bims-proteo Biomed News
on Proteostasis
Issue of 2025–09–21
fifty papers selected by
Eric Chevet, INSERM
  1. Polyubiquitin architecture editing on collided ribosomes maintains persistent RQC activity.
  2. Processing bodies promote lysosomal quality control and cell survival during recovery from lysosomal damage.
  3. Mutation-induced filaments of folded proteins are inert and non-toxic in a cellular system.
  4. Aggregate fragmentation: the ticket to aggrephagy.
  5. A role for CASM in the repair of damaged Golgi architecture.
  6. Endoplasmic reticulum junctions serve as a platform for endosome-lysosome interactions through their stop-and-go motion switching.
  7. Discovery of BRD9 Molecular Glue Degraders That Spare Cardiomyocytes.
  8. Covariation MS uncovers a protein that controls cysteine catabolism.
  9. ATG8ylation-orchestrated vacuolar membrane remodeling facilitates plant alkaline stress tolerance.
  10. The human ribosome modulates multidomain protein biogenesis by delaying cotranslational domain docking.
  11. Two microbiome metabolites compete for tRNA modification to impact mammalian cell proliferation and translation quality control.
  12. Pathogenic phosphorylation of linear ubiquitin machinery causes inflammasome sensor degradation.
  13. Cellular mechanism linking endoplasmic reticulum inheritance and cell cycle regulation of the nuclear genome.
  14. Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation.
  15. Focus on ubiquitylation and protein degradation.
  16. Cryo-EM Reveals Regulatory Mechanisms Governing Substrate Selection and Activation of Human LONP1.
  17. SCAMP5 regulates AP-4-dependent sorting and trafficking of ATG9A for presynaptic autophagy via PI4KB/PI4KIIIβ recruitment and PtdInsP4 production at the TGN.
  18. InsP3R signaling mediates mitochondrial stress-induced longevity through actomyosin-dependent mitochondrial dynamics.
  19. STK4 inhibits the E3 activity of HOIP by phosphorylating its allosteric ubiquitin-binding site.
  20. The serine protease HTRA1 targets Tau fibrils and provides a proteolytic barrier against pathogenic protein conformations.
  21. ATG16L strikes again! New findings link lysosome stress and physiology.
  22. More 26S and 30S proteasomes are beneficial in proteinopathy.
  23. Protocol for in vivo BONCAT labeling of nascent proteins in Xenopus laevis tadpole brains with high temporal precision for quantitative analysis.
  24. Atg18 facilitates autophagosome formation via its Atg8-interacting motif in Saccharomyces cerevisiae.
  25. Astrocytes mobilize a broader repertoire of lysosomal repair mechanisms than neurons.
  26. Dynamic recruitment of Munc13 primes docked secretory granules for exocytosis.
  27. A simple, fast, and cost-efficient protocol for ultra-sensitive ribosome profiling.
  28. SMURF2 inhibits autophagic control of Mycobacterium tuberculosis in macrophages.
  29. Chemical Proteomics-Guided Discovery of Covalent Ligands for Cancer Proteins.
  30. Neurons and astrocytes have distinct organelle signatures and responses to stress.
  31. Phase separation promotes Atg8 lipidation and vesicle condensation for autophagy progression.
  32. MondoA mediates transcriptional coordination between the MYC network and the integrated stress response in pancreatic ductal adenocarcinoma.
  33. Activity-dependent localization and dynamics of STIM1 and STIM2 at ER-PM contacts in hippocampal neurons.
  34. A cytokine receptor-targeting chimera (kineTAC) toolbox for expanding extracellular targeted protein degradation.
  35. A widespread family of molecular chaperones promotes the intracellular stability of type VIIb secretion system-exported toxins.
  36. tDR-quant: a reliable electroporation-based approach for quantifying tRNA-derived fragments binding to ribosomes.
  37. The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking.
  38. NMR Insights Into Stress-Induced Modulation of the Monomer-Dimer Equilibrium in a Small Heat Shock Protein.
  39. Organelle activity organized by the endoplasmic reticulum-mitochondria encounter structure -ERMES- is essential for Podospora anserina development.
  40. STING signals to NF-κB from late endolysosomal compartments using IRF3 as an adaptor.
  41. High-throughput diversification of protein-ligand surfaces to discover chemical inducers of proximity.
  42. Unfolded protein response signaling promotes myeloid cell production and cooperates with oncogenic mutation.
  43. Linear ubiquitination prevents lipodystrophy and obesity-associated metabolic syndrome.
  44. Cysteine redoxome landscape in the liver of male mice fed a high-fat high-sucrose diet.
  45. OGFOD1 enables AML chemo- and nutrient stress resistance by regulating protein synthesis.
  46. Regulation of host immunity by a novel Legionella pneumophila E3 ubiquitin ligase.
  47. ERAD machinery controls the conditional turnover of PIN-LIKES in plants.
  48. Nanoscale Structural and Functional Impacts of Disease-Associated Collagen Mutations.
  49. Network-driven discovery of repurposable drugs targeting hallmarks of aging.
  50. Non-canonical role of ATG4B in PRMT1-mediated DNA repair and leukemia progression.
  1. EMBO J. 2025 Sep 16.
    Polyubiquitin architecture editing on collided ribosomes maintains persistent RQC activity.
    Shota Tomomatsu, Yoshitaka Matsuo, Fumiaki Ohtake, Takuya Tomita, Yasushi Saeki, Toshifumi Inada.
      In ribosome-associated quality control (RQC), K63-linked polyubiquitination of ribosomal protein uS10 on the stalled ribosome is crucial for recruiting the RQC-trigger (RQT) complex. However, the mechanisms governing the maintenance and recycling of polyubiquitin architecture on colliding ribosomes remain unclear. Here we demonstrate that two deubiquitinating enzymes (DUBs), Ubp2 and Ubp3, play key roles in editing and recycling polyubiquitin chains on yeast uS10, thereby contributing to the promotion of RQC activity. Specifically, Ubp2 eliminates K63-linked polyubiquitin chains from uS10 on the free 40S subunit for recycling, while Ubp3 predominantly cleaves K48-linked di-ubiquitin and K48/K63-mixed-linkage polyubiquitin chains from uS10 on the translating ribosomes. We further demonstrate that K48-linkage-containing ubiquitin chains on uS10 of the colliding ribosome act as a negative signal for the RQT-mediated ribosome dissociation process. Collectively, our findings provide insight into the ubiquitin code in RQC, and define positive functions of two DUBs in maintaining persistent RQC activity.
    Keywords:  Deubiquitinase; Quality Control; Ribosome; Ubiquitin Code; Ubiquitination
    DOI:  https://doi.org/10.1038/s44318-025-00568-0
  2. Res Sq. 2025 Sep 09. pii: rs.3.rs-7474186. [Epub ahead of print]
    Processing bodies promote lysosomal quality control and cell survival during recovery from lysosomal damage.
    Jingyue Cassano, Jacob Duran, Li Chen, Jing Pu, Pavel Ivanov.
      Lysosomes are essential for cell survival but are highly susceptible to diverse physical and pathological stressors. Thus, the ability to initiate an acute damage response and promote recovery after stressor resolution is critical for maintaining cellular homeostasis and viability. Although recent studies have advanced our understanding of acute responses to lysosomal injury, the molecular mechanisms governing the recovery stage and distinguishing it from the acute phase remain poorly defined. Here, we delineate a key difference between these two stages in translational regulation and uncover lysosomal recovery from acute damage as a novel trigger for processing body (PB) formation. PBs are membraneless biomolecular condensates involved in RNA metabolism and translational reprogramming. We provide the first evidence that PBs are critical for lysosomal quality control and cell survival during recovery. Mechanistically, PBs are induced selectively during the recovery phase, but not during the acute damage response, through interactions with stress granules (SGs), distinct membraneless biomolecular condensates formed upon acute injury to stabilize damaged lysosomal membranes for repair. Functional analyses reveal that PBs promote lysosomal quality control by collaborating with SG-mediated membrane stabilization, while independently recruiting released cathepsins, thereby collectively supporting cell survival. Together, these findings establish PBs as central effectors of the lysosomal recovery program and underscore the broader relevance of biomolecular condensates in cellular responses to lysosomal damage and related disease processes.
    DOI:  https://doi.org/10.21203/rs.3.rs-7474186/v1
  3. Mol Syst Biol. 2025 Sep 15.
    Mutation-induced filaments of folded proteins are inert and non-toxic in a cellular system.
    Tal Levin, Hector Garcia-Seisdedos, Arseniy Lobov, Matthias Wojtynek, Alexander Alexandrov, Ghil Jona, Dikla Levi, Ohad Medalia, Emmanuel D Levy.
      Filamentous protein assemblies are essential for cellular functions but can also form aberrantly through mutations that induce self-interactions between folded protein subunits. These assemblies, which we refer to as agglomerates, differ from aggregates and amyloids that arise from protein misfolding. While cells have quality control mechanisms to identify, buffer, and eliminate aggregates, it is unknown whether similar mechanisms exist for agglomerates. Here, we define and characterize this distinct class of assemblies formed by the polymerization of folded proteins. To systematically assess their cellular impact, we developed a simple in-cell assay that distinguishes agglomerates from aggregates based on co-assembly with wild-type subunits. Unlike misfolded aggregates, we show that agglomerates retain their folded state, do not colocalize with the proteostasis machinery, and are not ubiquitinated. Moreover, agglomerates cause no detectable growth defects. Quantitative proteomics also revealed minor changes in protein abundance in cells expressing agglomerates. These results position agglomerates as a structurally and functionally distinct class of protein assemblies that are largely inert in cells, highlighting their potential as building blocks for intracellular engineering and synthetic biology.
    Keywords:  Cell Fitness; Protein Filamentation; Protein Self-assembly; Proteome; Yeast Biology
    DOI:  https://doi.org/10.1038/s44320-025-00144-y
  4. Autophagy. 2025 Sep 17.
    Aggregate fragmentation: the ticket to aggrephagy.
    Mario Mauthe, Harm Kampinga, Fulvio Reggiori.
      Our recent study identifies a previously unrecognized requirement for protein aggregate fragmentation as a prerequisite for autophagic clearance of amorphous aggregates, a process that has been termed aggrephagy. We show that aggregate fragmentation depends on two distinct but cooperative components: the DNAJB6 (DnaJ heat shock protein family (Hsp40) member B6)-HSPA/HSP70 (heat shock protein family A (Hsp70))-HSPH1/HSP110 chaperone module and the 19S regulatory particles (RPs) of the proteasome. These factors act together to not only to fragment protein aggregates but also to compact them, enabling clustering of selective autophagy receptors (SARs) and subsequent local phagophore formation. Our results show that this fragmentase activity plays a role in the aggrephagic clearance of different aggregate species, including disease-related HTT (huntingtin) aggregates.
    Keywords:  Amorphous; amyloid; chaperone; clustering; proteasome; selective autophagy receptors
    DOI:  https://doi.org/10.1080/15548627.2025.2562893
  5. bioRxiv. 2025 Sep 04. pii: 2025.09.04.674289. [Epub ahead of print]
    A role for CASM in the repair of damaged Golgi architecture.
    Seeun Oh, Saif Ullah, Bhaskar Saha, Michael A Mandell.
      The term CASM describes a process in which LC3 and other Atg8 proteins are covalently ligated to lipids in damaged endomembranes. While CASM is commonly described as a cytoprotective response to multiple types of membrane damage, the ways in which CASM helps cells maintain homeostasis are still unclear. Here, we show that CASM contributes to the maintenance or repair of Golgi apparatus architecture following the loss of TRIM46, a ubiquitin ligase with roles in microtubule organization. TRIM46-deficient cells were notable for enhanced TFEB-driven lysosomal biogenesis and Golgi ribbon fragmentation, with colocalization between the trans-Golgi marker TGN46 and the Atg8 proteins LC3B and GABARAP. Similar results were seen when Golgi architecture was disrupted by inhibitors of microtubule assembly or of vesicle trafficking. Further studies revealed that the Golgi atg8ylation seen in TRIM46 knockout cells was not degradative and mechanistically resembled CASM. Genetic inhibition of CASM in TRIM46-deficent cells reduced TFEB activation and exacerbated the Golgi morphology defects. Together, these studies reveal that lysosomal biogenesis and CASM are common features of a Golgi damage response, with CASM acting to preserve Golgi integrity.
    Keywords:  CASM; Golgi damage; TFEB; TRIM46; Tripartite motif; VAIL; atg8ylation; autophagy; lysosomal biogenesis; microtubule
    DOI:  https://doi.org/10.1101/2025.09.04.674289
  6. Sci Adv. 2025 Sep 19. 11(38): eadv4437
    Endoplasmic reticulum junctions serve as a platform for endosome-lysosome interactions through their stop-and-go motion switching.
    Wenjing Li, Yuanhao Guo, Qi Wang, Mengxuan Qiu, Yudong Zhang, Yutong Yang, Junjie Hu, Ge Yang.
      Endosomes and lysosomes (collectively termed "endolysosomes") traverse the cytoplasm in a stop-and-go manner, but the mechanisms underlying this motion remain poorly understood. Using deep learning-based image analyses, including particle tracking, spatial distribution, and endoplasmic reticulum (ER) morphology analysis, we found that ER junctions facilitate stop-and-go motion switching and serve as platforms for endolysosome interactions. Within the ER network, endolysosomes exhibit three dynamic states: fast movement, local slow movement, and pausing. Pauses occur mainly at ER junctions, where transient endosome-lysosome interactions often coincide with organelle fission and are followed by departure. Disruption of ER junctions impairs lysosomal motility and maturation. We further show that actin condensation around endolysosomes mediates motion switching, involving VAP-STARD3 interaction and the actin regulator YWHAH. Other organelles, such as lipid droplets and peroxisomes, also pause near ER junctions. These findings highlight ER junctions as regulatory hubs that orchestrate organelle dynamics, contributing to the spatial coordination of organelle distribution and interactions within the cytoplasm.
    DOI:  https://doi.org/10.1126/sciadv.adv4437
  7. J Am Chem Soc. 2025 Sep 17.
    Discovery of BRD9 Molecular Glue Degraders That Spare Cardiomyocytes.
    Woong Sub Byun, Zhe Zhuang, Anna P Hnatiuk, Cyrus Jin, Zixuan Jiang, Kheewoong Baek, Evelyn Chao, Katherine A Donovan, Eric S Fischer, Mark Mercola, Nathanael S Gray.
      Molecular glue degraders (MGDs) represent a class of drug-like small molecules that induce targeted protein degradation (TPD) by promoting selective protein-protein interactions. MGDs offer a promising therapeutic approach by selectively eliminating disease-associated proteins; however, their rational design and discovery have historically remained a significant challenge. The field remains constrained by a lack of strategies to effectively utilize ubiquitin ligases (E3s) for TPD, thus missing the therapeutic potential offered by tissue-specific E3 expression. In this study, we developed ZZ7, a molecular glue degrader that selectively degrades BRD9, a critical component of the SWI/SNF chromatin remodeling complex, specifically in synovial sarcoma cells, while sparing cardiomyocytes. The discovery of ZZ7 was driven by a "chemocentric" approach, incorporating a cysteine-reactive, reversible covalent warhead into a BRD9 inhibitor to transform its function from inhibition to degradation. ZZ7 covalently engages DCAF16 at Cys178, an E3 ligase that is highly expressed in synovial sarcoma cells but relatively underexpressed in human iPSC-derived cardiomyocytes, leveraging a cysteine residue that has not been previously exploited. These findings pave the way for new strategies in tissue- and disease-specific precision therapies, particularly for malignancies characterized by an elevated level of DCAF16 expression.
    DOI:  https://doi.org/10.1021/jacs.5c09857
  8. Nature. 2025 Sep 17.
    Covariation MS uncovers a protein that controls cysteine catabolism.
    Haopeng Xiao, Martha Ordonez, Emma C Fink, Taylor A Covington, Hilina B Woldemichael, Junyi Chen, Mika Sarkin Jain, Milan H Rohatgi, Shelley M Wei, Nils Burger, Muneeb A Sharif, Julius Jan, Yaoyu Wang, Jonathan J Petrocelli, Katherine Blackmore, Amanda L Smythers, Bingsen Zhang, Matthew Gilbert, Hakyung Cheong, Sumeet A Khetarpal, Arianne Smith, Dina Bogoslavski, Yu Lei, Laura Pontano Vaites, Fiona E McAllister, Nick Van Bruggen, Katherine A Donovan, Edward L Huttlin, Evanna L Mills, Eric S Fischer, Edward T Chouchani.
      The regulation of metabolic processes by proteins is fundamental to biology and yet is incompletely understood. Here we develop a mass spectrometry (MS)-based approach that leverages genetic diversity to nominate functional relationships between 285 metabolites and 11,868 proteins in living tissues. This method recapitulates protein-metabolite functional relationships mediated by direct physical interactions and local metabolic pathway regulation while nominating 3,542 previously undescribed relationships. With this foundation, we identify a mechanism of regulation over liver cysteine utilization and cholesterol handling, regulated by the poorly characterized protein LRRC58. We show that LRRC58 is the substrate adaptor of an E3 ubiquitin ligase that mediates proteasomal degradation of CDO1, the rate-limiting enzyme of the catabolic shunt of cysteine to taurine1. Cysteine abundance regulates LRRC58-mediated CDO1 degradation, and depletion of LRRC58 is sufficient to stabilize CDO1 to drive consumption of cysteine to produce taurine. Taurine has a central role in cholesterol handling, promoting its excretion from the liver2, and we show that depletion of LRRC58 in hepatocytes increases cysteine flux to taurine and lowers hepatic cholesterol in mice. Uncovering the mechanism of LRRC58 control over cysteine catabolism exemplifies the utility of covariation MS to identify modes of protein regulation of metabolic processes.
    DOI:  https://doi.org/10.1038/s41586-025-09535-5
  9. Autophagy. 2025 Sep 17.
    ATG8ylation-orchestrated vacuolar membrane remodeling facilitates plant alkaline stress tolerance.
    Jianxiong Wu, Jun Luo, Chunya Nie, Xuanang Zheng, Caiji Gao, Jun Zhou.
      While ATG8ylation, the C-terminal lipidation of mammalian and plant Atg8 (ATG8)-family proteins, is a well-established driver of autophagosome formation, emerging evidence reveals its non-canonical role in modifying single-membrane organelles under diverse environmental stresses. In a recent study, we found that disruption of the vacuolar proton gradient by alkaline stress rapidly triggers the translocation of ATG8 to the vacuolar membrane in plants. ATG8ylation facilitates membrane invagination through a mechanism independent of both ESCRT and the cytoskeleton. Concurrently, ATG8 recruits ATG2 to endoplasmic reticulum (ER)-vacuolar membrane contact sites, a process that may contribute to damaged membrane repair. Together, these processes enable plants to rapidly recover from vacuolar pH imbalance and adapt to alkaline conditions. Our findings advance the understanding of ATG8ylation in vacuolar membrane homeostasis and damage response, highlighting its conserved role in organellar stability and stress adaptation.
    Keywords:  ATG8ylation; Alkaline stress; monensin; non-canonical autophagy; vacuole
    DOI:  https://doi.org/10.1080/15548627.2025.2562885
  10. Nat Struct Mol Biol. 2025 Sep 19.
    The human ribosome modulates multidomain protein biogenesis by delaying cotranslational domain docking.
    Grant A Pellowe, Tomas B Voisin, Laura Karpauskaite, Sarah L Maslen, Alžběta Roeselová, J Mark Skehel, Chloe Roustan, Roger George, Andrea Nans, Svend Kjær, Ian A Taylor, David Balchin.
      Proteins with multiple domains are intrinsically prone to misfold, yet fold efficiently during their synthesis on the ribosome. This is especially important in eukaryotes, where multidomain proteins predominate. Here we sought to understand how multidomain protein folding is modulated by the eukaryotic ribosome. We used hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy to characterize the structure and dynamics of partially synthesized intermediates of a model multidomain protein. We find that nascent subdomains fold progressively during synthesis on the human ribosome, templated by interactions across domain interfaces. The conformational ensemble of the nascent chain is tuned by its unstructured C-terminal segments, which keep interfaces between folded domains in dynamic equilibrium until translation termination. This contrasts with the bacterial ribosome, on which domain interfaces form early and remain stable during synthesis. Delayed domain docking may avoid interdomain misfolding to promote the maturation of multidomain proteins in eukaryotes.
    DOI:  https://doi.org/10.1038/s41594-025-01676-5
  11. Nat Cell Biol. 2025 Sep 16.
    Two microbiome metabolites compete for tRNA modification to impact mammalian cell proliferation and translation quality control.
    Wen Zhang, Kuldeep Lahry, Denis Cipurko, Sihao Huang, Olivia Zbihley, Amanda M Sevilleja, Dominika Rudzka, Luke R Frietze, Mahdi Assari, Christopher D Katanski, Marisha Singh, Aurore Attina, Hélène Guillorit, Christopher P Watkins, Delphine Gourlain, Didier Varlet, Jennifer Falconi, Alexandre Djiane, Christophe Hirtz, Hankui Chen, Françoise Macari, Katherine Johnson, Nicolas Chevrier, Alexandre David, Tao Pan.
      The microbiome affects eukaryotic host cells via many metabolites, including the well-studied queuine as substrate for host tRNA queuosine modification. The microbial metabolite pre-queuosine 1 (preQ1) is produced in the bacterial tRNA queuosine biosynthesis pathway, with unknown effects on host cell biology. Here we show that preQ1 strongly represses cell proliferation in both human and mouse cells. Queuine reverses this effect by competing with preQ1 to modify the same tRNA. PreQ1 is detectable in the plasma and tissues of mice, and its injection suppresses tumour growth in a mouse cancer model. Mechanistically, preQ1 reduces cognate tRNA levels specifically, as well as codon-dependent translation of housekeeping genes. We identify the endoplasmic reticulum-localized inositol-requiring enzyme 1 (IRE1) ribonuclease as the enzyme responsible for the selective degradation of preQ1-modified tRNAs on translating ribosomes. Our results identify two microbial metabolites competing for host tRNA modification, which elicits translation quality control and impacts cell proliferation.
    DOI:  https://doi.org/10.1038/s41556-025-01750-6
  12. Cell Rep. 2025 Sep 12. pii: S2211-1247(25)01057-5. [Epub ahead of print]44(9): 116286
    Pathogenic phosphorylation of linear ubiquitin machinery causes inflammasome sensor degradation.
    Yang Yu, Shanshan Yu, Zhe Lu, Lihua Qiang, Yanzhao Zhong, Pupu Ge, Zehui Lei, Changgen Qiu, Yingxu Fang, Xinwen Zhang, Bingxi Li, Yu Pang, Jing Wang, Lingqiang Zhang, Cui Hua Liu, Qiyao Chai.
      Host immune cells are equipped with cytosolic sensors to detect invading pathogens and initiate anti-infectious responses. However, how pathogens undermine host intracellular surveillance for persistent infection is not fully understood. Here, we identify that Mycobacterium tuberculosis protein kinase PknG subverts inflammasome sensor NLRP3-mediated cytokine release and pyroptosis by targeting host linear ubiquitin chain assembly complex (LUBAC). Mechanistically, PknG phosphorylates the LUBAC subunit HOIL-1L to prevent it from engaging in LUBAC formation, thereby suppressing linear ubiquitination of inflammasome adaptor ASC to dampen NLRP3 inflammasome assembly. Meanwhile, this phosphorylation stabilizes and activates HOIL-1L, which, in turn, exerts ubiquitin ligase activity to mediate K48-linked ubiquitination of NLRP3 for degradation. Disrupting the kinase activity or HOIL-1L-interacting region of PknG facilitates host NLRP3-dependent anti-Mtb immunity in mice. Thus, the bacterial kinase disrupts host linear ubiquitin machinery and coopts its ubiquitin ligase subunit to constitute an inter-species enzymatic cascade that drives inflammasome sensor degradation for counteracting immune surveillance.
    Keywords:  CP: Molecular biology; Mycobacterium tuberculosis; inflammasome; intracellular immune surveillance; linear ubiquitin chain assembly complex, LUBAC; protein kinase G, PknG; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2025.116286
  13. bioRxiv. 2025 Sep 04. pii: 2025.09.04.674221. [Epub ahead of print]
    Cellular mechanism linking endoplasmic reticulum inheritance and cell cycle regulation of the nuclear genome.
    Ya-Shiuan Lai, Jesse Chao, Maho Niwa.
      Endoplasmic reticulum (ER) stress triggers activation of the ER surveillance (ERSU) pathway- a critical protective mechanism that transiently halts cortical ER inheritance to daughter cells and arrests cytokinesis by septin ring subunit Shs1 re-localization to the bud scar in response to ER stress. Once ER functional homeostasis is re-established, cells resume normal cell cycle progression; however, the molecular circuitry linking ER integrity to cell cycle regulation has remained largely unresolved. Here, we show that ER stress selectively disperse Bud2, a GAP for Bud1/Rsr1, severing its canonical role in cell polarity while integrating it into ER homeostasis signaling. Bud2 dispersion results in accelerated spindle pole body (SPB) duplication, spindle misorientation, defects in nuclear migration, and genome segregation errors under ER stress. Strikingly, a C-terminal truncation of Shs1 ( shs1-ΔCTD ) recapitulated the ER stress-induced dispersion of Bud2 phenotype even in the absence of ER stress, and delayed cell-cycle re-entry after ER homeostasis was regained-despite normal occurrence of typical ERSU hallmark events. Notably, Bud2 overexpression rescued the growth defects of shs1-ΔCTD mutants after ER homeostasis was re-established. Collectively, our findings reveal a new mechanistic axis whereby ER integrity coordinates organelle inheritance, cytoskeletal organization, and nuclear division via selective control of Bud2 and Shs1, establishing a direct regulatory bridge between ER status and mitotic fidelity.
    DOI:  https://doi.org/10.1101/2025.09.04.674221
  14. bioRxiv. 2025 Sep 09. pii: 2025.09.09.674968. [Epub ahead of print]
    Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation.
    G Jordan Ray, Eleonora Nardini, Heather R Keys, Daniel H Lin, David M Sabatini, David P Bartel.
      Autophagy targets a wide variety of substrates for degradation within lysosomes 1 . While lysosomes are known to possess RNase activity 2 , the role of lysosomal RNA degradation in post-transcriptional gene regulation is not well understood. Here, we define RNASET2, PLD3, and both endogenous and exogenous RNase A family members as lysosomal RNases. Cells lacking these RNases accumulated large amounts of lysosomal RNA. Although all types of RNA can be found within lysosomes, SRP RNAs, Y RNAs, 5' TOP mRNAs, long-lived mRNAs, and mRNAs encoding membrane and secreted proteins were specifically enriched. All types of RNA depend on autophagy for lysosomal targeting, but the lysosomally-enriched RNAs are more sensitive to loss of autophagy, implying that selective mechanisms mediate their lysosomal entry. RNA stability measurements revealed that lysosomally-degraded transcripts also had autophagy-dependent changes in stability. In exploring how specific RNAs are targeted for lysosomal degradation, we found that the Alu domain of SRP RNAs is sufficient for targeting these RNAs to lysosomes in fashion that depends on its interactions with the SRP9 and SRP14 proteins. For mRNAs, 5' TOP motifs are sufficient to increase their targeting to lysosomes for degradation in a LARP1-dependent manner. Altogether, our results establish lysosomes as selective modulators of cellular RNA content.
    DOI:  https://doi.org/10.1101/2025.09.09.674968
  15. Nat Struct Mol Biol. 2025 Sep;32(9): 1581
    Focus on ubiquitylation and protein degradation.
      
    DOI:  https://doi.org/10.1038/s41594-025-01672-9
  16. bioRxiv. 2025 Sep 06. pii: 2025.09.05.674599. [Epub ahead of print]
    Cryo-EM Reveals Regulatory Mechanisms Governing Substrate Selection and Activation of Human LONP1.
    Jeffrey T Mindrebo, Lauren Alexandrescu, Jennifer R Baker, Garret Wang, Gabriel C Lander.
      The human AAA+ protease LONP1 plays a central role in maintaining mitochondrial proteostasis. LONP1 processes a vast array of substrates, ranging from damaged or unfolded proteins to specific subunits stably integrated into respiratory complexes. Previous cryo-EM studies of LONP1 uncovered two distinct conformational states corresponding to inactive or active forms of the enzyme. While these states have shed light on the intricacies of LONP1 substrate translocation and proteolytic processing, little is known about the decision-making involved in LONP1 substrate engagement and subsequent initiation of its unfoldase activity. Here, we use cryo-EM to determine a novel ADP-bound, C3-symmetric intermediate state of LONP1 (LONP1C3) with putative substrate "fold-sensing" capabilities. Our biochemical and structural data indicate that LONP1C3 is an on-pathway intermediate and that is stabilized by interaction with folded substrates. Moreover, we identify additional symmetric and asymmetric conformational states, including a two-fold symmetric split-hexamer conformation, that we associate with the transition from LONP1C3 to LONP1ENZ. We propose that the C3-state regulates substrate selection and enables LONP1 to efficiently surveil the matrix proteome to ensure selective removal of damaged and dysfunctional proteins as well as privileged LONP1 substrates. These findings collectively provide further mechanistic insights into LONP1 substrate recruitment and engagement and inform on its diverse roles in maintaining homeostasis within the mitochondria.
    DOI:  https://doi.org/10.1101/2025.09.05.674599
  17. Autophagy. 2025 Sep 16. 1-20
    SCAMP5 regulates AP-4-dependent sorting and trafficking of ATG9A for presynaptic autophagy via PI4KB/PI4KIIIβ recruitment and PtdInsP4 production at the TGN.
    Seung Hyun Ryu, Jungmihn Lee, Unghwi Lee, Kitae Kim, Go-Eun Jun, Jeongmin Oh, Sang-Eun Lee, Sunghoe Chang.
      Neuronal autophagosome formation at distant presynaptic sites relies on ATG9A trafficking, a process mediated by AP-4 at the trans-Golgi network (TGN), but the molecular mechanisms governing its sorting for presynaptic delivery have remained elusive. Here, we uncover an unexpected role for SCAMP5, a key regulator of synaptic vesicle dynamics, in orchestrating presynaptic macroautophagy/autophagy through its actions at the TGN. SCAMP5 depletion severely impairs autophagosome formation at presynaptic boutons. Mechanistically, we identify SCAMP5 as a novel binding partner of PI4KB/PI4KIIIβ (phosphatidylinositol 4-kinase beta), where it controls PI4KB recruitment to the TGN and subsequent phosphatidylinositol-4-phosphate (PtdIns4P) production. As PtdIns4P is essential for AP-4 recruitment, SCAMP5 depletion disrupts AP-4-mediated ATG9A trafficking to presynaptic sites, thereby compromising presynaptic autophagy and subsequent protein turnover. Our findings establish that SCAMP5 coordinates ATG9A-dependent presynaptic autophagy through PI4KB recruitment and PtdIns4P production at the TGN, revealing a novel pathway critical for maintaining presynaptic protein homeostasis.Abbreviations: AP-4: adaptor protein 4; ATG9A: autophagy related 9A; PI4KB/PI4KIIIβ: phosphatidylinositol 4-kinase beta; PtdIns4P: phosphatidylinositol-4-phosphate; SCAMP5: secretory carrier membrane protein 5; TGN: trans-Golgi network.
    Keywords:  AP-4; ATG9A; PI4KB/PI4KIIIβ; SCAMP5; presynaptic autophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2559689
  18. bioRxiv. 2025 Sep 04. pii: 2025.08.30.673265. [Epub ahead of print]
    InsP3R signaling mediates mitochondrial stress-induced longevity through actomyosin-dependent mitochondrial dynamics.
    Gaomin Feng, Elizabeth M Ruark, Alexandra G Mulligan, Eric K F Donahue, Anthony Hoang, Brianne Jacquet-Cribe, Li Peng, Kristopher Burkewitz.
      Certain forms of mitochondrial impairment confer longevity, while mitochondrial dysfunction arising from aging and disease-associated mutations triggers severe pathogenesis. The adaptive pathways that distinguish benefit from pathology remain unclear. Here we reveal that longevity induced by mitochondrial Complex I/ nuo-6 mutation in C. elegans is dependent on the endoplasmic reticulum (ER) Ca 2+ channel, InsP3R. We find that the InsP3R promotes mitochondrial respiration, but the mitochondrial calcium uniporter is dispensable for both respiration and lifespan extension in Complex I mutants, suggesting InsP3R action is independent of matrix Ca 2+ flux. Transcriptomic profiling and imaging reveal a previously unrecognized role for the InsP3R in regulating mitochondrial scaling, where InsP3R impairment results in maladaptive hyper-expansion of dysfunctional mitochondrial networks. We reveal a conserved InsP3R signaling axis through which calmodulin and actomyosin remodeling machineries, including Arp2/3, formin FHOD-1, and MLCK, constrain mitochondrial expansion and promote longevity. Disruption of actin remodeling or autophagy mimics InsP3R loss. Conversely, driving fragmentation ameliorates mitochondrial expansion and rescues longevity, supporting a model in which InsP3R-dependent actin remodeling sustains mitochondrial turnover. These findings establish an inter-organelle signaling axis by which ER calcium release orchestrates mitochondrial-based longevity through cytoskeletal effectors.
    DOI:  https://doi.org/10.1101/2025.08.30.673265
  19. Cell Discov. 2025 Sep 16. 11(1): 75
    STK4 inhibits the E3 activity of HOIP by phosphorylating its allosteric ubiquitin-binding site.
    Yaru Wang, Xindi Zhou, Zhiqiao Lin, Yichao Huang, Yuchao Zhang, Haobo Liu, Yuqian Zhou, Jianping Liu, Lifeng Pan.
      HOIP, an RBR-type E3 ligase and the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), plays crucial roles in various cellular processes, including the NF-κB signaling pathway. The E3 activity of HOIP can be inhibited by the kinase STK4-mediated phosphorylation, although the mechanism is poorly understood. In this study, using biochemical, mass spectrometry and structural approaches, we systemically characterize the association of STK4 with HOIP, and unveil that STK4 can directly bind to the RING2-LDD module of HOIP through its kinase domain. The determined crystal structure of STK4 in complex with HOIP RING2-LDD not only elucidates the detailed binding mechanism of STK4 with HOIP, but also uncovers, for the first time, a unique binding mode of STK4 with its substrate. Moreover, we reveal that STK4 can directly phosphorylate the T786 residue of HOIP that is located in the allosteric ubiquitin-binding site of HOIP. Importantly, the phosphorylation of HOIP T786 mediated by STK4 can block the binding of ubiquitin to the allosteric site of HOIP, thereby attenuating the E3 activity of HOIP. In all, our findings provide mechanistic insights into the interaction between STK4 and HOIP as well as the negative regulation of HOIP's E3 activity by STK4-mediated phosphorylation, which are valuable for further understanding the regulatory modes of RBR-type E3 ligases.
    DOI:  https://doi.org/10.1038/s41421-025-00824-x
  20. J Biol Chem. 2025 Sep 16. pii: S0021-9258(25)02581-5. [Epub ahead of print] 110729
    The serine protease HTRA1 targets Tau fibrils and provides a proteolytic barrier against pathogenic protein conformations.
    Birte Hagemeier, Kamilla Ripkens, Nina Schulze, Anika Bluemke, Michal Strzala, Michelle Koci, Farnusch Kaschani, Markus Kaiser, Michael Erkelenz, Sebastian Schluecker, Melisa Merdanovic, Simon Poepsel, Doris Hellerschmied, Steven G Burston, Michael Ehrmann.
      Tauopathies such as Alzheimer's disease, frontotemporal dementia with Parkinsonism, and other neurodegenerative diseases are classified as protein folding diseases because they share amyloid fibrils as a hallmark. Typically, amyloid fibrils accumulate and spread through tissue over time. It is assumed that this process is accelerated as protein quality control becomes overwhelmed in aged tissues. However, a deep understanding of how specific protein quality control factors interfere with fibril accumulation and thereby delay disease onset is lacking. Here, we show that the widely conserved serine protease HTRA1 is activated by tau fibrils and provide quantitative, topological, and temporal insights into the proteolytic degradation of soluble and fibrillar tau. Live cell fluorescence microscopy demonstrates the interaction of HTRA1 with tau fibrils and their proteolytic degradation in cells. Our data highlight the potential of HTRA1 to act in a cell non-autonomous defense mechanism against the intercellular spread of pathogenic protein conformations.
    Keywords:  Alzheimer's disease; HTRA1; PDZ proteases; amyloid fibrils; tau
    DOI:  https://doi.org/10.1016/j.jbc.2025.110729
  21. J Cell Biol. 2025 Oct 06. pii: e202509030. [Epub ahead of print]224(10):
    ATG16L strikes again! New findings link lysosome stress and physiology.
    Alison D Klein, Michael Overholtzer.
      Lysosome stress responses are emerging, but their connections to normal physiology are not well understood. In this issue, Duque et al. (https://doi.org/10.1083/jcb.202503166) discover that the autophagy protein ATG16L, a mediator of a stress response called CASM, also regulates normal lysosome function.
    DOI:  https://doi.org/10.1083/jcb.202509030
  22. Proc Natl Acad Sci U S A. 2025 Sep 23. 122(38): e2422570122
    More 26S and 30S proteasomes are beneficial in proteinopathy.
    Youngwon Kim, Namhoon Kim, WonJae Lee, Youbin Kim, Donghyeon Kim, Jisu Park, Yong-Keun Jung.
      Despite the many studies on the ubiquitin-proteasome system, our understanding of the proteasome itself is limited. The balance and regulation of 26S and 30S proteasomes are not yet known. Here, we show that among the proteasome base assembly chaperones, only S5b/PSMD5 determines the levels of proteasome holoenzymes, especially the 30S proteasome, in mammals. In a variety of cell lines and mouse tissues, we found that apart from its role in yeast 19S proteasome assembly, loss of S5b/PSMD5 increased assembly toward the 26S and 30S proteasomes. During the process, we identified proteasome complexes that may represent alternative assembly intermediates, including the 19S base complex and 20S complexes harboring 19S subunits, eventually leading to a shift in the overall steady-state level of the 26S and 30S proteasomes. Intriguingly, the addition of the S5b/PSMD5 protein in vitro and its increase in cells efficiently disassembled the 30S proteasome into the 20S and 19S complexes. Increase in the 26S and 30S proteasomes over the 20S proteasome enhances the degradation of the aggregation-prone proteins and ubiquitinated proteins in cells and ameliorates cognitive impairment through the reduction of tau pathology in PS19 mice. These results suggest that 26S and 30S proteasomes are manipulated by S5b/PSMD5 and are beneficial for mitigating proteinopathy in mammals.
    Keywords:  26S and 30S proteasomes; S5b/PSMD5; aggregation-prone protein; assembly; proteinopathy
    DOI:  https://doi.org/10.1073/pnas.2422570122
  23. STAR Protoc. 2025 Sep 15. pii: S2666-1667(25)00492-7. [Epub ahead of print]6(4): 104086
    Protocol for in vivo BONCAT labeling of nascent proteins in Xenopus laevis tadpole brains with high temporal precision for quantitative analysis.
    Arifa Ahsan, Reshmi Bera, Benjamin Baah Konadu, Yuan Gao, Hai-Yan He.
      Proteostasis is critical for neuronal function and circuit plasticity. We present here an in vivo bio-orthogonal non-canonical amino acid tagging (BONCAT) protocol optimized to selectively label newly synthesized proteins in Xenopus laevis tadpole brains in live animals. We describe steps for the quantitative analysis of both total nascent proteins and specific nascent proteins of interest, using dot blotting and NeutrAvidin purification followed by western blotting. This approach allows for the quantitative analysis of the nascent proteome in intact neural circuits with high temporal precision. For complete details on the use and execution of this protocol, please refer to He et al.1.
    Keywords:  Cell Biology; Neuroscience; model Organisms
    DOI:  https://doi.org/10.1016/j.xpro.2025.104086
  24. FEBS J. 2025 Sep 16.
    Atg18 facilitates autophagosome formation via its Atg8-interacting motif in Saccharomyces cerevisiae.
    Tianzhi Li, Xiazhen Li, Miaomiao Li, Tao Yuan, Cong Ma.
      Autophagy, an essential process in eukaryotic cells, entails the sequestration and degradation of cytosolic components and organelles following fusion with the lysosome or vacuole. Autophagy-related protein 18 (Atg18), a key autophagy-related protein, binds phosphatidylinositol-3-phosphate (PI3P) to localize to autophagosomal membranes, where it recruits Atg2 to mediate lipid transfer during autophagosome biogenesis. Although the roles of Atg18 in autophagy are well established, whether this protein exerts additional regulatory functions in this process remains to be elucidated. Here, we report the weak interactions between Atg18 and Atg8 or Atg16 mediated by the Atg8-interacting motif (AIM) within Atg18. Disruption of the AIM in Atg18 leads to reduced autophagosome formation and diminished autophagic activity. Moreover, we demonstrate that Atg18 is involved in the recruitment of Atg8 to the autophagosome and facilitates the C-terminal cleavage of Atg8 by Atg4. Furthermore, the Atg18-Atg8 complex can be dissociated by Atg3, enabling free Atg18 to subsequently recruit Atg16 to the autophagosome, preparing for Atg8 lipidation. Thus, our findings unveil previously unknown roles for Atg18 in downstream factor recruitment and Atg4 cleavage during autophagosome formation via its AIM.
    Keywords:  AIM motif; Atg16; Atg18; Atg8 C‐terminal cleavage; autophagy
    DOI:  https://doi.org/10.1111/febs.70257
  25. bioRxiv. 2025 Sep 08. pii: 2025.09.07.674666. [Epub ahead of print]
    Astrocytes mobilize a broader repertoire of lysosomal repair mechanisms than neurons.
    Erin M Smith, Natali L Chanaday, Sandra Maday.
      Lysosomal damage impairs proteostasis and contributes to neurodegenerative diseases, yet cell-type-specific differences in lysosomal repair remain unclear. Using a neuron-astrocyte coculture system, we compared responses to lysosomal injury induced by a lysosomotropic methyl ester. Both neurons and astrocytes showed lysosomal damage, marked by galectin-3 recruitment to lumenal lysosomal β-galactosides, elevated lysosomal pH, and engagement of lysophagy receptors TAX1BP1 and p62. However, astrocytes showed a preferential recruitment of ESCRT repair machinery to damaged lysosomes. Additionally, the lysosomal membrane reformation pathway regulated by the RAB7-GAP, TBC1D15, was more robustly activated in astrocytes. By contrast, the PITT pathway, mediating lipid transfer between the ER and damaged lysosomes, was engaged in both cell types. Our data reveal a divergence in how neurons and astrocytes mobilize repair pathways to manage lysosomal damage. These data may reflect differences in lysosomal resilience between astrocytes and neurons and inform therapeutic strategies to correct lysosomal dysfunction in neurodegenerative diseases.
    DOI:  https://doi.org/10.1101/2025.09.07.674666
  26. Cell Rep. 2025 Sep 17. pii: S2211-1247(25)01072-1. [Epub ahead of print]44(10): 116301
    Dynamic recruitment of Munc13 primes docked secretory granules for exocytosis.
    Santiago Echeverry, Jan Saras, Per-Eric Lund, Johan Dunevall, Nikhil R Gandasi, Sebastian Barg.
      Munc13 proteins are essential for regulated exocytosis in neurons and endocrine cells. They consist of an elongated MUN domain that templates SNARE complex formation during priming, flanked by regulatory membrane-associated C1 and C2 domains. Here, we show, using quantitative high-resolution imaging, that priming of insulin granules coincides with recruitment of on average six copies of Munc13 to individual docked granules, similar to estimates of SNARE complexes formed during exocytosis. Intracellular Ca2+- or lipid-signaling accelerates granule priming by promoting C2B-dependent translocation of Munc13 to the plasma membrane, followed by slower (tens of seconds) C2A-domain dependent accumulation at docked granules. Exocytosis in human β-cells also exhibits rapid Ca2+-dependent short-term facilitation that involves Ca2+/C2B-dependent activation of Munc13 but not further accumulation at the release site. Thus, Munc13 controls secretory granule release probability by two separate C2B-dependent mechanisms that affect its recruitment to the release site and its subsequent activation by Ca2+.
    Keywords:  CP: Cell biology; Ca2+-dependent facilitation; Munc13; SNARE; exocytosis; insulin release; β cells
    DOI:  https://doi.org/10.1016/j.celrep.2025.116301
  27. Nucleic Acids Res. 2025 Sep 05. pii: gkaf902. [Epub ahead of print]53(17):
    A simple, fast, and cost-efficient protocol for ultra-sensitive ribosome profiling.
    Jiří Koubek, Katharina Jetzinger, Shiran Dror, Mikel Irastortza-Olaziregi, Dana Frank, Ilgin Kotan, Jaime Santos, Frank Tippmann, Pascal Lafrenz, Henrik Kaessmann, Orna Amster-Choder, Bernd Bukau, Günter Kramer.
      Ribosome profiling has become an essential tool for studying messenger RNA (mRNA) translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency, and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol, we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as those derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.
    DOI:  https://doi.org/10.1093/nar/gkaf902
  28. bioRxiv. 2025 Sep 05. pii: 2025.09.02.673599. [Epub ahead of print]
    SMURF2 inhibits autophagic control of Mycobacterium tuberculosis in macrophages.
    Priscila C Campos, Kathryn C Rahlwes, Victoria A Eknitphong, Beatriz R S Dias, Kubra F Naqvi, Samuel Alvarez-Arguedas, Michael U Shiloh.
      Autophagy is a critical host defense mechanism that restricts intracellular pathogens such as Mycobacterium tuberculosis (Mtb). A key step in this process is the ubiquitination of Mtb or Mtb-associated structures. The E3 ligase SMURF1 catalyzes K48-linked ubiquitination, promoting bacterial clearance. However, the function of its homolog, SMURF2, in host defense remains undefined. Here, we demonstrate that Smurf2 deletion in murine macrophages increases SMURF1 levels, enhances LC3B lipidation, augments K48 ubiquitination of Mtb-associated structures, and reduces intracellular Mtb replication. These effects are reversed by Smurf1 deletion, indicating that SMURF2 restricts autophagy in a SMURF1-dependent manner. Mice with myeloid-specific Smurf2 deletion exhibit modestly prolonged survival following aerosol Mtb infection. In human macrophages, SMURF2 knockdown or its pharmacological inhibition with the HECT ligase inhibitor Heclin reduces Mtb replication. Together, our findings identify SMURF2 as a negative regulator of selective autophagy and host immunity to Mtb and suggest that targeting SMURF2 may represent a novel host-directed therapeutic strategy for tuberculosis.
    DOI:  https://doi.org/10.1101/2025.09.02.673599
  29. Annu Rev Cancer Biol. 2024 Jun;8 155-175
    Chemical Proteomics-Guided Discovery of Covalent Ligands for Cancer Proteins.
    Xiaoyu Zhang, Benjamin F Cravatt.
      Advances in genome sequencing and editing technologies have enriched our understanding of the biochemical pathways that drive tumorigenesis. Translating this knowledge into new medicines for cancer treatment, however, remains challenging, and many oncogenic proteins have proven recalcitrant to conventional approaches for chemical probe and drug discovery. Here, we discuss how innovations in chemical proteomics and covalent chemistry are being integrated to identify and advance first-in-class small molecules that target cancer-relevant proteins. Mechanistic studies have revealed that covalent compounds perturb protein functions in cancer cells in diverse ways that include the remodeling of protein-protein and protein-RNA complexes, as well as through alterations in post-translational modification. We speculate on the attributes of chemical proteomics and covalent chemistry that have enabled targeting of previously inaccessible cancer-relevant pathways and consider technical challenges that remain to be addressed in order to fully realize the druggability of the cancer proteome.
    DOI:  https://doi.org/10.1146/annurev-cancerbio-061421-041946
  30. Cell Rep. 2025 Sep 15. pii: S2211-1247(25)01051-4. [Epub ahead of print]44(9): 116280
    Neurons and astrocytes have distinct organelle signatures and responses to stress.
    Shannon N Rhoads, Weizhen Dong, Chih-Hsuan Hsu, Ngudiankama R Mfulama, Ridhi Yarlagadda, Joey V Ragusa, Michael Ye, Andy Henrie, Maria Clara Zanellati, Graham H Diering, Todd J Cohen, Sarah Cohen.
      Neurons and astrocytes play critical yet divergent roles in brain physiology and neurological conditions. Intracellular organelles are integral to cellular function. However, an in-depth characterization of organelles in live neural cells has not been performed. Here, we use multispectral imaging to simultaneously visualize six organelles-endoplasmic reticulum (ER), lysosomes, mitochondria, peroxisomes, Golgi, and lipid droplets-in live primary rodent neurons and astrocytes. We generate a dataset of 173 z stack and 98 time-lapse images, accompanied by quantitative "organelle signature" analysis. Comparative analysis reveals a clear cell-type specificity in organelle morphology and interactions. Neurons are characterized by prominent mitochondrial composition and interactions, while astrocytes contain more lysosomes and lipid droplet interactions. Additionally, neurons display a more robust organelle response than astrocytes to acute oxidative or ER stress. Our data provide a systems-level characterization of neuron and astrocyte organelles that can be a reference for understanding cell-type-specific physiology and disease.
    Keywords:  CP: Cell biology; CP: Neuroscience; Golgi; astrocytes; endoplasmic reticulum; lipid droplets; lysosomes; microscopy; mitochondria; neurons; organelles; peroxisomes
    DOI:  https://doi.org/10.1016/j.celrep.2025.116280
  31. Nat Struct Mol Biol. 2025 Sep 16.
    Phase separation promotes Atg8 lipidation and vesicle condensation for autophagy progression.
    Yuko Fujioka, Takuma Tsuji, Tetsuya Kotani, Hiroyuki Kumeta, Chika Kakuta, Junko Shimasaki, Toyoshi Fujimoto, Hitoshi Nakatogawa, Nobuo N Noda.
      Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the preautophagosomal structure (PAS) in Saccharomyces cerevisiae, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets through specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution demonstrates that E3-enriched early PAS droplets promote Atg8 lipidation and that Atg8 coating of the vesicle membrane is both necessary and sufficient for their condensation into the droplets. These data suggest that the PAS functions as an efficient production site for lipidated Atg8 and pools membrane seeds to drive autophagosome formation.
    DOI:  https://doi.org/10.1038/s41594-025-01678-3
  32. bioRxiv. 2025 Sep 08. pii: 2025.09.03.674106. [Epub ahead of print]
    MondoA mediates transcriptional coordination between the MYC network and the integrated stress response in pancreatic ductal adenocarcinoma.
    Erin L Ramsey, Stephanie Dobersch, Brian Freie, Nan Hyung Hong, Xiaoying Wu, Sita Kugel, Robert N Eisenman, Patrick A Carroll.
      MYC amplification contributes to poor survival and outcome in pancreatic ductal adenocarcinoma (PDAC). Here we show that in PDAC cell lines with amplified MYC, MondoA is required for viability, facilitating proliferation while suppressing apoptosis in vitro and in vivo . Transcriptional and genomic profiling demonstrates that loss of MondoA leads to altered expression of direct MondoA targets as well as MYC target genes and is accompanied by shifts in genomic occupancy of MYC, MNT, and the MondoA paralog ChREBP. This altered genomic binding by MYC network members is associated with transcriptional perturbation of multiple metabolic and stress pathways, as well as global changes in N6-methyladenosine modification (m 6 A) of mRNA. MondoA inhibition disrupts coordination between MYC network members and the Integrated Stress Response (ISR), resulting in decreased translation of ATF4 mRNA, discordant gene regulation of shared targets of MYC and ATF4 and, ultimately, apoptosis. Re-establishing ATF4 protein expression rescues the diminished viability due to loss of MondoA expression or activity, providing direct evidence of a link between deregulated MYC and the transcriptional machinery of the ISR. Lastly, we find that small-molecule inhibition of MondoA is lethal in a subset of PDAC cell lines, including patient-derived organoids, suggesting that the ability to target MYC via chemical inhibition of MondoA transcriptional activity may have broad efficacy.
    Significance Statement: This report investigates mechanisms underlying the dependence of MYC-amplified pancreatic cancer cells on the MYC network member MondoA which, as a heterodimer with MLX, is a nutrient-sensing transcription factor. We show this dependency is linked to genomic crosstalk between MYC, components of the proximal MYC network, and the master regulator of the integrated stress response, ATF4. Moreover, we find that small molecule inhibitors of MondoA-MLX transcriptional activity abrogate survival of MYC-amplified PDAC lines and patient derived organoids. The significance of this work relates to its focus on a unique vulnerability intrinsic to MYC, an oncogenic driver associated with a wide range of cancers, which is considered to be "undruggable".
    DOI:  https://doi.org/10.1101/2025.09.03.674106
  33. Cell Rep. 2025 Sep 16. pii: S2211-1247(25)01061-7. [Epub ahead of print]44(10): 116290
    Activity-dependent localization and dynamics of STIM1 and STIM2 at ER-PM contacts in hippocampal neurons.
    Arun Chhikara, Filip Maciąg, Nasrin Sorusch, Martin Heine.
      Stromal interaction molecules (STIMs) are Ca2+ sensors within the endoplasmic reticulum (ER) plasma membrane (PM) that contribute to homeostatic functions in neurons. Upon depletion of Ca2+ from the ER, STIMs translocate to ER-PM junctions to contact the inner leaflet of the PM. Using single-particle tracking, we characterize the dynamic properties of endogenous STIM1 and STIM2 proteins in hippocampal neurons. STIMs form clusters in the somato-dendritic compartment but only transiently visit synapses. A substantial fraction of STIM2 proteins define ER-PM contacts under resting conditions and is dependent on the constitutive activity of NMDARs. STIM1 proteins are transiently recruited to ER-PM junctions only during strong activation of NMDARs. Activity-dependent confinement of STIM proteins is not influenced by CaV1.2 channel activity. We propose that STIM proteins fulfill a dominant structural function in neurons by regulating the size and frequency of ER-PM contacts to promote ER-PM communication along dendrites.
    Keywords:  CP: Cell biology; CP: Neuroscience; Ca(V)1.2; ER-PM contacts; K(V)2.1; NMDARs; SPT; STIMs; endoplasmatic reticulum; hippocampal neurons
    DOI:  https://doi.org/10.1016/j.celrep.2025.116290
  34. bioRxiv. 2025 Sep 02. pii: 2025.09.02.673832. [Epub ahead of print]
    A cytokine receptor-targeting chimera (kineTAC) toolbox for expanding extracellular targeted protein degradation.
    Kaan Kumru, Zi Yao, Brandon B Holmes, Fangzhu Zhao, Yun Zhang, Emilio Ferrara, Trenton M Peters-Clarke, Kevin K Leung, James A Wells.
      Extracellular targeted protein degradation (eTPD) is as an important new modality for manipulating the extracellular proteome. However, most eTPD receptors are expressed broadly or are restricted to the liver. Cytokine receptor targeting chimeras (kineTACs) are genetically encoded bispecifics for eTPD that fuse a natural ligand like CXCL12 to an antibody, directing soluble or membrane proteins for lysosomal degradation using the widely expressed chemokine receptor CXCR7 (Pance K, Gramespacher JA., Byrnes, JR, Salangsang F., Serrano JAC, Cotton AD, Steri V, and Wells JA, Nat. Biotechnol. 2023, 41, 273-281). Here, we dramatically expand the kineTAC toolbox by constructing 81 new kineTACs based on an unbiased list of cytokines, chemokines and growth factors. Remarkably, 55 of these expressed at suitable levels for analysis without any optimization. Many of these kineTACs bind receptors that have unique cell-type expression profiles, allowing for eTPD in specific cells and tissues and some were more potent than the original CXCL12-based kineTAC. We further show the internalizing capability of a kineTAC can enhance the performance of antibody drug conjugates. We believe these simple, genetically encoded tools will be useful for expanding the applications for optimized or cell type-selective eTPD.
    DOI:  https://doi.org/10.1101/2025.09.02.673832
  35. Proc Natl Acad Sci U S A. 2025 Sep 23. 122(38): e2503581122
    A widespread family of molecular chaperones promotes the intracellular stability of type VIIb secretion system-exported toxins.
    Polyniki Gkragkopoulou, Stephen R Garrett, Prakhar Y Shah, Dirk W Grebenc, Timothy A Klein, Youngchang Kim, John C Whitney.
      To survive in highly competitive environments, bacteria use specialized secretion systems to deliver antibacterial toxins into neighboring cells, thereby inhibiting their growth. In many Gram-positive bacteria, the export of such toxins requires a membrane-bound molecular apparatus known as the type VIIb secretion system (T7SSb). Recently, it was shown that toxin recruitment to the T7SSb requires a physical interaction between a toxin and two or more so-called targeting factors, which harbor key residues required for T7SS-dependent protein export. However, in addition to these targeting factors, some toxins additionally require a protein belonging to the DUF4176 protein family. Here, by examining two toxin-DUF4176 protein pairs, we demonstrate that DUF4176 constitutes a family of toxin-specific molecular chaperones. In addition to being required for toxin stability in producing cells, we find that DUF4176 proteins facilitate toxin export by specifically interacting with a previously uncharacterized intrinsically disordered region found in many T7SS toxins. Using X-ray crystallography, we determine structures of several DUF4176 chaperones in their unbound state, and of a DUF4176 chaperone in complex with the binding site of its cognate toxin. These structures reveal that this binding site consists of a disordered amphipathic α-helix that requires interaction with its cognate chaperone for proper folding. Overall, we have identified a family of secretion system associated molecular chaperones found throughout T7SSb-containing Gram-positive bacteria.
    Keywords:  bacterial protein export; bacterial toxins; molecular chaperones; protein–protein interactions; type VII secretion system
    DOI:  https://doi.org/10.1073/pnas.2503581122
  36. FEMS Yeast Res. 2025 Sep 16. pii: foaf051. [Epub ahead of print]
    tDR-quant: a reliable electroporation-based approach for quantifying tRNA-derived fragments binding to ribosomes.
    Kamilla Bąkowska-Żywicka, Agata Tyczewska.
      Ribosome-associated non-coding RNAs (rancRNAs), particularly tRNA-derived fragments (tDRs), have emerged as key regulators of translation, especially under stress conditions. In Saccharomyces cerevisiae, tDRs interact with small ribosomal subunits to modulate protein biosynthesis, yet methods to quantitatively assess these interactions have been lacking. Here, we present tDR-quant, a robust technique for in vivo quantification of tDR/ribosome associations using electroporation of radiolabeled tDRs into yeast spheroplasts, followed by polysome profiling and radioactivity detection. We show that tDR interactions with ribosomes are stress- and dose-dependent, primarily associating with the 40S subunit but also with 60S, monosomes, and polysomes under specific conditions. Translation assays revealed that increased tDR levels inhibit protein synthesis without altering polysome profiles. Northern blot and qRT-PCR validated tDR-quant results, confirming its reliability. Stress-specific association patterns suggest that tDRs dynamically regulate translation by interacting with different ribosomal components in response to environmental cues. Importantly, these interactions do not correlate directly with tDR abundance, indicating selective ribosome binding. This study provides the first comprehensive method to quantify tDR-ribosome interactions in vivo and demonstrates that tDRs act as regulatory elements fine-tuning translation during cellular stress in yeast.
    Keywords:  Saccharomyces cerevisiae; abiotic stress; protein biosynthesis regulation; ribosome; ribosome-associated noncoding RNAs; tRNA-derived fragments
    DOI:  https://doi.org/10.1093/femsyr/foaf051
  37. J Biol Chem. 2025 Sep 15. pii: S0021-9258(25)02573-6. [Epub ahead of print] 110721
    The RING-finger domain of Arabidopsis RMR functions as an E3 ligase essential for post-Golgi trafficking.
    Shuai Chen, Yonglun Zeng, Hiu Yan Wong, Yihong Chen, Lei Yang, Fang Luo, Caiji Gao, Liwen Jiang, Kam-Bo Wong.
      Receptor-homology-transmembrane-RING-H2 (RMR) sorting receptors are essential for directing soluble cargo proteins to protein storage vacuoles in plants. These type I integral membrane proteins comprise a single transmembrane domain, an N-terminal lumenal region containing a protease-associated domain for cargo recognition, and a C-terminal cytoplasmic region (CT) with a Really-Interesting-New-Gene-H2 (RING-H2) domain. Here, we determined the crystal structure of the RING-H2 domain of Arabidopsis RMR isoform-1 (AtRMR1-RING), where the conserved C3H2C3 motif coordinates two Zn ions, a feature typical of RING-type E3 ligases. AtRMR1-RING was shown to interact with Arabidopsis E2 ubiquitin-conjugating enzyme, and exhibits E3 ligase activity in an in vitro ubiquitination assay. Biochemical analysis reveals that I234Y substitution disrupted the E2/E3 interaction and greatly reduced E3 ligase activity. Furthermore, we showed that the conserved RING-H2 domains of AtRMR isoform 2, 3 and 4 are also E3 ligases. Inactivation of E3 ligase activity by the I234Y mutation resulted in Golgi retention of AtRMR1-CT and AtRMR2. These findings suggest that the E3 ligase activity is essential for post-Golgi trafficking of RMR receptors, providing new insights into receptor-mediated protein sorting in plants.
    Keywords:  E3 ligase; RING structure; RMR; protein trafficking; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2025.110721
  38. Magn Reson Chem. 2025 Sep 16.
    NMR Insights Into Stress-Induced Modulation of the Monomer-Dimer Equilibrium in a Small Heat Shock Protein.
    Zainab Amin, Jeetender Chugh.
      Small heat shock proteins (sHSPs) are essential molecular chaperones that play a crucial role in maintaining protein homeostasis and protecting cells from stress-induced damage. HSPB8 (Small heat shock protein B8), in particular, plays a crucial role in protein folding and degradation pathways and has been associated with protein aggregation disorders. However, its structural and dynamic behavior under different environmental stress conditions remains poorly defined. In particular, the effect of pH, temperature, and concentration on its oligomeric state and structural integrity needs further investigation. In this study, we performed the biophysical characterization of full-length HSPB8 and its α-crystallin domain (ACD) using solution-state nuclear magnetic resonance (NMR) spectroscopy under different environmental perturbations. The effect on the monomer-dimer equilibrium of the ACD was characterized by monitoring changes in chemical shifts and linewidths in response to the perturbations, including protein concentration, pH, and temperature. It was observed that at low pH, reduced protein concentrations, and elevated temperatures, the ACD favored sharper resonances, reflecting a monomeric state. The relative contribution of disulfide bond and noncovalent interactions in stabilizing the dimer form of ACD was also established. These results provide NMR-based molecular insights into the dynamic monomer-dimer equilibrium, crucial for HSPB8 function in protein folding.
    Keywords:  HSPB8; exchange broadening; monomer–dimer equilibrium; nuclear magnetic resonance (NMR) spectroscopy; small heat shock proteins; α‐crystallin domain (ACD)
    DOI:  https://doi.org/10.1002/mrc.70042
  39. Microb Cell. 2025 ;12 255-273
    Organelle activity organized by the endoplasmic reticulum-mitochondria encounter structure -ERMES- is essential for Podospora anserina development.
    Melisa Álvarez-Sánchez, Matías Ramírez-Noguez, Beatriz Aguirre-López, Leonardo Peraza-Reyes.
      Eucaryotic cell functioning and development depend on the concerted activity of its organelles. In the model fungus Podospora anserina, sexual development involves a dynamic regulation of mitochondria, peroxisomes and the endoplasmic reticulum (ER), suggesting that their activity during this process is coordinated. The ER-Mitochondria Encounter Structure (ERMES) is a tether complex composed of the ER protein Mmm1 and the mitochondrial proteins Mdm10, Mdm12 and Mdm34, which mediates membrane contact-site formation between these organelles. This complex also mediates interactions between mitochondria and peroxisomes. Here we analyzed the role of the ERMES complex during P. anserina development. By studying a thermosensitive MDM10 mutant, we show that MDM10 is required for mitochondrial morphology and distribution, as well as for peroxisome membrane-remodeling and motility. We discovered that lipid droplets exhibit a subapical hyphal localization, which depends on MDM10. MDM10 is also required for ER shaping and dynamics, notably of the apical ER domains of the polarized-growing hyphal region, in a process that involves the activity of the protein YOP1. We also show that apical ER shaping involves a Spitzenkörper-associated membrane traffic, which implicates MDM10, and that the mycelial growth defect of mdm10 mutants is exacerbated when the ER-shaping proteins YOP1 or RTN1 are loss. Finaly, we show that MMM1 is strictly required for mycelial growth and sexual development, suggesting that its activity is essential. Our results show that the activity of distinct organelles depends on the ERMES complex, and that the function of this complex is critical for P. anserina growth and development.
    Keywords:  endoplasmic reticulum; fungi; lipid droplets; mitochondria; organelle interactions; peroxisomes; sexual development
    DOI:  https://doi.org/10.15698/mic2025.09.860
  40. Nat Immunol. 2025 Sep 19.
    STING signals to NF-κB from late endolysosomal compartments using IRF3 as an adaptor.
    Bao-Cun Zhang, Alice Pedersen, Line S Reinert, Ying Li, Ryo Narita, Manja Idorn, Lili Hu, Morten K Skouboe, Sirui Li, Muyesier Maimaitili, Xiangning Ding, Yingying Cong, Jian Zhao, Marie-Louise Frémond, Kasper Mikkelsen, Zongliang Gao, Jin-Rong Huang, Emil A Thomsen, Jakob H Mikkelsen, Rajan Venkatraman, Martin K Thomsen, Marie B Iversen, Sonia Assil, Ran Zhang, Linda Henneman, Martin R Jakobsen, Claus Oxvig, Tina S Dalgaard, Peter Møller, Angela Fago, Tobias Wang, Christian B F Andersen, Dominic De Nardo, Fulvio Reggiori, Jenny P-Y Ting, Jacob G Mikkelsen, Rasmus O Bak, Trine H Mogensen, Pingwei Li, Søren R Paludan.
      NF-κB is central for activation of immune responses. Cytosolic DNA activates the cGAS-STING pathway to induce type I interferons (IFNs) and signaling through NF-κB, thus instigating host defenses and pathological inflammation. However, the mechanism underlying STING-induced NF-κB activation is unknown. Here we report that STING activates NF-κB in a delayed manner, following exit from the Golgi to endolysosomal compartments. Activation of NF-κB is dependent on the IFN-inducing transcription factor IRF3 but is independent of type I IFN signaling. This activation pattern is evolutionarily conserved in tetrapods. Mechanistically, the monomer IRF3 is recruited to STING pS358, with delayed kinetics relative to IRF3 recruitment to STING pS366, which promotes type I IFN responses. IRF3 engagement with STING pS358 induces trafficking to late endolysosomal compartments, supporting recruitment of TRAF6 and activation of NF-κB. We identify a TRAF6 binding motif in IRF3 that facilitates recruitment of TRAF6. This work defines a signaling surface on STING and a function for IRF3 as an adaptor in immune signaling. These findings indicate that STING signaling to NF-κB is enabled only within a short time window between exit from the Golgi and lysosomal degradation, possibly limiting inflammation under homeostatic and danger-sensing conditions.
    DOI:  https://doi.org/10.1038/s41590-025-02283-8
  41. bioRxiv. 2025 Sep 04. pii: 2024.09.30.615685. [Epub ahead of print]
    High-throughput diversification of protein-ligand surfaces to discover chemical inducers of proximity.
    James B Shaum, Miquel Muñoz I Ordoño, Erica A Steen, Daniela V Wenge, Hakyung Cheong, Moritz Hunkeler, Eric M Bilotta, Zoe Rutter, Paige A Barta, Abby M Thornhill, Natalia Milosevich, Lauren M Hargis, Jordan Janowski, Timothy R Bishop, Trever R Carter, Bryce da Camara, Matthias Hinterndorfer, Lucas Dada, Wen-Ji He, Fabian Offensperger, Hirotake Furihata, Sydney R Schweber, Charlie Hatton, Yanhe Wen, Benjamin F Cravatt, Keary M Engle, Katherine A Donovan, Bruno Melillo, Seiya Kitamura, Alessio Ciulli, Scott A Armstrong, Eric S Fischer, Georg E Winter, Michael A Erb.
      Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4 CRBN to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCF FBXO3 , an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.
    DOI:  https://doi.org/10.1101/2024.09.30.615685
  42. bioRxiv. 2025 Sep 08. pii: 2025.09.07.674755. [Epub ahead of print]
    Unfolded protein response signaling promotes myeloid cell production and cooperates with oncogenic mutation.
    Hyunjoo Choi, Sang-Eun Jung, Hyojung Paik, Maggie J Cox, Stephen T Oh, Yoon-A Kang.
      Unfolded protein response (UPR) is an evolutionally conserved adaptive mechanism that promotes protein homeostasis under endoplasmic reticulum (ER) stress. UPR signaling has numerous functions in metabolism, cancer, immunology, and neurodegenerative diseases. Recent studies also showed that UPR signaling has important roles in hematopoietic stem and progenitor cell biology. However, whether UPR signaling regulates hematopoietic lineage fate decision remains elusive. Here, we found that FcγR - MPP3 generates erythroid lineage and Jak2 V617F mutation leads to overproduction of erythroid cells by expanding FcγR - MPP3. We showed that UPR signaling increases myeloid cell production through promoting FcγR - MPP3 transition to granulocyte/macrophage progenitor (GMP) producing FcγR + MPP3. Under a disease condition, UPR signaling cooperates with Jak2 V617F mutation and exacerbates disease phenotype as increasing red blood cells in a mouse model of polycythemia vera (PV). Activation of UPR signaling also increased myeloid output in healthy donor bone marrow MPP cells while skewing the output towards erythroid lineage in PV patient bone marrow MPP cells. Together, our results identify a novel function of UPR signaling in hematopoietic lineage specification and provide critical insights into targeting UPR signaling in hematological malignancies.
    Highlights: UPR signaling promotes myeloid cell production. UPR signaling collaborates with Jak2 V617F mutation and increases red blood cell production.
    DOI:  https://doi.org/10.1101/2025.09.07.674755
  43. Sci Adv. 2025 Sep 19. 11(38): eadw2539
    Linear ubiquitination prevents lipodystrophy and obesity-associated metabolic syndrome.
    Ximena Hildebrandt, Önay Veli, Armel Hyoubi, Julia Zinngrebe, Ali T Abdallah, Julian Rodefeld, Anne Hoffmann, Liane Gardeweg, Öykü Kaya, Elena Wagner, Andreas Lindhorst, Matea Poggenberg, Yuan Wang, Joëlle Dimmler, Jutta Schillings, Pegi Koci, Francesca Bonechi, Lucas Valdez Capuccino, Christine Kiefer, Konstantinos Kelepouras, Adhideb Ghosh, Falko Noé, Christian Wolfrum, Michael Singer, Gianmaria Liccardi, Tom Luedde, Aslihan Yavas, Ahmed Ghallab, Jan G Hengsler, Philipp Antczak, Martin Gericke, Holger Winkels, Matthias Blüher, Henning Walczak, Alessandro Annibaldi, Pamela Fischer-Posovszky, Nieves Peltzer.
      Adipocyte hypertrophy during obesity triggers chronic inflammation, leading to metabolic disorders. However, the role of adipocyte-specific inflammatory signaling in metabolic syndrome remains unclear. The linear ubiquitin chain assembly complex, LUBAC, is an E3-ligase that generates nondegradative linear ubiquitination (Lin-Ub). LUBAC regulates NF-κB/MAPK-driven inflammation and prevents cell death triggered by immune receptors like TNF receptor-1. Here, we show that mice lacking HOIP, the Lin-E3 ligase catalytic subunit of LUBAC, in adipocytes (HoipA-KO) display lipodystrophy and heightened susceptibility to obesity-induced metabolic syndrome, particularly metabolic dysfunction-associated steatotic liver disease (MASLD). Mechanistically, loss of HOIP attenuates TNF-induced NF-κB activation and promotes cell death in human adipocytes. Inhibiting caspase-8-mediated cell death is sufficient to prevent lipodystrophy and MASLD in HoipA-KO obese mice. HOIP expression in adipose tissue positively correlates with metabolic fitness in obese individuals. Overall, our findings reveal a fundamental developmental role for Lin-Ub in adipocytes by mitigating cell death-driven adipose tissue inflammation and protecting against obesity-related metabolic syndrome.
    DOI:  https://doi.org/10.1126/sciadv.adw2539
  44. J Biol Chem. 2025 Sep 16. pii: S0021-9258(25)02582-7. [Epub ahead of print] 110730
    Cysteine redoxome landscape in the liver of male mice fed a high-fat high-sucrose diet.
    Cynthia M Galicia-Medina, Hein Ko Oo, Takumi Nishiuchi, Ryota Tanida, Tuerdiguli Abuduyimiti, Hisanori Goto, Yujiro Nakano, Yumie Takeshita, Kiyo-Aki Ishii, Takashi Toyama, Yoshiro Saito, Hiroaki Takayama, Toshinari Takamura.
      Reversible cysteine post-translational modifications serve as a "switch" for protein structure-function dynamics. Herein, we applied a comprehensive strategy to map the cysteine redoxome by pinpointing over 5,000 oxidized and reduced cysteine residues in the liver of male mice fed either a normal chow diet (NCD) or a high-fat/high-sucrose diet (HFHSD). The global and subcellular distribution of oxidized and reduced cysteine residues remained stable across both diet groups, indicating that HFHSD does not induce widespread shifts in cysteine redox equilibrium. Proteomic analyses revealed that HFHSD upregulates proteins involved in genomic stability, lipid detoxification, and energy regulation, while downregulating those linked to detoxification and metabolic flexibility. Notably, 169 cysteine residues exhibited dynamic redox changes in response to HFHSD, mapping to 35 KEGG pathways central to redox balance and energy homeostasis. Motif and structural analyses demonstrated that the reactivity of cysteine residues sensitive to redox stress is dictated by distinct electrostatic microenvironments and subcellular localization. Cysteine residues sensitive to HFHSD-induced oxidation were enriched in mitochondria and cytosol, and cysteine residues sensitive to HFHSD-induced reduction in extracellular regions. Furthermore, cysteine residues sensitive to HFHSD-induced reduction mainly participate in disulfide bond formation and are exposed to the surface of the protein, suggesting roles as molecular switches in protein function. The current cysteine redoxome strategy broadens the disease-associated proteome landscape and provides potential therapeutic target cysteine residues critical for regulating protein functions and interactions relevant to pathophysiology.
    Keywords:  liver metabolism; oxidation-reduction (redox); post-translational modification (PTM); protein motif; proteomics
    DOI:  https://doi.org/10.1016/j.jbc.2025.110730
  45. Cell Metab. 2025 Sep 16. pii: S1550-4131(25)00381-X. [Epub ahead of print]
    OGFOD1 enables AML chemo- and nutrient stress resistance by regulating protein synthesis.
    Christina Mayerhofer, Dan Li, Trine Kristiansen, Ernst Mayerhofer, Azeem Sharda, Giulia Schiroli, Karin Gustafsson, Lingli He, Michael Mazzola, Sam Keyes, Anna Kiem, Eve Crompton, Yanxin Xu, Sovannarith Korm, Zhixun Dou, Charles Vidoudez, Peter G Miller, Nick van Gastel, Timothy A Graubert, David T Scadden.
      Acute myeloid leukemia (AML) commonly relapses after initial chemotherapy response. We assessed metabolic adaptations in chemoresistant cells in vivo before overt relapse, identifying altered branched-chain amino acid (BCAA) levels in patient-derived xenografts (PDXs) and immunophenotypically identified leukemia stem cells from AML patients. Notably, this was associated with increased BCAA transporter expression with low BCAA catabolism. Restricting BCAAs further reduced chemoresistant AML cells, but relapse still occurred. Among the persisting cells, we found an unexpected increase in protein production. This was accompanied by elevated translation of 2-oxoglutarate- and iron-dependent oxygenase 1 (OGFOD1), a known ribosomal dioxygenase that adjusts the fidelity of tRNA anticodon pairing with coding mRNA. We found that OGFOD1 upregulates protein synthesis in AML, driving disease aggressiveness. Inhibiting OGFOD1 impaired translation processing, decreased protein synthesis and improved animal survival even with chemoresistant AML while sparing normal hematopoiesis. Leukemic cells can therefore persist despite the stress of chemotherapy and nutrient deprivation through adaptive control of translation. Targeting OGFOD1 may offer a distinctive, translation-modifying means of reducing the chemopersisting cells that drive relapse.
    Keywords:  BCAA; OGFOD1; Ribo-seq; acute myeloid leukemia; chemoresistance; metabolism; protein biosynthesis; ribosome pausing; translation accuracy
    DOI:  https://doi.org/10.1016/j.cmet.2025.08.008
  46. PLoS Pathog. 2025 Sep;21(9): e1013522
    Regulation of host immunity by a novel Legionella pneumophila E3 ubiquitin ligase.
    Shuxin Liu, Chunlin He, Yong Zhang, Siyao Liu, Tao-Tao Chen, Chunxiuli Li, Dong Chen, Songying Ouyang, Lei Song, Jiaqi Fu, Zhao-Qing Luo.
      Legionella pneumophila, the causative agent of Legionnaires' disease, exists ubiquitously in natural and artificial water systems. This pathogen poses serious threat to human health. One salient feature of L. pneumophila pathogenesis is the hundreds of effectors delivered into host cells by its Dot/Icm transporter. These virulence factors interfere with multiple hosts signaling pathways to subvert host defense. The ubiquitin network is essential in host signaling involved in immunity and thus is a common target of L. pneumophila effectors. At least thirteen Dot/Icm effectors have been shown to function as E3 ubiquitin ligases that cooperate with the host ubiquitination machinery by distinct mechanisms. In addition, seven deubiquitinases (DUBs) have been characterized. Furthermore, effectors that utilize catalysis mechanisms that are chemically distinct from the canonical one found in eukaryotes have been reported, indicating that hijacking of the host ubiquitin network by L. pneumophila is extensive and complex. Here, we identified ubiquitin interacting proteins with a proximity labeling method and found that the effector Lug14 (Lpg1106) functions as a novel ubiquitin ligase. Lug14 works with the E2 UbcH5c to catalyze ubiquitination with a preference for K11-linked chains by a mechanism that does not require a cysteine residue. Finally, we found that Lug14 targets ARIH2, a member of the host RBR E3 ligase family, leading to increased activation of the NLRP3 inflammasome in macrophages.
    DOI:  https://doi.org/10.1371/journal.ppat.1013522
  47. Sci Adv. 2025 Sep 19. 11(38): eadx5027
    ERAD machinery controls the conditional turnover of PIN-LIKES in plants.
    Seinab Noura, Jonathan Ferreira Da Silva Santos, Elena Feraru, Sebastian N W Hoernstein, Mugurel I Feraru, Laura Montero-Morales, Ann-Kathrin Rößling, David Scheuring, Richard Strasser, Pitter F Huesgen, Sascha Waidmann, Jürgen Kleine-Vehn.
      Auxin is a crucial phytohormone that regulates plant development and facilitates dynamic responses to environmental changes through subcellular control mechanisms. PIN-LIKES (PILS) are auxin transport facilitators at the endoplasmic reticulum (ER) that mediate nuclear auxin abundance and signaling. Although the posttranslational regulation of PILS is important for acclimating growth responses, the molecular mechanisms involved remain largely unknown. This study demonstrates that components of the ER-associated degradation (ERAD) machinery regulate the proteasome-dependent degradation of functional PILS proteins under nonstressed conditions. We further reveal that both internal and external signals use the ERAD complex to differentially modulate the turnover rates of PILS proteins. Our findings uncover an additional physiological role of the ERAD complex in regulating PILS protein turnover. This finding uncovers the interplay between protein homeostasis at the ER and growth regulation, opening unexplored molecular avenues into how plants acclimate to internal and external cues.
    DOI:  https://doi.org/10.1126/sciadv.adx5027
  48. bioRxiv. 2025 Sep 04. pii: 2025.09.03.673992. [Epub ahead of print]
    Nanoscale Structural and Functional Impacts of Disease-Associated Collagen Mutations.
    Caitlyn A Tobita, Siddhartha Banerjee, Jonathan Roth, Emma K Larson, Abuzar Nikzad, Abdullah Naiyer, Cody L Hoop, Jean Baum.
      Collagen is the most abundant structural protein in the human body, and its supramolecular organization is central to tissue mechanics and cell-matrix interactions. Integrins, key mediators of these interactions, are essential for key biological processes including adhesion, migration, differentiation, and platelet aggregation. While mutations in collagen are known to cause connective tissue disorders such as Osteogenesis Imperfecta (OI) with phenotypes ranging from mild to perinatal lethal, how these mutations alter fibril level architecture, dynamics and integrin-mediated interactions remains poorly understood. Here, we generated collagen-rich extra-cellular matrix (ECM) from primary dermal fibroblasts of a healthy donor (WT) and from two OI patients carrying distinct glycine mutations: G610C, associated with moderate disease, and G907D, linked to perinatal lethality. Comparative biophysical studies reveal that both mutants retain the canonical D-banding of collagen I fibrils but differ markedly at the nanoscale. G907D fibrils exhibit greater local structural perturbations and increased molecular mobility relative to the non-lethal G610C. Importantly, integrin binding also diverges between mutants: G610C displays reduced affinity, whereas G907D exhibits enhanced affinity compared to WT. Together, these findings establish a mechanistic link between single-residue mutations, nanoscale fibril architecture and collagen-receptor interactions, and highlight how genetic or acquired collagen defects can drive ECM dysregulation.
    DOI:  https://doi.org/10.1101/2025.09.03.673992
  49. ArXiv. 2025 Sep 03. pii: arXiv:2509.03330v1. [Epub ahead of print]
    Network-driven discovery of repurposable drugs targeting hallmarks of aging.
    Bnaya Gross, Joseph Ehlert, Vadim N Gladyshev, Joseph Loscalzo, Albert-László Barabási.
      Despite the thousands of genes implicated in age-related phenotypes, effective interventions for aging remain elusive, a lack of advance rooted in the multifactorial nature of longevity and the functional interconnectedness of the molecular components implicated in aging. Here, we introduce a network medicine framework that integrates 2,358 longevity-associated genes onto the human interactome to identify existing drugs that can modulate aging processes. We find that genes associated with each hallmark of aging form a connected subgraph, or hallmark module, a discovery enabling us to measure the proximity of 6,442 clinically approved or experimental compounds to each hallmark. We then introduce a transcription-based metric, pAGE, which evaluates whether the drug-induced expression shifts reinforce or counteract known age-related expression changes. By integrating network proximity and pAGE, we identify multiple drug repurposing candidate that not only target specific hallmarks but act to reverse their aging-associated transcriptional changes. Our findings are interpretable, revealing for each drug the molecular mechanisms through which it modulates the hallmark, offering an experimentally falsifiable framework to leverage genomic discoveries to accelerate drug repurposing for longevity.
  50. Autophagy. 2025 Sep 19.
    Non-canonical role of ATG4B in PRMT1-mediated DNA repair and leukemia progression.
    Zhenkun Wang, Yuting Fu, Shuyi Lin, Yuanyuan Zhou, Qiongdan Gao, YanHong Xiao, Zhenyu Ju, Bo Liu.
      Accumulating evidence indicates that many ATG (autophagy related) proteins perform non-canonical functions beyond their canonical roles in autophagy, particularly when they localize to subcellular compartments outside the cytoplasm. Although the autophagic functions of ATG4B (autophagy related 4B, cysteine peptidase) are well established, its potential non-canonical roles, especially under metabolic stress, remain largely unexplored. In our recent study, we show that energy deprivation induces autophagy-independent nuclear translocation of ATG4B. In the nucleus, ATG4B interacts with and cleaves PRMT1 (protein arginine methyltransferase 1), thereby reducing PRMT1-mediated methylation of the DNA-repair nuclease MRE11 and consequently impairing DNA repair. Notably, ATG4B is significantly upregulated in acute myeloid leukemia (AML) and shows prominent nuclear accumulation. Genetic knockdown or pharmacological inhibition of ATG4B in AML cells restores DNA repair capacity, activates the cell-cycle checkpoint kinase CHEK1/CHK1, attenuates malignant progression, and ultimately delays leukemia progression. These findings reveal an autophagy-independent role for nuclear ATG4B that links metabolic stress to the suppression of DNA repair and identify ATG4B as a potential therapeutic target in AML.
    Keywords:  ATG4B; DNA repair; PRMT1; energy metabolism; leukemia
    DOI:  https://doi.org/10.1080/15548627.2025.2564225
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