bims-proteo Biomed News
on Proteostasis
Issue of 2025–02–16
forty-one papers selected by
Eric Chevet, INSERM
  1. The integrated stress response regulates 18S nonfunctional rRNA decay in mammals.
  2. RIOK3 mediates the degradation of 40S ribosomes.
  3. Selective clearance of aberrant membrane proteins by TORC1-mediated micro-ER-phagy.
  4. Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.
  5. Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.
  6. Initiation of ERAD by the bifunctional complex of Mnl1/Htm1 mannosidase and protein disulfide isomerase.
  7. De novo designed Hsp70 activator dissolves intracellular condensates.
  8. Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.
  9. Small molecule targeted protein degradation via the UPS: venturing beyond E3 substrate receptors.
  10. Workflow for E3 Ligase Ligand Validation for PROTAC Development.
  11. Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.
  12. UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.
  13. The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.
  14. Potential ER tubular lumen-sensing intrinsically disordered regions.
  15. The ribosome as a platform to coordinate mRNA decay.
  16. Unveiling FKBP7 as an Early Endoplasmic Reticulum Sentinel in Pancreatic Stellate Cell Activation, Collagen Remodelling and Tumor Progression.
  17. Discovery of 2,4-quinazolinedione derivatives as LC3B recruiters in the facilitation of protein complex degradations.
  18. Listerin promotes α-synuclein degradation to alleviate Parkinson's disease through the ESCRT pathway.
  19. Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum.
  20. Computational design of serine hydrolases.
  21. Protocol for the purification and analysis of nuclear UFMylated proteins.
  22. Diverse routes to mitophagy governed by ubiquitylation and mitochondrial import.
  23. Co-translational protein aggregation and ribosome stalling as a broad-spectrum antibacterial mechanism.
  24. Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation.
  25. The phenylalanine-and-glycine repeats of NUP98 oncofusions form condensates that selectively partition transcriptional coactivators.
  26. Impairment of DET1 causes neurological defects and lethality in mice and humans.
  27. Dynamic global acetylation remodeling during the yeast heat shock response.
  28. Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.
  29. The RNA helicase HrpA rescues collided ribosomes in E. coli.
  30. E3 ligase SYVN1-mediated polyubiquitination of CPSF6 promotes alternative polyadenylation and antivirus effects of macrophages.
  31. Autophagy-Activating Nanoautophagosome-Tethering Compounds for Targeted Protein Degradation Specifically in Tumor Cells.
  32. Loss of ATG7 in microglia impairs UPR, triggers ferroptosis, and weakens amyloid pathology control.
  33. Rpl12 is a conserved ribophagy receptor.
  34. Essential roles of the unfolded protein response in intestinal physiology.
  35. Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations and its dynamics.
  36. An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4.
  37. Human and mouse proteomics reveals the shared pathways in Alzheimer's disease and delayed protein turnover in the amyloidome.
  38. Transcriptional repression of autophagy and lysosome biogenesis.
  39. Transcriptomic Analysis Uncovers an Unfolded Protein Response in ADNP Syndrome.
  40. Free introns of tRNAs as complementarity-dependent regulators of gene expression.
  41. Targeted approach to determine the impact of cancer-associated protease variants.
  1. Mol Cell. 2025 Feb 07. pii: S1097-2765(25)00047-4. [Epub ahead of print]
    The integrated stress response regulates 18S nonfunctional rRNA decay in mammals.
    Aaztli R Coria, Akruti Shah, Mohammad Shafieinouri, Sarah J Taylor, Emilien Orgebin, Wilfried Guiblet, Jennifer T Miller, Indra Mani Sharma, Colin Chih-Chien Wu.
      18S nonfunctional rRNA decay (NRD) detects and eliminates translationally nonfunctional 18S rRNA. Although this process is critical for ribosome quality control, the mechanisms underlying nonfunctional 18S rRNA turnover remain elusive, particularly in mammals. Here, we show that mammalian 18S NRD initiates through the integrated stress response (ISR) via GCN2. Nonfunctional 18S rRNA induces translational arrest at start sites. Biochemical analyses demonstrate that ISR activation limits translation initiation and attenuates collisions between scanning 43S preinitiation complexes and stalled nonfunctional ribosomes. The ISR promotes 18S NRD and 40S ribosomal protein turnover by RNF10-mediated ubiquitination. Ultimately, RIOK3 binds the resulting ubiquitinated 40S subunits and facilitates 18S rRNA decay. Overall, mammalian 18S NRD acts through GCN2, followed by ubiquitin-dependent 18S rRNA degradation involving the ubiquitin E3 ligase RNF10 and the atypical protein kinase RIOK3. These findings establish a dynamic feedback mechanism by which the GCN2-RNF10-RIOK3 axis surveils ribosome functionality at the translation initiation step.
    Keywords:  GCN2; RIOK3; integrated stress response; nonfunctional 18S rRNA; ribosome stalling
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.017
  2. Mol Cell. 2025 Feb 06. pii: S1097-2765(25)00048-6. [Epub ahead of print]
    RIOK3 mediates the degradation of 40S ribosomes.
    Zixuan Huang, Frances F Diehl, Mengjiao Wang, Yi Li, Aixia Song, Fei Xavier Chen, Nicolle A Rosa-Mercado, Roland Beckmann, Rachel Green, Jingdong Cheng.
      Cells tightly regulate ribosome homeostasis to adapt to changing environments. Ribosomes are degraded during stress, but the mechanisms responsible remain unclear. Here, we show that starvation induces the selective depletion of 40S ribosomes following their ubiquitylation by the E3 ligase RNF10. The atypical kinase RIOK3 specifically recognizes these ubiquitylated 40S ribosomes through a unique ubiquitin-interacting motif, visualized by cryoelectron microscopy (cryo-EM). RIOK3 binding and ubiquitin recognition are essential for 40S ribosome degradation during starvation. RIOK3 induces the degradation of ubiquitylated 40S ribosomes through progressive decay of their 18S rRNA beginning at the 3' end, as revealed by cryo-EM structures of degradation intermediates. Together, these data define a pathway and mechanism for stress-induced degradation of 40S ribosomes, directly connecting ubiquitylation to regulation of ribosome homeostasis.
    Keywords:  40S; RIOK3; RNA degradation; cryo-EM; homeostasis; ribosome; starvation; stress response; translation; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.013
  3. Cell Rep. 2025 Feb 12. pii: S2211-1247(25)00053-1. [Epub ahead of print]44(2): 115282
    Selective clearance of aberrant membrane proteins by TORC1-mediated micro-ER-phagy.
    Valeriya Gyurkovska, Yaneris M Alvarado Cartagena, Rakhilya Murtazina, Sarah F Zhao, Candela Ximenez de Olaso, Nava Segev.
      Aberrant accumulation and clearance of membrane proteins is associated with disease. Membrane proteins are inserted first to the endoplasmic reticulum (ER). During normal growth, two quality control (QC) processes, ER-associated degradation and macro-ER-phagy, deliver misfolded and excess membrane proteins for degradation in the proteasome and lysosome, respectively. We show that in yeast during normal growth, ER-QC is constitutive, since none of the stress-induced signaling pathways-nutritional, proteotoxic, or heat-are involved. In mutant cells defective in ER-QC, misfolded or excess proteins accumulate and nutritional stress, but not proteotoxic or heat stress, can stimulate their clearance. Early during nutritional stress, clearance occurs in the lysosome through a selective micro-ER-phagy pathway dependent on the ubiquitin ligase Rsp5, its Ssh4 adaptor, and ESCRT. In contrast, only a fraction of normal membrane proteins is degraded much later via macro-autophagy. Because the pathways explored here are conserved, nutritional stress emerges as a possible way for clearing disease-associated membrane proteins.
    Keywords:  CP: Cell biology; ER-quality control; ERAD; Endoplasmic reticulum; HSR; TORC1; UPR; macro-ER-phagy; micro-ER-phagy; nutritional stress
    DOI:  https://doi.org/10.1016/j.celrep.2025.115282
  4. Nat Commun. 2025 Feb 10. 16(1): 1501
    Mitofusin 2 displays fusion-independent roles in proteostasis surveillance.
    Mariana Joaquim, Selver Altin, Maria-Bianca Bulimaga, Tânia Simões, Hendrik Nolte, Verian Bader, Camilla Aurora Franchino, Solenn Plouzennec, Karolina Szczepanowska, Elena Marchesan, Kay Hofmann, Marcus Krüger, Elena Ziviani, Aleksandra Trifunovic, Arnaud Chevrollier, Konstanze F Winklhofer, Elisa Motori, Margarete Odenthal, Mafalda Escobar-Henriques.
      Mitochondria are essential organelles and their functional state dictates cellular proteostasis. However, little is known about the molecular gatekeepers involved, especially in absence of external stress. Here we identify a role of MFN2 in quality control independent of its function in organellar shape remodeling. MFN2 ablation alters the cellular proteome, marked for example by decreased levels of the import machinery and accumulation of the kinase PINK1. Moreover, MFN2 interacts with the proteasome and cytosolic chaperones, thereby preventing aggregation of newly translated proteins. Similarly to MFN2-KO cells, patient fibroblasts with MFN2-disease variants recapitulate excessive protein aggregation defects. Restoring MFN2 levels re-establishes proteostasis in MFN2-KO cells and rescues fusion defects of MFN1-KO cells. In contrast, MFN1 loss or mitochondrial shape alterations do not alter protein aggregation, consistent with a fusion-independent role of MFN2 in cellular homeostasis. In sum, our findings open new possibilities for therapeutic strategies by modulation of MFN2 levels.
    DOI:  https://doi.org/10.1038/s41467-025-56673-5
  5. Elife. 2025 Feb 12. pii: RP100541. [Epub ahead of print]13
    Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.
    Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prerona Bora, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy E Shutt, Danielle A Grotjahn, R Luke Wiseman.
      Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.
    Keywords:  cell biology; human; integrated stress response; mitochondrial fragmentation; mitochondrial morphology; mouse; stress signaling
    DOI:  https://doi.org/10.7554/eLife.100541
  6. Nat Struct Mol Biol. 2025 Feb 10.
    Initiation of ERAD by the bifunctional complex of Mnl1/Htm1 mannosidase and protein disulfide isomerase.
    Dan Zhao, Xudong Wu, Tom A Rapoport.
      Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In Saccharomyces cerevisiae, the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a degradation signal. Interestingly, Mnl1 is associated with protein disulfide isomerase (Pdi1). Here we used cryo-electron microscopy, biochemical and in vivo experiments to elucidate how this complex initiates ERAD. The Mnl1-Pdi1 complex first demannosylates misfolded, globular proteins that are recognized through the C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex. Mnl1 blocks the canonical oxidative function of Pdi1, allowing it to function as a disulfide reductase in ERAD. The generated unfolded polypeptides can then be translocated across the membrane into the cytosol.
    DOI:  https://doi.org/10.1038/s41594-025-01491-y
  7. Cell Chem Biol. 2025 Feb 04. pii: S2451-9456(25)00029-7. [Epub ahead of print]
    De novo designed Hsp70 activator dissolves intracellular condensates.
    Jason Z Zhang, Nathan Greenwood, Jason Hernandez, Josh T Cuperus, Buwei Huang, Bryan D Ryder, Christine Queitsch, Jason E Gestwicki, David Baker.
      Protein quality control (PQC) is carried out in part by the chaperone Hsp70 in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of Hsp70 binding proteins that either inhibit or stimulate Hsp70 ATPase activity. An ATPase stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution and revealed roles as cell growth promoting signaling hubs. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to regulate condensates and other cellular targets.
    Keywords:  Hsp70; chaperones; condensates; protein design
    DOI:  https://doi.org/10.1016/j.chembiol.2025.01.006
  8. Cell Rep. 2025 Feb 12. pii: S2211-1247(25)00068-3. [Epub ahead of print]44(2): 115297
    Membrane-tethered SCOTIN condensates elicit an endoplasmic reticulum stress response by sequestering luminal BiP.
    Areum Jo, Minkyo Jung, Ji Young Mun, Young Jin Kim, Joo-Yeon Yoo.
      The endoplasmic reticulum (ER) stress response controls the balance between cellular survival and death. Here, we implicate SCOTIN, an interferon-inducible ER protein, in activating the ER stress response and modulating cell fate through its proline-rich domain (PRD)-mediated cytosolic condensation. SCOTIN overexpression leads to the formation of condensates enveloping multiple layers of the ER, accompanied by morphological signs of organelle stress. Luminal BiP chaperone proteins are sequestered within these SCOTIN condensates, which elicit ER stress responses. The colocalization of luminal BiP with SCOTIN is strictly contingent upon the PRD-mediated condensation of SCOTIN in the cytosolic compartment, closely associated with the ER membrane. The cysteine-rich domain (CRD) of SCOTIN, along with the condensation-prone PRD domain, is required for ER stress induction. We propose that membrane-associated condensation transduces signals across the ER membrane, leading to the induction of BiP assembly and the ER stress response.
    Keywords:  CP: Cell biology; ER stress; SCOTIN; SHISA-5; biomolecular condensate; cell death; endoplasmic reticulum; phase separation; signal transmission; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2025.115297
  9. RSC Med Chem. 2025 Feb 12.
    Small molecule targeted protein degradation via the UPS: venturing beyond E3 substrate receptors.
    Renyu Guo, Fukang Yang, Emily C Cherney.
      The ubiquitin proteasome system (UPS) has been successfully hi-jacked by both bifunctional and monovalent small molecules to affect the degradation of proteins that were once considered undruggable. This field has primarily focused on the targeted recruitment of proteins to substrate receptors on E3 ubiquitin ligases, which are only one part of the UPS. More recently, the field has begun to explore recruitment to other types of UPS proteins including E2 ubiquitin-conjugating enzymes, substrate adaptor proteins within the E3 complex, chaperone proteins that associate with E3s, proteasomal subunits, and proteasome-associated proteins. While these approaches are relatively nascent compared to more traditional E3 substrate receptor-based degradation, these approaches are starting to show promise and could offer unique advantages. This review will cover key findings in small molecule UPS-mediated targeted protein degradation (TPD) affected by co-opting proteins beyond traditional E3 substrate receptors.
    DOI:  https://doi.org/10.1039/d4md00718b
  10. ACS Chem Biol. 2025 Feb 11.
    Workflow for E3 Ligase Ligand Validation for PROTAC Development.
    Nebojša Miletić, Janik Weckesser, Thorsten Mosler, Rajeshwari Rathore, Marina E Hoffmann, Paul Gehrtz, Sarah Schlesiger, Ingo V Hartung, Nicola Berner, Stephanie Wilhelm, Juliane Müller, Bikash Adhikari, Václav Němec, Saran Aswathaman Sivashanmugam, Lewis Elson, Hanna Holzmann, Martin P Schwalm, Lasse Hoffmann, Kamal Rayees Abdul Azeez, Susanne Müller, Bernhard Kuster, Elmar Wolf, Ivan Đikić, Stefan Knapp.
      Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding "degradable" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development.
    DOI:  https://doi.org/10.1021/acschembio.4c00812
  11. Nature. 2025 Feb 12.
    Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.
    Xiaowen Xie, Olivia Zhang, Megan J R Yeo, Ceejay Lee, Ran Tao, Stefan A Harry, N Connor Payne, Eunju Nam, Leena Paul, Yiran Li, Hui Si Kwok, Hanjie Jiang, Haibin Mao, Jennifer L Hadley, Hong Lin, Melissa Batts, Pallavi M Gosavi, Vincenzo D'Angiolella, Philip A Cole, Ralph Mazitschek, Paul A Northcott, Ning Zheng, Brian B Liau.
      Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1,2. As a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma3, the most common embryonal brain tumour in children4. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST5. However, their mechanism remains unresolved. Here we establish that KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2 as the direct target of the mutant substrate receptor. Using deep mutational scanning, we chart the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat β-propeller domains. The interface between HDAC1 and one of the KBTBD4 β-propellers is stabilized by the medulloblastoma mutations, which insert a bulky side chain into the HDAC1 active site pocket. Our structural and mutational analyses inform how this hotspot E3-neosubstrate interface can be chemically modulated. First, we unveil a converging shape-complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface and proliferation of KBTBD4-mutant medulloblastoma cells. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions.
    DOI:  https://doi.org/10.1038/s41586-024-08533-3
  12. Nature. 2025 Feb 12.
    UM171 glues asymmetric CRL3-HDAC1/2 assembly to degrade CoREST corepressors.
    Megan J R Yeo, Olivia Zhang, Xiaowen Xie, Eunju Nam, N Connor Payne, Pallavi M Gosavi, Hui Si Kwok, Irtiza Iram, Ceejay Lee, Jiaming Li, Nicholas J Chen, Khanh Nguyen, Hanjie Jiang, Zhipeng A Wang, Kwangwoon Lee, Haibin Mao, Stefan A Harry, Idris A Barakat, Mariko Takahashi, Amanda L Waterbury, Marco Barone, Andrea Mattevi, Steven A Carr, Namrata D Udeshi, Liron Bar-Peled, Philip A Cole, Ralph Mazitschek, Brian B Liau, Ning Zheng.
      UM171 is a potent agonist of ex vivo human haematopoietic stem cell self-renewal1. By co-opting KBTBD4, a substrate receptor of the CUL3-RING E3 ubiquitin ligase (CRL3) complex, UM171 promotes the degradation of the LSD1-CoREST corepressor complex, thereby limiting haematopoietic stem cell attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here we show that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1/2 to promote corepressor degradation. Through proteomics and chemical inhibitor studies, we identify the principal target of UM171 as HDAC1/2. Cryo-electron microscopy analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex identifies an asymmetric assembly in which a single UM171 molecule enables a pair of KELCH-repeat propeller domains to recruit the HDAC1 catalytic domain. One KBTBD4 propeller partially masks the rim of the HDAC1 active site, which is exploited by UM171 to extend the E3-neosubstrate interface. The other propeller cooperatively strengthens HDAC1 binding through a distinct interface. The overall CoREST-HDAC1/2-KBTBD4 interaction is further buttressed by the endogenous cofactor inositol hexakisphosphate, which acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces is demonstrated by base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, we reveal how the cooperativity offered by a dimeric CRL3 E3 can be leveraged by a small molecule degrader.
    DOI:  https://doi.org/10.1038/s41586-024-08532-4
  13. J Cell Biol. 2025 Apr 07. pii: e202407105. [Epub ahead of print]224(4):
    The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.
    Brian J North, Amelia E Ohnstad, Michael J Ragusa, Christopher J Shoemaker.
      During autophagy, toxic cargo is encapsulated by autophagosomes and trafficked to lysosomes for degradation. NBR1, an autophagy receptor targeting ubiquitinated aggregates, serves as a model for studying the multivalent, heterotypic interactions of cargo-bound receptors. Here, we find that three critical NBR1 partners-ATG8-family proteins, FIP200, and TAX1BP1-each bind to distinct, overlapping determinants within a short linear interaction motif (SLiM). To explore whether overlapping SLiMs extend beyond NBR1, we analyzed >100 LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Phosphomimetic peptides demonstrate that phosphorylation generally enhances FIP200 and ATG8-family binding but not TAX1BP1, indicating differential regulation. In vivo, LIR-mediated interactions with TAX1BP1 promote optimal NBR1 flux by leveraging additional functionalities from TAX1BP1. These findings reveal a one-to-many binding modality in the LIR motif of NBR1, illustrating the cooperative mechanisms of autophagy receptors and the regulatory potential of multifunctional SLiMs.
    DOI:  https://doi.org/10.1083/jcb.202407105
  14. J Cell Sci. 2025 Feb 10. pii: jcs.263696. [Epub ahead of print]
    Potential ER tubular lumen-sensing intrinsically disordered regions.
    Tomohiro Yorimitsu, Ken Sato.
      Intrinsically disordered regions (IDRs) are known to sense the positive membrane curvature of vesicles and tubules. However, whether IDRs can sense the negative curvature of their luminal surfaces remains elusive. Here, we show that IDRs direct specific localization to ER tubules. In Saccharomyces cerevisiae, Sed4 interacts with Sec16 at the ER exit site (ERES) to promote ER export. Upon loss of this interaction, Sed4 failed to assemble at the ERES but was enriched in the ER tubules in a luminal region-dependent manner. Fusion of the Sed4 luminal region with Sec12 and Sec22, which localize throughout the ER, resulted in their enrichment in the tubules. The luminal regions of Sed4 or its homologs, predicted to be IDRs, localized to tubules when translocated alone into the ER lumen. The lumen-imported IDRs derived from cytosol-localizing Sec16 and Atg13 also exhibited tubule localization. Furthermore, Sed4 constructs with the luminal region replaced by these IDRs were concentrated at the ERES. Collectively, we suggest that the IDRs may sense the properties of the tubule lumen, such as its surface, and facilitate Sed4 assembly at the ERES.
    Keywords:  ER exit site; ER lumen; ER tubules; Intrinsically disordered region; Sed4
    DOI:  https://doi.org/10.1242/jcs.263696
  15. Nucleic Acids Res. 2025 Feb 08. pii: gkaf049. [Epub ahead of print]53(4):
    The ribosome as a platform to coordinate mRNA decay.
    Martin B D Müller, Thomas Becker, Timo Denk, Satoshi Hashimoto, Toshifumi Inada, Roland Beckmann.
      Messenger RNA (mRNA) homeostasis is a critical aspect of cellular function, involving the dynamic interplay between transcription and decay processes. Recent advances have revealed that the ribosome plays a central role in coordinating mRNA decay, challenging the traditional view that free mRNA is the primary substrate for degradation. This review examines the mechanisms whereby ribosomes facilitate both the licensing and execution of mRNA decay. This involves factors such as the Ccr4-Not complex, small MutS-related domain endonucleases, and various quality control pathways. We discuss how translational fidelity, as well as the presence of nonoptimal codons and ribosome collisions, can trigger decay pathways such as nonstop decay and no-go decay. Furthermore, we highlight the direct association of canonical exonucleases, such as Xrn1 and the Ski-exosome system, with the ribosome, underscoring the ribosome's multifaceted role as a platform for regulatory processes governing mRNA stability. By integrating recent findings, this review offers a comprehensive overview of the structural basis of how ribosomes not only facilitate translation but also serve as critical hubs for mRNA decay coordination.
    DOI:  https://doi.org/10.1093/nar/gkaf049
  16. Cancer Lett. 2025 Feb 07. pii: S0304-3835(25)00102-8. [Epub ahead of print] 217538
    Unveiling FKBP7 as an Early Endoplasmic Reticulum Sentinel in Pancreatic Stellate Cell Activation, Collagen Remodelling and Tumor Progression.
    Christophe Quemerais, Christine Jean, Alexia Brunel, Emilie Decaup, Guillaume Labrousse, Hippolyte Audureau, Jérôme Raffenne, Ismahane Belhabib, Jérôme Cros, Aurélie Perraud, Nelson Dusetti, Remy Nicolle, Muriel Mathonnet, Stéphane Pyronnet, Yvan Martineau, Marjorie Fanjul, Corinne Bousquet.
      In pancreatic ductal adenocarcinoma (PDAC), fibroblast activation leads to excessive secretion of extracellular matrix (ECM) and soluble factors that regulate tumor progression, prompting investigation into endoplasmic reticulum (ER)-resident proteins that may support this activation. We identified FKBP7, a peptidyl-prolyl isomerase in the ER, as overexpressed in PDAC stroma compared to cancer cells, and in patients with favorable prognosis. Analysis of single-cell RNA sequencing databases revealed FKBP7 expression in pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs). When analyzed by immunohistochemistry on PDAC patient tissues, FKBP7 emerged as an early activation marker in the preneoplastic stroma, preceding αSMA expression, and responding to FAK- and TGFβ-induced stiffening and pro-fibrotic programs in PSCs. Functional analyses revealed that FKBP7 knockdown in PSCs enhanced contractility, Rho/ FAK signaling, and secretion of pro-inflammatory cytokines as well as remodeling of type I collagen, promoting an activated phenotype and accelerating tumor growth in vivo. Conversely, FKBP7 expression supported a tumor-restraining (i.e. encapsulating) ECM characterized by type IV collagen. Mechanistically, FKBP7 interacts with BiP, and blocking this interaction instead leads to increased PSC secretion of type I collagen. Thus, FKBP7 serves as a novel PSC marker and ER regulator in a complex with BiP of the secretion of specific collagen subtypes, highlighting its potential to mediate ECM normalization and constrain PDAC tumorigenesis.
    Keywords:  BiP; Cancer-associated fibroblasts; Collagen; Endoplasmic reticulum; Extracellular matrix; Pancreatic Stellate Cells; Pancreatic cancer
    DOI:  https://doi.org/10.1016/j.canlet.2025.217538
  17. Eur J Med Chem. 2025 Jan 19. pii: S0223-5234(25)00058-3. [Epub ahead of print]287 117293
    Discovery of 2,4-quinazolinedione derivatives as LC3B recruiters in the facilitation of protein complex degradations.
    Yanping Zeng, Jian Xiao, Li Shi, Yangsha Li, Yuanxin Xu, Jiayun Zhou, Xiao Dong, Haiyang Hou, Chao Zhong, Gang Cheng, Yi Chen, Naixia Zhang, Yanfen Fang, Youhong Hu.
      Targeted protein degradation through autophagosome-tethering compounds (ATTECs) that bypasses the ubiquitination process has garnered increasing attention. LC3B, a key protein in autophagosome formation, recruits substrates into the autophagy-lysosome system for degradation. In this study, we systematically optimized 2,4-quinazolinedione derivatives as LC3B-recruiting fragments, utilizing the CDK9 indicator. By attaching the designed LC3B-recruiting fragment to CDK9 inhibitor SNS-032 through a linker, the resulting bifunctional ATTEC molecule simultaneously degraded CDK9 and its associated Cyclin T1. Two-dimensional NMR experiments confirmed the direct interaction between the novel LC3B-recruiting fragments and LC3B. Mechanistic studies elucidated that degradation occurred via an LC3B-dependent autophagy-lysosomal pathway. Additionally, the general applicability of leveraging LC3B-recruiting fragments linked to inhibitors for the targeted degradation of protein complexes was validated with PRC2 and CDK2/4/6 along with their respective Cyclins. This work provides a series of novel LC3B-recruiting fragments that enrich the ATTEC toolbox and can be applied to the degradation of diverse intracellular disease-causing proteins.
    Keywords:  CDK9; Degradation; LC3B; Protein complex
    DOI:  https://doi.org/10.1016/j.ejmech.2025.117293
  18. Sci Adv. 2025 Feb 14. 11(7): eadp3672
    Listerin promotes α-synuclein degradation to alleviate Parkinson's disease through the ESCRT pathway.
    Fei Qin, Runyu Cao, Wenjing Cui, Xuemei Bai, Jiahua Yuan, Yuling Zhang, Yaxing Liu, Nan Cao, Na Dong, Min Zhou, Tian Chen, Feng Liu, Wanwei Sun, Yi Zheng, Wei Zhao, Bingyu Liu, Chengjiang Gao.
      Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive accumulation of abnormal α-synuclein (α-syn) within dopaminergic neurons in the substantia nigra region of the brain. Despite excessive accumulation of α-syn being key to the pathogenesis of PD, the mechanisms governing its clearance remain elusive. In this study, we found that the endosomal sorting complex required for transport (ESCRT) system plays a crucial role in capturing and facilitating the degradation of ubiquitinated α-syn. The E3 ubiquitin ligase Listerin was found to promote K27-linked polyubiquitination of α-syn, directing it to the endosome for subsequent degradation. We showed that the deletion of the Listerin gene exacerbates the neurodegenerative progression in a mouse model of PD, whereas the overexpression of Listerin effectively mitigates disease progression in PD mice. Consequently, our study reveals a mechanism for α-syn degradation and identifies Listerin as a promising therapeutic target for the treatment of PD.
    DOI:  https://doi.org/10.1126/sciadv.adp3672
  19. Mol Biol Cell. 2025 Feb 12. mbcE24120542
    Orchestration of SARS-CoV-2 Nsp4 and host-cell ESCRT proteins induces morphological changes of the endoplasmic reticulum.
    Allison Kifer, Franciso Pina, Nicholas Codallos, Anita Hermann, Lauren Ziegler, Maho Niwa.
      Upon entry into the host cell, the non-structural proteins 3, 4, and 6 (Nsp3, Nsp 4, and Nsp6) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate the formation of double-membrane vesicles (DMVs) through extensive rearrangement of the host cell endoplasmic reticulum (ER) to replicate the viral genome and translate viral proteins. To dissect the functional roles of each Nsp and the molecular mechanisms underlying the ER changes, we exploited both yeast S. cerevisiae and human cell experimental systems. Our results demonstrate that Nsp4 alone is sufficient to induce ER structural changes. Nsp4 expression led to robust activation of both the unfolded protein response (UPR) and the ER surveillance (ERSU) cell cycle checkpoint, resulting in cortical ER inheritance block and septin ring mislocalization. Interestingly, these ER morphological changes occurred independently of the canonical UPR and ERSU components but were mediated by the endosomal sorting complex for transport (ESCRT) proteins Vps4 and Vps24 in yeast. Similarly, ER structural changes occurred in human cells upon Nsp4 expression, providing a basis for a minimal experimental system for testing the involvement of human ESCRT proteins and ultimately advancing our understanding of DMV formation.
    DOI:  https://doi.org/10.1091/mbc.E24-12-0542
  20. Science. 2025 Feb 13. eadu2454
    Computational design of serine hydrolases.
    Anna Lauko, Samuel J Pellock, Kiera H Sumida, Ivan Anishchenko, David Juergens, Woody Ahern, Jihun Jeung, Alex Shida, Andrew Hunt, Indrek Kalvet, Christoffer Norn, Ian R Humphreys, Cooper Jamieson, Rohith Krishna, Yakov Kipnis, Alex Kang, Evans Brackenbrough, Asim K Bera, Banumathi Sankaran, K N Houk, David Baker.
      The design of enzymes with complex active sites that mediate multistep reactions remains an outstanding challenge. With serine hydrolases as a model system, we combined the generative capabilities of RFdiffusion with an ensemble generation method for assessing active site preorganization to design enzymes starting from minimal active site descriptions. Experimental characterization revealed catalytic efficiencies (kcat/Km) up to 2.2x105 M-1 s-1 and crystal structures that closely match the design models (Cα RMSDs < 1 Å). Selection for structural compatibility across the reaction coordinate enabled identification of new catalysts in low-throughput screens with five different folds distinct from those of natural serine hydrolases. Our de novo approach provides insight into the geometric basis of catalysis and a roadmap for designing enzymes that catalyze multistep transformations.
    DOI:  https://doi.org/10.1126/science.adu2454
  21. STAR Protoc. 2025 Feb 10. pii: S2666-1667(25)00040-1. [Epub ahead of print]6(1): 103634
    Protocol for the purification and analysis of nuclear UFMylated proteins.
    Pudchalaluck Panichnantakul, Marlene Oeffinger.
      Protein UFMylation regulates numerous cellular processes including ribosome quality control and nuclear DNA repair. Here, we present a technique to isolate nuclei and purify UFMylated proteins under denaturing non-reducing conditions from commonly used mammalian cell line models such as hTERT-RPE1, HEK293, U2OS, and HCT116 cells. We then describe procedures for identifying and analyzing purified UFMylated proteins using mass spectrometry and western blot. For complete details on the use and execution of this protocol, please refer to Panichnantakul et al.1.
    Keywords:  Cell Biology; Cell separation/fractionation; Proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2025.103634
  22. Trends Cell Biol. 2025 Feb 07. pii: S0962-8924(25)00003-0. [Epub ahead of print]
    Diverse routes to mitophagy governed by ubiquitylation and mitochondrial import.
    Michael J Clague, Sylvie Urbé.
      The selective removal of mitochondria by mitophagy proceeds via multiple mechanisms and is essential for human well-being. The PINK1/Parkin and NIX/BNIP3 pathways are strongly linked to mitochondrial dysfunction and hypoxia, respectively. Both are regulated by ubiquitylation and mitochondrial import. Recent studies have elucidated how the ubiquitin kinase PINK1 acts as a sensor of mitochondrial import stress through stable interaction with a mitochondrial import supercomplex. The stability of BNIP3 and NIX is regulated by the SCFFBXL4 ubiquitin ligase complex. Substrate recognition requires an adaptor molecule, PPTC7, whose availability is limited by mitochondrial import. Unravelling the functional implications of each mode of mitophagy remains a critical challenge. We propose that mitochondrial import stress prompts a switch between these two pathways.
    Keywords:  BNIP3; FBXL4; PINK1; PPTC7; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1016/j.tcb.2025.01.003
  23. Nat Commun. 2025 Feb 12. 16(1): 1561
    Co-translational protein aggregation and ribosome stalling as a broad-spectrum antibacterial mechanism.
    Laleh Khodaparast, Ladan Khodaparast, Ramon Duran-Romaña, Guiqin Wu, Bert Houben, Wouter Duverger, Matthias De Vleeschouwer, Katerina Konstantoulea, Fleur Nysen, Thomas Schalck, Daniel J Curwen, Lisandra L Martin, Sebastien Carpentier, Bernard Scorneaux, Jan Michiels, Joost Schymkowitz, Frederic Rousseau.
      Drug-resistant bacteria pose an urgent global health threat, necessitating the development of antibacterial compounds with novel modes of action. Protein biosynthesis accounts for up to half of the energy expenditure of bacterial cells, and consequently inhibiting the efficiency or fidelity of the bacterial ribosome is a major target of existing antibiotics. Here, we describe an alternative mode of action that affects the same process: allowing translation to proceed but causing co-translational aggregation of the nascent peptidic chain. We show that treatment with an aggregation-prone peptide induces formation of polar inclusion bodies and activates the SsrA ribosome rescue pathway in bacteria. The inclusion bodies contain ribosomal proteins and ribosome hibernation factors, as well as mRNAs and cognate nascent chains of many proteins in amyloid-like structures, with a bias for membrane proteins with a fold rich in long-range beta-sheet interactions. The peptide is bactericidal against a wide range of pathogenic bacteria in planktonic growth and in biofilms, and reduces bacterial loads in mouse models of Escherichia coli and Acinetobacter baumannii infections. Our results indicate that disrupting protein homeostasis via co-translational aggregation constitutes a promising strategy for development of broad-spectrum antibacterials.
    DOI:  https://doi.org/10.1038/s41467-025-56873-z
  24. PLoS Pathog. 2025 Feb;21(2): e1012934
    Inhibition of the Integrated stress response by Epstein-Barr virus oncoprotein LMP1 attenuates epithelial cell differentiation and lytic viral reactivation.
    Deo R Singh, Yitao Zhang, Sophia J White, Scott E Nelson, Stuart A Fogarty, Abigail S Pawelski, Alisha S Kansra, Shannon C Kenney.
      EBV infects normal oral keratinocytes (NOKs) and plays an essential role in undifferentiated nasopharyngeal carcinoma (NPC). We previously showed that the EBV oncogene, LMP1, promotes proliferation and inhibits spontaneous differentiation in telomerase-immortalized NOKs grown in growth factor-restricted conditions. Here we have further examined the phenotypes of NOKs infected with wild-type EBV (WT EBV) versus an LMP1-deleted EBV mutant (ΔLMP1 EBV) in growth factor-restricted conditions. RNA-seq results show that WT EBV-infected NOKs not only have reduced differentiation, but also decreased expression of genes activated by the integrated stress response (ISR) pathway, in comparison to the ΔLMP1 EBV-infected cells. The ISR pathway is mediated by increased phosphorylation of the eIF2α translation initiation factor, leading to decreased translation of most cellular proteins but increased expression of some proteins, including ATF4 and CHOP. Immunoblot analyses confirmed that WT EBV-infected NOKs have decreased phosphorylation of eIF2α in comparison to uninfected and ΔLMP1 EBV-infected cells and showed that expression of LMP1 alone is sufficient to inhibit eIF2α phosphorylation. We found that LMP1 decreases the activity of two different eIF2α kinases, PERK and GCN2, in WT EBV-infected NOKs, resulting in decreased expression of the ISR-induced transcription factors, ATF4 and CHOP, in WT EBV-infected versus uninfected and ΔLMP1 EBV-infected NOKs. Furthermore, we found that both GCN2 and PERK activity are required for efficient TPA-induced lytic EBV reactivation and TPA-mediated epithelial cell differentiation. In addition, we demonstrate that over-expression of CHOP is sufficient to induce both lytic EBV reactivation and epithelial cell differentiation in WT EBV-infected NOKs and NPC cells and show that this effect is mediated by CHOP activation of the differentiation-inducing transcription factors, KLF4 and BLIMP1. Our results suggest that inhibition of the ISR pathway by the EBV oncoprotein, LMP1, may promote early NPC development by preventing epithelial cell differentiation and lytic EBV reactivation.
    DOI:  https://doi.org/10.1371/journal.ppat.1012934
  25. Mol Cell. 2025 Feb 04. pii: S1097-2765(24)01067-0. [Epub ahead of print]
    The phenylalanine-and-glycine repeats of NUP98 oncofusions form condensates that selectively partition transcriptional coactivators.
    Jeong Hyun Ahn, Yiran Guo, Heankel Lyons, Samuel G Mackintosh, Benjamin K Lau, Ricky D Edmondson, Stephanie D Byrum, Aaron J Storey, Alan J Tackett, Ling Cai, Benjamin R Sabari, Gang Greg Wang.
      Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remains poorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the disordered phenylalanine-and-glycine (FG)-repeat-rich region of NUP98 and the H3K4me3/2-binding plant homeodomain (PHD) finger domain of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD domain targets condensate to the H3K4me3/2-demarcated developmental genes, while FG repeats determine the condensate composition and gene activation. FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the KMT2/MLL H3K4 methyltransferases, histone acetyltransferases, and BRD4. FG repeats are sufficient to partition transcriptional regulators and activate a reporter when tethered to a genomic locus. NUP98-PHF23 assembles the chromatin-bound condensates that partition multiple positive regulators, initiating a feedforward loop of reading-and-writing the active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs.
    Keywords:  MLL; NUP98; WDR5; biomolecular condensate; coactivator; fusion protein; leukemia; phase separation; selective partitioning; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molcel.2024.12.026
  26. Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2422631122
    Impairment of DET1 causes neurological defects and lethality in mice and humans.
    Ozge Karayel, Allison Soung, Hem Gurung, Alexander F Schubert, Susan Klaeger, Marc Kschonsak, Aljazi Al-Maraghi, Ajaz A Bhat, Ammira S Alshabeeb Akil, Debra L Dugger, Joshua D Webster, Dorothy M French, Dhullipala Anand, Naharmal Soni, Khalid A Fakhro, Christopher M Rose, Seth F Harris, Ada Ndoja, Kim Newton, Vishva M Dixit.
      COP1 and DET1 are components of an E3 ubiquitin ligase that is conserved from plants to humans. Mammalian COP1 binds to DET1 and is a substrate adaptor for the CUL4A-DDB1-RBX1 RING E3 ligase. Transcription factor substrates, including c-Jun, ETV4, and ETV5, are targeted for proteasomal degradation to effect rapid transcriptional changes in response to cues such as growth factor deprivation. Here, we link a homozygous DET1R26W mutation to lethal developmental abnormalities in humans. Experimental cryo-electron microscopy of the DET1 complex with DDB1 and DDA1, as well as co-immunoprecipitation experiments, revealed that DET1R26W impairs binding to DDB1, thereby compromising E3 ligase function. Accordingly, human-induced pluripotent stem cells homozygous for DET1R26W expressed ETV4 and ETV5 highly, and exhibited defective mitochondrial homeostasis and aberrant caspase-dependent cell death when differentiated into neurons. Neuronal cell death was increased further in the presence of Det1-deficient microglia as compared to WT microglia, indicating that the deleterious effects of the DET1 p.R26W mutation may stem from the dysregulation of multiple cell types. Mice lacking Det1 died during embryogenesis, while Det1 deletion just in neural stem cells elicited hydrocephalus, cerebellar dysplasia, and neonatal lethality. Our findings highlight an important role for DET1 in the neurological development of mice and humans.
    Keywords:  COP1; DET1; E3 ligase; neurodevelopment; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2422631122
  27. bioRxiv. 2025 Jan 10. pii: 2025.01.10.632339. [Epub ahead of print]
    Dynamic global acetylation remodeling during the yeast heat shock response.
    Rebecca E Hardman-Kavanaugh, Aaron J Storey, Tara N Stuecker, Stephanie E Hood, Gregory A Barrett-Wilt, Venkata R Krishnamurthi, Yong Wang, Stephanie D Byrum, Samuel G Mackintosh, Rick D Edmondson, Wayne P Wahls, Alan J Tackett, Jeffrey A Lewis.
      All organisms experience stress and must rapidly respond to changing conditions. Thus, cells have evolved sophisticated rapid-response mechanisms such as post-translational protein modification to rapidly and reversibly modulate protein activity. One such post-translational modification is reversible lysine acetylation, where proteomic studies have identified thousands of acetylated proteins across diverse organisms. While the sheer size of the 'acetylome' is striking, the function of acetylation for the vast majority of proteins remains largely obscure. Here, we show that global acetylation plays a previously unappreciated role in the heat shock response of Saccharomyces cerevisiae. We find that dysregulated acetylation renders cells heat sensitive, and moreover, that the acetylome is globally remodeled during heat shock over time. Using quantitative acetyl-proteomics, we identified ∼400 high-confidence acetyl marks across ∼200 proteins that significantly change in acetylation when cells are shifted to elevated temperature. Proteins with significant changes in lysine acetylation during heat shock strongly overlap with genes induced or repressed by stress. Thus, we hypothesize that protein acetylation augments the heat shock response by activating induced proteins and inactivating repressed proteins. Intriguingly, we find nearly 40 proteins with at least two acetyl marks that significantly change in the opposite directions. These proteins are strongly enriched for chaperones and ribosomal proteins, suggesting that these two key processes are coordinately regulated by protein acetylation during heat shock. Moreover, we hypothesize that the same type of activating and inactivating marks that exist on histones may be a general feature of proteins regulated by acetylation. Overall, this work has identified a new layer of post-translational regulation that likely augments the classic heat shock response.
    DOI:  https://doi.org/10.1101/2025.01.10.632339
  28. Cell Mol Life Sci. 2025 Feb 13. 82(1): 76
    Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.
    Flavien Picard, Takashi Nonaka, Edwige Belotti, Alexis Osseni, Elisabeth Errazuriz-Cerda, Coline Jost-Mousseau, Emilien Bernard, Agnès Conjard-Duplany, Delphine Bohl, Masato Hasegawa, Cédric Raoul, Thierry Galli, Laurent Schaeffer, Pascal Leblanc.
      Proteinopathies, such as amyotrophic lateral sclerosis (ALS), are marked by the accumulation of misfolded proteins that disrupt cellular processes. Eukaryotic cells have developed protein quality control systems to eliminate these aberrant proteins, but these systems often fail to differentiate between normal and misfolded proteins. In ALS, pathological inclusions primarily composed of misfolded TDP-43 are a hallmark of the disease. Recently, a novel unconventional secretion process called misfolding-associated protein secretion (MAPS) has been discovered to selectively export misfolded proteins. USP19, an Endoplasmic Reticulum-associated ubiquitin peptidase, plays a crucial role in this process. In this study, we investigated the impact of ER-anchored USP19 on the secretion of misfolded TDP-43. Here we found that USP19 overexpression significantly promotes the secretion of soluble and aggregated misfolded TDP-43, requiring both ER anchoring and ubiquitin peptidase activity. Characterization of the cellular and molecular mechanisms involved in this process highlighted the importance of early autophagosomal and late endosomal/amphisomal compartments, while lysosomes did not play a key role. By using dominant-negative mutants and small interfering RNAs, we identified that USP19-mediated secretion of misfolded TDP-43 is modulated by key factors involved in cellular trafficking and secretion pathways, such as ATG7, the ESCRT-O HGS/HRS, the Rab GTPases RAB11A, RAB8A, and RAB27A, and the v-SNARE VAMP7. We also confirmed the crucial role of the DNAJC5/CSPα cochaperone. Overall, this study provides new insights into how cells manage the secretion of misfolded TDP-43 proteins and potentially opens new avenues for therapeutic interventions in ALS and related disorders.
    Keywords:  ALS; Aggregates; Autophagosomes; Autophagy; Endosomes; Misfolding; Release/secretion; TDP-43; USP19; Ubiquitin peptidase
    DOI:  https://doi.org/10.1007/s00018-025-05589-w
  29. Mol Cell. 2025 Jan 30. pii: S1097-2765(25)00052-8. [Epub ahead of print]
    The RNA helicase HrpA rescues collided ribosomes in E. coli.
    Annabelle Campbell, Hanna F Esser, A Maxwell Burroughs, Otto Berninghausen, L Aravind, Thomas Becker, Rachel Green, Roland Beckmann, Allen R Buskirk.
      Although many antibiotics inhibit bacterial ribosomes, the loss of known factors that rescue stalled ribosomes does not lead to robust antibiotic sensitivity in E. coli, suggesting the existence of additional mechanisms. Here, we show that the RNA helicase HrpA rescues stalled ribosomes in E. coli. Acting selectively on ribosomes that have collided, HrpA uses ATP hydrolysis to split stalled ribosomes into subunits. Cryoelectron microscopy (cryo-EM) structures reveal how HrpA simultaneously binds to two collided ribosomes, explaining its selectivity, and how its helicase module engages downstream mRNA such that, by exerting a pulling force on the mRNA, it would destabilize the stalled ribosome. These studies show that ribosome splitting is a conserved mechanism that allows proteobacteria to tolerate ribosome-targeting antibiotics.
    Keywords:  DEAH-box protein; HrpA; SmrB; cryo-EM; ribosome collisions; ribosome splitting
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.018
  30. Cell Rep. 2025 Feb 13. pii: S2211-1247(25)00047-6. [Epub ahead of print]44(2): 115276
    E3 ligase SYVN1-mediated polyubiquitination of CPSF6 promotes alternative polyadenylation and antivirus effects of macrophages.
    Xin Lu, Chao Liu, Runze Wu, Zhijie Hu, Susu Liu, Xuening Li, Yuchi Liu, Mengxia Li, Jingting Liang, Yingye Huang, Yuting Han, Xin Ou, Ke Deng, Cheng Liang, Shangwu Chen, Yonggui Fu, Anlong Xu.
      Transcriptome-wide alternative polyadenylation (APA) is involved in both innate and adaptive immune responses of immune cells. Downregulation of the CPSF6 protein, one of the 3' end-processing factors, mediates APA in macrophages with responses to virus infection and plays an important role in its anti-virus effect. However, the signaling pathway and molecular mechanism underlying the downregulation of the CPSF6 protein remain elusive. Here, we found that MAVS triggers the nuclear import of the E3 ligase SYVN1 mediated by NUP153 in response to vesicular stomatitis virus infection. Then, SYVN1 catalyzes K48-linked polyubiquitination of CPSF6, resulting in degradation of CPSF6 via the proteasome and then transcriptome-wide APA and anti-virus effects. Our results identify an antiviral mechanism via APA regulation based on ubiquitination modification of the CPSF6 protein, which may serve as a target for developing immune interventions.
    Keywords:  CP: Immunology; CP: Molecular biology; CPSF6; SYVN1; alternative polyadenylation; antiviral immunity; ubiquitinated proteasome pathway
    DOI:  https://doi.org/10.1016/j.celrep.2025.115276
  31. ACS Macro Lett. 2025 Feb 11. 250-257
    Autophagy-Activating Nanoautophagosome-Tethering Compounds for Targeted Protein Degradation Specifically in Tumor Cells.
    Mengchen Xu, Jiajing Chen, Shuyu Wang, Linlin Xu, Xiaohui Wu, Jinpu Yu, Feihe Ma, Linqi Shi.
      Autophagosome-tethering compounds (ATTECs) represent an emerging targeted protein degradation (TPD) technology that directly draws intracellular proteins of interest (POIs) into autolysosomes. Although ATTECs are currently dominated by small molecules, the poor cell-type specificity and pharmacokinetic profile limit their applications in certain diseases. Moreover, the suboptimal intrinsic autophagic activity of cells affects the ATTECs-mediated degradation capability. Here we develop a nano-ATTEC system using our unique mixed-shell polymeric micelle (MSPM)-based nanoplatform for tumor-specific degradation of POIs. We demonstrate that the MSPMs-based nano-ATTEC is efficiently taken up by tumor cells in the acidic tumor microenvironment and to degrade POIs, rather than by normal cells under physiological conditions. More importantly, we find that this nano-ATTEC can not only target autolysosomes but also robustly enhance the autophagy activity, thereby establishing a positive feedback mechanism based on the autophagy pathway for efficient degradation of POIs. We believe that this MSPMs-based nano-ATTEC will find broad applications in tumor therapy.
    DOI:  https://doi.org/10.1021/acsmacrolett.4c00789
  32. J Exp Med. 2025 Apr 07. pii: e20230173. [Epub ahead of print]222(4):
    Loss of ATG7 in microglia impairs UPR, triggers ferroptosis, and weakens amyloid pathology control.
    Zhangying Cai, Shoutang Wang, Siyan Cao, Yun Chen, Silvia Penati, Vincent Peng, Carla M Yuede, Wandy L Beatty, Kent Lin, Yiyang Zhu, Yingyue Zhou, Marco Colonna.
      Microglia impact brain development, homeostasis, and pathology. One important microglial function in Alzheimer's disease (AD) is to contain proteotoxic amyloid-β (Aβ) plaques. Recent studies reported the involvement of autophagy-related (ATG) proteins in this process. Here, we found that microglia-specific deletion of Atg7 in an AD mouse model impaired microglia coverage of Aβ plaques, increasing plaque diffusion and neurotoxicity. Single-cell RNA sequencing, biochemical, and immunofluorescence analyses revealed that Atg7 deficiency reduces unfolded protein response (UPR) while increasing oxidative stress. Cellular assays demonstrated that these changes lead to lipoperoxidation and ferroptosis of microglia. In aged mice without Aβ buildup, UPR reduction and increased oxidative damage induced by Atg7 deletion did not impact microglia numbers. We conclude that reduced UPR and increased oxidative stress in Atg7-deficient microglia lead to ferroptosis when exposed to proteotoxic stress from Aβ plaques. However, these microglia can still manage misfolded protein accumulation and oxidative stress as they age.
    DOI:  https://doi.org/10.1084/jem.20230173
  33. Nat Cell Biol. 2025 Feb 11.
    Rpl12 is a conserved ribophagy receptor.
    Yuting Chen, Jiaxin Hu, Pengwei Zhao, Jie Fang, Yingqi Kuang, Zhaojie Liu, Shuling Dong, Weijing Yao, Yuanyuan Ding, Xinhui Wang, Yibin Pan, Jianbin Wu, Jingwei Zhao, Jing Yang, Zhenzhong Xu, Xiaodi Liu, Yi Zhang, Choufei Wu, Liqin Zhang, Mingzhu Fan, Shan Feng, Zhi Hong, Zhangming Yan, Hongguang Xia, Kaiyue Tang, Bing Yang, Wei Liu, Qiming Sun, Kunrong Mei, Wei Zou, Yunpeng Huang, Du Feng, Cong Yi.
      Ribophagy is a selective autophagic process that regulates ribosome turnover. Although NUFIP1 has been identified as a mammalian receptor for ribophagy, its homologues do not exist in yeast and nematodes. Here we demonstrate that Rpl12, a ribosomal large subunit protein, functions as a conserved ribophagy receptor in multiple organisms. Disruption of Rpl12-Atg8s binding leads to significant accumulation of ribosomal proteins and rRNA, while Atg1-mediated Rpl12 phosphorylation enhances its association with Atg11, thus triggering ribophagy during starvation. Ribophagy deficiency accelerates cell death induced by starvation and pathogen infection, leading to impaired growth and development and a shortened lifespan in both Caenorhabditis elegans and Drosophila melanogaster. Moreover, ribophagy deficiency results in motor impairments associated with ageing, while the overexpression of RPL12 significantly improves movement defects induced by starvation, ageing and Aβ accumulation in fly models. Our findings suggest that Rpl12 functions as a conserved ribophagy receptor vital for ribosome metabolism and cellular homeostasis.
    DOI:  https://doi.org/10.1038/s41556-024-01598-2
  34. eGastroenterology. 2024 Oct;2(4): e100129
    Essential roles of the unfolded protein response in intestinal physiology.
    Claudio Hetz, Juan Francisco Silva-Agüero, Lisa M Ellerby.
      The intestinal epithelium serves as an essential interface between the host and microbiota, regulating innate and adaptive immunity, absorption of nutrients and systemic metabolism, and mediating bidirectional communication with the nervous system. The intestinal epithelium suffers constant challenges to the proteostasis machinery due to its exposure to the dynamically changing and microbial laden lumenal gut environment and to the high secretory demand placed on multiple epithelial cell types to accommodate gut and systemic physiology-especially goblet, enteroendocrine and Paneth cells. In all cases, intestinal cells require an active unfolded protein response (UPR) to sustain their physiological function, the main pathway that monitors and adjusts secretory function changes in the environment. A specialised endoplasmic reticulum (ER) stress sensor uniquely expressed in epithelial cells lining mucosal surfaces, termed inositol-requiring transmembrane kinase/endoribonuclease β, has specific roles in intestinal epithelial homeostasis, regulating mucus production and communication with microbiota. Chronic ER stress or genetic mutations affecting key UPR mediators contribute to the occurrence of inflammatory bowel disease and ulcerative colitis, in addition to colon cancer. Here, we review recent advances linking the UPR and ER stress with gut physiology and intestinal disease. Therapeutic strategies to alleviate ER stress or enforce UPR function to improve intestinal function in ageing and in bowel diseases are also discussed.
    Keywords:  Crohn's Disease; Digestive System Diseases; Gastrointestinal Diseases; Inflammation; Microbiota
    DOI:  https://doi.org/10.1136/egastro-2024-100129
  35. Proc Natl Acad Sci U S A. 2025 Feb 18. 122(7): e2412117122
    Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations and its dynamics.
    Toma Ikeda, Tatsuya Nojima, Souma Yamamoto, Ryusei Yamada, Tatsuya Niwa, Hiroki Konno, Hideki Taguchi.
      According to classical Anfinsen's dogma, a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, certain proteins may fold into two or more conformations from single amino acid sequences. Here, we designed a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal β-strand and dihydrofolate reductase (DHFR) lacking the N-terminal β-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analyses, including atomic force microscopy (AFM) imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. The drastic conformational change upon the ligand binding was directly visualized by high-speed AFM. Furthermore, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a unique direction for creating artificial proteins with on-off behaviors.
    Keywords:  DHFR; GFP; high-speed AFM; metamorphic protein; protein folding
    DOI:  https://doi.org/10.1073/pnas.2412117122
  36. J Biol Chem. 2025 Feb 06. pii: S0021-9258(25)00123-1. [Epub ahead of print] 108275
    An in vitro platform for the enzymatic characterization of the rhomboid protease RHBDL4.
    Satarupa Bhaduri, Mac Kevin E Braza, Stancho Stanchev, Marina Tauber, Raghad Al-Bawab, Lawrence J Liu, Diego F Trujillo, Kristina Solorio-Kirpichyan, Ambuj Srivastava, Javier Sanlley-Hernandez, Anthony J O'Donoghue, Marius K Lemberg, Rommie Amaro, Kvido Strisovsky, Sonya E Neal.
      Rhomboid proteases are ubiquitous intramembrane serine proteases that can cleave transmembrane substrates within lipid bilayers. They exhibit many and diverse functions, such as but not limited to, growth factor signaling, immune and inflammatory response, protein quality control, and parasitic invasion. Human rhomboid protease RHBDL4 has been demonstrated to play a critical role in removing misfolded proteins from the Endoplasmic Reticulum and is implicated in severe diseases such as various cancers and Alzheimer's disease. Therefore, RHBDL4 is expected to constitute an important therapeutic target for such devastating diseases. Despite its critical role in many biological processes, the enzymatic properties of RHBDL4 remain largely unknown. To enable a comprehensive characterization of RHBDL4's kinetics, catalytic parameters, substrate specificity, and binding modality we expressed and purified recombinant RHBDL4, and employed it in a Förster Resonance Energy Transfer-based cleavage assay. Until now, kinetic studies have been limited mostly to bacterial rhomboid proteases. Our in vitro platform offers a new method for studying RHBDL4's enzymatic function and substrate preferences. Furthermore, we developed and tested potential inhibitors using our assay and successfully identified peptidyl α-ketoamide inhibitors of RHBDL4 that are highly effective against recombinant RHBDL4. We utilize ensemble docking and molecular dynamics (MD) simulations to explore the binding modality of substrate-derived peptides bound to RHBDL4. Our analysis focused on key interactions and dynamic movements within RHBDL4's active site that contributed to binding stability, offering valuable insights for optimizing the non-prime side of RHBDL4 ketoamide inhibitors. In summary, our study offers fundamental insights into RHBDL4's catalytic activities and substrate preferences, laying the foundation for downstream applications such as drug inhibitor screenings and structure-function studies, which will enable the identification of lead drug compounds for RHBDL4.
    Keywords:  endoplasmic reticulum stress; endoplasmic‐reticulum‐associated protein degradation; enzyme inhibitor; enzyme kinetics; enzyme purification; enzyme structure; protein misfolding; rhomboid protease; serine protease
    DOI:  https://doi.org/10.1016/j.jbc.2025.108275
  37. Nat Commun. 2025 Feb 11. 16(1): 1533
    Human and mouse proteomics reveals the shared pathways in Alzheimer's disease and delayed protein turnover in the amyloidome.
    Jay M Yarbro, Xian Han, Abhijit Dasgupta, Ka Yang, Danting Liu, Him K Shrestha, Masihuz Zaman, Zhen Wang, Kaiwen Yu, Dong Geun Lee, David Vanderwall, Mingming Niu, Huan Sun, Boer Xie, Ping-Chung Chen, Yun Jiao, Xue Zhang, Zhiping Wu, Surendhar R Chepyala, Yingxue Fu, Yuxin Li, Zuo-Fei Yuan, Xusheng Wang, Suresh Poudel, Barbora Vagnerova, Qianying He, Andrew Tang, Patrick T Ronaldson, Rui Chang, Gang Yu, Yansheng Liu, Junmin Peng.
      Murine models of Alzheimer's disease (AD) are crucial for elucidating disease mechanisms but have limitations in fully representing AD molecular complexities. Here we present the comprehensive, age-dependent brain proteome and phosphoproteome across multiple mouse models of amyloidosis. We identified shared pathways by integrating with human metadata and prioritized components by multi-omics analysis. Collectively, two commonly used models (5xFAD and APP-KI) replicate 30% of the human protein alterations; additional genetic incorporation of tau and splicing pathologies increases this similarity to 42%. We dissected the proteome-transcriptome inconsistency in AD and 5xFAD mouse brains, revealing that inconsistent proteins are enriched within amyloid plaque microenvironment (amyloidome). Our analysis of the 5xFAD proteome turnover demonstrates that amyloid formation delays the degradation of amyloidome components, including Aβ-binding proteins and autophagy/lysosomal proteins. Our proteomic strategy defines shared AD pathways, identifies potential targets, and underscores that protein turnover contributes to proteome-transcriptome discrepancies during AD progression.
    DOI:  https://doi.org/10.1038/s41467-025-56853-3
  38. Autophagy. 2025 Feb 12.
    Transcriptional repression of autophagy and lysosome biogenesis.
    Jaebeom Kim, Young Suk Yu, Keun Il Kim, Sung Hee Baek.
      The microphthalmia/transcription factor E (MiT/TFE) family activates macroautophagy/autophagy and lysosomal genes during acute nutrient deficiency. However, the mechanisms that suppress transcription of these genes under steady-state, nutrient-rich conditions to prevent unnecessary expression remain unclear. In this study, we identified a previously unrecognized mechanism of transcriptional repression for autophagy and lysosomal genes. Under nutrient-rich conditions, USF2 (upstream transcription factor 2) binds to the coordinated lysosomal expression and regulation (CLEAR) motif, recruiting a repressive complex containing HDAC (histone deacetylase). In contrast, during nutrient deficiency, TFEB (transcription factor EB) displaces USF2 at the same motif, activating transcription. This switch is regulated by USF2 phosphorylation at serine 155 by GSK3B (glycogen synthase kinase 3 beta). Reduced phosphorylation under nutrient-deprived conditions weakens USF2's DNA binding affinity, allowing TFEB to competitively bind and activate target genes. Knockdown or knockout of Usf2 upregulates specific autophagy and lysosomal genes, leading to enhanced lysosomal functionality and increased autophagic flux. In USF2-deficient cells, the SERPINA1 Z variant/antitrypsin Z - an aggregation-prone mutant protein used as a model - is rapidly cleared via the autophagy-lysosome pathway. Therefore, modulation of USF2 activity may be a therapeutic strategy for managing diseases associated with autophagy and lysosomal dysfunction.
    Keywords:  Autophagy; MiT/TFE; TFEB; USF2; lysosome; transcriptional repressor
    DOI:  https://doi.org/10.1080/15548627.2025.2465404
  39. Mol Cell Biol. 2025 Feb 14. 1-11
    Transcriptomic Analysis Uncovers an Unfolded Protein Response in ADNP Syndrome.
    Anna Bieluszewska, Phillip Wulfridge, Kuo-Chen Fang, Yan Hong, Tomoyo Sawada, Jennifer Erwin, Hongjun Song, Guo-Li Ming, Kavitha Sarma.
      Chromatin regulators are frequently mutated in autism spectrum disorders, but in most cases how they cause disease is unclear. Mutations in the activity dependent neuroprotective protein (ADNP) causes ADNP syndrome, which is characterized by intellectual deficiency and developmental delays. To identify mechanisms that contribute to ADNP syndrome, we used induced pluripotent stem cells derived from ADNP syndrome patients as a model to test the effects of syndromic ADNP mutations on gene expression and neurodifferentiation. We found that some ADNP mutations result in truncated ADNP proteins, which displayed aberrant subcellular localization. Gene expression analyses revealed widespread transcriptional deregulation in all tested mutants. Interestingly, mutants that show presence of ADNP fragments show ER stress as evidenced by activation of the unfolded protein response (UPR). The mutants showing the greatest UPR pathway activation associated with the most severe neurodifferentiation and survival defects. Our results reveal the potential to explore UPR activation as a new biomarker for ADNP syndrome severity and perhaps also in other ASDs where mutations result in presence of truncated proteins.
    Keywords:  ADNP syndrome; neurodifferentiation; patient-derived induced pluripotent stem cells; transcriptomics; unfolded protein response
    DOI:  https://doi.org/10.1080/10985549.2025.2463892
  40. Mol Cell. 2025 Feb 06. pii: S1097-2765(25)00053-X. [Epub ahead of print]
    Free introns of tRNAs as complementarity-dependent regulators of gene expression.
    Regina T Nostramo, Paolo L Sinopoli, Alicia Bao, Sara Metcalf, Lauren M Peltier, Anita K Hopper.
      From archaea to humans, a subset of transfer RNA (tRNA) genes possesses an intron that must be removed from transcribed pre-tRNAs to generate mature, functional tRNAs. Evolutionary conservation of tRNA intron sequences suggests that tRNA introns perform sequence-dependent cellular functions, which are presently unknown. Here, we demonstrate that free introns of tRNAs (fitRNAs) in Saccharomyces cerevisiae serve as small regulatory RNAs that inhibit mRNA levels via long (13-15 nt) statistically improbable stretches of (near) perfect complementarity to mRNA coding regions. The functions of fitRNAs are both constitutive and inducible because genomic deletion or inducible overexpression of tRNAIle introns led to corresponding increases or decreases in levels of complementary mRNAs. Remarkably, although tRNA introns are usually rapidly degraded, fitRNATrp selectively accumulates following oxidative stress, and target mRNA levels decrease. Thus, fitRNAs serve as gene regulators that fine-tune basal mRNA expression and alter the network of mRNAs that respond to oxidative stress.
    Keywords:  Los1; Sen2; Tom70; fitRNAs; gene regulation; introns; oxidative stress; tRNA intron sequence conservation; tRNA intron-ORF complementarity; tet-on tRNA intron expression
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.019
  41. Sci Adv. 2025 Feb 14. 11(7): eadp5958
    Targeted approach to determine the impact of cancer-associated protease variants.
    Kira Bickenbach, Nele David, Tomas Koudelka, Corentin Joos, Franka Scharfenberg, Malina Rüffer, Fred Armbrust, Dimitris Georgiadis, Fabrice Beau, Lea Stahmer, Sascha Rahn, Andreas Tholey, Claus Pietrzik, Christoph Becker-Pauly.
      Several steps of cancer progression, from tumor onset to metastasis, critically involve proteolytic activity. To elucidate the role of proteases in cancer, it is particularly important to consider single-nucleotide variants (SNVs) that affect the active site of proteases, thereby influencing cleavage specificity, substrate processing, and thus cancer cell behavior. To facilitate systematic studies, we here present a targeted approach to determine the impact of cancer-associated protease variants (TACAP). Starting with the semiautomated identification of potential specificity-modulating SNVs, our workflow comprises mass spectrometry-based cleavage specificity profiling and substrate identification, localization, and inhibitor studies, followed by functional analyses investigating cancer cell properties. To demonstrate the feasibility of TACAP, we analyzed the meprin β R238Q variant. This amino acid exchange R238Q leads to a loss of meprin β's characteristic cleavage preference for acidic amino acids at P1' position, accompanied with changes in substrate pool and inhibitor affinity compared to meprin β wild type.
    DOI:  https://doi.org/10.1126/sciadv.adp5958
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