bims-proteo Biomed News
on Proteostasis
Issue of 2022‒05‒29
23 papers selected by
Eric Chevet

  1. Cells. 2022 May 17. pii: 1661. [Epub ahead of print]11(10):
      The extracellular aggregation of destabilized transthyretin (TTR) variants is implicated in the onset and pathogenesis of familial TTR-related amyloid diseases. One strategy to reduce the toxic, extracellular aggregation of TTR is to decrease the population of aggregation-prone proteins secreted from mammalian cells. The stress-independent activation of the unfolded protein response (UPR)-associated transcription factor ATF6 preferentially decreases the secretion and subsequent aggregation of destabilized, aggregation-prone TTR variants. However, the mechanism of this reduced secretion was previously undefined. Here, we implement a mass-spectrometry-based interactomics approach to identify endoplasmic reticulum (ER) proteostasis factors involved in ATF6-dependent reductions in destabilized TTR secretion. We show that ATF6 activation reduces amyloidogenic TTR secretion and subsequent aggregation through a mechanism involving ER retention that is mediated by increased interactions with ATF6-regulated ER proteostasis factors including BiP and PDIA4. Intriguingly, the PDIA4-dependent retention of TTR is independent of both the single TTR cysteine residue and the redox activity of PDIA4, indicating that PDIA4 retains destabilized TTR in the ER through a redox-independent mechanism. Our results define a mechanistic basis to explain the ATF6 activation-dependent reduction in destabilized, amyloidogenic TTR secretion that could be therapeutically accessed to improve treatments of TTR-related amyloid diseases.
    Keywords:  ATF6; ER proteostasis; amyloid disease; extracellular proteostasis; protein aggregation; protein disulfide isomerase (PDI); unfolded protein response (UPR)
  2. J Biol Chem. 2022 May 21. pii: S0021-9258(22)00501-4. [Epub ahead of print] 102061
      The concentration of Ca2+ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca2+ concentrations and the dynamic Ca2+ flux between the different subcellular compartments are tightly controlled. Influx of Ca2+ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase (SERCA) pump, building the Ca2+ gradient across the ER membrane required for ATP import. Meanwhile, Ca2+ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review we provide an overview of the complex regulation of Ca2+ homeostasis, Ca2+ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca2+ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident co-chaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca2+ concentrations.
    Keywords:  BiP/Grp78/HspA5; DnaJ; J-proteins; UPR; chaperone; diabetes; stress; thioredoxin; translocon
  3. Cell Rep. 2022 May 24. pii: S2211-1247(22)00633-7. [Epub ahead of print]39(8): 110860
      Protein ubiquitination is an essential process that rapidly regulates protein synthesis, function, and fate in dynamic environments. Within its non-proteolytic functions, we showed that K63-linked polyubiquitinated conjugates heavily accumulate in yeast cells exposed to oxidative stress, stalling ribosomes at elongation. K63-ubiquitinated conjugates accumulate mostly because of redox inhibition of the deubiquitinating enzyme Ubp2; however, the role and regulation of ubiquitin-conjugating enzymes (E2) in this pathway remained unclear. Here, we show that the E2 Rad6 associates and modifies ribosomes during stress. We further demonstrate that Rad6 and its human homolog UBE2A are redox regulated by forming a reversible disulfide with the E1 ubiquitin-activating enzyme (Uba1). This redox regulation is part of a negative feedback regulation, which controls the levels of K63 ubiquitination under stress. Finally, we show that Rad6 activity is necessary to regulate translation, antioxidant defense, and adaptation to stress, thus providing an additional physiological role for this multifunctional enzyme.
    Keywords:  CP: Molecular biology; K63-linked ubiquitination; Rad6; oxidative stress; ribosome ubiquitination; stress response; translation
  4. Biochim Biophys Acta Mol Cell Res. 2022 May 22. pii: S0167-4889(22)00091-X. [Epub ahead of print] 119299
      The membrane-bound transcription factor Nrf1 (encoded by Nfe2l1) is activated by sensing glucose deprivation, cholesterol abundance, proteasomal inhibition and oxidative stress and then mediates distinct signaling responses to maintain cellular homeostasis. Herein, we found that Nrf1 stability and transactivity are both enhanced by USP19, a ubiquitin-specific protease tail-anchored in the endoplasmic reticulum (ER) through its C-terminal transmembrane domain. Further experiments revealed that USP19 directly interacts with Nrf1 in proximity to the ER and topologically acts as a deubiquitinating enzyme to remove ubiquitin moieties from this protein, which allow it to circumvent potential proteasomal degradation. This USP19-mediated effect takes place only after Nrf1 is retro-translocated by p97 out of the ER membrane to dislocate the cytoplasmic side. Conversely, knockout of USP19 causes significant decreases in the abundance of Nrf1 and the entrance of its active isoform into the nucleus, which result in the downregulation of its target proteasomal subunits and a modest reduction in USP19-/--derived tumor growth in xenograft mice when compared with wild-type controls. Altogether, these results demonstrate that USP19 serves as a novel mechanistic modulator of Nrf1, but not Nrf2, thereby enabling Nrf1 to be rescued from the putative ubiquitin-directed ER-associated degradation pathway. In turn, our additional experimental evidence has revealed that transcriptional expression of endogenous USP19 and its promoter-driven reporter genes is differentially regulated by Nrf2, as well by Nrf1, at distinct layers within a complex hierarchical regulatory network.
    Keywords:  Deubiquitination; Endoplasmic reticulum; Nrf1; Nrf2; Proteasome; USP19; Ubiquitination
  5. Dev Cell. 2022 May 17. pii: S1534-5807(22)00330-6. [Epub ahead of print]
      The endoplasmic reticulum (ER) confronts a challenge to accommodate long, smooth ER tubules into the structural complexity of the axonal compartment. Here, we describe a morphological feature for the axonal ER network in developing neurons we termed the ER ladder. Axonal ER ladders are composed of rungs that wrap tightly around the microtubule bundle and dynamic rails, which slide across microtubules. We found that the ER-shaping protein Reticulon 2 determines the architecture and dynamics of the axonal ER ladder by modulating its interaction with microtubules. Moreover, we show that ER ladder depletion impairs the trafficking of associated vesicular axonal cargoes. Finally, we demonstrate that stromal interaction molecule 1 (Stim1) localizes to ER rungs and translocates to ER-plasma membrane contact sites upon depletion of luminal Ca2+. Our findings uncover fundamental insights into the structural and functional organization of the axonal ER network in developing mammalian neurons.
    Keywords:  axon development; cryo-ET; endoplasmic reticulum; microtubule bundle; neurodegeneration; organelle dynamics; organelle shape
  6. J Biol Chem. 2022 May 18. pii: S0021-9258(22)00487-2. [Epub ahead of print] 102047
      Thrombospondin Type-1 Repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding, and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization is poorly understood. Here we conducted molecular dynamics simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.
    Keywords:  O-fucose; extracellular matrix; glycosylation; protein folding; thrombospondin type 1 repeats
  7. Elife. 2022 May 27. pii: e73466. [Epub ahead of print]11
      Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2a phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.
    Keywords:  S. cerevisiae; biochemistry; chemical biology
  8. Biochem J. 2022 May 27. 479(10): 1103-1119
      Apoptosis, pyroptosis, and necroptosis are distinct forms of programmed cell death that eliminate infected, damaged, or obsolete cells. Many proteins that regulate or are a part of the cell death machinery undergo ubiquitination, a post-translational modification made by ubiquitin ligases that modulates protein abundance, localization, and/or activity. For example, some ubiquitin chains target proteins for degradation, while others function as scaffolds for the assembly of signaling complexes. Deubiquitinases (DUBs) are the proteases that counteract ubiquitin ligases by cleaving ubiquitin from their protein substrates. Here, we review the DUBs that have been found to suppress or promote apoptosis, pyroptosis, or necroptosis.
    Keywords:  apoptosis; deubiquitinase; necroptosis; pyroptosis
  9. EMBO Mol Med. 2022 May 25. e15373
      Sigma-1 receptor (Sigmar1) is a specific chaperone located in the mitochondria-associated endoplasmic reticulum membrane (MAM) and plays a role in several physiological processes. However, the role of Sigmar1 in bone homeostasis remains unknown. Here, we show that mice lacking Sigmar1 exhibited severe osteoporosis in an ovariectomized model. In contrast, overexpression of Sigmar1 locally alleviated the osteoporosis phenotype. Treatment with Sigmar1 agonists impaired both human and mice osteoclast formation in vitro. Mechanistically, SERCA2 was identified to interact with Sigmar1 based on the immunoprecipitation-mass spectrum (IP-MS) and co-immunoprecipitation (co-IP) assays, and Q615 of SERCA2 was confirmed to be the critical residue for their binding. Furthermore, Sigmar1 promoted SERCA2 degradation through Hrd1/Sel1L-dependent ER-associated degradation (ERAD). Ubiquitination of SERCA2 at K460 and K541 was responsible for its proteasomal degradation. Consequently, inhibition of SERCA2 impeded Sigmar1 deficiency enhanced osteoclastogenesis. Moreover, we found that dimemorfan, an FDA-approved Sigmar1 agonist, effectively rescued bone mass in various established bone-loss models. In conclusion, Sigmar1 is a negative regulator of osteoclastogenesis, and activation of Sigmar1 by dimemorfan may be a potential treatment for osteoporosis in clinical practice.
    Keywords:  ER-associated degradation; SERCA2; Sigma-1 receptor; dimemorfan; osteoporosis
  10. Cancers (Basel). 2022 May 15. pii: 2436. [Epub ahead of print]14(10):
      SQSTM1/p62 is a multitasking protein that functions as an autophagy receptor, but also as a signaling hub regulating diverse cellular pathways. p62 accumulation in mice with autophagy-deficient hepatocytes mediates liver damage and hepatocarcinogenesis through Nrf2 overactivation, yet the role of the p62-Keap1-Nrf2 axis in cell death and hepatocarcinogenesis in the absence of underlying autophagy defects is less clear. Here, we addressed the role of p62 and Nrf2 activation in a chronic liver disease model, namely mice with liver parenchymal cell-specific knockout of NEMO (NEMOLPC-KO), in which we demonstrate that they show no inherent autophagy impairment. Unexpectedly, systemic p62 ablation aggravated the phenotype and caused early postnatal lethality in NEMOLPC-KO mice. Expression of a p62 mutant (p62ΔEx2-5), which retains the ability to form aggregates and activate Nrf2 signaling, did not cause early lethality, but exacerbated hepatocarcinogenesis in these mice. Our immunohistological and molecular analyses showed that the increased tumor burden was only consistent with increased expression/stability of p62ΔEx2-5 driving Nrf2 hyperactivation, but not with other protumorigenic functions of p62, such as mTOR activation, cMYC upregulation or increased fibrosis. Surprisingly, forced activation of Nrf2 per se did not increase liver injury or tumor burden in NEMOLPC-KO mice, suggesting that autophagy impairment is a necessary prerequisite to unleash the Nrf2 oncogenic potential in mice with autophagy-competent hepatocytes.
    Keywords:  IκB kinase (IKK) complex; Keap1-Nrf2 activation; autophagy; chronic liver disease; genetic mouse models; hepatocarcinogenesis; liver injury
  11. J Cell Sci. 2022 May 27. pii: jcs.259811. [Epub ahead of print]
      Acetyl-CoA participates in post-translational modification of proteins, central carbon and lipid metabolism in several cell compartments. In mammals, the acetyl-CoA transporter 1 (AT1) facilitates the flux of cytosolic acetyl-CoA into the endoplasmic reticulum (ER), enabling the acetylation of proteins of the secretory pathway, in concert with dedicated acetyltransferases including NAT8. However, the implication of the ER acetyl-CoA pool in acetylation of ER-transiting proteins in Apicomplexa is unknown. We identify homologues of AT1 and NAT8 in Toxoplasma gondii and Plasmodium berghei. Proteome-wide analyses revealed widespread N-terminal acetylation marks of secreted proteins in both parasites. Such acetylation profile of N-terminally processed proteins was never observed so far in any other organisms. AT1 deletion resulted in a considerable reduction of parasite fitness. In P. berghei, AT1 is important for growth of asexual blood stages and production of female gametocytes and male gametocytogenesis impaling its requirement for transmission. In the absence of AT1, the lysine and N-terminal acetylation sites remained globally unaltered, suggesting an uncoupling between the role of AT1 in development and active acetylation occurring along the secretory pathway.
    Keywords:  Acetyl-CoA transporter; Acetylation; Endoplasmic Reticulum; Plasmodium berghei; Secretory pathway; Toxoplasma gondii
  12. Hepatol Commun. 2022 May 27.
      Alpha-1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full-length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R-like ER kinase and inositol-requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro-apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.
  13. Cell Rep. 2022 May 24. pii: S2211-1247(22)00628-3. [Epub ahead of print]39(8): 110855
      Hypusine is a post-translational modification on eukaryotic translation initiation factor 5A (eIF5A). The last step of hypusine biosynthesis, deoxyhypusine hydroxylation, is an oxygen-dependent reaction. Here we show that deletion of the deoxyhypusine hydroxylase Lia1 compromises yeast respiration through translation downregulation of selected proteins in the respiration pathway. The translation suppression, because of the lack of deoxyhypusine hydroxylation, mainly affects translation of the N termini of the proteins, independent of the presence of proline residues but likely dependent on the interaction between the N-terminal nascent peptide and the ribosomal peptide exit tunnel. Proteomics and biochemical studies reveal that Lia1 deletion decreases N-terminal translation of proteins involved in mitochondrial respiration, oxidative stress response, and protein folding. Our work uncovers functions of the hypusine modification by considering the substrate requirement of the post-translational modification, highlights the unique challenges of translating the N termini of proteins, and reveals an oxygen-sensing mechanism in eukaryotic cells.
    Keywords:  CP: Metabolism; CP: Molecular biology; deoxyhypusine hydroxylase; hypusine; oxidative phosphorylation; oxygen sensing; translation
  14. Mol Microbiol. 2022 May 26.
      The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterised a small orphan protein, Q9UZY3 (Uniprot ID) conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (PAN) as bait. X-ray crystallography and SAXS experiments revealed that the protein is monomeric and adopts a β-barrel core structure with an Oligonucleotide/oligosaccharide-Binding (OB) fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays tRNA binding properties. Pull-downs, co-immunoprecipitation and ITC studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi cellular extracts. The protein was therefore named Pbp11, for Proteasome Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.
    Keywords:  Archaea; OB-fold; Proteasome; protein-protein interaction; ribosome-associated quality control; tRNA binding
  15. J Cell Biol. 2022 Jul 04. pii: e202201071. [Epub ahead of print]221(7):
      The mitochondrial unfolded protein response (UPRmt) is dedicated to promoting mitochondrial proteostasis and is linked to extreme longevity. The key regulator of this process is the transcription factor ATFS-1, which, upon UPRmt activation, is excluded from the mitochondria and enters the nucleus to regulate UPRmt genes. However, the repair proteins synthesized as a direct result of UPRmt activation must be transported into damaged mitochondria that had previously excluded ATFS-1 owing to reduced import efficiency. To address this conundrum, we analyzed the role of the import machinery when the UPRmt was induced. Using in vitro and in vivo analysis of mitochondrial proteins, we surprisingly find that mitochondrial import increases when the UPRmt is activated in an ATFS-1-dependent manner, despite reduced mitochondrial membrane potential. The import machinery is upregulated, and an intact import machinery is essential for UPRmt-mediated lifespan extension. ATFS-1 has a weak mitochondrial targeting sequence (MTS), allowing for dynamic subcellular localization during the initial stages of UPRmt activation.
  16. Dev Cell. 2022 May 15. pii: S1534-5807(22)00306-9. [Epub ahead of print]
      The changes that drive differentiation facilitate the emergence of abnormal cells that need to be removed before they contribute to further development or the germline. Consequently, in mice in the lead-up to gastrulation, ∼35% of embryonic cells are eliminated. This elimination is caused by hypersensitivity to apoptosis, but how it is regulated is poorly understood. Here, we show that upon exit of naive pluripotency, mouse embryonic stem cells lower their mitochondrial apoptotic threshold, and this increases their sensitivity to cell death. We demonstrate that this enhanced apoptotic response is induced by a decrease in mitochondrial fission due to a reduction in the activity of dynamin-related protein 1 (DRP1). Furthermore, we show that in naive pluripotent cells, DRP1 prevents apoptosis by promoting mitophagy. In contrast, during differentiation, reduced mitophagy levels facilitate apoptosis. Together, these results indicate that during early mammalian development, DRP1 regulation of mitophagy determines the apoptotic response.
    Keywords:  apoptosis; early development; embryonic stem cell differentiation; mitochondrial dynamics; mitophagy; pluripotency
  17. Biochem J. 2022 May 27. 479(10): 1059-1082
      Control of protein synthesis (mRNA translation) plays key roles in shaping the proteome and in many physiological, including homeostatic, responses. One long-known translational control mechanism involves phosphorylation of initiation factor, eIF2, which is catalysed by any one of four protein kinases, which are generally activated in response to stresses. They form a key arm of the integrated stress response (ISR). Phosphorylated eIF2 inhibits eIF2B (the protein that promotes exchange of eIF2-bound GDP for GTP) and thus impairs general protein synthesis. However, this mechanism actually promotes translation of certain mRNAs by virtue of specific features they possess. Recent work has uncovered many previously unknown features of this regulatory system. Several studies have yielded crucial insights into the structure and control of eIF2, including that eIF2B is regulated by several metabolites. Recent studies also reveal that control of eIF2 and the ISR helps determine organismal lifespan and surprising roles in sensing mitochondrial stresses and in controlling the mammalian target of rapamycin (mTOR). The latter effect involves an unexpected role for one of the eIF2 kinases, HRI. Phosphoproteomic analysis identified new substrates for another eIF2 kinase, Gcn2, which senses the availability of amino acids. Several genetic disorders arise from mutations in genes for eIF2α kinases or eIF2B (i.e. vanishing white matter disease, VWM and microcephaly, epileptic seizures, microcephaly, hypogenitalism, diabetes and obesity, MEHMO). Furthermore, the eIF2-mediated ISR plays roles in cognitive decline associated with Alzheimer's disease. New findings suggest potential therapeutic value in interfering with the ISR in certain settings, including VWM, for example by using compounds that promote eIF2B activity.
    Keywords:  mRNA; phosphorylation/dephosphorylation; protein kinase; stress response; translation; translation factors
  18. iScience. 2022 Jun 17. 25(6): 104356
      Hsp70 is a key molecular chaperone in the protein quality control system to safeguard protein homeostasis in cells. Previous studies have shown that Hsp70 chaperones TDP-43, a pathogenic protein associated with amyotrophic lateral sclerosis (ALS), in nuclear bodies and prevents it from the pathological aggregation. In this work, we report that Hsp70 undergoes liquid-liquid phase separation, chaperones FUS, another ALS-linked pathogenic protein, in stress granules (SGs), and prevents condensed FUS from amyloid aggregation. Knock-down of Hsp70 does not influence SG assembly but results in the liquid-to-solid transition in SGs. NMR experiments further reveal Hsp70 predominantly uses its C-terminal substrate-binding domain to interact with the low complexity domain of FUS, which represents a mechanism distinct from that interacting with TDP-43. These findings suggest that Hsp70 is widely involved in chaperoning the physiological dynamics of various membrane-less organelles and adopts different mechanisms to prevent the pathological aggregation of different proteins.
    Keywords:  Biophysical chemistry; Biophysics; Cell biology; Organizational aspects of cell biology
  19. EMBO J. 2022 May 25. e110155
      Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses.
    Keywords:  dual leucine zipper kinase; endocytosis; mitogen-activated protein kinase; neurodegeneration; sphingomyelin
  20. bioRxiv. 2022 May 17. pii: 2022.05.17.492198. [Epub ahead of print]
      Major histocompatibility complex class I (MHC-I) molecules, which are dimers of a glycosylated polymorphic transmembrane heavy chain and the small protein β 2 -microglobulin (β 2 m), bind peptides in the endoplasmic reticulum that are generated by the cytosolic turnover of cellular proteins. In virus-infected cells these peptides may include those derived from viral proteins. Peptide-MHC-I complexes then traffic through the secretory pathway and are displayed at the cell surface where those containing viral peptides can be detected by CD8 + T lymphocytes that kill infected cells. Many viruses enhance their in vivo survival by encoding genes that downregulate MHC-I expression to avoid CD8 + T cell recognition. Here we report that two accessory proteins encoded by SARS-CoV-2, the causative agent of the ongoing COVID-19 pandemic, downregulate MHC-I expression using distinct mechanisms. One, ORF3a, a viroporin, reduces global trafficking of proteins, including MHC-I, through the secretory pathway. The second, ORF7a, interacts specifically with the MHC-I heavy chain, acting as a molecular mimic of β 2 m to inhibit its association. This slows the exit of properly assembled MHC-I molecules from the endoplasmic reticulum. We demonstrate that ORF7a reduces antigen presentation by the human MHC-I allele HLA-A*02:01. Thus, both ORF3a and ORF7a act post-translationally in the secretory pathway to lower surface MHC-I expression, with ORF7a exhibiting a novel and specific mechanism that allows immune evasion by SARS-CoV-2.Significance Statement: Viruses may down-regulate MHC class I expression on infected cells to avoid elimination by cytotoxic T cells. We report that the accessory proteins ORF7a and ORF3a of SARS-CoV-2 mediate this function and delineate the two distinct mechanisms involved. While ORF3a inhibits global protein trafficking to the cell surface, ORF7a acts specifically on MHC-I by competing with β 2 m for binding to the MHC-I heavy chain. This is the first account of molecular mimicry of β 2 m as a viral mechanism of MHC-I down-regulation to facilitate immune evasion.
  21. EMBO J. 2022 May 27. e110352
      Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.
    Keywords:  autophagy; effectors; immunity; ubiquitination; xenophagy
  22. Adv Sci (Weinh). 2022 May 26. e2105469
      Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA-like ER kinase (PERK) and inositol-required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF-κB mediated senescence-associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP-homologous protein (CHOP), while IRE1α-induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α-XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.
    Keywords:  AZD1775; UPR; WEE1; mutant TP53; ovarian cancer
  23. Commun Biol. 2022 May 26. 5(1): 505
      Due to the inherent toxicity of protein aggregates, the propensity of natural, functional amyloidogenic proteins to aggregate must be tightly controlled to avoid negative consequences on cellular viability. The importance of controlled aggregation in biological processes is illustrated by spidroins, which are functional amyloidogenic proteins that form the basis for spider silk. Premature aggregation of spidroins is prevented by the N-terminal NT domain. Here we explored the potential of the engineered, spidroin-based NT* domain in preventing protein aggregation in the intracellular environment of human cells. We show that the NT* domain increases the soluble pool of a reporter protein carrying a ligand-regulatable aggregation domain. Interestingly, the NT* domain prevents the formation of aggregates independent of its position in the aggregation-prone protein. The ability of the NT* domain to inhibit ligand-regulated aggregation was evident both in the cytosolic and nuclear compartments, which are both highly relevant for human disorders linked to non-physiological protein aggregation. We conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins.