bims-proteo Biomed News
on Proteostasis
Issue of 2024‒07‒14
29 papers selected by
Eric Chevet, INSERM
  1. The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress.
  2. VCP/p97-associated proteins are binders and debranching enzymes of K48-K63-branched ubiquitin chains.
  3. Resolving chaperone-assisted protein folding on the ribosome at the peptide level.
  4. Transport of Protein Disulfide Isomerase from the Endoplasmic Reticulum to the Extracellular Space without Passage through the Golgi Complex.
  5. Deubiquitylase OTUD3 regulates integrated stress response to suppress progression and sorafenib resistance of liver cancer.
  6. Endoplasmic reticulum exit sites are segregated for secretion based on cargo size.
  7. Human DDX6 regulates translation and decay of inefficiently translated mRNAs.
  8. TFG regulates inner COPII coat recruitment to facilitate anterograde secretory protein transport.
  9. SigmaR1 shapes rough endoplasmic reticulum membrane sheets.
  10. ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.
  11. An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress.
  12. Short transmembrane domains target type II proteins to the Golgi apparatus and type I to the endoplasmic reticulum.
  13. Ca2+-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy upon glucose starvation.
  14. Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis.
  15. Chaperones in concert: Orchestrating co-translational protein folding in the cell.
  16. The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.
  17. Characterization of ribosome stalling and no-go mRNA decay stimulated by the Fragile X protein, FMRP.
  18. Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.
  19. Protein Condensate Atlas from predictive models of heteromolecular condensate composition.
  20. The caspase-activated DNase promotes cellular senescence.
  21. A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.
  22. Pressure sensing of lysosomes enables control of TFEB responses in macrophages.
  23. Neutrophil-derived migrasomes are an essential part of the coagulation system.
  24. Protocol to detect and quantify interactions between proteins expressed in Drosophila S2 cells.
  25. Mast cell secretory granule fusion with amphisomes coordinates their homotypic fusion and release of exosomes.
  26. Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.
  27. Bombyx mori UFL1 facilitates BmNPV proliferation by regulating host cell apoptosis through PERK.
  28. DrugDomain: The evolutionary context of drugs and small molecules bound to domains.
  29. Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.
  1. iScience. 2024 Jul 19. 27(7): 110196
    The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress.
    Wenbo Shi, Ran Ding, Yilin Chen, Fubo Ji, Junfang Ji, Weirui Ma, Jianping Jin.
      Stress granules (SGs) are membrane-less cellular compartments which are dynamically assembled via biomolecular condensation mechanism when eukaryotic cells encounter environmental stresses. SGs are important for gene expression and cell fate regulation. Dysregulation of SG homeostasis has been linked to human neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we report that the HRD1-SEL1L ubiquitin ligase complex specifically regulates the homeostasis of heat shock-induced SGs through the ubiquitin-proteasome system (UPS) and the UPS-associated ATPase p97. Mechanistically, the HRD1-SEL1L complex mediates SG homeostasis through the BiP-coupled PERK-eIF2α signaling axis of endoplasmic reticulum (ER) stress, thereby coordinating the unfolded protein response (UPR) with SG dynamics. Furthermore, we show that the distinctive branches of ER stress play differential roles in SG homeostasis. Our study indicates that the UPS and the UPR together via the HRD1-SEL1L ubiquitin ligase to maintain SG homeostasis in a stressor-dependent manner.
    Keywords:  Cell biology; Cellular physiology; Functional aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2024.110196
  2. Nat Struct Mol Biol. 2024 Jul 08.
    VCP/p97-associated proteins are binders and debranching enzymes of K48-K63-branched ubiquitin chains.
    Sven M Lange, Matthew R McFarland, Frederic Lamoliatte, Thomas Carroll, Logesvaran Krshnan, Anna Pérez-Ràfols, Dominika Kwasna, Linnan Shen, Iona Wallace, Isobel Cole, Lee A Armstrong, Axel Knebel, Clare Johnson, Virginia De Cesare, Yogesh Kulathu.
      Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48-K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48-K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48-K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48-K63-linked Ub, these results suggest a function for K48-K63-branched chains in VCP/p97-related processes.
    DOI:  https://doi.org/10.1038/s41594-024-01354-y
  3. Nat Struct Mol Biol. 2024 Jul 10.
    Resolving chaperone-assisted protein folding on the ribosome at the peptide level.
    Thomas E Wales, Aleksandra Pajak, Alžběta Roeselová, Santosh Shivakumaraswamy, Steven Howell, Svend Kjær, F Ulrich Hartl, John R Engen, David Balchin.
      Protein folding in vivo begins during synthesis on the ribosome and is modulated by molecular chaperones that engage the nascent polypeptide. How these features of protein biogenesis influence the maturation pathway of nascent proteins is incompletely understood. Here, we use hydrogen-deuterium exchange mass spectrometry to define, at peptide resolution, the cotranslational chaperone-assisted folding pathway of Escherichia coli dihydrofolate reductase. The nascent polypeptide folds along an unanticipated pathway through structured intermediates not populated during refolding from denaturant. Association with the ribosome allows these intermediates to form, as otherwise destabilizing carboxy-terminal sequences remain confined in the ribosome exit tunnel. Trigger factor binds partially folded states without disrupting their structure, and the nascent chain is poised to complete folding immediately upon emergence of the C terminus from the exit tunnel. By mapping interactions between the nascent chain and ribosomal proteins, we trace the path of the emerging polypeptide during synthesis. Our work reveals new mechanisms by which cellular factors shape the conformational search for the native state.
    DOI:  https://doi.org/10.1038/s41594-024-01355-x
  4. J Biol Chem. 2024 Jul 04. pii: S0021-9258(24)02037-4. [Epub ahead of print] 107536
    Transport of Protein Disulfide Isomerase from the Endoplasmic Reticulum to the Extracellular Space without Passage through the Golgi Complex.
    Percillia Victoria Santos Oliveira, Marco Dalla Torre, Victor Debbas, Andrea Orsi, Francisco Rafael Martins Laurindo, Roberto Sitia.
      Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N- glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route, nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107536
  5. Cell Rep. 2024 Jul 11. pii: S2211-1247(24)00816-7. [Epub ahead of print]43(7): 114487
    Deubiquitylase OTUD3 regulates integrated stress response to suppress progression and sorafenib resistance of liver cancer.
    Hongmiao Dai, Bo Wu, Yingwei Ge, Yang Hao, Lijie Zhou, Ruolin Hong, Jinhao Zhang, Wenli Jiang, Yuting Zhang, Hongchang Li, Lingqiang Zhang.
      The integrated stress response (ISR) is activated in response to intrinsic and extrinsic stimuli, playing a role in tumor progression and drug resistance. The regulatory role and mechanism of ISR in liver cancer, however, remain largely unexplored. Here, we demonstrate that OTU domain-containing protein 3 (OTUD3) is a deubiquitylase of eukaryotic initiation factor 2α (eIF2α), antagonizing ISR and suppressing liver cancer. OTUD3 decreases interactions between eIF2α and the kinase EIF2ΑK3 by removing K27-linked polyubiquitylation on eIF2α. OTUD3 deficiency in mice leads to enhanced ISR and accelerated progression of N-nitrosodiethylamine-induced hepatocellular carcinoma. Additionally, decreased OTUD3 expression associated with elevated eIF2α phosphorylation correlates with the progression of human liver cancer. Moreover, ISR activation due to decreased OTUD3 expression renders liver cancer cells resistant to sorafenib, while the combined use of the ISR inhibitor ISRIB significantly improves their sensitivity to sorafenib. Collectively, these findings illuminate the regulatory mechanism of ISR in liver cancer and provide a potential strategy to counteract sorafenib resistance.
    Keywords:  CP: Cancer; CP: Molecular biology
    DOI:  https://doi.org/10.1016/j.celrep.2024.114487
  6. Dev Cell. 2024 Jul 04. pii: S1534-5807(24)00387-3. [Epub ahead of print]
    Endoplasmic reticulum exit sites are segregated for secretion based on cargo size.
    Sonashree Saxena, Ombretta Foresti, Aofei Liu, Stefania Androulaki, Maria Pena Rodriguez, Ishier Raote, Meir Aridor, Bianxiao Cui, Vivek Malhotra.
      TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.
    Keywords:  COPII; ER export; ERES; TANGO1; collagens; endoplasmic reticulum; membrane traffic; optogenetics; protein secretion; secretory cargo
    DOI:  https://doi.org/10.1016/j.devcel.2024.06.009
  7. Elife. 2024 Jul 11. pii: RP92426. [Epub ahead of print]13
    Human DDX6 regulates translation and decay of inefficiently translated mRNAs.
    Ramona Weber, Chung-Te Chang.
      Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.
    Keywords:  CCR4-NOT complex; DDX6; biochemistry; chemical biology; chromosomes; codon optimization; gene expression; human; mRNA decay; ribosome stalling
    DOI:  https://doi.org/10.7554/eLife.92426
  8. Mol Biol Cell. 2024 Jul 10. mbcE24060282
    TFG regulates inner COPII coat recruitment to facilitate anterograde secretory protein transport.
    William Kasberg, Peter Luong, Kayla Minushkin, Iryna Pustova, Kevin A Swift, Meixian Zhao, Anjon Audhya.
      Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared to control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export.
    DOI:  https://doi.org/10.1091/mbc.E24-06-0282
  9. Dev Cell. 2024 Jul 02. pii: S1534-5807(24)00382-4. [Epub ahead of print]
    SigmaR1 shapes rough endoplasmic reticulum membrane sheets.
    Eric M Sawyer, Liv E Jensen, Janet B Meehl, Kevin P Larsen, Daniel A Petito, James H Hurley, Gia K Voeltz.
      Rough endoplasmic reticulum (ER) sheets are a fundamental domain of the ER and the gateway into the secretory pathway. Although reticulon proteins stabilize high-curvature ER tubules, it is unclear whether other proteins scaffold the flat membranes of rough ER sheets. Through a proteomics screen using ER sheet-localized RNA-binding proteins as bait, we identify the sigma-1 receptor (SigmaR1) as an ER sheet-shaping factor. High-resolution live cell imaging and electron tomography assign SigmaR1 as an ER sheet-localized factor whose levels determine the amount of rough ER sheets in cells. Structure-guided mutagenesis and in vitro reconstitution on giant unilamellar vesicles further support a mechanism whereby SigmaR1 oligomers use their extended arrays of amphipathic helices to bind and flatten the lumenal leaflet of ER membranes to oppose membrane curvature and stabilize rough ER sheets.
    Keywords:  SigmaR1; endoplasmic reticulum; membrane; organelle; polysome; translation
    DOI:  https://doi.org/10.1016/j.devcel.2024.06.005
  10. Life Sci Alliance. 2024 Sep;pii: e202402842. [Epub ahead of print]7(9):
    ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.
    Kathryn M Yammine, Sophia Mirda Abularach, Seo-Yeon Kim, Agata A Bikovtseva, Jinia Lilianty, Vincent L Butty, Richard P Schiavoni, John F Bateman, Shireen R Lamandé, Matthew D Shoulders.
      Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.
    DOI:  https://doi.org/10.26508/lsa.202402842
  11. Proc Natl Acad Sci U S A. 2024 Jul 16. 121(29): e2313370121
    An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress.
    Milad J Alasady, Martina Koeva, Seesha R Takagishi, Dmitri Segal, David R Amici, Roger S Smith, Daniel J Ansel, Susan Lindquist, Luke Whitesell, Elizabeth T Bartom, Mikko Taipale, Marc L Mendillo.
      Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.
    Keywords:  HSF1; JMJD6; proteostasis
    DOI:  https://doi.org/10.1073/pnas.2313370121
  12. J Cell Sci. 2024 Jul 08. pii: jcs.261738. [Epub ahead of print]
    Short transmembrane domains target type II proteins to the Golgi apparatus and type I to the endoplasmic reticulum.
    Claudie Bian, Anna Marchetti, Marco Dias, Jackie Perrin, Pierre Cosson.
      Transmembrane domains (TMDs) contain information targeting membrane proteins to various compartments of the secretory pathway. In previous studies, short or hydrophilic TMDs have been shown to target membrane proteins either to the endoplasmic reticulum (ER), or to the Golgi apparatus. The basis for differential sorting to the ER and to the Golgi apparatus remained however unclear. To clarify this point, we analyzed quantitatively the intracellular targeting of a collection of proteins exhibiting a single TMD. Our results reveal that membrane topology is a major targeting element in the early secretory pathway: type I proteins with a short transmembrane domain are targeted to the ER, and type II proteins to the Golgi apparatus. A combination of three features accounts for the sorting of simple membrane proteins in the secretory pathway: membrane topology, length and hydrophilicity of the TMD, and size of the cytosolic domain. By clarifying the rules governing sorting to the ER and to the Golgi apparatus, our study may revive the search for sorting mechanisms in the early secretory pathway.
    Keywords:  Endoplasmic reticulum; Golgi apparatus; Transmembrane domain; Type I protein; Type II protein
    DOI:  https://doi.org/10.1242/jcs.261738
  13. J Cell Biol. 2024 Sep 02. pii: e202310049. [Epub ahead of print]223(9):
    Ca2+-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy upon glucose starvation.
    Weijing Yao, Yingcong Chen, Yi Zhang, Shu Zhong, Miaojuan Ye, Yuting Chen, Siyu Fan, Miao Ye, Huan Yang, Yixing Li, Choufei Wu, Mingzhu Fan, Shan Feng, Zhaoxiang He, Long Zhou, Liqin Zhang, Yigang Wang, Wei Liu, Jingjing Tong, Du Feng, Cong Yi.
      Autophagy is essential for maintaining glucose homeostasis. However, the mechanism by which cells sense and respond to glucose starvation to induce autophagy remains incomplete. Here, we show that calcium serves as a fundamental triggering signal that connects environmental sensing to the formation of the autophagy initiation complex during glucose starvation. Mechanistically, glucose starvation instigates the release of vacuolar calcium into the cytoplasm, thus triggering the activation of Rck2 kinase. In turn, Rck2-mediated Atg11 phosphorylation enhances Atg11 interactions with Bmh1/2 bound to the Snf1-Sip1-Snf4 complex, leading to recruitment of vacuolar membrane-localized Snf1 to the PAS and subsequent Atg1 activation, thereby initiating autophagy. We also identified Glc7, a protein phosphatase-1, as a critical regulator of the association between Bmh1/2 and the Snf1 complex. We thus propose that calcium-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy by controlling Snf1-mediated Atg1 activation in response to glucose starvation.
    DOI:  https://doi.org/10.1083/jcb.202310049
  14. Nat Commun. 2024 Jul 10. 15(1): 5804
    Programmed cell death regulator BAP2 is required for IRE1-mediated unfolded protein response in Arabidopsis.
    Noelia Pastor-Cantizano, Evan R Angelos, Cristina Ruberti, Tao Jiang, Xiaoyu Weng, Brandon C Reagan, Taslima Haque, Thomas E Juenger, Federica Brandizzi.
      Environmental and physiological situations can challenge the balance between protein synthesis and folding capacity of the endoplasmic reticulum (ER) and cause ER stress, a potentially lethal condition. The unfolded protein response (UPR) restores ER homeostasis or actuates programmed cell death (PCD) when ER stress is unresolved. The cell fate determination mechanisms of the UPR are not well understood, especially in plants. Here, we integrate genetics and ER stress profiling with natural variation and quantitative trait locus analysis of 350 natural accessions of the model species Arabidopsis thaliana. Our analyses implicate a single nucleotide polymorphism to the loss of function of the general PCD regulator BON-ASSOCIATED PROTEIN2 (BAP2) in UPR outcomes. We establish that ER stress-induced BAP2 expression is antagonistically regulated by the UPR master regulator, inositol-requiring enzyme 1 (IRE1), and that BAP2 controls adaptive UPR amplitude in ER stress and ignites pro-death mechanisms in conditions of UPR insufficiency.
    DOI:  https://doi.org/10.1038/s41467-024-50105-6
  15. Mol Cell. 2024 Jul 11. pii: S1097-2765(24)00525-2. [Epub ahead of print]84(13): 2403-2404
    Chaperones in concert: Orchestrating co-translational protein folding in the cell.
    Bob Schiffrin, Antonio N Calabrese.
      In this issue of Molecular Cell, Roeselová et al.1 provide insights into co-translational folding of a multidomain protein in bacteria, revealing how the chaperones Trigger Factor, DnaJ, and DnaK work together to facilitate the folding of nascent chains.
    DOI:  https://doi.org/10.1016/j.molcel.2024.06.018
  16. EMBO Rep. 2024 Jul 09.
    The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.
    Aniket S Joshi, Meiricris Tomaz da Silva, Anirban Roy, Tatiana E Koike, Mingfu Wu, Micah B Castillo, Preethi H Gunaratne, Yu Liu, Takao Iwawaki, Ashok Kumar.
      Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.
    Keywords:  IRE1; Muscle Regeneration; Myoblast Fusion; XBP1; and Myomaker
    DOI:  https://doi.org/10.1038/s44319-024-00197-4
  17. J Biol Chem. 2024 Jul 04. pii: S0021-9258(24)02041-6. [Epub ahead of print] 107540
    Characterization of ribosome stalling and no-go mRNA decay stimulated by the Fragile X protein, FMRP.
    MaKenzie R Scarpitti, Benjamin Pastore, Wen Tang, Michael G Kearse.
      Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA•ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not poly(A)-binding protein, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of 4 putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes but only stimulates NGD of a small select set of transcripts, revealing a minor role of FMRP that would be misregulated in FXS.
    Keywords:  RNA binding protein; neurological disease; ribosome; translation control; translation regulation
    DOI:  https://doi.org/10.1016/j.jbc.2024.107540
  18. PLoS Biol. 2024 Jul 11. 22(7): e3002720
    Mutations in nucleotide metabolism genes bypass proteasome defects in png-1/NGLY1-deficient Caenorhabditis elegans.
    Katherine S Yanagi, Briar Jochim, Sheikh Omar Kunjo, Peter Breen, Gary Ruvkun, Nicolas Lehrbach.
      The conserved SKN-1A/Nrf1 transcription factor regulates the expression of proteasome subunit genes and is essential for maintenance of adequate proteasome function in animal development, aging, and stress responses. Unusual among transcription factors, SKN-1A/Nrf1 is a glycoprotein synthesized in the endoplasmic reticulum (ER). N-glycosylated SKN-1A/Nrf1 exits the ER and is deglycosylated in the cytosol by the PNG-1/NGLY1 peptide:N-glycanase. Deglycosylation edits the protein sequence of SKN-1A/Nrf1 by converting N-glycosylated asparagine residues to aspartate, which is necessary for SKN-1A/Nrf1 transcriptional activation of proteasome subunit genes. Homozygous loss-of-function mutations in the peptide:N-glycanase (NGLY1) gene cause NGLY1 deficiency, a congenital disorder of deglycosylation. There are no effective treatments for NGLY1 deficiency. Since SKN-1A/Nrf1 is a major client of NGLY1, the resulting proteasome deficit contributes to NGLY1 disease. We sought to identify targets for mitigation of proteasome dysfunction in NGLY1 deficiency that might indicate new avenues for treatment. We isolated mutations that suppress the sensitivity to proteasome inhibitors caused by inactivation of the NGLY1 ortholog PNG-1 in Caenorhabditis elegans. We identified multiple suppressor mutations affecting 3 conserved genes: rsks-1, tald-1, and ent-4. We show that the suppressors act through a SKN-1/Nrf-independent mechanism and confer proteostasis benefits consistent with amelioration of proteasome dysfunction. ent-4 encodes an intestinal nucleoside/nucleotide transporter, and we show that restriction of nucleotide availability is beneficial, whereas a nucleotide-rich diet exacerbates proteasome dysfunction in PNG-1/NGLY1-deficient C. elegans. Our findings suggest that dietary or pharmacological interventions altering nucleotide availability have the potential to mitigate proteasome insufficiency in NGLY1 deficiency and other diseases associated with proteasome dysfunction.
    DOI:  https://doi.org/10.1371/journal.pbio.3002720
  19. Nat Commun. 2024 Jul 10. 15(1): 5418
    Protein Condensate Atlas from predictive models of heteromolecular condensate composition.
    Kadi L Saar, Rob M Scrutton, Kotryna Bloznelyte, Alexey S Morgunov, Lydia L Good, Alpha A Lee, Sarah A Teichmann, Tuomas P J Knowles.
      Biomolecular condensates help cells organise their content in space and time. Cells harbour a variety of condensate types with diverse composition and many are likely yet to be discovered. Here, we develop a methodology to predict the composition of biomolecular condensates. We first analyse available proteomics data of cellular condensates and find that the biophysical features that determine protein localisation into condensates differ from known drivers of homotypic phase separation processes, with charge mediated protein-RNA and hydrophobicity mediated protein-protein interactions playing a key role in the former process. We then develop a machine learning model that links protein sequence to its propensity to localise into heteromolecular condensates. We apply the model across the proteome and find many of the top-ranked targets outside the original training data to localise into condensates as confirmed by orthogonal immunohistochemical staining imaging. Finally, we segment the condensation-prone proteome into condensate types based on an overlap with biomolecular interaction profiles to generate a Protein Condensate Atlas. Several condensate clusters within the Atlas closely match the composition of experimentally characterised condensates or regions within them, suggesting that the Atlas can be valuable for identifying additional components within known condensate systems and discovering previously uncharacterised condensates.
    DOI:  https://doi.org/10.1038/s41467-024-48496-7
  20. EMBO J. 2024 Jul 08.
    The caspase-activated DNase promotes cellular senescence.
    Aladin Haimovici, Valentin Rupp, Tarek Amer, Abdul Moeed, Arnim Weber, Georg Häcker.
      Cellular senescence is a response to many stressful insults. DNA damage is a consistent feature of senescent cells, but in many cases its source remains unknown. Here, we identify the cellular endonuclease caspase-activated DNase (CAD) as a critical factor in the initiation of senescence. During apoptosis, CAD is activated by caspases and cleaves the genomic DNA of the dying cell. The CAD DNase is also activated by sub-lethal signals in the apoptotic pathway, causing DNA damage in the absence of cell death. We show that sub-lethal signals in the mitochondrial apoptotic pathway induce CAD-dependent senescence. Inducers of cellular senescence, such as oncogenic RAS, type-I interferon, and doxorubicin treatment, also depend on CAD presence for senescence induction. By directly activating CAD experimentally, we demonstrate that its activity is sufficient to induce senescence in human cells. We further investigate the contribution of CAD to senescence in vivo and find substantially reduced signs of senescence in organs of ageing CAD-deficient mice. Our results show that CAD-induced DNA damage in response to various stimuli is an essential contributor to cellular senescence.
    Keywords:  Ageing; Apoptosis; Caspase-activated DNase; Senescence
    DOI:  https://doi.org/10.1038/s44318-024-00163-9
  21. Cell. 2024 Jun 28. pii: S0092-8674(24)00693-7. [Epub ahead of print]
    A non-canonical role for a small nucleolar RNA in ribosome biogenesis and senescence.
    Yujing Cheng, Siwen Wang, He Zhang, Jong-Sun Lee, Chunyang Ni, Jason Guo, Eric Chen, Shenming Wang, Asha Acharya, Tsung-Cheng Chang, Michael Buszczak, Hao Zhu, Joshua T Mendell.
      Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.
    Keywords:  RPL23; SNORA13; nucleolar stress; p53; ribosome biogenesis; senescence; snoRNA
    DOI:  https://doi.org/10.1016/j.cell.2024.06.019
  22. Nat Cell Biol. 2024 Jul 12.
    Pressure sensing of lysosomes enables control of TFEB responses in macrophages.
    Ruiqi Cai, Ori Scott, Gang Ye, Trieu Le, Ekambir Saran, Whijin Kwon, Subothan Inpanathan, Blayne A Sayed, Roberto J Botelho, Amra Saric, Stefan Uderhardt, Spencer A Freeman.
      Polymers are endocytosed and hydrolysed by lysosomal enzymes to generate transportable solutes. While the transport of diverse organic solutes across the plasma membrane is well studied, their necessary ongoing efflux from the endocytic fluid into the cytosol is poorly appreciated by comparison. Myeloid cells that employ specialized types of endocytosis, that is, phagocytosis and macropinocytosis, are highly dependent on such transport pathways to prevent the build-up of hydrostatic pressure that otherwise offsets lysosomal dynamics including vesiculation, tubulation and fission. Without undergoing rupture, we found that lysosomes incurring this pressure owing to defects in solute efflux, are unable to retain luminal Na+, which collapses its gradient with the cytosol. This cation 'leak' is mediated by pressure-sensitive channels resident to lysosomes and leads to the inhibition of mTORC1, which is normally activated by Na+-coupled amino acid transporters driven by the Na+ gradient. As a consequence, the transcription factors TFEB/TFE3 are made active in macrophages with distended lysosomes. In addition to their role in lysosomal biogenesis, TFEB/TFE3 activation causes the release of MCP-1/CCL2. In catabolically stressed tissues, defects in efflux of solutes from the endocytic pathway leads to increased monocyte recruitment. Here we propose that macrophages respond to a pressure-sensing pathway on lysosomes to orchestrate lysosomal biogenesis as well as myeloid cell recruitment.
    DOI:  https://doi.org/10.1038/s41556-024-01459-y
  23. Nat Cell Biol. 2024 Jul 12.
    Neutrophil-derived migrasomes are an essential part of the coagulation system.
    Dong Jiang, Lin Jiao, Qing Li, Renxiang Xie, Haohao Jia, ShiHui Wang, Yining Chen, Siyuan Liu, Dandan Huang, Jiajia Zheng, Wenhao Song, Ying Li, JianFeng Chen, Jinsong Li, Binwu Ying, Li Yu.
      Migrasomes are organelles that are generated by migrating cells. Here we report the key role of neutrophil-derived migrasomes in haemostasis. We found that a large number of neutrophil-derived migrasomes exist in the blood of mice and humans. Compared with neutrophil cell bodies and platelets, these migrasomes adsorb and enrich coagulation factors on the surface. Moreover, they are highly enriched with adhesion molecules, which enable them to preferentially accumulate at sites of injury, where they trigger platelet activation and clot formation. Depletion of neutrophils, or genetic reduction of the number of these migrasomes, significantly decreases platelet plug formation and impairs coagulation. These defects can be rescued by intravenous injection of purified neutrophil-derived migrasomes. Our study reveals neutrophil-derived migrasomes as a previously unrecognized essential component of the haemostasis system, which may shed light on the cause of various coagulation disorders and open therapeutic possibilities.
    DOI:  https://doi.org/10.1038/s41556-024-01440-9
  24. STAR Protoc. 2024 Jul 05. pii: S2666-1667(24)00336-8. [Epub ahead of print]5(3): 103171
    Protocol to detect and quantify interactions between proteins expressed in Drosophila S2 cells.
    Cristina C DeOliveira, Changfan Lin, Brian R Crane.
      Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer. For complete details on the use and execution of this protocol, please refer to Lin et al.1.
    Keywords:  Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification
    DOI:  https://doi.org/10.1016/j.xpro.2024.103171
  25. Cell Rep. 2024 Jul 09. pii: S2211-1247(24)00811-8. [Epub ahead of print]43(7): 114482
    Mast cell secretory granule fusion with amphisomes coordinates their homotypic fusion and release of exosomes.
    Sewar Omari, Amit Roded, Maggie Eisenberg, Hydar Ali, Mitsunori Fukuda, Stephen J Galli, Ronit Sagi-Eisenberg.
      Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3+ late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P3 to PI(4,5)P2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes.
    Keywords:  CP: Cell biology; CP: Immunology; amphisomes; exosomes; lysosome related organelles; mast cells; phosphoinositide conversion; secretory granules
    DOI:  https://doi.org/10.1016/j.celrep.2024.114482
  26. FEBS J. 2024 Jul 08.
    Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.
    Matteo Costacurta, Jarrod J Sandow, Belinda Maher, Olivia Susanto, Stephin J Vervoort, Jennifer R Devlin, Daniel Garama, Mark R Condina, Joel R Steele, Hossein V Kahrood, Daniel Gough, Ricky W Johnstone, Jake Shortt.
      Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.
    Keywords:  BioID2; IMiDs; bortezomib; cereblon; myosin
    DOI:  https://doi.org/10.1111/febs.17196
  27. Arch Insect Biochem Physiol. 2024 Jul;116(3): e22127
    Bombyx mori UFL1 facilitates BmNPV proliferation by regulating host cell apoptosis through PERK.
    Yijun Zheng, Haonan Meng, Xiaochun Jiang, Shoujun Huang.
      Ubiquitin-fold modifier 1 (UFM1) is attached to protein substrates through the sequential activity of an E1 (UBA5)-E2 (UFC1)-E3 (UFL1) cascade. UFL1 is the E3 ligase for UFMylation in vertebrates. However, there have been no studies on UFL1 in silkworm to date. In this study, we identified a UFL1 ortholog in Bombyx mori genome. Spatio-temporal expression profiles showed that BmUFL1 expression was high in the midgut, epidermis, and testis and in the pupa-adult stage. BmUFL1 knockdown inhibited B. mori nucleopolyhedrovirus (BmNPV) proliferation, while BmUFL1 overexpression promoted BmNPV proliferation. Mechanically, protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling and cell apoptosis are involved in BmUFL1-regulated BmNPV proliferation. Overall, these results suggest that BmUFL1 facilitates BmNPV proliferation in silkworm.
    Keywords:  BmNPV; BmUFL1; ER stress; apoptosis
    DOI:  https://doi.org/10.1002/arch.22127
  28. Protein Sci. 2024 Aug;33(8): e5116
    DrugDomain: The evolutionary context of drugs and small molecules bound to domains.
    Kirill E Medvedev, R Dustin Schaeffer, Nick V Grishin.
      Interactions between proteins and small organic compounds play a crucial role in regulating protein functions. These interactions can modulate various aspects of protein behavior, including enzymatic activity, signaling cascades, and structural stability. By binding to specific sites on proteins, small organic compounds can induce conformational changes, alter protein-protein interactions, or directly affect catalytic activity. Therefore, many drugs available on the market today are small molecules (72% of all approved drugs in the last 5 years). Proteins are composed of one or more domains: evolutionary units that convey function or fitness either singly or in concert with others. Understanding which domain(s) of the target protein binds to a drug can lead to additional opportunities for discovering novel targets. The evolutionary classification of protein domains (ECOD) classifies domains into an evolutionary hierarchy that focuses on distant homology. Previously, no structure-based protein domain classification existed that included information about both the interaction between small molecules or drugs and the structural domains of a target protein. This data is especially important for multidomain proteins and large complexes. Here, we present the DrugDomain database that reports the interaction between ECOD of human target proteins and DrugBank molecules and drugs. The pilot version of DrugDomain describes the interaction of 5160 DrugBank molecules associated with 2573 human proteins. It describes domains for all experimentally determined structures of these proteins and incorporates AlphaFold models when such structures are unavailable. The DrugDomain database is available online: http://prodata.swmed.edu/DrugDomain/.
    Keywords:  domain; drugs; protein structure; small molecules; target
    DOI:  https://doi.org/10.1002/pro.5116
  29. Biochim Biophys Acta Mol Basis Dis. 2024 Jul 05. pii: S0925-4439(24)00327-2. [Epub ahead of print] 167334
    Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells.
    Xiaofei Zou, Xiuru Liang, Wangjuan Dai, Ting Zhu, Chaoyi Wang, Yutian Zhou, Yi Qian, Zhengjie Yan, Chao Gao, Li Gao, Yugui Cui, Jiayin Liu, Yan Meng.
      Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4-/- mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4-/- mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.
    Keywords:  Follicle-stimulating hormone receptor (FSHR); Granulosa cells; Ovarian aging; Oxidative protein folding; Peroxiredoxin 4 (PRDX4); Protein homeostasis
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167334
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