bims-proteo Biomed News
on Proteostasis
Issue of 2021‒04‒04
forty-nine papers selected by
Eric Chevet
INSERM
  1. Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins.
  2. Silencing of the proteasome and oxidative stress impair endoplasmic reticulum targeting and signal cleavage of a prostate carcinoma antigen.
  3. The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore.
  4. ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.
  5. The CDC37-HSP90 chaperone complex co-translationally degrades the nascent kinase-dead mutant of HIPK2.
  6. Structure of Gcn1 bound to stalled and colliding 80S ribosomes.
  7. Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.
  8. Does Saccharomyces cerevisiae Require Specific Post-Translational Silencing against Leaky Translation of Hac1up?
  9. USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.
  10. New answers to the old RIDDLE: RNF168 and the DNA damage response pathway.
  11. How does Sec63 affect the conformation of Sec61 in yeast?
  12. The ménage à trois of autophagy, lipid droplets and liver disease.
  13. Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.
  14. Multiple ways for stress sensing and regulation of the endoplasmic reticulum-stress sensors.
  15. Negative Regulation of the Innate Immune Response through Proteasomal Degradation and Deubiquitination.
  16. Dysregulation of the Ubiquitin Proteasome System in Human Malignancies: A Window for Therapeutic Intervention.
  17. Acute ethanol stress induces sumoylation of conserved chromatin structural proteins in Saccharomyces cerevisiae.
  18. Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling.
  19. Effectors Targeting the Unfolded Protein Response during Intracellular Bacterial Infection.
  20. A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer.
  21. DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.
  22. Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.
  23. The Vhl E3 ubiquitin ligase complex regulates melanisation via sima, cnc and the copper import protein Ctr1A.
  24. A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.
  25. Differential impact of imipramine on thapsigargin- and tunicamycin-induced endoplasmic reticulum stress and mitochondrial dysfunction in neuroblastoma SH-SY5Y cells.
  26. Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex.
  27. Interactome Mapping of eIF3A in a Colon Cancer and an Immortalized Embryonic Cell Line Using Proximity-Dependent Biotin Identification.
  28. New functions of a known autophagy regulator: VCP and autophagy initiation.
  29. TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88.
  30. Disrupting progression of the yeast Hsp90 folding pathway at different transition points results in client-specific maturation defects.
  31. Emerging Roles of TRIM8 in Health and Disease.
  32. Mitochondrial quality control: from molecule to organelle.
  33. K29/K48-branched ubiquitin chains TRIP the alarm fueling neo-substrate degradation via the CRL2VHL.
  34. OsCBE1, a Substrate Receptor of Cullin4-Based E3 Ubiquitin Ligase, Functions as a Regulator of Abiotic Stress Response and Productivity in Rice.
  35. Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.
  36. Emc3 maintains intestinal homeostasis by preserving secretory lineages.
  37. The Role of Post-Translational Modifications in Targeting Protein Cargo to Extracellular Vesicles.
  38. Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.
  39. Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism.
  40. Ubiquitin-dependent regulation of transcription in development and disease.
  41. Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control.
  42. Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses.
  43. Development of a High-Throughput, Compound-Multiplexed Fluorescence Polarization Assay to Identify ATG5-ATG16L1 Protein-Protein Interaction Inhibitors.
  44. Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta.
  45. Quantification of eIF2α Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy.
  46. Loss of TANGO1 Leads to Absence of Bone Mineralization.
  47. OTUD1 Regulates Antifungal Innate Immunity through Deubiquitination of CARD9.
  48. Proteostatic imbalance and protein spreading in amyotrophic lateral sclerosis.
  49. Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells.
  1. J Cell Biol. 2021 May 03. pii: e202004086. [Epub ahead of print]220(5):
    Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins.
    Jacob A Culver, Malaiyalam Mariappan.
      Numerous proteins that have hydrophobic transmembrane domains (TMDs) traverse the cytosol and posttranslationally insert into cellular membranes. It is unclear how these hydrophobic membrane proteins evade recognition by the cytosolic protein quality control (PQC), which typically recognizes exposed hydrophobicity in misfolded proteins and marks them for proteasomal degradation by adding ubiquitin chains. Here, we find that tail-anchored (TA) proteins, a vital class of membrane proteins, are recognized by cytosolic PQC and are ubiquitinated as soon as they are synthesized in cells. Surprisingly, the ubiquitinated TA proteins are not routed for proteasomal degradation but instead are handed over to the targeting factor, TRC40, and delivered to the ER for insertion. The ER-associated deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins after their insertion into the ER. Thus, our data suggest that deubiquitinases rescue posttranslationally targeted membrane proteins that are inappropriately ubiquitinated by PQC in the cytosol.
    DOI:  https://doi.org/10.1083/jcb.202004086
  2. Biochem Biophys Res Commun. 2021 Mar 25. pii: S0006-291X(21)00405-8. [Epub ahead of print]554 56-62
    Silencing of the proteasome and oxidative stress impair endoplasmic reticulum targeting and signal cleavage of a prostate carcinoma antigen.
    Franziska Hoefer, Marcus Groettrup.
      The endoplasmic reticulum (ER) is an organelle with high protein density and therefore prone to be damaged by protein aggregates. One proposed preventive measure is a pre-emptive quality control pathway that attenuates ER import during protein folding stress. ER resident proteins are targeted into the ER via signal peptides cleaved rapidly upon ER insertion by the ER signal peptidase. Here we show that the ER insertion and cleavage of the ER-targeting peptide of the prostate carcinoma antigen prostate stem cell antigen (PSCA) is retarded and strongly reduced when the proteasome is inhibited or genetically silenced. Also overexpression of the C-terminally extended ubiquitin variant Ub2-UBB+1 or oxidative stress attenuated signal peptide processing. Proteasome inhibition likewise protracted ER signal processing of the ER targeted hormone leptin and the MHC class I molecule H-2Dd. These findings, which are consistent with a pre-emptive ER quality control pathway, may explain why an immunodominant MHC class I peptide ligand of PSCA spanning its ER signal peptidase cleavage site is efficiently generated in the cytoplasm from PSCA precursors that fail to reach the ER.
    Keywords:  Antigen processing; Endoplasmic reticulum; Prostate stem cell antigen; Proteostasis; Signal peptidase
    DOI:  https://doi.org/10.1016/j.bbrc.2021.03.023
  3. Autophagy. 2021 Mar 30. 1-15
    The BAX-binding protein MOAP1 associates with LC3 and promotes closure of the phagophore.
    Hao-Chun Chang, Ran N Tao, Chong Teik Tan, Ya Jun Wu, Boon Huat Bay, Victor C Yu.
      MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12-ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.Abbreviations CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.
    Keywords:  Autophagosome formation; LC3-binding protein; LIR motif; autophagy; cell death; nutrient deprivation
    DOI:  https://doi.org/10.1080/15548627.2021.1896157
  4. Mol Cell. 2021 Mar 25. pii: S1097-2765(21)00169-6. [Epub ahead of print]
    ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.
    Thanh Ngoc Nguyen, Benjamin Scott Padman, Susanne Zellner, Grace Khuu, Louise Uoselis, Wai Kit Lam, Marvin Skulsuppaisarn, Runa S J Lindblom, Emily M Watts, Christian Behrends, Michael Lazarou.
      The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.
    Keywords:  ATG4; ATG9a; FIB-SEM; LRBA; PINK1; Parkin; autophagosome; autophagy; mitochondria; mitophagy
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.001
  5. FEBS Lett. 2021 Mar 31.
    The CDC37-HSP90 chaperone complex co-translationally degrades the nascent kinase-dead mutant of HIPK2.
    Jan Paul Müller, Karl-Heinz Klempnauer.
      Homeodomain-interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto-phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin-dependent degradation. By comparing the expression of wild-type and kinase-defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co-translational degradation of kinase-defective versus wild-type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37-HSP90 chaperone complex and points to a novel role of CDC37 in the co-translational degradation of a client protein.
    DOI:  https://doi.org/10.1002/1873-3468.14080
  6. Proc Natl Acad Sci U S A. 2021 Apr 06. pii: e2022756118. [Epub ahead of print]118(14):
    Structure of Gcn1 bound to stalled and colliding 80S ribosomes.
    Agnieszka A Pochopien, Bertrand Beckert, Sergo Kasvandik, Otto Berninghausen, Roland Beckmann, Tanel Tenson, Daniel N Wilson.
      The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.
    Keywords:  Gcn1; disome; ribosome; stress; translation
    DOI:  https://doi.org/10.1073/pnas.2022756118
  7. FASEB J. 2021 May;35(5): e21515
    Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.
    Yumeng Huang, Xiaoxi Xu, Peter Arvan, Ming Liu.
      The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPβ/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPβ/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic β-cells with deficient expression of either TRAPβ/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPβ/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPβ/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPβ/SSR2 or TRAPδ/SSR4 null mutant β-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in β-cells with diminished expression of TRAPβ/SSR2, strongly suggesting that TRAPβ/SSR2 is needed to support TRAPα/SSR1 function.
    Keywords:  diabetes; insulin secretion; pancreatic beta cell; preproinsulin; translocation
    DOI:  https://doi.org/10.1096/fj.202002774R
  8. Microorganisms. 2021 Mar 17. pii: 620. [Epub ahead of print]9(3):
    Does Saccharomyces cerevisiae Require Specific Post-Translational Silencing against Leaky Translation of Hac1up?
    Ali Tehfe, Talia Roseshter, Yulong Wei, Xuhua Xia.
      HAC1 encodes a key transcription factor that transmits the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus and regulates downstream UPR genes in Saccharomyces cerevisiae. In response to the accumulation of unfolded proteins in the ER, Ire1p oligomers splice HAC1 pre-mRNA (HAC1u) via a non-conventional process and allow the spliced HAC1 (HAC1i) to be translated efficiently. However, leaky splicing and translation of HAC1u may occur in non-UPR cells to induce undesirable UPR. To control accidental UPR activation, multiple fail-safe mechanisms have been proposed to prevent leaky HAC1 splicing and translation and to facilitate rapid degradation of translated Hac1up and Hac1ip. Among proposed regulatory mechanisms is a degron sequence encoded at the 5' end of the HAC1 intron that silences Hac1up expression. To investigate the necessity of an intron-encoded degron sequence that specifically targets Hac1up for degradation, we employed publicly available transcriptomic data to quantify leaky HAC1 splicing and translation in UPR-induced and non-UPR cells. As expected, we found that HAC1u is only efficiently spliced into HAC1i and efficiently translated into Hac1ip in UPR-induced cells. However, our analysis of ribosome profiling data confirmed frequent occurrence of leaky translation of HAC1u regardless of UPR induction, demonstrating the inability of translation fail-safe to completely inhibit Hac1up production. Additionally, among 32 yeast HAC1 surveyed, the degron sequence is highly conserved by Saccharomyces yeast but is poorly conserved by all other yeast species. Nevertheless, the degron sequence is the most conserved HAC1 intron segment in yeasts. These results suggest that the degron sequence may indeed play an important role in mitigating the accumulation of Hac1up to prevent accidental UPR activation in the Saccharomyces yeast.
    Keywords:  HAC1; degron; non-spliceosome splicing; unfolded protein response
    DOI:  https://doi.org/10.3390/microorganisms9030620
  9. EMBO Rep. 2021 Mar 30. e51415
    USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.
    Nicole Giebel, Anchel de Jaime-Soguero, Ana García Del Arco, Jonathan J M Landry, Marlene Tietje, Laura Villacorta, Vladimir Benes, Vanesa Fernández-Sáiz, Sergio P Acebrón.
      The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R-spondin induces auto-ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R-spondin by protecting ZNRF3/RNF43 from ubiquitin-dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R-spondin-LGR4-ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R-spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.
    Keywords:  EMT; LGR4/5/6; colorectal cancer; deubiquitination; mouse intestinal organoids
    DOI:  https://doi.org/10.15252/embr.202051415
  10. FEBS J. 2021 Apr 02.
    New answers to the old RIDDLE: RNF168 and the DNA damage response pathway.
    Jessica Kelliher, Gargi Ghosal, Justin Wai Chung Leung.
      The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination plays an integral role in regulating cellular processes including DNA damage signaling and repair. The ubiquitin E3 ligase RNF168 is essential in assembling a cohort of DNA repair proteins at the damaged chromatin via its enzymatic activity. RNF168 ubiquitinates histone H2A(X) at the N-terminus and generates a specific docking scaffold for ubiquitin binding motif-containing proteins. The regulation of RNF168 at damaged chromatin and the mechanistic implication in the recruitment of DNA repair proteins to the damaged sites remains an area of active investigation. Here, we review the function and regulation of RNF168 in the context of ubiquitin-mediated DNA damage signaling and repair. We will also discuss the unanswered questions that require further investigation and how understanding RNF168 targeting specificity could benefit the therapeutic development for cancer treatment.
    Keywords:  DNA double strand break; DNA repair; RIDDLE syndrome; RIDDLIN; ubiquitin
    DOI:  https://doi.org/10.1111/febs.15857
  11. PLoS Comput Biol. 2021 Mar 29. 17(3): e1008855
    How does Sec63 affect the conformation of Sec61 in yeast?
    Pratiti Bhadra, Lalitha Yadhanapudi, Karin Römisch, Volkhard Helms.
      The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the accessory membrane protein Sec62 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.
    DOI:  https://doi.org/10.1371/journal.pcbi.1008855
  12. Autophagy. 2021 Apr 02. 1-24
    The ménage à trois of autophagy, lipid droplets and liver disease.
    Yasmina Filali-Mouncef, Catherine Hunter, Federica Roccio, Stavroula Zagkou, Nicolas Dupont, Charlotte Primard, Tassula Proikas-Cezanne, Fulvio Reggiori.
      Autophagic pathways cross with lipid homeostasis and thus provide energy and essential building blocks that are indispensable for liver functions. Energy deficiencies are compensated by breaking down lipid droplets (LDs), intracellular organelles that store neutral lipids, in part by a selective type of autophagy, referred to as lipophagy. The process of lipophagy does not appear to be properly regulated in fatty liver diseases (FLDs), an important risk factor for the development of hepatocellular carcinomas (HCC). Here we provide an overview on our current knowledge of the biogenesis and functions of LDs, and the mechanisms underlying their lysosomal turnover by autophagic processes. This review also focuses on nonalcoholic steatohepatitis (NASH), a specific type of FLD characterized by steatosis, chronic inflammation and cell death. Particular attention is paid to the role of macroautophagy and macrolipophagy in relation to the parenchymal and non-parenchymal cells of the liver in NASH, as this disease has been associated with inappropriate lipophagy in various cell types of the liver.Abbreviations: ACAT: acetyl-CoA acetyltransferase; ACAC/ACC: acetyl-CoA carboxylase; AKT: AKT serine/threonine kinase; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BECN1/Vps30/Atg6: beclin 1; BSCL2/seipin: BSCL2 lipid droplet biogenesis associated, seipin; CMA: chaperone-mediated autophagy; CREB1/CREB: cAMP responsive element binding protein 1; CXCR3: C-X-C motif chemokine receptor 3; DAGs: diacylglycerols; DAMPs: danger/damage-associated molecular patterns; DEN: diethylnitrosamine; DGAT: diacylglycerol O-acyltransferase; DNL: de novo lipogenesis; EHBP1/NACSIN (EH domain binding protein 1); EHD2/PAST2: EH domain containing 2; CoA: coenzyme A; CCL/chemokines: chemokine ligands; CCl4: carbon tetrachloride; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FA: fatty acid; FFAs: free fatty acids; FFC: high saturated fats, fructose and cholesterol; FGF21: fibroblast growth factor 21; FITM/FIT: fat storage inducing transmembrane protein; FLD: fatty liver diseases; FOXO: forkhead box O; GABARAP: GABA type A receptor-associated protein; GPAT: glycerol-3-phosphate acyltransferase; HCC: hepatocellular carcinoma; HDAC6: histone deacetylase 6; HECT: homologous to E6-AP C-terminus; HFCD: high fat, choline deficient; HFD: high-fat diet; HSCs: hepatic stellate cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; ITCH/AIP4: itchy E3 ubiquitin protein ligase; KCs: Kupffer cells; LAMP2A: lysosomal associated membrane protein 2A; LDs: lipid droplets; LDL: low density lipoprotein; LEP/OB: leptin; LEPR/OBR: leptin receptor; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIR: LC3-interacting region; LPS: lipopolysaccharide; LSECs: liver sinusoidal endothelial cells; MAGs: monoacylglycerols; MAPK: mitogen-activated protein kinase; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCD: methionine-choline deficient; MGLL/MGL: monoglyceride lipase; MLXIPL/ChREBP: MLX interacting protein like; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver disease; NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; NPC: NPC intracellular cholesterol transporter; NR1H3/LXRα: nuclear receptor subfamily 1 group H member 3; NR1H4/FXR: nuclear receptor subfamily 1 group H member 4; PDGF: platelet derived growth factor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA: patatin like phospholipase domain containing; PNPLA2/ATGL: patatin like phospholipase domain containing 2; PNPLA3/adiponutrin: patatin like phospholipase domain containing 3; PPAR: peroxisome proliferator activated receptor; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARD/PPARδ: peroxisome proliferator activated receptor delta; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SE: sterol esters; SIRT1: sirtuin 1; SPART/SPG20: spartin; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; TAGs: triacylglycerols; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TGFB1/TGFβ: transforming growth factor beta 1; Ub: ubiquitin; UBE2G2/UBC7: ubiquitin conjugating enzyme E2 G2; ULK1/Atg1: unc-51 like autophagy activating kinase 1; USF1: upstream transcription factor 1; VLDL: very-low density lipoprotein; VPS: vacuolar protein sorting; WIPI: WD-repeat domain, phosphoinositide interacting; WDR: WD repeat domain.
    Keywords:  Chaperone-mediated autophagy; fibrosis; hepatocellular carcinoma; macroautophagy; macrolipophagy; microautophagy; microlipophagy; nafld; nash; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis
    DOI:  https://doi.org/10.1080/15548627.2021.1895658
  13. FASEB J. 2021 May;35(5): e21349
    Pathological mechanisms of vacuolar aggregate myopathy arising from a Casq1 mutation.
    Amy D Hanna, Chang Seok Lee, Lyle Babcock, Hui Wang, Joseph Recio, Susan L Hamilton.
      Mice with a mutation (D244G, DG) in calsequestrin 1 (CASQ1), analogous to a human mutation in CASQ1 associated with a delayed onset human myopathy (vacuolar aggregate myopathy), display a progressive myopathy characterized by decreased activity, decreased ability of fast twitch muscles to generate force and low body weight after one year of age. The DG mutation causes CASQ1 to partially dissociate from the junctional sarcoplasmic reticulum (SR) and accumulate in the endoplasmic reticulum (ER). Decreased junctional CASQ1 reduces SR Ca2+ release. Muscles from older DG mice display ER stress, ER expansion, increased mTOR signaling, inadequate clearance of aggregated proteins by the proteasomes, and elevation of protein aggregates and lysosomes. This study suggests that the myopathy associated with the D244G mutation in CASQ1 is driven by CASQ1 mislocalization, reduced SR Ca2+ release, CASQ1 misfolding/aggregation and ER stress. The subsequent maladaptive increase in protein synthesis and decreased protein aggregate clearance are likely to contribute to disease progression.
    Keywords:  EC coupling; ER stress; calsequestrin; myopathy; sarcoplasmic reticulum
    DOI:  https://doi.org/10.1096/fj.202001653RR
  14. Cell Struct Funct. 2021 Mar 26.
    Multiple ways for stress sensing and regulation of the endoplasmic reticulum-stress sensors.
    Quynh Giang Le, Yukio Kimata.
      Dysfunction of the endoplasmic reticulum (ER), so-called ER stress, is accompanied with accumulation of unfolded proteins in the ER. Eukaryotic cells commonly have an ER-located transmembrane protein, Ire1, which triggers cellular protective events against ER stress. In animal cells, PERK and ATF6 also initiate the ER-stress response. As a common strategy to control the activity of these ER-stress sensors, an ER-resident molecular chaperone, BiP, serves as their negative regulator, and dissociates from them in response to ER stress. Although it sounds reasonable that unfolded proteins and Ire1 compete for BiP association, some publications argue against this competition model. Moreover, yeast Ire1 (and possibly also the mammalian major Ire1 paralogue IRE1α) directly detects ER-accumulated unfolded proteins, and subsequently oligomerizes for its further activation. Apart from protein misfolding, the saturation of membrane phospholipids is another outcome of ER-stressing stimuli, which is sensed by the transmembrane domain of Ire1. This review describes the canonical and up-to-date insights concerning stress-sensing and regulatory mechanisms of yeast Ire1 and metazoan ER-stress sensors.Key words: endoplasmic reticulum, stress, unfolded protein response, molecular chaperone.
    Keywords:  endoplasmic reticulum; molecular chaperone; stress; unfolded protein response
    DOI:  https://doi.org/10.1247/csf.21015
  15. Viruses. 2021 Mar 30. pii: 584. [Epub ahead of print]13(4):
    Negative Regulation of the Innate Immune Response through Proteasomal Degradation and Deubiquitination.
    Valentina Budroni, Gijs A Versteeg.
      The rapid and dynamic activation of the innate immune system is achieved through complex signaling networks regulated by post-translational modifications modulating the subcellular localization, activity, and abundance of signaling molecules. Many constitutively expressed signaling molecules are present in the cell in inactive forms, and become functionally activated once they are modified with ubiquitin, and, in turn, inactivated by removal of the same post-translational mark. Moreover, upon infection resolution a rapid remodeling of the proteome needs to occur, ensuring the removal of induced response proteins to prevent hyperactivation. This review discusses the current knowledge on the negative regulation of innate immune signaling pathways by deubiquitinating enzymes, and through degradative ubiquitination. It focusses on spatiotemporal regulation of deubiquitinase and E3 ligase activities, mechanisms for re-establishing proteostasis, and degradation through immune-specific feedback mechanisms vs. general protein quality control pathways.
    Keywords:  E3 ligase; cytokine; deubiquitinase; innate immune system; proteasome; ubiquitin
    DOI:  https://doi.org/10.3390/v13040584
  16. Cancers (Basel). 2021 Mar 25. pii: 1513. [Epub ahead of print]13(7):
    Dysregulation of the Ubiquitin Proteasome System in Human Malignancies: A Window for Therapeutic Intervention.
    Chee Wai Fhu, Azhar Ali.
      The ubiquitin proteasome system (UPS) governs the non-lysosomal degradation of oxidized, damaged, or misfolded proteins in eukaryotic cells. This process is tightly regulated through the activation and transfer of polyubiquitin chains to target proteins which are then recognized and degraded by the 26S proteasome complex. The role of UPS is crucial in regulating protein levels through degradation to maintain fundamental cellular processes such as growth, division, signal transduction, and stress response. Dysregulation of the UPS, resulting in loss of ability to maintain protein quality through proteolysis, is closely related to the development of various malignancies and tumorigenesis. Here, we provide a comprehensive general overview on the regulation and roles of UPS and discuss functional links of dysregulated UPS in human malignancies. Inhibitors developed against components of the UPS, which include U.S. Food and Drug Administration FDA-approved and those currently undergoing clinical trials, are also presented in this review.
    Keywords:  cancer; chemoresistance; dysregulation; inhibitors; therapy; ubiquitin proteasome system
    DOI:  https://doi.org/10.3390/cancers13071513
  17. Mol Biol Cell. 2021 Mar 31. mbcE20110715
    Acute ethanol stress induces sumoylation of conserved chromatin structural proteins in Saccharomyces cerevisiae.
    Amanda I Bradley, Nicole M Marsh, Heather R Borror, Kaitlyn E Mostoller, Amber I Gama, Richard G Gardner.
      Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the post-translational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. We found that cells exhibit a transient sumoylation response after acute exposure to ≤ 7.5% ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin-structural proteins.
    DOI:  https://doi.org/10.1091/mbc.E20-11-0715
  18. Cell Rep. 2021 Mar 30. pii: S2211-1247(21)00219-9. [Epub ahead of print]34(13): 108905
    Ogt controls neural stem/progenitor cell pool and adult neurogenesis through modulating Notch signaling.
    Junchen Chen, Xiaoxue Dong, Xuejun Cheng, Qiang Zhu, Jinyu Zhang, Qian Li, Xiaoli Huang, Min Wang, Liping Li, Weixiang Guo, Binggui Sun, Qiang Shu, Wen Yi, Xuekun Li.
      Ogt catalyzed O-linked N-acetylglucosamine (O-GlcNAcylation, O-GlcNAc) plays an important function in diverse biological processes and diseases. However, the roles of Ogt in regulating neurogenesis remain largely unknown. Here, we show that Ogt deficiency or depletion in adult neural stem/progenitor cells (aNSPCs) leads to the diminishment of the aNSPC pool and aberrant neurogenesis and consequently impairs cognitive function in adult mice. RNA sequencing reveals that Ogt deficiency alters the transcription of genes relating to cell cycle, neurogenesis, and neuronal development. Mechanistic studies show that Ogt directly interacts with Notch1 and catalyzes the O-GlcNAc modification of Notch TM/ICD fragment. Decreased O-GlcNAc modification of TM/ICD increases the binding of E3 ubiquitin ligase Itch to TM/ICD and promotes its degradation. Itch knockdown rescues neurogenic defects induced by Ogt deficiency in vitro and in vivo. Our findings reveal the essential roles and mechanisms of Ogt and O-GlcNAc modification in regulating mammalian neurogenesis and cognition.
    Keywords:  O-GlcNAcylation; Ogt; cognition; neural stem/progenitor cells; neurogenesis; notch; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.celrep.2021.108905
  19. Microorganisms. 2021 Mar 29. pii: 705. [Epub ahead of print]9(4):
    Effectors Targeting the Unfolded Protein Response during Intracellular Bacterial Infection.
    Manal H Alshareef, Elizabeth L Hartland, Kathleen McCaffrey.
      The unfolded protein response (UPR) is a homeostatic response to endoplasmic reticulum (ER) stress within eukaryotic cells. The UPR initiates transcriptional and post-transcriptional programs to resolve ER stress; or, if ER stress is severe or prolonged, initiates apoptosis. ER stress is a common feature of bacterial infection although the role of the UPR in host defense is only beginning to be understood. While the UPR is important for host defense against pore-forming toxins produced by some bacteria, other bacterial effector proteins hijack the UPR through the activity of translocated effector proteins that facilitate intracellular survival and proliferation. UPR-mediated apoptosis can limit bacterial replication but also often contributes to tissue damage and disease. Here, we discuss the dual nature of the UPR during infection and the implications of UPR activation or inhibition for inflammation and immunity as illustrated by different bacterial pathogens.
    Keywords:  ER stress; UPR; bacteria; effector proteins; infection; secretion systems
    DOI:  https://doi.org/10.3390/microorganisms9040705
  20. Methods Mol Biol. 2021 ;2267 91-102
    A Novel Strategy to Track Lysine-48 Ubiquitination by Fluorescence Resonance Energy Transfer.
    Kenneth Wu, Zhen-Qiang Pan.
      Posttranslational modification of protein by lysine-48 (K48) linked ubiquitin (Ub) chains is the major cellular mechanism for selective protein degradation that critically impacts biological processes such as cell cycle checkpoints. In this chapter, we describe an in vitro biochemical approach to detect a K48-linked di-Ub chain by fluorescence resonance energy transfer (FRET). To this end, we detail methods for the preparation of the relevant enzymes and substrates, as well as for the execution of the reaction with high efficiency. Tracking K48 polyubiquitination using this sensitive and highly reproducible format provides an opportunity for high-throughput screening that leads to identification of small molecule modulators capable of changing ubiquitination for improving human health.
    Keywords:  Cullin-RING E3 ubiquitin ligase; E2 Cdc34; FRET; Fluorescently labeled ubiquitin; K48 ubiquitination
    DOI:  https://doi.org/10.1007/978-1-0716-1217-0_7
  21. J Am Chem Soc. 2021 Mar 30.
    DCAF11 Supports Targeted Protein Degradation by Electrophilic Proteolysis-Targeting Chimeras.
    Xiaoyu Zhang, Lena M Luukkonen, Christie L Eissler, Vincent M Crowley, Yu Yamashita, Michael A Schafroth, Shota Kikuchi, David S Weinstein, Kent T Symons, Brian E Nordin, Joe L Rodriguez, Thomas G Wucherpfennig, Ludwig G Bauer, Melissa M Dix, Dean Stamos, Todd M Kinsella, Gabriel M Simon, Kristen A Baltgalvis, Benjamin F Cravatt.
      Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.
    DOI:  https://doi.org/10.1021/jacs.1c00990
  22. Int J Mol Sci. 2021 Mar 27. pii: 3463. [Epub ahead of print]22(7):
    Feedback Regulation of O-GlcNAc Transferase through Translation Control to Maintain Intracellular O-GlcNAc Homeostasis.
    Chia-Hung Lin, Chen-Chung Liao, Mei-Yu Chen, Teh-Ying Chou.
      Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.
    Keywords:  O-GlcNAc homeostasis; O-GlcNAc transferase (OGT); O-GlcNAcase (OGA); O-GlcNAcylation; O-linked N-acetylglucosamine (O-GlcNAc); epigenetics; eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1); histone deacetylase (HDAC); post-translational modification; translation control
    DOI:  https://doi.org/10.3390/ijms22073463
  23. Biochim Biophys Acta Mol Cell Res. 2021 Mar 25. pii: S0167-4889(21)00076-8. [Epub ahead of print] 119022
    The Vhl E3 ubiquitin ligase complex regulates melanisation via sima, cnc and the copper import protein Ctr1A.
    Bichao Zhang, Lauren Kirn, Richard Burke.
      VHL encodes a tumour suppressor, which possesses E3 ubiquitin ligase activity in complex with EloC and Cul2. In tumour cells or in response to hypoxia, VHL activity is lost, causing accumulation of the transcription factor HIF-1alpha. In this study, we demonstrated that in Drosophila, Rpn9, a regulatory component of the 26 s proteasome, participates in the Vhl-induced proteasomal degradation of sima, the Drosophila orthologue of HIF-1alpha. Knockdown of Vhl induces increased melanisation in the adult fly thorax and concurrent decrease in pigmentation in the abdomen. Both these defects are rescued by knockdown of sima and partially by knockdown of cnc, which encodes the fly orthologue of the transcription factor Nrf2, the master regulator of oxidative stress response. We further show that sima overexpression and Rpn9 knockdown both result in post-translational down-regulation of the copper uptake transporter Ctr1A in the fly eye and that Ctr1A expression exacerbates Vhl knockdown defects in the thorax and rescues these defects in the abdomen. We conclude that Vhl negatively regulates both sima and cnc and that in the absence of Vhl, these transcription factors interact to regulate Ctr1A, copper uptake and consequently melanin formation. We propose a model whereby the co-regulatory relationship between sima and cnc flips between thorax and abdomen: in the thorax, sima is favoured leading to upregulation of Ctr1A; in the abdomen, cnc dominates, resulting in the post-translational downregulation of Ctr1A.
    Keywords:  Ctr1A copper transporter; HIF-1 / sima; Melanisation; Nrf2 / cnc; Rpn9; Vhl E3 ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.bbamcr.2021.119022
  24. Proc Natl Acad Sci U S A. 2021 Apr 06. pii: e2018089118. [Epub ahead of print]118(14):
    A molecular link between cell wall biosynthesis, translation fidelity, and stringent response in Streptococcus pneumoniae.
    Surya D Aggarwal, Adrian J Lloyd, Saigopalakrishna S Yerneni, Ana Rita Narciso, Jennifer Shepherd, David I Roper, Christopher G Dowson, Sergio R Filipe, N Luisa Hiller.
      Survival in the human host requires bacteria to respond to unfavorable conditions. In the important Gram-positive pathogen Streptococcus pneumoniae, cell wall biosynthesis proteins MurM and MurN are tRNA-dependent amino acyl transferases which lead to the production of branched muropeptides. We demonstrate that wild-type cells experience optimal growth under mildly acidic stressed conditions, but ΔmurMN strain displays growth arrest and extensive lysis. Furthermore, these stress conditions compromise the efficiency with which alanyl-tRNAAla synthetase can avoid noncognate mischarging of tRNAAla with serine, which is toxic to cells. The observed growth defects are rescued by inhibition of the stringent response pathway or by overexpression of the editing domain of alanyl-tRNAAla synthetase that enables detoxification of tRNA misacylation. Furthermore, MurM can incorporate seryl groups from mischarged Seryl-tRNAAla UGC into cell wall precursors with exquisite specificity. We conclude that MurM contributes to the fidelity of translation control and modulates the stress response by decreasing the pool of mischarged tRNAs. Finally, we show that enhanced lysis of ΔmurMN pneumococci is caused by LytA, and the murMN operon influences macrophage phagocytosis in a LytA-dependent manner. Thus, MurMN attenuates stress responses with consequences for host-pathogen interactions. Our data suggest a causal link between misaminoacylated tRNA accumulation and activation of the stringent response. In order to prevent potential corruption of translation, consumption of seryl-tRNAAla by MurM may represent a first line of defense. When this mechanism is overwhelmed or absent (ΔmurMN), the stringent response shuts down translation to avoid toxic generation of mistranslated/misfolded proteins.
    Keywords:  Streptococcus pneumoniae; autolysis; cell wall; stringent response; translation quality control
    DOI:  https://doi.org/10.1073/pnas.2018089118
  25. Eur J Pharmacol. 2021 Mar 30. pii: S0014-2999(21)00226-0. [Epub ahead of print] 174073
    Differential impact of imipramine on thapsigargin- and tunicamycin-induced endoplasmic reticulum stress and mitochondrial dysfunction in neuroblastoma SH-SY5Y cells.
    Maria Brodnanova, Zuzana Hatokova, Andrea Evinova, Michal Cibulka, Peter Racay.
      The aim of our work was to study effect of antidepressant imipramine on both thapsigargin- and tunicamycin-induced ER stress and mitochondrial dysfunction in neuroblastoma SH-SY5Y cells. ER stress in SH-SY5Y cells was induced by either tunicamycin or thapsigargin in the presence or absence of imipramine. Cell viability was tested by the MTT assay. Splicing of XBP1 mRNA was studied by RT-PCR. Finally, expression of Hrd1 and Hsp60 was determined by western blot analysis. Our findings provide evidence that at high concentrations imipramine potentiates ER stress-induced death of SH-SY5Y cells. The effect of imipramine on ER stress-induced death of SH-SY5Y cells was stronger in combination of imipramine with thapsigargin. In addition, we have found that treatment of SH-SY5Y cells with imipramine in combination of either thapsigargin or tunicamycin is associated with the alteration of ER stress-induced IRE1α-XBP1 signalling. Despite potentiation of ER stress-induced XBP1 splicing, imipramine suppresses both thapsigargin- and tunicamycin-induced expression of Hrd1. Finally, imipramine in combination with thapsigargin-but not tunicamycin-aggravates ER stress-induced mitochondrial dysfunction without significant impact on intracellular mitochondrial content as indicated by the unaltered expression of Hsp60. Our results indicate the possibility that chronic treatment with imipramine might be associated with a higher risk of development and progression of neurodegenerative disorders, in particular those allied with ER stress and mitochondrial dysfunction like Parkinson's and Alzheimer's disease.
    Keywords:  cell death; endoplasmic reticulum; imipramine; mitochondria; neurodegenerative disorders; unfolded protein response
    DOI:  https://doi.org/10.1016/j.ejphar.2021.174073
  26. J Biol Chem. 2021 Mar 24. pii: S0021-9258(21)00366-5. [Epub ahead of print] 100586
    Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex.
    Géssica C Barros, Rodrigo D Requião, Rodolfo L Carneiro, Claudio A Masuda, Mariana H Moreira, Silvana Rossetto, Tatiana Domitrovic, Fernando L Palhano.
      Previous work has suggested that highly positively charged protein segments coded by rare codons or poly (A) stretches induce ribosome stalling and translational arrest through electrostatic interactions with the negatively charged ribosome exit tunnel, leading to inefficient elongation. This arrest leads to the activation of the Ribosome Quality Control (RQC) pathway and results in low expression of these reporter proteins. However, the only endogenous yeast proteins known to activate the RQC are Rqc1, a protein essential for RQC function, and Sdd1, a protein with unknown function, both of which contain polybasic sequences. To explore the generality of this phenomenon, we investigated whether the RQC complex controls the expression of other proteins with polybasic sequences. We showed by ribosome profiling data analysis and western blot that proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, were not targeted by the RQC complex. We also observed that the previously reported Ltn1-dependent regulation of Rqc1 is post-translational, independent of the RQC activity. Taken together, our results suggest that RQC should not be regarded as a general regulatory pathway for the expression of highly positively charged proteins in yeast.
    Keywords:  poly (A) tracts; polybasic sequences; protein synthesis; ribosome; ribosome profiling; ribosome quality control; stalled polypeptides; translation; translation control; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.jbc.2021.100586
  27. Cancers (Basel). 2021 Mar 14. pii: 1293. [Epub ahead of print]13(6):
    Interactome Mapping of eIF3A in a Colon Cancer and an Immortalized Embryonic Cell Line Using Proximity-Dependent Biotin Identification.
    Diep-Khanh Vo, Alexander Engler, Darko Stoimenovski, Roland Hartig, Thilo Kaehne, Thomas Kalinski, Michael Naumann, Johannes Haybaeck, Norbert Nass.
      Translation initiation comprises complex interactions of eukaryotic initiation factor (eIF) subunits and the structural elements of the mRNAs. Translation initiation is a key process for building the cell's proteome. It not only determines the total amount of protein synthesized but also controls the translation efficiency for individual transcripts, which is important for cancer or ageing. Thus, understanding protein interactions during translation initiation is one key that contributes to understanding how the eIF subunit composition influences translation or other pathways not yet attributed to eIFs. We applied the BioID technique to two rapidly dividing cell lines (the immortalized embryonic cell line HEK-293T and the colon carcinoma cell line HCT-166) in order to identify interacting proteins of eIF3A, a core subunit of the eukaryotic initiation factor 3 complex. We identified a total of 84 interacting proteins, with very few proteins being specific to one cell line. When protein biosynthesis was blocked by thapsigargin-induced endoplasmic reticulum (ER) stress, the interacting proteins were considerably smaller in number. In terms of gene ontology, although eIF3A interactors are mainly part of the translation machinery, protein folding and RNA binding were also found. Cells suffering from ER-stress show a few remaining interactors which are mainly ribosomal proteins or involved in RNA-binding.
    Keywords:  BioID; ER-stress; cancer; eukaryotic translation initiation factor; interactome
    DOI:  https://doi.org/10.3390/cancers13061293
  28. Autophagy. 2021 Mar 31. 1-2
    New functions of a known autophagy regulator: VCP and autophagy initiation.
    Yuchen Lei, Daniel J Klionsky.
      VCP, a conserved ATPase, is involved in several cellular processes, and mutations in this protein are associated with various diseases. VCP also plays a role in autophagosome maturation. However, because a deficiency in autophagosome maturation presents a readily observable phenotype, other roles of VCP in autophagy regulation, in particular in the initial steps of autophagosome formation, may have been overlooked. In a recently published paper, using small-molecule inhibitors, Hill et al. showed that VCP regulates autophagy initiation through both stabilization of BECN1 and enhancement of phosphati-dylinositol 3-kinase (PtdIns3K) complex assembly.
    Keywords:  ATXN3; BECN1; Ptdins3K; VCP; autophagy initiation
    DOI:  https://doi.org/10.1080/15548627.2021.1905974
  29. PLoS Pathog. 2021 Mar 31. 17(3): e1009481
    TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88.
    Jia-Qi Fang, Qian Ou, Jun Pan, Jie Fang, Da-Yong Zhang, Miao-Qi Qiu, Yue-Qi Li, Xiao-Hui Wang, Xue-Yu Yang, Zhe Chi, Wei Gao, Jun-Ping Guo, Thomas Miethke, Jian-Ping Pan.
      TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.
    DOI:  https://doi.org/10.1371/journal.ppat.1009481
  30. Genetics. 2021 Mar 31. pii: iyab009. [Epub ahead of print]217(3):
    Disrupting progression of the yeast Hsp90 folding pathway at different transition points results in client-specific maturation defects.
    Kaitlyn Hohrman, Davi Gonçalves, Kevin A Morano, Jill L Johnson.
      The protein molecular chaperone Hsp90 (Heat shock protein, 90 kilodalton) plays multiple roles in the biogenesis and regulation of client proteins impacting myriad aspects of cellular physiology. Amino acid alterations located throughout Saccharomyces cerevisiae Hsp90 have been shown to result in reduced client activity and temperature-sensitive growth defects. Although some Hsp90 mutants have been shown to affect activity of particular clients more than others, the mechanistic basis of client-specific effects is unknown. We found that Hsp90 mutants that disrupt the early step of Hsp70 and Sti1 interaction, or show reduced ability to adopt the ATP-bound closed conformation characterized by Sba1 and Cpr6 interaction, similarly disrupt activity of three diverse clients, Utp21, Ssl2, and v-src. In contrast, mutants that appear to alter other steps in the folding pathway had more limited effects on client activity. Protein expression profiling provided additional evidence that mutants that alter similar steps in the folding cycle cause similar in vivo consequences. Our characterization of these mutants provides new insight into how Hsp90 and cochaperones identify and interact with diverse clients, information essential for designing pharmaceutical approaches to selectively inhibit Hsp90 function.
    Keywords:  Hop/Sti1; cochaperone; heat shock factor; molecular chaperone; tetratricopeptide repeats
    DOI:  https://doi.org/10.1093/genetics/iyab009
  31. Cells. 2021 Mar 05. pii: 561. [Epub ahead of print]10(3):
    Emerging Roles of TRIM8 in Health and Disease.
    Flaviana Marzano, Luisa Guerrini, Graziano Pesole, Elisabetta Sbisà, Apollonia Tullo.
      The superfamily of TRIM (TRIpartite Motif-containing) proteins is one of the largest groups of E3 ubiquitin ligases. Among them, interest in TRIM8 has greatly increased in recent years. In this review, we analyze the regulation of TRIM8 gene expression and how it is involved in many cell reactions in response to different stimuli such as genotoxic stress and attacks by viruses or bacteria, playing a central role in the immune response and orchestrating various fundamental biological processes such as cell survival, carcinogenesis, autophagy, apoptosis, differentiation and inflammation. Moreover, we show how TRIM8 functions are not limited to ubiquitination, and contrasting data highlight its role either as an oncogene or as a tumor suppressor gene, acting as a "double-edged weapon". This is linked to its involvement in the selective regulation of three pivotal cellular signaling pathways: the p53 tumor suppressor, NF-κB and JAK-STAT pathways. Lastly, we describe how TRIM8 dysfunctions are linked to inflammatory processes, autoimmune disorders, rare developmental and cardiovascular diseases, ischemia, intellectual disability and cancer.
    Keywords:  JAK-STAT; NF-κB; TRIM8; p53
    DOI:  https://doi.org/10.3390/cells10030561
  32. Cell Mol Life Sci. 2021 Mar 29.
    Mitochondrial quality control: from molecule to organelle.
    Alba Roca-Portoles, Stephen W G Tait.
      Mitochondria are organelles central to myriad cellular processes. To maintain mitochondrial health, various processes co-operate at both the molecular and organelle level. At the molecular level, mitochondria can sense imbalances in their homeostasis and adapt to these by signaling to the nucleus. This mito-nuclear communication leads to the expression of nuclear stress response genes. Upon external stimuli, mitochondria can also alter their morphology accordingly, by inducing fission or fusion. In an extreme situation, mitochondria are degraded by mitophagy. Adequate function and regulation of these mitochondrial quality control pathways are crucial for cellular homeostasis. As we discuss, alterations in these processes have been linked to several pathologies including neurodegenerative diseases and cancer.
    Keywords:  ISR; Mitochondrial diseases; Mitochondrial dysfunction; Mitochondrial fission; Mitochondrial fusion; Mitophagy; PINK1; Parkin; UPRmt
    DOI:  https://doi.org/10.1007/s00018-021-03775-0
  33. Mol Cell. 2021 Apr 01. pii: S1097-2765(21)00140-4. [Epub ahead of print]81(7): 1363-1365
    K29/K48-branched ubiquitin chains TRIP the alarm fueling neo-substrate degradation via the CRL2VHL.
    Charlene M Magtoto, Rebecca Feltham.
      In this issue of Molecular Cell, Kaiho-Soma et al. (2021) demonstrate that the HECT-type E3 ubiquitin ligase TRIP12 cooperates with CRL complexes to promote PROTAC-induced degradation of neo-substrates by generating K29/K48-branched ubiquitin chains.
    DOI:  https://doi.org/10.1016/j.molcel.2021.02.030
  34. Int J Mol Sci. 2021 Mar 02. pii: 2487. [Epub ahead of print]22(5):
    OsCBE1, a Substrate Receptor of Cullin4-Based E3 Ubiquitin Ligase, Functions as a Regulator of Abiotic Stress Response and Productivity in Rice.
    Juyoung Choi, Wonkyung Lee, Gynheung An, Seong-Ryong Kim.
      Ubiquitination is an important environmental stress response, and E3 ubiquitin ligases play a major role in the process. T-DNA insertion mutants of rice, Oscbe1-1, and Oscbe1-2, were identified through the screening of cold stress tolerance at seedling stage. Oscbe1 mutants showed a significantly higher cold stress tolerance in the fresh weight, chlorophyll content, and photosynthetic efficiency than wild type. Molecular prediction showed that OsCBE1 (Oryza sativa Cullin4-Based E3 ubiquitin ligase1) encoded a novel substrate receptor of Cullin4-based E3 ubiquitin ligase complex (C4E3). Whereas Oscbe1 mutants had fewer panicles and grains than wild type in the paddy field, the overexpression lines of OsCBE1 had more panicles and grains, suggesting that OsCBE1 is involved in the regulation of both abiotic stress response and development. Oscbe1 mutants also showed ABA hypersensitivity during seed germination, suggesting OsCBE1 function for the stress response via ABA signaling. In silico analysis of OsCBE1 activity predicted a CCCH-type transcription factor, OsC3H32, as a putative substrate. Co-IP (Co-immunoprecipitation) study showed that OsCBE1 interacts with OsDDB1, an expected binding component of OsCBE1 and OsC3H32. Additionally, expression of OsOLE16, OsOLE18, and OsBURP5 were negatively related with expression of OsCBE1. These results suggest that OsCBE1 functions as a regulator of the abiotic stress response via CCCH as a member of the C4E3.
    Keywords:  E3 ubiquitin ligase; abiotic stress; crop productivity; cullin4; rice
    DOI:  https://doi.org/10.3390/ijms22052487
  35. Proc Natl Acad Sci U S A. 2021 Apr 06. pii: e2020215118. [Epub ahead of print]118(14):
    Selective autophagy of AKAP11 activates cAMP/PKA to fuel mitochondrial metabolism and tumor cell growth.
    Zhiqiang Deng, Xianting Li, Marian Blanca Ramirez, Kerry Purtell, Insup Choi, Jia-Hong Lu, Qin Yu, Zhenyu Yue.
      Autophagy is a catabolic pathway that provides self-nourishment and maintenance of cellular homeostasis. Autophagy is a fundamental cell protection pathway through metabolic recycling of various intracellular cargos and supplying the breakdown products. Here, we report an autophagy function in governing cell protection during cellular response to energy crisis through cell metabolic rewiring. We observe a role of selective type of autophagy in direct activation of cyclic AMP protein kinase A (PKA) and rejuvenation of mitochondrial function. Mechanistically, autophagy selectively degrades the inhibitory subunit RI of PKA holoenzyme through A-kinase-anchoring protein (AKAP) 11. AKAP11 acts as an autophagy receptor that recruits RI to autophagosomes via LC3. Glucose starvation induces AKAP11-dependent degradation of RI, resulting in PKA activation that potentiates PKA-cAMP response element-binding signaling, mitochondria respiration, and ATP production in accordance with mitochondrial elongation. AKAP11 deficiency inhibits PKA activation and impairs cell survival upon glucose starvation. Our results thus expand the view of autophagy cytoprotection mechanism by demonstrating selective autophagy in RI degradation and PKA activation that fuels the mitochondrial metabolism and confers cell resistance to glucose deprivation implicated in tumor growth.
    Keywords:  AKAP11; PKA; autophagy; cell survival; mitochondrial metabolism
    DOI:  https://doi.org/10.1073/pnas.2020215118
  36. Mucosal Immunol. 2021 Mar 30.
    Emc3 maintains intestinal homeostasis by preserving secretory lineages.
    Meina Huang, Li Yang, Ning Jiang, Quanhui Dai, Runsheng Li, Zhaocai Zhou, Bing Zhao, Xinhua Lin.
      Intestinal exocrine secretory lineages, including goblet cells and Paneth cells, provide vital innate host defense to pathogens. However, how these cells are specified and maintained to ensure intestinal barrier function remains poorly defined. Here we show that endoplasmic reticulum membrane protein complex subunit 3 (Emc3) is essential for differentiation and function of exocrine secretory lineages. Deletion of Emc3 in intestinal epithelium decreases mucus production by goblet cells and Paneth cell population, along with gut microbial dysbiosis, which result in spontaneous inflammation and increased susceptibility to DSS-induced colitis. Moreover, Emc3 deletion impairs stem cell niche function of Paneth cells, thus resulting in intestinal organoid culture failure. Mechanistically, Emc3 deficiency leads to increased endoplasmic reticulum (ER) stress. Mitigating ER stress with tauroursodeoxycholate acid alleviates secretory dysfunction and restores organoid formation. Our study identifies a dominant role of Emc3 in maintaining intestinal mucosal homeostasis.
    DOI:  https://doi.org/10.1038/s41385-021-00399-2
  37. Subcell Biochem. 2021 ;97 45-60
    The Role of Post-Translational Modifications in Targeting Protein Cargo to Extracellular Vesicles.
    Ishara Atukorala, Suresh Mathivanan.
      Extracellular vesicles (EVs) are naturally occurring nanoparticles that contain proteins and nucleic acids. It is speculated that cells release EVs loaded with a selective cargo of proteins through highly regulated processes. Several proteomic and biochemical studies have highlighted phosphorylated, glycosylated, ubiquitinated, SUMOylated, oxidated and palmitoylated proteins within the EVs. Emerging evidences suggest that post-translational modifications (PTMs) can regulate the sorting of specific proteins into EVs and such proteins with specific PTMs have also been identified in clinical samples. Hence, it has been proposed that EV proteins with PTMs could be used as potential biomarkers of disease conditions. Among the other cellular mechanisms, the endosomal sorting complex required for transport (ESCRT) is also implicated in cargo sorting into EVs. In this chapter, various PTMs that are shown to regulate protein cargo sorting into EVs will be discussed.
    Keywords:  Cargo sorting; Exosomes; Extracellular vesicles; Glycosylation; ISGylation; Myristoylation; Oxidation; Palmitoylation; Phosphorylation; Post-translational modifications; SUMOylation; Ubiquitination
    DOI:  https://doi.org/10.1007/978-3-030-67171-6_3
  38. Nat Commun. 2021 Apr 01. 12(1): 2034
    Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.
    Joshua Hutchings, Viktoriya G Stancheva, Nick R Brown, Alan C M Cheung, Elizabeth A Miller, Giulia Zanetti.
      COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.
    DOI:  https://doi.org/10.1038/s41467-021-22110-6
  39. J Biol Chem. 2021 Mar 30. pii: S0021-9258(21)00394-X. [Epub ahead of print] 100613
    Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism.
    Jiahui Tao, Amandine Berthet, Y Rose Citron, Paraskevi L Tsiolaki, Robert Stanley, Jason E Gestwicki, David A Agard, Lisa McConlogue.
      Over-expression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same non-canonical mechanism.
    Keywords:  Hsp70; Lewy body dementia; Parkinson disease; chaperone; oligomer; protein folding; synucleinopathy; α-Synuclein
    DOI:  https://doi.org/10.1016/j.jbc.2021.100613
  40. EMBO Rep. 2021 Mar 28. e51078
    Ubiquitin-dependent regulation of transcription in development and disease.
    Kevin G Mark, Michael Rape.
      Transcription is an elaborate process that is required to establish and maintain the identity of the more than two hundred cell types of a metazoan organism. Strict regulation of gene expression is therefore vital for tissue formation and homeostasis. An accumulating body of work found that ubiquitylation of histones, transcription factors, or RNA polymerase II is crucial for ensuring that transcription occurs at the right time and place during development. Here, we will review principles of ubiquitin-dependent control of gene expression and discuss how breakdown of these regulatory circuits leads to a wide array of human diseases.
    Keywords:  RNA polymerase II; histone modification; transcription; ubiquitin
    DOI:  https://doi.org/10.15252/embr.202051078
  41. Autophagy. 2021 Mar 28. 1-19
    Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control.
    Anna Manzéger, Kinga Tagscherer, Péter Lőrincz, Henrik Szaker, Tamás Lukácsovich, Petra Pilz, Regina Kméczik, George Csikós, Miklós Erdélyi, Miklós Sass, Tibor Kovács, Tibor Vellai, Viktor A Billes.
      Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. Here we examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively, and found that these enzymes affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy.Abbreviations: Atg, autophagy-related; BDSC, Bloomington Drosophila Stock Center; DGRC, Drosophila Genetic Resource Center; EDTP, Egg-derived tyrosine phosphatase; FYVE, zinc finger domain from Fab1 (yeast ortholog of PIKfyve), YOTB, Vac1 (vesicle transport protein) and EEA1 cysteine-rich proteins; LTR, LysoTracker Red; MTM, myotubularin; MTMR, myotubularin-related; PI, phosphatidylinositol; Pi3K59F, Phosphotidylinositol 3 kinase 59F; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns5P, phosphatidylinositol-5-phosphate; ref(2)P, refractory to sigma P; Syx17, Syntaxin 17; TEM, transmission electron microscopy; UAS, upstream activating sequence; Uvrag, UV-resistance associated gene; VDRC, Vienna Drosophila RNAi Center; Vps34, Vacuolar protein sorting 34.
    Keywords:  Autophagy; edtp; mtmr6; myotubularins; phosphoinositides
    DOI:  https://doi.org/10.1080/15548627.2021.1899681
  42. Viruses. 2021 Mar 23. pii: 528. [Epub ahead of print]13(3):
    Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses.
    Marie Lork, Gauthier Lieber, Benjamin G Hale.
      SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses.
    Keywords:  ISG15; SUMO; TRIM28; endogenous retroelements; influenza; innate immunity; interferon; proteomics; ubiquitin-like modification; virus infection
    DOI:  https://doi.org/10.3390/v13030528
  43. SLAS Discov. 2021 Mar 30. 24725552211000679
    Development of a High-Throughput, Compound-Multiplexed Fluorescence Polarization Assay to Identify ATG5-ATG16L1 Protein-Protein Interaction Inhibitors.
    Maryna Salkovski, Ivan Pavlinov, Qiwen Gao, Leslie N Aldrich.
      Macroautophagy is a catabolic process wherein cytosolic cargo is engulfed in an autophagosome that fuses with a lysosome to degrade the cargo for recycling. Autophagy maintains cellular homeostasis and is involved in a myriad of illnesses ranging from cancer to neurodegenerative diseases, but its therapeutic potential remains elusive due to a lack of potent and specific autophagy modulators. To identify specific inhibitors of early autophagy, a target-based, compound-multiplexed, fluorescence polarization, high-throughput screen that targets the ATG5-ATG16L1 protein-protein interaction was developed. This interaction is critical for the formation of LC3-II, which is involved in phagophore maturation, and its disruption should inhibit autophagy. This assay is based on the polarization of light emitted by a fluorescent rhodamine tag conjugated to a peptide corresponding to the N-terminal region of ATG16L1 (ATG16L1-N). It was confirmed that this peptide binds specifically to ATG5, and the assay was validated by rapidly screening 4800 molecules through compound multiplexing. Through these initial screening efforts, a molecule was identified that disrupts the ATG5-ATG16L1 protein-protein interaction with micromolar potency, and this molecule will serve as a starting point for chemical optimization as an autophagy inhibitor.
    Keywords:  ATG5–ATG16L1 protein–protein interaction; assay development; autophagy; fluorescence polarization; inhibition
    DOI:  https://doi.org/10.1177/24725552211000679
  44. Matrix Biol. 2021 Mar 30. pii: S0945-053X(21)00037-8. [Epub ahead of print]
    Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta.
    Nadia Garibaldi, Barbara M Contento, Gabriele Babini, Jacopo Morini, Stella Siciliani, Marco Biggiogera, Mario Raspanti, Joan C Marini, Antonio Rossi, Antonella Forlino, Roberta Besio.
      Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.
    Keywords:  chemical chaperone; collagen; endoplasmic reticulum stress; osteogenesis imperfecta; unfolded protein response
    DOI:  https://doi.org/10.1016/j.matbio.2021.03.001
  45. Methods Mol Biol. 2021 ;2267 217-226
    Quantification of eIF2α Phosphorylation Associated with Mitotic Catastrophe by Immunofluorescence Microscopy.
    Juliette Humeau, Lucillia Bezu, Oliver Kepp, Laura Senovilla, Peng Liu, Guido Kroemer.
      Mitotic catastrophe is an oncosuppressive mechanism that drives cells toward senescence or death when an error occurs during mitosis. Eukaryotic cells have developed adaptive signaling pathways to cope with stress. The phosphorylation on serine 51 of the eukaryotic translation initiation factor (eIF2α) is a highly conserved event in stress responses, including the one that is activated upon treatment with mitotic catastrophe inducing agents, such as microtubular poisons or actin blockers. The protocol described herein details a method to quantify the phosphorylation of eIF2α by high-throughput immunofluorescence microscopy. This method is useful to capture the 'integrated stress response', which is characterized by eIF2α phosphorylation in the context of mitotic catastrophe.
    Keywords:  Endoplasmic reticulum; Immunofluorescence; Mitotic catastrophe; eIF2α
    DOI:  https://doi.org/10.1007/978-1-0716-1217-0_15
  46. JBMR Plus. 2021 Mar;5(3): e10451
    Loss of TANGO1 Leads to Absence of Bone Mineralization.
    Brecht Guillemyn, Sheela Nampoothiri, Delfien Syx, Fransiska Malfait, Sofie Symoens.
      TANGO1 (transport and Golgi organization-1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER-Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild-to-moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near-total bone loss and phenocopies the situation of Mia3 -/- mice. Whole-exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4-base pair (4-bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1-related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe-to-lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
    Keywords:  COLLAGEN SECRETION; COLLAGENOPATHY; COPII VESICLES; OSTEOGENESIS IMPERFECTA; TANGO1
    DOI:  https://doi.org/10.1002/jbm4.10451
  47. J Immunol. 2021 Mar 31. pii: ji2001253. [Epub ahead of print]
    OTUD1 Regulates Antifungal Innate Immunity through Deubiquitination of CARD9.
    Xiaorong Chen, Honghai Zhang, Xueer Wang, Zhugui Shao, Yanqi Li, Guimin Zhao, Feng Liu, Bingyu Liu, Yi Zheng, Tian Chen, Hui Zheng, Lei Zhang, Chengjiang Gao.
      CARD9 is an essential adaptor protein in antifungal innate immunity mediated by C-type lectin receptors. The activity of CARD9 is critically regulated by ubiquitination; however, the deubiquitinases involved in CARD9 regulation remain incompletely understood. In this study, we identified ovarian tumor deubiquitinase 1 (OTUD1) as an essential regulator of CARD9. OTUD1 directly interacted with CARD9 and cleaved polyubiquitin chains from CARD9, leading to the activation of the canonical NF-κB and MAPK pathway. OTUD1 deficiency impaired CARD9-mediated signaling and inhibited the proinflammatory cytokine production following fungal stimulation. Importantly, Otud1 -/- mice were more susceptible to fungal infection than wild-type mice in vivo. Collectively, our results identify OTUD1 as an essential regulatory component for the CARD9 signaling pathway and antifungal innate immunity through deubiquitinating CARD9.
    DOI:  https://doi.org/10.4049/jimmunol.2001253
  48. EMBO J. 2021 Mar 31. e106389
    Proteostatic imbalance and protein spreading in amyotrophic lateral sclerosis.
    Maria Elena Cicardi, Lara Marrone, Mimoun Azzouz, Davide Trotti.
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder whose exact causative mechanisms are still under intense investigation. Several lines of evidence suggest that the anatomical and temporal propagation of pathological protein species along the neural axis could be among the main driving mechanisms for the fast and irreversible progression of ALS pathology. Many ALS-associated proteins form intracellular aggregates as a result of their intrinsic prion-like properties and/or following impairment of the protein quality control systems. During the disease course, these mutated proteins and aberrant peptides are released in the extracellular milieu as soluble or aggregated forms through a variety of mechanisms. Internalization by recipient cells may seed further aggregation and amplify existing proteostatic imbalances, thus triggering a vicious cycle that propagates pathology in vulnerable cells, such as motor neurons and other susceptible neuronal subtypes. Here, we provide an in-depth review of ALS pathology with a particular focus on the disease mechanisms of seeding and transmission of the most common ALS-associated proteins, including SOD1, FUS, TDP-43, and C9orf72-linked dipeptide repeats. For each of these proteins, we report historical, biochemical, and pathological evidence of their behaviors in ALS. We further discuss the possibility to harness pathological proteins as biomarkers and reflect on the implications of these findings for future research.
    Keywords:  ALS; C9orf72; impaired proteostasis; misfolded proteins; spreading pathology
    DOI:  https://doi.org/10.15252/embj.2020106389
  49. Bio Protoc. 2021 Mar 05. 11(5): e3936
    Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Protein Vesicle Tracking in HeLa Cells.
    Natalia Pacheco-Fernandez, Mehrshad Pakdel, Julia Von Blume.
      Monitoring vesicle trafficking is an excellent tool for the evaluation of protein dynamics in living cells. Such study is key for the understanding of protein sorting and secretion. Recent developments in microscopy, as well as new methodologies developed to study synchronized trafficking of proteins, allowed a better understanding of signaling, regulation and trafficking dynamics at the secretory pathway. One of the most helpful tools so far developed is the Retention Using Selective Hooks (RUSH) system, a methodology that facilitates the evaluation of synchronized cargo trafficking by monitoring fluorescent vesicles in cells upon biotin addition. Here we present a protocol that allows the quantitative evaluation of protein cargo trafficking at different fixed time points and an analytic approach that enables a better examination of specific cargo trafficking dynamics at the secretory pathway. Graphic abstract: Schematic representation of RUSH sorting assay in mammalian cells.
    Keywords:  Cargo sorting; Confocal microscopy; Protein trafficking; RUSH; Vesicle tracking
    DOI:  https://doi.org/10.21769/BioProtoc.3936
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