bims-proteo Biomed News
on Proteostasis
Issue of 2023‒11‒26
25 papers selected by
Eric Chevet, INSERM
  1. The ribosome-associated chaperone Zuo1 controls translation upon TORC1 inhibition.
  2. Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.
  3. Molecular view of ER membrane remodeling by the Sec61/TRAP translocon.
  4. BioE3 identifies specific substrates of ubiquitin E3 ligases.
  5. An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.
  6. Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.
  7. Reticulons bind sphingolipids to activate the endoplasmic reticulum cell cycle checkpoint, the ER surveillance pathway.
  8. LUBAC is required for RIG-I sensing of RNA viruses.
  9. Hydrolytic activity of yeast oligosaccharyltransferase is enhanced when misfolded proteins accumulate in the endoplasmic reticulum.
  10. The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.
  11. Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.
  12. ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions.
  13. Structural study of UFL1-UFC1 interaction uncovers the role of UFL1 N-terminal helix in ufmylation.
  14. Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.
  15. Protein disaggregation machineries in the human cytosol.
  16. DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.
  17. Genetic retargeting of E3 ligases to enhance CAR T cell therapy.
  18. Autophagy drives the conversion of developmental neural stem cells to the adult quiescent state.
  19. A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.
  20. Mitochondrial phosphoproteomes are functionally specialized across tissues.
  21. The tRNA-GCN2-FBXO22-axis-mediated mTOR ubiquitination senses amino acid insufficiency.
  22. Stress granule formation helps to mitigate neurodegeneration.
  23. Deep topographic proteomics of a human brain tumour.
  24. GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.
  25. E3 ligase Trim35 inhibits LSD1 demethylase activity through K63-linked ubiquitination and enhances anti-tumor immunity in NSCLC.
  1. EMBO J. 2023 Nov 20. e113240
    The ribosome-associated chaperone Zuo1 controls translation upon TORC1 inhibition.
    Ailsa Black, Thomas D Williams, Flavie Soubigou, Ifeoluwapo M Joshua, Houjiang Zhou, Frederic Lamoliatte, Adrien Rousseau.
      Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed of the HSP70 chaperone Ssb and its HSP40 co-chaperone Zuo1, is required to maintain proteostasis and cell viability under TORC1 inhibition in Saccharomyces cerevisiae. In the absence of Zuo1, translation does not decrease in response to the loss of TORC1 activity. A functional interaction between Zuo1 and Ssb is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Furthermore, we have shown that the rapid degradation of eIF4G following TORC1 inhibition is mediated by autophagy and is prevented in zuo1Δ cells, contributing to decreased survival in these conditions. We found that autophagy is defective in zuo1Δ cells, which impedes eIF4G degradation upon TORC1 inhibition. Our findings identify an essential role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling.
    Keywords:  TORC1; proteostasis; ribosome-associated chaperones; stress; translation
    DOI:  https://doi.org/10.15252/embj.2022113240
  2. Cell Rep. 2023 Nov 17. pii: S2211-1247(23)01463-8. [Epub ahead of print]42(11): 113451
    Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.
    Brian G Peterson, Jiwon Hwang, Jennifer E Russ, Jeremy W Schroeder, P Lydia Freddolino, Ryan D Baldridge.
      Misfolded endoplasmic reticulum (ER) proteins are degraded through a process called ER-associated degradation (ERAD). Soluble, lumenal ERAD targets are recognized, retrotranslocated across the ER membrane, ubiquitinated, extracted from the membrane, and degraded by the proteasome using an ERAD pathway containing a ubiquitin ligase called Hrd1. To determine how Hrd1 mediates these processes, we developed a deep mutational scanning approach to identify residues involved in Hrd1 function, including those exclusively required for lumenal degradation. We identify several regions required for different Hrd1 functions. Most surprisingly, we find two cytosolic regions of Hrd1 required for lumenal ERAD substrate degradation. Using in vivo and in vitro approaches, we define roles for disordered regions between structural elements that are required for Hrd1 autoubiquitination and substrate interaction. Our results demonstrate that disordered cytosolic regions promote substrate retrotranslocation by controlling Hrd1 activation and establishing directionality of retrotranslocation for lumenal substrate across the ER membrane.
    Keywords:  CP: Cell biology; ERAD; UPS; deep mutational scanning; endoplasmic reticulum-associated degradation; multiplexed high-throughput screening; protein degradation; protein disorder; retrotranslocation; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.celrep.2023.113451
  3. EMBO Rep. 2023 Nov 20. e57910
    Molecular view of ER membrane remodeling by the Sec61/TRAP translocon.
    Sudeep Karki, Matti Javanainen, Shahid Rehan, Dale Tranter, Juho Kellosalo, Juha Huiskonen, Lotta Happonen, Ville Paavilainen.
      Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.
    Keywords:  cryo-EM; membrane proteins; protein translocation; secretory proteins; structural biology
    DOI:  https://doi.org/10.15252/embr.202357910
  4. Nat Commun. 2023 Nov 23. 14(1): 7656
    BioE3 identifies specific substrates of ubiquitin E3 ligases.
    Orhi Barroso-Gomila, Laura Merino-Cacho, Veronica Muratore, Coralia Perez, Vincenzo Taibi, Elena Maspero, Mikel Azkargorta, Ibon Iloro, Fredrik Trulsson, Alfred C O Vertegaal, Ugo Mayor, Felix Elortza, Simona Polo, Rosa Barrio, James D Sutherland.
      Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.
    DOI:  https://doi.org/10.1038/s41467-023-43326-8
  5. Cell Rep. 2023 Nov 22. pii: S2211-1247(23)01496-1. [Epub ahead of print]42(12): 113484
    An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.
    Hannah Mende, Anshu Khatri, Carolin Lange, Sergio Alejandro Poveda-Cuevas, Georg Tascher, Adriana Covarrubias-Pinto, Frank Löhr, Sebastian E Koschade, Ivan Dikic, Christian Münch, Anja Bremm, Lorenzo Brunetti, Christian H Brandts, Hannah Uckelmann, Volker Dötsch, Vladimir V Rogov, Ramachandra M Bhaskara, Stefan Müller.
      The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.
    Keywords:  AML; ATG8; Autophagy; CP: Cancer; CP: Molecular biology; GABARAP; LC3; NPM1; NPM1c; TFEB; atypical LIR; lysosome
    DOI:  https://doi.org/10.1016/j.celrep.2023.113484
  6. EMBO Rep. 2023 Nov 21. e57300
    Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.
    Monami Ogura, Tatsuya Kaminishi, Takayuki Shima, Miku Torigata, Nao Bekku, Keisuke Tabata, Satoshi Minami, Kohei Nishino, Akiko Nezu, Maho Hamasaki, Hidetaka Kosako, Tamotsu Yoshimori, Shuhei Nakamura.
      Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.
    Keywords:  ESCRT; lysosome; microautophagy; non-canonical ATG8 lipidation
    DOI:  https://doi.org/10.15252/embr.202357300
  7. Cell Rep. 2023 Nov 17. pii: S2211-1247(23)01415-8. [Epub ahead of print]42(12): 113403
    Reticulons bind sphingolipids to activate the endoplasmic reticulum cell cycle checkpoint, the ER surveillance pathway.
    Francisco Piña, Bing Yan, Junjie Hu, Maho Niwa.
      The inheritance of a functional endoplasmic reticulum (ER) is ensured by the ER stress surveillance (ERSU) pathway. Here, we made the unexpected discovery that reticulon 1 (Rtn1) and Yop1, well-known ER-curvature-generating proteins, each possess two sphingolipid-binding motifs within their transmembrane domains and that these motifs recognize the ER-stress-induced sphingolipid phytosphingosine (PHS), resulting in an ER inheritance block. Upon binding PHS, Rtn1/Yop1 accumulate on the ER tubule, poised to enter the emerging daughter cell, and cause its misdirection to the bud scars (i.e., previous cell division sites). Amino acid changes in the conserved PHS-binding motifs preclude Rtn1 or Yop1 from binding PHS and diminish their enrichment on the tubular ER, ultimately preventing the ER-stress-induced inheritance block. Conservation of these sphingolipid-binding motifs in human reticulons suggests that sphingolipid binding to Rtn1 and Yop1 represents an evolutionarily conserved mechanism that enables cells to respond to ER stress.
    Keywords:  CP: Cell biology; ER; ER inheritance; ER stress; ER surveillance; cell cycle checkpoint; cytokinesis block; endoplasmic reticulum; phytosphingosine; reticulons; sphingolipids
    DOI:  https://doi.org/10.1016/j.celrep.2023.113403
  8. Cell Death Differ. 2023 Nov 24.
    LUBAC is required for RIG-I sensing of RNA viruses.
    Helena C Teague, Charlotte Lefevre, Eva Rieser, Lina Wolfram, Diego de Miguel, Daniel Patricio de Oliveira, Marisa Oliveira, Daniel S Mansur, Nerea Irigoyen, Henning Walczak, Brian J Ferguson.
      The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.
    DOI:  https://doi.org/10.1038/s41418-023-01233-x
  9. FEBS J. 2023 Nov 24.
    Hydrolytic activity of yeast oligosaccharyltransferase is enhanced when misfolded proteins accumulate in the endoplasmic reticulum.
    Sheng-Tao Li, Hiroto Hirayama, Chengcheng Huang, Tsugiyo Matsuda, Ritsuko Oka, Takahiro Yamasaki, Daisuke Kohda, Tadashi Suzuki.
      It is known that oligosaccharyltransferase (OST) has hydrolytic activity towards dolichol-linked oligosaccharides, which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation (ERAD). Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of dolichol-linked oligosaccharides, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the endoplasmic reticulum in yeast. The possible role of free N-glycans on protein folding is discussed.
    Keywords:  free N-glycans; oligosaccharyltransferase; protein folding; ubiquitin ligase
    DOI:  https://doi.org/10.1111/febs.17011
  10. Nat Commun. 2023 Nov 22. 14(1): 7636
    The heat shock protein LarA activates the Lon protease in response to proteotoxic stress.
    Deike J Omnus, Matthias J Fink, Aswathy Kallazhi, Maria Xandri Zaragoza, Axel Leppert, Michael Landreh, Kristina Jonas.
      The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.
    DOI:  https://doi.org/10.1038/s41467-023-43385-x
  11. bioRxiv. 2023 Oct 08. pii: 2023.10.06.561292. [Epub ahead of print]
    Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.
    Swati Gaikwad, Fardin Ghobakhlou, Hongen Zhang, Alan G Hinnebusch.
      Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met- tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'- untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative IRES elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met- tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.
    DOI:  https://doi.org/10.1101/2023.10.06.561292
  12. PLoS Biol. 2023 Nov 20. 21(11): e3002290
    ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusions.
    Sílvia Vale-Costa, Temitope Akhigbe Etibor, Daniela Brás, Ana Laura Sousa, Mariana Ferreira, Gabriel G Martins, Victor Hugo Mello, Maria João Amorim.
      It is now established that many viruses that threaten public health establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol and reduces genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling pathway endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.
    DOI:  https://doi.org/10.1371/journal.pbio.3002290
  13. EMBO Rep. 2023 Nov 21. e56920
    Structural study of UFL1-UFC1 interaction uncovers the role of UFL1 N-terminal helix in ufmylation.
    Sayanika Banerjee, Julia K Varga, Manoj Kumar, Guy Zoltsman, Shahar Rotem-Bamberger, Einav Cohen-Kfir, Michail N Isupov, Rina Rosenzweig, Ora Schueler-Furman, Reuven Wiener.
      Ufmylation plays a crucial role in various cellular processes including DNA damage response, protein translation, and ER homeostasis. To date, little is known about how the enzymes responsible for ufmylation coordinate their action. Here, we study the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling, X-ray crystallography, NMR, and biochemical assays. Guided by Alphafold2 models, we generate an active UFL1 fusion construct that includes its partner DDRGK1 and solve the crystal structure of this critical interaction. This fusion construct also unveiled the importance of the UFL1 N-terminal helix for binding to UFC1. The binding site suggested by our UFL1-UFC1 model reveals a conserved interface, and competition between UFL1 and UBA5 for binding to UFC1. This competition changes in the favor of UFL1 following UFM1 charging of UFC1. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism, which coordinates the cascade of E1-E2-E3-mediated transfer of UFM1 to its substrate and provides new leads to target this modification.
    Keywords:  AlphaFold2; DDRGK1; UFC1; UFL1; ufmylation
    DOI:  https://doi.org/10.15252/embr.202356920
  14. Cell. 2023 Nov 17. pii: S0092-8674(23)01177-7. [Epub ahead of print]
    Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.
    Xuewei Zhao, Ding Ma, Kensuke Ishiguro, Hironori Saito, Shinichiro Akichika, Ikuya Matsuzawa, Mari Mito, Toru Irie, Kota Ishibashi, Kimi Wakabayashi, Yuriko Sakaguchi, Takeshi Yokoyama, Yuichiro Mishima, Mikako Shirouzu, Shintaro Iwasaki, Takeo Suzuki, Tsutomu Suzuki.
      Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.
    Keywords:  RNA modification; galQ; galactosylqueuosine; manQ; mannosyl-queuosine; proteostasis; queuosine; ribosome; tRNA; translation
    DOI:  https://doi.org/10.1016/j.cell.2023.10.026
  15. Curr Opin Struct Biol. 2023 Nov 23. pii: S0959-440X(23)00209-9. [Epub ahead of print]83 102735
    Protein disaggregation machineries in the human cytosol.
    Anne Wentink, Rina Rosenzweig.
      Proteins carry out the vast majority of functions in cells, but can only do so when properly folded. Following stress or mutation, proteins can lose their proper fold, resulting in misfolding, inactivity, and aggregation-posing a threat to cellular health. In order to counteract protein aggregation, cells have evolved a remarkable subset of molecular chaperones, called protein disaggregases, which collaboratively possess the ability to forcibly untangle protein aggregates. Here, we review the different chaperone disaggregation machineries present in the human cytosol and their mechanisms of action. Understanding, how these disaggregases function, is both universally and clinically important, as protein aggregation has been linked to multiple, debilitating neurodegenerative diseases.
    Keywords:  Hsp110; Hsp70; J-domain proteins (JDPs); Protein disaggregation; amyloid fibers; molecular chaperones; protein refolding
    DOI:  https://doi.org/10.1016/j.sbi.2023.102735
  16. Nucleic Acids Res. 2023 Nov 24. pii: gkad1119. [Epub ahead of print]
    DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.
    Kang Zhu, Marcin J Suskiewicz, Chatrin Chatrin, Øyvind Strømland, Bryan W Dorsey, Vincent Aucagne, Dragana Ahel, Ivan Ahel.
      Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.
    DOI:  https://doi.org/10.1093/nar/gkad1119
  17. Cell Chem Biol. 2023 Nov 14. pii: S2451-9456(23)00386-0. [Epub ahead of print]
    Genetic retargeting of E3 ligases to enhance CAR T cell therapy.
    Isabel C Lane, Gabriele Kembuan, Jeannie Carreiro, Michael C Kann, William Lin, Amanda A Bouffard, Johannes Kreuzer, Robert Morris, Emily M Schneider, Joanna Y Kim, Charles Zou, Diego Salas-Benito, Jessica A Gasser, Mark B Leick, Mikołaj Słabicki, Wilhelm Haas, Marcela V Maus, Max Jan.
      Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, treatment failure is often caused by CAR T cell dysfunction. Additional approaches are needed to overcome inhibitory signals that limit anti-tumor potency. Here, we developed bifunctional fusion "degrader" proteins that bridge one or more target proteins and an E3 ligase complex to enforce target ubiquitination and degradation. Conditional degradation strategies were developed using inducible degrader transgene expression or small molecule-dependent E3 recruitment. We further engineered degraders to block SMAD-dependent TGFβ signaling using a domain from the SARA protein to target both SMAD2 and SMAD3. SMAD degrader CAR T cells were less susceptible to suppression by TGFβ and demonstrated enhanced anti-tumor potency in vivo. These results demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligase target specificity for conditional, multi-specific endogenous protein degradation, with promising applications including enhancing the potency of CAR T cell therapy.
    Keywords:  CAR T cell; cancer immunotherapy; cellular immunotherapy; synthetic biology; targeted protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.chembiol.2023.10.024
  18. Nat Commun. 2023 Nov 24. 14(1): 7541
    Autophagy drives the conversion of developmental neural stem cells to the adult quiescent state.
    Isabel Calatayud-Baselga, Lucía Casares-Crespo, Carmina Franch-Ibáñez, José Guijarro-Nuez, Pascual Sanz, Helena Mira.
      Neurogenesis in the adult mammalian brain relies on the lifelong persistence of quiescent neural stem cell (NSC) reservoirs. Little is known about the mechanisms that lead to the initial establishment of quiescence, the main hallmark of adult stem cells, during development. Here we show that protein aggregates and autophagy machinery components accumulate in developmental radial glia-like NSCs as they enter quiescence and that pharmacological or genetic blockade of autophagy disrupts quiescence acquisition and maintenance. Conversely, increasing autophagy through AMPK/ULK1 activation instructs the acquisition of the quiescent state without affecting BMP signaling, a gatekeeper of NSC quiescence during adulthood. Selective ablation of Atg7, a critical gene for autophagosome formation, in radial glia-like NSCs at early and late postnatal stages compromises the initial acquisition and maintenance of quiescence during the formation of the hippocampal dentate gyrus NSC niche. Therefore, we demonstrate that autophagy is cell-intrinsically required to establish NSC quiescence during hippocampal development. Our results uncover an important role of autophagy in the transition of developmental NSCs into their dormant adult form, paving the way for studies directed at further understanding the mechanisms of stem cell niche formation and maintenance in the mammalian brain.
    DOI:  https://doi.org/10.1038/s41467-023-43222-1
  19. Clin Cancer Res. 2023 Nov 20. OF1-OF18
    A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response-Dependent Survival of Quiescent Cancer Cells.
    Veronica Calvo, Wei Zheng, Anna Adam-Artigues, Kirk A Staschke, Xin Huang, Julie F Cheung, Ana Rita Nobre, Sho Fujisawa, David Liu, Maria Fumagalli, David Surguladze, Michael E Stokes, Ari Nowacek, Mark Mulvihill, Eduardo F Farias, Julio A Aguirre-Ghiso.
      PURPOSE: The integrated stress response (ISR) kinase PERK serves as a survival factor for both proliferative and dormant cancer cells. We aim to validate PERK inhibition as a new strategy to specifically eliminate solitary disseminated cancer cells (DCC) in secondary sites that eventually reawake and originate metastasis.EXPERIMENTAL DESIGN: A novel clinical-grade PERK inhibitor (HC4) was tested in mouse syngeneic and PDX models that present quiescent/dormant DCCs or growth-arrested cancer cells in micro-metastatic lesions that upregulate ISR.
    RESULTS: HC4 significantly blocks metastasis, by killing quiescent/slow-cycling ISRhigh, but not proliferative ISRlow DCCs. HC4 blocked expansion of established micro-metastasis that contained ISRhigh slow-cycling cells. Single-cell gene expression profiling and imaging revealed that a significant proportion of solitary DCCs in lungs were indeed dormant and displayed an unresolved ER stress as revealed by high expression of a PERK-regulated signature. In human breast cancer metastasis biopsies, GADD34 expression (PERK-regulated gene) and quiescence were positively correlated. HC4 effectively eradicated dormant bone marrow DCCs, which usually persist after rounds of therapies. Importantly, treatment with CDK4/6 inhibitors (to force a quiescent state) followed by HC4 further reduced metastatic burden. In HNSCC and HER2+ cancers HC4 caused cell death in dormant DCCs. In HER2+ tumors, PERK inhibition caused killing by reducing HER2 activity because of sub-optimal HER2 trafficking and phosphorylation in response to EGF.
    CONCLUSIONS: Our data identify PERK as a unique vulnerability in quiescent or slow-cycling ISRhigh DCCs. The use of PERK inhibitors may allow targeting of pre-existing or therapy-induced growth arrested "persister" cells that escape anti-proliferative therapies.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-23-1427
  20. Life Sci Alliance. 2024 Feb;pii: e202302147. [Epub ahead of print]7(2):
    Mitochondrial phosphoproteomes are functionally specialized across tissues.
    Fynn M Hansen, Laura S Kremer, Ozge Karayel, Isabell Bludau, Nils-Göran Larsson, Inge Kühl, Matthias Mann.
      Mitochondria are essential organelles whose dysfunction causes human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Increasing evidence suggests that phosphorylation may play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. Building on recent advances in mass spectrometry (MS)-based proteomics, we here quantitatively profile mitochondrial tissue proteomes along with their matching phosphoproteomes. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and performed high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue-specific mitochondrial proteins (mitophos.de). Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on dynamin-related protein 1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.
    DOI:  https://doi.org/10.26508/lsa.202302147
  21. Cell Metab. 2023 Nov 11. pii: S1550-4131(23)00385-6. [Epub ahead of print]
    The tRNA-GCN2-FBXO22-axis-mediated mTOR ubiquitination senses amino acid insufficiency.
    Meng-Kai Ge, Cheng Zhang, Na Zhang, Ping He, Hai-Yan Cai, Song Li, Shuai Wu, Xi-Li Chu, Yu-Xue Zhang, Hong-Ming Ma, Li Xia, Shuo Yang, Jian-Xiu Yu, Shi-Ying Yao, Xiao-Long Zhou, Bing Su, Guo-Qiang Chen, Shao-Ming Shen.
      Mammalian target of rapamycin complex 1 (mTORC1) monitors cellular amino acid changes for function, but the molecular mediators of this process remain to be fully defined. Here, we report that depletion of cellular amino acids, either alone or in combination, leads to the ubiquitination of mTOR, which inhibits mTORC1 kinase activity by preventing substrate recruitment. Mechanistically, amino acid depletion causes accumulation of uncharged tRNAs, thereby stimulating GCN2 to phosphorylate FBXO22, which in turn accrues in the cytoplasm and ubiquitinates mTOR at Lys2066 in a K27-linked manner. Accordingly, mutation of mTOR Lys2066 abolished mTOR ubiquitination in response to amino acid depletion, rendering mTOR insensitive to amino acid starvation both in vitro and in vivo. Collectively, these data reveal a novel mechanism of amino acid sensing by mTORC1 via a previously unknown GCN2-FBXO22-mTOR pathway that is uniquely controlled by uncharged tRNAs.
    Keywords:  FBXO22; GCN2; amino acids; mTOR; mTORC1; ubiquitination; uncharged tRNA
    DOI:  https://doi.org/10.1016/j.cmet.2023.10.016
  22. bioRxiv. 2023 Nov 11. pii: 2023.11.07.566060. [Epub ahead of print]
    Stress granule formation helps to mitigate neurodegeneration.
    M Rebecca Glineburg, Evrim Yildirim, Nicolas Gomez, Xingli Li, Jaclyn Pak, Christopher Altheim, Jacob Waksmacki, Gerald McInerney, Sami J Barmada, Peter K Todd.
      Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP ( rin in Drosophila ) and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.
    DOI:  https://doi.org/10.1101/2023.11.07.566060
  23. Nat Commun. 2023 Nov 24. 14(1): 7710
    Deep topographic proteomics of a human brain tumour.
    Simon Davis, Connor Scott, Janina Oetjen, Philip D Charles, Benedikt M Kessler, Olaf Ansorge, Roman Fischer.
      The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour at three spatial resolutions, the highest being 40 µm, to reveal distinct abundance patterns of thousands of proteins. We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns and correlate proteins in the context of tissue heterogeneity and cellular features such as extracellular matrix or proximity to blood vessels. We identify PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, we demonstrate spatially-aware deep proteo-phenotyping of tissue heterogeneity, to re-define understanding tissue biology and pathology at the molecular level.
    DOI:  https://doi.org/10.1038/s41467-023-43520-8
  24. Cell Rep Med. 2023 Nov 21. pii: S2666-3791(23)00491-3. [Epub ahead of print]4(11): 101297
    GRP78-CAR T cell effector function against solid and brain tumors is controlled by GRP78 expression on T cells.
    Jorge Ibanez, Nikhil Hebbar, Unmesha Thanekar, Zhongzhen Yi, Haley Houke, Meghan Ward, Chris Nevitt, Liqing Tian, Stephen C Mack, Heather Sheppard, Jason Chiang, M Paulina Velasquez, Giedre Krenciute.
      Lack of targetable antigens is a key limitation for developing successful T cell-based immunotherapies. Members of the unfolded protein response (UPR) represent ideal immunotherapy targets because the UPR regulates the ability of cancer cells to resist cell death, sustain proliferation, and metastasize. Glucose-regulated protein 78 (GRP78) is a key UPR regulator that is overexpressed and translocated to the cell surface of a wide variety of cancers in response to elevated endoplasmic reticulum (ER) stress. We show that GRP78 is highly expressed on the cell surface of multiple solid and brain tumors, making cell surface GRP78 a promising chimeric antigen receptor (CAR) T cell target. We demonstrate that GRP78-CAR T cells can recognize and kill GRP78+ brain and solid tumors in vitro and in vivo. Additionally, our findings demonstrate that GRP78 is upregulated on CAR T cells upon T cell activation; however, this expression is tumor-cell-line specific and results in heterogeneous GRP78-CAR T cell therapeutic response.
    Keywords:  CAR T cells; ER stress; GRP78; brain tumors; immune synapse; immunotherapy; solid tumors
    DOI:  https://doi.org/10.1016/j.xcrm.2023.101297
  25. Cell Rep. 2023 Nov 17. pii: S2211-1247(23)01489-4. [Epub ahead of print]42(12): 113477
    E3 ligase Trim35 inhibits LSD1 demethylase activity through K63-linked ubiquitination and enhances anti-tumor immunity in NSCLC.
    Feiyu Tang, Can Lu, Xiang He, Wei Lin, Bowen Xie, Xing Gao, Yang Peng, Desong Yang, Lunquan Sun, Liang Weng.
      Targeting lysine-specific histone demethylase 1A (LSD1) can improve tumor immunogenicity of poorly immunogenic tumors, such as non-small cell lung cancer (NSCLC), with elevated T cell infiltration and sensitize tumors to anti-PD-1 therapy. However, the lack of reliable biomarkers limits utilization of LSD1 inhibitors in cancer therapy. Here, we identify an E3 ligase, Trim35, as an effective biomarker for high activity of LSD1 to predict prognosis of LSD1-targeted therapy as well as immunotherapy. Mechanistically, Trim35 represses LSD1 demethylase activity by mediating K63 ubiquitination at lysine site 422 of LSD1. Suppressed LSD1 activity facilitates ERGIC1 transcription, followed by autophagy inhibition and IFNGR1 stabilization to activate IFN-γ signaling, leading to increased MHC class I expression and immune surveillance of NSCLC cells. Furthermore, combinational use of an LSD1 inhibitor and anti-PD-1 therapy can significantly eradicate poorly immunogenic lung cancer with low Trim35. These findings strongly suggest that Trim35 is a promising biomarker for prediction of immunotherapy outcome in NSCLC.
    Keywords:  CP: Cancer; CP: Immunology; LSD1; NSCLC; Trim35; immunogenicity; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2023.113477
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