bims-proteo Biomed News
on Proteostasis
Issue of 2022‒10‒23
29 papers selected by
Eric Chevet
INSERM
  1. Heat Shock Protein Hspa13 Regulates Endoplasmic Reticulum and Cytosolic Proteostasis Through Modulation of Protein Translocation.
  2. Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.
  3. Substrate-driven assembly of a translocon for multipass membrane proteins.
  4. The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics.
  5. Parkin coordinates mitochondrial lipid remodeling to execute mitophagy.
  6. The unfolded protein response reverses the effects of glucose on lifespan in chemically-sterilized C. elegans.
  7. Mechanism of an intramembrane chaperone for multipass membrane proteins.
  8. N-myc mediated translation control is a therapeutic vulnerability in medulloblastoma.
  9. Shielding the mRNA-translation factor eIF2B from inhibitory p-eIF2 as a viral strategy to evade protein kinase R-mediated innate immunity.
  10. A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70.
  11. The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.
  12. Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain.
  13. XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.
  14. The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.
  15. Clathrin-associated AP-1 controls termination of STING signalling.
  16. Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila.
  17. Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life.
  18. Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair.
  19. Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators.
  20. Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.
  21. Localization matters in targeted protein degradation.
  22. HDLBP promotes hepatocellular carcinoma proliferation and sorafenib resistance by suppressing Trim71-dependent RAF1 degradation.
  23. LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface.
  24. MTCH2 is a mitochondrial outer membrane protein insertase.
  25. Proteolysis Targeting Chimeras (PROTACs): A Perspective on Integral Membrane Protein Degradation.
  26. Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.
  27. Loss of heat shock factor initiates intracellular lipid surveillance by actin destabilization.
  28. Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole.
  29. Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.
  1. J Biol Chem. 2022 Oct 13. pii: S0021-9258(22)01040-7. [Epub ahead of print] 102597
    Heat Shock Protein Hspa13 Regulates Endoplasmic Reticulum and Cytosolic Proteostasis Through Modulation of Protein Translocation.
    Mateo F Espinoza, Khanh K Nguyen, Melody M Sycks, Ziqi Lyu, Guy M Quanrud, Maureen R Montoya, Joseph C Genereux.
      Most eukaryotic secretory proteins are co-translationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER Hsp70 Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the HEK293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin (TTR), leading to accumulation and aggregation of immature TTR in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this manuscript has been deposited in the PRIDE archive and can be accessed at PXD033498.
    Keywords:  Endoplasmic Reticulum; HSP70; Import; Molecular Chaperones; Proteostasis; Proximity Labeling; Secretion; Translocation
    DOI:  https://doi.org/10.1016/j.jbc.2022.102597
  2. Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2205314119
    Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.
    Hao Ye, Jiayang Gao, Zizhen Liang, Youshun Lin, Qianyi Yu, Shuxian Huang, Liwen Jiang.
      Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.
    Keywords:  PI3P; autophagy; membrane contact site; oxysterol-binding protein–related protein
    DOI:  https://doi.org/10.1073/pnas.2205314119
  3. Nature. 2022 Oct 19.
    Substrate-driven assembly of a translocon for multipass membrane proteins.
    Arunkumar Sundaram, Melvin Yamsek, Frank Zhong, Yogesh Hooda, Ramanujan S Hegde, Robert J Keenan.
      Most membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes docked at the translocon, a heterogeneous ensemble of transmembrane factors operating on the nascent chain1,2. How the translocon coordinates the actions of these factors to accommodate its different substrates is not well understood. Here we define the composition, function and assembly of a translocon specialized for multipass membrane protein biogenesis3. This 'multipass translocon' is distinguished by three components that selectively bind the ribosome-Sec61 complex during multipass protein synthesis: the GET- and EMC-like (GEL), protein associated with translocon (PAT) and back of Sec61 (BOS) complexes. Analysis of insertion intermediates reveals how features of the nascent chain trigger multipass translocon assembly. Reconstitution studies demonstrate a role for multipass translocon components in protein topogenesis, and cells lacking these components show reduced multipass protein stability. These results establish the mechanism by which nascent multipass proteins selectively recruit the multipass translocon to facilitate their biogenesis. More broadly, they define the ER translocon as a dynamic assembly whose subunit composition adjusts co-translationally to accommodate the biosynthetic needs of its diverse range of substrates.
    DOI:  https://doi.org/10.1038/s41586-022-05330-8
  4. Nat Commun. 2022 Oct 21. 13(1): 6283
    The cholesterol transport protein GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics.
    Matthew Yoke Wui Ng, Chara Charsou, Ana Lapao, Sakshi Singh, Laura Trachsel-Moncho, Sebastian W Schultz, Sigve Nakken, Michael J Munson, Anne Simonsen.
      During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The contributions of specific lipids, such as cholesterol, to the membranes that form the autophagosome, remain to be fully characterized. Here, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and a corresponding increase in autophagy initiation events. We further show that the ER-localized cholesterol transport protein GRAMD1C functions as a negative regulator of starvation-induced autophagy and that both its cholesterol transport VASt domain and membrane binding GRAM domain are required for GRAMD1C-mediated suppression of autophagy initiation. Similar to its yeast orthologue, GRAMD1C associates with mitochondria through its GRAM domain. Cells lacking GRAMD1C or its VASt domain show increased mitochondrial cholesterol levels and mitochondrial oxidative phosphorylation, suggesting that GRAMD1C may facilitate cholesterol transfer at ER-mitochondria contact sites. Finally, we demonstrate that expression of GRAMD family proteins is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.
    DOI:  https://doi.org/10.1038/s41467-022-33933-2
  5. EMBO Rep. 2022 Oct 18. e55191
    Parkin coordinates mitochondrial lipid remodeling to execute mitophagy.
    Chao-Chieh Lin, Jin Yan, Meghan D Kapur, Kristi L Norris, Cheng-Wei Hsieh, De Huang, Nicolas Vitale, Kah-Leong Lim, Ziqiang Guan, Xiao-Fan Wang, Jen-Tsan Chi, Wei-Yuan Yang, Tso-Pang Yao.
      Autophagy has emerged as the prime machinery for implementing organelle quality control. In the context of mitophagy, the ubiquitin E3 ligase Parkin tags impaired mitochondria with ubiquitin to activate autophagic degradation. Although ubiquitination is essential for mitophagy, it is unclear how ubiquitinated mitochondria activate autophagosome assembly locally to ensure efficient destruction. Here, we report that Parkin activates lipid remodeling on mitochondria targeted for autophagic destruction. Mitochondrial Parkin induces the production of phosphatidic acid (PA) and its subsequent conversion to diacylglycerol (DAG) by recruiting phospholipase D2 and activating the PA phosphatase, Lipin-1. The production of DAG requires mitochondrial ubiquitination and ubiquitin-binding autophagy receptors, NDP52 and optineurin (OPTN). Autophagic receptors, via Golgi-derived vesicles, deliver an autophagic activator, EndoB1, to ubiquitinated mitochondria. Inhibition of Lipin-1, NDP52/OPTN, or EndoB1 results in a failure to produce mitochondrial DAG, autophagosomes, and mitochondrial clearance, while exogenous cell-permeable DAG can induce autophagosome production. Thus, mitochondrial DAG production acts downstream of Parkin to enable the local assembly of autophagosomes for the efficient disposal of ubiquitinated mitochondria.
    Keywords:  Lipin-1; PLD2; Parkin; diacylglycerol; mitophagy
    DOI:  https://doi.org/10.15252/embr.202255191
  6. Nat Commun. 2022 Oct 19. 13(1): 5889
    The unfolded protein response reverses the effects of glucose on lifespan in chemically-sterilized C. elegans.
    Caroline Beaudoin-Chabot, Lei Wang, Cenk Celik, Aishah Tul-Firdaus Abdul Khalid, Subhash Thalappilly, Shiyi Xu, Jhee Hong Koh, Venus Wen Xuan Lim, Ann Don Low, Guillaume Thibault.
      Metabolic diseases often share common traits, including accumulation of unfolded proteins in the endoplasmic reticulum (ER). Upon ER stress, the unfolded protein response (UPR) is activated to limit cellular damage which weakens with age. Here, we show that Caenorhabditis elegans fed a bacterial diet supplemented high glucose at day 5 of adulthood (HGD-5) extends their lifespan, whereas exposed at day 1 (HGD-1) experience shortened longevity. We observed a metabolic shift only in HGD-1, while glucose and infertility synergistically prolonged the lifespan of HGD-5, independently of DAF-16. Notably, we identified that UPR stress sensors ATF-6 and PEK-1 contributed to the longevity of HGD-5 worms, while ire-1 ablation drastically increased HGD-1 lifespan. Together, we postulate that HGD activates the otherwise quiescent UPR in aged worms to overcome ageing-related stress and restore ER homeostasis. In contrast, young animals subjected to HGD provokes unresolved ER stress, conversely leading to a detrimental stress response.
    DOI:  https://doi.org/10.1038/s41467-022-33630-0
  7. Nature. 2022 Oct 19.
    Mechanism of an intramembrane chaperone for multipass membrane proteins.
    Luka Smalinskaitė, Min Kyung Kim, Aaron J O Lewis, Robert J Keenan, Ramanujan S Hegde.
      Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity11. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endopasmic reticulum.
    DOI:  https://doi.org/10.1038/s41586-022-05336-2
  8. Cancer Res. 2022 Oct 20. pii: CAN-22-0945. [Epub ahead of print]
    N-myc mediated translation control is a therapeutic vulnerability in medulloblastoma.
    Duygu Kuzuoglu-Ozturk, Ozlem Aksoy, Christin Schmidt, Robin Lea, Jon D Larson, Ryan R L Phelps, Nicole Nasholm, Megan Holt, Adrian Contreras, Miller Huang, Shannon Wong-Michalak, Hao Shao, Robert Wechsler-Reya, Joanna J Phillips, Jason E Gestwicki, Davide Ruggero, William A Weiss.
      Deregulation of N-myc is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data unexpectedly revealed that N-myc regulates the expression of specific components (~5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-22-0945
  9. Curr Opin Immunol. 2022 Oct 12. pii: S0952-7915(22)00098-X. [Epub ahead of print]78 102251
    Shielding the mRNA-translation factor eIF2B from inhibitory p-eIF2 as a viral strategy to evade protein kinase R-mediated innate immunity.
    Jennifer D Wuerth, Friedemann Weber.
      The interferon-regulated kinase PKR (protein kinase RNA-activated) is a potent innate immune factor against a broad range of viruses. Being part of the integrated stress response (ISR), its restrictive effect is predominantly exerted by phosphorylating the eukaryotic translation-initiation factor eIF2, thereby turning it into an inhibitor of translation-initiation factor eIF2B. A plethora of viruses are known to evade the shutdown of cellular mRNA translation by interfering either with PKR activation or with eIF2 phosphorylation. Recently, a novel PKR evasion strategy was described: proteins from three taxonomically distinct RNA viruses allow for full PKR activation and eIF2 phosphorylation in the infected cell, but protect eIF2B from inhibition by phosphorylated eIF2, thus enabling mRNA translation in the presence of an activated ISR.
    DOI:  https://doi.org/10.1016/j.coi.2022.102251
  10. Nat Chem Biol. 2022 Oct 20.
    A fluorescent multi-domain protein reveals the unfolding mechanism of Hsp70.
    Satyam Tiwari, Bruno Fauvet, Salvatore Assenza, Paolo De Los Rios, Pierre Goloubinoff.
      Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins.
    DOI:  https://doi.org/10.1038/s41589-022-01162-9
  11. Life Sci Alliance. 2023 Jan;pii: e202201526. [Epub ahead of print]6(1):
    The metabolite-controlled ubiquitin conjugase Ubc8 promotes mitochondrial protein import.
    Saskia Rödl, Fabian den Brave, Markus Räschle, Büsra Kizmaz, Svenja Lenhard, Carina Groh, Hanna Becker, Jannik Zimmermann, Bruce Morgan, Elke Richling, Thomas Becker, Johannes M Herrmann.
      Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.
    DOI:  https://doi.org/10.26508/lsa.202201526
  12. Elife. 2022 Oct 20. pii: e79855. [Epub ahead of print]11
    Molecular mechanism of Afadin substrate recruitment to the receptor phosphatase PTPRK via its pseudophosphatase domain.
    Iain M Hay, Katie E Mulholland, Tiffany Lai, Stephen C Graham, Hayley J Sharpe, Janet E Deane.
      Protein tyrosine phosphatase receptor-type kappa (PTPRK) is a transmembrane receptor that links extracellular homophilic interactions to intracellular catalytic activity. Previously we showed that PTPRK promotes cell-cell adhesion by selectively dephosphorylating several cell junction regulators including the protein Afadin (Fearnley et al., 2019). Here we demonstrate that Afadin is recruited for dephosphorylation by directly binding to the PTPRK D2 pseudophosphatase domain. We mapped this interaction to a putative coiled coil (CC) domain in Afadin that is separated by more than 100 amino acids from the substrate pTyr residue. We identify the residues that define PTP specificity, explaining how Afadin is selectively dephosphorylated by PTPRK yet not by the closely related receptor tyrosine phosphatase PTPRM. Our work demonstrates that PTP substrate specificity can be determined by protein-protein interactions distal to the active site. This explains how PTPRK and other PTPs achieve substrate specificity despite a lack of specific sequence context at the substrate pTyr. Furthermore, by demonstrating that these interactions are phosphorylation-independent and mediated via binding to a non-catalytic domain, we highlight how receptor PTPs could function as intracellular scaffolds in addition to catalyzing protein dephosphorylation.
    Keywords:  biochemistry; cell biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.79855
  13. J Am Soc Nephrol. 2022 Oct 21. pii: ASN.2021091180. [Epub ahead of print]
    XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.
    Matteus Krappitz, Rishi Bhardwaj, Ke Dong, Tobias Staudner, Duygu Elif Yilmaz, Carlotta Pioppini, Parisa Westergerling, David Ruemmele, Till Hollmann, Thuy Anh Nguyen, Yiqiang Cai, Anna-Rachel Gallagher, Stefan Somlo, Sorin Fedeles.
      BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1α-XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1.METHODS: We engineered a Pkd1 REJ domain missense murine model, Pkd1R2216W , on the basis of the orthologous human hypomorphic allele Pkd1R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression.
    RESULTS: Expression of active XBP1 in cultured cells bearing PC1R2216W mutations increased levels and ciliary trafficking of PC1R2216W. Mice homozygous for Pkd1R2216W or heterozygous for Pkd1R2216W in trans with a conditional Pkd1fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters.
    CONCLUSIONS: Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.
    Keywords:  Ire1α-XBP1 pathway; chaperone; genetic renal disease; mutation; polycystic kidney disease; polycystin-1; protein folding; unfolded protein response
    DOI:  https://doi.org/10.1681/ASN.2021091180
  14. Nature. 2022 Oct 19.
    The E3 ligase adapter cereblon targets the C-terminal cyclic imide degron.
    Saki Ichikawa, Hope A Flaxman, Wenqing Xu, Nandini Vallavoju, Hannah C Lloyd, Binyou Wang, Dacheng Shen, Matthew R Pratt, Christina M Woo.
      The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.
    DOI:  https://doi.org/10.1038/s41586-022-05333-5
  15. Nature. 2022 Oct 19.
    Clathrin-associated AP-1 controls termination of STING signalling.
    Ying Liu, Pengbiao Xu, Sophie Rivara, Chong Liu, Jonathan Ricci, Xuefeng Ren, James H Hurley, Andrea Ablasser.
      Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.
    DOI:  https://doi.org/10.1038/s41586-022-05354-0
  16. EMBO Rep. 2022 Oct 17. e202153552
    Parkin drives pS65-Ub turnover independently of canonical autophagy in Drosophila.
    Joanne L Usher, Alvaro Sanchez-Martinez, Ana Terriente-Felix, Po-Lin Chen, Juliette J Lee, Chun-Hong Chen, Alexander J Whitworth.
      Parkinson's disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1-Parkin pathway operates in vivo, we developed methods to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production, while pS65-Ub accumulates in unstimulated parkin-null flies, consistent with blocked degradation. Additionally, we show that pS65-Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat-induced pS65-Ub in an Atg5-null background. Thus, we have established that pS65-Ub immunodetection can be used to analyse Pink1-Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1-Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.
    Keywords:   in vivo ; Parkinson's disease; mitochondria; mitophagy; phospho-ubiquitin
    DOI:  https://doi.org/10.15252/embr.202153552
  17. Nat Commun. 2022 Oct 21. 13(1): 6271
    Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life.
    Kaushik Bhattacharya, Samarpan Maiti, Szabolcs Zahoran, Lorenz Weidenauer, Dina Hany, Diana Wider, Lilia Bernasconi, Manfredo Quadroni, Martine Collart, Didier Picard.
      The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life. Although reduced Hsp90 levels correlate with aging, it was unknown whether eukaryotic cells and organisms can tune the basal Hsp90 levels to alleviate physiologically accumulated stress. We have investigated whether and how mice adapt to the deletion of three out of four alleles of the two genes encoding cytosolic Hsp90, with one Hsp90β allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90β protein to at least wild-type levels. Our studies reveal an internal ribosome entry site in the 5' untranslated region of the Hsp90β mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We find that the minimum amount of total Hsp90 required to support viability of mammalian cells and organisms is 50-70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced ≥100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have markedly increased during eukaryotic evolution.
    DOI:  https://doi.org/10.1038/s41467-022-33916-3
  18. Nat Commun. 2022 Oct 18. 13(1): 6174
    Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair.
    Ganga B Vamisetti, Abhishek Saha, Yichao J Huang, Rajeshwer Vanjari, Guy Mann, Julia Gutbrod, Nabieh Ayoub, Hiroaki Suga, Ashraf Brik.
      Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.
    DOI:  https://doi.org/10.1038/s41467-022-33808-6
  19. Sci Adv. 2022 Oct 21. 8(42): eabn4466
    Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators.
    Zhishuo Wang, Beatriz Orosa-Puente, Mika Nomoto, Heather Grey, Thomas Potuschak, Takakazu Matsuura, Izumi C Mori, Yasuomi Tada, Pascal Genschik, Steven H Spoel.
      The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis, the salicylic acid- and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCFEBF2 and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates.
    DOI:  https://doi.org/10.1126/sciadv.abn4466
  20. Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2123187119
    Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.
    Jeremy Katzen, Luis Rodriguez, Yaniv Tomer, Apoorva Babu, Ming Zhao, Aditi Murthy, Paige Carson, Matthew Barrett, Maria C Basil, Justine Carl, John P Leach, Michael Morley, Matthew D McGraw, Surafel Mulugeta, Timothy Pelura, Glenn Rosen, Edward E Morrisey, Michael F Beers.
      Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.
    Keywords:  ER stress; quality control; surfactant protein C; transitional cell; unfolded protein response
    DOI:  https://doi.org/10.1073/pnas.2123187119
  21. Cell Chem Biol. 2022 Oct 20. pii: S2451-9456(22)00354-3. [Epub ahead of print]29(10): 1465-1466
    Localization matters in targeted protein degradation.
    Varun Jayeshkumar Shah, Ivan Đikić.
      Many factors influence the efficiency of targeted protein degradation induced by proteolysis targeting chimeras (PROTACs). In this issue of Cell Chemical Biology,Simpson et al. (2022) highlight the impact of subcellular localization. Their study elucidates that the protein of interest exhibits different amenability to PROTAC-mediated degradation at different cellular compartments.
    DOI:  https://doi.org/10.1016/j.chembiol.2022.09.006
  22. Cell Mol Gastroenterol Hepatol. 2022 Oct 13. pii: S2352-345X(22)00216-8. [Epub ahead of print]
    HDLBP promotes hepatocellular carcinoma proliferation and sorafenib resistance by suppressing Trim71-dependent RAF1 degradation.
    Jingsheng Yuan, Tao Lv, Jian Yang, Zhenru Wu, Lvnan Yan, Jiayin Yang, Yujun Shi, Li Jiang.
      BACKGROUND & AIMS: The contribution of abnormal metabolic targets to hepatocellular carcinoma (HCC) progression and the associated regulatory mechanisms are attractive research areas. High-density lipoprotein binding protein (HDLBP) is an important transporter that protects cells from excessive cholesterol accumulation, but few studies have identified a role for HDLBP in HCC progression.METHODS: HDLBP expression was determined in HCC tissues and published datasets. The biological roles of HDLBP in vitro and in vivo were examined by performing a series of functional experiments.
    RESULTS: An integrated analysis confirmed that HDLBP expression was significantly elevated in HCC compared to noncancerous liver tissues. The knockdown or overexpression of HDLBP substantially inhibited or enhanced, respectively, HCC proliferation and sorafenib resistance. Subsequently, a mass spectrometry screen identified RAF1 as a potential downstream target of HDLBP. Mechanistically, when RAF1 was stabilized by HDLBP, MEKK1 continuously induced RAF1Ser259-dependent MAPK signalling. Meanwhile, HDLBP interacted with RAF1 by competing with the TRIM71 E3 ligase and inhibited RAF1 degradation through the ubiquitin-proteasome pathway.
    CONCLUSIONS: Our study reveals that HDLBP is an important mediator that stabilizes the RAF1 protein and maintains its activity, leading to HCC progression and sorafenib resistance. Thus, HDLBP might represent a potential biomarker and future therapeutic target for HCC.
    Keywords:  MEKK1; tripartite motif containing 71; ubiquitination; vigilin
    DOI:  https://doi.org/10.1016/j.jcmgh.2022.10.005
  23. Biochemistry. 2022 Oct 20.
    LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface.
    Yinyan Tang, Amber Kay, Ziwen Jiang, Michelle R Arkin.
      Autophagy is a catabolic cellular process in which unwanted proteins and organelles are degraded by lysosomes. It is characterized by the formation of the double-membrane autophagosome decorated with LC3B, a protein that mediates autophagosomal fusion with lysosomes. The cysteine protease ATG4b acts at two stages in the life cycle of LC3B. We set out to characterize the protein-protein interaction between LC3B and ATG4b. Through biochemical and biophysical studies, we show that the ubiquitin-like core of LC3B (residues 1-115; "LC3B-115"), which lacks the C-terminal cleavage site (between residue 120 and 121), binds to full-length ATG4b with a surprisingly tight dissociation constant (KD) in the low nanomolar range; 10-30-fold tighter than that of the substrate pro-LC3B (residues 1-125) or the product LC3B-I (residues 1-120). Consequently, LC3B-115 is a potent inhibitor of the ATG4b-mediated cleavage of pro-LC3B (IC50 = 15 nM). Binding of the LC3B-115 has no effect on the conformation of the active site of ATG4b, as judged by the turnover of a peptide substrate ("substrate-33"), derived from LC3B-I residues 116-120. Conversely, truncations of ATG4b show that binding and proteolysis of LC3B critically depend on the C-terminal tail of ATG4b, whereas proteolysis of the peptide substrate-33 does not require the C-terminal tail of ATG4b. These results support a bipartite model for LC3B-ATG4b binding in which the core of LC3B binds to ATG4b and the C-terminal tail of pro-LC3B organizes the ATG4b active site; additionally, the C-terminal tail of ATG4b contributes at least 1000-fold higher binding affinity to the LC3B-ATG4b interaction and likely wraps around the LC3B-ubiquitin core. PPIs are often described as containing an energetic "hot spot" for binding; in the case of LC3B-ATG4b, however, the substrate-enzyme complex contains multiple, energetically relevant domains that differentially affect binding affinity and catalytic efficiency.
    DOI:  https://doi.org/10.1021/acs.biochem.2c00482
  24. Science. 2022 Oct 21. 378(6617): 317-322
    MTCH2 is a mitochondrial outer membrane protein insertase.
    Alina Guna, Taylor A Stevens, Alison J Inglis, Joseph M Replogle, Theodore K Esantsi, Gayathri Muthukumar, Kelly C L Shaffer, Maxine L Wang, Angela N Pogson, Jeff J Jones, Brett Lomenick, Tsui-Fen Chou, Jonathan S Weissman, Rebecca M Voorhees.
      In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified mitochondrial carrier homolog 2 (MTCH2), and its paralog MTCH1, and showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, and multipass proteins, but not outer membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate insertion into reconstituted proteoliposomes. Functional and mutational studies suggested that MTCH2 has evolved from a solute carrier transporter. MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for the outer membrane, controlling mislocalization of TAs into the endoplasmic reticulum and modulating the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction.
    DOI:  https://doi.org/10.1126/science.add1856
  25. ACS Pharmacol Transl Sci. 2022 Oct 14. 5(10): 849-858
    Proteolysis Targeting Chimeras (PROTACs): A Perspective on Integral Membrane Protein Degradation.
    Camilla Ruffilli, Sascha Roth, Monica Rodrigo, Helen Boyd, Noam Zelcer, Kevin Moreau.
      Targeted protein degradation (TPD) is a promising therapeutic modality to modulate protein levels and its application promises to reduce the "undruggable" proteome. Among TPD strategies, Proteolysis TArgeting Chimera (PROTAC) technology has shown a tremendous potential with attractive advantages when compared to the inhibition of the same target. While PROTAC technology has had a significant impact in scientific research, its application to degrade integral membrane proteins (IMPs) is still in its beginnings. Among the 15 compounds having entered clinical trials by the end of 2021, only two targets are membrane-associated proteins. In this review we are discussing the potential reasons which may underlie this, and we are presenting new tools that have been recently developed to solve these limitations and to empower the use of PROTACs to target IMPs.
    DOI:  https://doi.org/10.1021/acsptsci.2c00142
  26. Acta Neuropathol. 2022 Oct 20.
    Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.
    Conrad C Weihl, Ana Töpf, Rocio Bengoechea, Jennifer Duff, Richard Charlton, Solange Kapetanovic Garcia, Cristina Domínguez-González, Abdulaziz Alsaman, Aurelio Hernández-Laín, Luis Varona Franco, Monica Elizabeth Ponce Sanchez, Sarah J Beecroft, Hayley Goullee, Jil Daw, Ankan Bhadra, Heather True, Michio Inoue, Andrew R Findlay, Nigel Laing, Montse Olivé, Gianina Ravenscroft, Volker Straub.
      DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.
    Keywords:  Chaperone; Congenital myopathy; Myofibrillar myopathy; Myopathy; Protein aggregation
    DOI:  https://doi.org/10.1007/s00401-022-02510-8
  27. Cell Rep. 2022 Oct 18. pii: S2211-1247(22)01343-2. [Epub ahead of print]41(3): 111493
    Loss of heat shock factor initiates intracellular lipid surveillance by actin destabilization.
    Abigail Watterson, Sonja L B Arneaud, Naureen Wajahat, Jordan M Wall, Lexus Tatge, Shaghayegh T Beheshti, Melina Mihelakis, Nicholas Y Cheatwood, Jacob McClendon, Atossa Ghorashi, Ishmael Dehghan, Chase D Corley, Jeffrey G McDonald, Peter M Douglas.
      Cells sense stress and initiate response pathways to maintain lipid and protein homeostasis. However, the interplay between these adaptive mechanisms is unclear. Herein, we demonstrate how imbalances in cytosolic protein homeostasis affect intracellular lipid surveillance. Independent of its ancient thermo-protective properties, the heat shock factor, HSF-1, modulates lipid metabolism and age regulation through the metazoan-specific nuclear hormone receptor, NHR-49. Reduced hsf-1 expression destabilizes the Caenorhabditis elegans enteric actin network, subsequently disrupting Rab GTPase-mediated trafficking and cell-surface residency of nutrient transporters. The ensuing malabsorption limits lipid availability, thereby activating the intracellular lipid surveillance response through vesicular release and nuclear translocation of NHR-49 to both increase nutrient absorption and restore lipid homeostasis. Overall, cooperation between these regulators of cytosolic protein homeostasis and lipid surveillance ensures metabolic health and age progression through actin integrity, endocytic recycling, and lipid sensing.
    Keywords:  CP: Cell biology; NHR-49; Rab GTPase; absorption; actin cytoskeleton; geranylgeranylation; heat shock factor; lipid sensing; lipid surveillance; nuclear hormone receptor; protein homeostasis
    DOI:  https://doi.org/10.1016/j.celrep.2022.111493
  28. J Cell Biol. 2022 Dec 05. pii: e202203139. [Epub ahead of print]221(12):
    Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole.
    Jierui Zhao, Mai Thu Bui, Juncai Ma, Fabian Künzl, Lorenzo Picchianti, Juan Carlos De La Concepcion, Yixuan Chen, Sofia Petsangouraki, Azadeh Mohseni, Marta García-Leon, Marta Salas Gomez, Caterina Giannini, Dubois Gwennogan, Roksolana Kobylinska, Marion Clavel, Swen Schellmann, Yvon Jaillais, Jiri Friml, Byung-Ho Kang, Yasin Dagdas.
      Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1's function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.
    DOI:  https://doi.org/10.1083/jcb.202203139
  29. Cell Rep. 2022 Oct 18. pii: S2211-1247(22)01324-9. [Epub ahead of print]41(3): 111474
    Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.
    Darshan Sapkota, Mandy S J Kater, Kristina Sakers, Kayla R Nygaard, Yating Liu, Sarah K Koester, Stuart B Fass, Allison M Lake, Rohan Khazanchi, Rana R Khankan, Mitchell C Krawczyk, August B Smit, Susan E Maloney, Mark H G Verheijen, Ye Zhang, Joseph D Dougherty.
      Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.
    Keywords:  CP: Neuroscience; TRAP; astrocyte; regulation; seizure; translation
    DOI:  https://doi.org/10.1016/j.celrep.2022.111474
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