bims-proteo Biomed News
on Proteostasis
Issue of 2022‒06‒19
eighteen papers selected by
Eric Chevet

  1. Proc Natl Acad Sci U S A. 2022 Jun 21. 119(25): e2122482119
      Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes' peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5'-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.
    Keywords:  26S proteasome; arsenite; heat shock; proteotoxic stress; ubiquitin conjugates
  2. J Cell Biol. 2022 Jul 04. pii: e202203083. [Epub ahead of print]221(7):
      The process of membrane atg8ylation, defined herein as the conjugation of the ATG8 family of ubiquitin-like proteins to membrane lipids, is beginning to be appreciated in its broader manifestations, mechanisms, and functions. Classically, membrane atg8ylation with LC3B, one of six mammalian ATG8 family proteins, has been viewed as the hallmark of canonical autophagy, entailing the formation of characteristic double membranes in the cytoplasm. However, ATG8s are now well described as being conjugated to single membranes and, most recently, proteins. Here we propose that the atg8ylation is coopted by multiple downstream processes, one of which is canonical autophagy. We elaborate on these biological outputs, which impact metabolism, quality control, and immunity, emphasizing the context of inflammation and immunological effects. In conclusion, we propose that atg8ylation is a modification akin to ubiquitylation, and that it is utilized by different systems participating in membrane stress responses and membrane remodeling activities encompassing autophagy and beyond.
  3. J Clin Invest. 2022 Jun 14. pii: e157504. [Epub ahead of print]
      Mitochondrial stress triggers a response in the cell's mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DELE1. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 is critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.
    Keywords:  Cell Biology; Cell stress; Genetics; Mitochondria; Proteases
  4. Nat Commun. 2022 Jun 14. 13(1): 3410
      Ribosome associated complex (RAC), an obligate heterodimer of HSP40 and HSP70 (Zuo1 and Ssz1 in yeast), is conserved in eukaryotes and functions as co-chaperone for another HSP70 (Ssb1/2 in yeast) to facilitate co-translational folding of nascent polypeptides. Many mechanistic details, such as the coordination of one HSP40 with two HSP70s and the dynamic interplay between RAC-Ssb and growing nascent chains, remain unclear. Here, we report three sets of structures of RAC-containing ribosomal complexes isolated from Saccharomyces cerevisiae. Structural analyses indicate that RAC on the nascent-chain-free ribosome is in an autoinhibited conformation, and in the presence of a nascent chain at the peptide tunnel exit (PTE), RAC undergoes large-scale structural remodeling to make Zuo1 J-Domain more accessible to Ssb. Our data also suggest a role of Zuo1 in orienting Ssb-SBD proximal to the PTE for easy capture of the substrate. Altogether, in accordance with previous data, our work suggests a sequence of structural remodeling events for RAC-Ssb during co-translational folding, triggered by the binding and passage of growing nascent chain from one to another.
  5. Mol Biol Cell. 2022 Jun 15. mbcE21100505
      We report on how the ER-associated-autophagy pathway (ERAA) delivers P23H-rhodopsin (P23H-R) to the lysosome. P23H-R accumulates in an ERAD-resistant conformation that is stabilized in a detergent-soluble state by DNAJB12 and Hsp70. P23H-R, DNAJB12, and FIP200 colocalize in discrete foci that punctuate the rim of omegasome rings coated by WIPI1. Loss of DNAJB12 function prevents the association of P23H-R containing ER-tubules with omegasomes. P23H-R tubules thread through the wall of WIPI1 rings into their central cavity. Transfer of P23H-R from ER-connected phagophores to lysosomes requires GABARAP, and is associated with the transient docking of lysosomes to WIPI1 rings. After departure from WIPI1 rings, new patches of P23H-R are seen in the membranes of lysosomes. The absence of GABARAP prevents transfer of P23H-R from phagophores to lysosomes without interfering with docking. These data identify lysosome docking to omegasomes as an important step in the DNAJB12 and GABARAP-dependent autophagic disposal of dominantly toxic P23H-R.
  6. PLoS Genet. 2022 Jun 13. 18(6): e1010275
      Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.
  7. EMBO J. 2022 Jun 13. e110410
      Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.
    Keywords:  atomic force microscopy; cryo-electron tomography; disaggregation; molecular chaperones
  8. Structure. 2022 Jun 01. pii: S0969-2126(22)00190-3. [Epub ahead of print]
      More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. Degrons are stretches of amino acids that help mediate recognition by E3 ligases and thus confer protein degradation via the ubiquitin-proteasome system. While degrons that mediate controlled degradation of, for example, signaling components and cell-cycle regulators are well described, so-called protein-quality-control degrons that mediate the degradation of destabilized proteins are poorly understood. Here, we show that disease-linked dihydrofolate reductase (DHFR) missense variants are structurally destabilized and chaperone-dependent proteasome targets. We find two regions in DHFR that act as degrons, and the proteasomal turnover of one of these was dependent on the molecular chaperone Hsp70. Structural analyses by nuclear magnetic resonance (NMR) and hydrogen/deuterium exchange revealed that this degron is buried in wild-type DHFR but becomes transiently exposed in the disease-linked missense variants.
    Keywords:  chaperone; conformational diseases; proteasome; protein degradation; protein misfolding; protein quality control; protein stability; protein unfolding; proteostasis; ubiquitin
  9. J Biol Chem. 2022 Jun 08. pii: S0021-9258(22)00555-5. [Epub ahead of print] 102114
      Parkin and PINK1 regulate a mitochondrial quality control system that is mutated in some early onset forms of Parkinson's disease. Parkin is an E3 ubiquitin ligase and regulated by the mitochondrial kinase PINK1 via a two-step cascade. PINK1 first phosphorylates ubiquitin, which binds a recruitment site on parkin to localize parkin to damaged mitochondria. In the second step, PINK1 phosphorylates parkin on its ubiquitin-like domain (Ubl) domain, which binds a regulatory site to release ubiquitin ligase activity. Recently, an alternative feed-forward mechanism was identified that bypasses the need for parkin phosphorylation through the binding of a second phospho-ubiquitin (pUb) molecule. Here, we report the structure of parkin activated through this feed-forward mechanism. The crystal structure of parkin with pUb bound to both the recruitment and regulatory sites reveals the molecular basis for differences in specificity and affinity of the two sites. We use isothermal titration calorimetry measurements to reveal cooperativity between the two binding sites and the role of linker residues for pUbl binding to the regulatory site. The observation of flexibility in the process of parkin activation offers hope for the future design of small molecules for the treatment of Parkinson's disease.
  10. Brain Pathol. 2022 Jun 15. e13084
      Patients suffering from immune-mediated necrotizing myopathies (IMNM) harbor, the pathognomonic myositis-specific auto-antibodies anti-SRP54 or -HMGCR, while about one third of them do not. Activation of chaperone-assisted autophagy was described as being part of the molecular etiology of IMNM. Endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR)-stress accompanied by activation of the unfolded protein response (UPR) often precedes activation of the protein clearance machinery and represents a cellular defense mechanism toward restoration of proteostasis. Here, we show that ER/SR-stress may be part of the molecular etiology of IMNM. To address this assumption, ER/SR-stress related key players covering the three known branches (PERK-mediated, IRE1-mediated, and ATF6-mediated) were investigated on both, the transcript and the protein levels utilizing 39 muscle biopsy specimens derived from IMNM-patients. Our results demonstrate an activation of all three UPR-branches in IMNM, which most likely precedes the activation of the protein clearance machinery. In detail, we identified increased phosphorylation of PERK and eIF2a along with increased expression and protein abundance of ATF4, all well-documented characteristics for the activation of the UPR. Further, we identified increased general XBP1-level, and elevated XBP1 protein levels. Additionally, our transcript studies revealed an increased ATF6-expression, which was confirmed by immunostaining studies indicating a myonuclear translocation of the cleaved ATF6-form toward the forced transcription of UPR-related chaperones. In accordance with that, our data demonstrate an increase of downstream factors including ER/SR co-chaperones and chaperones (e.g., SIL1) indicating an UPR-activation on a broader level with no significant differences between seropositive and seronegative patients. Taken together, one might assume that UPR-activation within muscle fibers might not only serve to restore protein homeostasis, but also enhance sarcolemmal presentation of proteins crucial for attracting immune cells. Since modulation of ER-stress and UPR via application of chemical chaperones became a promising therapeutic treatment approach, our findings might represent the starting point for new interventional concepts.
    Keywords:  ER-stress; HMGCR; IMNM; SRP; UPR; auto-antibodies
  11. Mol Neurobiol. 2022 Jun 14.
      Synuclein aggregation in neuronal cells is the primary underlying cause of synucleinopathies. Changes in gene expression patterns, structural modifications, and altered interactions with other cellular proteins often trigger aggregation of α-synuclein, which accumulates as oligomers or fibrils in Lewy bodies. Although fibrillar forms of α-synuclein are primarily considered pathological, recent studies have revealed that even the intermediate states of aggregates are neurotoxic, complicating the development of therapeutic interventions. Autophagy and ubiquitin-proteasome pathways play a significant role in maintaining the soluble levels of α-synuclein inside cells; however, the heterogeneous nature of the aggregates presents a significant bottleneck to its degradation by these cellular pathways. With studies focused on identifying the proteins that modulate synuclein aggregation and clearance, detailed mechanistic insights are emerging about the individual and synergistic effects of these degradation pathways in regulating soluble α-synuclein levels. In this article, we discuss the impact of α-synuclein aggregation on autophagy-lysosome and ubiquitin-proteasome pathways and the therapeutic strategies that target various aspects of synuclein aggregation or degradation via these pathways. Additionally, we also highlight the natural and synthetic compounds that have shown promise in alleviating the cellular damage caused due to synuclein aggregation.
    Keywords:  Aggregate clearance; Autophagy; Chaperone-mediated autophagy; Neurodegenerative diseases; Oligomers; Parkinson’s disease; Proteasome; Small molecules; Synucleinopathies; Ubiquitin; α-Synuclein
  12. Cell Rep. 2022 Jun 14. pii: S2211-1247(22)00732-X. [Epub ahead of print]39(11): 110950
      Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.
    Keywords:  CP: Molecular biology; EGF receptor; endocytosis; proximity labeling; signaling
  13. Sci Adv. 2022 Jun 17. 8(24): eabn6153
      In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated Nα-acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana. However, the relevance of HsHYPK for determining the human N-acetylome is unclear. Here, we identify the AtHYPK protein as the first in vivo regulator of NatA activity in plants. AtHYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes Nα-terminal acetylation of diverse NatA substrates. Loss-of-AtHYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of HsHYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X2/N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.
  14. Nat Chem Biol. 2022 Jun 13.
      Regenerating pancreatic β-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of β-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased β-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and β-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in β-cell regeneration, a role that warrants further investigation in diabetes.
  15. FASEB J. 2022 07;36(7): e22396
      Dietary removal of an essential amino acid (EAA) triggers the integrated stress response (ISR) in liver. Herein, we explored the mechanisms that activate the ISR and execute changes in transcription and translation according to the missing EAA. Wild-type mice and mice lacking general control nonderepressible 2 (Gcn2) were fed an amino acid complete diet or a diet devoid of either leucine or sulfur amino acids (methionine and cysteine). Serum and liver leucine concentrations were significantly reduced within the first 6 h of feeding a diet lacking leucine, corresponding with modest, GCN2-dependent increases in Atf4 mRNA translation and induction of selected ISR target genes (Fgf21, Slc7a5, Slc7a11). In contrast, dietary removal of the sulfur amino acids lowered serum methionine, but not intracellular methionine, and yet hepatic mRNA abundance of Atf4, Fgf21, Slc7a5, Slc7a11 substantially increased regardless of GCN2 status. Liver tRNA charging levels did not correlate with intracellular EAA concentrations or GCN2 status and remained similar to mice fed a complete diet. Furthermore, loss of Gcn2 increased the occurrence of ribosome collisions in liver and derepressed mechanistic target of rapamycin complex 1 signal transduction, but these changes did not influence execution of the ISR. We conclude that ISR activation is directed by intracellular EAA concentrations, but ISR execution is not. Furthermore, a diet devoid of sulfur amino acids does not require GCN2 for the ISR to execute changes to the transcriptome.
    Keywords:  dietary restriction; feeding; mammalian; polysomes; postprandial period; protein synthesis
  16. Chem Soc Rev. 2022 Jun 17.
      Proteolysis Targeting Chimeras (PROTACs), an emerging therapeutic entity designed to degrade target proteins by hijacking the ubiquitin-proteasome system, have the potential to revolutionize the healthcare industry. The broad applicability of this protein degradation strategy has been verified with a few E3 ligases and a variety of distinct targets through the construction of modular chimeric structures. Despite recent efforts to promote the use of PROTACs for clinical applications, most PROTACs do not make it beyond the preclinical stage of drug development. There are several reasons that prevent PROTACs from reaching the market, and the inadequate delivery to the target site is one of the most challenging hurdles. With the increasing need for accelerating the translational process, combining the concepts of PROTACs and delivery systems has been explored to enhance the in vivo performance of PROTACs. These improved delivery strategies can eliminate unfavorable physicochemical properties of PROTACs, improve their targetability, and decrease their off-target side effects. The integration of powerful PROTACs and versatile delivery systems will inaugurate a burgeoning orientation for the field of targeted protein degradation. In this review, we will survey the latest progress in improving the in vivo degradation efficacy of PROTACs through delivery strategies, outline design principles for PROTAC-based delivery systems, discuss the current challenges with PROTACs, and outlook future opportunities in this field.
  17. Life Sci Alliance. 2022 Oct;pii: e202201478. [Epub ahead of print]5(10):
      Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. Here, we demonstrate that overexpressed TMBIM5 can mediate mitochondrial calcium uptake. Under steady-state conditions, loss of TMBIM5 results in increased potassium and reduced proton levels in the mitochondrial matrix caused by attenuated exchange of these ions. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore. These mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.
  18. Autophagy. 2022 Jun 16.
      Macroautophagy/autophagy defects are a risk factor for intestinal bowel disease (IBD), but the mechanism remains unclear. We previously demonstrated that conditional whole-body deletion of the essential Atg7 (autophagy related 7) gene in adult mice (atg7Δ/Δ) causes specific tissue damage and shortens lifespan to three months primarily due to neurodegeneration with surprisingly no disturbing effects on the intestine. In contrast, we recently found that conditional whole-body deletion of other essential autophagy genes, Atg5 or Rb1cc1/Fip200 (atg5Δ/Δ or rb1cc1Δ/Δ), cause death within five days due to rapid inhibition of autophagy, elimination of intestinal stem cells, and loss of barrier function in the ileum. atg5Δ/Δ mice lose PDGFRA/PDGFRα+ mesenchymal cells (PMCs) and WNT signaling essential for stem cell renewal. Depletion of aspartate and nucleotides in atg5Δ/Δ ileum was revealed by novel mass-spectrometry imaging (MALDI-MSI), consistent with metabolic insufficiency underlying PMCs loss. The difference in the autophagy gene knockout phenotypes is likely due to distinct kinetics of autophagy loss because gradual whole-body atg5 deletion extends lifespan, phenocopying deletion of Atg7 or Atg12. Therefore, we established that autophagy is required for ileum PMC metabolism, stem cell maintenance and mammalian survival. PMC loss caused by autophagy deficiency may therefore contribute to IBD.
    Keywords:  Autophagy; IBD; PDGFRα+ mesenchymal cells; WNT signaling; intestinal stem cells