bims-proteo Biomed News
on Proteostasis
Issue of 2020‒08‒16
twenty-nine papers selected by
Eric Chevet
INSERM


  1. Mol Cell Proteomics. 2020 Aug 11. pii: mcp.RA120.002228. [Epub ahead of print]
    Hannigan MM, Hoffman AM, Thompson W, Zheng T, Nicchitta CV.
      Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.
    Keywords:  Affinity proteomics; Mass Spectrometry; Membranes*; Protein Synthesis*; RNA binding protein; Ribosomes*; endoplasmic reticulum; protein translocation; quantitative proteomics
    DOI:  https://doi.org/10.1074/mcp.RA120.002228
  2. Sci Signal. 2020 Aug 11. pii: eabb4749. [Epub ahead of print]13(644):
    Hughes DT, Halliday M, Smith HL, Verity NC, Molloy C, Radford H, Butcher AJ, Mallucci GR.
      Chronic activation of the unfolded protein response (UPR), notably the branch comprising the kinase PERK and the translation initiation factor eIF2α, is a pathological feature of many neurodegenerative diseases caused by protein misfolding. Partial reduction of UPR signaling at the level of phosphorylated eIF2α is neuroprotective and avoids the pancreatic toxicity caused by full inhibition of PERK kinase activity. However, other stress pathways besides the UPR converge on phosphorylated eIF2α in the integrated stress response (ISR), which is critical to normal cellular function. We explored whether partial inhibition of PERK signaling may be a better therapeutic option. PERK-mediated phosphorylation of eIF2α requires its binding to the insert loop within PERK's kinase domain, which is, itself, phosphorylated at multiple sites. We found that, as expected, Akt mediates the phosphorylation of Thr799 in PERK. This phosphorylation event reduced eIF2α binding to PERK and selectively attenuated downstream signaling independently of PERK activity and the broader ISR. Induction of Thr799 phosphorylation with a small-molecule activator of Akt similarly reduced PERK signaling and increased both neuronal and animal survival without measurable pancreatic toxicity in a mouse model of prion disease. Thus, promoting PERK phosphorylation at Thr799 to partially down-regulate PERK-eIF2α signaling while avoiding widespread ISR inhibition may be a safe therapeutic approach in neurodegenerative disease.
    DOI:  https://doi.org/10.1126/scisignal.abb4749
  3. Structure. 2020 Aug 05. pii: S0969-2126(20)30244-6. [Epub ahead of print]
    Chen X, Dorris Z, Shi D, Huang RK, Khant H, Fox T, de Val N, Williams D, Zhang P, Walters KJ.
      The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Its diverse substrates unify as proteasome targets by ubiquitination. We used cryogenic electron microscopy (cryo-EM) to study how human 26S proteasome interacts with M1-linked hexaubiquitin (M1-Ub6) unanchored to a substrate and E3 ubiquitin ligase E6AP/UBE3A. Proteasome structures are available with model substrates extending through the RP ATPase ring and substrate-conjugated K63-linked ubiquitin chains present at inhibited deubiquitinating enzyme hRpn11 and the nearby ATPase hRpt4/hRpt5 coiled coil. In this study, we find M1-Ub6 at the hRpn11 site despite the absence of conjugated substrate, indicating that ubiquitin binding at this location does not require substrate interaction with the RP. Moreover, unanchored M1-Ub6 binds to this hRpn11 site of the proteasome with the CP gating residues in both the closed and opened conformational states.
    Keywords:  E6AP; Rpn11; UBE3A; cryo-EM; deubiquitinating enzyme; linear ubiquitin chains; proteasome; protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.str.2020.07.011
  4. Nat Commun. 2020 Aug 13. 11(1): 4056
    Bosc C, Broin N, Fanjul M, Saland E, Farge T, Courdy C, Batut A, Masoud R, Larrue C, Skuli S, Espagnolle N, Pagès JC, Carrier A, Bost F, Bertrand-Michel J, Tamburini J, Récher C, Bertoli S, Mansat-De Mas V, Manenti S, Sarry JE, Joffre C.
      Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid β-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.
    DOI:  https://doi.org/10.1038/s41467-020-17882-2
  5. J Cell Sci. 2020 Aug 11. pii: jcs218107. [Epub ahead of print]133(15):
    Urra H, Pihán P, Hetz C.
      Different perturbations alter the function of the endoplasmic reticulum (ER), resulting in the accumulation of misfolded proteins in its lumen, a condition termed ER stress. To restore ER proteostasis, a highly conserved pathway is engaged, known as the unfolded protein response (UPR), triggering adaptive programs or apoptosis of terminally damaged cells. IRE1α (also known as ERN1), the most conserved UPR sensor, mediates the activation of responses to determine cell fate under ER stress. The complexity of IRE1α regulation and its signaling outputs is mediated in part by the assembly of a dynamic multi-protein complex, named the UPRosome, that regulates IRE1α activity and the crosstalk with other pathways. We discuss several studies identifying components of the UPRosome that have illuminated novel functions in cell death, autophagy, DNA damage, energy metabolism and cytoskeleton dynamics. Here, we provide a theoretical analysis to assess the biological significance of the UPRosome and present the results of a systematic bioinformatics analysis of the available IRE1α interactome data sets followed by functional enrichment clustering. This in silico approach decoded that IRE1α also interacts with proteins involved in the cell cycle, transport, differentiation, response to viral infection and immune response. Thus, defining the spectrum of IRE1α-binding partners will reveal novel signaling outputs and the relevance of the pathway to human diseases.
    Keywords:  Cell fate; ER stress; IRE1α; UPRosome
    DOI:  https://doi.org/10.1242/jcs.218107
  6. Int J Mol Sci. 2020 Aug 06. pii: E5657. [Epub ahead of print]21(16):
    He J, Cheng J, Wang T.
      Mitochondrial stress is considered as a factor that reprograms the mitochondrial biogenesis and metabolism. As known, SUMOylation occurs through a series of stress-induced biochemical reactions. During the process of SUMOylation, the small ubiquitin-like modifier (SUMO) and its specific proteases (SENPs) are key signal molecules. Furthermore, they are considered as novel mitochondrial stress sensors that respond to the signals produced by various stresses. The responses are critical for mitochondrial homeostasis. The scope of this review is to provide an overview of the function of SUMOylation in the mitochondrial stress response, to delineate a SUMOylation-involved signal network diagram, and to highlight a number of key questions that remain answered.
    Keywords:  SENPs; SUMO; SUMOylation; mitochondrion; stress response
    DOI:  https://doi.org/10.3390/ijms21165657
  7. Cells. 2020 Aug 08. pii: E1858. [Epub ahead of print]9(8):
    Jha S, Holmberg CI.
      The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.
    Keywords:  C. elegans; autophagy; crosstalk; tissue specificity; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/cells9081858
  8. EMBO Rep. 2020 Aug 11. e48260
    Xu R, Jones W, Wilcz-Villega E, Costa AS, Rajeeve V, Bentham RB, Bryson K, Nagano A, Yaman B, Olendo Barasa S, Wang Y, Chelala C, Cutillas P, Szabadkai G, Frezza C, Bianchi K.
      IκB kinase ε (IKKε) is a key molecule at the crossroads of inflammation and cancer. Known to regulate cytokine secretion via NFκB and IRF3, the kinase is also a breast cancer oncogene, overexpressed in a variety of tumours. However, to what extent IKKε remodels cellular metabolism is currently unknown. Here, we used metabolic tracer analysis to show that IKKε orchestrates a complex metabolic reprogramming that affects mitochondrial metabolism and consequently serine biosynthesis independently of its canonical signalling role. We found that IKKε upregulates the serine biosynthesis pathway (SBP) indirectly, by limiting glucose-derived pyruvate utilisation in the TCA cycle, inhibiting oxidative phosphorylation. Inhibition of mitochondrial function induces activating transcription factor 4 (ATF4), which in turn drives upregulation of the expression of SBP genes. Importantly, pharmacological reversal of the IKKε-induced metabolic phenotype reduces proliferation of breast cancer cells. Finally, we show that in a highly proliferative set of ER negative, basal breast tumours, IKKε and PSAT1 are both overexpressed, corroborating the link between IKKε and the SBP in the clinical context.
    Keywords:  ATF4; IKKε; breast cancer; mitochondrial metabolism; serine biosynthesis
    DOI:  https://doi.org/10.15252/embr.201948260
  9. Cell Rep. 2020 Aug 11. pii: S2211-1247(20)31010-X. [Epub ahead of print]32(6): 108025
    Kweon HJ, Gu S, Witham E, Dhara M, Yu H, Mandon ED, Jawhari A, Bredt DS.
      The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
    Keywords:  NACHO; assembly; chaperone; nAChR; nicotinic acetylcholine receptor α7; trafficking
    DOI:  https://doi.org/10.1016/j.celrep.2020.108025
  10. Antiviral Res. 2020 Aug 05. pii: S0166-3542(20)30295-3. [Epub ahead of print] 104881
    DeWald LE, Starr C, Butters T, Treston A, Warfield KL.
      N-linked glycosylation is the most common form of protein glycosylation and is required for the proper folding, trafficking, and/or receptor binding of some host and viral proteins. As viruses lack their own glycosylation machinery, they are dependent on the host's machinery for these processes. Certain iminosugars are known to interfere with the N-linked glycosylation pathway by targeting and inhibiting α-glucosidases I and II in the endoplasmic reticulum (ER). Perturbing ER α-glucosidase function can prevent these enzymes from removing terminal glucose residues on N-linked glycans, interrupting the interaction between viral glycoproteins and host chaperone proteins that is necessary for proper folding of the viral protein. Iminosugars have demonstrated broad-spectrum antiviral activity in vitro and in vivo against multiple viruses. This review discuses the broad activity of iminosugars against Flaviviridae. Iminosugars have shown favorable activity against multiple members of the Flaviviridae family in vitro and in murine models of disease, although the activity and mechanism of inhibition can be virus specific. While iminosugars are not currently approved for the treatment of viral infections, their potential use as future host-targeted antiviral (HTAV) therapies continues to be investigated.
    Keywords:  ER α-glucosidases; Flaviviridae; Iminosugars; antiviral therapy; flavivirus; glycosylation
    DOI:  https://doi.org/10.1016/j.antiviral.2020.104881
  11. Rev Physiol Biochem Pharmacol. 2020 Aug 14.
    Danese A, Marchi S, Vitto VAM, Modesti L, Leo S, Wieckowski MR, Giorgi C, Pinton P.
      Endoplasmic reticulum (ER)-mitochondria regions are specialized subdomains called also mitochondria-associated membranes (MAMs). MAMs allow regulation of lipid synthesis and represent hubs for ion and metabolite signaling. As these two organelles can module both the amplitude and the spatiotemporal patterns of calcium (Ca2+) signals, this particular interaction controls several Ca2+-dependent pathways well known for their contribution to tumorigenesis, such as metabolism, survival, sensitivity to cell death, and metastasis. Mitochondria-mediated apoptosis arises from mitochondrial Ca2+ overload, permeabilization of the mitochondrial outer membrane, and the release of mitochondrial apoptotic factors into the cytosol. Decreases in Ca2+ signaling at the ER-mitochondria interface are being studied in depth as failure of apoptotic-dependent cell death is one of the predominant characteristics of cancer cells. However, some recent papers that linked MAMs Ca2+ crosstalk-related upregulation to tumor onset and progression have aroused the interest of the scientific community.In this review, we will describe how different MAMs-localized proteins modulate the effectiveness of Ca2+-dependent apoptotic stimuli by causing both increases and decreases in the ER-mitochondria interplay and, specifically, by modulating Ca2+ signaling.
    Keywords:  Calcium; Calcium signaling; Cancer; Downregulation; MAMs; Upregulation
    DOI:  https://doi.org/10.1007/112_2020_43
  12. Cell Rep. 2020 Aug 11. pii: S2211-1247(20)31006-8. [Epub ahead of print]32(6): 108021
    Wang C, Wan X, Yu T, Huang Z, Shen C, Qi Q, Xiang S, Chen X, Arbely E, Ling ZQ, Liu CY, Yu W.
      Phosphoglycerate dehydrogenase (PHGDH) is the first enzyme in the serine synthesis pathway in which it is also the rate-limiting enzyme. It is significantly upregulated in many cancers, especially breast cancer. However, the posttranslational mechanism of PHGDH upregulation in breast cancer is unknown. In this study, we find that RNF5, an E3 ubiquitin ligase, is essential for the degradation of PHGDH protein. PHGDH is degraded by RNF5 to prevent the proliferation of breast cancer cells. The acetylation of PHGDH at K58 is able to disrupt the interaction of RNF5-PHGDH and promote the proliferation of breast cancer cells. Tip60 and SIRT2 regulate the reversible acetylation modification of PHGDH in response to glucose alteration. Moreover, PHGDH is significantly upregulated in samples of human breast cancer and is associated with decreased RNF5 expression. This implies a potential therapeutic target through the interference interaction of PHGDH-RNF5 to degrade PHGDH in breast cancer.
    DOI:  https://doi.org/10.1016/j.celrep.2020.108021
  13. Prog Neurobiol. 2020 Aug 11. pii: S0301-0082(20)30147-7. [Epub ahead of print] 101892
    Lanzillotta C, Zuliani I, Tramutola A, Barone E, Blarzino C, Folgiero V, Caforio M, Valentini D, Villani A, Locatelli F, Butterfield DA, Head E, Perluigi M, Abisambra JF, Di Domenico F.
      A major challenge in neurobiology is the identification of the mechanisms by which protein misfolding leads to cellular toxicity. Many neurodegenerative disorders, in which aberrant protein conformers aggregate into pathological inclusions, present the chronic activation of the PERK branch of the unfolded protein response. The adaptive effects of the PERK pathway include reduction of translation by transient inhibition of eIF2α and antioxidant protein production via induction of Nrf2 transcription factor. In contrast, PERK prolonged activation leads to sustained reduction in protein synthesis and induction of cell death pathways. To further investigate the role of the PERK pathway in neurodegenerative disorders, we focused on Down syndrome (DS), in which aging confers a high risk of Alzheimer disease (AD). By investigating human DS frontal cortices, we found early and sustained PERK activation associated with the induction of eIF2α and ATF4 downstream signals. We also observed that the Nrf2 response is uncoupled from PERK and its antioxidant effects are repressed in a mechanism implicating the transcription repressor Bach1. The pharmacological inhibition of PERK in DS mice reduced eIF2α-related translational repression and promoted Nrf2 nuclear translocation, favoring the rescue of Nrf2/Bach1 imbalance. The further analysis of peripheral cells from living DS individuals provided strong support of the pathological link between PERK and trisomy 21. Our results suggest that failure to regulate the PERK pathway is a peculiar characteristic of DS pathology and it may represent an essential step to promote cellular dysfunction, which actively contributes in the brain to the early development of AD.
    Keywords:  Down syndrome; Nrf2; PERK; Protein translation; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.pneurobio.2020.101892
  14. J Biol Chem. 2020 Aug 12. pii: jbc.RA119.011006. [Epub ahead of print]
    Xie S, Zhang L, Dong D, Ge R, He Q, Fan C, Xie W, Zhou J, Li D, Liu M.
      Tripartite motif-containing protein 21 (TRIM21) is a cytosolic antibody receptor that targets the internalized virus-antibody complex to the proteasome for degradation. However, the precise mechanism regulating TRIM21 activity is unknown. Here we show that TRIM21 is a substrate of histone deacetylase 6 (HDAC6) and that its function is regulated by acetylation. HDAC6 interacts with TRIM21 through its PRYSPRY motif and deacetylates TRIM21 at lysine 385 and lysine 387, thus promoting its homodimerization. Inhibiting HDAC6 activity increases TRIM21 acetylation, and hyperacetylation blocks TRIM21 dimerization and ubiquitination, preventing its binding to the virus-antibody complex and its degradation via the ubiquitin-proteasome pathway. HDAC6 depletion or inhibition increases virus accumulation in cells, indicative of an impaired capacity for antibody-dependent intracellular neutralization of viruses, whereas TRIM21 acetylation-deficient K385/387R mutant rescues HDAC6 depletion-caused ADIN impairment. These findings provide evidence for HDAC6 as a novel regulator of TRIM21-mediated intracellular innate immunity.
    Keywords:  TRIM21; acetylation; antibody-dependent intracellular neutralization; histone deacetylase 6 (HDAC6); infection; ubiquitin; virus
    DOI:  https://doi.org/10.1074/jbc.RA119.011006
  15. J Biol Chem. 2020 Aug 11. pii: jbc.RA120.014173. [Epub ahead of print]
    Sharma RB, Darko C, Alonso LC.
      Success or failure of pancreatic beta cell adaptation to ER stress is a determinant of diabetes susceptibility. The ATF6 and IRE1/XBP1 pathways are separate ER stress response effectors important to beta cell health and function. ATF6 and XBP1 direct overlapping transcriptional responses in some cell types. However, the signaling dynamics and interdependence of ATF6α and XBP1 in pancreatic beta cells have not been explored. To assess pathway-specific signal onset, we performed timed exposures of primary mouse islet cells to ER stressors and measured the early transcriptional response. Comparing the time course of induction of ATF6 and XBP1 targets suggested the two pathways have similar response dynamics. The role of ATF6α in target induction was assessed by acute knockdown using islet cells from Atf6αflox/flox mice transduced with adenovirus expressing Cre recombinase. Surprisingly given the mild impact of chronic deletion in mice, acute ATF6α knockdown markedly reduced ATF6-pathway target gene expression under both basal and stressed conditions. Intriguingly, while ATF6α knockdown did not alter Xbp1 splicing dynamics or intensity, it did reduce induction of XBP1 targets. Inhibition of Xbp1 splicing did not decrease induction of ATF6α targets. Taken together, these data suggest that the XBP1 and ATF6 pathways are simultaneously activated in islet cells in response to acute stress, and that ATF6α is required for full activation of XBP1 targets, but XBP1 is not required for activation of ATF6α targets. These observations improve understanding of the ER stress transcriptional response in pancreatic islets.
    Keywords:  diabetes; endoplasmic reticulum stress (ER stress); insulin synthesis; pancreatic beta cell; pancreatic islet; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1074/jbc.RA120.014173
  16. Cancer Metab. 2020 ;8 7
    Panarsky R, Crooks DR, Lane AN, Yang Y, Cassel TA, Fan TW, Linehan WM, Moscow JA.
      Background: The loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT).Methods: We determined growth rates and survival of cell lines in response to amino acid depletion and supplementation. RNAseq was used to determine the transcription changes contingent on Asn and Gln supplementation, which was further followed with stable isotope resolved metabolomics (SIRM) using both [U- 13C,15N] Gln and Asn.
    Results: We found that Asn increased the growth rate of both cell lines in vitro. Gln, but not Asn, increased oxygen consumption rates and glycolytic reserve of both cell lines. Although Asn was taken up by the cells, there was little evidence of Asn-derived label in cellular metabolites, indicating that Asn was not catabolized. However, Asn strongly stimulated Gln labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway (HBP) by Asn. Asn in combination with Gln, but not Asn or Gln alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. Asn exposure also increased both the RNA and protein expression of the HBP regulator GFPT2, which is a transcriptional target of sXBP1.
    Conclusions: Asn in the presence of Gln induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of HBP activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.
    Keywords:  Asparagine metabolism; Fumarate hydratase; Renal cell carcinoma; Unfolded protein response, SIRM
    DOI:  https://doi.org/10.1186/s40170-020-00214-9
  17. Plant Physiol. 2020 Aug 11. pii: pp.00799.2020. [Epub ahead of print]
    Zhao CY, Xue HW.
      Root development is important for normal plant growth and nutrient absorption. Studies have revealed the involvement of various factors in this complex process, improving our understanding of the relevant regulatory mechanisms. Here, we functionally characterize the role of Arabidopsis phosphatidylinositol 4-kinase γ2 (PI4Kγ2) in root elongation regulation, which functions to modulate stability of the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) and auxin metabolism. Mutant plants deficient in PI4Kγ2 (pi4kγ2) exhibited a shortened root length and elongation zone due to reduced auxin level. PI4Kγ2 was shown to interact with MIEL1, regulating its degradation and further the stability of transcription factor MYB30, which suppresses auxin metabolism by directly binding to promoter regions of GH3.2 and GH3.6. Interestingly, pi4kγ2 plants presented altered hypersensitive response, indicating that PI4Kγ2 regulates synergetic growth and defense of plants through modulating auxin metabolism. These results reveal the importance of protein interaction in regulating ubiquitin-mediated protein degradation in eukaryotic cells, and illustrate a mechanism coordinating plant growth and biotic stress response.
    DOI:  https://doi.org/10.1104/pp.20.00799
  18. EMBO Rep. 2020 Aug 12. e50718
    Malaquin N, Olivier MA, Martinez A, Nadeau S, Sawchyn C, Coppé JP, Cardin G, Mallette FA, Campisi J, Rodier F.
      Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.
    Keywords:  DNA damage response; MRN complex; NF-κB; chromatin; senescence secretome
    DOI:  https://doi.org/10.15252/embr.202050718
  19. Cell Death Differ. 2020 Aug 07.
    Chanda A, Ikeuchi Y, Karve K, Sarkar A, Chandhoke AS, Deng L, Bonni A, Bonni S.
      SUMO E3 ligases specify protein substrates for SUMOylation. The SUMO E3 ligases PIAS1 and TIF1γ target the transcriptional regulator SnoN for SUMOylation leading to suppression of epithelial-mesenchymal transition (EMT). Whether and how TIF1γ and PIAS1 might coordinate SnoN SUMOylation and regulation of EMT remained unknown. Here, we reveal that SnoN associates simultaneously with both TIF1γ and PIAS1, leading to a trimeric protein complex. Hence, PIAS1 and TIF1γ collaborate to promote the SUMOylation of SnoN. Importantly, loss of function studies of PIAS1 and TIF1γ suggest that these E3 ligases act in an interdependent manner to suppress EMT of breast cell-derived tissue organoids. Collectively, our findings unveil a novel mechanism by which SUMO E3 ligases coordinate substrate SUMOylation with biological implications.
    DOI:  https://doi.org/10.1038/s41418-020-0599-8
  20. Sci Adv. 2020 Jul;6(31): eabb8725
    Xu F, Li X, Huang X, Pan J, Wang Y, Zhou S.
      Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.
    DOI:  https://doi.org/10.1126/sciadv.abb8725
  21. Biol Aujourdhui. 2020 ;214(1-2): 15-23
    Lachkar F, Papaioannou A, Ferré P, Foufelle F.
      Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent pathology associated with obesity. It encompasses a spectrum of hepatic disorders ranging from steatosis to non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress has been widely involved to drive in NAFLD progression through the activation of the unfolded protein response (UPR). While transient UPR activation can boost hepatic ER functions, its continuous activation upon a chronic ER stress contributes to lipid accumulation, inflammation and hepatocyte death, which are determinant factors for the progression to more severe stages. The aim of this review is to describe the mechanisms through which the UPR can take part in the transition from a healthy to a diseased liver and to report on possible ways of pharmacological manipulation against these pathological mechanisms.
    Keywords:  ER stress; UPR; inflammation; lipides; lipids; obesity; obésité; stress du réticulum endoplasmique
    DOI:  https://doi.org/10.1051/jbio/2020007
  22. Cell Chem Biol. 2020 Aug 07. pii: S2451-9456(20)30293-2. [Epub ahead of print]
    Nalawansha DA, Crews CM.
      Targeted protein degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the ubiquitin-proteasome system. Over the last 5 years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine.
    Keywords:  E3 ligase; PROTACs; PhotoPROTACs; covalent ligands; proteasome; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.chembiol.2020.07.020
  23. Cell Rep. 2020 Aug 11. pii: S2211-1247(20)30986-4. [Epub ahead of print]32(6): 108001
    Tsvetkov P, Eisen TJ, Heinrich SU, Brune Z, Hallacli E, Newby GA, Kayatekin C, Pincus D, Lindquist S.
      The heat shock protein 90 (Hsp90) chaperone functions as a protein-folding buffer and plays a role promoting the evolution of new heritable traits. To better understand how Hsp90 can affect mRNA translation, we screen more than 1,600 factors involved in mRNA regulation for physical interactions with Hsp90 in human cells. The mRNA binding protein CPEB2 strongly binds Hsp90 via its prion domain. In a yeast model, transient inhibition of Hsp90 results in persistent activation of a CPEB translation reporter even in the absence of exogenous CPEB that persists for 30 generations after the inhibitor is removed. Ribosomal profiling reveals that some endogenous yeast mRNAs, including HAC1, show a persistent change in translation efficiency following transient Hsp90 inhibition. Thus, transient loss of Hsp90 function can promote a nongenetic inheritance of a translational state affecting specific mRNAs, introducing a mechanism by which Hsp90 can promote phenotypic variation.
    Keywords:  3′ UTR regulation; FET5; HAC1; Hsp90; V-ATPase; cellular memory; epigentic regualtion; mRNA translation; prions; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2020.108001
  24. iScience. 2020 Jul 24. pii: S2589-0042(20)30600-3. [Epub ahead of print]23(8): 101410
    Shimizu T, Taguchi A, Higashijima Y, Takubo N, Kanki Y, Urade Y, Wada Y.
      Oxidative/nitrosative stress is a major trigger of cardiac dysfunction, involving the unfolded protein response and mitochondrial dysfunction. Activation of nitric oxide-cyclic guanosine monophosphate-protein kinase G signaling by sildenafil improves cardiac mal-remodeling during pressure-overload-induced heart failure. Transcriptome analysis was conducted in failing hearts with or without sildenafil treatment. Protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) downstream signaling pathways, EIF2 and NRF2, were significantly altered. Although EIF2 signaling was suppressed, NRF2 signaling was upregulated, inhibiting the maturation of miR 24-3p through EGFR-mediated Ago2 phosphorylation. To study the effect of sildenafil on these pathways, we generated cardiac-specific PERK knockout mice. In these mice, sildenafil could not inhibit the maturations, the nuclear translocation of NRF2 was suppressed, and mitochondrial dysfunction advanced. Altogether, these results show that PERK-mediated suppression of miRNAs by sildenafil is vital for maintaining mitochondrial homeostasis through NRF2-mediated oxidative stress response.
    Keywords:  Biological Sciences; Cell Biology; Molecular Biology
    DOI:  https://doi.org/10.1016/j.isci.2020.101410
  25. Genes Dev. 2020 Aug 13.
    Ding X, Jo J, Wang CY, Cristobal CD, Zuo Z, Ye Q, Wirianto M, Lindeke-Myers A, Choi JM, Mohila CA, Kawabe H, Jung SY, Bellen HJ, Yoo SH, Lee HK.
      Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology.
    Keywords:  CNS development; multiple sclerosis; oligodendrocyte; remyelination
    DOI:  https://doi.org/10.1101/gad.338046.120
  26. Sci Rep. 2020 Aug 14. 10(1): 13831
    Nitika , Blackman JS, Knighton LE, Takakuwa JE, Calderwood SK, Truman AW.
      Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 is regulated by a suite of co-chaperone molecules that bring "clients" to the primary chaperone for efficient folding. Rather than targeting Hsp70 itself, here we have examined the feasibility of inhibiting the Hsp70 co-chaperone DNAJA1 as a novel anticancer strategy. We found DNAJA1 to be upregulated in a variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the NIH Approved Oncology collection for chemical-genetic interactions with loss of DNAJA1 in cancer. 41 compounds showed strong synergy with DNAJA1 loss, whereas 18 dramatically lost potency. Several hits were validated using a DNAJA1 inhibitor (116-9e) in castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, these results confirm that DNAJA1 is a hub for anticancer drug resistance and that DNAJA1 inhibition is a potent strategy to sensitize cancer cells to current and future therapeutics. The large change in drug efficacy linked to DNAJA1 suggests a personalized medicine approach where tumor DNAJA1 status may be used to optimize therapeutic strategy.
    DOI:  https://doi.org/10.1038/s41598-020-70764-x
  27. J Integr Plant Biol. 2020 Aug 12.
    Xiao Z, Yang C, Liu C, Yang L, Yang S, Zhou J, Li F, Jiang L, Xiao S, Gao C, Shen W.
      The endosomal sorting complex required for transport (ESCRT) machinery is an ancient, evolutionarily conserved membrane remodeling complex that is essential for multivesicular body (MVB) biogenesis in eukaryotes. FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific ESCRT component, modulates MVB-mediated endosomal sorting and autophagic degradation. Although the basic cellular functions of FREE1 as an ESCRT component have been described, the regulators that control FREE1 turnover remain unknown. Here, we analyzed how FREE1 homeostasis is mediated by the RING-finger E3 ubiquitin ligases, SINA of Arabidopsis thaliana (SINATs), in response to iron deficiency. Under iron-deficient growth conditions, SINAT1-4 were induced and ubiquitinated FREE1, thereby promoting its degradation and relieving the repressive effect of FREE1 on iron absorption. By contrast, SINAT5, another SINAT member that lacks ubiquitin ligase activity due to the absence of the RING domain, functions as a protector protein that stabilizes FREE1. Collectively, our findings uncover a hitherto unknown mechanism of homeostatic regulation of FREE1, and demonstrate a unique regulatory SINAT-FREE1 module that subtly regulates plant response to iron deficiency stress. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1111/jipb.13005
  28. Cancers (Basel). 2020 Aug 07. pii: E2213. [Epub ahead of print]12(8):
    Villemain L, Prigent S, Abou-Lovergne A, Pelletier L, Chiral M, Pontoglio M, Foufelle F, Caruso S, Pineau R, Rebouissou S, Chevet E, Zucman-Rossi J, Combettes L.
      Sigma receptor 1 (SigR1) is an endoplasmic reticulum resident integral membrane protein whose functions remain unclear. Although the liver shows the highest expression of SigR1, its role in this organ is unknown. SigR1 is overexpressed in many cancers and its expression is correlated to hormonal status in hormone-dependent cancers. To better understand the role of SigR1 in hepatocytes we focused our work on the regulation of its expression in tumoral liver. In this context, hepatocellular adenomas, benign hepatic tumors associated with estrogen intake are of particular interest. The expression of SigR1 mRNA was assessed in hepatocellular adenoma (HCA) patients using qPCR. The impact of estrogen on the expression of SigR1 was studied in vivo (mice) and in vitro (HepG2 and Huh7 cells). The effect of HNF1α on the expression of SigR1 was studied in vivo by comparing wild type mice to HNF1 knockout mice. Estrogen enhanced SigR1 expression through its nuclear receptor ERα. HNF1α mutated HCA (H-HCA) significantly overexpressed SigR1 compared to all other HCA subtypes. HNF1 knockout mice showed an increase in SigR1 expression. Overexpressing SigR1 in cellular models increases proliferation rate and storage of lipid droplets, which phenocopies the H-HCA phenotype. SigR1 is involved in hepatocyte proliferation and steatosis and may play an important role in the control of the H-HCA phenotype.
    Keywords:  hepatocyte; liver tumor; proliferation; steatosis
    DOI:  https://doi.org/10.3390/cancers12082213
  29. ACS Chem Biol. 2020 Jul 31.
    Brüninghoff K, Aust A, Taupitz KF, Wulff S, Dörner W, Mootz HD.
      Post-translational modification with the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome and is implicated in numerous cellular processes. The main outcome of SUMO conjugation is a re-wiring of protein-protein interactions through recognition of the modifier's surface by SUMO binding proteins. The SUMO-interacting motif (SIM) mediates binding to a groove on SUMO, however, the low affinity of this interaction and the poor conservation of SIM sequences complicates the isolation and identification of SIM proteins. To address these challenges, we have designed and biochemically characterized monomeric and multimeric SUMO-2 probes with a genetically encoded photo-crosslinker positioned next to the SIM binding groove. Following photo-induced covalent capture, even weak SUMO binders are not washed away during the enrichment procedure, and very stringent washing conditions can be applied to remove nonspecifically binding proteins. A total of 329 proteins were isolated from nuclear HeLa cell extracts and identified using mass spectrometry. We found the molecular design of our probes corroborated by the presence of many established SUMO interacting proteins and the high percentage (> 90%) of hits containing a potential SIM sequence, as predicted by bioinformatic analyses. Notably, 266 of the 329 proteins have not been previously reported as SUMO binders using traditional non-covalent enrichment procedures. We confirmed SUMO binding with purified proteins and mapped the position of the covalent crosslinks for selected cases. We postulate a new SIM in MRE11, involved in DNA repair. The identified SUMO binding candidates will help to reveal the complex SUMO-mediated protein network.
    DOI:  https://doi.org/10.1021/acschembio.0c00609