bims-proteo Biomed News
on Proteostasis
Issue of 2023–11–12
thirty-two papers selected by
Eric Chevet, INSERM
  1. V-ATPase recruitment to ER exit sites switches COPII-mediated transport to lysosomal degradation.
  2. TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.
  3. UFMylation: a ubiquitin-like modification.
  4. TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein.
  5. Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells.
  6. Autophagy-mediated control of ribosome homeostasis in oncogene-induced senescence.
  7. Multiple E3 ligases control tankyrase stability and function.
  8. DNAJB12 and DNJB14 are non-redundant Hsp40 redox chaperones involved in endoplasmic reticulum protein reflux.
  9. Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor.
  10. Deubiquitinases in cancer.
  11. A conserved requirement for RME-8/DNAJC13 in neuronal autophagic lysosome reformation.
  12. The p97/VCP adaptor UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
  13. The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.
  14. Calmodulin regulates protease versus co-chaperone activity of a metacaspase.
  15. Spatially resolved mapping of proteome turnover dynamics with subcellular precision.
  16. Differential Ire1 determines loser cell fate in tumor-suppressive cell competition.
  17. A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.
  18. Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.
  19. Regulation of eDHFR-tagged proteins with trimethoprim PROTACs.
  20. FMRP phosphorylation modulates neuronal translation through YTHDF1.
  21. IST1 regulates select recycling pathways.
  22. Visualization of conformational transition of GRP94 in solution.
  23. ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.
  24. A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation.
  25. The proteasome component PSMD14 drives myelomagenesis through a histone deubiquitinase activity.
  26. Stress-induced β cell early senescence confers protection against type 1 diabetes.
  27. Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.
  28. HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation.
  29. Hypomorphic variants of SEL1L-HRD1 ER-associated degradation are associated with neurodevelopmental disorders.
  30. DNAJB6 mutants display toxic gain of function through unregulated interaction with Hsp70 chaperones.
  31. Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence.
  32. ATM-CHK2-TRIM32 axis regulates ATG7 ubiquitination to initiate autophagy under oxidative stress.
  1. Dev Cell. 2023 Oct 29. pii: S1534-5807(23)00526-9. [Epub ahead of print]
    V-ATPase recruitment to ER exit sites switches COPII-mediated transport to lysosomal degradation.
    Yiwei Sun, Xi'e Wang, Xiaotong Yang, Lei Wang, Jingjin Ding, Chih-Chen Wang, Hong Zhang, Xi Wang.
      Endoplasmic reticulum (ER)-phagy is crucial to regulate the function and homeostasis of the ER via lysosomal degradation, but how it is initiated is unclear. Here we discover that Z-AAT, a disease-causing mutant of α1-antitrypsin, induces noncanonical ER-phagy at ER exit sites (ERESs). Accumulation of misfolded Z-AAT at the ERESs impairs coat protein complex II (COPII)-mediated ER-to-Golgi transport and retains V0 subunits that further assemble V-ATPase at the arrested ERESs. V-ATPase subsequently recruits ATG16L1 onto ERESs to mediate in situ lipidation of LC3C. FAM134B-II is then recruited by LC3C via its LIR motif and elicits ER-phagy leading to efficient lysosomal degradation of Z-AAT. Activation of this ER-phagy mediated by the V-ATPase-ATG16L1-LC3C axis (EVAC) is also triggered by blocking ER export. Our findings identify a pathway which switches COPII-mediated transport to lysosomal degradation for ER quality control.
    Keywords:  ATG16L1; COPII; ER exit sites; ER-phagy; LC3C; Sec24C; V-ATPase; autophagy; protein quality control; α1 antitrypsin
    DOI:  https://doi.org/10.1016/j.devcel.2023.10.007
  2. EMBO J. 2023 Nov 06. e114272
    TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.
    Yuki Hayashi, Sho Takatori, Waleed Y Warsame, Taisuke Tomita, Takao Fujisawa, Hidenori Ichijo.
      Endoplasmic reticulum (ER) proteostasis is maintained by various catabolic pathways. Lysosomes clear entire ER portions by ER-phagy, while proteasomes selectively clear misfolded or surplus aberrant proteins by ER-associated degradation (ERAD). Recently, lysosomes have also been implicated in the selective clearance of aberrant ER proteins, but the molecular basis remains unclear. Here, we show that the phosphatidylinositol-3-phosphate (PI3P)-binding protein TOLLIP promotes selective lysosomal degradation of aberrant membrane proteins, including an artificial substrate and motoneuron disease-causing mutants of VAPB and Seipin. These cargos are recognized by TOLLIP through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain. In contrast to ER-phagy receptors, which clear both native and aberrant proteins by ER-phagy, TOLLIP selectively clears aberrant cargos by coupling them with the PI3P-dependent lysosomal trafficking without promoting bulk ER turnover. Moreover, TOLLIP depletion augments ER stress after ERAD inhibition, indicating that TOLLIP and ERAD cooperatively safeguard ER proteostasis. Our study identifies TOLLIP as a unique type of cargo-specific adaptor dedicated to the clearance of aberrant ER cargos and provides insights into molecular mechanisms underlying lysosome-mediated quality control of membrane proteins.
    Keywords:  ER-phagy; TOLLIP; cargo adaptor; intrinsically disordered region; lysosome
    DOI:  https://doi.org/10.15252/embj.2023114272
  3. Trends Biochem Sci. 2023 Nov 07. pii: S0968-0004(23)00259-1. [Epub ahead of print]
    UFMylation: a ubiquitin-like modification.
    Xingchen Zhou, Sayyed J Mahdizadeh, Matthieu Le Gallo, Leif A Eriksson, Eric Chevet, Elodie Lafont.
      Post-translational modifications (PTMs) add a major degree of complexity to the proteome and are essential controllers of protein homeostasis. Amongst the hundreds of PTMs identified, ubiquitin and ubiquitin-like (UBL) modifications are recognized as key regulators of cellular processes through their ability to affect protein-protein interactions, protein stability, and thus the functions of their protein targets. Here, we focus on the most recently identified UBL, ubiquitin-fold modifier 1 (UFM1), and the machinery responsible for its transfer to substrates (UFMylation) or its removal (deUFMylation). We first highlight the biochemical peculiarities of these processes, then we develop on how UFMylation and its machinery control various intertwined cellular processes and we highlight some of the outstanding research questions in this emerging field.
    Keywords:  UFL1; UFM1; UFMylation; cellular stress; proteostasis
    DOI:  https://doi.org/10.1016/j.tibs.2023.10.004
  4. J Biol Chem. 2023 Nov 08. pii: S0021-9258(23)02478-X. [Epub ahead of print] 105450
    TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein.
    Nathan P Canniff, Jill B Graham, Kevin P Guay, Daniel A Lubicki, Stephen J Eyles, Jennifer N Rauch, Daniel N Hebert.
      Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determined that TTC17 is a large, soluble, ER localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and co-chaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of IGF1R, GPNMB, CLU and UGGT1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.
    Keywords:  ER quality control; TPR; UGGT; adaptor protein; cell compartmentalization; chaperone; endoplasmic reticulum (ER); endoplasmic reticulum stress (ER stress); intracellular trafficking; molecular chaperones; protein trafficking; secretion
    DOI:  https://doi.org/10.1016/j.jbc.2023.105450
  5. Cell Chem Biol. 2023 Oct 26. pii: S2451-9456(23)00367-7. [Epub ahead of print]
    Pharmacologic activation of a compensatory integrated stress response kinase promotes mitochondrial remodeling in PERK-deficient cells.
    Valerie Perea, Kelsey R Baron, Vivian Dolina, Giovanni Aviles, Grace Kim, Jessica D Rosarda, Xiaoyan Guo, Martin Kampmann, R Luke Wiseman.
      The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly selective ISR activators.
    Keywords:  ISR; UPR; integrated stress response; pharmacologic activator; stress-responsive signaling pathway; unfolded protein response
    DOI:  https://doi.org/10.1016/j.chembiol.2023.10.006
  6. Cell Rep. 2023 Nov 05. pii: S2211-1247(23)01393-1. [Epub ahead of print]42(11): 113381
    Autophagy-mediated control of ribosome homeostasis in oncogene-induced senescence.
    Aida Rodríguez López, Maria H Jørgensen, Jesper F Havelund, Frederic S Arendrup, Srinivasa Prasad Kolapalli, Thorbjørn M Nielsen, Eva Pais, Carsten Jörn Beese, Ahmad Abdul-Al, Anna Constance Vind, Jiri Bartek, Simon Bekker-Jensen, Marta Montes, Panagiotis Galanos, Nils Faergeman, Lotta Happonen, Lisa B Frankel.
      Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.
    Keywords:  CP: Cell biology; CP: Molecular biology; USP10; autophagy; oncogene-induced senescence; ribosomes; selective autophagy; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2023.113381
  7. Nat Commun. 2023 Nov 08. 14(1): 7208
    Multiple E3 ligases control tankyrase stability and function.
    Jerome Perrard, Susan Smith.
      Tankyrase 1 and 2 are ADP-ribosyltransferases that catalyze formation of polyADP-Ribose (PAR) onto themselves and their binding partners. Tankyrase protein levels are regulated by the PAR-binding E3 ligase RNF146, which promotes K48-linked polyubiquitylation and proteasomal degradation of tankyrase and its partners. We identified a novel interaction between tankyrase and a distinct class of E3 ligases: the RING-UIM (Ubiquitin-Interacting Motif) family. We show that RNF114 and RNF166 bind and stabilize monoubiquitylated tankyrase and promote K11-linked diubiquitylation. This action competes with RNF146-mediated degradation, leading to stabilization of tankyrase and its binding partner, Angiomotin, a cancer cell signaling protein. Moreover, we identify multiple PAR-binding E3 ligases that promote ubiquitylation of tankyrase and induce stabilization or degradation. Discovery of K11 ubiquitylation that opposes degradation, along with identification of multiple PAR-binding E3 ligases that ubiquitylate tankyrase, provide insights into mechanisms of tankyrase regulation and may offer additional uses for tankyrase inhibitors in cancer therapy.
    DOI:  https://doi.org/10.1038/s41467-023-42939-3
  8. Biochim Biophys Acta Gen Subj. 2023 Nov 03. pii: S0304-4165(23)00200-3. [Epub ahead of print]1868(1): 130502
    DNAJB12 and DNJB14 are non-redundant Hsp40 redox chaperones involved in endoplasmic reticulum protein reflux.
    Aline Dias da Purificação, Victor Debbas, Leonardo Yuji Tanaka, Gabriele Verônica de Mello Gabriel, João Wosniak Júnior, Tiphany Coralie De Bessa, Sheila Garcia-Rosa, Francisco Rafael Martins Laurindo, Percillia Victoria Santos Oliveira.
       BACKGROUND: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux.
    METHODS: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI).
    RESULTS: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells.
    CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.
    Keywords:  DNAJB12; DNAJB14; ER protein reflux; Endoplasmic reticulum stress
    DOI:  https://doi.org/10.1016/j.bbagen.2023.130502
  9. Nat Struct Mol Biol. 2023 Nov 09.
    Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor.
    Chari M Noddings, Jill L Johnson, David A Agard.
      Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR). Previously, we revealed that Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding, aided by co-chaperones. In vivo, the co-chaperones FKBP51 and FKBP52 antagonistically regulate GR activity, but a molecular understanding is lacking. Here we present a 3.01 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP52 complex, revealing how FKBP52 integrates into the GR chaperone cycle and directly binds to the active client, potentiating GR activity in vitro and in vivo. We also present a 3.23 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP51 complex, revealing how FKBP51 competes with FKBP52 for GR:Hsp90 binding and demonstrating how FKBP51 can act as a potent antagonist to FKBP52. Altogether, we demonstrate how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation.
    DOI:  https://doi.org/10.1038/s41594-023-01128-y
  10. Nat Rev Cancer. 2023 Nov 07.
    Deubiquitinases in cancer.
    Grant Dewson, Pieter J A Eichhorn, David Komander.
      Ubiquitination is an essential regulator of most, if not all, signalling pathways, and defects in cellular signalling are central to cancer initiation, progression and, eventually, metastasis. The attachment of ubiquitin signals by E3 ubiquitin ligases is directly opposed by the action of approximately 100 deubiquitinating enzymes (DUBs) in humans. Together, DUBs and E3 ligases coordinate ubiquitin signalling by providing selectivity for different substrates and/or ubiquitin signals. The balance between ubiquitination and deubiquitination is exquisitely controlled to ensure properly coordinated proteostasis and response to cellular stimuli and stressors. Not surprisingly, then, DUBs have been associated with all hallmarks of cancer. These relationships are often complex and multifaceted, highlighted by the implication of multiple DUBs in certain hallmarks and by the impact of individual DUBs on multiple cancer-associated pathways, sometimes with contrasting cancer-promoting and cancer-inhibiting activities, depending on context and tumour type. Although it is still understudied, the ever-growing knowledge of DUB function in cancer physiology will eventually identify DUBs that warrant specific inhibition or activation, both of which are now feasible. An integrated appreciation of the physiological consequences of DUB modulation in relevant cancer models will eventually lead to the identification of patient populations that will most likely benefit from DUB-targeted therapies.
    DOI:  https://doi.org/10.1038/s41568-023-00633-y
  11. Autophagy. 2023 Nov 09. 1-17
    A conserved requirement for RME-8/DNAJC13 in neuronal autophagic lysosome reformation.
    Sierra B Swords, Nuo Jia, Anne Norris, Jil Modi, Qian Cai, Barth D Grant.
      Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autophagic lysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. The DNA-J domain HSC70 co-chaperone RME-8/DNAJC13 has been linked to endosomal coat protein regulation and to neurological disease. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on intact C. elegans mechanosensory neurons, and primary mouse cortical neurons in culture. Loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including mutants lacking bec-1/BECN1/Beclin1 and vps-15/PIK3R4/p150 that regulate the class III phosphatidylinositol 3-kinase (PtdIns3K) VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a previously unrecognized role in ALR, likely affecting autolysosome tubule severing. Additionally, in both systems, loss of RME-8/DNAJC13 reduced macroautophagic/autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.Abbreviation: ALR, autophagic lysosome reformation; ATG-13/EPG-1, AuTophaGy (yeast Atg homolog)-13; ATG-18, AuTophaGy (yeast Atg homolog)-18; AV, autophagic vacuole; CLIC-1, Clathrin Light Chain-1; EPG-3, Ectopic P Granules-3; EPG-6, Ectopic P Granules-6; LGG-1, LC3, GABARAP and GATE-16 family-1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; PD, Parkinson disease; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; RME-8, Receptor Mediated Endocytosis-8; SNX-1, Sorting NeXin-1; VPS-34, related to yeast Vacuolar Protein Sorting factor-34.
    Keywords:  Autophagy; clathrin; endocytosis; lysosomes; neurodegeneration; trafficking
    DOI:  https://doi.org/10.1080/15548627.2023.2269028
  12. Nat Struct Mol Biol. 2023 Nov 09.
    The p97/VCP adaptor UBXD1 drives AAA+ remodeling and ring opening through multi-domain tethered interactions.
    Julian R Braxton, Chad R Altobelli, Maxwell R Tucker, Eric Tse, Aye C Thwin, Michelle R Arkin, Daniel R Southworth.
      p97, also known as valosin-containing protein, is an essential cytosolic AAA+ (ATPases associated with diverse cellular activities) hexamer that unfolds substrate polypeptides to support protein homeostasis and macromolecular disassembly. Distinct sets of p97 adaptors guide cellular functions but their roles in direct control of the hexamer are unclear. The UBXD1 adaptor localizes with p97 in critical mitochondria and lysosome clearance pathways and contains multiple p97-interacting domains. Here we identify UBXD1 as a potent p97 ATPase inhibitor and report structures of intact human p97-UBXD1 complexes that reveal extensive UBXD1 contacts across p97 and an asymmetric remodeling of the hexamer. Conserved VIM, UBX and PUB domains tether adjacent protomers while a connecting strand forms an N-terminal domain lariat with a helix wedged at the interprotomer interface. An additional VIM-connecting helix binds along the second (D2) AAA+ domain. Together, these contacts split the hexamer into a ring-open conformation. Structures, mutagenesis and comparisons to other adaptors further reveal how adaptors containing conserved p97-remodeling motifs regulate p97 ATPase activity and structure.
    DOI:  https://doi.org/10.1038/s41594-023-01126-0
  13. PLoS Biol. 2023 Nov 08. 21(11): e3002372
    The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast.
    Chen-Xi Zou, Zhu-Hui Ma, Zhao-Di Jiang, Zhao-Qian Pan, Dan-Dan Xu, Fang Suo, Guang-Can Shao, Meng-Qiu Dong, Li-Lin Du.
      Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.
    DOI:  https://doi.org/10.1371/journal.pbio.3002372
  14. Cell Rep. 2023 Nov 07. pii: S2211-1247(23)01384-0. [Epub ahead of print]42(11): 113372
    Calmodulin regulates protease versus co-chaperone activity of a metacaspase.
    Anna Maria Eisele-Bürger, Frederik Eisele, Sandra Malmgren Hill, Xinxin Hao, Kara L Schneider, Rahmi Imamoglu, David Balchin, Beidong Liu, F Ulrich Hartl, Peter V Bozhkov, Thomas Nyström.
      Metacaspases are ancestral homologs of caspases that can either promote cell death or confer cytoprotection. Furthermore, yeast (Saccharomyces cerevisiae) metacaspase Mca1 possesses dual biochemical activity: proteolytic activity causing cell death and cytoprotective, co-chaperone-like activity retarding replicative aging. The molecular mechanism favoring one activity of Mca1 over another remains elusive. Here, we show that this mechanism involves calmodulin binding to the N-terminal pro-domain of Mca1, which prevents its proteolytic activation and promotes co-chaperone-like activity, thus switching from pro-cell death to anti-aging function. The longevity-promoting effect of Mca1 requires the Hsp40 co-chaperone Sis1, which is necessary for Mca1 recruitment to protein aggregates and their clearance. In contrast, proteolytically active Mca1 cleaves Sis1 both in vitro and in vivo, further clarifying molecular mechanism behind a dual role of Mca1 as a cell-death protease versus gerontogene.
    Keywords:  CP: Cell biology; Cmd1; Mca1; Sis1; aging; calmodulin; chaperone; longevity; metacaspase; protein quality control; proteostasis
    DOI:  https://doi.org/10.1016/j.celrep.2023.113372
  15. Nat Commun. 2023 Nov 08. 14(1): 7217
    Spatially resolved mapping of proteome turnover dynamics with subcellular precision.
    Feng Yuan, Yi Li, Xinyue Zhou, Peiyuan Meng, Peng Zou.
      Cellular activities are commonly associated with dynamic proteomic changes at the subcellular level. Although several techniques are available to quantify whole-cell protein turnover dynamics, such measurements often lack sufficient spatial resolution at the subcellular level. Herein, we report the development of prox-SILAC method that combines proximity-dependent protein labeling (APEX2/HRP) with metabolic incorporation of stable isotopes (pulse-SILAC) to map newly synthesized proteins with subcellular spatial resolution. We apply prox-SILAC to investigate proteome dynamics in the mitochondrial matrix and the endoplasmic reticulum (ER) lumen. Our analysis reveals a highly heterogeneous distribution in protein turnover dynamics within macromolecular machineries such as the mitochondrial ribosome and respiratory complexes I-V, thus shedding light on their mechanism of hierarchical assembly. Furthermore, we investigate the dynamic changes of ER proteome when cells are challenged with stress or undergoing stimulated differentiation, identifying subsets of proteins with unique patterns of turnover dynamics, which may play key regulatory roles in alleviating stress or promoting differentiation. We envision that prox-SILAC could be broadly applied to profile protein turnover at various subcellular compartments, under both physiological and pathological conditions.
    DOI:  https://doi.org/10.1038/s41467-023-42861-8
  16. Cell Rep. 2023 Nov 03. pii: S2211-1247(23)01315-3. [Epub ahead of print]42(11): 113303
    Differential Ire1 determines loser cell fate in tumor-suppressive cell competition.
    Jiadong Zheng, Yifan Guo, Changyi Shi, Shuai Yang, Wenyan Xu, Xianjue Ma.
      Tumor-suppressive cell competition (TSCC) is a conserved surveillance mechanism in which neighboring cells actively eliminate oncogenic cells. Despite overwhelming studies showing that the unfolded protein response (UPR) is dysregulated in various tumors, it remains debatable whether the UPR restrains or promotes tumorigenesis. Here, using Drosophila eye epithelium as a model, we uncover a surprising decisive role of the Ire1 branch of the UPR in regulating cell polarity gene scribble (scrib) loss-induced TSCC. Both mutation and hyperactivation of Ire1 accelerate elimination of scrib clones via inducing apoptosis and autophagy, respectively. Unexpectedly, relative Ire1 activity is also crucial for determining loser cell fate, as dysregulating Ire1 signaling in the surrounding healthy cells reversed the "loser" status of scrib clones by decreasing their apoptosis. Furthermore, we show that Ire1 is required for cell competition in mammalian cells. Together, these findings provide molecular insights into scrib-mediated TSCC and highlight Ire1 as a key determinant of loser cell fate.
    Keywords:  CP: Cell biology; Drosophila; E-cad; Ire1; UPR; apoptosis; autophagy; cell competition; scribble; tumor
    DOI:  https://doi.org/10.1016/j.celrep.2023.113303
  17. Nat Chem Biol. 2023 Nov 09.
    A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.
    Rosalie E Lawrence, Sophie R Shoemaker, Aniliese Deal, Smriti Sangwan, Aditya A Anand, Lan Wang, Susan Marqusee, Peter Walter.
      The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this 'switch-helix' controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.
    DOI:  https://doi.org/10.1038/s41589-023-01453-9
  18. Nat Commun. 2023 Nov 08. 14(1): 7212
    Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation.
    Betsegaw Lemma, Di Zhang, Ganga B Vamisetti, Bryan G Wentz, Hiroaki Suga, Ashraf Brik, Jacek Lubkowski, David Fushman.
      Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide's affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.
    DOI:  https://doi.org/10.1038/s41467-023-43025-4
  19. Nat Commun. 2023 Nov 03. 14(1): 7071
    Regulation of eDHFR-tagged proteins with trimethoprim PROTACs.
    Jean M Etersque, Iris K Lee, Nitika Sharma, Kexiang Xu, Andrew Ruff, Justin D Northrup, Swarbhanu Sarkar, Tommy Nguyen, Richard Lauman, George M Burslem, Mark A Sellmyer.
      Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.
    DOI:  https://doi.org/10.1038/s41467-023-42820-3
  20. Mol Cell. 2023 Nov 02. pii: S1097-2765(23)00865-1. [Epub ahead of print]
    FMRP phosphorylation modulates neuronal translation through YTHDF1.
    Zhongyu Zou, Jiangbo Wei, Yantao Chen, Yunhee Kang, Hailing Shi, Fan Yang, Zhuoyue Shi, Shijie Chen, Ying Zhou, Caraline Sepich-Poore, Xiaoxi Zhuang, Xiaoming Zhou, Hualiang Jiang, Zhexing Wen, Peng Jin, Cheng Luo, Chuan He.
      RNA-binding proteins (RBPs) control messenger RNA fate in neurons. Here, we report a mechanism that the stimuli-induced neuronal translation is mediated by phosphorylation of a YTHDF1-binding protein FMRP. Mechanistically, YTHDF1 can condense with ribosomal proteins to promote the translation of its mRNA targets. FMRP regulates this process by sequestering YTHDF1 away from the ribosome; upon neuronal stimulation, FMRP becomes phosphorylated and releases YTHDF1 for translation upregulation. We show that a new small molecule inhibitor of YTHDF1 can reverse fragile X syndrome (FXS) developmental defects associated with FMRP deficiency in an organoid model. Our study thus reveals that FMRP and its phosphorylation are important regulators of activity-dependent translation during neuronal development and stimulation and identifies YTHDF1 as a potential therapeutic target for FXS in which developmental defects caused by FMRP depletion could be reversed through YTHDF1 inhibition.
    Keywords:  FMRP; RNA-binding proteins; YTHDF1; m(6)A; neuronal translation control; protein condensates
    DOI:  https://doi.org/10.1016/j.molcel.2023.10.028
  21. Traffic. 2023 Nov 05.
    IST1 regulates select recycling pathways.
    Amy K Clippinger, Teresa V Naismith, Wonjin Yoo, Silvia Jansen, David J Kast, Phyllis I Hanson.
      ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.
    Keywords:  AP-1; clathrin; endosomal sorting complex required for transport; sorting nexin; transferrin receptor
    DOI:  https://doi.org/10.1111/tra.12921
  22. Life Sci Alliance. 2024 Feb;pii: e202302051. [Epub ahead of print]7(2):
    Visualization of conformational transition of GRP94 in solution.
    Shangwu Sun, Rui Zhu, Mengyao Zhu, Qi Wang, Na Li, Bei Yang.
      GRP94, an ER paralog of the heat-shock protein 90 family, binds and hydrolyses ATP to chaperone the folding and maturation of its selected clients. Compared with other hsp90 proteins, the in-solution conformational dynamics of GRP94 along the ATP hydrolysis cycle are less understood, hindering our understanding of its chaperoning mechanism. Leveraging small-angle X-ray scattering, negative-staining EM, and hydrogen-deuterium exchange coupled mass-spec, here we show that in its apo form, ∼60% of mouse GRP94 (mGRP94) populates an "extended" conformation, whereas the rest exist in either "close V" or "twist V" like "compact" conformations. Different from other hsp90 proteins, the presence of AMPPNP only impacts the relative abundance of the two compact conformations, rather than shifting the equilibrium between the "extended" and "compact" conformations of mGRP94. HDX-MS study of apo, AMPPNP-bound, and ADP-bound mGRP94 suggests a conformational transition from "twist V" to "close V" upon ATP binding and a back transition from "close V" to "twist V" upon ATP hydrolysis. These results illustrate the dissimilarities of GRP94 in conformation transition during ATP hydrolysis from other hsp90 paralogs.
    DOI:  https://doi.org/10.26508/lsa.202302051
  23. Life Sci Alliance. 2024 Jan;pii: e202302335. [Epub ahead of print]7(1):
    ESYT1 tethers the ER to mitochondria and is required for mitochondrial lipid and calcium homeostasis.
    Alexandre Janer, Jordan L Morris, Michiel Krols, Hana Antonicka, Mari J Aaltonen, Zhen-Yuan Lin, Hanish Anand, Anne-Claude Gingras, Julien Prudent, Eric A Shoubridge.
      Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1-SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria-ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.
    DOI:  https://doi.org/10.26508/lsa.202302335
  24. Nat Commun. 2023 Nov 10. 14(1): 7281
    A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation.
    Alireza Ghanbarpour, Steven E Cohen, Xue Fei, Laurel F Kinman, Tristan A Bell, Jia Jia Zhang, Tania A Baker, Joseph H Davis, Robert T Sauer.
      AAA+ proteases degrade intracellular proteins in a highly specific manner. E. coli ClpXP, for example, relies on a C-terminal ssrA tag or other terminal degron sequences to recognize proteins, which are then unfolded by ClpX and subsequently translocated through its axial channel and into the degradation chamber of ClpP for proteolysis. Prior cryo-EM structures reveal that the ssrA tag initially binds to a ClpX conformation in which the axial channel is closed by a pore-2 loop. Here, we show that substrate-free ClpXP has a nearly identical closed-channel conformation. We destabilize this closed-channel conformation by deleting residues from the ClpX pore-2 loop. Strikingly, open-channel ClpXP variants degrade non-native proteins lacking degrons faster than the parental enzymes in vitro but degraded GFP-ssrA more slowly. When expressed in E. coli, these open channel variants behave similarly to the wild-type enzyme in assays of filamentation and phage-Mu plating but resulted in reduced growth phenotypes at elevated temperatures or when cells were exposed to sub-lethal antibiotic concentrations. Thus, channel closure is an important determinant of ClpXP degradation specificity.
    DOI:  https://doi.org/10.1038/s41467-023-43145-x
  25. Mol Cell. 2023 Nov 02. pii: S1097-2765(23)00856-0. [Epub ahead of print]
    The proteasome component PSMD14 drives myelomagenesis through a histone deubiquitinase activity.
    Lin He, Chunyu Yu, Sen Qin, Enrun Zheng, Xinhua Liu, Yanhua Liu, Shimiao Yu, Yang Liu, Xuelin Dou, Zesen Shang, Yizhou Wang, Yue Wang, Xuehong Zhou, Boning Liu, Yuping Zhong, Zhiqiang Liu, Jin Lu, Luyang Sun.
      While 19S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14, forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma.
    Keywords:  19S regulatory particle; 26S proteasome; NSD2; PSMD14; RELA; bortezomib resistance; histone methylation; histone ubiquitination; multiple myeloma; t(4;14) translocation
    DOI:  https://doi.org/10.1016/j.molcel.2023.10.019
  26. Cell Metab. 2023 Nov 05. pii: S1550-4131(23)00383-2. [Epub ahead of print]
    Stress-induced β cell early senescence confers protection against type 1 diabetes.
    Hugo Lee, Gulcan Semra Sahin, Chien-Wen Chen, Shreyash Sonthalia, Sandra Marín Cañas, Hulya Zeynep Oktay, Alexander T Duckworth, Gabriel Brawerman, Peter J Thompson, Maria Hatzoglou, Decio L Eizirik, Feyza Engin.
      During the progression of type 1 diabetes (T1D), β cells are exposed to significant stress and, therefore, require adaptive responses to survive. The adaptive mechanisms that can preserve β cell function and survival in the face of autoimmunity remain unclear. Here, we show that the deletion of the unfolded protein response (UPR) genes Atf6α or Ire1α in β cells of non-obese diabetic (NOD) mice prior to insulitis generates a p21-driven early senescence phenotype and alters the β cell secretome that significantly enhances the leukemia inhibitory factor-mediated recruitment of M2 macrophages to islets. Consequently, M2 macrophages promote anti-inflammatory responses and immune surveillance that cause the resolution of islet inflammation, the removal of terminally senesced β cells, the reduction of β cell apoptosis, and protection against T1D. We further demonstrate that the p21-mediated early senescence signature is conserved in the residual β cells of T1D patients. Our findings reveal a previously unrecognized link between β cell UPR and senescence that, if leveraged, may represent a novel preventive strategy for T1D.
    Keywords:  ER stress; M2 macrophage; NOD mice; UPR; immune surveillance; secretome; senescence; type 1 diabetes; β cells
    DOI:  https://doi.org/10.1016/j.cmet.2023.10.014
  27. Cell Rep Methods. 2023 Nov 02. pii: S2667-2375(23)00290-4. [Epub ahead of print] 100626
    Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.
    Birthe Meineke, Johannes Heimgärtner, Rozina Caridha, Matthias F Block, Kyle J Kimler, Maria F Pires, Michael Landreh, Simon J Elsässer.
      Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.
    Keywords:  CP: Biotechnology; CP: Molecular biology; Genetic code expansion; amber suppression; bioorthogonal labeling; click chemistry; mammalian cell culture; non-canonical amino acids; random genomic integration; stop codon suppression; unnatural amino acids
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100626
  28. Cell Rep. 2023 Nov 08. pii: S2211-1247(23)01364-5. [Epub ahead of print]42(11): 113352
    HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation.
    Wei Zhang, Jiegang Yang, Beike Wang, Youtao Lu, Jingbo Yang, Wenqun Zhong, Ziyan Yu, Zhiyuan Qin, Bolin Xiao, Kuiming Wang, Yi Y Ma, Ravi Amaravadi, Meenhard Herlyn, Junhyong Kim, Xiaowei Xu, Wei Guo.
      By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ T cell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.
    Keywords:  CP: Cancer; CP: Immunology; ▪▪▪
    DOI:  https://doi.org/10.1016/j.celrep.2023.113352
  29. J Clin Invest. 2023 Nov 09. pii: e170054. [Epub ahead of print]
    Hypomorphic variants of SEL1L-HRD1 ER-associated degradation are associated with neurodevelopmental disorders.
    Huilun Helen Wang, Liangguang Leo Lin, Zexin Jason Li, Xiaoqiong Wei, Omar Askander, Gerarda Cappuccio, Mais O Hashem, Laurence Hubert, Arnold Munnich, Mashael Alqahtani, Qi Pang, Margit Burmeister, You Lu, Karine Poirier, Claude Besmond, Shengyi Sun, Nicola Brunetti-Pierri, Fowzan S Alkuraya, Ling Qi.
      Recent studies using cell type-specific knockout mouse models have improved our understanding of the pathophysiological relevance of SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD); however, its importance in humans remains unclear as no disease variant has been identified. Here we report the identification of three bi-allelic missense variants of SEL1L and HRD1 (or SYVN1) in six children from three independent families presenting with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, hypotonia and/or ataxia. These SEL1L (p.Gly585Asp, p.Met528Arg) and HRD1 (p.Pro398Leu) variants were hypomorphic and impaired ERAD function at distinct steps of ERAD including substrate recruitment (SEL1L p.Gly585Asp), SEL1L-HRD1 complex formation (SEL1L p.Met528Arg), and HRD1 activity (HRD1 p.Pro398Leu). Our study not only provide new insights into the structure-function relationship of SEL1L-HRD1 ERAD, but also establish the importance of SEL1L-HRD1 ERAD in humans.
    Keywords:  Cell Biology; Genetic variation; Neurological disorders; Protein misfolding
    DOI:  https://doi.org/10.1172/JCI170054
  30. Nat Commun. 2023 Nov 03. 14(1): 7066
    DNAJB6 mutants display toxic gain of function through unregulated interaction with Hsp70 chaperones.
    Meital Abayev-Avraham, Yehuda Salzberg, Dar Gliksberg, Meital Oren-Suissa, Rina Rosenzweig.
      Molecular chaperones are essential cellular components that aid in protein folding and preventing the abnormal aggregation of disease-associated proteins. Mutations in one such chaperone, DNAJB6, were identified in patients with LGMDD1, a dominant autosomal disorder characterized by myofibrillar degeneration and accumulations of aggregated protein within myocytes. The molecular mechanisms through which such mutations cause this dysfunction, however, are not well understood. Here we employ a combination of solution NMR and biochemical assays to investigate the structural and functional changes in LGMDD1 mutants of DNAJB6. Surprisingly, we find that DNAJB6 disease mutants show no reduction in their aggregation-prevention activity in vitro, and instead differ structurally from the WT protein, affecting their interaction with Hsp70 chaperones. While WT DNAJB6 contains a helical element regulating its ability to bind and activate Hsp70, in LGMDD1 disease mutants this regulation is disrupted. These variants can thus recruit and hyperactivate Hsp70 chaperones in an unregulated manner, depleting Hsp70 levels in myocytes, and resulting in the disruption of proteostasis. Interfering with DNAJB6-Hsp70 binding, however, reverses the disease phenotype, suggesting future therapeutic avenues for LGMDD1.
    DOI:  https://doi.org/10.1038/s41467-023-42735-z
  31. Cell Rep. 2023 Nov 07. pii: S2211-1247(23)01383-9. [Epub ahead of print]42(11): 113371
    Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence.
    Radoslav Savić, Jialiang Yang, Simon Koplev, Mahru C An, Priyanka L Patel, Robert N O'Brien, Brittany N Dubose, Tetyana Dodatko, Eduard Rogatsky, Katyayani Sukhavasi, Raili Ermel, Arno Ruusalepp, Sander M Houten, Jason C Kovacic, Andrew F Stewart, Christopher B Yohn, Eric E Schadt, Remi-Martin Laberge, Johan L M Björkegren, Zhidong Tu, Carmen Argmann.
      Senescent cells are a major contributor to age-dependent cardiovascular tissue dysfunction, but knowledge of their in vivo cell markers and tissue context is lacking. To reveal tissue-relevant senescence biology, we integrate the transcriptomes of 10 experimental senescence cell models with a 224 multi-tissue gene co-expression network based on RNA-seq data of seven tissues biopsies from ∼600 coronary artery disease (CAD) patients. We identify 56 senescence-associated modules, many enriched in CAD GWAS genes and correlated with cardiometabolic traits-which supports universality of senescence gene programs across tissues and in CAD. Cross-tissue network analyses reveal 86 candidate senescence-associated secretory phenotype (SASP) factors, including COL6A3. Experimental knockdown of COL6A3 induces transcriptional changes that overlap the majority of the experimental senescence models, with cell-cycle arrest linked to modulation of DREAM complex-targeted genes. We provide a transcriptomic resource for cellular senescence and identify candidate biomarkers, SASP factors, and potential drivers of senescence in human tissues.
    Keywords:  COL6A3; CP: Cell biology; cellular senescence; collagen; disease; human; integration; network; transcriptomic
    DOI:  https://doi.org/10.1016/j.celrep.2023.113371
  32. Cell Rep. 2023 Nov 08. pii: S2211-1247(23)01414-6. [Epub ahead of print]42(11): 113402
    ATM-CHK2-TRIM32 axis regulates ATG7 ubiquitination to initiate autophagy under oxidative stress.
    Jingwei Liu, Songming Lu, Lixia Zheng, Qiqiang Guo, Liangzi Cao, Yutong Xiao, Di Chen, Yu Zou, Xu Liu, Chengsi Deng, Siyi Zhang, Ruohan Yang, Yubang Wang, Ying Zhang, Naijin Zhang, Xiaoyu Song, Chengzhong Xing, Zhenning Wang, Liu Cao.
      Oxidative stress-induced autophagy helps to prevent cellular damage and to maintain homeostasis. However, the regulatory pathway that initiates autophagy remains unclear. We previously showed that reactive oxygen species (ROS) function as signaling molecules to activate the ATM-CHK2 pathway and promote autophagy. Here, we find that the E3 ubiquitin ligase TRIM32 functions downstream of ATM-CHK2 to regulate ATG7 ubiquitination. Under metabolic stress, ROS induce ATM phosphorylation at S1981, which in turn phosphorylates CHK2 at T68. We show that CHK2 binds and phosphorylates TRIM32 at the S55 site, which then mediates K63-linked ubiquitination of ATG7 at the K45 site to initiate autophagy. In addition, Chk2-/- mice show an aggravated infarction phenotype and reduced phosphorylation of TRIM32 and ubiquitination of ATG7 in a stroke model. We propose a molecular mechanism for autophagy initiation by ROS via the ATM-CHK2-TRIM32-ATG7 axis to maintain intracellular homeostasis and to protect cells exposed to pathological conditions from stress-induced tissue damage.
    Keywords:  ATG7; CP: Cell biology; CP: Molecular biology; ROS; autophagy; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2023.113402
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