bims-proteo Biomed News
on Proteostasis
Issue of 2021‒02‒21
fifty-eight papers selected by
Eric Chevet
INSERM


  1. J Clin Invest. 2021 Feb 16. pii: 143988. [Epub ahead of print]
    Yoshida S, Wei X, Zhang G, O'Connor CL, Torres M, Zhou Z, Lin L, Menon R, Xu X, Zheng W, Xiong Y, Otto EA, Tang CA, Hua R, Verma R, Mori H, Zhang Y, Hu CA, Liu M, Garg P, Hodgin JB, Sun S, Bitzer M, Qi L.
      Podocytes are key to kidney glomerular filtration barrier by forming slit diaphragm between interdigitating foot processes; however, molecular details and functional importance of protein folding and degradation in the ER remain unknown. Here we show that SEL1L-HRD1 protein complex of endoplasmic reticulum (ER)-associated degradation (ERAD) is required for slit diaphragm formation and glomerular filtration function. SEL1L-HRD1 ERAD is highly expressed in podocytes of both mouse and human kidneys. Mice with podocyte-specific Sel1L deficiency develop podocytopathy and severe congenital nephrotic syndrome shortly after weaning with impaired slit diaphragm, and die prematurely with a median life span of ~3 months. Mechanistically, we show that nephrin, a type-1 membrane protein causally linked to congenital nephrotic syndrome, is an endogenous ERAD substrate. ERAD deficiency attenuates the maturation of nascent nephrin, leading to its retention in the ER. Lastly, we show that various autosomal-recessive nephrin disease mutants are highly unstable and degraded by Sel1L-Hrd1 ERAD, which attenuates the pathogenicity of the mutants towards the wildtype allele. Hence, this study uncovers a critical role of Sel1L-Hrd1 ERAD in glomerular filtration barrier function and provides new insights into the pathogenesis associated with autosomal recessive disease mutants.
    Keywords:  Cell Biology; Nephrology; Protein misfolding; Protein traffic; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI143988
  2. Exp Neurol. 2021 Feb 15. pii: S0014-4886(21)00053-4. [Epub ahead of print] 113648
    Wang L, Bianchi L.
      Mounting evidence support that glia play a key role in organismal ageing. However, the mechanisms by which glia impact ageing are not understood. One of the processes that has significant impact on the rate of ageing is the unfolded protein response. The more robust the UPR, the more the organism can counteract the effect of environmental and genetic stressors. However, how decline of cellular UPR translates into organismal ageing and eventual death is not fully understood. Here we discuss recent findings highlighting that neuropeptides released by glia act long distance to regulate ageing in C. elegans. Taking advantage of the short life span and the genetic amenability of this organism, the endoplasmic reticulum unfolded protein responses (UPRER) can be activated in C. elegans glia. This leads to cell-nonautonomous activation of the UPRER in the intestine. Activation of intestinal UPRER requires the function of genes involved in neuropeptide processing and release, suggesting that neuropeptides signal from glia to the intestine to regulate ER stress response. Importantly, the cell-nonautonomous activation of UPRER leads to extension of life span. Taken together, these data suggest that environmental and genetic factors that impact the response of glia to stress have the potential to influence organismal ageing. Further research on the specific neuropeptides involved should cast new light on the mechanism of ageing and may suggest novel anti-ageing therapies.
    Keywords:  Ageing; C. elegans; Glia; Neuropeptides; Stress; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.expneurol.2021.113648
  3. Curr Biol. 2021 Feb 09. pii: S0960-9822(20)31829-7. [Epub ahead of print]
    Zang J, Klemm S, Pain C, Duckney P, Bao Z, Stamm G, Kriechbaumer V, Bürstenbinder K, Hussey PJ, Wang P.
      In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.
    Keywords:  ER network; ER-PM contact sites; IQD family; NET family; cytoskeleton; kinesin-light-chain-related proteins; plant cell morphogenesis
    DOI:  https://doi.org/10.1016/j.cub.2020.12.009
  4. Mol Cell Proteomics. 2020 Dec 08. pii: S1535-9476(20)35122-7. [Epub ahead of print]20 100008
    Wright MT, Kouba L, Plate L.
      Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for triiodothyronine and thyroxine hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism. Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several congenital hypothyroidism variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for endoplasmic reticulum-associated degradation. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for 1 Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.
    Keywords:  affinity purification - mass spectrometry; cell secretion; congenital hypothyroidism; protein folding; protein quality control; protein-protein interactions; tandem mass tags
    DOI:  https://doi.org/10.1074/mcp.RA120.002168
  5. EMBO Rep. 2021 Feb 15. e49617
    Poncet AF, Bosteels V, Hoffmann E, Chehade S, Rennen S, Huot L, Peucelle V, Maréchal S, Khalife J, Blanchard N, Janssens S, Marion S.
      The unfolded protein response (UPR) has emerged as a central regulator of immune cell responses in several pathologic contexts including infections. However, how intracellular residing pathogens modulate the UPR in dendritic cells (DCs) and thereby affect T cell-mediated immunity remains uncharacterized. Here, we demonstrate that infection of DCs with Toxoplasma gondii (T. gondii) triggers a unique UPR signature hallmarked by the MyD88-dependent activation of the IRE1α pathway and the inhibition of the ATF6 pathway. Induction of XBP1s controls pro-inflammatory cytokine secretion in infected DCs, while IRE1α promotes MHCI antigen presentation of secreted parasite antigens. In mice, infection leads to a specific activation of the IRE1α pathway, which is restricted to the cDC1 subset. Mice deficient for IRE1α and XBP1 in DCs display a severe susceptibility to T. gondii and succumb during the acute phase of the infection. This early mortality is correlated with increased parasite burden and a defect in splenic T-cell responses. Thus, we identify the IRE1α/XBP1s branch of the UPR as a key regulator of host defense upon T. gondii infection.
    Keywords:  Toxoplasma gondii; UPR; antigen presentation; cytokines; dendritic cells
    DOI:  https://doi.org/10.15252/embr.201949617
  6. Cancer Res. 2021 Feb 18. pii: canres.2642.2020. [Epub ahead of print]
    Yu R, Longo J, van Leeuwen JE, Zhang C, Branchard E, Elbaz M, Cescon DW, Drake RR, Dennis JW, Penn LZ.
      Aberrant N-glycan Golgi remodeling and metabolism are associated with epithelial-mesenchymal transition (EMT) and metastasis in breast cancer patients. Despite this association, the N-glycosylation pathway has not been successfully targeted in cancer. Here we show that inhibition of the mevalonate pathway with fluvastatin, a clinically approved drug, reduces both N-glycosylation and N-glycan-branching, essential components of the EMT program and tumor metastasis. This indicates novel crosstalk between N-glycosylation at the endoplasmic reticulum (ER) and N-glycan remodeling at the Golgi. Consistent with this cooperative model between the two spatially separated levels of protein N-glycosylation, fluvastatin-induced tumor cell death was enhanced by loss of Golgi-associated N-acetylglucosaminyltransferases MGAT1 or MGAT5. In a mouse model of post-surgical metastatic breast cancer, adjuvant fluvastatin treatment reduced metastatic burden and improved overall survival. Collectively, these data support the immediate repurposing of fluvastatin as an adjuvant therapeutic to combat metastatic recurrence in breast cancer by targeting protein N-glycosylation at both the ER and Golgi.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-2642
  7. Mol Metab. 2021 Feb 13. pii: S2212-8778(21)00032-6. [Epub ahead of print] 101192
    Kim H, Wei J, Song Z, Mottillo E, Samavati L, Zhang R, Li L, Chen X, Jena BP, Lin JD, Fang D, Zhang K.
      OBJECTIVE: The Endoplasmic Reticulum (ER)-resident E3 ligase HRD1 and its co-activator Sel1L are major components of ER-Associated Degradation (ERAD) machinery. Here, we investigated the molecular mechanism and functional significance underlying the circadian regulation of HRD1/Sel1L-mediated protein degradation program in hepatic energy metabolism.METHODS: Genetically engineered animal models as well as gain- and loss-of function studies were employed to address the circadian regulatory mechanism and functional significance. Gene expression, transcriptional activation, protein-protein interaction, and animal metabolic phenotyping analyses were performed to dissect the molecular network and metabolic pathways.
    RESULTS: Hepatic HRD1 and Sel1L expression exhibits circadian rhythmicity that is controlled by the ER-tethered transcriptional activator CREBH, the nuclear receptor PPARα, and the core clock oscillator BMAL1 in mouse livers. HRD1/Sel1L mediates polyubiquitination and degradation of the CREBH protein across the circadian cycle to modulate rhythmic expression of the genes encoding the rate-limiting enzymes or regulators in fatty acid (FA) oxidation, triglycerides (TG) lipolysis, lipophagy, and gluconeogenesis. HRD1 liver-specific knockout (LKO) mice displayed increased expression of the genes involved in lipid and glucose metabolism and impaired circadian profiles of circulating TG, FA, and glucose due to over-production of CREBH. The circadian metabolic activities of HRD1 LKO mice were inversely correlated with those of CREBH KO mice. Suppressing CREBH over-production in the livers of HRD1 LKO mice restored the diurnal levels of circulating TG and FA of HRD1 LKO mice.
    CONCLUSION: Our work revealed a key circadian-regulated molecular network through which the E3 ubiquitin ligase HRD1 and its co-activator Sel1L regulate hepatic circadian metabolism.
    Keywords:  ER-associated degradation; Endoplasmic reticulum; circadian metabolism; lipid metabolism; transcriptional regulation
    DOI:  https://doi.org/10.1016/j.molmet.2021.101192
  8. Plant Cell. 2020 Sep 04. 32(9): 2964-2978
    Sun J, Movahed N, Zheng H.
      ROOT HAIR DEFECTIVE3 (RHD3) is an atlastin GTPase involved in homotypic fusion of endoplasmic reticulum (ER) tubules in the formation of the interconnected ER network. Because excessive fusion of ER tubules will lead to the formation of sheet-like ER, the action of atlastin GTPases must be tightly regulated. We show here that RHD3 physically interacts with two Arabidopsis (Arabidopsis thaliana) LUNAPARK proteins, LNP1 and LNP2, at three-way junctions of the ER, the sites where different ER tubules fuse. Recruited by RHD3 to newly formed three-way junctions, LNPs act negatively with RHD3 to stabilize the nascent three-way junctions of the ER. Without this LNP-mediated stabilization, in Arabidopsis lnp1-1 lnp2-1 mutant cells, the ER becomes a dense tubular network. Interestingly, in lnp1-1 lnp2-1 mutant cells, the expression level of RHD3 is higher than that in wild-type plants. RHD3 is degraded more slowly in the absence of LNPs as well as in the presence of MG132 and concanamycin A. However, in the presence of LNPs, the degradation of RHD3 is promoted. We have provided in vitro evidence that Arabidopsis LNPs have E3 ubiquitin ligase activity and that LNP1 can directly ubiquitinate RHD3. Our data show that after ER fusion is completed, RHD3 is degraded by LNPs so that nascent three-way junctions can be stabilized and a tubular ER network can be maintained.
    DOI:  https://doi.org/10.1105/tpc.18.00937
  9. Nat Commun. 2021 Feb 19. 12(1): 1194
    Gao P, Ma X, Yuan M, Yi Y, Liu G, Wen M, Jiang W, Ji R, Zhu L, Tang Z, Yu Q, Xu J, Yang R, Xia S, Yang M, Pan J, Yuan H, An H.
      Ubiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.
    DOI:  https://doi.org/10.1038/s41467-021-21456-1
  10. Exp Cell Res. 2021 Feb 11. pii: S0014-4827(21)00046-X. [Epub ahead of print] 112515
    Sekine Y, Houston R, Sekine S.
      Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.
    Keywords:  membrane; membraneless organelles; metabolic stress; metabolite; organelle; stress response
    DOI:  https://doi.org/10.1016/j.yexcr.2021.112515
  11. Cell Metab. 2021 Feb 09. pii: S1550-4131(21)00013-9. [Epub ahead of print]
    Latorre-Muro P, O'Malley KE, Bennett CF, Perry EA, Balsa E, Tavares CDJ, Jedrychowski M, Gygi SP, Puigserver P.
      The architecture of cristae provides a spatial mitochondrial organization that contains functional respiratory complexes. Several protein components including OPA1 and MICOS complex subunits organize cristae structure, but upstream regulatory mechanisms are largely unknown. Here, in vivo and in vitro reconstitution experiments show that the endoplasmic reticulum (ER) kinase PERK promotes cristae formation by increasing TOM70-assisted mitochondrial import of MIC19, a critical subunit of the MICOS complex. Cold stress or β-adrenergic stimulation activates PERK that phosphorylates O-linked N-acetylglucosamine transferase (OGT). Phosphorylated OGT glycosylates TOM70 on Ser94, enhancing MIC19 protein import into mitochondria and promoting cristae formation and respiration. In addition, PERK-activated OGT O-GlcNAcylates and attenuates CK2α activity, which mediates TOM70 Ser94 phosphorylation and decreases MIC19 mitochondrial protein import. We have identified a cold-stress inter-organelle PERK-OGT-TOM70 axis that increases cell respiration through mitochondrial protein import and subsequent cristae formation. These studies have significant implications in cellular bioenergetics and adaptations to stress conditions.
    Keywords:  MIC19; PERK-OGT axis; TOM70; brown adipocytes; cold stress; cristae biogenesis; mitochondrial protein import; respiration
    DOI:  https://doi.org/10.1016/j.cmet.2021.01.013
  12. Blood Adv. 2021 Feb 23. 5(4): 1037-1049
    Rius B, Mesgarzadeh JS, Romine IC, Paxman RJ, Kelly JW, Wiseman RL.
      Light chain (LC) amyloidosis (AL) involves the toxic aggregation of amyloidogenic immunoglobulin LCs secreted from a clonal expansion of diseased plasma cells. Current AL treatments use chemotherapeutics to ablate the AL plasma cell population. However, no treatments are available that directly reduce the toxic LC aggregation involved in AL pathogenesis. An attractive strategy to reduce toxic LC aggregation in AL involves enhancing endoplasmic reticulum (ER) proteostasis in plasma cells to reduce the secretion and subsequent aggregation of amyloidogenic LCs. Here, we show that the ER proteostasis regulator compound 147 reduces secretion of an amyloidogenic LC as aggregation-prone monomers and dimers in AL patient-derived plasma cells. Compound 147 was established to promote ER proteostasis remodeling by activating the ATF6 unfolded protein response signaling pathway through a mechanism involving covalent modification of ER protein disulfide isomerases (PDIs). However, we show that 147-dependent reductions in amyloidogenic LCs are independent of ATF6 activation. Instead, 147 reduces amyloidogenic LC secretion through the selective, on-target covalent modification of ER proteostasis factors, including PDIs, revealing an alternative mechanism by which this compound can influence ER proteostasis of amyloidogenic proteins. Importantly, compound 147 does not interfere with AL plasma cell toxicity induced by bortezomib, a standard chemotherapeutic used to ablate the underlying diseased plasma cells in AL. This shows that pharmacologic targeting of ER proteostasis through selective covalent modification of ER proteostasis factors is a strategy that can be used in combination with chemotherapeutics to reduce the LC toxicity associated with AL pathogenesis.
    DOI:  https://doi.org/10.1182/bloodadvances.2020002813
  13. Biochem J. 2021 Feb 19. pii: BCJ20200676. [Epub ahead of print]
    Shao R, Shi J, Du K, Wang N, Cai W, Liu S, Ding Z, Wang Y, Li D.
      Abnormal lipid accumulation is associated to the development of metabolic diseases such as hepatic steatosis and lipid storage diseases. Pharmacological agents that can attenuate lipid accumulation therefore have therapeutic potentials for these diseases. Resveratrol (RSV), a natural active substance found in fruits and nuts, has been reported to effectively reduce the intracellular lipid accumulation, but the underlying mechanisms of RSV remain elusive. Here, we show that RSV triggers an endoplasmic reticulum (ER)- Ca2+ signaling that activates transcriptional factor EB (TFEB), a master transcriptional regulator of autophagic and lysosomal biogenesis. Moreover, RSV activates protein phosphatase 2A (PP2A), which binds and dephosphorylates TFEB, promoting its nuclear translocation and the expression of TFEB target genes required for autophagosome and lysosomal biogenesis. Notably, genetic inhibition of TFEB significantly ameliorates RSV-mediated lipid clearance. Taken together, these data link RSV-induced ER calcium signaling, PP2A and TFEB activation to promote autophagy and lysosomal function, by which RSV may trigger a cellular self-defense mechanism that effectively mitigate lipid accumulation commonly associated with many metabolic diseases.
    Keywords:  Lysosome; PP2A; Resveratrol; TFEB
    DOI:  https://doi.org/10.1042/BCJ20200676
  14. EMBO Rep. 2021 Feb 15. e51349
    Montoliu-Gaya L, Tietze D, Kaminski D, Mirgorodskaya E, Tietze AA, Sterky FH.
      Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse-specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post-translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin-HS formation. CA10 is exclusively found on non-HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS-modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site-specific glycosylations.
    Keywords:  CA10; carbonic anhydrase-related protein; heparan sulfate; neurexin; synapse
    DOI:  https://doi.org/10.15252/embr.202051349
  15. PLoS Genet. 2021 Feb 16. 17(2): e1009357
    Wu JX, He KY, Zhang ZZ, Qu YL, Su XB, Shi Y, Wang N, Wang L, Han ZG.
      The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.
    DOI:  https://doi.org/10.1371/journal.pgen.1009357
  16. Proc Natl Acad Sci U S A. 2021 Feb 23. pii: e2017497118. [Epub ahead of print]118(8):
    Liu G, Yan J, Wang X, Chen J, Wang X, Dong Y, Zhang S, Gan X, Huang J, Chen X.
      The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial-temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.
    Keywords:  Bre1; DNA replication; H2B ubiquitination; RPA; homologous recombination
    DOI:  https://doi.org/10.1073/pnas.2017497118
  17. PLoS One. 2021 ;16(2): e0247132
    Suhandynata RT, Gao YQ, Zhou AL, Yang Y, Wang PC, Zhou H.
      Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.
    DOI:  https://doi.org/10.1371/journal.pone.0247132
  18. EMBO J. 2021 Feb 15. e105543
    Wang Y, Sharma P, Jefferson M, Zhang W, Bone B, Kipar A, Bitto D, Coombes JL, Pearson T, Man A, Zhekova A, Bao Y, Tripp RA, Carding SR, Yamauchi Y, Mayer U, Powell PP, Stewart JP, Wileman T.
      Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.
    Keywords:  ATG16L1 WD Domain; cytokine storm; influenza; intrinsic defence; non-canonical autophagy
    DOI:  https://doi.org/10.15252/embj.2020105543
  19. Oncogene. 2021 Feb 18.
    Chen L, Zhang Y, Shu X, Chen Q, Wei T, Wang H, Wang X, Wu Q, Zhang X, Liu X, Zheng S, Huang L, Xiao J, Jiang C, Yang B, Wang Z, Guo X.
      Reversible phosphorylation has emerged as an important mechanism for regulating 26S proteasome function in health and disease. Over 100 phospho-tyrosine sites of the human proteasome have been detected, and yet their function and regulation remain poorly understood. Here we show that the 19S subunit Rpt2 is phosphorylated at Tyr439, a strictly conserved residue within the C-terminal HbYX motif of Rpt2 that is essential for 26S proteasome assembly. Unexpectedly, we found that Y439 phosphorylation depends on Rpt2 membrane localization mediated by its N-myristoylation. Multiple receptors tyrosine kinases can trigger Rpt2-Y439 phosphorylation by activating Src, a N-myristoylated tyrosine kinase. Src directly phosphorylates Rpt2-Y439 in vitro and negatively regulates 26S proteasome activity at cellular membranes, which can be reversed by the membrane-associated isoform of protein tyrosine phosphatase nonreceptor type 2 (PTPN2). In H1975 lung cancer cells with activated Src, blocking Rpt2-Y439 phosphorylation by the Y439F mutation conferred partial resistance to the Src inhibitor saracatinib both in vitro and in a mouse xenograft tumor model, and caused significant changes of cellular responses to saracatinib at the proteome level. Our study has defined a novel mechanism involved in the spatial regulation of proteasome function and provided new insights into tyrosine kinase inhibitor-based anticancer therapies.
    DOI:  https://doi.org/10.1038/s41388-021-01674-z
  20. Annu Rev Biochem. 2021 Jan 27.
    Raote I, Malhotra V.
      The functions of coat protein complex II (COPII) coats in cargo packaging and the creation of vesicles at the endoplasmic reticulum are conserved in eukaryotic protein secretion. Standard COPII vesicles, however, cannot handle the secretion of metazoan-specific cargoes such as procollagens, apolipoproteins, and mucins. Metazoans have thus evolved modules centered on proteins like TANGO1 (transport and Golgi organization 1) to engage COPII coats and early secretory pathway membranes to engineer a novel mode of cargo export at the endoplasmic reticulum. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
    DOI:  https://doi.org/10.1146/annurev-biochem-080120-022017
  21. J Biol Chem. 2021 Feb 10. pii: S0021-9258(21)00180-0. [Epub ahead of print] 100408
    Ramírez J, Morales M, Osinalde N, Martínez-Padrón I, Mayor U, Ferrús A.
      Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, we used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. We identified sixteen candidates that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, we identified Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, we validate Comt/NSF to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. We tested the possible functional relevance of this modification using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca2+ dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. Our results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination.
    Keywords:  Ariadne-1; Drosophila; E3 ubiquitin ligase; NSF; neurotransmitter release; synapse; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2021.100408
  22. Glia. 2021 Feb 20.
    Smolič T, Tavčar P, Horvat A, Černe U, Halužan Vasle A, Tratnjek L, Kreft ME, Scholz N, Matis M, Petan T, Zorec R, Vardjan N.
      When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia-like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+ , likely to enhance the LD-organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress-induced lipotoxicity.
    Keywords:  adrenergic receptors; astrocytes; lipid droplets; metabolic/hypoxic stress; noradrenaline
    DOI:  https://doi.org/10.1002/glia.23978
  23. Nat Struct Mol Biol. 2021 Feb 15.
    Witus SR, Burrell AL, Farrell DP, Kang J, Wang M, Hansen JM, Pravat A, Tuttle LM, Stewart MD, Brzovic PS, Chatterjee C, Zhao W, DiMaio F, Kollman JM, Klevit RE.
      Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
    DOI:  https://doi.org/10.1038/s41594-020-00556-4
  24. Proc Natl Acad Sci U S A. 2021 Feb 23. pii: e2007328118. [Epub ahead of print]118(8):
    Wu K, Huynh KQ, Lu I, Moustakim M, Miao H, Yu C, Haeusgen MJ, Hopkins BD, Huang L, Zheng N, Sanchez R, DeVita RJ, Pan ZQ.
      Cullin-RING (really intersting new gene) E3 ubiquitin ligases (CRLs) are the largest E3 family and direct numerous protein substrates for proteasomal degradation, thereby impacting a myriad of physiological and pathological processes including cancer. To date, there are no reported small-molecule inhibitors of the catalytic activity of CRLs. Here, we describe high-throughput screening and medicinal chemistry optimization efforts that led to the identification of two compounds, 33-11 and KH-4-43, which inhibit E3 CRL4 and exhibit antitumor potential. These compounds bind to CRL4's core catalytic complex, inhibit CRL4-mediated ubiquitination, and cause stabilization of CRL4's substrate CDT1 in cells. Treatment with 33-11 or KH-4-43 in a panel of 36 tumor cell lines revealed cytotoxicity. The antitumor activity was validated by the ability of the compounds to suppress the growth of human tumor xenografts in mice. Mechanistically, the compounds' cytotoxicity was linked to aberrant accumulation of CDT1 that is known to trigger apoptosis. Moreover, a subset of tumor cells was found to express cullin4 proteins at levels as much as 70-fold lower than those in other tumor lines. The low-cullin4-expressing tumor cells appeared to exhibit increased sensitivity to 33-11/KH-4-43, raising a provocative hypothesis for the role of low E3 abundance as a cancer vulnerability.
    Keywords:  E3 CRL4; cullin4; protein degradation; small-molecule inhibitors; tumor inhibition
    DOI:  https://doi.org/10.1073/pnas.2007328118
  25. Talanta. 2021 Apr 01. pii: S0039-9140(20)31311-4. [Epub ahead of print]225 122020
    Lu Y, Wang R, Sun Y, Tian M, Dong B.
      ER stress has close relation with various metabolic diseases including obesity and insulin resistance, and could result in the abnormal production of ROS including O2-. Real-time and in situ detection of endogenous O2- in ER is vitally important for revealing the physiological roles of O2- during ER stress. Herein, we present an ER-specific two-photon probe (ER-Rs) for the detection of endogenous O2- in living cells and zebrafishes. The probe ER-Rs employed triflate as the response site for O2-, and used p-methylsulfonamide as the ER-specific moiety. In response to O2-, the triflate group of the probe ER-Rs was transformed to hydroxyl and the turn-on fluorescence was produced. The probe ER-Rs displayed highly sensitive and selective response to O2-, and could be employed as an ER-specific two-photon probe for the visualization of endogenous O2- in live cells, tissues and zebrafishes.
    Keywords:  Endoplasmic reticulum; In vivo imaging; Superoxide anion; Two-photon fluorescence
    DOI:  https://doi.org/10.1016/j.talanta.2020.122020
  26. Oncogene. 2021 Feb 18.
    Fletcher R, Tong J, Risnik D, Leibowitz BJ, Wang YJ, Concha-Benavente F, DeLiberty JM, Stolz DB, Pai RK, Ferris RL, Schoen RE, Yu J, Zhang L.
      Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.
    DOI:  https://doi.org/10.1038/s41388-021-01687-8
  27. Cell Rep. 2021 Jan 26. pii: S2211-1247(21)00004-8. [Epub ahead of print]34(4): 108691
    González-Prieto R, Eifler-Olivi K, Claessens LA, Willemstein E, Xiao Z, Talavera Ormeno CMP, Ovaa H, Ulrich HD, Vertegaal ACO.
      In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors.
    Keywords:  DNA damage response; SUMO; SUMO Interaction Motif; SUMO1; SUMO2; chain; mass spectrometry; non-homologous end joining; small ubiquitin-like modifier; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2021.108691
  28. Pharmacol Ther. 2021 Feb 16. pii: S0163-7258(21)00010-3. [Epub ahead of print] 107809
    Singh S, Ng J, Sivaraman J.
      The HECT E3 ligase family regulates key cellular signaling pathways, with its 28 members divided into three subfamilies: NEDD4 subfamily (9 members), HERC subfamily (6 members) and "Other" subfamily (13 members). Here, we focus on the less-explored "Other" subfamily and discuss the recent findings pertaining to their biological roles. The N-terminal regions preceding the conserved HECT domains are significantly diverse in length and sequence composition, and are mostly unstructured, except for short regions that incorporate known substrate-binding domains. In some of the better-characterized "Other" members (e.g., HUWE1, AREL1 and UBE3C), structure analysis shows that the extended region (~aa 50) adjacent to the HECT domain affects the stability and activity of the protein. The enzymatic activity is also influenced by interactions with different adaptor proteins and inter/intramolecular interactions. Primarily, the "Other" subfamily members assemble atypical ubiquitin linkages, with some cooperating with E3 ligases from the other subfamilies to form branched ubiquitin chains on substrates. Viruses and pathogenic bacteria target and hijack the activities of "Other" subfamily members to evade host immune responses and cause diseases. As such, these HECT E3 ligases have thus emerged as potential candidates for therapeutic drug development.
    Keywords:  Cancer; E3 ubiquitin ligase; Posttranslational modification; Protein Degradation; Ubiquitination; “Other” subfamily HECT
    DOI:  https://doi.org/10.1016/j.pharmthera.2021.107809
  29. EMBO J. 2021 Feb 19. e106524
    Ercan B, Naito T, Koh DHZ, Dharmawan D, Saheki Y.
      Cholesterol is essential for cell physiology. Transport of the "accessible" pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER-localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell-free reconstitution and cell-based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine-tune the non-vesicular transport of PM cholesterol to the ER via GRAMD1s.
    Keywords:  GRAM domain; cholesterol; lipid sensor; membrane contact sites; plasma membrane
    DOI:  https://doi.org/10.15252/embj.2020106524
  30. J Cell Sci. 2021 Feb 15. pii: jcs.248203. [Epub ahead of print]
    Fujita T, Kubo S, Shioda T, Tokumura A, Minami S, Tsuchiya M, Isaka Y, Ogawa H, Hamasaki M, Yu L, Yoshimori T, Nakamura S.
      TFEB, a bHLH transcription factor, is a master regulator of autophagy, lysosome biogenesis, and lipid catabolism. Compared to the post-translational regulation, the regulation of TFEB mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to TFEB mRNA and stabilized it after transcription through maintaining polyA length. We further found that this mode of regulation was conserved in C. elegans and was essential for TFEB mediated lipid break-down which becomes overrepresented during prolonged starvation. Taken together our study reveals the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function TFEB mediating autophagy and lipid metabolism.
    Keywords:  : TFEB; Autophagy; Lipid catabolism; MRNA stability; THOC4
    DOI:  https://doi.org/10.1242/jcs.248203
  31. Science. 2021 Feb 19. 371(6531): 846-849
    Itoh Y, Andréll J, Choi A, Richter U, Maiti P, Best RB, Barrientos A, Battersby BJ, Amunts A.
      Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.
    DOI:  https://doi.org/10.1126/science.abe0763
  32. Exp Ther Med. 2021 Mar;21(3): 248
    Obert DP, Wolpert AK, Grimm NL, Korff S.
      The mismatch of oxygen supply and demand during hemorrhagic shock disturbs endoplasmic reticulum (ER) homeostasis. The resulting accumulation of unfolded proteins in the ER lumen, which is a condition that is defined as ER stress, triggers the unfolded protein response (UPR). Since the UPR influences the extent of organ damage following hemorrhagic shock/reperfusion (HS/R) and mediates the protective effects of stress preconditioning before ischemia-reperfusion injury, the current study investigated the mechanisms of ER stress preconditioning and its impact on post-hemorrhagic liver damage. Male C56BL/6-mice were injected intraperitoneally with the ER stress inductor tunicamycin (TM) or its drug vehicle 48 h prior to being subjected to a 90 min pressure-controlled hemorrhagic shock (30±5 mmHg). A period of 14 h after hemorrhagic shock induction, mice were sacrificed. Hepatocellular damage was quantified by analyzing hepatic transaminases and hematoxylin-eosin stained liver tissue sections. Additionally, the topographic expression patterns of the ER stress marker binding immunoglobulin protein (BiP), UPR signaling pathways, and the autophagy marker Beclin1 were evaluated. TM injection significantly increased BiP expression and modified the topographic expression patterns of the UPR signaling proteins. In addition, immunohistochemical analysis of Beclin1 revealed an increased pericentral staining intensity following TM pretreatment. The histologic analysis of hepatocellular damage demonstrated a significant reduction in cell death areas in HS/R+TM (P=0.024). ER stress preconditioning influences the UPR and alleviates post-hemorrhagic liver damage. The beneficial effects were, at least partially, mediated by the upregulation of BiP and autophagy induction. These results underscore the importance of the UPR in the context of HS/R and may help identify novel therapeutic targets.
    Keywords:  binding immunoglobulin protein; hemorrhage; ischemia-reperfusion injury; tunicamycin; unfolded protein response
    DOI:  https://doi.org/10.3892/etm.2021.9679
  33. Matrix Biol. 2021 Feb 15. pii: S0945-053X(21)00027-5. [Epub ahead of print]
    Juretschke T, Beli P.
      Autophagy is a quality control pathway that maintains cellular homeostasis by recycling surplus and dysregulated cell organelles. Identification of selective autophagy receptors demonstrated the existence of pathways that selectively degrade organelles, protein aggregates or pathogens. Interestingly, different types of DNA damage can induce autophagy and autophagy-deficiency leads to genomic instability. Recent studies provided first insights into the pathways that connect autophagy with the DNA damage response. However, the physiological role of autophagy and the identity of its targets after DNA damage remain enigmatic. In this review, we summarize recent literature on the targets of autophagy and mechanisms that lead to its activation after DNA damage, and discuss potential consequences of DNA damage-induced autophagy.
    Keywords:  DNA damage response (DDR); DNA repair; autophagy; genomic stability; selective autophagy
    DOI:  https://doi.org/10.1016/j.matbio.2021.02.004
  34. Cell Signal. 2021 Feb 16. pii: S0898-6568(21)00044-9. [Epub ahead of print] 109956
    Li X, Sun L, Yan G, Yan X.
      Atg4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of Atg4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how Atg4B phosphorylation is regulated by amino acid deprivation is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) to identify the Atg4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between Atg4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP KO reduced degradation of LC3-II and p62 due to a partial block in autophagic flux. MS analysis of Flag-tagged Atg4B identified phosphorylation of Atg4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated Atg4B at S34. This phosphorylation could enhance Atg4B activity and p62 degradation. In addition, PFKP S386 phosphorylation abundance correlates with Atg4B S34 phosphorylation abundance and autophagy in HEK293T cells. In brief, our findings described that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for Atg4B to regulate Atg4B activity and autophagy under amino acid deprivation condition.
    Keywords:  Amino acid deprivation; Atg4B; Autophagy; PFKP; Phosphorylation
    DOI:  https://doi.org/10.1016/j.cellsig.2021.109956
  35. Mol Biol Cell. 2021 Feb 17. mbcE20110748
    Laborenz J, Bykov YS, Knöringer K, Räschle M, Filker S, Prescianotto-Baschong C, Spang A, Tatsuta T, Langer T, Storchová Z, Schuldiner M, Herrmann JM.
      For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of non-productive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homolog is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting.
    DOI:  https://doi.org/10.1091/mbc.E20-11-0748
  36. Front Oncol. 2020 ;10 592501
    Rong C, Zhou R, Wan S, Su D, Wang SL, Hess J.
      Ubiquitin C-terminal hydrolases (UCHs), a subfamily of deubiquitinating enzymes (DUBs), have been found in a variety of tumor entities and play distinct roles in the pathogenesis and development of various cancers including head and neck cancer (HNC). HNC is a heterogeneous disease arising from the mucosal epithelia of the upper aerodigestive tract, including different anatomic sites, distinct histopathologic types, as well as human papillomavirus (HPV)-positive and negative subgroups. Despite advances in multi-disciplinary treatment for HNC, the long-term survival rate of patients with HNC remains low. Emerging evidence has revealed the members of UCHs are associated with the pathogenesis and clinical prognosis of HNC, which highlights the prognostic and therapeutic implications of UCHs for patients with HNC. In this review, we summarize the physiological and pathological functions of the UCHs family, which provides enlightenment of potential mechanisms of UCHs family in HNC pathogenesis and highlights the potential consideration of UCHs as attractive drug targets.
    Keywords:  clinical relevance; deubiquitinating enzymes; genomic alteration; head and neck cancer; ubiquitin C-terminal hydrolases
    DOI:  https://doi.org/10.3389/fonc.2020.592501
  37. Mol Cell Neurosci. 2021 Feb 10. pii: S1044-7431(21)00015-4. [Epub ahead of print] 103602
    Kawabe H, Stegmüller J.
      Ubiquitination is a key posttranslational modification for the controlled protein degradation and proteostasis. The substrate specificity is determined by a family of E3 ubiquitin ligases, which are encoded by more than 600 genes in the mammalian genome. Gain- or loss-of-function of a number of E3 genes results in neurodegeneration or neurodevelopmental disorders, affecting synapse function. This implies that the specific ubiquitination of synaptic substrates are of crucial importance for the normal neuronal network. In this review, we will summerize the history, current topics, and challenges in the field of ubiquitination-dependent regulations of synaptogenesis and synaptic transmission.
    Keywords:  E3 ligase; Neuron; Synapse; Ubiquitin
    DOI:  https://doi.org/10.1016/j.mcn.2021.103602
  38. Life Sci Alliance. 2021 05;pii: e202000811. [Epub ahead of print]4(5):
    Silveira MA, Tav C, Bérube-Simard FA, Cuppens T, Leclercq M, Fournier É, Côté MC, Droit A, Bilodeau S.
      Master transcription factors control the transcriptional program and are essential to maintain cellular functions. Among them, steroid nuclear receptors, such as the estrogen receptor α (ERα), are central to the etiology of hormone-dependent cancers which are accordingly treated with corresponding endocrine therapies. However, resistance invariably arises. Here, we show that high levels of the stress response master regulator, the heat shock factor 1 (HSF1), are associated with antiestrogen resistance in breast cancer cells. Indeed, overexpression of HSF1 leads to ERα degradation, decreased expression of ERα-activated genes, and antiestrogen resistance. Furthermore, we demonstrate that reducing HSF1 levels reinstates expression of the ERα and restores response to antiestrogens. Last, our results establish a proof of concept that inhibition of HSF1, in combination with antiestrogens, is a valid strategy to tackle resistant breast cancers. Taken together, we are proposing a mechanism where high HSF1 levels interfere with the ERα-dependent transcriptional program leading to endocrine resistance in breast cancer.
    DOI:  https://doi.org/10.26508/lsa.202000811
  39. J Cell Physiol. 2021 Feb 15.
    Kalimuthu K, Kim JH, Park YS, Luo X, Zhang L, Ku JL, Choudry MHA, Lee YJ.
      Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis, consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.
    Keywords:  TRAIL cytotoxicity; endoplasmic reticulum stress; glucose deprivation
    DOI:  https://doi.org/10.1002/jcp.30329
  40. PLoS Pathog. 2021 Feb;17(2): e1009317
    Lu LF, Zhang C, Li ZC, Zhou XY, Jiang JY, Chen DD, Zhang YA, Xiong F, Zhou F, Li S.
      The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process.
    DOI:  https://doi.org/10.1371/journal.ppat.1009317
  41. Redox Biol. 2021 Feb 10. pii: S2213-2317(21)00036-7. [Epub ahead of print] 101888
    Khoder-Agha F, Kietzmann T.
      Reactive oxygen species (ROS) carry out prime physiological roles as intracellular signaling agents, yet pathologically high concentrations of ROS cause irreversible damage to biomolecules, alter cellular programs and contribute to various diseases. While decades of intensive research have identified redox-related patterns and signaling pathways, very few addressed how the glycosylation machinery senses and responds to oxidative stress. A common trait among ROS and glycans residing on glycoconjugates is that they are both highly dynamic, as they are quickly fine-tuned in response to stressors such as inflammation, cancer and infectious diseases. On this account, the delicate balance of the redox potential, which is tightly regulated by dozens of enzymes including NOXs, and the mitochondrial electron transport chain as well as the fluidity of glycan biosynthesis resulting from the cooperation of glycosyltransferases, glycosidases, and nucleotide sugar transporters, is paramount to cell survival. Here, we review the broad spectrum of the interplay between redox changes and glycosylation with respect to their principle consequences on human physiology.
    Keywords:  CGD; Endoplasmic reticulum; Glycosylation; Golgi; Hypoxia; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.redox.2021.101888
  42. Sci Adv. 2021 Feb;pii: eabe3610. [Epub ahead of print]7(8):
    Kearney CJ, Vervoort SJ, Ramsbottom KM, Todorovski I, Lelliott EJ, Zethoven M, Pijpers L, Martin BP, Semple T, Martelotto L, Trapani JA, Parish IA, Scott NE, Oliaro J, Johnstone RW.
      Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
    DOI:  https://doi.org/10.1126/sciadv.abe3610
  43. Am J Transl Res. 2021 ;13(2): 732-742
    Wu MY, Gao F, Tang JF, Shen JC, Gao R, Dang BQ, Chen G.
      Protein kinase R-like endoplasmic reticulum kinase (PERK) is an important transmembrane protein in the endoplasmic reticulum (ER). PERK signaling has a critical function in neuronal apoptosis. This work aimed to assess PERK signaling for its function in surgical brain injury (SBI) and to explore the underlying mechanisms. Totally 120 male Sprague Dawley (SD) rats were assessed in an SBI model. The effects of the PERK inhibitor GSK2606414 were examined by Western-blot, immunofluorescent staining, TUNEL staining, fluoro-jade C (FJC) staining and neurological assays in rats with SBI. In this study, p-PERK and p-eIF2α protein amounts were increased upon SBI establishment, peaking at 24 h. Meanwhile, administration of GSK2606414 reversed these effects and prevented neuronal apoptosis. The PERK pathway has a significant function in neuronal apoptosis, and its suppression after SBI promotes the alleviation of brain injury. This suggests that targeting the PERK signaling pathway may represent an efficient therapeutic option for improving prognosis in SBI patients.
    Keywords:  ER stress; PERK; SBI; UPR; apoptosis; eIF2α
  44. J Cell Biol. 2021 Mar 01. pii: e202012058. [Epub ahead of print]220(3):
    Reinisch KM, Prinz WA.
      We have long known that lipids traffic between cellular membranes via vesicles but have only recently appreciated the role of nonvesicular lipid transport. Nonvesicular transport can be high volume, supporting biogenesis of rapidly expanding membranes, or more targeted and precise, allowing cells to rapidly alter levels of specific lipids in membranes. Most such transport probably occurs at membrane contact sites, where organelles are closely apposed, and requires lipid transport proteins (LTPs), which solubilize lipids to shield them from the aqueous phase during their transport between membranes. Some LTPs are cup like and shuttle lipid monomers between membranes. Others form conduits allowing lipid flow between membranes. This review describes what we know about nonvesicular lipid transfer mechanisms while also identifying many remaining unknowns: How do LTPs facilitate lipid movement from and into membranes, do LTPs require accessory proteins for efficient transfer in vivo, and how is directionality of transport determined?
    DOI:  https://doi.org/10.1083/jcb.202012058
  45. Sci Adv. 2021 Feb;pii: eabc8310. [Epub ahead of print]7(8):
    Kim YJ, Kong Q, Yamamoto S, Kuramoto K, Huang M, Wang N, Hong JH, Xiao T, Levine B, Qiu X, Zhao Y, Miller RJ, Dong H, Meltzer HY, Xu M, He C.
      Drug abuse is a foremost public health problem. Cocaine is a widely abused drug worldwide that produces various reward-related behaviors. The mechanisms that underlie cocaine-induced disorders are unresolved, and effective treatments are lacking. Here, we found that an autophagy-related protein Becn2 is a previously unidentified regulator of cocaine reward behaviors. Becn2 deletion protects mice from cocaine-stimulated locomotion and reward behaviors, as well as cocaine-induced dopamine accumulation and signaling, by increasing presynaptic dopamine receptor 2 (D2R) autoreceptors in dopamine neurons. Becn2 regulates D2R endolysosomal trafficking, degradation, and cocaine-induced behaviors via interacting with a D2R-bound adaptor GASP1. Inactivating Becn2 by upstream autophagy inhibitors stabilizes striatal presynaptic D2R, reduces dopamine release and signaling, and prevents cocaine reward in normal mice. Thus, the autophagy protein Becn2 is essential for cocaine psychomotor stimulation and reward through regulating dopamine neurotransmission, and targeting Becn2 by autophagy inhibitors is a potential strategy to prevent cocaine-induced behaviors.
    DOI:  https://doi.org/10.1126/sciadv.abc8310
  46. Nat Commun. 2021 02 16. 12(1): 1055
    Li T, Wang X, Ju E, da Silva SR, Chen L, Zhang X, Wei S, Gao SJ.
      mTORC1, a central controller of cell proliferation in response to growth factors and nutrients, is dysregulated in cancer. Whereas arginine activates mTORC1, it is overridden by high expression of cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1). Because cancer cells often encounter low levels of nutrients, an alternative mechanism might exist to regulate CASTOR1 expression. Here we show K29-linked polyubiquitination and degradation of CASTOR1 by E3 ubiquitin ligase RNF167. Furthermore, AKT phosphorylates CASTOR1 at S14, significantly increasing its binding to RNF167, and hence its ubiquitination and degradation, while simultaneously decreasing its affinity to MIOS, leading to mTORC1 activation. Therefore, AKT activates mTORC1 through both TSC2- and CASTOR1-dependent pathways. Several cell types with high CASTOR1 expression are insensitive to arginine regulation. Significantly, AKT and RNF167-mediated CASTOR1 degradation activates mTORC1 independent of arginine and promotes breast cancer progression. These results illustrate a mTORC1 regulating mechanism and identify RNF167 as a therapeutic target for mTORC1-dysregulated diseases.
    DOI:  https://doi.org/10.1038/s41467-021-21206-3
  47. EMBO Rep. 2021 Feb 15. e52164
    Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie-A-Ling M, Lacaud G, Labib KP.
      The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
    Keywords:  CMG helicase; CUL2LRR1; DNA replication; TRAIP; p97 ATPase
    DOI:  https://doi.org/10.15252/embr.202052164
  48. EMBO Rep. 2021 Feb 15. e50852
    Melixetian M, Bossi D, Mihailovich M, Punzi S, Barozzi I, Marocchi F, Cuomo A, Bonaldi T, Testa G, Marine JC, Leucci E, Minucci S, Pelicci PG, Lanfrancone L.
      Transition from proliferative-to-invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down-regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR-depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re-expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor-initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient-rich conditions by repressing translation of selected ISR RNAs.
    Keywords:  ATF4; integrated stress response; lncRNAs; melanoma; translational reprogramming
    DOI:  https://doi.org/10.15252/embr.202050852
  49. Cancer Lett. 2021 Feb 16. pii: S0304-3835(21)00074-4. [Epub ahead of print]
    Huang M, Zhou Y, Duan D, Yang C, Zhou Z, Li F, Kong Y, Hsieh YC, Zhang R, Ding W, Xiao W, Puno P, Chen C.
      Increasing evidence suggested that a number of ubiquitin enzymes, including ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, E3 ubiquitin ligases and deubiquitination enzymes contribute to therapeutic resistance in triple-negative breast cancer (TNBC) cells. Inhibition of these enzymes with small molecule inhibitors may restore therapeutic sensitivity. Here, we demonstrated ubiquitin conjugating enzyme UbcH5b strongly supports HECTD3 auto-ubiquitination in vitro. Based on this, we developed a Fluorescence Resonance Energy Transfer (FRET) assay and identified three Schisandraceae triterpenoids, including PC3-15, to block HECTD3/UbcH5b auto-ubiquitination. Furthermore, we revealed that PC3-15 directly binds to UbcH5b and also inhibits UbcH5b-mediated p62 ubiquitination. We found that the UbcH5b-p62 axis confers TNBC cells resistance to lapatinib by promoting autophagy. Consistently, PC3-15 inhibits lapatinib-induced autophagy and increases lapatinib sensitivity in TNBC in vitro and in mouse xenografts. These findings suggest that the UbcH5b-p62 axis provides potential therapeutic targets and that Schisandraceae triterpenoids may be used for TNBC treatment in combination with lapatinib.
    Keywords:  Autophagy; HECTD3; Schisandraceae triterpenoid; UbcH5b; Ubiquitination
    DOI:  https://doi.org/10.1016/j.canlet.2021.02.009
  50. Trends Biochem Sci. 2021 Feb 13. pii: S0968-0004(21)00020-7. [Epub ahead of print]
    King KE, Losier TT, Russell RC.
      Autophagy is the primary catabolic program of the cell that promotes survival in response to metabolic stress. It is tightly regulated by a suite of kinases responsive to nutrient status, including mammalian target of rapamycin complex 1 (mTORC1), AMP-activated protein kinase (AMPK), protein kinase C-α (PKCα), MAPK-activated protein kinases 2/3 (MAPKAPK2/3), Rho kinase 1 (ROCK1), c-Jun N-terminal kinase 1 (JNK), and Casein kinase 2 (CSNK2). Here, we highlight recently uncovered mechanisms linking amino acid, glucose, and oxygen levels to autophagy regulation through mTORC1 and AMPK. In addition, we describe new pathways governing the autophagic machinery, including the Unc-51-like (ULK1), vacuolar protein sorting 34 (VPS34), and autophagy related 16 like 1 (ATG16L1) enzyme complexes. Novel downstream targets of ULK1 protein kinase are also discussed, such as the ATG16L1 subunit of the microtubule-associated protein 1 light chain 3 (LC3)-lipidating enzyme and the ATG14 subunit of the VPS34 complex. Collectively, we describe the complexities of the autophagy pathway and its role in maintaining cellular nutrient homeostasis during times of starvation.
    Keywords:  AMPK; ATG complexes; amino acids; glucose; mTORC1; oxygen
    DOI:  https://doi.org/10.1016/j.tibs.2021.01.006
  51. Biochemistry. 2021 Feb 14.
    Sheedlo MJ, Kenny S, Podkorytov IS, Brown K, Ma J, Iyer S, Hewitt CS, Arbough T, Mikhailovskii O, Flaherty DP, Wilson MA, Skrynnikov NR, Das C.
      We report the co-crystal structure of the (catalytic Cys)-to-Ala mutant of the deubiquitinase domain of the Legionella pneumophila effector SdeA (SdeADUB) with its ubiquitin (Ub) product. Most of the intermolecular interactions are preserved in this product-bound structure compared to that of the previously characterized complex of SdeADUB with the suicide inhibitor ubiquitin vinylmethyl ester (Ub-VME), whose structure models the acyl-enzyme thioester intermediate. Nuclear magnetic resonance (NMR) titration studies show a chemical shift perturbation pattern that suggests that the same interactions also exist in solution. Isothermal titration calorimetry and NMR titration data reveal that the affinity of wild-type (WT) SdeADUB for Ub is significantly lower than that of the Cys-to-Ala mutant. This is potentially due to repulsive interaction between the thiolate ion of the catalytic Cys residue in WT SdeADUB and the carboxylate group of the C-terminal Gly76 residue in Ub. In the context of SdeADUB catalysis, this electrostatic repulsion arises after the hydrolysis of the scissile isopeptide bond in the acyl-enzyme intermediate and the consequent formation of the C-terminal carboxylic group in the Ub fragment. We hypothesize that this electrostatic repulsion may expedite the release of the Ub product by SdeADUB. We note that similar repulsive interactions may also occur in other deubiquitinases and hydrolases of ubiquitin-like protein modifiers and may constitute a fairly general mechanism of product release within this family. This is a potentially important feature for a family of enzymes that form extensive protein-protein interactions during enzyme-substrate engagement.
    DOI:  https://doi.org/10.1021/acs.biochem.0c00760
  52. Thromb Haemost. 2021 Jan 27.
    Li T, Chen H, Shi X, Yin L, Tan C, Gu J, Liu Y, Li C, Xiao G, Liu K, Liu M, Tan S, Xiao Z, Zhang H, Xiao X.
      Sepsis is a life-threatening complication of infection closely associated with coagulation abnormalities. Heat shock factor 1 (HSF1) is an important transcription factor involved in many biological processes, but its regulatory role in blood coagulation remained unclear. We generated a sepsis model in HSF1-knockout mice to evaluate the role of HSF1 in microthrombosis and multiple organ dysfunction. Compared with septic wild-type mice, septic HSF1-knockout mice exhibited a greater degree of lung, liver, and kidney tissue damage, increased fibrin/: fibrinogen deposition in the lungs and kidneys, and increased coagulation activity. RNA-seq analysis revealed that tissue-type plasminogen activator (t-PA) was upregulated in the lung tissues of septic mice, and the level of t-PA was significantly lower in HSF1-knockout mice than in wild-type mice in sepsis. The effects of HSF1 on t-PA expression were further validated in HSF1-knockout mice with sepsis and in vitro in mouse brain microvascular endothelial cells using HSF1 RNA interference or overexpression under lipopolysaccharide stimulation. Bioinformatics analysis, combined with electromobility shift and luciferase reporter assays, indicated that HSF1 directly upregulated t-PA at the transcriptional level. Our results reveal, for the first time, that HSF1 suppresses coagulation activity and microthrombosis by directly upregulating t-PA, thereby exerting protective effects against multiple organ dysfunction in sepsis.
    DOI:  https://doi.org/10.1055/s-0040-1722627
  53. J Transl Med. 2021 Feb 16. 19(1): 77
    Tian H, Shi S, You B, Zhang Q, Gu M, You Y.
      BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common malignancy in head and neck. With the development of treatments, the prognosis has improved these years, but metastasis is still the main cause of treatment failure. The endoplasmic reticulum (ER) resident protein 44 is a UPR-induced ER protein of the protein disulphide isomerase (PDI) family. This study investigated the role of ERp44 in NPC progression.METHODS: Firstly, immunohistochemistry, western blot and qRT-PCR were used to investigate the expression of ERp44 in NPC samples and cell lines. We analyzed 44 NPC samples for ERp44 expression and investigated the association between its expression level with clinicopathologic parameters. Then we took CCK8, Transwell migration assay and used the zebrafish model to access the role of ERp44 on the malignant phenotype in NPC cells. Secondly, we used co-IP to gain the proteins that interact with ERp44 and took proteomic analysis. Furthermore, we successfully constructed the mutant variants of ERp44 and found the interaction domain with ATP citrate lyase(ACLY). Lastly, we subcutaneously injected NPC cells into nude mice and took immunohistochemistry to exam the expression of ACLY and ERp44. Then we used western blot to detect the expression level of epithelial-mesenchymal transition (EMT) markers.
    RESULTS: In the present study, we found ERp44 was elevated in NPC tissues and correlated with clinical stages and survive state of the patients. In vitro, the downregulation of ERp44 in NPC cells (CNE2, 5-8F) could suppress cells proliferation and migration. After that, we recognized that ACLY might be a potential target that could interact with ERp44. We further constructed the mutant variants of ERp44 and found the interaction domain with ACLY. The promotion of ERp44 on cell migration could be inhibited when ACLY was knocked down. More importantly, we also observed that the interaction of ERp44 with ACLY, especially the thioredoxin region in ERp44 play a vital role in regulating EMT. Lastly, we found ERp44 was positively correlated with the expression of ACLY and could promote NPC cells growth in nude mice.
    CONCLUSION: Our data indicated that ERp44 participates in promoting NPC progression through the interaction with ACLY and regulation of EMT.
    Keywords:  ACLY; EMT; ERp44; Metastasis; Nasopharyngeal carcinoma
    DOI:  https://doi.org/10.1186/s12967-020-02694-1
  54. Plant Cell. 2020 Feb 05. 32(2): 470-485
    Nagashima Y, Ma Z, Liu X, Qian X, Zhang X, von Schaewen A, Koiwa H.
      Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-β-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1's sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L48L49→A48A49 variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.
    DOI:  https://doi.org/10.1105/tpc.19.00714
  55. Plant Cell Physiol. 2021 Feb 17. pii: pcab029. [Epub ahead of print]
    Li N, Nakamura S, Ramundo S, Nishimura Y, Hagihara S, Izumi M.
      Intraorganellar proteases and cytoplasmic proteolytic systems such as autophagy orchestrate the degradation of organellar proteins to ensure organelle homeostasis in eukaryotic cells. The green alga Chlamydomonas reinhardtii is an ideal unicellular model organism for elucidating the mechanisms maintaining proteostasis in chloroplasts. However, the autophagic pathways targeting the photosynthetic organelles of these algae have not been clearly elucidated. Here, we explored the role of autophagy in chloroplast protein degradation in Chlamydomonas cells. We labeled the chloroplast protein Rubisco small subunit (RBCS) with the yellow fluorescent protein Venus in a Chlamydomonas strain in which expression of the chloroplast gene clpP1, encoding a major catalytic subunit of the chloroplast Clp protease, can be conditionally repressed to selectively perturb chloroplast protein homeostasis. We observed transport of both nucleus-encoded RBCS-Venus fusion protein and chloroplast-encoded Rubisco large subunit (rbcL) from the chloroplast to the vacuoles in response to chloroplast proteotoxic stress induced by clpP1 inhibition. This process was retarded by the addition of autophagy inhibitors. Biochemical detection of lytic cleavage of RBCS-Venus supported the notion that Rubisco is degraded in the vacuoles via autophagy. Electron microscopy revealed vacuolar accumulation of autophagic vesicles and exposed their ultrastructure during repression of clpP1 expression. Treatment with an autophagy activator also induced chloroplast autophagy. These results indicate that autophagy contributes to chloroplast protein degradation in Chlamydomonas cells.
    Keywords:  Autophagy; Chlamydomonas (Chlamydomonas reinhardtii); Chloroplast; Organelle quality control; Proteotoxic stress
    DOI:  https://doi.org/10.1093/pcp/pcab029
  56. Histochem Cell Biol. 2021 Feb 18.
    Bruno SR, Anathy V.
      It has long been appreciated that the endoplasmic reticulum (ER) and mitochondria, organelles important for regular cell function and survival, also play key roles in pathogenesis of various lung diseases, including asthma, fibrosis, and infections. Alterations in processes regulated within these organelles, including but not limited to protein folding in the ER and oxidative phosphorylation in the mitochondria, are important in disease pathogenesis. In recent years it has also become increasingly apparent that organelle structure dictates function. It is now clear that organelles must maintain precise organization and localization for proper function. Newer microscopy capabilities have allowed the scientific community to reveal, via 3D imaging, that the structure of these organelles and their interactions with each other are a main component of regulating function and, therefore, effects on the disease state. In this review, we will examine how 3D imaging through techniques could allow advancements in knowledge of how the ER and mitochondria function and the roles they may play in lung epithelia in progression of lung disease.
    Keywords:  3D; Endoplasmic reticulum; Epithelial; Lung; Mitochondria; Structure
    DOI:  https://doi.org/10.1007/s00418-020-01950-1
  57. Mol Cell Proteomics. 2018 Nov;pii: S1535-9476(20)32019-3. [Epub ahead of print]17(11): 2132-2145
    Shraibman B, Barnea E, Kadosh DM, Haimovich Y, Slobodin G, Rosner I, López-Larrea C, Hilf N, Kuttruff S, Song C, Britten C, Castle J, Kreiter S, Frenzel K, Tatagiba M, Tabatabai G, Dietrich PY, Dutoit V, Wick W, Platten M, Winkler F, von Deimling A, Kroep J, Sahuquillo J, Martinez-Ricarte F, Rodon J, Lassen U, Ottensmeier C, van der Burg SH, Thor Straten P, Poulsen HS, Ponsati B, Okada H, Rammensee HG, Sahin U, Singh H, Admon A.
      Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.
    Keywords:  CTA - cancer/testis antigens; Cancer biomarker(s); Cancer therapeutics; Glioblastoma; HLA - human leukocytes antigen; Immunoaffinity; Immunology; MHC - major histocompatibility complex; Peptidomics; Plasma or serum analysis; immunotherapy
    DOI:  https://doi.org/10.1074/mcp.RA118.000792
  58. Biol Cell. 2021 Feb 18.
    Caldas LA, Carneiro FA, Monteiro FL, Augusto I, Higa LM, Miranda K, Tanuri A, de Souza W.
      BACKGROUND INFORMATION: SARS-CoV-2 infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil).RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space.
    CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles, and cell components, in a context of a virus-induced membrane remodelling. This article is protected by copyright. All rights reserved.
    Keywords:  Coronavirus morphogenesis; Electron microscopy; SARS-CoV-2
    DOI:  https://doi.org/10.1111/boc.202000146