bims-proteo Biomed News
on Proteostasis
Issue of 2024‒02‒25
35 papers selected by
Eric Chevet, INSERM
  1. UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.
  2. Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.
  3. The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.
  4. Targeted protein degradation via intramolecular bivalent glues.
  5. Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis.
  6. Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs.
  7. Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.
  8. Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress.
  9. Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.
  10. Selective degradation of ribosomes during oncogene-induced senescence: molecular insights and biological perspectives.
  11. Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers.
  12. Targeted protein degradation directly engaging lysosomes or proteasomes.
  13. Mouse oocytes sequester aggregated proteins in degradative super-organelles.
  14. ER remodeling via lipid metabolism.
  15. Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.
  16. HRD1-induced TMEM2 ubiquitination promotes ER stress-mediated apoptosis through a non-canonical pathway in intestinal ischemia/reperfusion.
  17. RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis.
  18. Neuronal potassium channel activity triggers initiation of mRNA translation through binding of translation regulators.
  19. A mutational atlas for Parkin proteostasis.
  20. TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux.
  21. UFL1 ablation in T cells suppresses PD-1 UFMylation to enhance anti-tumor immunity.
  22. Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity.
  23. IRE1 RNase controls CD95-mediated cell death.
  24. BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.
  25. Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.
  26. SUMO Modifies GβL and Mediates mTOR Signaling.
  27. Ageing-dependent thiol oxidation reveals early oxidation of proteins with core proteostasis functions.
  28. UDP-glucose dehydrogenase supports autophagy-deficient PDAC growth via increasing hyaluronic acid biosynthesis.
  29. A new era of cysteine proteomics - Technological advances in thiol biology.
  30. ATG5 attenuates inflammatory signaling in mouse embryonic stem cells to control differentiation.
  31. O-GlcNAcylation of E3 ubiquitin ligase SKP2 promotes hepatocellular carcinoma proliferation.
  32. Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.
  33. Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.
  34. Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.
  35. Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release.
  1. Nature. 2024 Feb 21.
    UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.
    Paul A DaRosa, Ivan Penchev, Samantha C Gumbin, Francesco Scavone, Magda Wąchalska, Joao A Paulo, Alban Ordureau, Joshua J Peter, Yogesh Kulathu, J Wade Harper, Thomas Becker, Roland Beckmann, Ron R Kopito.
      Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.
    DOI:  https://doi.org/10.1038/s41586-024-07073-0
  2. Mol Cell. 2024 Feb 20. pii: S1097-2765(24)00089-3. [Epub ahead of print]
    Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.
    Jerry Li, Nicholas Purser, Joanna Liwocha, Daniel C Scott, Holly A Byers, Barbara Steigenberger, Spencer Hill, Ishita Tripathi-Giesgen, Trent Hinkle, Fynn M Hansen, J Rajan Prabu, Senthil K Radhakrishnan, Donald S Kirkpatrick, Kurt M Reichermeier, Brenda A Schulman, Gary Kleiger.
      Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
    Keywords:  E2; E3; PROTAC; cryo-EM; proteolysis targeting chimera; targeted protein degradation; ubiquitin; ubiquitin ligase; ubiquitin-carrying enzyme; ubiquitin-conjugating enzyme
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.022
  3. Nature. 2024 Feb 21.
    The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.
    Linda Makhlouf, Joshua J Peter, Helge M Magnussen, Rohan Thakur, David Millrine, Thomas C Minshull, Grace Harrison, Joby Varghese, Frederic Lamoliatte, Martina Foglizzo, Thomas Macartney, Antonio N Calabrese, Elton Zeqiraj, Yogesh Kulathu.
      Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.
    DOI:  https://doi.org/10.1038/s41586-024-07093-w
  4. Nature. 2024 Feb 21.
    Targeted protein degradation via intramolecular bivalent glues.
    Oliver Hsia, Matthias Hinterndorfer, Angus D Cowan, Kentaro Iso, Tasuku Ishida, Ramasubramanian Sundaramoorthy, Mark A Nakasone, Hana Imrichova, Caroline Schätz, Andrea Rukavina, Koraljka Husnjak, Martin Wegner, Alejandro Correa-Sáez, Conner Craigon, Ryan Casement, Chiara Maniaci, Andrea Testa, Manuel Kaulich, Ivan Dikic, Georg E Winter, Alessio Ciulli.
      Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
    DOI:  https://doi.org/10.1038/s41586-024-07089-6
  5. Nat Commun. 2024 Feb 22. 15(1): 1637
    Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis.
    Ji Geng, Shuangxi Li, Yu Li, Zhihao Wu, Sunil Bhurtel, Suman Rimal, Danish Khan, Rani Ohja, Onn Brandman, Bingwei Lu.
      Translational control exerts immediate effect on the composition, abundance, and integrity of the proteome. Ribosome-associated quality control (RQC) handles ribosomes stalled at the elongation and termination steps of translation, with ZNF598 in mammals and Hel2 in yeast serving as key sensors of translation stalling and coordinators of downstream resolution of collided ribosomes, termination of stalled translation, and removal of faulty translation products. The physiological regulation of RQC in general and ZNF598 in particular in multicellular settings is underexplored. Here we show that ZNF598 undergoes regulatory K63-linked ubiquitination in a CNOT4-dependent manner and is upregulated upon mitochondrial stresses in mammalian cells and Drosophila. ZNF598 promotes resolution of stalled ribosomes and protects against mitochondrial stress in a ubiquitination-dependent fashion. In Drosophila models of neurodegenerative diseases and patient cells, ZNF598 overexpression aborts stalled translation of mitochondrial outer membrane-associated mRNAs, removes faulty translation products causal of disease, and improves mitochondrial and tissue health. These results shed lights on the regulation of ZNF598 and its functional role in mitochondrial and tissue homeostasis.
    DOI:  https://doi.org/10.1038/s41467-024-45525-3
  6. bioRxiv. 2024 Feb 08. pii: 2024.02.04.578812. [Epub ahead of print]
    Serine mistranslation induces the integrated stress response without accumulation of uncharged tRNAs.
    Hong Zhang, Jiqiang Ling.
      Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that support robust and accurate protein synthesis. A rapidly expanding number of studies show that mutations in aaRSs lead to multiple human diseases, including neurological disorders and cancer. Much remains unknown about how aaRS mutations impact human health. In particular, how aminoacylation errors affect stress responses and fitness in eukaryotic cells remains poorly understood. The integrated stress response (ISR) is an adaptive mechanism in response to multiple stresses. However, chronic activation of the ISR contributes to the development of multiple diseases (e.g., neuropathies). Here we show that Ser misincorporation into Ala and Thr codons, resulting from aaRS editing defects or mutations in tRNAs, constitutively active the ISR. Such activation does not appear to depend on the accumulation of uncharged tRNAs, implicating that Ser mistranslation may lead to ribosome stalling and collision.
    DOI:  https://doi.org/10.1101/2024.02.04.578812
  7. Mol Biol Cell. 2024 Feb 21. mbcE23070296
    Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.
    Davi Goncalves, Duong Long Duy, Sara Peffer, Kevin A Morano.
      Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a process of long-term sequestration.
    DOI:  https://doi.org/10.1091/mbc.E23-07-0296
  8. bioRxiv. 2024 Feb 07. pii: 2024.02.06.579149. [Epub ahead of print]
    Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress.
    José M Liboy-Lugo, Carla A Espinoza, Jessica Sheu-Gruttadauria, Jesslyn E Park, Albert Xu, Ziad Jowhar, Angela L Gao, José A Carmona-Negrón, Torsten Wittmann, Natalia Jura, Stephen N Floor.
      Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.
    Keywords:  G3BPs; Stress granules; integrated stress response; ribosome profiling
    DOI:  https://doi.org/10.1101/2024.02.06.579149
  9. Proc Natl Acad Sci U S A. 2024 Feb 27. 121(9): e2318046121
    Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.
    Sankalp Shukla, Wei Chen, Shanlin Rao, Serim Yang, Chenxi Ou, Kevin P Larsen, Gerhard Hummer, Phyllis I Hanson, James H Hurley.
      Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.
    Keywords:  ESCRT; calcium-binding protein; lysosome; membrane repair; reconstitution
    DOI:  https://doi.org/10.1073/pnas.2318046121
  10. Autophagy. 2024 Feb 21. 1-3
    Selective degradation of ribosomes during oncogene-induced senescence: molecular insights and biological perspectives.
    Aida Rodríguez López, Lisa B Frankel.
      Ribosomes are conserved macromolecular machines that are responsible for protein synthesis in all cells. While our knowledge of ribosome biogenesis and function has increased significantly in recent years, little is known about how ribosomes are degraded under specific cellular conditions. We recently uncovered that ribosomes are efficiently turned over by selective macroautophagy/autophagy during oncogene-induced senescence (OIS). By profiling the ribosome interactome in human fibroblasts undergoing OIS, we discovered a key role for the de-ubiquitinating enzyme USP10 in guiding this process. Release of USP10 from ribosomes during senescence leads to their enhanced ubiquitination and selective sequestering by autophagy through the SQSTM1/p62 receptor protein. This process is important for sustaining senescence-associated metabolome and secretome alterations.
    Keywords:  Oncogene-induced senescence; USP10; ribosomes; selective autophagy; translation; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2024.2319022
  11. Cell Rep. 2024 Feb 17. pii: S2211-1247(24)00131-1. [Epub ahead of print]43(2): 113803
    Protein-folding chaperones predict structure-function relationships and cancer risk in BRCA1 mutation carriers.
    Brant Gracia, Patricia Montes, Angelica Maria Gutierrez, Banu Arun, Georgios Ioannis Karras.
      Predicting the risk of cancer mutations is critical for early detection and prevention, but differences in allelic severity of human carriers confound risk predictions. Here, we elucidate protein folding as a cellular mechanism driving differences in mutation severity of tumor suppressor BRCA1. Using a high-throughput protein-protein interaction assay, we show that protein-folding chaperone binding patterns predict the pathogenicity of variants in the BRCA1 C-terminal (BRCT) domain. HSP70 selectively binds 94% of pathogenic BRCA1-BRCT variants, most of which engage HSP70 more than HSP90. Remarkably, the magnitude of HSP70 binding linearly correlates with loss of folding and function. We identify a prevalent class of human hypomorphic BRCA1 variants that bind moderately to chaperones and retain partial folding and function. Furthermore, chaperone binding signifies greater mutation penetrance and earlier cancer onset in the clinic. Our findings demonstrate the utility of chaperones as quantitative cellular biosensors of variant folding, phenotypic severity, and cancer risk.
    Keywords:  BRCA1; CP: Cancer; HSP70; HSP90; cancer; expressivity; genetic variation; hypomorphism; penetrance; protein-folding chaperones; variants of uncertain significance
    DOI:  https://doi.org/10.1016/j.celrep.2024.113803
  12. Chem Soc Rev. 2024 Feb 19.
    Targeted protein degradation directly engaging lysosomes or proteasomes.
    Jiseong Kim, Insuk Byun, Do Young Kim, Hyunhi Joh, Hak Joong Kim, Min Jae Lee.
      Targeted protein degradation (TPD) has been established as a viable alternative to attenuate the function of a specific protein of interest in both biological and clinical contexts. The unique TPD mode-of-action has allowed previously undruggable proteins to become feasible targets, expanding the landscape of "druggable" properties and "privileged" target proteins. As TPD continues to evolve, a range of innovative strategies, which do not depend on recruiting E3 ubiquitin ligases as in proteolysis-targeting chimeras (PROTACs), have emerged. Here, we present an overview of direct lysosome- and proteasome-engaging modalities and discuss their perspectives, advantages, and limitations. We outline the chemical composition, biochemical activity, and pharmaceutical characteristics of each degrader. These alternative TPD approaches not only complement the first generation of PROTACs for intracellular protein degradation but also offer unique strategies for targeting pathologic proteins located on the cell membrane and in the extracellular space.
    DOI:  https://doi.org/10.1039/d3cs00344b
  13. Cell. 2024 Feb 09. pii: S0092-8674(24)00068-0. [Epub ahead of print]
    Mouse oocytes sequester aggregated proteins in degradative super-organelles.
    Gabriele Zaffagnini, Shiya Cheng, Marion C Salzer, Barbara Pernaute, Juan Manuel Duran, Manuel Irimia, Melina Schuh, Elvan Böke.
      Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.
    Keywords:  RUFY1; embryo; female fertility; lysosomal acidification; membraneless organelles; oocyte; oocyte quality; protein aggregation; proteostasis; super-organelles
    DOI:  https://doi.org/10.1016/j.cell.2024.01.031
  14. Trends Cell Biol. 2024 Feb 22. pii: S0962-8924(24)00023-0. [Epub ahead of print]
    ER remodeling via lipid metabolism.
    Wonyul Jang, Volker Haucke.
      Unlike most other organelles found in multiple copies, the endoplasmic reticulum (ER) is a unique singular organelle within eukaryotic cells. Despite its continuous membrane structure, encompassing more than half of the cellular endomembrane system, the ER is subdivided into specialized sub-compartments, including morphological, membrane contact site (MCS), and de novo organelle biogenesis domains. In this review, we discuss recent emerging evidence indicating that, in response to nutrient stress, cells undergo a reorganization of these sub-compartmental ER domains through two main mechanisms: non-destructive remodeling of morphological ER domains via regulation of MCS and organelle hitchhiking, and destructive remodeling of specialized domains by ER-phagy. We further highlight and propose a critical role of membrane lipid metabolism in this ER remodeling during starvation.
    Keywords:  endoplasmic reticulum; hitchhiking; lipids; membrane contact sites; membrane remodeling; metabolism; nutrient stress
    DOI:  https://doi.org/10.1016/j.tcb.2024.01.011
  15. FEBS Lett. 2024 Feb 23.
    Protein sorting upon exit from the endoplasmic reticulum dominates Golgi biogenesis in budding yeast.
    Saku Sasaki, Philipp Schlarmann, Kazuki Hanaoka, Hinako Nishii, Hisao Moriya, Manuel Muñiz, Kouichi Funato.
      Cells sense and control the number and quality of their organelles, but the underlying mechanisms of this regulation are not understood. Our recent research in the yeast Saccharomyces cerevisiae has shown that long acyl chain ceramides in the endoplasmic reticulum (ER) membrane and the lipid moiety of glycosylphosphatidylinositol (GPI) anchor determine the sorting of GPI-anchored proteins in the ER. Here, we show that a mutant strain, which produces shorter ceramides than the wild-type strain, displays a different count of Golgi cisternae. Moreover, deletions of proteins that remodel the lipid portion of GPI anchors resulted in an abnormal number of Golgi cisternae. Thus, our study reveals that protein sorting in the ER plays a critical role in maintaining Golgi biogenesis.
    Keywords:  GPI; Golgi biogenesis; ceramide; endoplasmic reticulum; lipid remodeling; protein sorting
    DOI:  https://doi.org/10.1002/1873-3468.14830
  16. Cell Death Dis. 2024 Feb 20. 15(2): 154
    HRD1-induced TMEM2 ubiquitination promotes ER stress-mediated apoptosis through a non-canonical pathway in intestinal ischemia/reperfusion.
    Xuzi Zhao, Deshun Liu, Yan Zhao, Zhecheng Wang, Yue Wang, Zhao Chen, Shili Ning, Guangzhi Wang, Lu Meng, Jihong Yao, Xiaofeng Tian.
      Intestinal ischemia/reperfusion (I/R) injury is a typical pathological course in the clinic with a high morbidity rate. Recent research has pointed out the critical role of ubiquitination during the occurrence and development of intestinal I/R by precisely mediating protein quality control and function. Here, we conducted an integrated multiomic analysis to identify critical ubiquitination-associated molecules in intestinal I/R and identified endoplasmic reticulum-located HRD1 as a candidate molecule. During intestinal I/R, excessive ER stress plays a central role by causing apoptotic pathway activation. In particular, we found that ER stress-mediated apoptosis was mitigated by HRD1 knockdown in intestinal I/R mice. Mechanistically, TMEM2 was identified as a new substrate of HRD1 in intestinal I/R by mass spectrometry analysis, which has a crucial role in attenuating apoptosis and promoting non-canonical ER stress resistance. A strong negative correlation was found between the protein levels of HRD1 and TMEM2 in human intestinal ischemia samples. Specifically, HRD1 interacted with the lysine 42 residue of TMEM2 and reduced its stabilization by K48-linked polyubiquitination. Furthermore, KEGG pathway analysis revealed that TMEM2 regulated ER stress-mediated apoptosis in association with the PI3k/Akt signaling pathway rather than canonical ER stress pathways. In summary, HRD1 regulates ER stress-mediated apoptosis through a non-canonical pathway by ubiquitinating TMEM2 and inhibiting PI3k/Akt activation during intestinal I/R. The current study shows that HRD1 is an intestinal I/R critical regulator and that targeting the HRD1/TMEM2 axis may be a promising therapeutic approach.
    DOI:  https://doi.org/10.1038/s41419-024-06504-0
  17. Cell Rep. 2024 Feb 16. pii: S2211-1247(24)00130-X. [Epub ahead of print]43(2): 113802
    RNA helicase IGHMBP2 regulates THO complex to ensure cellular mRNA homeostasis.
    Archana Bairavasundaram Prusty, Anja Hirmer, Julieth Andrea Sierra-Delgado, Hannes Huber, Ulf-Peter Guenther, Andreas Schlosser, Olexandr Dybkov, Ezgi Yildirim, Henning Urlaub, Kathrin C Meyer, Sibylle Jablonka, Florian Erhard, Utz Fischer.
      RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5' UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export. Accordingly, failure of IGHMBP2 regulation of THOC causes perturbations of the transcriptome and its encoded proteome, and ablation of THOC subunits phenocopies these changes. Thus, IGHMBP2 is an upstream regulator of THOC. Of note, IGHMBP2-dependent regulation of THOC is also observed in astrocytes derived from patients with SMARD1 disease, suggesting that deregulated mRNA metabolism contributes to SMARD1 etiology and may enable alternative therapeutic avenues.
    Keywords:  CP: Molecular biology; CP: Neuroscience; IGHMBP2; RNA helicase; RNA regulon; SMARD1; THOC; mRNA homeostasis; translation regulation
    DOI:  https://doi.org/10.1016/j.celrep.2024.113802
  18. bioRxiv. 2024 Feb 07. pii: 2024.02.07.579306. [Epub ahead of print]
    Neuronal potassium channel activity triggers initiation of mRNA translation through binding of translation regulators.
    Taylor J Malone, Jing Wu, Yalan Zhang, Pawel Licznerski, Rongmin Chen, Sheikh Nahiyan, Maysam Pedram, Elizabeth A Jonas, Leonard K Kaczmarek.
      Neuronal activity stimulates mRNA translation crucial for learning and development. While FMRP (Fragile X Mental Retardation Protein) and CYFIP1 (Cytoplasmic FMR1 Interacting Protein 1) regulate translation, the mechanism linking translation to neuronal activity is not understood. We now find that translation is stimulated when FMRP and CYFIP1 translocate to the potassium channel Slack (KCNT1, Slo2.2). When Slack is activated, both factors are released from eIF4E (Eukaryotic Initiation Factor 4E), where they normally inhibit translation initiation. A constitutively active Slack mutation and pharmacological stimulation of the wild-type channel both increase binding of FMRP and CYFIP1 to the channel, enhancing the translation of a reporter for β-actin mRNA in cell lines and the synthesis of β-actin in neuronal dendrites. Slack activity-dependent translation is abolished when both FMRP and CYFIP1 expression are suppressed. The effects of Slack mutations on activity-dependent translation may explain the severe intellectual disability produced by these mutations in humans.HIGHLIGHTS: Activation of Slack channels triggers translocation of the FMRP/CYFIP1 complexSlack channel activation regulates translation initiation of a β-actin reporter constructA Slack gain-of-function mutation increases translation of β-actin reporter construct and endogenous cortical β-actinFMRP and CYFIP1 are required for Slack activity-dependent translation.
    IN BRIEF: Malone et al . show that the activation of Slack channels triggers translocation of the FMRP/CYFIP1 complex from the translation initiation factor eIF4E to the channel. This translocation releases eIF4E and stimulates mRNA translation of a reporter for β-actin and cortical β-actin mRNA, elucidating the mechanism that connects neuronal activity with translational regulation.
    DOI:  https://doi.org/10.1101/2024.02.07.579306
  19. Nat Commun. 2024 Feb 20. 15(1): 1541
    A mutational atlas for Parkin proteostasis.
    Lene Clausen, Vasileios Voutsinos, Matteo Cagiada, Kristoffer E Johansson, Martin Grønbæk-Thygesen, Snehal Nariya, Rachel L Powell, Magnus K N Have, Vibe H Oestergaard, Amelie Stein, Douglas M Fowler, Kresten Lindorff-Larsen, Rasmus Hartmann-Petersen.
      Proteostasis can be disturbed by mutations affecting folding and stability of the encoded protein. An example is the ubiquitin ligase Parkin, where gene variants result in autosomal recessive Parkinsonism. To uncover the pathological mechanism and provide comprehensive genotype-phenotype information, variant abundance by massively parallel sequencing (VAMP-seq) is leveraged to quantify the abundance of Parkin variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, shows that most low abundance variants are proteasome targets and are located within the structured domains of the protein. Half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) reveals an exposed degron region proximal to the so-called "activation element". This work provides examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation.
    DOI:  https://doi.org/10.1038/s41467-024-45829-4
  20. Autophagy. 2024 Feb 23.
    TRIM27 elicits protective immunity against tuberculosis by activating TFEB-mediated autophagy flux.
    Dongdong Zhao, Lihua Qiang, Zehui Lei, Pupu Ge, Zhe Lu, Yiru Wang, Xinwen Zhang, Yuyun Qiang, Bingxi Li, Yu Pang, Lingqiang Zhang, Cui Hua Liu, Jing Wang.
      Infectious diseases, such as Mycobacterium tuberculosis (Mtb)-caused tuberculosis (TB), remain a global threat exacerbated by increasing drug resistance. Host-directed therapy (HDT) is a promising strategy for infection treatment through targeting host immunity. However, the limited understanding of the function and regulatory mechanism of host factors involved in immune defense against infections has impeded HDT development. Here, we identify the ubiquitin ligase (E3) TRIM27 (tripartite motif-containing 27) as a host protective factor against Mtb by enhancing host macroautophagy/autophagy flux in an E3 ligase activity-independent manner. Mechanistically, upon Mtb infection, nuclear-localized TRIM27 increases and functions as a transcription activator of TFEB (transcription factor EB). Specifically, TRIM27 binds to the TFEB promoter and the TFEB transcription factor CREB1 (cAMP responsive element binding protein 1), thus enhancing CREB1-TFEB promoter binding affinity and promoting CREB1 transcription activity toward TFEB, eventually inducing autophagy-related gene expression as well as autophagy flux activation to clear the pathogen. Furthermore, TFEB activator 1 can rescue TRIM27 deficiency-caused decreased autophagy-related gene transcription and attenuated autophagy flux, and accordingly suppressed the intracellular survival of Mtb in cell and mouse models. Taken together, our data reveal that TRIM27 is a host defense factor against Mtb, and the TRIM27-CREB1-TFEB axis is a potential HDT-based TB target that can enhance host autophagy flux.
    Keywords:  Autophagy flux; Transcription factor EB; cAMP responsive element binding protein 1; mycobacterium tuberculosis; tripartite motif-containing 27; tuberculosis
    DOI:  https://doi.org/10.1080/15548627.2024.2321831
  21. Mol Cell. 2024 Feb 09. pii: S1097-2765(24)00091-1. [Epub ahead of print]
    UFL1 ablation in T cells suppresses PD-1 UFMylation to enhance anti-tumor immunity.
    Chuan He, Xixin Xing, Hsin-Yi Chen, Minling Gao, Jie Shi, Bolin Xiang, Xiangling Xiao, Yishuang Sun, Haisheng Yu, Gaoshan Xu, Yingmeng Yao, Zuosong Xie, Yujie Xing, Bugi Ratno Budiarto, Shih-Yu Chen, Yang Gao, Yu-Ru Lee, Jinfang Zhang.
      UFMylation is an emerging ubiquitin-like post-translational modification that regulates various biological processes. Dysregulation of the UFMylation pathway leads to human diseases, including cancers. However, the physiological role of UFMylation in T cells remains unclear. Here, we report that mice with conditional knockout (cKO) Ufl1, a UFMylation E3 ligase, in T cells exhibit effective tumor control. Single-cell RNA sequencing analysis shows that tumor-infiltrating cytotoxic CD8+ T cells are increased in Ufl1 cKO mice. Mechanistically, UFL1 promotes PD-1 UFMylation to antagonize PD-1 ubiquitination and degradation. Furthermore, AMPK phosphorylates UFL1 at Thr536, disrupting PD-1 UFMylation to trigger its degradation. Of note, UFL1 ablation in T cells reduces PD-1 UFMylation, subsequently destabilizing PD-1 and enhancing CD8+ T cell activation. Thus, Ufl1 cKO mice bearing tumors have a better response to anti-CTLA-4 immunotherapy. Collectively, our findings uncover a crucial role of UFMylation in T cells and highlight UFL1 as a potential target for cancer treatment.
    Keywords:  AMPK; PD-1; UFL1; UFMylation; tumor immunotherapy
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.024
  22. Cell Rep Methods. 2024 Feb 13. pii: S2667-2375(24)00027-4. [Epub ahead of print] 100712
    Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity.
    Ryan Traynor, Jennifer Moran, Michael Stevens, Odetta Antico, Axel Knebel, Bahareh Behrouz, Kalpana Merchant, C James Hastie, Paul Davies, Miratul M K Muqit, Virginia De Cesare.
      Parkinson's disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.
    Keywords:  CP: Molecular biology; CP: Neuroscience; MALDI-TOF/MS; PINK1/Parkin pathway; Parkin E3 ligase; Parkinson's disease; drug discovery; high-throughput screening; ubiquitin
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100712
  23. EMBO Rep. 2024 Feb 21.
    IRE1 RNase controls CD95-mediated cell death.
    Diana Pelizzari-Raymundo, Victoria Maltret, Manon Nivet, Raphael Pineau, Alexandra Papaioannou, Xingchen Zhou, Flavie Caradec, Sophie Martin, Matthieu Le Gallo, Tony Avril, Eric Chevet, Elodie Lafont.
      Signalling by the Unfolded Protein Response (UPR) or by the Death Receptors (DR) are frequently activated towards pro-tumoral outputs in cancer. Herein, we demonstrate that the UPR sensor IRE1 controls the expression of the DR CD95/Fas, and its cell death-inducing ability. Both genetic and pharmacologic blunting of IRE1 activity increased CD95 expression and exacerbated CD95L-induced cell death in glioblastoma (GB) and Triple-Negative Breast Cancer (TNBC) cell lines. In accordance, CD95 mRNA was identified as a target of Regulated IRE1-Dependent Decay of RNA (RIDD). Whilst CD95 expression is elevated in TNBC and GB human tumours exhibiting low RIDD activity, it is surprisingly lower in XBP1s-low human tumour samples. We show that IRE1 RNase inhibition limited CD95 expression and reduced CD95-mediated hepatic toxicity in mice. In addition, overexpression of XBP1s increased CD95 expression and sensitized GB and TNBC cells to CD95L-induced cell death. Overall, these results demonstrate the tight IRE1-mediated control of CD95-dependent cell death in a dual manner through both RIDD and XBP1s, and they identify a novel link between IRE1 and CD95 signalling.
    Keywords:  CD95; Cell Death; ER Stress; IRE1; Unfolded Protein Response
    DOI:  https://doi.org/10.1038/s44319-024-00095-9
  24. Commun Biol. 2024 Feb 20. 7(1): 209
    BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.
    Sharon Tran, Juliani Juliani, Tiffany J Harris, Marco Evangelista, Julian Ratcliffe, Sarah L Ellis, David Baloyan, Camilla M Reehorst, Rebecca Nightingale, Ian Y Luk, Laura J Jenkins, Sonia Ghilas, Marina H Yakou, Chantelle Inguanti, Chad Johnson, Michael Buchert, James C Lee, Peter De Cruz, Kinga Duszyc, Paul A Gleeson, Benjamin T Kile, Lisa A Mielke, Alpha S Yap, John M Mariadason, W Douglas Fairlie, Erinna F Lee.
      Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.
    DOI:  https://doi.org/10.1038/s42003-024-05890-7
  25. Nat Commun. 2024 Feb 20. 15(1): 1531
    Genome-wide screens identify SEL1L as an intracellular rheostat controlling collagen turnover.
    Michael J Podolsky, Benjamin Kheyfets, Monika Pandey, Afaq H Beigh, Christopher D Yang, Carlos O Lizama, Ritwik Datta, Liangguang L Lin, Zhihong Wang, Paul J Wolters, Michael T McManus, Ling Qi, Kamran Atabai.
      Accumulating evidence has implicated impaired extracellular matrix (ECM) clearance as a key factor in fibrotic disease. Despite decades of research elucidating the effectors of ECM clearance, relatively little is understood regarding the upstream regulation of this process. Collagen is the most abundant constituent of normal and fibrotic ECM in mammalian tissues. Its catabolism occurs through extracellular proteolysis and cell-mediated uptake of collagen fragments for intracellular degradation. Given the paucity of information regarding the regulation of this latter process, here we execute unbiased genome-wide screens to understand the molecular underpinnings of cell-mediated collagen clearance. Using this approach, we discover a mechanism through which collagen biosynthesis is sensed by cells internally and directly regulates clearance of extracellular collagen. The sensing mechanism appears to be dependent on endoplasmic reticulum-resident protein SEL1L and occurs via a noncanonical function of this protein. This pathway functions as a homeostatic negative feedback loop that limits collagen accumulation in tissues. In human fibrotic lung disease, the induction of this collagen clearance pathway by collagen synthesis is impaired, thereby contributing to the pathological accumulation of collagen in lung tissue. Thus, we describe cell-autonomous, rheostatic collagen clearance as an important pathway of tissue homeostasis.
    DOI:  https://doi.org/10.1038/s41467-024-45817-8
  26. J Biol Chem. 2024 Feb 21. pii: S0021-9258(24)00154-6. [Epub ahead of print] 105778
    SUMO Modifies GβL and Mediates mTOR Signaling.
    Sophia Louise Lucille Park, Uri Nimrod Ramírez-Jarquín, Neelam Shahani, Oscar Rivera, Manish Sharma, Preksha Sandipkumar Joshi, Aayushi Hansalia, Sunayana Dagar, Francis P McManus, Pierre Thibault, Srinivasa Subramaniam.
      The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications, however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GβL, is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GβL is SUMOylated at lysine sites K86, K215, K245, K261 and K305. We found that SUMO depletion reduces mTOR-Raptor and mTOR-Rictor complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GβL but not SUMOylation defective KR mutant GβL promote mTOR signaling in GβL-depleted cells. Furthermore, we found that amino acids-induced mTORC1 signaling is diminished in the SUMO1-KO mice. Taken together, we report for the very first time that SUMO modifies GβL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.
    Keywords:  Amino acid stimulation; Kinase signaling; Lysine-site regulation; Nutrient signaling; Post-translational modification; Protein-protein interaction; SUMO isoforms; SUMO mechanism; Sumo interactive motif (SIM)
    DOI:  https://doi.org/10.1016/j.jbc.2024.105778
  27. Life Sci Alliance. 2024 May;pii: e202302300. [Epub ahead of print]7(5):
    Ageing-dependent thiol oxidation reveals early oxidation of proteins with core proteostasis functions.
    Katarzyna Jonak, Ida Suppanz, Julian Bender, Agnieszka Chacinska, Bettina Warscheid, Ulrike Topf.
      Oxidative post-translational modifications of protein thiols are well recognized as a readily occurring alteration of proteins, which can modify their function and thus control cellular processes. The development of techniques enabling the site-specific assessment of protein thiol oxidation on a proteome-wide scale significantly expanded the number of known oxidation-sensitive protein thiols. However, lacking behind are large-scale data on the redox state of proteins during ageing, a physiological process accompanied by increased levels of endogenous oxidants. Here, we present the landscape of protein thiol oxidation in chronologically aged wild-type Saccharomyces cerevisiae in a time-dependent manner. Our data determine early-oxidation targets in key biological processes governing the de novo production of proteins, protein folding, and degradation, and indicate a hierarchy of cellular responses affected by a reversible redox modification. Comparison with existing datasets in yeast, nematode, fruit fly, and mouse reveals the evolutionary conservation of these oxidation targets. To facilitate accessibility, we integrated the cross-species comparison into the newly developed OxiAge Database.
    DOI:  https://doi.org/10.26508/lsa.202302300
  28. Cell Rep. 2024 Feb 16. pii: S2211-1247(24)00136-0. [Epub ahead of print]43(2): 113808
    UDP-glucose dehydrogenase supports autophagy-deficient PDAC growth via increasing hyaluronic acid biosynthesis.
    Minghe Fan, Sihan Huo, Yuyao Guo, Ruoxuan Wang, Wenqin Hao, Ziyang Zhang, Lina Wang, Ying Zhao.
      Autophagy is an essential degradation and recycling process that maintains cellular homeostasis during stress or nutrient deprivation. However, certain types of tumors such as pancreatic cancers can circumvent autophagy inhibition to sustain growth. The mechanism that autophagy-deficient pancreatic ductal adenocarcinoma (PDAC) uses to grow under nutrient deprivation is poorly understood. Our data show that nutrient deprivation in PDAC results in UDP-glucose dehydrogenase (UGDH) degradation, which is dependent on autophagic cargo receptor sequestosome 1 (p62). Moreover, we demonstrate that accumulated UGDH is indispensable for autophagy-deficient PDAC cells proliferation by promoting hyaluronic acid (HA) synthesis upon energy deprivation. Using an orthotopic mouse model of PDAC, we find that inhibition of HA synthesis by targeting UGDH in PDAC reduces tumor weight. Thus, the combined inhibition of HA and autophagy might be an attractive strategy for PDAC treatment.
    Keywords:  CP: Cancer; CP: Metabolism; HA; PDAC; UDP-glucose dehydrogenase; UGDH; autophagy; hyaluronic acid; p62; pancreatic ductal adenocarcinoma; sequestosome 1
    DOI:  https://doi.org/10.1016/j.celrep.2024.113808
  29. Curr Opin Chem Biol. 2024 Feb 20. pii: S1367-5931(24)00011-5. [Epub ahead of print]79 102435
    A new era of cysteine proteomics - Technological advances in thiol biology.
    Nils Burger, Edward T Chouchani.
      Cysteines are amenable to a diverse set of modifications that exhibit critical regulatory functions over the proteome and thereby control a wide range of cellular processes. Proteomic technologies have emerged as a powerful strategy to interrogate cysteine modifications across the proteome. Recent advancements in enrichment strategies, multiplexing capabilities and increased analytical sensitivity have enabled deeper quantitative cysteine profiling, capturing a substantial proportion of the cysteine proteome. This is complemented by a rapidly growing repertoire of analytical strategies illuminating the diverse landscape of cysteine modifications. Cysteine chemoproteomics technologies have evolved into a powerful strategy to facilitate the development of covalent drugs, opening unprecedented opportunities to target the extensive undrugged proteome. Herein we review recent technological and scientific advances that shape the cysteine proteomics field.
    DOI:  https://doi.org/10.1016/j.cbpa.2024.102435
  30. Dev Cell. 2024 Feb 14. pii: S1534-5807(24)00046-7. [Epub ahead of print]
    ATG5 attenuates inflammatory signaling in mouse embryonic stem cells to control differentiation.
    Sheng Li, Jin Sun, Bo-Wen Zhang, Lu Yang, Ying-Cui Wan, Bei-Bei Chen, Nan Xu, Qian-Ru Xu, Juan Fan, Jia-Ni Shang, Rui Li, Chen-Ge Yu, Yan Xi, Su Chen.
      Attenuated inflammatory response is a property of embryonic stem cells (ESCs). However, the underlying mechanisms are unclear. Moreover, whether the attenuated inflammatory status is involved in ESC differentiation is also unknown. Here, we found that autophagy-related protein ATG5 is essential for both attenuated inflammatory response and differentiation of mouse ESCs and that attenuation of inflammatory signaling is required for mouse ESC differentiation. Mechanistically, ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of β-TrCP1, resulting in the inhibition of nuclear factor κB (NF-κB) signaling and inflammatory response. Moreover, differentiation defects observed in ATG5-depleted mouse ESCs are due to β-TrCP1 accumulation and hyperactivation of NF-κB signaling, as loss of β-TrCP1 and inhibition of NF-κB signaling rescued the differentiation defects. Therefore, this study reveals a previously uncharacterized mechanism maintaining the attenuated inflammatory response in mouse ESCs and further expands the understanding of the biological roles of ATG5.
    Keywords:  ATG5; FBXW7; NF-κB signaling; embryonic stem cells; inflammatory response; β-TrCP1
    DOI:  https://doi.org/10.1016/j.devcel.2024.01.026
  31. Oncogene. 2024 Feb 23.
    O-GlcNAcylation of E3 ubiquitin ligase SKP2 promotes hepatocellular carcinoma proliferation.
    Zhongqi Feng, Jiaxin Yin, Zhirong Zhang, Zhen Chen, Luyi Huang, Ni Tang, Kai Wang.
      O-linked-β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) and ubiquitination are critical posttranslational modifications that regulate tumor development and progression. The continuous progression of the cell cycle is the fundamental cause of tumor proliferation. S-phase kinase-associated protein 2 (SKP2), an important E3 ubiquitin ligase, assumes a pivotal function in the regulation of the cell cycle. However, it is still unclear whether SKP2 is an effector of O-GlcNAcylation that affects tumor progression. In this study, we found that SKP2 interacted with O-GlcNAc transferase (OGT) and was highly O-GlcNAcylated in hepatocellular carcinoma (HCC). Mechanistically, the O-GlcNAcylation at Ser34 stabilized SKP2 by reducing its ubiquitination and degradation mediated by APC-CDH1. Moreover, the O-GlcNAcylation of SKP2 enhanced its binding ability with SKP1, thereby enhancing its ubiquitin ligase function. Consequently, SKP2 facilitated the transition from the G1-S phase of the cell cycle by promoting the ubiquitin degradation of cell cycle-dependent kinase inhibitors p27 and p21. Additionally, targeting the O-GlcNAcylation of SKP2 significantly suppressed the proliferation of HCC. Altogether, our findings reveal that O-GlcNAcylation, a novel posttranslational modification of SKP2, plays a crucial role in promoting HCC proliferation, and targeting the O-GlcNAcylation of SKP2 may become a new therapeutic strategy to impede the progression of HCC.
    DOI:  https://doi.org/10.1038/s41388-024-02977-7
  32. Mol Cell. 2024 Feb 20. pii: S1097-2765(24)00093-5. [Epub ahead of print]
    Denaturing purifications demonstrate that PRC2 and other widely reported chromatin proteins do not appear to bind directly to RNA in vivo.
    Jimmy K Guo, Mario R Blanco, Ward G Walkup, Grant Bonesteele, Carl R Urbinati, Abhik K Banerjee, Amy Chow, Olivia Ettlin, Mackenzie Strehle, Parham Peyda, Enrique Amaya, Vickie Trinh, Mitchell Guttman.
      Polycomb repressive complex 2 (PRC2) is reported to bind to many RNAs and has become a central player in reports of how long non-coding RNAs (lncRNAs) regulate gene expression. Yet, there is a growing discrepancy between the biochemical evidence supporting specific lncRNA-PRC2 interactions and functional evidence demonstrating that PRC2 is often dispensable for lncRNA function. Here, we revisit the evidence supporting RNA binding by PRC2 and show that many reported interactions may not occur in vivo. Using denaturing purification of in vivo crosslinked RNA-protein complexes in human and mouse cell lines, we observe a loss of detectable RNA binding to PRC2 and chromatin-associated proteins previously reported to bind RNA (CTCF, YY1, and others), despite accurately mapping bona fide RNA-binding sites across others (SPEN, TET2, and others). Taken together, these results argue for a critical re-evaluation of the broad role of RNA binding to orchestrate various chromatin regulatory mechanisms.
    Keywords:  CLIP; PRC2; RNA; RNA-binding proteins; chromatin
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.026
  33. Cell Rep. 2024 Feb 17. pii: S2211-1247(24)00117-7. [Epub ahead of print]43(2): 113789
    Shigella induces stress granule formation by ADP-riboxanation of the eIF3 complex.
    Qinxin Zhang, Wei Xian, Zilin Li, Qian Lu, Xindi Chen, Jinli Ge, Zhiheng Tang, Bohao Liu, Zhe Chen, Xiang Gao, Michael O Hottiger, Peipei Zhang, Jiazhang Qiu, Feng Shao, Xiaoyun Liu.
      Under stress conditions, translationally stalled mRNA and associated proteins undergo liquid-liquid phase separation and condense into cytoplasmic foci called stress granules (SGs). Many viruses hijack SGs for their pathogenesis; however, whether pathogenic bacteria also exploit this pathway remains unknown. Here, we report that members of the OspC family of Shigella flexneri induce SG formation in infected cells. Mechanistically, the OspC effectors target multiple subunits of the host translation initiation factor 3 complex by ADP-riboxanation. The modification of eIF3 leads to translational arrest and thus the formation of SGs. Furthermore, OspC-mediated SGs are beneficial for S. flexneri replication within infected host cells, and bacterial strains unable to induce SGs are attenuated for virulence in a murine model of infection. Our findings reveal a mechanism by which bacterial pathogens induce SG assembly by inactivating host translational machinery and promote bacterial proliferation in host cells.
    Keywords:  ADP-riboxanation; CP: Molecular biology; OspC; Shigella flexneri; eIF3; stress granules
    DOI:  https://doi.org/10.1016/j.celrep.2024.113789
  34. PLoS Biol. 2024 Feb 20. 22(2): e3002508
    Proteins that carry dual targeting signals can act as tethers between peroxisomes and partner organelles.
    Elena Bittner, Thorsten Stehlik, Jason Lam, Lazar Dimitrov, Thomas Heimerl, Isabelle Schöck, Jannik Harberding, Anita Dornes, Nikola Heymons, Gert Bange, Maya Schuldiner, Einat Zalckvar, Michael Bölker, Randy Schekman, Johannes Freitag.
      Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.
    DOI:  https://doi.org/10.1371/journal.pbio.3002508
  35. Nat Commun. 2024 Feb 20. 15(1): 1534
    Chronic endoplasmic reticulum stress in myotonic dystrophy type 2 promotes autoimmunity via mitochondrial DNA release.
    Sarah Rösing, Fabian Ullrich, Susann Meisterfeld, Franziska Schmidt, Laura Mlitzko, Marijana Croon, Ryan G Nattrass, Nadia Eberl, Julia Mahlberg, Martin Schlee, Anja Wieland, Philipp Simon, Daniel Hilbig, Ulrike Reuner, Alexander Rapp, Julia Bremser, Peter Mirtschink, Stephan Drukewitz, Thomas Zillinger, Stefan Beissert, Katrin Paeschke, Gunther Hartmann, Aleksandra Trifunovic, Eva Bartok, Claudia Günther.
      Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.
    DOI:  https://doi.org/10.1038/s41467-024-45535-1
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