bims-proteo Biomed News
on Proteostasis
Issue of 2021‒01‒24
fifty-four papers selected by
Eric Chevet
INSERM


  1. J Virol. 2021 Jan 20. pii: JVI.01990-20. [Epub ahead of print]
    Hinte F, Müller J, Brune W.
      The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated degradation (ERAD) are two essential components of the quality control system for proteins in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an ERAD substrate, indicating that the UPR and ERAD regulate each other. Viruses are intracellular parasites that exploit the host cell for their own benefit. Cytomegaloviruses selectively modulate the UPR to take advantage of beneficial and inhibit detrimental effects on viral replication. We have previously shown that murine and human cytomegaloviruses express homologous proteins (M50 and UL50, respectively) that dampen the UPR at late times post infection by inducing IRE1 degradation. However, the degradation mechanism has remained uncertain. Here we show that the cytomegalovirus M50 protein mediates IRE1 degradation by the proteasome. M50-dependent IRE1 degradation can be blocked by pharmacological inhibition of p97/VCP or by genetic ablation of SEL1L, both of which are components of the ERAD machinery. SEL1L acts as a cofactor of the E3 ubiquitin ligase HRD1, while p97/VCP is responsible for the extraction of ubiquitylated proteins from the ER to the cytosol. We further show that M50 facilitates the IRE1-SEL1L interaction by binding to both, IRE1 and SEL1L. These results indicate that the viral M50 protein dampens the UPR by tethering IRE1 to SEL1L, thereby promoting its degradation by the ERAD machinery.IMPORTANCE Viruses infect cells of their host and force them to produce virus progeny. This can impose stress on the host cell and activate counter-regulatory mechanisms. Protein overload in the endoplasmic reticulum (ER) leads to ER stress and triggers the unfolded protein response, which in turn upregulates protein folding and increases the degradation of proteins in the ER. Previous work has shown that cytomegaloviruses interfere with the unfolded protein response by degrading the sensor molecule IRE1. Herein we demonstrate how the cytomegalovirus M50 protein exploits the ER-associated degradation machinery to dispose of IRE1. Degradation of IRE1 curbs the unfolded protein response and helps the virus to increase the synthesis of its own proteins and the production of virus progeny.
    DOI:  https://doi.org/10.1128/JVI.01990-20
  2. Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31648-X. [Epub ahead of print]34(3): 108659
    Cremer T, Jongsma MLM, Trulsson F, Vertegaal ACO, Neefjes J, Berlin I.
      The endolysosomal system fulfills a wide variety of cellular functions, many of which are modulated through interactions with other organelles. In particular, the ER exerts spatiotemporal constraints on the organization and motility of endosomes and lysosomes. We have recently described the ER transmembrane E3 ubiquitin ligase RNF26 as a regulator of endolysosomal perinuclear positioning and transport dynamics. Here, we report that the ubiquitin conjugating enzyme UBE2J1, also anchored in the ER membrane, partners with RNF26 in this context, and that the cellular activity of the resulting E2/E3 pair is localized in a perinuclear ER subdomain and supported by transmembrane interactions. Through modification of SQSTM1/p62 on lysine 435, the ER-embedded UBE2J1/RNF26 ubiquitylation complex recruits endosomal adaptors to immobilize their cognate vesicles in the perinuclear region of the cell. The resulting spatiotemporal compartmentalization promotes the trafficking of activated EGFR to lysosomes and facilitates the termination of EGF-induced AKT signaling.
    Keywords:  Akt; EGFR; RNF26; UBE2J1; endoplasmic reticulum; endosomes; p62; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2020.108659
  3. Metabolites. 2021 Jan 14. pii: E52. [Epub ahead of print]11(1):
    Xu J, Taubert S.
      Biological membranes are not only essential barriers that separate cellular and subcellular structures, but also perform other critical functions such as the initiation and propagation of intra- and intercellular signals. Each membrane-delineated organelle has a tightly regulated and custom-made membrane lipid composition that is critical for its normal function. The endoplasmic reticulum (ER) consists of a dynamic membrane network that is required for the synthesis and modification of proteins and lipids. The accumulation of unfolded proteins in the ER lumen activates an adaptive stress response known as the unfolded protein response (UPR-ER). Interestingly, recent findings show that lipid perturbation is also a direct activator of the UPR-ER, independent of protein misfolding. Here, we review proteostasis-independent UPR-ER activation in the genetically tractable model organism Caenorhabditis elegans. We review the current knowledge on the membrane lipid composition of the ER, its impact on organelle function and UPR-ER activation, and its potential role in human metabolic diseases. Further, we summarize the bi-directional interplay between lipid metabolism and the UPR-ER. We discuss recent progress identifying the different respective mechanisms by which disturbed proteostasis and lipid bilayer stress activate the UPR-ER. Finally, we consider how genetic and metabolic disturbances may disrupt ER homeostasis and activate the UPR and discuss how using -omics-type analyses will lead to more comprehensive insights into these processes.
    Keywords:  endoplasmic reticulum; lipid bilayer stress; lipidomics; phosphatidylcholine; unfolded protein response; unsaturated fatty acid
    DOI:  https://doi.org/10.3390/metabo11010052
  4. J Biol Chem. 2020 Nov 24. pii: S0021-9258(20)00085-X. [Epub ahead of print]296 100095
    Deng J, Bai X, Tang H, Pang S.
      DNA damage triggers the cellular adaptive response to arrest proliferation and repair DNA damage; when damage is too severe to be repaired, apoptosis is initiated to prevent the spread of genomic insults. However, how cells endure DNA damage to maintain cell function remains largely unexplored. By using Caenorhabditis elegans as a model, we report that DNA damage elicits cell maintenance programs, including the unfolded protein response of the endoplasmic reticulum (UPRER). Mechanistically, sublethal DNA damage unexpectedly suppresses apoptotic genes in C. elegans, which in turn increases the activity of the inositol-requiring enzyme 1/X-box binding protein 1 (IRE-1/XBP-1) branch of the UPRER by elevating unsaturated phosphatidylcholine. In addition, UPRER activation requires silencing of the lipid regulator skinhead-1 (SKN-1). DNA damage suppresses SKN-1 activity to increase unsaturated phosphatidylcholine and activate UPRER. These findings reveal the UPRER activation as an organismal adaptive response that is important to maintain cell function during DNA damage.
    Keywords:  C. elegans; DNA damage response; ER stress response; SKN-1; apoptotic genes; fatty acid; phosphatidylcholine
    DOI:  https://doi.org/10.1074/jbc.RA120.016083
  5. Mol Neurobiol. 2021 Jan 18.
    Wang SM, Goguadze N, Kimura Y, Yasui Y, Pan B, Wang TY, Nakamura Y, Lin YT, Hogan QH, Wilson KL, Su TP, Wu HE.
      Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER) chaperones implicated in neuropathic pain. Here we examine if the Sig-1R may relate to neuropathic pain at the level of dorsal root ganglia (DRG). We focus on the neuronal excitability of DRG in a "spare nerve injury" (SNI) model of neuropathic pain in rats and find that Sig-1Rs likely contribute to the genesis of DRG neuronal excitability by decreasing the protein level of voltage-gated Cav2.2 as a translational inhibitor of mRNA. Specifically, during SNI, Sig-1Rs translocate from ER to the nuclear envelope via a trafficking protein Sec61β. At the nucleus, the Sig-1R interacts with cFos and binds to the promoter of 4E-BP1, leading to an upregulation of 4E-BP1 that binds and prevents eIF4E from initiating the mRNA translation for Cav2.2. Interestingly, in Sig-1R knockout HEK cells, Cav2.2 is upregulated. In accordance with those findings, we find that intra-DRG injection of Sig-1R agonist (+)pentazocine increases frequency of action potentials via regulation of voltage-gated Ca2+ channels. Conversely, intra-DRG injection of Sig-1R antagonist BD1047 attenuates neuropathic pain. Hence, we discover that the Sig-1R chaperone causes neuropathic pain indirectly as a translational inhibitor.
    Keywords:  4E-BP1; Dorsal root ganglia; Neuropathic pain; Sigma-1 receptor; Spare nerve injury; Translational inhibition
    DOI:  https://doi.org/10.1007/s12035-020-02276-8
  6. Cell Death Differ. 2021 Jan 20.
    Fiil BK, Gyrd-Hansen M.
      Ubiquitination is an essential post-translational modification that regulates most cellular processes. The assembly of ubiquitin into polymeric chains by E3 ubiquitin ligases underlies the pleiotropic functions ubiquitin chains regulate. Ubiquitin chains assembled via the N-terminal methionine, termed Met1-linked ubiquitin chains or linear ubiquitin chains, have emerged as essential signalling scaffolds that regulate pro-inflammatory responses, anti-viral interferon responses, cell death and xenophagy of bacterial pathogens downstream of innate immune receptors. Met1-linked ubiquitin chains are exclusively assembled by the linear ubiquitin chain assembly complex, LUBAC, and are disassembled by the deubiquitinases OTULIN and CYLD. Genetic defects that perturb the regulation of Met1-linked ubiquitin chains causes severe immune-related disorders, illustrating their potent signalling capacity. Here, we review the current knowledge about the cellular machinery that conjugates, recognises, and disassembles Met1-linked ubiquitin chains, and discuss the function of this unique posttranslational modification in regulating inflammation, cell death and immunity to pathogens.
    DOI:  https://doi.org/10.1038/s41418-020-00702-x
  7. J Cell Sci. 2021 Jan 19. pii: jcs.257758. [Epub ahead of print]
    O'Keefe S, Roboti P, Duah KB, Zong G, Schneider H, Shi WQ, High S.
      In order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2 the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation/insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum, antiviral agents.
    Keywords:  Cell-free translation; ER membrane complex (EMC); Endoplasmic reticulum (ER); Sec61 translocon; Viral protein biogenesis
    DOI:  https://doi.org/10.1242/jcs.257758
  8. J Lipid Res. 2020 Dec;pii: S0022-2275(20)60027-X. [Epub ahead of print]61(12): 1675-1686
    De Giorgi M, Jarrett KE, Burton JC, Doerfler AM, Hurley A, Li A, Hsu RH, Furgurson M, Patel KR, Han J, Borchers CH, Lagor WR.
      HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.
    Keywords:  3-hydroxy-3-methylglutaryl-coenzyme A; adeno-associated virus; animal models; cholesterol synthesis and regulation; clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; dehydrodolichyl diphosphate synthase subunit; dolichol; endoplasmic reticulum; liver
    DOI:  https://doi.org/10.1194/jlr.RA120001006
  9. Int J Mol Sci. 2021 Jan 14. pii: E791. [Epub ahead of print]22(2):
    Liu Q, Aminu B, Roscow O, Zhang W.
      Tumor microenvironments are composed of a myriad of elements, both cellular (immune cells, cancer-associated fibroblasts, mesenchymal stem cells, etc.) and non-cellular (extracellular matrix, cytokines, growth factors, etc.), which collectively provide a permissive environment enabling tumor progression. In this review, we focused on the regulation of tumor microenvironment through ubiquitination. Ubiquitination is a reversible protein post-translational modification that regulates various key biological processes, whereby ubiquitin is attached to substrates through a catalytic cascade coordinated by multiple enzymes, including E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. In contrast, ubiquitin can be removed by deubiquitinases in the process of deubiquitination. Here, we discuss the roles of E3 ligases and deubiquitinases as modulators of both cellular and non-cellular components in tumor microenvironment, providing potential therapeutic targets for cancer therapy. Finally, we introduced several emerging technologies that can be utilized to develop effective therapeutic agents for targeting tumor microenvironment.
    Keywords:  E3 ligase; cancer-associated fibroblasts; deubiquitinase; extracellular matrix; hypoxia; immune cells; inhibitors; tumor microenvironment; ubiquitin variants; ubiquitination
    DOI:  https://doi.org/10.3390/ijms22020791
  10. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50480-9. [Epub ahead of print]295(49): 16630-16642
    Kaur S, Chen Y, Shenoy SK.
      The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein-coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule-binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.
    Keywords:  G protein-coupled receptor; G protein-coupled receptor (GPCR); deubiquitylation (deubiquitination); endocytosis; glucagon; receptor recycling; ubiquitin-specific protease; ubiquitination
    DOI:  https://doi.org/10.1074/jbc.RA120.014532
  11. Cell Death Differ. 2021 Jan 18.
    Habel N, El-Hachem N, Soysouvanh F, Hadhiri-Bzioueche H, Giuliano S, Nguyen S, Horák P, Gay AS, Debayle D, Nottet N, Béranger G, Paillerets BB, Bertolotto C, Ballotti R.
      Ubiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.
    DOI:  https://doi.org/10.1038/s41418-020-00710-x
  12. J Biol Chem. 2021 Jan 15. pii: S0021-9258(21)00063-6. [Epub ahead of print] 100295
    Eesmaa A, Yu LY, Göös H, Nõges K, Kovaleva V, Hellman M, Zimmermann R, Jung M, Permi P, Varjosalo M, Lindholm P, Saarma M.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an ER-stress regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone GRP78. We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling towards a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, PDIA1 and PDIA6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and NMR spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.
    Keywords:  ATP; GRP78; Mesencephalic astrocyte-derived neurotrophic factor (MANF); apoptosis; dopamine neurons; endoplasmic reticulum stress (ER stress); neuronal cell death; neuroprotection; protein-protein interaction; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1016/j.jbc.2021.100295
  13. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50490-1. [Epub ahead of print]295(49): 16754-16772
    Mohan HM, Yang B, Dean NA, Raghavan M.
      α1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A mutant of AAT (E342K) called ATZ forms polymers that are present at only low levels in the serum and induce intracellular protein inclusions, causing lung emphysema and liver cirrhosis. An understanding of factors that can reduce the intracellular accumulation of ATZ is of great interest. We now show that calreticulin (CRT), an endoplasmic reticulum (ER) glycoprotein chaperone, promotes the secretory trafficking of ATZ, enhancing the media:cell ratio. This effect is more pronounced for ATZ than with AAT and is only partially dependent on the glycan-binding site of CRT, which is generally relevant to substrate recruitment and folding by CRT. The CRT-related chaperone calnexin does not enhance ATZ secretory trafficking, despite the higher cellular abundance of calnexin-ATZ complexes. CRT deficiency alters the distributions of ATZ-ER chaperone complexes, increasing ATZ-BiP binding and inclusion body formation and reducing ATZ interactions with components required for ER-Golgi trafficking, coincident with reduced levels of the protein transport protein Sec31A in CRT-deficient cells. These findings indicate a novel role for CRT in promoting the secretory trafficking of a protein that forms polymers and large intracellular inclusions. Inefficient secretory trafficking of ATZ in the absence of CRT is coincident with enhanced accumulation of ER-derived ATZ inclusion bodies. Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung and liver diseases linked to AAT deficiency.
    Keywords:  COPII; calnexin; calreticulin; chaperone; endoplasmic reticulum (ER); glycoprotein secretion; intracellular trafficking; mass spectrometry (MS); secretory trafficking; α1-antitrypsin
    DOI:  https://doi.org/10.1074/jbc.RA120.014372
  14. J Biol Chem. 2020 Nov 22. pii: S0021-9258(20)00005-8. [Epub ahead of print]296 100019
    Arystarkhova E, Ozelius LJ, Brashear A, Sweadner KJ.
      Missense mutations in ATP1A3, the α3 isoform of Na,K-ATPase, cause neurological phenotypes that differ greatly in symptoms and severity. A mechanistic basis for differences is lacking, but reduction of activity alone cannot explain them. Isogenic cell lines with endogenous α1 and inducible exogenous α3 were constructed to compare mutation properties. Na,K-ATPase is made in the endoplasmic reticulum (ER), but the glycan-free catalytic α subunit complexes with glycosylated β subunit in the ER to proceed through Golgi and post-Golgi trafficking. We previously observed classic evidence of protein misfolding in mutations with severe phenotypes: differences in ER retention of endogenous β1 subunit, impaired trafficking of α3, and cytopathology, suggesting that they misfold during biosynthesis. Here we tested two mutations associated with different phenotypes: D923N, which has a median age of onset of hypotonia or dystonia at 3 years, and L924P, with severe infantile epilepsy and profound impairment. Misfolding during biosynthesis in the ER activates the unfolded protein response, a multiarmed program that enhances protein folding capacity, and if that fails, triggers apoptosis. L924P showed more nascent protein retention in ER than D923N; more ER-associated degradation of α3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2α, a major defensive step of the unfolded protein response. In L924P there was also altered subcellular distribution of endogenous α1 subunit, analogous to a dominant negative effect. Both mutations showed pro-apoptotic sensitization by reduced phosphorylation of BAD. Encouragingly, however, 4-phenylbutyrate, a pharmacological corrector, reduced L924P ER retention, increased α3 expression, and restored morphology.
    Keywords:  4-phenylbutyrate (4PBA); N-linked glycosylation; Na(+)/K(+)-ATPase; endoplasmic reticulum associated protein degradation (ERAD); endoplasmic reticulum stress (ER stress); eukaryotic initiation factor 2alpha (eIF2 α); genetic disease; protein misfolding; subcellular fractionation; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1074/jbc.RA120.015271
  15. EMBO Rep. 2021 Jan 20. e51063
    Joshi KK, Matlack TL, Pyonteck S, Vora M, Menzel R, Rongo C.
      Metazoans use protein homeostasis (proteostasis) pathways to respond to adverse physiological conditions, changing environment, and aging. The nervous system regulates proteostasis in different tissues, but the mechanism is not understood. Here, we show that Caenorhabditis elegans employs biogenic amine neurotransmitters to regulate ubiquitin proteasome system (UPS) proteostasis in epithelia. Mutants for biogenic amine synthesis show decreased poly-ubiquitination and turnover of a GFP-based UPS substrate. Using RNA-seq and mass spectrometry, we found that biogenic amines promote eicosanoid production from poly-unsaturated fats (PUFAs) by regulating expression of cytochrome P450 monooxygenases. Mutants for one of these P450s share the same UPS phenotype observed in biogenic amine mutants. The production of n-6 eicosanoids is required for UPS substrate turnover, whereas accumulation of n-6 eicosanoids accelerates turnover. Our results suggest that sensory neurons secrete biogenic amines to modulate lipid signaling, which in turn activates stress response pathways to maintain UPS proteostasis.
    Keywords:  dopamine; eicosanoid; protein homeostasis; serotonin; ubiquitin
    DOI:  https://doi.org/10.15252/embr.202051063
  16. Sci Rep. 2021 Jan 21. 11(1): 2013
    Dieterich IA, Cui Y, Braun MM, Lawton AJ, Robinson NH, Peotter JL, Yu Q, Casler JC, Glick BS, Audhya A, Denu JM, Li L, Puglielli L.
      Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.
    DOI:  https://doi.org/10.1038/s41598-021-81447-6
  17. J Biol Chem. 2020 Dec 04. pii: S0021-9258(17)50489-5. [Epub ahead of print]295(49): 16743-16753
    Liu Q, Yang X, Long G, Hu Y, Gu Z, Boisclair YR, Long Q.
      Mitochondrial dysfunction is associated with a variety of human diseases including neurodegeneration, diabetes, nonalcohol fatty liver disease (NAFLD), and cancer, but its underlying causes are incompletely understood. Using the human hepatic cell line HepG2 as a model, we show here that endoplasmic reticulum-associated degradation (ERAD), an ER protein quality control process, is critically required for mitochondrial function in mammalian cells. Pharmacological inhibition or genetic ablation of key proteins involved in ERAD increased cell death under both basal conditions and in response to proinflammatory cytokines, a situation frequently found in NAFLD. Decreased viability of ERAD-deficient HepG2 cells was traced to impaired mitochondrial functions including reduced ATP production, enhanced reactive oxygen species (ROS) accumulation, and increased mitochondrial outer membrane permeability. Transcriptome profiling revealed widespread down-regulation of genes underpinning mitochondrial functions, and up-regulation of genes associated with tumor growth and aggression. These results highlight a critical role for ERAD in maintaining mitochondrial functional and structural integrity and raise the possibility of improving cellular and organismal mitochondrial function via enhancing cellular ERAD capacity.
    Keywords:  ERAD; ROS; SEL1L; calcium; cell death; cytochrome c; endoplasmic reticulum; endoplasmic reticulum stress (ER stress); endoplasmic reticulum-associated protein degradation (ERAD); hepatocyte death; liver; mitochondria; mitochondrial disease; mitochondrial permeability transition (MPT)
    DOI:  https://doi.org/10.1074/jbc.RA120.013987
  18. J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50639-0. [Epub ahead of print]295(51): 17549-17559
    Sharpe LJ, Coates HW, Brown AJ.
      The synthesis of cholesterol requires more than 20 enzymes, many of which are intricately regulated. Post-translational control of these enzymes provides a rapid means for modifying flux through the pathway. So far, several enzymes have been shown to be rapidly degraded through the ubiquitin-proteasome pathway in response to cholesterol and other sterol intermediates. Additionally, several enzymes have their activity altered through phosphorylation mechanisms. Most work has focused on the two rate-limiting enzymes: 3-hydroxy-3-methylglutaryl CoA reductase and squalene monooxygenase. Here, we review current literature in the area to define some common themes in the regulation of the entire cholesterol synthesis pathway. We highlight the rich variety of inputs controlling each enzyme, discuss the interplay that exists between regulatory mechanisms, and summarize findings that reveal an intricately coordinated network of regulation along the cholesterol synthesis pathway. We provide a roadmap for future research into the post-translational control of cholesterol synthesis, and no doubt the road ahead will reveal further twists and turns for this fascinating pathway crucial for human health and disease.
    Keywords:  E3 ubiquitin ligase; HMGCR; SM; acetylation; cholesterol; cholesterol metabolism; cholesterol regulation; cholesterol synthesis; phosphorylation; post-translational modification; post-translational modification (PTM); post-translational regulation; proteasome; protein degradation; ubiquitin ligase; ubiquitination; ubiquitylation (ubiquitination)
    DOI:  https://doi.org/10.1074/jbc.REV120.010723
  19. Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2006947118. [Epub ahead of print]118(4):
    De Cesare V, Carbajo Lopez D, Mabbitt PD, Fletcher AJ, Soetens M, Antico O, Wood NT, Virdee S.
      The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado-Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.
    Keywords:  DUBs; non-lysine ubiquitination; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2006947118
  20. Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31626-0. [Epub ahead of print]34(3): 108637
    Tosal-Castano S, Peselj C, Kohler V, Habernig L, Berglund LL, Ebrahimi M, Vögtle FN, Höög J, Andréasson C, Büttner S.
      Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.
    Keywords:  Nvj1; SND pathway; Saccharomyces cerevisiae; Snd3; Tsc13; Vac8; glucose metabolism; interorganellar connectivity; membrane contact sites; nucleus-vacuole junction
    DOI:  https://doi.org/10.1016/j.celrep.2020.108637
  21. Cell Mol Gastroenterol Hepatol. 2021 Jan 19. pii: S2352-345X(21)00013-8. [Epub ahead of print]
    Wang F, Zhang Y, Shen J, Yang B, Dai W, Yan J, Maimouni S, Daguplo HQ, Coppola S, Gao Y, Wang Y, Du Z, Peng K, Liu H, Zhang Q, Tang F, Wang P, Gao S, Wang Y, Ding WX, Guo G, Wang F, Zong WX.
      BACKGROUND AND AIMS: TRIM21 is a ubiquitin E3 ligase that is implicated in numerous biological processes including immune response, cell metabolism, redox homeostasis, and cancer development. We recently reported that TRIM21 can negatively regulate the p62-Keap1-Nrf2 antioxidant pathway by ubiquitylating p62 and prevents its oligomerization and protein sequestration function. As redox homeostasis plays a pivotal role in many cancers including liver cancer, we sought to determine the role of TRIM21 in hepatocarcinogenesis.METHODS: We examined the correlation between TRIM21 expression and the disease using publicly available data sets and 49 cases of HCC clinical samples. We used TRIM21 genetic knockout mice to determine how TRIM21 ablation impact HCC induced by the carcinogen DEN plus phenobarbital (PB). We explored the mechanism that loss of TRIM21 protects cells from DEN-induced oxidative damage and cell death.
    RESULTS: There is a positive correlation between TRIM21 expression and HCC. Consistently, TRIM21-knockout mice are resistant to DEN-induced hepatocarcinogenesis. This is accompanied by decreased cell death and tissue damage upon DEN treatment, hence reduced hepatic tissue repair response and compensatory proliferation. Cells deficient in TRIM21 display enhanced p62 sequestration of Keap1 and are protected from DEN-induced ROS induction and cell death. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient mutant TRIM21 impedes the protection from DEN-induced oxidative damage and cell death in TRIM21-deficient cells.
    CONCLUSIONS: Increased TRIM21 expression is associated with human HCC. Genetic ablation of TRIM21 leads to protection against oxidative hepatic damage and decreased hepatocarcinogenesis, suggesting TRIM21 as a preventive and therapeutic target.
    Keywords:  Nrf2; TRIM21; diethylnitrosamine; hepatocellular carcinoma; p62
    DOI:  https://doi.org/10.1016/j.jcmgh.2021.01.007
  22. J Biol Chem. 2021 Jan 19. pii: S0021-9258(21)00079-X. [Epub ahead of print] 100310
    Uchida Y, Yamamoto Y, Sakisaka T.
      The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is a FA reductase present in the ER membrane, and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), an ER Ca2+ pump responsible for Ca2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca2+ storage in the ER and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. Moreover, depletion of TER reduces the Ca2+-dependent nuclear translocation of nuclear factor of activated T cells 4 (NFAT4). These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.
    Keywords:  NFAT transcription factor; calcium; calcium ATPase; calcium transport; endoplasmic reticulum; fatty acid metabolism; protein-protein interaction
    DOI:  https://doi.org/10.1016/j.jbc.2021.100310
  23. Nat Commun. 2021 Jan 22. 12(1): 521
    Rasul F, Zheng F, Dong F, He J, Liu L, Liu W, Cheema JY, Wei W, Fu C.
      The endoplasmic reticulum-mitochondria encounter structure (ERMES) complex creates contact sites between the endoplasmic reticulum and mitochondria, playing crucial roles in interorganelle communication, mitochondrial fission, mtDNA inheritance, lipid transfer, and autophagy. The mechanism regulating the number of ERMES foci within the cell remains unclear. Here, we demonstrate that the mitochondrial membrane protein Emr1 contributes to regulating the number of ERMES foci. We show that the absence of Emr1 significantly decreases the number of ERMES foci. Moreover, we find that Emr1 interacts with the ERMES core component Mdm12 and colocalizes with Mdm12 on mitochondria. Similar to ERMES mutant cells, cells lacking Emr1 display defective mitochondrial morphology and impaired mitochondrial segregation, which can be rescued by an artificial tether capable of linking the endoplasmic reticulum and mitochondria. We further demonstrate that the cytoplasmic region of Emr1 is required for regulating the number of ERMES foci. This work thus reveals a crucial regulatory protein necessary for ERMES functions and provides mechanistic insights into understanding the dynamic regulation of endoplasmic reticulum-mitochondria communication.
    DOI:  https://doi.org/10.1038/s41467-020-20866-x
  24. J Biol Chem. 2020 Aug 21. pii: S0021-9258(17)50059-9. [Epub ahead of print]295(34): 11963-11970
    Marinko JT, Carter BD, Sanders CR.
      Charcot-Marie-Tooth disease (CMT) is a neuropathy of the peripheral nervous system that afflicts ∼1:2500 people. The most common form of this disease (CMT1A, 1:4000) is associated with duplication of chromosome fragment 17p11.2-12, which results in a third WT PMP22 allele. In rodent models overexpressing the PMP22 (peripheral myelin protein 22) protein and in dermal fibroblasts from patients with CMT1A, PMP22 aggregates have been observed. This suggests that overexpression of PMP22 under CMT1A conditions overwhelms the endoplasmic reticulum quality control system, leading to formation of cytotoxic aggregates. In this work, we used a single-cell flow-cytometry trafficking assay to quantitatively examine the relationship between PMP22 expression and trafficking efficiency in individual cells. We observed that as expression of WT or disease variants of PMP22 is increased, the amount of intracellular PMP22 increases to a greater extent than the amount of surface-trafficked protein. This was true for both transiently transfected cells and PMP22 stable expressing cells. Our results support the notion that overexpression of PMP22 in CMT1A leads to a disproportionate increase in misfolding and mistrafficking of PMP22, which is likely a contributor to disease pathology and progression.
    Keywords:  Charcot–Marie–Tooth disease (CMT); cell surface protein; flow cytometry; membrane protein; membrane trafficking; myelin; peripheral neuropathy; protein folding; protein misfolding
    DOI:  https://doi.org/10.1074/jbc.AC120.014940
  25. Sci Rep. 2021 Jan 18. 11(1): 1713
    Zhang Y, Lai X, Yang S, Ren H, Yuan J, Jin H, Shi C, Lai Z, Xia G.
      Plants have evolved genetic and physiological mechanisms to mitigate the adverse effects of high temperature. CARBOXYL TERMINUS OF THE HSC70-INTERACTING PROTEINS (CHIP) is a conserved chaperone-dependent ubiquitin E3 ligase that targets misfolded proteins. Here, we report functional analysis of the SlCHIP gene from tomato (Solanum lycopersicum) in heat tolerance. SlCHIP encodes a CHIP protein with three tandem tetracopeptide repeat (TPR) motifs and a C-terminal U box domain. Phylogenetic analysis of CHIP homologs from animals, spore-bearing and seed plants revealed a tree topology similar to the evolutionary tree of the organisms. Expression of SlCHIP was induced under high temperature and was also responsive to plant stress hormones. Silencing of SlCHIP in tomato reduced heat tolerance based on increased heat stress symptoms, reduced photosynthetic activity, elevated electrolyte leakage and accumulation of insoluble protein aggregates. The accumulated protein aggregates in SlCHIP-silenced plants were still highly ubiquitinated, suggesting involvement of other E3 ligases in ubiquitination. SlCHIP restored the heat tolerance of Arabidopsis chip mutant to the wild type levels. These results indicate that tomato SlCHIP plays a critical role in heat stress responses most likely by targeting degradation of misfolded proteins that are generated during heat stress.
    DOI:  https://doi.org/10.1038/s41598-021-81372-8
  26. Nat Commun. 2021 01 20. 12(1): 479
    Shpilka T, Du Y, Yang Q, Melber A, Uma Naresh N, Lavelle J, Kim S, Liu P, Weidberg H, Li R, Yu J, Zhu LJ, Strittmatter L, Haynes CM.
      As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.
    DOI:  https://doi.org/10.1038/s41467-020-20784-y
  27. Nature. 2020 Dec 09.
    Agudo-Canalejo J, Schultz SW, Chino H, Migliano SM, Saito C, Koyama-Honda I, Stenmark H, Brech A, May AI, Mizushima N, Knorr RL.
      Compartmentalization of cellular material in droplet-like structures is a hallmark of liquid-liquid phase separation1,2, but the mechanisms of droplet removal are poorly understood. Evidence suggests that droplets can be degraded by autophagy3,4, a highly conserved degradation system in which membrane sheets bend to isolate portions of the cytoplasm within double-membrane autophagosomes5-7. Here we examine how autophagosomes sequester droplets that contain the protein p62 (also known as SQSTM1) in living cells, and demonstrate that double-membrane, autophagosome-like vesicles form at the surface of protein-free droplets in vitro through partial wetting. A minimal physical model shows that droplet surface tension supports the formation of membrane sheets. The model also predicts that bending sheets either divide droplets for piecemeal sequestration or sequester entire droplets. We find that autophagosomal sequestration is robust to variations in the droplet-sheet adhesion strength. However, the two sides of partially wetted sheets are exposed to different environments, which can determine the bending direction of autophagosomal sheets. Our discovery of this interplay between the material properties of droplets and membrane sheets enables us to elucidate the mechanisms that underpin droplet autophagy, or 'fluidophagy'. Furthermore, we uncover a switching mechanism that allows droplets to act as liquid assembly platforms for cytosol-degrading autophagosomes8 or as specific autophagy substrates9-11. We propose that droplet-mediated autophagy represents a previously undescribed class of processes that are driven by elastocapillarity, highlighting the importance of wetting in cytosolic organization.
    DOI:  https://doi.org/10.1038/s41586-020-2992-3
  28. J Cell Sci. 2021 Jan 19. pii: jcs.250670. [Epub ahead of print]
    Ravussin A, Brech A, Tooze SA, Stenmark H.
      Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.
    Keywords:  Autophagy; Endosome; Phosphoinositide; Recycling
    DOI:  https://doi.org/10.1242/jcs.250670
  29. J Biol Chem. 2020 Dec 25. pii: S0021-9258(17)50688-2. [Epub ahead of print]295(52): 18148-18159
    Turan NN, Moshal KS, Roder K, Baggett BC, Kabakov AY, Dhakal S, Teramoto R, Chiang DY, Zhong M, Xie A, Lu Y, Dudley SC, MacRae CA, Karma A, Koren G.
      The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current INa and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on INa. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.
    Keywords:  E3 ubiquitin ligase; LITAF; NEDD4-2; Nav1.5; action potential duration; cardiomyocyte; computer modelling; sodium channel; ubiquitin; zebrafish
    DOI:  https://doi.org/10.1074/jbc.RA120.015216
  30. Autophagy. 2021 Jan 19. 1-20
    Meyer N, Henkel L, Linder B, Zielke S, Tascher G, Trautmann S, Geisslinger G, Münch C, Fulda S, Tegeder I, Kögel D.
      Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM. Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.
    Keywords:  Acid sphingomyelinase; autophagy-dependent cell death; brain tumors; cholesterol metabolism; drug repurposing; er stress; lysophagy
    DOI:  https://doi.org/10.1080/15548627.2021.1874208
  31. J Integr Plant Biol. 2021 Jan 20.
    Hong L, Niu F, Lin Y, Wang S, Chen L, Jiang L.
      Flowering time is crucial for successful reproduction in plants, whose onset and progression are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors (TFs) is a dynamic and essential mechanism for plant growth and development. CRL3BPM E3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T (FT) that is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays (EMSA), in vivo chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) assays and dual-luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3BPM E3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3BPM E3 ligases in Arabidopsis. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1111/jipb.13071
  32. Cell Death Differ. 2021 Jan 18.
    Chen X, Yu C, Kang R, Kroemer G, Tang D.
      In eukaryotic cells, macromolecular homeostasis requires selective degradation of damaged units by the ubiquitin-proteasome system (UPS) and autophagy. Thus, dysfunctional degradation systems contribute to multiple pathological processes. Ferroptosis is a type of iron-dependent oxidative cell death driven by lipid peroxidation. Various antioxidant systems, especially the system xc--glutathione-GPX4 axis, play a significant role in preventing lipid peroxidation-mediated ferroptosis. The endosomal sorting complex required for transport-III (ESCRT-III)-dependent membrane fission machinery counteracts ferroptosis by repairing membrane damage. Moreover, cellular degradation systems play a dual role in regulating the ferroptotic response, depending on the cargo they degrade. The key ferroptosis repressors, such as SLC7A11 and GPX4, are degraded by the UPS. In contrast, the overactivation of selective autophagy, including ferritinophagy, lipophagy, clockophagy and chaperone-mediated autophagy, promotes ferroptotic death by degrading ferritin, lipid droplets, circadian proteins, and GPX4, respectively. Autophagy modulators (e.g., BECN1, STING1/TMEM173, CTSB, HMGB1, PEBP1, MTOR, AMPK, and DUSP1) also determine the ferroptotic response in a context-dependent manner. In this review, we provide an updated overview of the signals and mechanisms of the degradation system regulating ferroptosis, opening new horizons for disease treatment strategies.
    DOI:  https://doi.org/10.1038/s41418-020-00728-1
  33. Chembiochem. 2021 Jan 22.
    D'Amico F, Mukhopadhyay R, Ovaa H, Mulder MPC.
      The ubiquitination machinery regulates several fundamental biological processes from protein homeostasis to a wide variety of cellular signalling pathways. As a consequence, its dysregulation is linked to diseases, including cancer, neurodegeneration, and autoimmunity. With this review, we aim to highlight the therapeutic potential of targeting E3 ligases with a special focus on an emerging class of RING ligases, named Tri-Partite Motif (TRIM) proteins, whose role as targets for drug development is currently gaining pharmaceutical attention. TRIM proteins exert their catalytic activity as scaffolds involved in many protein-protein interactions, whose multi-domains and adapter-like nature make their druggability very challenging. Here we report an overview of the current understanding of this class of single polypeptide RING E3 ligases and discuss potential targeting options.
    Keywords:  ligases inhibitors high throughput screening TRIM Ubiquitin
    DOI:  https://doi.org/10.1002/cbic.202000787
  34. EMBO Rep. 2021 Jan 22. e49651
    Barysch SV, Stankovic-Valentin N, Miedema T, Karaca S, Doppel J, Nait Achour T, Vasudeva A, Wolf L, Sticht C, Urlaub H, Melchior F.
      Molecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin-related modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compare the endogenous SUMO proteomes of HeLa cells before and after EGF stimulation. Thereby, we identify a small group of transcriptional coregulators including IRF2BP1, IRF2BP2, and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wild type or SUMOylation-deficient IRF2BP1 indicates that transient deSUMOylation of IRF2BP proteins is important for appropriate expression of immediate early genes including dual specificity phosphatase 1 (DUSP1, MKP-1) and the transcription factor ATF3. We find that IRF2BP1 is a repressor, whose transient deSUMOylation on the DUSP1 promoter allows-and whose timely reSUMOylation restricts-DUSP1 transcription. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription.
    Keywords:  ATF3; DUSP1; EGFR; IRF2BP1; SUMO
    DOI:  https://doi.org/10.15252/embr.201949651
  35. PLoS Biol. 2021 Jan 22. 19(1): e3000998
    Prasanna X, Salo VT, Li S, Ven K, Vihinen H, Jokitalo E, Vattulainen I, Ikonen E.
      Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.
    DOI:  https://doi.org/10.1371/journal.pbio.3000998
  36. EMBO J. 2021 Jan 20. e2020105375
    Yamanaka S, Murai H, Saito D, Abe G, Tokunaga E, Iwasaki T, Takahashi H, Takeda H, Suzuki T, Shibata N, Tamura K, Sawasaki T.
      Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.
    Keywords:  CRBN; protein degradation; thalidomide metabolite; thalidomide teratogenicity; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2020105375
  37. Cell Death Dis. 2021 Jan 22. 12(1): 114
    Chaumonnot K, Masson S, Sikner H, Bouchard A, Baverel V, Bellaye PS, Collin B, Garrido C, Kohli E.
      The role of GRP94, an endoplasmic reticulum (ER) stress protein with both pro- and anti-inflammatory functions, has not been investigated in macrophages during ER stress, whereas ER stress has been reported in many diseases involving macrophages. In this work, we studied GRP94 in M1/LPS + IFNγ and M2/IL-4 primary macrophages derived from human monocytes (isolated from buffy coats), in basal and ER stress conditions induced by thapsigargin (Tg), an inducer of ER calcium depletion and tunicamycin (Tm), an inhibitor of N-glycosylation. We found that GRP94 was expressed on the membrane of M2 but not M1 macrophages. In M2, Tg, but not Tm, while decreased GRP94 content in the membrane, it induced its secretion. This correlated with the induction of a pro-inflammatory profile, which was dependent on the UPR IRE1α arm activation and on a functional GRP94. As we previously reported that GRP94 associated with complement C3 at the extracellular level, we analyzed C3 and confirmed GRP94-C3 interaction in our experimental model. Further, Tg increased this interaction and, in these conditions, C3b and cathepsin L were detected in the extracellular medium where GRP94 co-immunoprecipitated with C3 and C3b. Finally, we showed that the C3b inactivated fragment, iC3b, only present on non-stressed M2, depended on functional GRP94, making both GRP94 and iC3b potential markers of M2 cells. In conclusion, our results show that GRP94 is co-secreted with C3 under ER stress conditions which may facilitate its cleavage by cathepsin L, thus contributing to the pro-inflammatory profile observed in stressed M2 macrophages.
    DOI:  https://doi.org/10.1038/s41419-020-03288-x
  38. Biochemistry. 2021 Jan 20.
    Jiang H, Dempsey DR, Cole PA.
      WWP1 is an E3 ubiquitin ligase that has been reported to target the tumor suppressor lipid phosphatase PTEN. K740N and N745S are recently identified germline variants of WWP1 that have been linked to PTEN-associated cancers [Lee, Y. R., et al. (2020) N. Engl. J. Med.]. These WWP1 variants have been suggested to release WWP1 from its native autoinhibited state, thereby promoting enhanced PTEN ubiquitination as a mechanism for driving cancer. Using purified proteins and in vitro enzymatic assays, we investigate the possibility that K740N and N745S WWP1 possess enhanced ubiquitin ligase activity and demonstrate that these variants are similar to the wild type (WT) in both autoubiquitination and PTEN ubiquitination. Furthermore, K740N and N745S WWP1 show dependencies similar to those of WT in terms of allosteric activation by an engineered ubiquitin variant, upstream E2 concentration, and substrate ubiquitin concentration. Transfected WWP1 WT and mutants demonstrate comparable effects on cellular PTEN levels. These findings challenge the idea that K740N and N745S WWP1 variants promote cancer by enhanced PTEN ubiquitination.
    DOI:  https://doi.org/10.1021/acs.biochem.0c00869
  39. Cell Rep. 2021 Jan 19. pii: S2211-1247(20)31624-7. [Epub ahead of print]34(3): 108635
    Lobato-Gil S, Heidelberger JB, Maghames C, Bailly A, Brunello L, Rodriguez MS, Beli P, Xirodimas DP.
      The ubiquitin-like molecule NEDD8 controls several biological processes and is a promising target for therapeutic intervention. NEDDylation occurs through specific NEDD8 enzymes (canonical) or enzymes of the ubiquitin system (atypical). Identification of NEDD8 sites on substrates is critical for delineating the processes controlled by NEDDylation. By combining the use of the NEDD8 R74K mutant with anti-di-glycine (anti-diGly) antibodies, we identified 1,101 unique NEDDylation sites in 620 proteins. Bioinformatics analysis reveals that canonical and atypical NEDDylation have distinct proteomes; the spliceosome/mRNA surveillance/DNA replication and ribosome/proteasome, respectively. The data also reveal the formation of poly-NEDD8, hybrid NEDD8-ubiquitin, and NEDD8-SUMO-2 chains as potential molecular signals. In particular, NEDD8-SUMO-2 chains are induced upon proteotoxic stress (atypical) through NEDDylation of K11 in SUMO-2, and conjugates accumulate in previously described nucleolus-related inclusions. The study uncovers a diverse proteome for NEDDylation and is consistent with the concept of extensive cross-talk between ubiquitin and Ubls under proteotoxic stress conditions.
    Keywords:  NEDD8; NEDD8-SUMO polymers; NEDP1; atypical; canonical; diGly signatures; hybrid chains; nucleolus-related inclusions; proteotoxic stress
    DOI:  https://doi.org/10.1016/j.celrep.2020.108635
  40. Cell Death Differ. 2021 Jan 19.
    Zheng Z, Zhang X, Huang B, Liu J, Wei X, Shan Z, Wu H, Feng Z, Chen Y, Fan S, Zhao F, Chen J.
      Site-1 protease (S1P) is a Golgi-located protein that activates unique membrane-bound latent transcription factors, and it plays an indispensable role in endoplasmic reticulum stress, lipid metabolism, inflammatory response and lysosome function. A patient with S1P mutation exhibits severe skeletal dysplasia with kyphoscoliosis, dysmorphic facial features and pectus carinatum. However, whether S1P regulates bone remodeling by affecting osteoclastogenesis remains elusive. Here, we show that S1P is indeed a positive regulator of osteoclastogenesis. S1P ablation in mice led to significant osteosclerosis compared with wild-type littermates. Mechanistically, S1P showed upregulated during osteoclastogenesis and was identified as a direct target of miR-9-5p. S1P deletion in bone marrow monocytes (BMMs) inhibited ATF6 and SREBP2 maturation, which subsequently impeded CHOP/SREBP2-complex-induced LC3 expression and autophagy flux. Consistently, transfection of LC3 adenovirus evidently rescued osteoclastogenesis in S1P-deficient BMMs. We then identified the interaction regions between CHOP and SREBP2 by Co-immunoprecipitation (Co-IP) and molecular docking. Furthermore, S1P deletion or inhibitor efficaciously rescued ovariectomized (OVX)- and LPS-induced bone loss in vivo. Collectively, we showed that S1P regulates osteoclast differentiation in a LC3 dependent manner and so is a potential therapy target for osteoporosis.
    DOI:  https://doi.org/10.1038/s41418-020-00731-6
  41. JCI Insight. 2021 Jan 21. pii: 141518. [Epub ahead of print]
    Barghout SH, Aman A, Nouri K, Blatman Z, Arevalo K, Thomas GE, MacLean N, Hurren R, Ketela T, Saini M, Abohawya M, Kiyota T, Al-Awar R, Schimmer AD.
      TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 (UBA1) that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase 1 clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ABC efflux transporter breast cancer resistance protein (BCRP; ABCG2), and reduced the intracellular levels of TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origin. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.
    Keywords:  Cancer; Drug screens; Oncology; Therapeutics; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/jci.insight.141518
  42. Nat Commun. 2021 01 21. 12(1): 513
    Johnson AE, Orr BO, Fetter RD, Moughamian AJ, Primeaux LA, Geier EG, Yokoyama JS, Miller BL, Davis GW.
      Missense mutations in Valosin-Containing Protein (VCP) are linked to diverse degenerative diseases including IBMPFD, amyotrophic lateral sclerosis (ALS), muscular dystrophy and Parkinson's disease. Here, we characterize a VCP-binding co-factor (SVIP) that specifically recruits VCP to lysosomes. SVIP is essential for lysosomal dynamic stability and autophagosomal-lysosomal fusion. SVIP mutations cause muscle wasting and neuromuscular degeneration while muscle-specific SVIP over-expression increases lysosomal abundance and is sufficient to extend lifespan in a context, stress-dependent manner. We also establish multiple links between SVIP and VCP-dependent disease in our Drosophila model system. A biochemical screen identifies a disease-causing VCP mutation that prevents SVIP binding. Conversely, over-expression of an SVIP mutation that prevents VCP binding is deleterious. Finally, we identify a human SVIP mutation and confirm the pathogenicity of this mutation in our Drosophila model. We propose a model for VCP disease based on the differential, co-factor-dependent recruitment of VCP to intracellular organelles.
    DOI:  https://doi.org/10.1038/s41467-020-20796-8
  43. EMBO J. 2021 Jan 20. e105846
    Yeh CW, Huang WC, Hsu PH, Yeh KH, Wang LC, Hsu PW, Lin HC, Chen YN, Chen SC, Yeang CH, Yen HS.
      Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino- or carboxyl-termini are eliminated by the N- or C-degron pathways, respectively. We aimed to elucidate the function of C-degron pathways and to unveil how normal proteomes are exempt from C-degron pathway-mediated destruction. Our data reveal that C-degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C-degron and N-degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C-degron pathways, but not of defective proteins, suggesting proteolysis-based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.
    Keywords:  C-degron pathway; CRL2 ubiquitin ligase; N-degron pathway; protein spatial quality control; protein termini
    DOI:  https://doi.org/10.15252/embj.2020105846
  44. Life Sci Alliance. 2021 Mar;pii: e202000918. [Epub ahead of print]4(3):
    Xiao T, Shakya VP, Hughes AL.
      Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins.
    DOI:  https://doi.org/10.26508/lsa.202000918
  45. Elife. 2021 Jan 21. pii: e60480. [Epub ahead of print]10
    Rodriguez KM, Buch-Larsen SC, Kirby IT, Siordia I, Hutin D, Rasmussen M, Grant DM, David LL, Matthews J, Nielsen ML, Cohen MS.
      Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.
    Keywords:  biochemistry; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.60480
  46. FASEB J. 2021 Feb;35(2): e21281
    Hirata H, Xu X, Nishioka K, Matsuhisa F, Kitajima S, Kukita T, Murayama M, Urano Y, Miyamoto H, Mawatari M, Kukita A.
      Osteoclast bone resorption activity is critically regulated to maintain bone homeostasis. Osteoclasts resorb bone by producing protons and acid hydrolase via lysosomal secretion, however, a detailed mechanism remains elusive. PMEPA1 is a vesicular membrane protein, which binds to the NEDD4 family member of ubiquitin ligases. We have previously reported that Pmepa1 is highly expressed in bone resorbing osteoclasts, and regulates bone resorption. Here, we investigated the mechanism of bone resorption regulated by PMEPA1. Mutant mice lacking NEDD4-binding domains of PMEPA1 displayed enhanced bone volume, and reduced bone resorption activity in comparison with those of WT mice. Analysis with pH-sensitive fluorescence probe revealed that proton secretion from osteoclasts significantly decreased in Pmepa1 mutant osteoclasts. Immunofluorescence analysis revealed that PMEPA1 was colocalized with NEDD4, V0A3, and V0D2 subunits of vacuolar ATPase, which regulate the proton production of osteoclasts. In addition, Nedd4 knockdown reduced bone resorption and proton secretion of osteoclasts. Furthermore, Pmepa1 mutation and Nedd4 knockdown altered the cytoplasmic distribution of components of V-ATPase and expression of autophagy-related proteins, suggesting that lysosomal secretion is affected. Collectively, these findings indicate that PMEPA1 controls proton secretion from osteoclasts via NEDD4 by regulating vesicular trafficking, and NEDD4 is an important regulator of bone resorption.
    Keywords:  NEDD4; V-ATPase; lysosomal secretion; osteoclast; proton production
    DOI:  https://doi.org/10.1096/fj.202001795R
  47. Biomolecules. 2021 Jan 05. pii: E60. [Epub ahead of print]11(1):
    Çetin G, Klafack S, Studencka-Turski M, Krüger E, Ebstein F.
      The ubiquitin-proteasome system (UPS) is the major intracellular and non-lysosomal protein degradation system. Thanks to its unique capacity of eliminating old, damaged, misfolded, and/or regulatory proteins in a highly specific manner, the UPS is virtually involved in almost all aspects of eukaryotic life. The critical importance of the UPS is particularly visible in immune cells which undergo a rapid and profound functional remodelling upon pathogen recognition. Innate and/or adaptive immune activation is indeed characterized by a number of substantial changes impacting various cellular processes including protein homeostasis, signal transduction, cell proliferation, and antigen processing which are all tightly regulated by the UPS. In this review, we summarize and discuss recent progress in our understanding of the molecular mechanisms by which the UPS contributes to the generation of an adequate immune response. In this regard, we also discuss the consequences of UPS dysfunction and its role in the pathogenesis of recently described immune disorders including cancer and auto-inflammatory diseases.
    Keywords:  autoinflammation; cancer; immunity; proteostasis; ubiquitin–proteasome system
    DOI:  https://doi.org/10.3390/biom11010060
  48. Proc Natl Acad Sci U S A. 2021 Jan 26. pii: e2021943118. [Epub ahead of print]118(4):
    Koh M, Ahmad I, Ko Y, Zhang Y, Martinez TF, Diedrich JK, Chu Q, Moresco JJ, Erb MA, Saghatelian A, Schultz PG, Bollong MJ.
      Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
    Keywords:  expanded genetic code; photo-crosslinking; short open reading frame-encoded peptide; transcriptional regulation
    DOI:  https://doi.org/10.1073/pnas.2021943118
  49. J Clin Invest. 2021 Jan 19. pii: 143645. [Epub ahead of print]
    Chen YT, Jhao PY, Hung CT, Wu YF, Lin SJ, Chiang WC, Lin SL, Yang KC.
      Renal fibrosis, a common pathological manifestation of virtually all types of chronic kidney diseases (CKD), often results in diffuse kidney scarring and predisposes to end-stage renal disease. Currently, there is no effective therapy against renal fibrosis. Recently, our laboratory identified an ER-resident protein, thioredoxin domain containing 5 (TXNDC5), as a critical mediator of cardiac fibrosis. Transcriptome analyses of renal biopsy specimens from CKD patients revealed marked TXNDC5 upregulation in fibrotic kidneys, suggesting a potential role of TXNDC5 in renal fibrosis. Employing multiple fluorescent reporter mouse lines, we showed that TXNDC5 was specifically upregulated in collagen-secreting fibroblasts in fibrotic mouse kidneys. In addition, we showed that TXNDC5 was required for TGFβ1-induced fibrogenic responses in human kidney fibroblasts (HKF), whereas TXNDC5 over-expression was sufficient to promote HKF activation, proliferation and collagen production. Mechanistically, we showed that TXNDC5, transcriptionally controlled by ATF6-dependent ER stress pathway, mediates its pro-fibrogenic effects by enforcing TGFβ signaling activity through post-translational stabilization and upregulation of type I TGFβ receptor in kidney fibroblasts. Using a tamoxifen-inducible, fibroblast-specific Txndc5 knockout mouse line, we demonstrated that deletion of Txndc5 in kidney fibroblasts mitigated the progression of established kidney fibrosis, suggesting the therapeutic potential of TXNDC5 targeting for renal fibrosis and CKD. .
    Keywords:  Cell Biology; Fibrosis; Nephrology; Protein misfolding
    DOI:  https://doi.org/10.1172/JCI143645
  50. Commun Biol. 2021 Jan 19. 4(1): 80
    Nakabayashi O, Takahashi H, Moriwaki K, Komazawa-Sakon S, Ohtake F, Murai S, Tsuchiya Y, Koyahara Y, Saeki Y, Yoshida Y, Yamazaki S, Tokunaga F, Sawasaki T, Nakano H.
      Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.
    DOI:  https://doi.org/10.1038/s42003-020-01603-y
  51. Cell Death Differ. 2021 Jan 18.
    Masclef L, Ahmed O, Estavoyer B, Larrivée B, Labrecque N, Nijnik A, Affar EB.
      The BAP1 gene has emerged as a major tumor suppressor mutated with various frequencies in numerous human malignancies, including uveal melanoma, malignant pleural mesothelioma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, and thymic epithelial tumors. BAP1 mutations are also observed at low frequency in other malignancies including breast, colorectal, pancreatic, and bladder cancers. BAP1 germline mutations are associated with high incidence of mesothelioma, uveal melanoma, and other cancers, defining the "BAP1 cancer syndrome." Interestingly, germline BAP1 mutations constitute an important paradigm for gene-environment interactions, as loss of BAP1 predisposes to carcinogen-induced tumorigenesis. Inactivating mutations of BAP1 are also identified in sporadic cancers, denoting the importance of this gene for normal tissue homeostasis and tumor suppression, although some oncogenic properties have also been attributed to BAP1. BAP1 belongs to the deubiquitinase superfamily of enzymes, which are responsible for the maturation and turnover of ubiquitin as well as the reversal of substrate ubiquitination, thus regulating ubiquitin signaling. BAP1 is predominantly nuclear and interacts with several chromatin-associated factors, assembling multi-protein complexes with mutually exclusive partners. BAP1 exerts its function through highly regulated deubiquitination of its substrates. As such, BAP1 orchestrates chromatin-associated processes including gene expression, DNA replication, and DNA repair. BAP1 also exerts cytoplasmic functions, notably in regulating Ca2+ signaling at the endoplasmic reticulum. This DUB is also subjected to multiple post-translational modifications, notably phosphorylation and ubiquitination, indicating that several signaling pathways tightly regulate its function. Recent progress indicated that BAP1 plays essential roles in multiple cellular processes including cell proliferation and differentiation, cell metabolism, as well as cell survival and death. In this review, we summarize the biological and molecular functions of BAP1 and explain how the inactivation of this DUB might cause human cancers. We also highlight some of the unresolved questions and suggest potential new directions.
    DOI:  https://doi.org/10.1038/s41418-020-00709-4
  52. Cancers (Basel). 2021 Jan 19. pii: E341. [Epub ahead of print]13(2):
    Ali MS, Panuzzo C, Calabrese C, Maglione A, Piazza R, Cilloni D, Saglio G, Pergolizzi B, Bracco E.
      HERC E3 subfamily members are parts of the E3 ubiquitin ligases and key players for a wide range of cellular functions. Though the involvement of the Ubiquitin Proteasome System in blood disorders has been broadly studied, so far the role of large HERCs in this context remains unexplored. In the present study we examined the expression of the large HECT E3 Ubiquitin Ligase, HERC1, in blood disorders. Our findings revealed that HERC1 gene expression was severely downregulated both in acute and in chronic myelogenous leukemia at diagnosis, while it is restored after complete remission achievement. Instead, in Philadelphia the negative myeloproliferative neoplasm HERC1 level was peculiarly controlled, being very low in Primary Myelofibrosis and significantly upregulated in those Essential Thrombocytemia specimens harboring the mutation in the calreticulin gene. Remarkably, in CML cells HERC1 mRNA level was associated with the BCR-ABL1 kinase activity and the HERC1 protein physically interacted with BCR-ABL1. Furthermore, we found that HERC1 was directly tyrosine phosphorylated by the ABL kinase. Overall and for the first time, we provide original evidence on the potential tumor-suppressing or -promoting properties, depending on the context, of HERC1 in myeloid related blood disorders.
    Keywords:  Bcr-Abl1; E3 ubiquitin ligases; HERC1; gene expression; myeloid neoplasms; protein–protein interaction; ubiquitin proteasome system
    DOI:  https://doi.org/10.3390/cancers13020341
  53. J Biol Chem. 2020 Dec 18. pii: S0021-9258(17)50648-1. [Epub ahead of print]295(51): 17672-17683
    Klickstein JA, Mukkavalli S, Raman M.
      Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates, limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use, and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user-defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percentage of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates, and (v) localization of aggregates (cytosol, perinuclear, or nuclear). A data table of aggregate information on a per cell basis, as well as a summary table, is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules, and inclusion bodies caused by huntingtin polyglutamine expansion.
    Keywords:  Huntington disease; ImageJ; aggregate; aggregation; aggresome; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); image-based analysis; inclusion body; microscopic imaging; misfolded protein; p97/valosin-containing protein; polyQ inclusion body; polyubiquitin chain; protein aggregation; protein misfolding p97/valosin-containing protein; protein quality control; proteostasis; stress granule; ubiquitin
    DOI:  https://doi.org/10.1074/jbc.RA120.015398
  54. Cell Death Dis. 2021 Jan 22. 12(1): 120
    Amer-Sarsour F, Kordonsky A, Berdichevsky Y, Prag G, Ashkenazi A.
      Ubiquitylation and deubiquitylation play a pivotal role in protein homeostasis (proteostasis). Proteostasis shapes the proteome landscape in the human brain and its impairment is linked to neurodevelopmental and neurodegenerative disorders. Here we discuss the emerging roles of deubiquitylating enzymes in neuronal function and survival. We provide an updated perspective on the genetics, physiology, structure, and function of deubiquitylases in neuronal health and disease.
    DOI:  https://doi.org/10.1038/s41419-020-03361-5