2): 100008
    High-throughput screening identifies a novel small-molecule modulator of Hsp70 that selectively enhances ubiquitination and degradation of misfolded neuronal NO synthase.
    Anthony M Garcia, Amanda K Davis, Cristian Martinez-Ramos, Yoshihiro Morishima, Miranda Lau, Emily Xu, Arya Sunil, Haoming Zhang, Andrew Alt, Andrew P Lieberman, Yoichi Osawa.
      The Hsp90 and Hsp70 chaperones act as a protein quality control system for several hundred client proteins, including many implicated in neurodegenerative disorders. Hsp90 and Hsp70 are widely thought to be important drug targets. Although many structurally distinct compounds have been developed to target Hsp90, relatively few are known to target Hsp70 and even fewer have been tested in protein quality control systems. To address this, we describe a high-throughput thermal shift-based screen to find compounds that bind and stabilize Hsp70 and then employ assays with misfolded forms of a well-established client protein, neuronal NO synthase (nNOS), to identify compounds that enhance ubiquitination of client proteins. The ubiquitination assay employed a quantitative ELISA method to measure Hsp70:CHIP-dependent ubiquitination of heme-deficient nNOS, which is a model of a misfolded client, in reaction mixtures containing purified E1, E2, Hsp70, CHIP, and ubiquitin. We screened 44,447 molecules from the Maybridge and ChemDiv libraries and found one compound, protein folding disease compound 15 (PFD-15), that enhanced in vitro nNOS ubiquitination with an EC50 of approximately 8 μM. PFD-15 was tested in human embryonic kidney 293 cells stably transfected with a C331A nNOS, a mutation that makes nNOS a preferred client protein for ubiquitination. In this model, PFD-15 decreased steady-state levels of C331A nNOS, but not the wild-type nNOS, in a time- and concentration-dependent manner by a process attenuated by lactacystin, an inhibitor to the proteasome. PFD-15 appears to enhance binding of Hsp70 and CHIP to client proteins without interference of protein quality control mechanisms, enabling the selective clearance of misfolded proteins. SIGNIFICANCE STATEMENT: There are few treatment options for neurodegenerative diseases, which are widely thought to be caused by formation of toxic misfolded proteins. One novel approach is to enhance the Hsp90/Hsp70 protein quality control machinery to remove these misfolded proteins. Targeting Hsp70 may have advantages over targeting Hsp90, but fewer compounds targeting Hsp70 have been developed relative to those for Hsp90. The current study provides a novel approach to enhance the number of compounds targeting the Hsp70's role in protein quality control.
    Keywords:  Chaperone; Degradation; Hsp70; Protein quality control; Ubiquitination
    DOI:  https://doi.org/10.1016/j.molpha.2024.100008
  21. Sci Adv. 2025 Feb 28. 11(9): eadr1938
    A long-lived pool of PINK1 imparts a molecular memory of depolarization-induced activity.
    Liam Pollock, Ioanna Ch Georgiou, Emma V Rusilowicz-Jones, Michael J Clague, Sylvie Urbé.
      The Parkinson's disease-linked kinase, PINK1, is a short-lived protein that undergoes cleavage upon mitochondrial import leading to its proteasomal degradation. Under depolarizing conditions, it accumulates on mitochondria where it becomes activated, phosphorylating both ubiquitin and the ubiquitin E3 ligase Parkin, at Ser65. Our experiments reveal that in retinal pigment epithelial cells, only a fraction of PINK1 becomes stabilized after depolarization by electron transport chain inhibitors. Furthermore, the observed accrual of PINK1 cannot be completely accounted for without an accompanying increase in biosynthesis. We have used a ubiquitylation inhibitor TAK-243 to accumulate cleaved PINK1. Under these conditions, generation of unconjugated "free" phospho-ubiquitin serves as a proxy readout for PINK1 activity. This has enabled us to find a preconditioning phenomenon, whereby an initial depolarizing treatment leaves a residual pool of active PINK1 that remains competent to seed the activation of nascent cleaved PINK1 following a 16-hour recovery period.
    DOI:  https://doi.org/10.1126/sciadv.adr1938
  22. Commun Biol. 2025 Mar 04. 8(1): 358
    Plant-specific tail-anchored coiled-coil protein MAG3 stabilizes Golgi-associated ERESs to facilitate protein exit from the ER.
    Junpei Takagi, Hideyuki Takahashi, Kenta C Moriya, Minoru Nagano, Yoichiro Fukao, Haruko Ueda, Kentaro Tamura, Tomoo Shimada, Ikuko Hara-Nishimura.
      Endoplasmic reticulum exit sites (ERESs) are ER subdomains where coat protein complex II carriers are assembled for ER-to-Golgi transport. We previously proposed a dynamic capture-and-release model of ERESs by Golgi stacks in plants. However, how ERESs and Golgi stacks maintain a stable interaction in plant cells with vigorous cytoplasmic streaming is unknown. Here, we show that a plant-specific ER transmembrane protein, which we designate as MAG3, plays a crucial role in mediating the capture-and-release of ERESs in Arabidopsis. We isolated a mutant (mag3) defective in protein exit from the ER in seeds. MAG3 localized specifically to the ER-Golgi interface with Golgi-associated ERESs and remained there after ERES release. MAG3 deficiency caused a reduction in the amount of ERESs associated with each Golgi stack. MAG3 interacted with WPP DOMAIN PROTEINs, which are also plant-specific. These results suggest that plants have evolved a unique system to support ER-to-Golgi transport despite intracellular motility.
    DOI:  https://doi.org/10.1038/s42003-025-07602-1
  23. Nat Commun. 2025 Mar 03. 16(1): 2138
    The pathways of secretory cargo export at the endoplasmic reticulum.
    Vivek Malhotra.
      Palade's original model proposed that secretory cargo is transported between stable compartments via vesicles. However, recent findings challenge this view, suggesting that secretory pathway compartments are dynamic, with cargo itself dictating whether transfer occurs via vesicles or through the continuity and maturation of compartmental structures. At the heart of this process is TANGO1, a key component of a molecular machine that works in concert with COPII proteins to construct export routes tailored to the size and quantity of secretory cargo.
    DOI:  https://doi.org/10.1038/s41467-025-57408-2
  24. Mol Cell. 2025 Feb 26. pii: S1097-2765(25)00107-8. [Epub ahead of print]
    A family of bacterial Josephin-like deubiquitinases with an irreversible cleavage mode.
    Thomas Hermanns, Susanne Kolek, Matthias Uthoff, Richard A de Heiden, Monique P C Mulder, Ulrich Baumann, Kay Hofmann.
      Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. This study describes a family of DUBs from several bacterial genera, including Simkania, Parachlamydia, Burkholderia, and Pigmentiphaga, which is structurally related to eukaryotic Josephin-type DUBs but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing this shift and found them to be conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This enzyme class has great potential to serve as tools for studying the ubiquitin system, particularly aspects involving branched chains.
    Keywords:  bacterial effectors; clippases; deubiquitinases; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2025.02.002
  25. Nat Immunol. 2025 Mar 06.
    The integrated stress response pathway controls cytokine production in tissue-resident memory CD4+ T cells.
    Nariaki Asada, Pauline Ginsberg, Hans-Joachim Paust, Ning Song, Jan-Hendrik Riedel, Jan-Eric Turner, Anett Peters, Anna Kaffke, Jonas Engesser, Huiying Wang, Yu Zhao, Robin Khatri, Philipp Gild, Roland Dahlem, Björn-Philipp Diercks, Sarada Das, Zoya Ignatova, Tobias B Huber, Immo Prinz, Nicola Gagliani, Hans-Willi Mittrücker, Christian F Krebs, Ulf Panzer.
      Tissue-resident memory T (TRM) cells are a specialized T cell population that reside in tissues and provide a rapid protective response upon activation. Here, we showed that human and mouse CD4+ TRM cells existed in a poised state and stored messenger RNAs encoding proinflammatory cytokines without protein production. At steady state, cytokine mRNA translation in TRM cells was suppressed by the integrated stress response (ISR) pathway. Upon activation, the central ISR regulator, eIF2α, was dephosphorylated and stored cytokine mRNA was translated for immediate cytokine production. Genetic or pharmacological activation of the ISR-eIF2α pathway reduced cytokine production and ameliorated autoimmune kidney disease in mice. Consistent with these results, the ISR pathway in CD4+ TRM cells was downregulated in patients with immune-mediated diseases of the kidney and the intestine compared to healthy controls. Our results indicated that stored cytokine mRNA and translational regulation in CD4+ TRM cells facilitate rapid cytokine production during local immune response.
    DOI:  https://doi.org/10.1038/s41590-025-02105-x
  26. ACS Omega. 2025 Feb 25. 10(7): 6650-6662
    Molecular Glue-Design-Evaluator (MOLDE): An Advanced Method for In-Silico Molecular Glue Design.
    A S Ben Geoffrey, Deepak Agrawal, Nagaraj M Kulkarni, Manonmani Gunasekaran.
      Protein function modulation using small-molecule binding is an important therapeutic strategy for many diseases. However, many proteins remain undruggable due to the lack of suitable binding pockets for small-molecule binding. Proximity-induced protein degradation using molecular glues has recently been identified as an important strategy to target undruggable proteins. Molecular glues were discovered serendipitously and as such currently lack an established approach for in-silico-driven rationale design. In this work, we aim to establish an in-silico method for designing molecular glues. To achieve this, we leverage known molecular glue-mediated ternary complexes and derive a rationale for the in-silico design of molecular glues. Establishing an in-silico rationale for molecular glue design would significantly contribute to the literature and accelerate the discovery of molecular glues for targeting previously undruggable proteins. Our work presented here and named Molecular Glue-Designer-Evaluator (MOLDE) contributes to the growing literature of in-silico approaches to drug design in-silico literature.
    DOI:  https://doi.org/10.1021/acsomega.4c08049
  27. J Mol Biol. 2025 Feb 28. pii: S0022-2836(25)00118-4. [Epub ahead of print] 169052
    A BiP-centric view of endoplasmic reticulum functions and of my career.
    Linda M Hendershot.
      After completing my post-doctoral training at the University of Alabama, Birmingham and a brief period on the faculty there, I joined the Department of Tumor Cell Biology at St. Jude Children's Research Hospital in 1987 as an Assistant Member and started my independent research program. For the following 37 years, I led a relatively small basic research group comprised at various times of post-doctoral fellows, graduate students, undergraduate students, and research technicians; many of whom I am still in contact. Last year I closed the lab and transitioned to an emeritus position at St. Jude. I continue to maintain several research collaborations covering areas of research that have long been dear to my heart. My post-doctoral studies on BiP revealed that it controlled immunoglobulin assembly and transport, and as such, played a critical role in fidelity of the immune response. My lab continued to define BiP's functions in protein folding and subunit assembly, as well as, in degradation using biochemical, cell-based, and biophysical analyses. Several ER localized co-factors that regulate the activity of BiP and allow it to contribute to its multiple ER functions were identified by our group. These include DnaJ family members and nucleotide change factors. Through a variety of collaborative studies, we pursued BiP's functions in maintaining the permeability barrier of the translocon, contributing to ER calcium stores, and regulating the up-stream transducers of the UPR, a stress response that is activated by the accumulation of unfolded proteins in the ER.
    Keywords:  ER stress; Endoplasmic Reticulum; Immunoglobulins; Molecular chaperones; Quality Control of Protein Maturation; Secretory pathway protein biosynthesis; UPR
    DOI:  https://doi.org/10.1016/j.jmb.2025.169052
  28. J Biol Chem. 2025 Feb 27. pii: S0021-9258(25)00214-5. [Epub ahead of print] 108365
    Substrate Recognition and Cleavage-Site Preferences of Lon Protease.
    Melanie Cragan, Neha Puri, A Wali Karzai.
      The evolutionarily conserved AAA+ Lon protease plays a pivotal role in protein homeostasis by precisely remodeling the proteome and specifically removing unfolded, damaged, and surplus natively folded regulatory proteins. Proteolysis by Lon comprises the three fundamental stages of substrate recognition via specific amino acid sequence motifs (degrons), ATP-fueled substrate unfolding and translocation into a sequestered proteolytic chamber, and cleavage of the translocated polypeptide by the peptidase domain. Although a plethora of Lon substrates have been identified in several bacterial species, broadly applicable rules that govern recognition of numerous substrates, and hence the ability to de novo identify new Lon substrates and regulatory pathways, has lagged behind. Similarly, cleavage-site preferences of Lon proteases, and whether these crucial enzymes from diverse bacterial species share similar preferences, has remained underexplored. In this study, we report the identification and characterization of a class of high-affinity autonomous C-terminal Yersinia Pestis (yp) Lon recognition degrons, variants of which are present in numerous known and new yp-Lon substrates and broadly distributed in diverse bacterial species. Moreover, the identification of this degron group offers the predictive power to discover new Lon substrates in eubacteria. Furthermore, cleavage-site preference analyses of multiple Lon substrates reveal that the Lon peptidase domain preferentially cleaves translocated polypeptides after Phenylalanine residues to generate peptides that range from 7 - 35 residues, with an average length of 11 residues, a general feature conserved amongst Lon proteases from phylogenetically distinct bacterial species.
    Keywords:  ATPases associated with diverse cellular activities (AAA+); Lon protease; Protein homeostasis; Proteolysis; Substrate specificity
    DOI:  https://doi.org/10.1016/j.jbc.2025.108365
  29. Cell Syst. 2025 Feb 21. pii: S2405-4712(25)00035-3. [Epub ahead of print] 101202
    Virus targeting as a dominant driver of interfacial evolution in the structurally resolved human-virus protein-protein interaction network.
    Wan-Chun Su, Yu Xia.
      Regions on a host protein that interact with virus proteins (exogenous interfaces) frequently overlap with those that interact with other host proteins (endogenous interfaces), resulting in competition between hosts and viruses for these shared interfaces (mimic-targeted interfaces). Yet, the evolutionary consequences of this competitive relationship on the host are not well understood. Here, we integrate experimentally determined structures and homology-based templates of protein complexes with protein-protein interaction networks to construct a high-resolution human-virus structural interaction network. We perform site-specific evolutionary rate analyses on this structural interaction network and find that exogenous-specific interfaces evolve faster than endogenous-specific interfaces. Mimic-targeted interfaces evolve as fast as exogenous-specific interfaces, despite being targeted by both human and virus proteins. Our findings suggest that virus targeting plays a dominant role in host interfacial evolution within the context of domain-domain interactions and that mimic-targeted interfaces on human proteins are the key battleground for a mammalian-specific host-virus evolutionary arms race.
    Keywords:  evolutionary arms race; evolutionary battleground; host-virus coevolution; host-virus interactions; interface mimicry; interfacial evolution; protein-protein interactions; residue-level evolutionary rates; structural biology; systems biology
    DOI:  https://doi.org/10.1016/j.cels.2025.101202
  30. PNAS Nexus. 2025 Mar;4(3): pgaf033
    How pore formation in complex biological membranes is governed by lipid composition, mechanics, and lateral sorting.
    Leonhard J Starke, Christoph Allolio, Jochen S Hub.
      The primary function of biological membranes is to enable compartmentalization among cells and organelles. Loss of integrity by the formation of membrane pores would trigger uncontrolled depolarization or influx of toxic compounds, posing a fatal threat to living cells. How the lipid complexity of biological membranes enables mechanical stability against pore formation while, simultaneously, allowing for ongoing membrane remodeling is largely enigmatic. We performed molecular dynamics simulations of eight complex lipid membranes including the plasma membrane and membranes of the organelles endoplasmic reticulum, Golgi, lysosome, and mitochondrion. To quantify the mechanical stability of these membranes, we computed the free energy of transmembrane pore nucleation as well as the line tension of the rim of open pores. Our simulations reveal that complex biological membranes are remarkably stable, however, with the plasma membrane standing out as exceptionally stable, which aligns with its crucial role as a protective layer. We observe that sterol content is a key regulator for biomembrane stability, and that lateral sorting among lipid mixtures influences the energetics of membrane pores. A comparison of 25 model membranes with varying sterol content, tail length, tail saturation, and head group type shows that the pore nucleation free energy is mostly associated with the lipid tilt modulus, whereas the line tension along the pore rim is determined by the lipid intrinsic curvature. Together, our study provides an atomistic and energetic view on the role of lipid complexity in biomembrane stability.
    Keywords:  Helfrich theory; lipid membranes; molecular dynamics simulations; pore formation
    DOI:  https://doi.org/10.1093/pnasnexus/pgaf033
  31. Cell. 2025 Feb 21. pii: S0092-8674(25)00109-6. [Epub ahead of print]
    RNA-binding proteins and glycoRNAs form domains on the cell surface for cell-penetrating peptide entry.
    Jonathan Perr, Andreas Langen, Karim Almahayni, Gianluca Nestola, Peiyuan Chai, Charlotta G Lebedenko, Regan F Volk, Diego Detrés, Reese M Caldwell, Malte Spiekermann, Helena Hemberger, Namita Bisaria, Toshihiko Aiba, Francisco J Sánchez-Rivera, Konstantinos Tzelepis, Eliezer Calo, Leonhard Möckl, Balyn W Zaro, Ryan A Flynn.
      The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA-binding proteins (RBPs) is present on the surface of living cells. These cell-surface RBPs (csRBPs) precisely organize into well-defined nanoclusters enriched for multiple RBPs and glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell-surface interaction for the cell-penetrating peptide trans-activator of transcription (TAT). Removal of RNA from the cell surface, or loss of RNA-binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.
    Keywords:  RNA-binding proteins; cell surface; cell-penetrating peptides; glycoRNA
    DOI:  https://doi.org/10.1016/j.cell.2025.01.040
  32. Cell Rep. 2025 Feb 26. pii: S2211-1247(25)00124-X. [Epub ahead of print]44(3): 115353
    Custom affinity probes reveal DNA-damage-induced, ssDNA-independent chromatin SUMOylation in budding yeast.
    Vera Tröster, Ronald P Wong, Arne Börgel, Baris Cakilkaya, Christian Renz, Martin M Möckel, Karolin Eifler-Olivi, Joana Marinho, Thomas Reinberg, Sven Furler, Jonas V Schaefer, Andreas Plückthun, Eva Wolf, Helle D Ulrich.
      The small ubiquitin-related modifier SUMO regulates cellular processes in eukaryotes either by modulating individual protein-protein interactions or with relaxed substrate selectivity by group modification. Here, we report the isolation and characterization of designed ankyrin repeat protein (DARPin)-based affinity probes directed against budding yeast SUMO (Smt3). We validate selected DARPins as compartment-specific inhibitors or neutral detection agents. Structural characterization reveals a recognition mode distinct from that of natural SUMO interactors. In vivo application pinpoints Smt3's essential function to the nucleus and demonstrates DARPin-mediated sensitization toward various stress conditions. A subset of selected clones is validated as SUMOylation reporters in cells. In this manner, we identify a DNA-damage-induced nuclear SUMOylation response that-in contrast to previously reported chromatin group SUMOylation-is independent of single-stranded DNA and the SUMO-E3 Siz2 but depends on Mms21 and likely reflects late intermediates of homologous recombination. Thus, Smt3-specific DARPins can provide insight into the dynamics of SUMOylation in defined subcellular structures.
    Keywords:  CP: Molecular biology; DARPin; DNA repair; SUMO-SIM interaction; SUMOylation; affinity probe; chromatin; genome stability; group SUMOylation; homologous recombination
    DOI:  https://doi.org/10.1016/j.celrep.2025.115353
  33. Cell Rep. 2025 Feb 27. pii: S2211-1247(25)00119-6. [Epub ahead of print]44(3): 115348
    IDR-driven TOLLIP condensates antagonize the innate antiviral immunity by promoting the deSUMOylation of MAVS.
    Jinxiu Hou, Shengnan Zheng, Xuejing Zhang, Mengwei Zhuang, Xianghe Zhao, Jian Deng, Huiyu Yang, Xiaojing Xia, Chengjiang Gao, Pei-Hui Wang, Yi Zheng.
      Mitochondrial antiviral signaling protein (MAVS) is a central adaptor protein in retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling against RNA viral infection. Posttranslational modifications (PTMs) play a critical role in modulating the activity of MAVS. However, how phase separation regulates the PTMs to fine-tune MAVS activation remains to be elucidated. In this study, we identify Toll-interacting protein (TOLLIP) as a negative regulator of RLR signaling. A deficiency of TOLLIP leads to an enhanced type I interferon response upon RNA viral infection. Mice with the deletion of TOLLIP are more resistant to lethal vesicular stomatitis virus (VSV) infection than wild-type counterparts. Mechanistically, TOLLIP forms condensates that rely on its intrinsically disordered region (IDR). TOLLIP condensates interact with SENP1, promote the aggregation of SENP1, and enhance the interaction between SENP1 and MAVS, consequently leading to deSUMOylation and less aggregation of MAVS. Overall, our study reveals the critical role of TOLLIP condensation in regulating the activation of MAVS, emphasizing the complexity of MAVS activity modulation.
    Keywords:  CP: Immunology; CP: Molecular biology; IDR; MAVS; SUMOylation; TOLLIP; antiviral innate immunity; phase separation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115348
  34. iScience. 2025 Mar 21. 28(3): 111946
    Alterations in ether phospholipids metabolism activate the conserved UPR-Xbp1- PDIA3/ERp60 signaling to maintain intestinal homeostasis.
    Stephanie Makdissi, Rihab Loudhaief, Smitha George, Tabatha Weller, Minna Salim, Ahsan Malick, Yizhu Mu, Brendon D Parsons, Francesca Di Cara.
      Intestinal epithelium regeneration and homeostasis must be tightly regulated. Alteration of epithelial homeostasis is a major contributing factor to diseases such as colorectal cancer and inflammatory bowel diseases. Many pathways involved in epithelial regeneration have been identified, but more regulators remain undiscovered. Metabolism has emerged as an overlooked regulator of intestinal epithelium homeostasis. Using the model organism Drosophila melanogaster, we found that ether lipids metabolism is required to maintain intestinal epithelial homeostasis. Its dysregulation in intestinal progenitors causes the activation of the unfolded protein response of the endoplasmic reticulum (UPR) that triggers Xbp1 and upregulates the conserved disulfide isomerase PDIA3/ERp60. Activation of the Xbp1-ERp60 signaling causes Jak/Stat-mediated increase in progenitor cells, compromising epithelial barrier function and survival in males but not females. This study identified ether lipids-PDIA3/ERp60 as a key regulator of intestinal progenitor homeostasis in health that, if altered, causes pathological conditions in the intestinal epithelium.
    Keywords:  Cell biology; Lipidomics; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2025.111946
  35. Nat Rev Drug Discov. 2025 Feb 28.
    Induced proximity pushes beyond protein degraders, as first RIPTAC moves into the clinic.
    Asher Mullard.
      
    DOI:  https://doi.org/10.1038/d41573-025-00037-7
  36. Science. 2025 Mar 07. 387(6738): 1063-1068
    Macroevolutionary divergence of gene expression driven by selection on protein abundance.
    Alexander L Cope, Joshua G Schraiber, Matt Pennell.
      The regulation of messenger RNA (mRNA) and protein abundances is well-studied, but less is known about the evolutionary processes shaping their relationship. To address this, we derived a new phylogenetic model and applied it to multispecies mammalian data. Our analyses reveal (i) strong stabilizing selection on protein abundances over macroevolutionary time, (ii) mutations affecting mRNA abundances minimally impact protein abundances, (iii) mRNA abundances evolve under selection to align with protein abundances, and (iv) mRNA abundances adapt faster than protein abundances owing to greater mutational opportunity. These conclusions are supported by comparisons of model parameters with independent functional genomic data. By decomposing mutational and selective influences on mRNA-protein dynamics, our approach provides a framework for discovering the evolutionary rules that drive divergence in gene expression.
    DOI:  https://doi.org/10.1126/science.ads2658
  37. Sci Adv. 2025 Mar 07. 11(10): eadr8146
    Differential regulation of BAX and BAK apoptotic activity revealed by small molecules.
    Kaiming Li, Yu Q Yap, Donia M Moujalled, Fransisca Sumardy, Yelena Khakham, Angela Georgiou, Michelle Jahja, Thomas E Lew, Melanie De Silva, Meng-Xiao Luo, Jia-Nan Gong, Zheng Yuan, Richard W Birkinshaw, Peter E Czabotar, Kym Lowes, David C S Huang, Benjamin T Kile, Andrew H Wei, Grant Dewson, Mark F van Delft, Guillaume Lessene.
      Defective apoptosis mediated by B cell lymphoma 2 antagonist/killer (BAK) or B cell lymphoma 2-associated X protein (BAX) underlies various pathologies including autoimmune and degenerative conditions. On mitochondria, voltage-dependent anion channel 2 (VDAC2) interacts with BAK and BAX through a common interface to inhibit BAK or to facilitate BAX apoptotic activity. We identified a small molecule (WEHI-3773) that inhibits interaction between VDAC2 and BAK or BAX revealing contrasting effects on their apoptotic activity. WEHI-3773 inhibits apoptosis mediated by BAX by blocking VDAC2-mediated BAX recruitment to mitochondria. Conversely, WEHI-3773 promotes BAK-mediated apoptosis by limiting inhibitory sequestration by VDAC2. In cells expressing both pro-apoptotic proteins, apoptosis promotion by WEHI-3773 dominates, because activated BAK activates BAX through a feed-forward mechanism. Loss of BAX drives resistance to the BCL-2 inhibitor venetoclax in some leukemias. WEHI-3773 overcomes this resistance by promoting BAK-mediated killing. This work highlights the coordination of BAX and BAK apoptotic activity through interaction with VDAC2 that may be targeted therapeutically.
    DOI:  https://doi.org/10.1126/sciadv.adr8146
  38. J Cell Biol. 2025 Apr 07. pii: e202407110. [Epub ahead of print]224(4):
    Cytoplasmic ribosomes on mitochondria alter the local membrane environment for protein import.
    Ya-Ting Chang, Benjamin A Barad, Juliette Hamid, Hamidreza Rahmani, Brian M Zid, Danielle A Grotjahn.
      Most of the mitochondria proteome is nuclear-encoded, synthesized by cytoplasmic ribosomes, and targeted to the mitochondria posttranslationally. However, a subset of mitochondrial-targeted proteins is imported co-translationally, although the molecular mechanisms governing this process remain unclear. We employ cellular cryo-electron tomography to visualize interactions between cytoplasmic ribosomes and mitochondria in Saccharomyces cerevisiae. We use surface morphometrics tools to identify a subset of ribosomes optimally oriented on mitochondrial membranes for protein import. This allows us to establish the first subtomogram average structure of a cytoplasmic ribosome at the mitochondrial surface in the native cellular context, which showed three distinct connections with the outer mitochondrial membrane surrounding the peptide exit tunnel. Further, this analysis demonstrated that cytoplasmic ribosomes primed for mitochondrial protein import cluster on the outer mitochondrial membrane at sites of local constrictions of the outer and inner mitochondrial membranes. Overall, our study reveals the architecture and the spatial organization of cytoplasmic ribosomes at the mitochondrial surface, providing a native cellular context to define the mechanisms that mediate efficient mitochondrial co-translational protein import.
    DOI:  https://doi.org/10.1083/jcb.202407110
  39. Cancer Discov. 2025 Mar 10.
    Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity in breast cancer.
    Aviad Ben-Shmuel, Yael Gruper, Coral Halperin, Oshrat Levi-Galibov, Hallel Rosenberg-Fogler, Debra Barki, Giulia Carradori, Yaniv Stein, Gal Yagel, Mariia Naumova, Shimrit Mayer, Maya Dadiani, Dana Morzaev-Sulzbach, Ofra Golani, Reinat Nevo, Ziv Porat, Einav Nili Gal-Yam, Ruth Scherz-Shouval.
      Cancer-associated fibroblasts (CAFs) are abundant components of the breast tumor microenvironment and major contributors to immune-modulation. CAFs regulate the activity of many immune cells including T-cells, macrophages and dendritic cells, however little is known about their interaction with natural killer (NK) cells, which constitute an important arm of anti-tumor immunity. Using mouse models of breast cancer and ex-vivo co-cultures, we find that CAFs inhibit NK cell cytotoxicity towards cancer cells. We unravel the mechanism by which suppression occurs, through ligand-receptor engagement between NK cells and CAFs, leading to CAF cytolysis and downregulation of activating receptor expression on NK cells, promoting cancer cell escape from NK cell surveillance. In triple negative breast cancer patients, we find enrichment of NK cells in CAF-rich regions, and upregulation of NK binding ligands on CAFs which correlates with poor disease outcome. These results reveal a CAF-mediated immunosuppressive decoy mechanism with implications for treatment of carcinomas.
    DOI:  https://doi.org/10.1158/2159-8290.CD-24-0131
  40. STAR Protoc. 2025 Mar 05. pii: S2666-1667(25)00072-3. [Epub ahead of print]6(1): 103666
    Protocol for multi-component reconstitution of stress granules in vitro.
    Zhiying Yao, Yi Liu, Peiguo Yang.
      To date, stress granule studies mainly focus on cell models. The diversity of molecules in stress granules makes it challenging to uncover the function of each molecule in stress granule regulation. Here, we provide a protocol to reconstitute stress granules with multi-components in vitro. We describe steps for strain amplification, protein purification, and liquid-liquid phase separation (LLPS). The multi-component reconstitution system constructed in this protocol also provides a technique for other condensate reconstitution studies in vitro. For complete details on the use and execution of this protocol, please refer to Yao et al.1.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.xpro.2025.103666
  41. bioRxiv. 2025 Feb 17. pii: 2025.02.12.637979. [Epub ahead of print]
    Mapping the nanoscale organization of the human cell surface proteome reveals new functional associations and surface antigen clusters.
    Brendan M Floyd, Elizabeth L Schmidt, Nicholas A Till, Jonathan L Yang, Pinyu Liao, Benson M George, Ryan A Flynn, Carolyn R Bertozzi.
      The cell surface is a dynamic interface that controls cell-cell communication and signal transduction relevant to organ development, homeostasis and repair, immune reactivity, and pathologies driven by aberrant cell surface phenotypes. The spatial organization of cell surface proteins is central to these processes. High-resolution fluorescence microscopy and proximity labeling have advanced studies of surface protein associations, but the spatial organization of the complete surface proteome remains uncharted. In this study, we systematically mapped the surface proteome of human T-lymphocytes and B-lymphoblasts using proximity labeling of 85 antigens, identified from over 100 antibodies tested for binding to surface-exposed proteins. These experiments were coupled with an optimized data-independent acquisition mass spectrometry workflow to generate a robust dataset. Unsupervised clustering of the resulting interactome revealed functional modules, including well-characterized complexes such as the T-cell receptor and HLA class I/II, alongside novel clusters. Notably, we identified mitochondrial proteins localized to the surface, including the transcription factor TFAM, suggesting previously unappreciated roles for mitochondrial proteins at the plasma membrane. A high-accuracy machine learning classifier predicted over 6,000 surface protein associations, highlighting functional associations such as IL10RB's role as a negative regulator of type I interferon signaling. Spatial modeling of the surface proteome provided insights into protein dispersion patterns, distinguishing widely distributed proteins, such as CD45, from localized antigens, such as CD226 pointing to active mechanisms of regulating surface organization. This work provides a comprehensive map of the human surfaceome and a resource for exploring the spatial and functional dynamics of the cell membrane proteome.
    DOI:  https://doi.org/10.1101/2025.02.12.637979
  42. Sci Adv. 2025 Mar 07. 11(10): eadq1047
    TBK1 and IKKε prevent premature cell death by limiting the activity of both RIPK1 and NLRP3 death pathways.
    Fabian A Fischer, Benjamin Demarco, Felicia Chan Hui Min, Hui Wen Yeap, Dominic De Nardo, Kaiwen W Chen, Jelena S Bezbradica.
      The loss of TBK1, or both TBK1 and the related kinase IKKε, results in uncontrolled cell death-driven inflammation. Here, we show that the pathway leading to cell death depends on the nature of the activating signal. Previous models suggest that in steady state, TBK1/IKKε-deficient cells die slowly and spontaneously predominantly by uncontrolled tumor necrosis factor-RIPK1-driven death. However, upon infection of cells that express the NLRP3 inflammasome, (e.g., macrophages), with pathogens that activate this pathway (e.g., Listeria monocytogenes), TBK1/IKKε-deficient cells die rapidly, prematurely, and exclusively by enhanced NLRP3-driven pyroptosis. Even infection with the RIPK1-activating pathogen, Yersinia pseudotuberculosis, results in enhanced RIPK1-caspase-8 activation and enhanced secondary NLRP3 activation. Mechanistically, TBK1/IKKε control endosomal traffic, and their loss disrupts endosomal homeostasis, thereby signaling cell stress. This results in premature NLRP3 activation even upon sensing "signal 2" alone, without the obligatory "signal 1." Collectively, TBK1/IKKε emerge as a central brake in limiting death-induced inflammation by both RIPK1 and NLRP3 death-inducing pathways.
    DOI:  https://doi.org/10.1126/sciadv.adq1047
  43. Commun Biol. 2025 Mar 05. 8(1): 373
    AFsample2 predicts multiple conformations and ensembles with AlphaFold2.
    Yogesh Kalakoti, Björn Wallner.
      Understanding protein dynamics and conformational states is crucial for insights into biological processes and disease mechanisms, which can aid drug development. Recently, several methods have been devised to broaden the conformational predictions made by AlphaFold2 (AF2). We introduce AFsample2, a method using random MSA column masking to reduce co-evolutionary signals, enhancing structural diversity in AF2-generated models. AFsample2 effectively predicts alternative states for various proteins, producing high-quality end states and diverse conformational ensembles. In the OC23 dataset, alternate state models improved (ΔTM>0.05) in 9 out of 23 cases without affecting preferred state generation. Similar results were seen in 16 membrane protein transporters, with 11 out of 16 targets showing improvement. TM-score improvements to experimental end states were substantial, sometimes exceeding 50%, improving from 0.58 to 0.98. Additionally, AFsample2 increased the diversity of intermediate conformations by 70% compared to standard AF2, producing highly confident models potentially representing intermediate states. For four targets, predicted intermediate states were structurally similar to known structural homologs in the PDB, suggesting that they are true intermediate states. These findings indicate that AFsample2 can used to provide structural insights into proteins with multiple states, as well as potential paths between the states.
    DOI:  https://doi.org/10.1038/s42003-025-07791-9
  44. Nature. 2025 Mar 05.
    Cell-autonomous innate immunity by proteasome-derived defence peptides.
    Karin Goldberg, Arseniy Lobov, Paola Antonello, Merav D Shmueli, Idan Yakir, Tal Weizman, Adi Ulman, Daoud Sheban, Einav Laser, Matthias P Kramer, Ronen Shteinvil, Guoyun Chen, Angham Ibraheem, Vera Sysoeva, Vered Fishbain-Yoskovitz, Gayatree Mohapatra, Anat Abramov, Sandy Shimshi, Kseniia Ogneva, Madhurima Nandy, Sivan Amidror, Hadar Bootz-Maoz, Shanny H Kuo, Nili Dezorella, Assaf Kacen, Aaron Javitt, Gee W Lau, Nissan Yissachar, Zvi Hayouka, Yifat Merbl.
      For decades, antigen presentation on major histocompatibility complex class I for T cell-mediated immunity has been considered the primary function of proteasome-derived peptides1,2. However, whether the products of proteasomal degradation play additional parts in mounting immune responses remains unknown. Antimicrobial peptides serve as a first line of defence against invading pathogens before the adaptive immune system responds. Although the protective function of antimicrobial peptides across numerous tissues is well established, the cellular mechanisms underlying their generation are not fully understood. Here we uncover a role for proteasomes in the constitutive and bacterial-induced generation of defence peptides that impede bacterial growth both in vitro and in vivo by disrupting bacterial membranes. In silico prediction of proteome-wide proteasomal cleavage identified hundreds of thousands of potential proteasome-derived defence peptides with cationic properties that may be generated en route to degradation to act as a first line of defence. Furthermore, bacterial infection induces changes in proteasome composition and function, including PSME3 recruitment and increased tryptic-like cleavage, enhancing antimicrobial activity. Beyond providing mechanistic insights into the role of proteasomes in cell-autonomous innate immunity, our study suggests that proteasome-cleaved peptides may have previously overlooked functions downstream of degradation. From a translational standpoint, identifying proteasome-derived defence peptides could provide an untapped source of natural antibiotics for biotechnological applications and therapeutic interventions in infectious diseases and immunocompromised conditions.
    DOI:  https://doi.org/10.1038/s41586-025-08615-w
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