bims-proteo Biomed News
on Proteostasis
Issue of 2023‒05‒28
24 papers selected by
Eric Chevet
INSERM
  1. Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.
  2. Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.
  3. A novel IRE1 kinase inhibitor for adjuvant glioblastoma treatment.
  4. Coupled protein quality control during nonsense-mediated mRNA decay.
  5. Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2.
  6. Lipid-anchored Proteasomes Control Membrane Protein Homeostasis.
  7. The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.
  8. Dysregulation of amino acid metabolism upon rapid depletion of cap-binding protein eIF4E.
  9. Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response.
  10. Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.
  11. Molecular mechanisms for activation of the 26S proteasome.
  12. Interplay between calcium and endoplasmic reticulum stress.
  13. Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.
  14. Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.
  15. An abundance of free regulatory (19S) proteasome particles regulates neuronal synapses.
  16. Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.
  17. Redox states in the endoplasmic reticulum directly regulate the activity of calcium channel, inositol 1,4,5-trisphosphate receptors.
  18. Surveying the global landscape of post-transcriptional regulators.
  19. Mechanobiology of organelles: illuminating their roles in mechanosensing and mechanotransduction.
  20. The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.
  21. New frontiers in quality control: the case of GPI-anchored proteins.
  22. TRABID inhibition activates cGAS/STING-mediated anti-tumor immunity through mitosis and autophagy dysregulation.
  23. Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence.
  24. Inhibition of endolysosome fusion increases exosome secretion.
  1. Nature. 2023 May 24.
    Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum.
    Alexis González, Adriana Covarrubias-Pinto, Ramachandra M Bhaskara, Marius Glogger, Santosh K Kuncha, Audrey Xavier, Eric Seemann, Mohit Misra, Marina E Hoffmann, Bastian Bräuning, Ashwin Balakrishnan, Britta Qualmann, Volker Dötsch, Brenda A Schulman, Michael M Kessels, Christian A Hübner, Mike Heilemann, Gerhard Hummer, Ivan Dikić.
      The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.
    DOI:  https://doi.org/10.1038/s41586-023-06089-2
  2. Nature. 2023 May 24.
    Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.
    Hector Foronda, Yangxue Fu, Adriana Covarrubias-Pinto, Hartmut T Bocker, Alexis González, Eric Seemann, Patricia Franzka, Andrea Bock, Ramachandra M Bhaskara, Lutz Liebmann, Marina E Hoffmann, Istvan Katona, Nicole Koch, Joachim Weis, Ingo Kurth, Joseph G Gleeson, Fulvio Reggiori, Gerhard Hummer, Michael M Kessels, Britta Qualmann, Muriel Mari, Ivan Dikić, Christian A Hübner.
      Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.
    DOI:  https://doi.org/10.1038/s41586-023-06090-9
  3. iScience. 2023 May 19. 26(5): 106687
    A novel IRE1 kinase inhibitor for adjuvant glioblastoma treatment.
    Diana Pelizzari-Raymundo, Dimitrios Doultsinos, Raphael Pineau, Chloé Sauzay, Thodoris Koutsandreas, Timothy Langlais, Antonio Carlesso, Elena Gkotsi, Luc Negroni, Tony Avril, Aristotelis Chatziioannou, Eric Chevet, Leif A Eriksson, Xavier Guillory.
      Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress overcome by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.
    Keywords:  Biological sciences; Medicine; Molecular neuroscience; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2023.106687
  4. J Cell Sci. 2023 05 15. pii: jcs261216. [Epub ahead of print]136(10):
    Coupled protein quality control during nonsense-mediated mRNA decay.
    Alison J Inglis, Alina Guna, Ángel Gálvez-Merchán, Akshaye Pal, Theodore K Esantsi, Heather R Keys, Evgeni M Frenkel, Robert Oania, Jonathan S Weissman, Rebecca M Voorhees.
      Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.
    Keywords:  Nonsense-mediated decay; Quality control; Ubiquitin-proteasome pathway; mRNA
    DOI:  https://doi.org/10.1242/jcs.261216
  5. Cell Rep. 2023 May 19. pii: S2211-1247(23)00545-4. [Epub ahead of print]42(5): 112534
    Activation of endoplasmic reticulum stress in premature aging via the inner nuclear membrane protein SUN2.
    Sandra Vidak, Leonid A Serebryannyy, Gianluca Pegoraro, Tom Misteli.
      One of the major cellular mechanisms to ensure cellular protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is triggered by accumulation of misfolded proteins in the ER lumen. The ER stress response is also activated in the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we explore the mechanism of activation of the ER stress response in HGPS. We find that aggregation of the diseases-causing progerin protein at the nuclear envelope triggers ER stress. Induction of ER stress is dependent on the inner nuclear membrane protein SUN2 and its ability to cluster in the nuclear membrane. Our observations suggest that the presence of nucleoplasmic protein aggregates can be sensed, and signaled to the ER lumen, via clustering of SUN2. These results identify a mechanism of communication between the nucleus and the ER and provide insight into the molecular disease mechanisms of HGPS.
    Keywords:  CP: Cell biology; ER stress; Hutchinson-Gilford progeria syndrome; chaperones; transmembrane proteins; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2023.112534
  6. bioRxiv. 2023 May 12. pii: 2023.05.12.540509. [Epub ahead of print]
    Lipid-anchored Proteasomes Control Membrane Protein Homeostasis.
    Ruizhu Zhang, Shuxian Pan, Suya Zheng, Qingqing Liao, Zhaodi Jiang, Dixian Wang, Xuemei Li, Ao Hu, Xinran Li, Yezhang Zhu, Xiaoqi Shen, Jing Lei, Siming Zhong, Xiaomei Zhang, Lingyun Huang, Xiaorong Wang, Lan Huang, Li Shen, Bao-Liang Song, Jingwei Zhao, Zhiping Wang, Bing Yang, Xing Guo.
      Protein degradation in eukaryotic cells is mainly carried out by the 26S proteasome, a macromolecular complex not only present in the cytosol and nucleus but also associated with various membranes. How proteasomes are anchored to the membrane and the biological meaning thereof have been largely unknown in higher organisms. Here we show that N-myristoylation of the Rpt2 subunit is a general mechanism for proteasome-membrane interaction. Loss of this modification in the Rpt2-G2A mutant cells leads to profound changes in the membrane-associated proteome, perturbs the endomembrane system and undermines critical cellular processes such as cell adhesion, endoplasmic reticulum-associated degradation (ERAD) and membrane protein trafficking. Rpt2 G2A/G2A homozygous mutation is embryonic lethal in mice and is sufficient to abolish tumor growth in a nude mice xenograft model. These findings have defined an evolutionarily conserved mechanism for maintaining membrane protein homeostasis and underscored the significance of compartmentalized protein degradation by m yristoyl- a nchored p roteasomes (MAPs) in health and disease.
    DOI:  https://doi.org/10.1101/2023.05.12.540509
  7. J Cell Sci. 2023 May 15. pii: jcs260657. [Epub ahead of print]136(10):
    The E2 ubiquitin-conjugating enzymes UBE2D1 and UBE2D2 regulate VEGFR2 dynamics and endothelial function.
    William R Critchley, Gina A Smith, Ian C Zachary, Michael A Harrison, Sreenivasan Ponnambalam.
      Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.
    Keywords:  Angiogenesis; Endothelial cells; Signalling; UBE2D1; UBE2D2; Ubiquitin; VEGFR2
    DOI:  https://doi.org/10.1242/jcs.260657
  8. bioRxiv. 2023 May 12. pii: 2023.05.11.540079. [Epub ahead of print]
    Dysregulation of amino acid metabolism upon rapid depletion of cap-binding protein eIF4E.
    Paige D Diamond, Nicholas J McGlincy, Nicholas T Ingolia.
      Protein synthesis is a crucial but metabolically costly biological process that must be tightly coordinated with cellular needs and nutrient availability. In response to environmental stress, translation initiation is modulated to control protein output while meeting new demands. The cap-binding protein eIF4E-the earliest contact between mRNAs and the translation machinery-serves as one point of control, but its contributions to mRNA-specific translation regulation remain poorly understood. To survey eIF4E-dependent translational control, we acutely depleted eIF4E and determined how this impacts protein synthesis. Despite its essentiality, eIF4E depletion had surprisingly modest effects on cell growth and protein synthesis. Analysis of transcript-level changes revealed that long-lived transcripts were downregulated, likely reflecting accelerated turnover. Paradoxically, eIF4E depletion led to simultaneous upregulation of genes involved in catabolism of aromatic amino acids, which arose as secondary effects of reduced protein biosynthesis on amino acid pools, and genes involved in the biosynthesis of amino acids. These futile cycles of amino acid synthesis and degradation were driven, in part, by translational activation of GCN4 , a transcription factor typically induced by amino acid starvation. Furthermore, we identified a novel regulatory mechanism governing translation of PCL5, a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This translational control was partial dependent on a uniquely long poly-(A) tract in the PCL5 5' UTR and on poly-(A) binding protein. Collectively, these results highlight how eIF4E connects translation to amino acid homeostasis and stress responses and uncovers new mechanisms underlying how cells tightly control protein synthesis during environmental challenges.
    DOI:  https://doi.org/10.1101/2023.05.11.540079
  9. Cell Rep. 2023 May 23. pii: S2211-1247(23)00568-5. [Epub ahead of print]42(6): 112557
    Heat shock factor 1 (HSF1) specifically potentiates c-MYC-mediated transcription independently of the canonical heat shock response.
    Meng Xu, Ling Lin, Babul Moni Ram, Omprakash Shriwas, Kun-Han Chuang, Siyuan Dai, Kuo-Hui Su, Zijian Tang, Chengkai Dai.
      Despite its pivotal roles in biology, how the transcriptional activity of c-MYC is tuned quantitatively remains poorly defined. Here, we show that heat shock factor 1 (HSF1), the master transcriptional regulator of the heat shock response, acts as a prime modifier of the c-MYC-mediated transcription. HSF1 deficiency diminishes c-MYC DNA binding and dampens its transcriptional activity genome wide. Mechanistically, c-MYC, MAX, and HSF1 assemble into a transcription factor complex on genomic DNAs, and surprisingly, the DNA binding of HSF1 is dispensable. Instead, HSF1 physically recruits the histone acetyltransferase general control nonderepressible 5 (GCN5), promoting histone acetylation and augmenting c-MYC transcriptional activity. Thus, we find that HSF1 specifically potentiates the c-MYC-mediated transcription, discrete from its canonical role in countering proteotoxic stress. Importantly, this mechanism of action engenders two distinct c-MYC activation states, primary and advanced, which may be important to accommodate diverse physiological and pathological conditions.
    Keywords:  CP: Molecular biology; CUT&RUN-seq; GCN5; HSF1; c-MYC; transcription factor complex
    DOI:  https://doi.org/10.1016/j.celrep.2023.112557
  10. Mol Cell. 2023 May 12. pii: S1097-2765(23)00321-0. [Epub ahead of print]
    Metamorphic proteins at the basis of human autophagy initiation and lipid transfer.
    Anh Nguyen, Francesca Lugarini, Céline David, Pouya Hosnani, Çağla Alagöz, Annabelle Friedrich, David Schlütermann, Barbora Knotkova, Anoshi Patel, Iwan Parfentev, Henning Urlaub, Michael Meinecke, Björn Stork, Alex C Faesen.
      Autophagy is a conserved intracellular degradation pathway that generates de novo double-membrane autophagosomes to target a wide range of material for lysosomal degradation. In multicellular organisms, autophagy initiation requires the timely assembly of a contact site between the ER and the nascent autophagosome. Here, we report the in vitro reconstitution of a full-length seven-subunit human autophagy initiation supercomplex built on a core complex of ATG13-101 and ATG9. Assembly of this core complex requires the rare ability of ATG13 and ATG101 to switch between distinct folds. The slow spontaneous metamorphic conversion is rate limiting for the self-assembly of the supercomplex. The interaction of the core complex with ATG2-WIPI4 enhances tethering of membrane vesicles and accelerates lipid transfer of ATG2 by both ATG9 and ATG13-101. Our work uncovers the molecular basis of the contact site and its assembly mechanisms imposed by the metamorphosis of ATG13-101 to regulate autophagosome biogenesis in space and time.
    Keywords:  autophagy; autophagy initiation; lipid transfer; membrane-contact site; metabolism; protein metamorphosis
    DOI:  https://doi.org/10.1016/j.molcel.2023.04.026
  11. bioRxiv. 2023 May 10. pii: 2023.05.09.540094. [Epub ahead of print]
    Molecular mechanisms for activation of the 26S proteasome.
    Donghoon Lee, Yanan Zhu, Louis Colson, Xiaorong Wang, Siyi Chen, Emre Tkacik, Lan Huang, Qi Ouyang, Alfred L Goldberg, Ying Lu.
      Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasome's protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.
    DOI:  https://doi.org/10.1101/2023.05.09.540094
  12. Cell Calcium. 2023 May 12. pii: S0143-4160(23)00065-9. [Epub ahead of print]113 102753
    Interplay between calcium and endoplasmic reticulum stress.
    Jody Groenendyk, Marek Michalak.
      Cellular homeostasis is crucial for the healthy functioning of the organism. Disruption of cellular homeostasis activates endoplasmic reticulum (ER) stress coping responses including the unfolded protein response (UPR). There are three ER resident stress sensors responsible for UPR activation - IRE1α, PERK and ATF6. Ca2+ signaling plays an important role in stress responses including the UPR and the ER is the main Ca2+ storage organelle and a source of Ca2+ for cell signaling. The ER contains many proteins involved in Ca2+ import/export/ storage, Ca2+ movement between different cellular organelles and ER Ca2+ stores refilling. Here we focus on selected aspects of ER Ca2+ homeostasis and its role in activation of the ER stress coping responses.
    Keywords:  Calcium homeostasis; Endoplasmic reticulum; Membrane contact sites; Sarcoplasmic reticulum; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.ceca.2023.102753
  13. PLoS Genet. 2023 May 25. 19(5): e1010772
    Hsp90 mutants with distinct defects provide novel insights into cochaperone regulation of the folding cycle.
    Rebecca Mercier, Danielle Yama, Paul LaPointe, Jill L Johnson.
      Molecular chaperones play a key role in maintaining proteostasis and cellular health. The abundant, essential, cytosolic Hsp90 (Heat shock protein, 90 kDa) facilitates the folding and activation of hundreds of newly synthesized or misfolded client proteins in an ATP-dependent folding pathway. In a simplified model, Hsp70 first helps load client onto Hsp90, ATP binding results in conformational changes in Hsp90 that result in the closed complex, and then less defined events result in nucleotide hydrolysis, client release and return to the open state. Cochaperones bind and assist Hsp90 during this process. We previously identified a series of yeast Hsp90 mutants that appear to disrupt either the 'loading', 'closing' or 'reopening' events, and showed that the mutants had differing effects on activity of some clients. Here we used those mutants to dissect Hsp90 and cochaperone interactions. Overexpression or deletion of HCH1 had dramatically opposing effects on the growth of cells expressing different mutants, with a phenotypic shift coinciding with formation of the closed conformation. Hch1 appears to destabilize Hsp90-nucleotide interaction, hindering formation of the closed conformation, whereas Cpr6 counters the effects of Hch1 by stabilizing the closed conformation. Hch1 and the homologous Aha1 share some functions, but the role of Hch1 in inhibiting progression through the early stages of the folding cycle is unique. Sensitivity to the Hsp90 inhibitor NVP-AUY922 also correlates with the conformational cycle, with mutants defective in the loading phase being most sensitive and those defective in the reopening phase being most resistant to the drug. Overall, our results indicate that the timing of transition into and out of the closed conformation is tightly regulated by cochaperones. Further analysis will help elucidate additional steps required for progression through the Hsp90 folding cycle and may lead to new strategies for modulating Hsp90 function.
    DOI:  https://doi.org/10.1371/journal.pgen.1010772
  14. Nat Commun. 2023 May 23. 14(1): 2978
    Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome.
    Fu Zheng, Xinyue Zhou, Peng Zou, Chenxin Yu.
      Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 477 mitochondrial proteins with 94% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum (ER). The temporal control of RinID enables pulse-chase labeling of ER proteome in HeLa cells, which reveals substantially higher clearance rate for secreted proteins than ER resident proteins.
    DOI:  https://doi.org/10.1038/s41467-023-38565-8
  15. Science. 2023 May 26. 380(6647): eadf2018
    An abundance of free regulatory (19S) proteasome particles regulates neuronal synapses.
    Chao Sun, Kristina Desch, Belquis Nassim-Assir, Stefano L Giandomenico, Paulina Nemcova, Julian D Langer, Erin M Schuman.
      The proteasome, the major protein-degradation machine in cells, regulates neuronal synapses and long-term information storage. Here, using super-resolution microscopy, we found that the two essential subcomplexes of the proteasome, the regulatory (19S) and catalytic (20S) particles, are differentially distributed within individual rat cortical neurons. We discovered an unexpected abundance of free 19S particles near synapses. The free neuronal 19S particles bind and deubiquitylate lysine 63-ubiquitin (Lys63-ub), a non-proteasome-targeting ubiquitin linkage. Pull-down assays revealed a significant overrepresentation of synaptic molecules as Lys63-ub interactors. Inhibition of the 19S deubiquitylase activity significantly altered excitatory synaptic transmission and reduced the synaptic availability of AMPA receptors at multiple trafficking points in a proteasome-independent manner. Together, these results reveal a moonlighting function of the regulatory proteasomal subcomplex near synapses.
    DOI:  https://doi.org/10.1126/science.adf2018
  16. Methods Enzymol. 2023 ;pii: S0076-6879(22)00387-1. [Epub ahead of print]684 1-38
    Selective ribosome profiling as a tool to study interactions of translating ribosomes in mammalian cells.
    Manuel Günnigmann, Jiří Koubek, Günter Kramer, Bernd Bukau.
      The processing, membrane targeting and folding of newly synthesized polypeptides is closely linked to their synthesis at the ribosome. A network of enzymes, chaperones and targeting factors engages ribosome-nascent chain complexes (RNCs) to support these maturation processes. Exploring the modes of action of this machinery is critical for our understanding of functional protein biogenesis. Selective ribosome profiling (SeRP) is a powerful method for interrogating co-translational interactions of maturation factors with RNCs. It provides proteome-wide information on the factor's nascent chain interactome, the timing of factor binding and release during the progress of translation of individual nascent chain species, and the mechanisms and features controlling factor engagement. SeRP is based on the combination of two ribosome profiling (RP) experiments performed on the same cell population. In one experiment the ribosome-protected mRNA footprints of all translating ribosomes of the cell are sequenced (total translatome), while the other experiment detects only the ribosome footprints of the subpopulation of ribosomes engaged by the factor of interest (selected translatome). The codon-specific ratio of ribosome footprint densities from selected over total translatome reports on the factor enrichment at specific nascent chains. In this chapter, we provide a detailed SeRP protocol for mammalian cells. The protocol includes instructions on cell growth and cell harvest, stabilization of factor-RNC interactions, nuclease digest and purification of (factor-engaged) monosomes, as well as preparation of cDNA libraries from ribosome footprint fragments and deep sequencing data analysis. Purification protocols of factor-engaged monosomes and experimental results are exemplified for the human ribosomal tunnel exit-binding factor Ebp1 and chaperone Hsp90, but the protocols are readily adaptable to other co-translationally acting mammalian factors.
    Keywords:  Chaperone; Nascent chains; Ribosome; Selective-ribosome profiling
    DOI:  https://doi.org/10.1016/bs.mie.2022.09.006
  17. Proc Natl Acad Sci U S A. 2023 05 30. 120(22): e2216857120
    Redox states in the endoplasmic reticulum directly regulate the activity of calcium channel, inositol 1,4,5-trisphosphate receptors.
    Shohei Fujii, Ryo Ushioda, Kazuhiro Nagata.
      Inositol 1,4,5-trisphosphate receptors (IP3Rs) are one of the two types of tetrameric ion channels that release calcium ion (Ca2+) from the endoplasmic reticulum (ER) into the cytosol. Ca2+ released via IP3Rs is a fundamental second messenger for numerous cell functions. Disturbances in the intracellular redox environment resulting from various diseases and aging interfere with proper calcium signaling, however, the details are unclear. Here, we elucidated the regulatory mechanisms of IP3Rs by protein disulfide isomerase family proteins localized in the ER by focusing on four cysteine residues residing in the ER lumen of IP3Rs. First, we revealed that two of the cysteine residues are essential for functional tetramer formation of IP3Rs. Two other cysteine residues, on the contrary, were revealed to be involved in the regulation of IP3Rs activity; its oxidation by ERp46 and the reduction by ERdj5 caused the activation and the inactivation of IP3Rs activity, respectively. We previously reported that ERdj5 can activate the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b) using its reducing activity [Ushioda et al., Proc. Natl. Acad. Sci. U.S.A. 113, E6055-E6063 (2016)]. Thus, we here established that ERdj5 exerts the reciprocal regulatory function for IP3Rs and SERCA2b by sensing the ER luminal Ca2+ concentration, which contributes to the calcium homeostasis in the ER.
    Keywords:  PDI family members; calcium homeostasis; redox regulation
    DOI:  https://doi.org/10.1073/pnas.2216857120
  18. Nat Struct Mol Biol. 2023 May 25.
    Surveying the global landscape of post-transcriptional regulators.
    Kendra Reynaud, Anna M McGeachy, David Noble, Zuriah A Meacham, Nicholas T Ingolia.
      Numerous proteins regulate gene expression by modulating mRNA translation and decay. To uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.
    DOI:  https://doi.org/10.1038/s41594-023-00999-5
  19. Trends Cell Biol. 2023 May 24. pii: S0962-8924(23)00084-3. [Epub ahead of print]
    Mechanobiology of organelles: illuminating their roles in mechanosensing and mechanotransduction.
    Santosh Phuyal, Patrizia Romani, Sirio Dupont, Hesso Farhan.
      Mechanobiology studies the mechanisms by which cells sense and respond to physical forces, and the role of these forces in shaping cells and tissues themselves. Mechanosensing can occur at the plasma membrane, which is directly exposed to external forces, but also in the cell's interior, for example, through deformation of the nucleus. Less is known on how the function and morphology of organelles are influenced by alterations in their own mechanical properties, or by external forces. Here, we discuss recent advances on the mechanosensing and mechanotransduction of organelles, including the endoplasmic reticulum (ER), the Golgi apparatus, the endo-lysosmal system, and the mitochondria. We highlight open questions that need to be addressed to gain a broader understanding of the role of organelle mechanobiology.
    Keywords:  Golgi apparatus; endoplasmic reticulum; endosomes; mechanosensing; mechanotransduction; mitochondria
    DOI:  https://doi.org/10.1016/j.tcb.2023.05.001
  20. Nat Cancer. 2023 May;4(5): 629-647
    The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.
    Aaron Javitt, Merav D Shmueli, Matthias P Kramer, Aleksandra A Kolodziejczyk, Ivan J Cohen, Lihi Radomir, Daoud Sheban, Iris Kamer, Kevin Litchfield, Elizabeta Bab-Dinitz, Oranit Zadok, Vanessa Neiens, Adi Ulman, Hila Wolf-Levy, Avital Eisenberg-Lerner, Assaf Kacen, Michal Alon, Ana Toste Rêgo, Elvira Stacher-Priehse, Michael Lindner, Ina Koch, Jair Bar, Charles Swanton, Yardena Samuels, Yishai Levin, Paula C A da Fonseca, Eran Elinav, Nir Friedman, Silke Meiners, Yifat Merbl.
      Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.
    DOI:  https://doi.org/10.1038/s43018-023-00557-4
  21. Nat Rev Mol Cell Biol. 2023 May 25.
    New frontiers in quality control: the case of GPI-anchored proteins.
    Leticia Lemus, Ramanujan S Hegde, Veit Goder.
      
    DOI:  https://doi.org/10.1038/s41580-023-00616-9
  22. Nat Commun. 2023 May 26. 14(1): 3050
    TRABID inhibition activates cGAS/STING-mediated anti-tumor immunity through mitosis and autophagy dysregulation.
    Yu-Hsuan Chen, Han-Hsiun Chen, Won-Jing Wang, Hsin-Yi Chen, Wei-Syun Huang, Chien-Han Kao, Sin-Rong Lee, Nai Yang Yeat, Ruei-Liang Yan, Shu-Jou Chan, Kuen-Phon Wu, Ruey-Hwa Chen.
      Activation of tumor-intrinsic innate immunity has been a major strategy for improving immunotherapy. Previously, we reported an autophagy-promoting function of the deubiquitinating enzyme TRABID. Here, we identify a critical role of TRABID in suppressing anti-tumor immunity. Mechanistically, TRABID is upregulated in mitosis and governs mitotic cell division by removing K29-linked polyubiquitin chain from Aurora B and Survivin, thereby stabilizing the entire chromosomal passenger complex. TRABID inhibition causes micronuclei through a combinatory defect in mitosis and autophagy and protects cGAS from autophagic degradation, thereby activating the cGAS/STING innate immunity pathway. Genetic or pharmacological inhibition of TRABID promotes anti-tumor immune surveillance and sensitizes tumors to anti-PD-1 therapy in preclinical cancer models in male mice. Clinically, TRABID expression in most solid cancer types correlates inversely with an interferon signature and infiltration of anti-tumor immune cells. Our study identifies a suppressive role of tumor-intrinsic TRABID in anti-tumor immunity and highlights TRABID as a promising target for sensitizing solid tumors to immunotherapy.
    DOI:  https://doi.org/10.1038/s41467-023-38784-z
  23. Nat Cell Biol. 2023 May 25.
    Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence.
    Insup Choi, Minghui Wang, Seungyeul Yoo, Peng Xu, Steven P Seegobin, Xianting Li, Xian Han, Qian Wang, Junmin Peng, Bin Zhang, Zhenyu Yue.
      Dysfunctional autophagy has been implicated in the pathogenesis of Alzheimer's disease (AD). Previous evidence suggested disruptions of multiple stages of the autophagy-lysosomal pathway in affected neurons. However, whether and how deregulated autophagy in microglia, a cell type with an important link to AD, contributes to AD progression remains elusive. Here we report that autophagy is activated in microglia, particularly of disease-associated microglia surrounding amyloid plaques in AD mouse models. Inhibition of microglial autophagy causes disengagement of microglia from amyloid plaques, suppression of disease-associated microglia, and aggravation of neuropathology in AD mice. Mechanistically, autophagy deficiency promotes senescence-associated microglia as evidenced by reduced proliferation, increased Cdkn1a/p21Cip1, dystrophic morphologies and senescence-associated secretory phenotype. Pharmacological treatment removes autophagy-deficient senescent microglia and alleviates neuropathology in AD mice. Our study demonstrates the protective role of microglial autophagy in regulating the homeostasis of amyloid plaques and preventing senescence; removal of senescent microglia is a promising therapeutic strategy.
    DOI:  https://doi.org/10.1038/s41556-023-01158-0
  24. J Cell Biol. 2023 06 05. pii: e202209084. [Epub ahead of print]222(6):
    Inhibition of endolysosome fusion increases exosome secretion.
    Ganesh Vilas Shelke, Chad D Williamson, Michal Jarnik, Juan S Bonifacino.
      Exosomes are small vesicles that are secreted from cells to dispose of undegraded materials and mediate intercellular communication. A major source of exosomes is intraluminal vesicles within multivesicular endosomes that undergo exocytic fusion with the plasma membrane. An alternative fate of multivesicular endosomes is fusion with lysosomes, resulting in degradation of the intraluminal vesicles. The factors that determine whether multivesicular endosomes fuse with the plasma membrane or with lysosomes are unknown. In this study, we show that impairment of endolysosomal fusion by disruption of a pathway involving the BLOC-one-related complex (BORC), the small GTPase ARL8, and the tethering factor HOPS increases exosome secretion by preventing the delivery of intraluminal vesicles to lysosomes. These findings demonstrate that endolysosomal fusion is a critical determinant of the amount of exosome secretion and suggest that suppression of the BORC-ARL8-HOPS pathway could be used to boost exosome yields in biotechnology applications.
    DOI:  https://doi.org/10.1083/jcb.202209084
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