bims-proteo Biomed News
on Proteostasis
Issue of 2020‒10‒18
thirty-four papers selected by
Eric Chevet
INSERM


  1. J Neurochem. 2020 Oct 16.
      Endoplasmic reticulum (ER) stress occurs when protein folding or maturation is disrupted. A malfunction in the ER stress response can lead to cell death and has been observed in many neurological diseases. However, how the ER stress response is regulated in neuronal cells remains largely unclear. Here, we studied an E3 ubiquitin ligase named neural precursor cell expressed developmentally downregulated protein 4-like (Nedd4-2). Nedd4-2 is highly expressed in the brain and has a high affinity toward ubiquitinating membrane-bound proteins. We first utilized unbiased proteomic profiling with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of isolated membrane fractions from mouse whole brains to identify novel targets of Nedd4-2. Through this screen, we found that the expression and ubiquitination of ribosomal proteins are regulated by Nedd4-2 and we confirmed an association between Nedd4-2 and ribosomes through ribosome sedimentation and polysome profiling. Further, we utilized immunoprecipitation and western blotting to show that induction of ER stress promotes an association between Nedd4-2 and ribosomal proteins, which is mediated through dephosphorylation of Nedd4-2 at serine-342. This increased interaction between Nedd4-2 and ribosomal proteins in turn mediates ER stress-associated translational suppression. In summary, the results of this study demonstrate a novel regulatory mechanism underlying the ER stress response and a novel function of Nedd4-2 in translational control. Our findings may shed light on neurological diseases in which the ER stress response or the function of Nedd4-2 is dysregulated.
    Keywords:  Nedd4-2; ribosome; stress; translation; ubiquitination
    DOI:  https://doi.org/10.1111/jnc.15219
  2. Biomol Ther (Seoul). 2020 Oct 13.
      Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitinproteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.
    Keywords:  Apoptosis; CRBN; Endoplasmic reticulum stress; Ubiquitination; c-Jun; p62
    DOI:  https://doi.org/10.4062/biomolther.2020.089
  3. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Oct 10. pii: S1388-1981(20)30229-8. [Epub ahead of print] 158837
      MARCHF6 is a large multi-pass E3 ubiquitin ligase embedded in the membranes of the endoplasmic reticulum. It participates in endoplasmic reticulum associated degradation, including autoubiquitination, and many of its identified substrates are involved in sterol and lipid metabolism. Post-translationally, MARCHF6 expression is attuned to cholesterol status, with high cholesterol preventing its degradation and hence boosting MARCHF6 levels. By modulating MARCHF6 activity, cholesterol may regulate other aspects of cell metabolism beyond the known repertoire. Whilst we have learnt much about MARCHF6 in the past decade, there are still many more mysteries to be unravelled to fully understand its regulation, substrates, and role in human health and disease.
    Keywords:  E3 ubiquitin ligase; MARCHF6; cholesterol; endoplasmic reticulum associated degradation (ERAD); protein degradation
    DOI:  https://doi.org/10.1016/j.bbalip.2020.158837
  4. Cell Rep. 2020 Oct 13. pii: S2211-1247(20)31251-1. [Epub ahead of print]33(2): 108262
      Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that 4EHP-GIGYF1/2 complexes trigger co-translational mRNA decay. Human cells lacking these proteins accumulate mRNAs with prominent ribosome pausing. They include, among others, transcripts encoding secretory and membrane-bound proteins or tubulin subunits. In addition, 4EHP-GIGYF1/2 complexes fail to reduce mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the cap structure, DDX6, and ZNF598. We further find that co-translational binding of GIGYF1/2 to the mRNA marks transcripts with perturbed elongation to decay. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.
    Keywords:  DDX6; GYF domain; endoplasmic reticulum; mRNA decay; nascent chain; ribosome pausing; signal peptide; translation; tubulin
    DOI:  https://doi.org/10.1016/j.celrep.2020.108262
  5. Neurobiol Dis. 2020 Oct 10. pii: S0969-9961(20)30410-1. [Epub ahead of print]146 105135
      Dystonia 16 (DYT16) is caused by mutations in PACT, the protein activator of interferon-induced double-stranded RNA-activated protein kinase (PKR). PKR regulates the integrated stress response (ISR) via phosphorylation of the translation initiation factor eIF2α. This post-translational modification attenuates general protein synthesis while concomitantly triggering enhanced translation of a few specific transcripts leading either to recovery and homeostasis or cellular apoptosis depending on the intensity and duration of stress signals. PKR plays a regulatory role in determining the cellular response to viral infections, oxidative stress, endoplasmic reticulum (ER) stress, and growth factor deprivation. In the absence of stress, both PACT and PKR are bound by their inhibitor transactivation RNA-binding protein (TRBP) thereby keeping PKR inactive. Under conditions of cellular stress these inhibitory interactions dissociate facilitating PACT-PACT interactions critical for PKR activation. While both PACT-TRBP and PKR-TRBP interactions are pro-survival, PACT-PACT and PACT-PKR interactions are pro-apoptotic. In this study we evaluate if five DYT16 substitution mutations alter PKR activation and ISR. Our results indicate that the mutant DYT16 proteins show stronger PACT-PACT interactions and enhanced PKR activation. In DYT16 patient derived lymphoblasts the enhanced PACT-PKR interactions and heightened PKR activation leads to a dysregulation of ISR and increased apoptosis. More importantly, this enhanced sensitivity to ER stress can be rescued by luteolin, which disrupts PACT-PKR interactions. Our results not only demonstrate the impact of DYT16 mutations on regulation of ISR and DYT16 etiology but indicate that therapeutic interventions could be possible after a further evaluation of such strategies.
    Keywords:  DYT16; Dystonia 16; ISR; PACT; PKR; Prkra; eIF2α
    DOI:  https://doi.org/10.1016/j.nbd.2020.105135
  6. Cancer Res. 2020 Oct 16. pii: canres.1871.2020. [Epub ahead of print]
      DZIP3/hRUL138 is a poorly characterized RNA binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G1 arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3'UTR of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G1 phase of the cell cycle where it is needed for cell cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 employs a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell cycle and cancer progression.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1871
  7. Biol Chem. 2020 Oct 01. pii: /j/bchm.just-accepted/hsz-2020-0286/hsz-2020-0286.xml. [Epub ahead of print]
      Maintenance of cellular redox control is pivotal for normal cellular functions and cell fate decisions including cell death. Among the key cellular redox systems in mammals, the glutathione peroxidase (GPX) family of proteins is the largest conferring multifaceted functions and affecting virtually all cellular processes. The endoplasmic reticulum (ER)- resident glutathione peroxidases, designated as GPX7 and GPX8, are the most recently added members of this family of enzymes. Recent studies have provided exciting insights how both enzymes support critical processes of the ER including oxidative protein folding, maintenance of ER redox control, by eliminating H2O2, and preventing palmitic acid-induced lipotoxicity. Consequently, numerous pathological conditions, such as neurodegeneration, cancer and metabolic diseases have been linked with altered GPX7 and GPX8 expression. Studies in mice have demonstrated that loss of GPX7 leads to increased differentiation of preadipocytes, increased tumorigenesis and shortened lifespan. By contrast, GPX8 deficiency in mice results in enhanced caspase-4/11 activation and increased endotoxic shock in colitis model. With the increasing recognition that both types of enzymes are dysregulated in various tumor entities in man, we deem a review of the emerging roles played by GPX7 and GPX8 in health and disease development timely and appropriate.
    Keywords:  Ca2+ signaling; ER stress; GPX; oxidative protein folding; oxidative stress
    DOI:  https://doi.org/10.1515/hsz-2020-0286
  8. Biology (Basel). 2020 Oct 12. pii: E332. [Epub ahead of print]9(10):
      Endoplasmic reticulum (ER) stress and its adaptive cellular response, the unfolded protein response (UPR), are involved in various diseases including neurodegenerative diseases, metabolic diseases, and even cancers. Here, we analyzed the novel function of ubiquitin-specific peptidase 14 (USP14) in ER stress. The overexpression of Drosophila USP14 protected the cells from ER stress without affecting the proteasomal activity. Null Hong Kong (NHK) and alpha-1-antitrypsin Z (ATZ) are ER-associated degradation substrates. The degradation of NHK, but not of ATZ, was delayed by USP14. USP14 restored the levels of rhodopsin-1 protein in a Drosophila model for autosomal dominant retinitis pigmentosa and suppressed the retinal degeneration in this model. In addition, we observed that proteasome complex is dynamically reorganized in response to ER stress in human 293T cells. These findings suggest that USP14 may be a therapeutic strategy in diseases associated with ER stress.
    Keywords:  Drosophila; ER stress; USP14; retinal degeneration
    DOI:  https://doi.org/10.3390/biology9100332
  9. Autophagy. 2020 Oct 10. 1-2
      Reticulophagy (or ER-phagy) is a type of selective autophagy that targets the endoplasmic reticulum (ER). In the process of reticulophagy, part of the ER is fragmented and packed within autophagosomes. However, the underlying mechanism that induces this local remodeling of ER subdomains was poorly understood. Our recent study showed that in the budding yeast Saccharomyces cerevisiae the reticulophagy receptor Atg40 plays an important role in ER remodeling beyond its role as a tether between the ER and the phagophore [1]. Atg40 has an ability to generate positive membrane curvature through the reticulon-like domain and locally forms a super assemblage though its binding to Atg8 at ER-phagophore contacts. These Atg40 assemblages cause folding of the ER subdomains to allow them to be efficiently packed into autophagosomes. Furthermore, our structural analysis identified an evolutionarily conserved short helix that assists strong Atg8-binding of reticulophagy receptors.
    Keywords:  Atg40; Atg8; endoplasmic reticulum; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1831801
  10. Front Cell Dev Biol. 2020 ;8 855
      Plasmalogens are a subclass of ether glycerophospholipids characterized by a vinyl-ether bond at the sn-1 position of the glycerol backbone. Plasmalogen biosynthesis is initiated in peroxisomes. At the third step of plasmalogen synthesis, alkyl-dihydroxyacetonephosphate (DHAP) is enzymatically reduced to 1-alkyl-sn-glycero-3-phospate by acyl/alkyl DHAP reductase (ADHAPR), whose activity is found in both peroxisomes and microsomes. We herein show that knockdown of ADHAPR in HeLa cells reduced the synthesis of ethanolamine plasmalogen (PlsEtn), similar to the Chinese hamster ovary cell mutant FAA.K1B deficient in ADHAPR activity. Endogenous ADHAPR and ectopically expressed FLAG-tagged ADHAPR were localized to peroxisomes and endoplasmic reticulum (ER) as a type I integral membrane protein in HeLa cells. ADHAPR targets to peroxisomes via a Pex19p-dependent class I pathway. In addition, it is also inserted into the ER via the SRP-dependent mechanism. The ADHAPR mutant lacking the N-terminal domain preferentially targets to the ER, restoring the reduced level of PlsEtn synthesis in FAA.K1B cell. In contrast, the expression of full-length ADHAPR in the mutant cells elevates the synthesis of phosphatidylethanolamine, but not PlsEtn. Taken together, these results suggest that the third step of plasmalogen synthesis is mediated by ER-localized ADHAPR.
    Keywords:  acyl/alkyl dihydroxyacetonephosphate reductase; endoplasmic reticulum; organelle targeting; peroxisome; plasmalogen
    DOI:  https://doi.org/10.3389/fcell.2020.00855
  11. J Biol Chem. 2020 10 13. pii: jbc.REV120.010723. [Epub ahead of print]
      The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.
    Keywords:  E3 ubiquitin ligase; cholesterol; cholesterol metabolism; cholesterol regulation; phosphorylation; post-transcriptional regulation; post-translational modification (PTM); protein degradation; ubiquitin ligase; ubiquitylation (ubiquitination)
    DOI:  https://doi.org/10.1074/jbc.REV120.010723
  12. Mol Cell. 2020 Oct 02. pii: S1097-2765(20)30651-1. [Epub ahead of print]
      Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.
    Keywords:  RNA-binding sites; UV crosslinking; mass spectrometry; protein-RNA interaction; stress responses; yeast
    DOI:  https://doi.org/10.1016/j.molcel.2020.09.021
  13. EMBO Rep. 2020 Oct 14. e50642
      The tumor suppressor Merlin/NF2, a key activator of the Hippo pathway in growth control, is regulated by phosphorylation. However, it is uncertain whether additional post-translational modifications regulate Merlin. Here, we show that ubiquitination is required to activate Merlin in the Hippo pathway. Ubiquitinated Merlin is mostly conjugated by one or two ubiquitin molecules. Such modification is promoted by serine 518 dephosphorylation in response to Ca2+ signaling or cell detachment. Merlin ubiquitination is mediated by the E3 ubiquitin ligase, NEDD4L, which requires a scaffold protein, AMOTL1, to approach Merlin. Several NF2-patient-derived Merlin mutations disrupt its binding to AMOTL1 and its regulation by the AMOTL1-NEDD4L apparatus. Lysine (K) 396 is the major ubiquitin conjugation residue. Disruption of Merlin ubiquitination by the K396R mutation or NEDD4L depletion diminishes its binding to Lats1 and inhibits Lats1 activation. These effects are also accompanied by loss of Merlin's anti-mitogenic and tumor suppressive properties. Thus, we propose that dephosphorylation and ubiquitination compose an intramolecular relay to activate Merlin functions in activating the Hippo pathway during growth control.
    Keywords:  AMOTL1; Hippo pathway; Merlin; NEDD4L; ubiquitination
    DOI:  https://doi.org/10.15252/embr.202050642
  14. Biomedicines. 2020 Oct 12. pii: E409. [Epub ahead of print]8(10):
      Upon increased demand for protein synthesis, accumulation of misfolded and/or unfolded proteins within the endoplasmic reticulum (ER), a pro-survival response is activated termed unfolded protein response (UPR), aiming at restoring the proper function of the ER. Prolonged activation of the UPR leads, however, to ER stress, a cellular state that contributes to the pathogenesis of various chronic diseases including obesity and diabetes. ER stress response by itself can result in endothelial dysfunction, a hallmark of cardiovascular disease, through various cellular mechanisms including apoptosis, insulin resistance, inflammation and oxidative stress. Extracellular vesicles (EVs), particularly large EVs (lEVs) commonly referred to as microparticles (MPs), are membrane vesicles. They are considered as a fingerprint of their originating cells, carrying a variety of molecular components of their parent cells. lEVs are emerging as major contributors to endothelial cell dysfunction in various metabolic disease conditions. However, the mechanisms underpinning the role of lEVs in endothelial dysfunction are not fully elucidated. Recently, ER stress emerged as a bridging molecular link between lEVs and endothelial cell dysfunction. Therefore, in the current review, we summarized the roles of lEVs and ER stress in endothelial dysfunction and discussed the molecular crosstalk and relationship between ER stress and lEVs in endothelial dysfunction.
    Keywords:  cardiovascular disease; diabetes; endoplasmic reticulum (ER) stress; endothelial dysfunction; extracellular vesicles (EVs); metabolic syndrome; microparticles (MPs)
    DOI:  https://doi.org/10.3390/biomedicines8100409
  15. J Cell Biochem. 2020 Oct 14.
      The Skp1-Cul1-F-box protein (SCF) E3 ligase complex is one of the largest ubiquitin E3 ligase families. FBXL19, a F-box protein in SCFFBXL19 E3 ligase complex, regulates a variety of cellular responses including cell migration. We have shown that FBXL19 is not stable and its degradation is mediated by the ubiquitin-proteasome system, while the ubiquitin E3 ligase for FBXL19 ubiquitination and degradation has not been identified. In the study, we discovered that a new ubiquitin E3 ligase, SCFFBXW17 , ubiquitinates and induces FBXL19 degradation. Exogenous FBXW17 targets FBXL19 for its ubiquitination and degradation. Lysine 114 in FBXL19 is a potential ubiquitin acceptor site. Acetylation of FBXL19 attenuated SCFFBXW17 -mediated FBXL19 degradation. SCFFBXL19 E3 ligase reduced Rac1 levels and cell migration, while the effects were attenuated by exogenous FBXW17. Downregulation of FBXW17 attenuated lysophosphatidic acid-induced lamellipodia formation and Rac1 accumulation at migration leading edge. Taken together with our previous studies, FBXL19 is degraded by the ubiquitin-proteasome system and its site-specific ubiquitination is mediated by SCFFBXW17 E3 ligase, which promotes cell migration.
    Keywords:  F-box protein; SCF E3 ligase; cell migration; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1002/jcb.29860
  16. J Cell Sci. 2020 Oct 16. pii: jcs.249045. [Epub ahead of print]
      Cytoskeleton-associated protein 4 (CKAP4) is palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Knockout (KO) of CKAP4 in HeLaS3 cells induced the alterations of mitochondrial structures and increased the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding of CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased, and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions, and in vivo xenograft tumor formation. The phenotypes were not rescued by a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.
    Keywords:  CKAP4; ER; MAM; Mitochondria; Palmitoylation; VDAC2
    DOI:  https://doi.org/10.1242/jcs.249045
  17. J Neurosci. 2020 Oct 12. pii: JN-RM-0940-20. [Epub ahead of print]
      Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Over-expression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition upon mTORC1 repression may be neuroprotective; however, whether 4E-BP1 over-expression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1 over-expressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1 over-expressing primary neurons. After documenting that 4E-BP1 over-expressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins, rotenone, maneb, or paraquat, and documented significant protection in neurons from newborn male and female 4E-BP1-OE transgenic mice. We observed 4E-BP1-dependent up-regulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 over-expression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested if 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1 over-expressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD.Significance Statement:In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated if neuronal over-expression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 over-expression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of alpha-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.
    DOI:  https://doi.org/10.1523/JNEUROSCI.0940-20.2020
  18. Protein Sci. 2020 Oct 11.
      The heat shock protein (Hsp) Hsp90 is one of the most abundant proteins in the cell. It controls the functional turnover of proteins being involved in protein folding, refolding, transport as well as protein degradation. Co-chaperones influence Hsp90's activity in different ways, among which the Hsp organizing protein (Hop) was found to inhibit its ATP hydrolysis upon binding. Despite the availability of a number of studies investigating the Hsp90:Hop complex, several aspects of the Hsp90:Hop interaction have remained unresolved. Here we employed a combinatory approach comprising native PAGE, ITC, MALS, SAXS, DLS and NMR spectroscopy to obtain a comprehensive picture about the human Hsp90β:Hop association in solution. Our data show that only one Hop molecule binds the Hsp90β dimer, Hop can interact with the open and closed state of Hsp90β, and Hop's TPR2A-2B domains determine the affinity for Hsp90's C-terminal and middle domain, whereby the interaction with the C-terminal domain of Hsp90β is sufficient to induce an allosteric conformational change between the two Hsp90β monomers in the Hsp902 :Hop1 complex. Together, this study highlights the important role of the co-chaperone Hop in reorganizing Hsp90 for efficient client loading. This article is protected by copyright. All rights reserved.
    Keywords:  Hop ; Hsp90 ; TPR domain; co-chaperone
    DOI:  https://doi.org/10.1002/pro.3969
  19. Am J Hum Genet. 2020 Oct 09. pii: S0002-9297(20)30329-3. [Epub ahead of print]
      Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.
    Keywords:  HSP47; KDELR2; osteogenesis imperfecta; retrograde Golgi-ER transport
    DOI:  https://doi.org/10.1016/j.ajhg.2020.09.009
  20. Mech Ageing Dev. 2020 Oct 09. pii: S0047-6374(20)30177-9. [Epub ahead of print] 111381
      Saccharomyces cerevisiae MED2/YDL005C is a subunit of the mediator complex (Mediator), which is responsible for tightly controlling the transcription of protein-coding genes by mediating the interaction of RNA polymerase II with gene-specific transcription factors. Although a high-throughput analysis in yeast showed that the MED2 protein exhibits altered cellular localization under hypoxic stress, no specific function of MED2 has been described to date. In this study, we first provided evidence that MED2 is involved in the endoplasmic reticulum (ER) stress response and modulation of the replicative life span. We showed that deletion of MED2 leads to sensitivity to the ER stress inducer tunicamycin (TM) as well as a shortened replicative lifespan (RLS), accompanied by increased intracellular ROS levels and hyperpolarization of mitochondria. On the other hand, overexpression of MED2 in wild-type (WT) yeast enhanced TM resistance and extended the RLS. In addition, the IRE1-HAC1 pathway was essential for the TM resistance of MED2-overexpressing cells. Moreover, we showed that MED2 deficiency enhances ER unfolded protein response (UPR) activity compared to that in WT cells. Collectively, these results suggest the novel role of MED2 as a regulator in maintaining ER homeostasis and longevity.
    Keywords:  MED2; endoplasmic reticulum; replicative lifespan
    DOI:  https://doi.org/10.1016/j.mad.2020.111381
  21. Nanotechnology. 2020 Oct 15. 32(2): 025708
      During neuronal development, neuronal cells read extracellular stimuli from the micro/nano-environment within which they exist, retrieving essential directionality and wiring information. Here, focal adhesions (FAs-protein clusters anchoring integrins to cytoskeleton) act as sensors, by integrating signals from both the extracellular matrix environment and chemotactic factors, contributing to the final neuronal pathfinding and migration. In the processes that orchestrate neuronal development, the important function of ubiquitin E3A ligase (UBE3A) is emerging. UBE3A has crucial functions in the brain and changes in its expression levels lead to neurodevelopmental disorders: the lack of UBE3A leads to Angelman syndrome (AS, OMIN 105830), while its increase causes autisms (Dup15q-autism). By using nano/micro-structured anisotropic substrates we previously showed that UBE3A-deficient neurons have deficits in contact guidance (Tonazzini et al, Mol Autism 2019). Here, we investigate the adhesion and migration dynamics of UBE3A-silenced SH-SY5Y neuroblastoma cells in vitro by exploiting nano/micro-grooved substrates. We analyze the molecular processes regulating the development of FAs by transfection with EGFP-vector encoding for paxillin, a protein of FA clusters, and by live-cell total-internal-reflection-fluorescence microscopy. We show that UBE3A-silenced SH-SY5Y cells have impaired FA morphological development and pathway activation, which lead to a delayed adhesion and also explain the defective contact guidance in response to directional topographical stimuli. However, UBE3A-silenced SH-SY5Y cells show an overall normal migration behavior, in terms of speed and ability to follow the GRs directional stimulus. Only the collective cell migration upon cell gaps was slightly delayed for UBE3Ash SHs. Overall, the deficits of UBE3Ash SHS-SY5Y cells in FA maturation/sensing and in collective migration may have patho-physiological implications, in AS condition, considering the much more complex stimuli that neurons find in vivo during the neurodevelopment.
    DOI:  https://doi.org/10.1088/1361-6528/abbb03
  22. J Cell Biol. 2020 Nov 02. pii: e202006178. [Epub ahead of print]219(11):
      The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.
    DOI:  https://doi.org/10.1083/jcb.202006178
  23. Dev Cell. 2020 Oct 12. pii: S1534-5807(20)30705-X. [Epub ahead of print]55(1): 97-107
      In the last decade, liquid-liquid phase separation has emerged as a fundamental principle in the organization of crowded cellular environments into functionally distinct membraneless compartments. It is now established that biomolecules can condense into various physical phases, traditionally defined for simple polymer systems, and more recently elucidated by techniques employed in life sciences. We review pioneering cryo-electron tomography studies that have begun to unravel a wide spectrum of molecular architectures, ranging from amorphous to crystalline assemblies, that underlie cellular condensates. These observations bring into question current interpretations of microscopic phase behavior. Furthermore, by examining emerging concepts of non-classical phase separation pathways in small-molecule crystallization, we draw parallels with biomolecular condensation that highlight aspects not yet fully explored. In particular, transient and metastable intermediates that might be challenging to capture experimentally inside cells could be probed through computational simulations and enable a multi-scale understanding of the subcellular organization governed by distinct phases.
    DOI:  https://doi.org/10.1016/j.devcel.2020.09.003
  24. Nat Commun. 2020 Oct 16. 11(1): 5226
      Signs of proteostasis failure often entwine with those of metabolic stress at the cellular level. Here, we study protein sequestration during glucose deprivation-induced ATP decline in Saccharomyces cerevisiae. Using live-cell imaging, we find that sequestration of misfolded proteins and nascent polypeptides into two distinct compartments, stress granules, and Q-bodies, is triggered by the exhaustion of ATP. Both compartments readily dissolve in a PKA-dependent manner within minutes of glucose reintroduction and ATP level restoration. We identify the ATP hydrolase activity of Hsp104 disaggregase as the critical ATP-consuming process determining compartments abundance and size, even in optimal conditions. Sequestration of proteins into distinct compartments during acute metabolic stress and their retrieval during the recovery phase provide a competitive fitness advantage, likely promoting cell survival during stress.
    DOI:  https://doi.org/10.1038/s41467-020-19104-1
  25. Proc Natl Acad Sci U S A. 2020 Oct 14. pii: 202005389. [Epub ahead of print]
      The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.
    Keywords:  ATG16L1; Chlamydia trachomatis; autophagy; host-pathogen interactions; intracellular traffic
    DOI:  https://doi.org/10.1073/pnas.2005389117
  26. J Mol Biol. 2020 Oct 12. pii: S0022-2836(20)30576-3. [Epub ahead of print]
      The AAA-ATPase VCP/p97 cooperates with the SEP-domain adapters p37, UBXN2A and p47 in stripping inhibitor-3 (I3) from protein phosphatase-1 (PP1) for activation. In contrast to p97-mediated degradative processes, PP1 complex disassembly is ubiquitin-independent. It is therefore unclear how selective targeting is achieved. Using biochemical reconstitution and crosslink mass spectrometry, we show here that SEP-domain adapters use a multivalent substrate recognition strategy. An N-terminal sequence element predicted to form a helix, together with the SEP-domain, binds and engages the direct target I3 in the central pore of p97 for unfolding, while its partner PP1 is held by a linker between SHP box and UBX domain locked onto the peripheral N-domain of p97. Although the I3-binding element is functional in p47, p47 in vitro requires a transplant of the PP1-binding linker from p37 for activity stressing that both sites are essential to control specificity. Of note, unfolding is then governed by an inhibitory segment in the N-terminal region of p47, suggesting a regulatory function. Together, this study reveals how p97 adapters engage a protein complex for ubiquitin-independent disassembly while ensuring selectivity for one subunit.
    Keywords:  AAA+ ATPases; chaperone; photo-crosslinking mass spectrometry; protein phosphatase-1; protein unfolding
    DOI:  https://doi.org/10.1016/j.jmb.2020.10.001
  27. FEBS J. 2020 Oct 15.
      Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70 % of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 µm2 /s and 0.7 µm2 /s. Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.
    Keywords:  fluorescence microscopy; membrane biophysics; protein folding; protein secretion; protein:lipid interactions; single-molecule analysis
    DOI:  https://doi.org/10.1111/febs.15596
  28. Cell Cycle. 2020 Oct 12. 1-12
      RFWD3 is an E3 ubiquitin ligase that plays important roles in DNA damage response and DNA replication. We have previously demonstrated that the stabilization of RFWD3 by PCNA at the replication fork enables ubiquitination of the single-stranded binding protein, RPA and its subsequent degradation for replication progression. Here, we report that RFWD3 associates with the Origin Recognition Complex (ORC) and ORC-Associated (ORCA/LRWD1), components of the pre-replicative complex required for the initiation of DNA replication. Overexpression of ORC/ORCA leads to the stabilization of RFWD3. Interestingly, RFWD3 seems to stabilize ORC/ORCA in cells expressing wild type p53, as the depletion of RFWD3 reduces the levels of ORC/ORCA. Further, the catalytic activity of RFWD3 is required for the stabilization of ORC. Our results indicate that the RFWD3 promotes the stability of ORC, enabling efficient pre-RC assembly.
    Keywords:  ORC; ORCA/LRWD1; RFWD3; p53; replication; ubiquitination
    DOI:  https://doi.org/10.1080/15384101.2020.1829823
  29. Trends Cancer. 2020 Oct 13. pii: S2405-8033(20)30261-2. [Epub ahead of print]
      Tumor progression is associated with dedifferentiated histopathologies concomitant with cancer cell survival within a changing, and often hostile, tumor microenvironment. These processes are enabled by cellular plasticity, whereby intracellular cues and extracellular signals are integrated to enable rapid shifts in cancer cell phenotypes. Cancer cell plasticity, at least in part, fuels tumor heterogeneity and facilitates metastasis and drug resistance. Protein synthesis is frequently dysregulated in cancer, and emerging data suggest that translational reprograming collaborates with epigenetic and metabolic programs to effectuate phenotypic plasticity of neoplasia. Herein, we discuss the potential role of mRNA translation in cancer cell plasticity, highlight emerging histopathological correlates, and deliberate on how this is related to efforts to improve understanding of the complex tumor ecology.
    Keywords:  cancer plasticity; mRNA translation; metabolism; protein synthesis; stromal–epithelial interactions; therapy resistance; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.trecan.2020.09.005
  30. J Cell Biol. 2020 Dec 07. pii: e202001165. [Epub ahead of print]219(12):
      The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.
    DOI:  https://doi.org/10.1083/jcb.202001165
  31. Aging (Albany NY). 2020 Oct 13. 12
      The dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of most cellular proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered potential therapeutic targets for many cancers. However, the role of deubiquitinase ubiquitin-specific protease 18 (USP18) in pancreatic cancer remains unknown. Here, we found that the deubiquitinase ubiquitin-specific protease 18 (USP18) is significantly upregulated in pancreatic cancer and is correlated with a shorter median overall and relapse-free survival. A functional assay demonstrated that overexpression of USP18 resulted in increased proliferation of pancreatic cancer cells. Conversely, these phenomena were reversed after USP18 was silenced in pancreatic cancer cells. Further investigation revealed that USP18 promoted cell progression by increasing c-Myc expression, which has been reported to control pancreatic cancer progression, and our data demonstrated that c-Myc is key for USP18-mediated pancreatic cancer cell progression in vitro and in vivo. Moreover, we found that USP18 promoted pancreatic cancer progression via upregulation of Notch-1-dependent c-Myc. Mechanistically, USP18 interacts with and removes K48-linked ubiquitin chains from Notch1, thereby stabilizing Notch1 and promoting the Notch1-c-Myc pathway. Our work identifies and validates USP18 as a pancreatic cancer oncogene and provides a potential druggable target for this intractable disease.
    Keywords:  Notch1; USP18; c-Myc; pancreatic cancer; ubiquitination
    DOI:  https://doi.org/10.18632/aging.103760
  32. Cell Death Dis. 2020 Oct 13. 11(10): 847
      Osteoclasts are multinucleated giant cells with the ability to degrade bone tissue, and are closely related to abnormal bone metabolic diseases. Endoplasmic reticulum (ER) is an organelle responsible for protein modification, quality control, and transportation. The accumulation of unfolded or misfolded proteins in ER cavity induces ER stress. Double-stranded RNA-dependent protein kinase-like ER kinase (PERK) is an ER stress-sensing protein, which is ubiquitous in eukaryotic cells. Systemic PERK knockout mice show severe bone loss, suggesting that PERK is of great significance for maintaining the normal growth and development of bone tissue, but the role of PERK in osteoclastogenesis is still unclear. In this study, we found that PERK was significantly activated during RANKL-induced osteoclast differentiation; knockdown of PERK by siRNA and inhibition of PERK by GSK2606414, respectively, had significant negative regulatory effects on the formation and bone resorption of osteoclasts. PERK inhibitor GSK2606414 down-regulated the mRNA levels and protein expression of osteoclast differentiation marker genes, and inhibited RANKL-induced activation of Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways. Treatment with PERK inhibitor GSK2606414 in ovariectomized mouse model significantly suppressed bone loss and osteoclast formation. Thapsigargin activated ER stress to enhance autophagy, while GSK2606414 had a significant inhibitory effect on autophagy flux and autophagosome formation. Antioxidant N-acetylcysteine (NAC) could inhibit the expression of PERK phosphorylation, osteoclast-related proteins and autophagy-related proteins, but the use of PERK activator CCT020312 can reverse inhibition effect of NAC. Our findings demonstrate a key role for PERK in osteoclast differentiation and suggest its therapeutic potential.
    DOI:  https://doi.org/10.1038/s41419-020-03046-z