bims-proteo Biomed News
on Proteostasis
Issue of 2021‒11‒07
thirty-five papers selected by
Eric Chevet

  1. MicroPubl Biol. 2021 ;2021
      The conserved Argonaute-family members ALG-1 and ALG-2 are known to regulate processing and maturation of microRNAs to target mRNAs for degradation or translational inhibition (Bouasker and Simard 2012; Meister 2013). Consequently, depletion of alg-1 and alg-2 results in multiple phenotypes. Our data describe a role of microRNA-regulation in stress resistance and proteostasis with special emphasis on ubiquitin-dependent degradation pathways, such as ubiquitin fusion degradation (UFD) and endoplasmic reticulum (ER)-associated protein degradation (ERAD).
  2. Autophagy. 2021 Nov 05. 1-17
      The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are the main proteolytic systems in eukaryotic cells for preserving protein homeostasis, i.e., proteostasis. By facilitating the timely destruction of aberrant proteins, these complementary pathways keep the intracellular environment free of inherently toxic protein aggregates. Chemical interference with the UPS or autophagy has emerged as a viable strategy for therapeutically targeting malignant cells which, owing to their hyperactive state, heavily rely on the sanitizing activity of these proteolytic systems. Here, we report on the discovery of CBK79, a novel compound that impairs both protein degradation by the UPS and autophagy. While CBK79 was identified in a high-content screen for drug-like molecules that inhibit the UPS, subsequent analysis revealed that this compound also compromises autophagic degradation of long-lived proteins. We show that CBK79 induces non-canonical lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) that requires ATG16L1 but is independent of the ULK1 (unc-51 like autophagy activating kinase 1) and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes. Thermal preconditioning of cells prevented CBK79-induced UPS impairment but failed to restore autophagy, indicating that activation of stress responses does not allow cells to bypass the inhibitory effect of CBK79 on autophagy. The identification of a small molecule that simultaneously impairs the two main proteolytic systems for protein quality control provides a starting point for the development of a novel class of proteostasis-targeting drugs.
    Keywords:  Autophagy; compound screen; inhibitor; proteostasis; stress response; ubiquitin-proteasome system
  3. Mol Cancer Res. 2021 Nov 02. pii: molcanres.0702.2021. [Epub ahead of print]
      The discovery of 17β-estradiol (E2)-induced apoptosis has clinical relevance. Mechanistically, E2 over activates nuclear estrogen receptor α (ERα) that results in stress responses. The unfolded protein response (UPR) is initiated by E2 in the endoplasmic reticulum after hours of treatment in endocrine-resistant breast cancer cells, thereby activating three UPR sensors-PRK-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) with different functions. Specifically, PERK plays a critical role in induction of apoptosis while IRE1α and ATF6 are involved in the endoplasmic reticulum stress-associated degradation (ERAD) of PI3K/Akt/mTOR pathways. In addition to attenuating protein translation, PERK increases the DNA-binding activity of nuclear factor-κB (NF-κB) and subsequent tumor necrosis factor α (TNFα) expression. Additionally, PERK communicates with the mitochondria to regulate oxidative stress at mitochondria-associated endoplasmic reticulum membranes (MAMs). Furthermore, PERK is a component enriched in MAMs that interacts with multifunctional MAM-tethering proteins and integrally modulates the exchange of metabolites such as lipids, reactive oxygen species (ROS), and Ca2+ at contact sites. MAMs are also critical sites for the initiation of autophagy to remove defective organelles and misfolded proteins through specific regulatory proteins. Thus, PERK conveys signals from nucleus to these membrane-structured organelles that form an interconnected network to regulate E2-induced apoptosis. Herein, we address the mechanistic progress on how PERK acts as a multifunctional molecule to commit E2 to inducing apoptosis in endocrine-resistant breast cancer.
  4. FEBS J. 2021 Nov 05.
      Monoubiquitination of histone H2B at lysine 120 plays a vital role in active transcription and DNA damage response pathways. UBR7 has been recently identified as an H2BK120 monoubiquitin ligase. However, the molecular details of its ubiquitin transfer mechanism are not well understood. Here, we report that PHD finger of UBR7 is essential for its association with E2 UbcH6 and consequent ubiquitin transfer to its substrate histone H2B. We have also identified the critical region of UbcH6 involved in this function and shown that the residues stretching from 114 to 125 of histone H2B C-terminal tail are sufficient for UBR7/UbcH6-mediated ubiquitin transfer. We also employed antibody-independent mass spectrometry to confirm UBR7 mediated ubiquitination of H2B C-terminal tail. We have demonstrated that the PHD finger of UBR7 forms a dimer and this dimerization is essential for ubiquitination of histone H2B. We have mapped the critical residues involved in dimerization and mutation of these residues abrogates E3 ligase activity and is associated with cancer. Furthermore, we have compared the mode of ubiquitin discharge from UbcH6 mediated by UBR7 and RNF20 through thioester hydrolysis assay. Interestingly, binding of substrate H2B to UBR7 induces conformational change in the PHD finger, which triggers ubiquitin transfer from UbcH6. However RNF20 RING finger alone is sufficient to promote the release of ubiquitin from UbcH6. Overall, the mechanism of ubiquitin transfer by the newly identified E3 ubiquitin ligase UBR7 is markedly different from that of RNF20.
    Keywords:  Chromatin; E3 ligase; Monoubiquitination; Oligomerization; PHD finger
  5. Cell Death Dis. 2021 Nov 01. 12(11): 1038
      Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.
  6. Mol Metab. 2021 Oct 30. pii: S2212-8778(21)00212-X. [Epub ahead of print] 101365
      Pancreatic β-cells are the insulin factory of the organism with a mission to regulate glucose homeostasis in the body. Due to their high secretory activity, β-cells rely on a functional and intact endoplasmic reticulum (ER). Perturbations to ER homeostasis and unmitigated stress lead to β-cell dysfunction and death. Type 1 diabetes (T1D) is a chronic inflammatory disease caused by autoimmune-mediated destruction of β-cells. Although autoimmunity is an essential component of T1D pathogenesis, accumulating evidence suggests that β-cell ER stress and aberrant unfolded protein response (UPR) can play an important role in disease initiation and progression. Here, we review β-cell ER stress in various mouse models, evaluate its involvement in inflammation, and discuss the effects of ER stress on β-cell plasticity and demise, and islet autoimmunity in T1D. Finally, we provide insight into therapeutic targeting of ER stress and the UPR for prevention or treatment of T1D.
    Keywords:  NOD mice; beta cell; er stress; human islets; type 1 diabetes
  7. Front Mol Biosci. 2021 ;8 788118
    Keywords:  autophagy; organismal aging; protein folding; protein modification; proteostasis; ubiquitin proteasome system
  8. Nat Commun. 2021 Nov 03. 12(1): 6321
      The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.
  9. Mol Cell Proteomics. 2021 Nov 02. pii: S1535-9476(21)00145-6. [Epub ahead of print] 100173
      RNF111/Arkadia is an E3 ubiquitin ligase that activates the TGF-β pathway by degrading transcriptional repressors SKIL/SnoN and SKI, and truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim we carried out label free quantitative proteomics after enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared to U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins prior to proteomics analysis by mass spectrometry, the diGly remnant peptide immunoprecipitation with a K-ε-GG antibody (diGly) and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody (pan UB) that recognizes all ubiquitin chains and monoubiquitylation. While we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the pan UB method also constitutes a powerful approach since it enabled detection of 52 potential RNF111 substrates including SKI, SKIL and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation.
  10. Sci Rep. 2021 Nov 02. 11(1): 20772
      The endoplasmic reticulum (ER) is the organelle responsible for the folding of secretory/membrane proteins and acts as a dynamic calcium ion (Ca2+) store involved in various cellular signalling pathways. Previously, we reported that the ER-resident disulfide reductase ERdj5 is involved in the ER-associated degradation (ERAD) of misfolded proteins in the ER and the activation of SERCA2b, a Ca2+ pump on the ER membrane. These results highlighted the importance of the regulation of redox activity in both Ca2+ and protein homeostasis in the ER. Here, we show that the deletion of ERdj5 causes an imbalance in intracellular Ca2+ homeostasis, the activation of Drp1, a cytosolic GTPase involved in mitochondrial fission, and finally the aberrant fragmentation of mitochondria, which affects cell viability as well as phenotype with features of cellular senescence. Thus, ERdj5-mediated regulation of intracellular Ca2+ is essential for the maintenance of mitochondrial homeostasis involved in cellular senescence.
  11. Biochem J. 2021 Nov 02. pii: BCJ20210345. [Epub ahead of print]
      The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon "breathing" during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.
    Keywords:  Mycolactone; Sec61; Translocon; calcium imaging; calcium signalling; molecular docking
  12. PLoS One. 2021 ;16(11): e0259556
      The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.
  13. Cancer Discov. 2021 Nov 05.
      Tumor-secreted lipids alter ER membrane composition to polarize tumor-associated macrophages (TAM).
  14. Autophagy. 2021 Oct 31. 1-16
      The phagophore expands into autophagosomes in close proximity to endoplasmic reticulum (ER) exit sites (ERESs). Here, we propose that a single-pass ER transmembrane protein, SHISA5/SCOTIN, acts as an autophagy suppressor under basal condition by blocking the contact between the phagophore and ERES. HeLa cells lacking SHISA5 displayed higher levels of macroautophagy/autophagy. The enhanced autophagy in SHISA5 KO cells requires class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) activity and functional assembly of ERES, but not ULK1 activity. A proximity ligation assay (PLA) of SEC16A (Sec16 homolog A, endoplasmic reticulum export factor)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) and SEC31A (Sec31 homolog A, COPII coat complex component)-MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) demonstrated that contact between the ERES and phagophore increased in SHISA5 KO cells, and the cytosolic domain of SHISA5 was sufficient to rescue this phenotype. Close proximity between ERES and phagophore in SHISA5 KO cells was also visualized by performing an ultrastructure correlative image analysis of SEC31A associated with LC3-positive membranes. Furthermore, we observed that SHISA5 was located near ERES under basal conditions, but displaced away from ERES under autophagy-inducing conditions. These data suggest that SHISA5 functions to block spontaneous contact between ERES and phagophore, and the blockage effect of SHISA5 should be relieved for the proper induction of autophagy.
    Keywords:  Constitutive autophagy; SHISA5/SCOTIN; endoplasmic reticulum exit sites; membrane contact; phagophore
  15. J Cell Sci. 2021 Nov 03. pii: jcs.258891. [Epub ahead of print]
      The WAVE regulatory complex (WRC) is the major Arp2/3 activator, promoting lamellipodial protrusions in migrating cells. The WRC is basally inactive but can be activated by Rac1 and phospholipids, and phosphorylation. However, the in vivo relevance of phosphorylation of WAVE remains largely unknown. Here, we identified the kinase CK1α as a novel regulator of WAVE controlling cell shape and cell motility in Drosophila macrophages. CK1α binds and phosphorylates WAVE in vitro. Phosphorylation of WAVE by CK1α appears not to be required for activation but rather regulates its stability. Pharmacologic inhibition of CK1α promotes ubiquitin-dependent degradation of WAVE. Consistently, loss of ck1α but not ck2 function phenocopies WAVE depletion. Phosphorylation-deficient mutations in the CK1α consensus sequences within the VCA domain of WAVE can neither rescue mutant lethality nor lamellipodia defects. By contrast, phosphomimetic mutations rescue all cellular and developmental defects. Finally, RNAi-mediated suppression of 26S proteasome or E3 ligase complexes substantially rescues lamellipodia defects in CK1α depleted macrophages. Thus, we conclude that the basal phosphorylation of WAVE by CK1α protects it from premature ubiquitin-dependent degradation, thus promoting WAVE function in vivo.
    Keywords:  Actin; Arp2/3; CK1α; CK2; Cell migration; Cell motility; Cell shape; Drosophila; Lamellipodia; Macrophages; Phosphorylation; Ubiquitin-dependent protein degradation; WAVE
  16. EMBO Rep. 2021 Nov 02. e53679
      The tumor suppressor BRCA1 accumulates at sites of DNA damage in a ubiquitin-dependent manner. In this work, we revisit the role of RAP80 in promoting BRCA1 recruitment to damaged chromatin. We find that RAP80 acts redundantly with the BRCA1 RING domain to promote BRCA1 recruitment to DNA damage sites. We show that that RNF8 E3 ligase acts upstream of both the RAP80- and RING-dependent activities, whereas RNF168 acts uniquely upstream of the RING domain. BRCA1 RING mutations that do not impact BARD1 interaction, such as the E2 binding-deficient I26A mutation, render BRCA1 unable to accumulate at DNA damage sites in the absence of RAP80. Cells that combine BRCA1 I26A and mutations that disable the RAP80-BRCA1 interaction are hypersensitive to PARP inhibition and are unable to form RAD51 foci. Our results suggest that in the absence of RAP80, the BRCA1 E3 ligase activity is necessary for recognition of histone H2A Lys13/Lys15 ubiquitylation by BARD1, although we cannot rule out the possibility that the BRCA1 RING facilitates ubiquitylated nucleosome recognition in other ways.
    Keywords:  BARD1; BRCA1; DNA damage; RAP80; ubiquitin
  17. Plant Cell. 2021 Oct 30. pii: koab263. [Epub ahead of print]
      Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase SAR1, which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.
  18. Hum Mol Genet. 2021 Oct 28. pii: ddab314. [Epub ahead of print]
      Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogenous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.
  19. Sci Rep. 2021 Nov 01. 11(1): 21346
      The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.
  20. J Cell Sci. 2021 Nov 04. pii: jcs.259340. [Epub ahead of print]
      N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.
    Keywords:  Disulfide formation; Glycosylation; MagT1; Oligosaccharyl transferase; STT3A/B; TUSC3
  21. Nat Commun. 2021 Nov 03. 12(1): 6322
      Molecular programs that underlie precursor progression in multiple myeloma are incompletely understood. Here, we report a disease spectrum-spanning, single-cell analysis of the Vκ*MYC myeloma mouse model. Using samples obtained from mice with serologically undetectable disease, we identify malignant cells as early as 30 weeks of age and show that these tumours contain subclonal copy number variations that persist throughout progression. We detect intratumoural heterogeneity driven by transcriptional variability during active disease and show that subclonal expression programs are enriched at different times throughout early disease. We then show how one subclonal program related to GCN2 stress response is progressively activated during progression in myeloma patients. Finally, we use chemical and genetic perturbation of GCN2 in vitro to support this pathway as a therapeutic target in myeloma. These findings therefore present a model of precursor progression in Vκ*MYC mice, nominate an adaptive mechanism important for myeloma survival, and highlight the need for single-cell analyses to understand the biological underpinnings of disease progression.
  22. Nucleic Acids Res. 2021 Oct 28. pii: gkab898. [Epub ahead of print]
      In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of Huntington's disease-causing alleles. We characterized a tRNAPro mutant that mistranslates proline codons with alanine, and tRNASer mutants, including a tRNASerAGA G35A variant with a phenylalanine anticodon (tRNASerAAA) found in ∼2% of the population. The tRNAPro mutant caused synthetic toxicity with a deleterious huntingtin poly-glutamine (polyQ) allele in neuronal cells. The tRNASerAAA variant showed synthetic toxicity with proteasome inhibition but did not enhance toxicity of the huntingtin allele. Cells mistranslating phenylalanine or proline codons with serine had significantly reduced rates of protein synthesis. Mistranslating cells were slow but effective in forming insoluble polyQ aggregates, defective in protein and aggregate degradation, and resistant to the neuroprotective integrated stress response inhibitor (ISRIB). Our findings identify mistranslating tRNA variants as genetic factors that slow protein aggregation kinetics, inhibit aggregate clearance, and increase drug resistance in cellular models of neurodegenerative disease.
  23. Science. 2021 Jun 25. 372(6549): eabc3593
      Tailoring stress responsesWhen faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393.
  24. Science. 2021 Jun 25. 372(6549): eabf6548
      Tailoring stress responsesWhen faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393.
  25. STAR Protoc. 2021 Dec 17. 2(4): 100896
      Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery. For complete details on the use and execution of this protocol, please refer to Varca et al. (2021).
    Keywords:  High Throughput Screening; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification
  26. Cancer Res. 2021 Nov 04. pii: canres.0384.2021. [Epub ahead of print]
      F-box and WD repeat domain containing 7 (FBXW7) is a substrate receptor of the ubiquitin ligase SKP1-Cullin1-F-box complex and a potent tumor suppressor that prevents unregulated cell growth and tumorigenesis. However, little is known about FBXW7-mediated control of cell metabolism and related functions in cancer therapy. Here, we report that FBXW7 expression inversely correlates with the expression levels of the key metabolic enzyme isocitrate dehydrogenase 1 (IDH1) in glioma patients and public glioma datasets. Deletion of FBXW7 significantly increased both wild type (WT) and mutant IDH1 expression, which was mediated by blocking degradation of sterol regulatory element binding protein 1 (SREBP1). The upregulation of neomorphic mutant IDH1 by FBXW7 deletion stimulated production of the oncometabolite 2-hydroxyglutarate (2-HG) at the expense of increasing pentose phosphate pathway (PPP) activity and NADPH consumption, limiting the buffering ability against radiation-induced oxidative stress. Additionally, FBXW7 knockout and IDH1 mutations induced non-homologous end joining (NHEJ) and homologous recombination (HR) defects, respectively. In vitro and in vivo, loss of FBXW7 dramatically enhanced the efficacy of radiation treatment in IDH1 mutant cancer cells. Taken together, this work identifies FBXW7 deficiency as a potential biomarker representing both DNA repair and metabolic vulnerabilities that sensitizes IDH1 mutant cancers to radiotherapy.
  27. Cell Rep. 2021 Nov 02. pii: S2211-1247(21)01426-1. [Epub ahead of print]37(5): 109949
      Tfcp2l1 can maintain mouse embryonic stem cell (mESC) self-renewal. However, it remains unknown how Tfcp2l1 protein stability is regulated. Here, we demonstrate that β-transducin repeat-containing protein (β-TrCP) targets Tfcp2l1 for ubiquitination and degradation in a mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-dependent manner. Specifically, β-TrCP1 and β-TrCP2 recognize and ubiquitylate Tfcp2l1 through the canonical β-TrCP-binding motif DSGDNS, in which the serine residues have been phosphorylated by MK2. Point mutation of serine-to-alanine residues reduces β-TrCP-mediated ubiquitylation and enhances the ability of Tfcp2l1 to promote mESC self-renewal while repressing the speciation of the endoderm, mesoderm, and trophectoderm. Similarly, inhibition of MK2 reduces the association of Tfcp2l1 with β-TrCP1 and increases the self-renewal-promoting effects of Tfcp2l1, whereas overexpression of MK2 or β-TrCP genes decreases Tfcp2l1 protein levels and induces mESC differentiation. Collectively, our study reveals a posttranslational modification of Tfcp2l1 that will expand our understanding of the regulatory network of stem cell pluripotency.
    Keywords:  MK2; Tfcp2l1; degradation; embryonic stem cells; self-renewal; β-TrCP1
  28. Mol Cell Biol. 2021 Nov 01. MCB0024421
      Ded1 is a conserved RNA helicase that promotes translation initiation in steady-state conditions. Ded1 has also been shown to regulate translation during cellular stress and affect the dynamics of stress granules (SGs), accumulations of RNA and protein linked to translation repression. To better understand its role in stress responses, we examined Ded1 function in two different models: DED1 overexpression and oxidative stress. DED1 overexpression inhibits growth and promotes the formation of SGs. A ded1 mutant lacking the low-complexity C-terminal region (ded1-ΔCT), which mediates Ded1 oligomerization and interaction with the translation factor eIF4G1, suppressed these phenotypes, consistent with other stresses. During oxidative stress, a ded1-ΔCT mutant was defective in growth and in SG formation compared to wild-type cells, although SGs were increased rather than decreased in these conditions. Unlike stress induced by direct TOR inhibition, the phenotypes in both models were only partially dependent on eIF4G1 interaction, suggesting an additional contribution from Ded1 oligomerization. Furthermore, examination of the growth defects and translational changes during oxidative stress suggested that Ded1 plays a role during recovery from stress. Integrating these disparate results, we propose that Ded1 controls multiple aspects of translation and RNP dynamics in both initial stress responses and during recovery.
  29. FEMS Yeast Res. 2021 Oct 28. pii: foab054. [Epub ahead of print]
      Heat-shock protein 90 (Hsp90) is a central regulator of cellular proteostasis. It stabilizes numerous proteins that are involved in fundamental processes of life, including cell growth, cell cycle progression, and the environmental response. In addition to stabilizing proteins, Hsp90 governs gene expression and controls the release of cryptic genetic variation. Given its central role in evolution and development, it is important to identify proteins and genes that interact with Hsp90. This requires sophisticated genetic and biochemical tools, including extensive mutant collections, suitable epitope tags, proteomics approaches and Hsp90 specific pharmacological inhibitors for chemogenomic screens. These usually only exist in model organisms, such as the yeast Saccharomyces cerevisiae. Yet, the importance of other fungal species, such as Candida albicans and Cryptococcus neoformans, as serious human pathogens accelerated the development of genetic tools to study their virulence and stress response pathways. These tools can also be exploited to map Hsp90 interaction networks. Here, we review tools and techniques for Hsp90 network mapping available in different fungi and provide a summary of existing mapping efforts. Mapping Hsp90 networks in fungal species spanning >500 million years of evolution provides a unique vantage point, allowing tracking of the evolutionary history of eukaryotic Hsp90 networks.
    Keywords:  Aspergillus fumigatus; Candida; Cryptococcus neoformans; Hsp90; chemogenomics; mutant libraries; protein-protein interactions; proteomics; synthetic lethality
  30. Nat Commun. 2021 Nov 02. 12(1): 6313
      Cross-talk between distinct protein post-translational modifications is critical for an effective DNA damage response. Arginine methylation plays an important role in maintaining genome stability, but how this modification integrates with other enzymatic activities is largely unknown. Here, we identify the deubiquitylating enzyme USP11 as a previously uncharacterised PRMT1 substrate, and demonstrate that the methylation of USP11 promotes DNA end-resection and the repair of DNA double strand breaks (DSB) by homologous recombination (HR), an event that is independent from another USP11-HR activity, the deubiquitylation of PALB2. We also show that PRMT1 is a ubiquitylated protein that it is targeted for deubiquitylation by USP11, which regulates the ability of PRMT1 to bind to and methylate MRE11. Taken together, our findings reveal a specific role for USP11 during the early stages of DSB repair, which is mediated through its ability to regulate the activity of the PRMT1-MRE11 pathway.
  31. J Clin Invest. 2021 Nov 01. pii: e146187. [Epub ahead of print]131(21):
      Although serine metabolism plays a crucial role in the proliferation and survival of tumor cells, how it supports tumor cell migration remains poorly understood. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the oxidation of 3-phosphoglycerate to 3-phosphonooxypyruvate, the first committed step in de novo serine biosynthesis. Here we show that PHGDH was monoubiquitinated by cullin 4A-based E3 ligase complex at lysine 146 in colorectal cancer (CRC) cells, which enhanced PHGDH activity by recruiting a chaperone protein, DnaJ homolog subfamily A member 1, to promote its tetrameric formation, thereby increasing the levels of serine, glycine, and S-adenosylmethionine (SAM). Increased levels of SAM upregulated the expression of cell adhesion genes (laminin subunit gamma 2 and cysteine rich angiogenic inducer 61) by initiating SET domain containing 1A-mediated trimethylation of histone H3K4, thereby promoting tumor cell migration and CRC metastasis. Intriguingly, SAM levels in tumors or blood samples correlated with the metastatic recurrence of patients with CRC. Our finding not only reveals a potentially new role and mechanism of SAM-promoted tumor metastasis but also demonstrates a regulatory mechanism of PHGDH activity by monoubiquitination.
    Keywords:  Amino acid metabolism; Cell migration/adhesion; Colorectal cancer; Metabolism; Oncology
  32. Nat Cancer. 2021 Sep;2 978-993
      Multi-tyrosine kinase inhibitors (MTKIs) have thus far had limited success in the treatment of castration-resistant prostate cancer (CRPC). Here, we report a phase I-cleared orally bioavailable MTKI, ESK981, with a novel autophagy inhibitory property that decreased tumor growth in diverse preclinical models of CRPC. The anti-tumor activity of ESK981 was maximized in immunocompetent tumor environments where it upregulated CXCL10 expression through the interferon gamma pathway and promoted functional T cell infiltration, which resulted in enhanced therapeutic response to immune checkpoint blockade. Mechanistically, we identify the lipid kinase PIKfyve as the direct target of ESK981. PIKfyve-knockdown recapitulated ESK981's anti-tumor activity and enhanced the therapeutic benefit of immune checkpoint blockade. Our study reveals that targeting PIKfyve via ESK981 turns tumors from cold into hot through inhibition of autophagy, which may prime the tumor immune microenvironment in advanced prostate cancer patients and be an effective treatment strategy alone or in combination with immunotherapies.
  33. EMBO Mol Med. 2021 Nov 02. e14824
      The cardinal stages of macroautophagy are driven by core autophagy-related (ATG) proteins, whose ablation largely abolishes intracellular turnover. Disrupting ATG genes is paradigmatic of studying autophagy deficiency, yet emerging data suggest that ATG proteins have extensive biological importance beyond autophagic elimination. An important example is ATG7, an essential autophagy effector enzyme that in concert with other ATG proteins, also regulates immunity, cell death and protein secretion, and independently regulates the cell cycle and apoptosis. Recently, a direct association between ATG7 dysfunction and disease was established in patients with biallelic ATG7 variants and childhood-onset neuropathology. Moreover, a prodigious body of evidence supports a role for ATG7 in protecting against complex disease states in model organisms, although how dysfunctional ATG7 contributes to manifestation of these diseases, including cancer, neurodegeneration and infection, in humans remains unclear. Here, we systematically review the biological functions of ATG7, discussing the impact of its impairment on signalling pathways and human pathology. Future studies illuminating the molecular relationship between ATG7 dysfunction and disease will expedite therapies for disorders involving ATG7 deficiency and/or impaired autophagy.
    Keywords:  ATG7; autophagy; disease; neurodegeneration; therapeutics
  34. Hepatology. 2021 Nov 04.
      BACKGROUND & AIMS: Hepatic ischemia reperfusion (HIR) injury, a common clinical complication of liver transplantation and resection, affects patient prognosis. RNF5 is an E3 ubiquitin ligase that plays important roles in endoplasmic reticulum stress, unfolded protein reactions, and inflammatory responses; however, its role in HIR is unclear.APPROACH & RESULTS: RNF5 expression was significantly downregulated during HIR in mice and hepatocytes. Subsequently, RNF5 knockdown and overexpression cell lines were subjected to hypoxia-reoxygenation challenge. The results shown that RNF5 knockdown significantly increased hepatocyte inflammation and apoptosis, while RNF5 overexpression had the opposite effect. Furthermore, hepatocyte-specific RNF5 knockout and transgenic mice were established and subjected to HIR, and RNF5 deficiency markedly aggravated liver damage, cell apoptosis, and activated hepatic inflammatory responses. While hepatic RNF5 transgenic mice had the opposite effect compared with RNF5 knockout mice. Mechanistically, RNF5 interacted with PGAM5 and mediated the degradation of PGAM5 through K48-linked ubiquitination, thereby inhibiting the activation of ASK1 and its downstream JNK/p38. This eventually suppresses the inflammatory response and cell apoptosis in HIR.
    CONCLUSION: We revealed that RNF5 protected against HIR via its interaction with PGAM5 to inhibit the activation of ASK1 and the downstream JNK/p38 signaling cascade. Our findings indicate that the RNF5-PGAM5 axis may be a promising therapeutic target for HIR.
    Keywords:  ASK1; JNK/p38 signaling; PGAM5; RNF5; hepatic IRI
  35. J Cell Biol. 2022 Jan 03. pii: e202103033. [Epub ahead of print]221(1):
      The ESCRT protein CHMP2B and the RNA-binding protein TDP-43 are both associated with ALS and FTD. The pathogenicity of CHMP2B has mainly been considered a consequence of autophagy-endolysosomal dysfunction, whereas protein inclusions containing phosphorylated TDP-43 are a pathological hallmark of ALS and FTD. Intriguingly, TDP-43 pathology has not been associated with the FTD-causing CHMP2BIntron5 mutation. In this study, we identify CHMP2B as a modifier of TDP-43-mediated neurodegeneration in a Drosophila screen. Down-regulation of CHMP2B reduces TDP-43 phosphorylation and toxicity in flies and mammalian cells. Surprisingly, although CHMP2BIntron5 causes dramatic autophagy dysfunction, disturbance of autophagy does not alter TDP-43 phosphorylation levels. Instead, we find that inhibition of CK1, but not TTBK1/2 (all of which are kinases phosphorylating TDP-43), abolishes the modifying effect of CHMP2B on TDP-43 phosphorylation. Finally, we uncover that CHMP2B modulates CK1 protein levels by negatively regulating ubiquitination and the proteasome-mediated turnover of CK1. Together, our findings propose an autophagy-independent role and mechanism of CHMP2B in regulating CK1 abundance and TDP-43 phosphorylation.