bims-proteo Biomed News
on Proteostasis
Issue of 2020‒08‒02
28 papers selected by
Eric Chevet

  1. Mol Cell. 2020 Jul 28. pii: S1097-2765(20)30510-4. [Epub ahead of print]
    Zhao D, Zou CX, Liu XM, Jiang ZD, Yu ZQ, Suo F, Du TY, Dong MQ, He W, Du LL.
      Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.
    Keywords:  ER stress; ER-phagy; ER-plasma membrane contact; UPR; autophagy; autophagy receptor
  2. Int J Mol Sci. 2020 Jul 28. pii: E5369. [Epub ahead of print]21(15):
    Lopata A, Kniss A, Löhr F, Rogov VV, Dötsch V.
      In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.
    Keywords:  CUE domain; ERAD; ubiquitin chain conformation; ubiquitination
  3. EMBO J. 2020 Jul 27. e105696
    Cinque L, De Leonibus C, Iavazzo M, Krahmer N, Intartaglia D, Salierno FG, De Cegli R, Di Malta C, Svelto M, Lanzara C, Maddaluno M, Wanderlingh LG, Huebner AK, Cesana M, Bonn F, Polishchuk E, Hübner CA, Conte I, Dikic I, Mann M, Ballabio A, Sacco F, Grumati P, Settembre C.
      Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.
    Keywords:   TFEB ; ER-phagy; FGF signaling; Fam134B; IRS1/PI3K signaling
  4. Elife. 2020 Jul 28. pii: e55596. [Epub ahead of print]9
    Galeone A, Adams JM, Matsuda S, Presa MF, Pandey A, Han SY, Tachida Y, Hirayama H, Vaccari T, Suzuki T, Lutz CM, Affolter M, Zuberi A, Jafar-Nejad H.
      During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.
    Keywords:  BMP signaling; D. melanogaster; ERAD; N-glycosylation; NGLY1; cell biology; deglycosylation; developmental biology; mouse; retrotranslocation
  5. Rev Med Chil. 2020 Feb;pii: S0034-98872020000200216. [Epub ahead of print]148(2): 216-223
    Rivera-Krstulović C, Duran-Aniotz C.
      The clinical features of Alzheimer's disease (AD), for example the progressive memory loss, are produced by neuronal loss and synaptic dysfunction. These events have been associated with histopathological alterations in AD brain, including the presence of amyloid plaques and neurofibrillary tangles. Recent studies suggest that cellular stress produced by the aggregation of misfolded proteins leads to alterations in protein homeostasis, that is regulated for the most part by endoplasmic reticulum (ER). The ER is the main compartment involved in the folding and secretion of proteins and is drastically affected in AD neurons. Recent evidence implicates the participation of adaptive responses to stress within the ER in the disease process through a signaling pathway known as the Unfolded Protein Response (UPR) which alleviates the protein aggregation and ER stress. Based on the involvement of ER stress in several diseases, efforts are being done to identify small molecules that can inhibit or activate selective UPR components. Here, we review the findings suggesting a functional role of ER stress in the etiology of AD. Possible therapeutic strategies to mitigate ER stress in the context of AD are discussed.
  6. EMBO J. 2020 Jul 27. e105965
    Fraiberg M, Elazar Z.
      The endoplasmic reticulum (ER) is a dynamic intracellular network responsible for folding and maturation of organellar and secreted proteins. Selective autophagy of ER (ER-phagy) is emerging as an essential process that maintains proteostasis in the ER and is regulated by growth conditions. In this issue, Cinque et al (2020) show that fibroblast growth factor 18 (FGF18) specifically activates ER-phagy through a TFEB/TFE-dependent transcriptional regulation of the ER-phagy receptor Fam134b, a process essential for bone ossification and skeletal development.
  7. Cell Calcium. 2020 Jul 17. pii: S0143-4160(20)30091-9. [Epub ahead of print]91 102249
    Pihán P, Hetz C.
      The endoplasmic reticulum (ER) is the source of lysosomal calcium. The finding that the protein TMBIM6 -a putative ER calcium channel and cell death regulator -promotes calcium transfer from the ER to lysosomes to induce autophagy uncovers a missing piece in the puzzle of inter-organelle communication.
    Keywords:  Autophagy; Calcium; ER stress; Lysosome; Protein misfolding; UPR
  8. Dev Cell. 2020 Jul 15. pii: S1534-5807(20)30546-3. [Epub ahead of print]
    Williams R, Laskovs M, Williams RI, Mahadevan A, Labbadia J.
      The loss of protein homeostasis (proteostasis) is a primary driver of age-related tissue dysfunction. Recent studies have revealed that the failure of proteostasis with age is triggered by developmental and reproductive cues that repress the activity of proteostasis-related pathways in early adulthood. In Caenorhabditis elegans, reduced mitochondrial electron transport chain (ETC) function during development can override signals that promote proteostasis collapse in aged tissues. However, it is unclear precisely how these beneficial effects are mediated. Here, we reveal that in response to ETC impairment, the PP2A complex generates a dephosphorylated, mitochondrial stress-specific variant of the transcription factor HSF-1. This results in the selective induction of small heat shock proteins in adulthood, thereby protecting against age-related proteostasis collapse. We propose that mitochondrial signals early in life can protect the aging cytosolic proteome by tailoring HSF-1 activity to preferentially drive the expression of non-ATP-dependent chaperones.
    Keywords:  HSF1; PP2A; aging; mitochondria; molecular chaperones; protein aggregation; proteostasis; stress responses
  9. Cell Stress Chaperones. 2020 Jul 28.
    Dores-Silva PR, Cauvi DM, Coto ALS, Kiraly VTR, Borges JC, De Maio A.
      Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.
    Keywords:  Charged phospholipids; HSPA5; Hsp70; Liposomes; Membranes
  10. J Biol Chem. 2020 07 28. pii: jbc.RA120.014307. [Epub ahead of print]
    Attanasio S, Ferriero R, Gernoux G, De Cegli R, Carissimo A, Nusco E, Campione S, Teckman J, Mueller C, Piccolo P, Brunetti-Pierri N.
      Alpha-1 antitrypsin (AAT) encoded by SERPINA1 gene is an acute phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, known as ATZ, that changes the protein structure and leads to its misfolding and polymerization that cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most upregulated genes in PiZ mice that express ATZ, and in human livers of patients homozygous for the Z allele. Compared to controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to upregulate SERPINA1 cooperating with c-JUN, that was previously shown to upregulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN upregulates SERPINA1 transcription and play an important role in the hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.
    Keywords:  alpha1 antitrypsin; c-Jun transcription factor; liver; liver injury; serpin; transcription regulation
  11. Plant Commun. 2020 Jul 13. 1(4): 100020
    McLellan H, Chen K, He Q, Wu X, Boevink PC, Tian Z, Birch PRJ.
      Ubiquitination is a post-translational modification that regulates many processes in plants. Several ubiquitin E3 ligases act as either positive or negative regulators of immunity by promoting the degradation of different substrates. StPUB17 is an E3 ligase that has previously been shown to positively regulate immunity to bacteria, fungi and oomycetes, including the late blight pathogen Phytophthora infestans. Silencing of StPUB17 promotes pathogen colonization and attenuates Cf4/avr4 cell death. Using yeast-2-hybrid and co-immunoprecipitation we identified the putative K-homology (KH) RNA-binding protein (RBP), StKH17, as a candidate substrate for degradation by StPUB17. StKH17 acts as a negative regulator of immunity that promotes P. infestans infection and suppresses specific immune pathways. A KH RBP domain mutant of StKH17 (StKH17GDDG) is no longer able to negatively regulate immunity, indicating that RNA binding is likely required for StKH17 function. As StPUB17 is a known target of the ubiquitin E3 ligase, StPOB1, we reveal an additional step in an E3 ligase regulatory cascade that controls plant defense.
    Keywords:  E3 ligase; KH RNA-binding protein; late blight; oomycete; plant disease
  12. Autophagy. 2020 Jul 27. 1-2
    Yin Z, Klionsky DJ.
      Selective autophagy is critical for the regulation of cellular homeostasis in organisms from yeast to humans. This process is a specific degradation pathway for a wide variety of substrates including unwanted cytosolic components, such as protein aggregates, damaged and/or superfluous organelles, and pathogens. However, it has been less clear as to whether a protein complex or substructure of an organelle can be targeted for removal by selective autophagy. One example of such a substrate is the nuclear pore complex (NPC), a large macromolecular assembly that is present throughout the nuclear envelope. Here, we highlight two recent studies that demonstrate for the first time that NPCs are targeted for vacuolar degradation through selective autophagy.ABBREVIATIONS: AIM: Atg8-interacting motif; NE: nuclear envelope; NPC: nuclear pore complex; Nup: nucleoporin; PMN/micronucleophagy: piecemeal microautophagy of the nucleus.
    Keywords:  Autophagy; NPC-phagy; Nup159; cargo receptor; nuclear pore complex; selective autophagy
  13. Cell Rep. 2020 Jul 28. pii: S2211-1247(20)30955-4. [Epub ahead of print]32(4): 107974
    Zhang F, Yan P, Yu H, Le H, Li Z, Chen J, Liang X, Wang S, Wei W, Liu L, Zhang Y, Ji X, Xie A, Chen W, Han Z, Pu WT, Chen S, Chen Y, Sun K, Ge B, Zhang B.
      Attenuated DNA repair leads to genomic instability and tumorigenesis. BRCA1/BARD1 are the best-known tumor suppressors that promote homology recombination (HR) and arrest cell cycle. However, it remains ambiguous whether and how their E3 ligase activity regulates HR. Here, we demonstrate that upon genotoxic stress, BRCA1 together with BARD1 catalyzes the K48 polyubiquitination on LARP7, a 7SK RNA binding protein known to control RNAPII pausing, and thereby degrades it through the 26S ubiquitin-proteasome pathway. Depleting LARP7 suppresses the expression of CDK1 complex, arrests the cell at the G2/M DNA damage checkpoint, and reduces BRCA2 phosphorylation, which thereby facilitates RAD51 recruitment to damaged DNA to enhance HR. Importantly, LARP7 depletion observed in breast cancer patients leads to chemoradiotherapy resistance both in vitro and in vivo. Altogether, this study unveils a mechanism by which BRCA1/BARD1 control HR and cell cycle, and highlights LARP7 as a potential target for cancer prevention and therapy.
    Keywords:  7SK snRNP; BRCA1; DNA damage response; DNA repair; LARP7; RNAPII pausing; breast cancer; therapy resistance
  14. Proc Natl Acad Sci U S A. 2020 Jul 28. pii: 201920327. [Epub ahead of print]
    Choi WH, Yun Y, Park S, Jeon JH, Lee J, Lee JH, Yang SA, Kim NK, Jung CH, Kwon YT, Han D, Lim SM, Lee MJ.
      The 26S proteasome, a self-compartmentalized protease complex, plays a crucial role in protein quality control. Multiple levels of regulatory systems modulate proteasomal activity for substrate hydrolysis. However, the destruction mechanism of mammalian proteasomes is poorly understood. We found that inhibited proteasomes are sequestered into the insoluble aggresome via HDAC6- and dynein-mediated transport. These proteasomes colocalized with the autophagic receptor SQSTM1 and cleared through selective macroautophagy, linking aggresomal segregation to autophagic degradation. This proteaphagic pathway was counterbalanced with the recovery of proteasomal activity and was critical for reducing cellular proteasomal stress. Changes in associated proteins and polyubiquitylation on inhibited 26S proteasomes participated in the targeting mechanism to the aggresome and autophagosome. The STUB1 E3 Ub ligase specifically ubiquitylated purified human proteasomes in vitro, mainly via Lys63-linked chains. Genetic and chemical inhibition of STUB1 activity significantly impaired proteasome processing and reduced resistance to proteasomal stress. These data demonstrate that aggresomal sequestration is the crucial upstream event for proteasome quality control and overall protein homeostasis in mammals.
    Keywords:  STUB1; aggresome; proteaphagy; proteasome; ubiquitin
  15. Cell Mol Life Sci. 2020 Jul 30.
    Weber-Boyvat M, Trimbuch T, Shah S, Jäntti J, Olkkonen VM, Rosenmund C.
      OSBP-homologous proteins (ORPs, Oshp) are lipid binding/transfer proteins. Several ORP/Oshp localize to membrane contacts between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate lipid transfer or regulate lipid-modifying enzymes. A common way in which they target contacts is by binding to the ER proteins, VAP/Scs2p, while the second membrane is targeted by other interactions with lipids or proteins.We have studied the cross-talk of secretory SNARE proteins and their regulators with ORP/Oshp and VAPA/Scs2p at ER-plasma membrane contact sites in yeast and murine primary neurons. We show that Oshp-Scs2p interactions depend on intact secretory SNARE proteins, especially Sec9p. SNAP-25/Sec9p directly interact with ORP/Osh proteins and their disruption destabilized the ORP/Osh proteins, associated with dysfunction of VAPA/Scs2p. Deleting OSH1-3 in yeast or knocking down ORP2 in primary neurons reduced the oligomerization of VAPA/Scs2p and affected their multiple interactions with SNAREs. These observations reveal a novel cross-talk between the machineries of ER-plasma membrane contact sites and those driving exocytosis.
    Keywords:  Membrane contact site; ORP; Osh; SNAP-25; SNARE; Sec9
  16. Nature. 2020 Jul 29.
    Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR.
      The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.
  17. J Mol Cell Cardiol. 2020 Jul 24. pii: S0022-2828(20)30231-5. [Epub ahead of print]
    Moyzis AG, Lally NS, Liang W, Leon LJ, Najor RH, Orogo AM, Gustafsson ÅB.
      Myeloid cell leukemia-1 (Mcl-1) is a structurally and functionally unique anti-apoptotic Bcl-2 protein. While elevated levels of Mcl-1 contribute to tumor cell survival and drug resistance, loss of Mcl-1 in cardiac myocytes leads to rapid mitochondrial dysfunction and heart failure development. Although Mcl-1 is an anti-apoptotic protein, previous studies indicate that its functions extend beyond regulating apoptosis. Mcl-1 is localized to both the mitochondrial outer membrane and matrix. Here, we have identified that Mcl-1 in the outer mitochondrial membrane mediates mitochondrial fission, which is independent of its anti-apoptotic function. We demonstrate that Mcl-1 interacts with Drp1 to promote mitochondrial fission in response to various challenges known to perturb mitochondria morphology. Induction of fission by Mcl-1 reduces nutrient deprivation-induced cell death and the protection is independent of its BH3 domain. Finally, cardiac-specific overexpression of Mcl-1OM, but not Mcl-1Matrix, contributes to a shift in the balance towards fission and leads to reduced exercise capacity, suggesting that a pre-existing fragmented mitochondrial network leads to decreased ability to adapt to an acute increase in workload and energy demand. Overall, these findings highlight the importance of Mcl-1 in maintaining mitochondrial health in cells.
    Keywords:  Drp1; Fission; Heart; Mcl-1; Mitochondria
  18. PLoS One. 2020 ;15(7): e0236520
    Miyasaka M, Mioka T, Kishimoto T, Itoh E, Tanaka K.
      In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.
  19. Theranostics. 2020 ;10(18): 8400-8414
    Zhang J, Yang J, Lin C, Liu W, Huo Y, Yang M, Jiang SH, Sun Y, Hua R.
      Rationale: Endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) is an endoplasmic reticulum (ER) luminal glycoprotein that has a role in the formation of disulfide bonds of secreted proteins and membrane proteins. Emerging data identify ERO1L as a tumor promoter in a wide spectrum of human malignancies. However, its molecular basis of oncogenic activities remains largely unknown. Methods: Pan-cancer analysis was performed to determine the expression profile and prognostic value of ERO1L in human cancers. The mechanism by which ERO1L promotes tumor growth and glycolysis in pancreatic ductal adenocarcinoma (PDAC) was investigated by cell biological, molecular, and biochemical approaches. Results: ERO1L was highly expressed in PDAC and its precursor pancreatic intraepithelial neoplasia and acts as an independent prognostic factor for patient survival. Hypoxia and ER stress contributed to the overexpression pattern of ERO1L in PDAC. ERO1L knockdown or pharmacological inhibition with EN460 suppressed PDAC cell proliferation in vitro and slowed tumor growth in vivo. Ectopic expression of wild type ERO1L but not its inactive mutant form EROL-C394A promoted tumor growth. Bioinformatics analyses and functional analyses confirmed a regulatory role of ERO1L on the Warburg effect. Notably, inhibition of tumor glycolysis partially abrogated the growth-promoting activity of ERO1L. Mechanistically, ERO1L-mediated ROS generation was essential for its oncogenic activities. In clinical samples, ERO1L expression was correlated with the maximum standard uptake value (SUVmax) in PDAC patients who received 18F-FDG PET/CT imaging preoperatively. Analysis of TCGA cohort revealed a specific glycolysis gene expression signature that is highly correlated with unfolded protein response-related gene signature. Conclusion: Our findings uncover a key function for ERO1L in Warburg metabolism and indicate that targeting this pathway may offer alternative therapeutic strategies for PDAC.
    Keywords:  Aerobic glycolysis; ERO1A; ERO1α; HIF1α; Pancreatic cancer
  20. J Cell Sci. 2020 Jul 21. pii: jcs246041. [Epub ahead of print]133(14):
    Aichem A, Groettrup M.
      Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) also called ubiquitin D (UBD) is a member of the ubiquitin-like modifier (ULM) family. The FAT10 gene is localized in the MHC class I locus and FAT10 protein expression is mainly restricted to cells and organs of the immune system. In all other cell types and tissues, FAT10 expression is highly inducible by the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF). Besides ubiquitin, FAT10 is the only ULM which directly targets its substrates for degradation by the 26S proteasome. This poses the question as to why two ULMs sharing the proteasome-targeting function have evolved and how they differ from each other. This Review summarizes the current knowledge of the special structure of FAT10 and highlights its differences from ubiquitin. We discuss how these differences might result in differential outcomes concerning proteasomal degradation mechanisms and non-covalent target interactions. Moreover, recent insights about the structural and functional impact of FAT10 interacting with specific non-covalent interaction partners are reviewed.
    Keywords:  Autophagy; FAT10; Proteasome; Proteostasis; Ubiquitin; Ubiquitin-like modifier; VCP
  21. Mol Biochem Parasitol. 2020 Jul 28. pii: S0166-6851(20)30077-3. [Epub ahead of print] 111313
    Garrison P, Bangs JD.
      Misfolded proteins trapped in the endoplasmic reticulum (ER) are specifically recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has off-target inhibitory effects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, no deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment offers clarity to the off-target effects of MG132 treatment.
    Keywords:  CB-5083; ERAD; MG132; p97; trypanosome; turnover
  22. EMBO J. 2020 Jul 26. e104415
    Muliyil S, Levet C, Düsterhöft S, Dulloo I, Cowley SA, Freeman M.
      Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here, we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.
    Keywords:  ADAM17; Glia; lipid droplet; neurodegeneration; reactive oxygen species
  23. Physiol Rev. 2020 Jul 30.
    Chatham JC, Zhang J, Wende AR.
      In the mid 1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by an O-linkage by a N-acetylglucosamine moiety (O-GlcNAc) overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery O-GlcNAcylation has been shown to contribute to numerous cellular functions including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.
    Keywords:  Calcium; Glucose; Insulin; O-GlcNAc; metabolism
  24. J Biol Chem. 2020 Jul 29. pii: jbc.RA120.014349. [Epub ahead of print]
    Martinelli L, Adamopoulos A, Johansson P, Wan PT, Gunnarsson J, Guo H, Boyd H, Zelcer N, Sixma TK.
      Hepatic abundance of the Low-Density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL-cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase Inducible Degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (F for 4.1 protein, E for ezrin, R for radixin and M for moesin) domain, responsible for substrate recognition and plasma-membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homo-dimerization. As IDOL is a putative lipid-lowering drug-target we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high auto-ubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this  we determined crystal structures of the extended FERM domain of IDOL and  multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 tri-lobed FERM domain structure but show that the F3c subdomain orientation obscures the target binding site. To substantiate this finding, we analyzed the full length FERM domain and a series of truncated FERM constructs by small angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
    Keywords:  E3 ubiquitin ligase; FERM domain; IDOL; LDL receptor; enzyme purification; low-density lipoprotein (LDL); protein structure; small-angle X-ray scattering (SAXS)
  25. Neuron. 2020 Jul 26. pii: S0896-6273(20)30491-8. [Epub ahead of print]
    Agarwal N, Taberner FJ, Rangel Rojas D, Moroni M, Omberbasic D, Njoo C, Andrieux A, Gupta P, Bali KK, Herpel E, Faghihi F, Fleming T, Dejean A, Lechner SG, Nawroth PP, Lewin GR, Kuner R.
      Diabetic peripheral neuropathy (DPN) is a highly frequent and debilitating clinical complication of diabetes that lacks therapies. Cellular oxidative stress regulates post-translational modifications, including SUMOylation. Here, using unbiased screens, we identified key enzymes in metabolic pathways and ion channels as novel molecular targets of SUMOylation that critically regulated their activity. Sensory neurons of diabetic patients and diabetic mice demonstrated changes in the SUMOylation status of metabolic enzymes and ion channels. In support of this, profound metabolic dysfunction, accelerated neuropathology, and sensory loss were observed in diabetic gene-targeted mice selectively lacking the ability to SUMOylate proteins in peripheral sensory neurons. TRPV1 function was impaired by diabetes-induced de-SUMOylation as well as by metabolic imbalance elicited by de-SUMOylation of metabolic enzymes, facilitating diabetic sensory loss. Our results unexpectedly uncover an endogenous post-translational mechanism regulating diabetic neuropathy in patients and mouse models that protects against metabolic dysfunction, nerve damage, and altered sensory perception.
  26. J Cell Biol. 2020 09 07. pii: e202006005. [Epub ahead of print]219(9):
    Sicari D, Chatziioannou A, Koutsandreas T, Sitia R, Chevet E.
      Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway-mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology that could also be targeted with therapeutic objectives. Finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications.
  27. EMBO J. 2020 Jul 28. e103009
    Fan SJ, Kroeger B, Marie PP, Bridges EM, Mason JD, McCormick K, Zois CE, Sheldon H, Khalid Alham N, Johnson E, Ellis M, Stefana MI, Mendes CC, Wainwright SM, Cunningham C, Hamdy FC, Morris JF, Harris AL, Wilson C, Goberdhan DC.
      Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.
    Keywords:  Rab11(a); exosome; extracellular vesicle; mechanistic Target of Rapamycin; multivesicular body
  28. Nat Commun. 2020 Jul 30. 11(1): 3807
    Murphy P, Xu Y, Rouse SL, Jaffray EG, Plechanovová A, Matthews SJ, Carlos Penedo J, Hay RT.
      The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.