bims-proteo Biomed News
on Proteostasis
Issue of 2020‒09‒27
fifty-three papers selected by
Eric Chevet
INSERM


  1. J Neurosci. 2020 Sep 21. pii: JN-RM-0604-20. [Epub ahead of print]
    Wu S, Stone S, Nave KA, Lin W.
      Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfold protein response (UPR) and ER-associated degradation (ERAD) is the primary ER quality control mechanism. The adaptor protein Sel1L (Suppressor/Enhancer of Lin-12-like) controls the stability of the E3 ubiquitin ligase Hrd1 (hydroxymethylglutaryl reductase degradation protein 1), and is necessary for the ERAD activity of the Sel1L-Hrd1 complex. Herein, we showed that Sel1L deficiency specifically in oligodendrocytes caused ERAD impairment, the UPR activation, and attenuation of myelin protein biosynthesis; and resulted in late-onset, progressive myelin thinning in the CNS of adult mice (both male and female). The pancreatic ER kinase (PERK) branch of the UPR functions as the master regulator of protein translation in ER-stressed cells. Importantly, PERK inactivation reversed attenuation of myelin protein biosynthesis in oligodendrocytes and restored myelin thickness in the CNS of oligodendrocyte-specific Sel1L deficient mice (both male and female). Conversely, blockage of proteolipid protein (PLP) production exacerbated myelin thinning in the CNS of oligodendrocyte-specific Sel1L deficient mice (both male and female). These findings suggest that impaired ERAD in oligodendrocytes reduces myelin thickness in the adult CNS through suppression of myelin protein translation by activating PERK.SIGNIFICANCE STATEMENTMyelin is an enormous extended plasma membrane of oligodendrocytes that wraps and insulates axons. Myelin structure, including thickness, was thought to be extraordinarily stable in adults. Myelin proteins, which are produced in the endoplasmic reticulum (ER), are essential and necessary for maintaining myelin structure. The integrated unfolded protein response (UPR) and ER-associated degradation (ERAD) is the primary mechanism that maintains ER protein homeostasis. Herein, we explored the role of the integrated UPR and ERAD in oligodendrocytes in regulating myelin protein production and maintaining myelin structure using mouse models. The results presented in this study imply that the integrated UPR and ERAD in oligodendrocytes maintains myelin thickness in adults by regulating myelin protein production.
    DOI:  https://doi.org/10.1523/JNEUROSCI.0604-20.2020
  2. Nat Cell Biol. 2020 Sep 21.
    Xu L, Liu X, Peng F, Zhang W, Zheng L, Ding Y, Gu T, Lv K, Wang J, Ortinau L, Hu T, Shi X, Shi G, Shang G, Sun S, Iwawaki T, Ji Y, Li W, Rosen JM, Zhang XH, Park D, Adoro S, Catic A, Tong W, Qi L, Nakada D, Chen X.
      Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life1-3. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)-niche interaction and determines the fate of HSCs. The SEL1L-HRD1 complex, the most conserved branch of ERAD4, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity5, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell-niche interaction.
    DOI:  https://doi.org/10.1038/s41556-020-00581-x
  3. Oncogene. 2020 Sep 21.
    Li LY, Chen XS, Wang KS, Guan YD, Ren XC, Cao DS, Sun XY, Li AX, Tao YG, Zhang Y, Yin MZ, Wang XL, Wu MH, Yang JM, Cheng Y.
      Autophagy can protect stressed cancer cell by degradation of damaged proteins and organelles. However, the regulatory mechanisms behind this cellular process remain incompletely understood. Here, we demonstrate that RSK2 (p90 ribosomal S6 kinase 2) plays a critical role in ER stress-induced autophagy in breast cancer cells. We demonstrated that the promotive effect of RSK2 on autophagy resulted from directly binding of AMPKα2 in nucleus and phosphorylating it at Thr172 residue. IRE1α, an ER membrane-associated protein mediating unfolded protein response (UPR), is required for transducing the signal for activation of ERK1/2-RSK2 under ER stress. Suppression of autophagy by knockdown of RSK2 enhanced the sensitivity of breast cancer cells to ER stress both in vitro and in vivo. Furthermore, we demonstrated that inhibition of RSK2-mediated autophagy rendered breast cancer cells more sensitive to paclitaxel, a chemotherapeutic agent that induces ER stress-mediated cell death. This study identifies RSK2 as a novel controller of autophagy in tumor cells and suggests that targeting RSK2 can be exploited as an approach to reinforce the efficacy of ER stress-inducing agents against cancer.
    DOI:  https://doi.org/10.1038/s41388-020-01447-0
  4. Traffic. 2020 Sep 25.
    Gomez-Navarro N, Boulanger J, Miller E.
      The appropriate delivery of secretory proteins to the correct subcellular destination is an essential cellular process. In the endoplasmic reticulum (ER), secretory proteins are captured into COPII vesicles, that generally exclude ER resident proteins and misfolded proteins. We previously characterized a collection of yeast mutants that fail to enforce this sorting stringency and improperly secrete the ER chaperone, Kar2 (Copic et al., Genetics 2009). Here, we used the emp24Δ mutant strain that secretes Kar2 to identify candidate proteins that might regulate ER export, reasoning that loss of regulatory proteins would restore sorting stringency. We find that loss of the deubiquitylation complex Ubp3/Bre5 reverses all of the known phenotypes of the emp24Δ mutant, and similarly reverses Kar2 secretion of many other ER retention mutants. Based on a combination of genetic interactions and live cell imaging, we conclude that Ubp3 and Bre5 modulate COPII coat assembly at ER exit sites and thus influence the rate of secretion. Therefore, we propose that Ubp3/Bre5 influences the rate of vesicle formation from the ER that in turn can impact ER quality control events. This article is protected by copyright. All rights reserved.
    Keywords:  Bre5; COPII; ER export quality control; Kar2; Ubp3
    DOI:  https://doi.org/10.1111/tra.12766
  5. Science. 2020 Sep 25. pii: eabc5809. [Epub ahead of print]369(6511):
    McKenna MJ, Sim SI, Ordureau A, Wei L, Harper JW, Shao S, Park E.
      Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.
    DOI:  https://doi.org/10.1126/science.abc5809
  6. J Biol Chem. 2020 Sep 25. pii: jbc.RA120.014372. [Epub ahead of print]
    Mohan HM, Yang B, Dean NA, Raghavan M.
      Alpha-1-antitrypsin (AAT) regulates the activity of multiple proteases in the lungs and liver. A E342K mutant of AAT called ATZ forms polymers present at only low levels in the serum that 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 to 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 (CNX) does not enhance ATZ secretory trafficking, despite the higher cellular abundance of CNX-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 (IB). Further understanding of the factors that control the secretory trafficking of ATZ and their regulation by CRT could lead to new therapies for lung diseases linked to AAT deficiency.
    Keywords:  COPII; alpha1-antitrypsin; calnexin; calreticulin; chaperone; endoplasmic reticulum (ER); glycoprotein secretion; intracellular trafficking; mass spectrometry (MS)
    DOI:  https://doi.org/10.1074/jbc.RA120.014372
  7. Int J Mol Sci. 2020 Sep 17. pii: E6815. [Epub ahead of print]21(18):
    Ghosh A, Shcherbik N.
      Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.
    Keywords:  Ssb/RAC triad; r-protein; rRNA; ribosome; ribosome-associated chaperones; ribosome-associated protein quality control (RQC); ribosome-bound nascent chains (RNCs); ubiquitin; ubiquitin ligase Ltn1; ubiquitination of polypeptides bound to a ribosome
    DOI:  https://doi.org/10.3390/ijms21186815
  8. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 201919013. [Epub ahead of print]
    Sun AH, Collette JR, Sifers RN.
      The failure of polypeptides to achieve conformational maturation following biosynthesis can result in the formation of protein aggregates capable of disrupting essential cellular functions. In the secretory pathway, misfolded asparagine (N)-linked glycoproteins are selectively sorted for endoplasmic reticulum-associated degradation (ERAD) in response to the catalytic removal of terminal alpha-linked mannose units. Remarkably, ER mannosidase I/Man1b1, the first alpha-mannosidase implicated in this conventional N-glycan-mediated process, can also contribute to ERAD in an unconventional, catalysis-independent manner. To interrogate this functional dichotomy, the intracellular fates of two naturally occurring misfolded N-glycosylated variants of human alpha1-antitrypsin (AAT), Null Hong Kong (NHK), and Z (ATZ), in Man1b1 knockout HEK293T cells were monitored in response to mutated or truncated forms of transfected Man1b1. As expected, the conventional catalytic system requires an intact active site in the Man1b1 luminal domain. In contrast, the unconventional system is under the control of an evolutionarily extended N-terminal cytoplasmic tail. Also, N-glycans attached to misfolded AAT are not required for accelerated degradation mediated by the unconventional system, further demonstrating its catalysis-independent nature. We also established that both systems accelerate the proteasomal degradation of NHK in metabolic pulse-chase labeling studies. Taken together, these results have identified the previously unrecognized regulatory capacity of the Man1b1 cytoplasmic tail and provided insight into the functional dichotomy of Man1b1 as a component in the mammalian proteostasis network.
    Keywords:  ERAD; Man1b1; alpha1-antitrypsin; mannosidase; proteostasis
    DOI:  https://doi.org/10.1073/pnas.1919013117
  9. Autophagy. 2020 Sep 24. 1-16
    Thirumalaikumar VP, Gorka M, Schulz K, Masclaux-Daubresse C, Sampathkumar A, Skirycz A, Vierstra RD, Balazadeh S.
      In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of an nbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression of HSP genes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS. Abbreviations: AIM: Atg8-interacting motif; ATG: autophagy-related; BiFC: bimolecular fluorescence complementation; ConA: concanamycinA; CoIP: co-immunoprecipitation; DMSO: dimethyl sulfoxide; FKBP: FK506-binding protein; FBPASE: fructose 1,6-bisphosphatase; GFP: green fluorescent protein; HS: heat stress; HSF: heat shock factor; HSFA2: heat shock factor A2; HSP: heat shock protein; HSP90: heat shock protein 90; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; 3-MA: 3-methyladenine; NBR1: next-to-BRCA1; PQC: protein quality control; RFP: red fluorescent protein; ROF1: rotamase FKBP1; TF: transcription factor; TUB: tubulin; UBA: ubiquitin-associated; YFP: yellow fluorescent protein.
    Keywords:   Arabidopsis thaliana ; HSFA2; HSP90.1; NBR1; ROF1; heat stress; selective autophagy; stress memory; stress recovery
    DOI:  https://doi.org/10.1080/15548627.2020.1820778
  10. Int J Mol Sci. 2020 Sep 18. pii: E6841. [Epub ahead of print]21(18):
    Escobar-Henriques M, Anton V.
      Cdc48/p97 is a ring-shaped, ATP-driven hexameric motor, essential for cellular viability. It specifically unfolds and extracts ubiquitylated proteins from membranes or protein complexes, mostly targeting them for proteolytic degradation by the proteasome. Cdc48/p97 is involved in a multitude of cellular processes, reaching from cell cycle regulation to signal transduction, also participating in growth or death decisions. The role of Cdc48/p97 in endoplasmic reticulum-associated degradation (ERAD), where it extracts proteins targeted for degradation from the ER membrane, has been extensively described. Here, we present the roles of Cdc48/p97 in mitochondrial regulation. We discuss mitochondrial quality control surveillance by Cdc48/p97 in mitochondrial-associated degradation (MAD), highlighting the potential pathologic significance thereof. Furthermore, we present the current knowledge of how Cdc48/p97 regulates mitofusin activity in outer membrane fusion and how this may impact on neurodegeneration.
    Keywords:  Cdc48; Fzo1; MAD; Mfn1/2; VCP; fusion; mitochondria; mitofusin; p97; ubiquitin
    DOI:  https://doi.org/10.3390/ijms21186841
  11. FEBS J. 2020 Sep 22.
    Vögtle FN.
      Mitochondrial protein homeostasis is crucial for cellular health and perturbations have been linked to a plethora of human diseases. Proteostasis is maintained mainly by a network of mitochondrial chaperones and proteases, that assist in protein folding and degrade non-functional or superfluous proteins. Upon proteomic imbalances or defects in mitochondrial functions protective cellular responses are activated to restore and maintain organellar integrity. This viewpoint describes our current knowledge and understanding of these protective pathways and addresses open questions and perspectives in the field of mitochondrial stress responses.
    Keywords:  Mitochondrial proteostasis; integrated stress response; mitochondrial dysfunction; mitochondrial protein biogenesis
    DOI:  https://doi.org/10.1111/febs.15569
  12. J Cell Sci. 2020 Sep 21. pii: jcs.244855. [Epub ahead of print]
    Lytridou AA, Demetriadou A, Christou M, Potamiti L, Mastroyiannopoulos NP, Kyriacou K, Phylactou LA, Drousiotou A, Petrou PP.
      Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionary conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing, ER structures. We demonstrate that, the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process which does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.
    Keywords:  Apoptosis; ER stress; Glycogen; Glycogen synthase; Glycogenin; UPR
    DOI:  https://doi.org/10.1242/jcs.244855
  13. Cancer Res. 2020 Sep 22. pii: canres.1357.2020. [Epub ahead of print]
    Manfrini N, Mancino M, Miluzio A, Oliveto S, Balestra M, Calamita P, Alfieri R, Rossi RL, Sassoè-Pognetto M, Salio C, Cuomo A, Bonaldi T, Manfredi M, Marengo E, Ranzato E, Martinotti S, Cittaro D, Tonon G, Biffo S.
      Multiple myeloma (MM) is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of MM is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of MM cells. One of these is FAM46C that is lost in more than 10% of MM patients. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in MM cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER-bound protein FNDC3A in order to reside in the cytoplasmic side of the ER. FNDC3A was lost in some MM cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells, and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that MM cells remodel their trafficking machinery to cope with ER stress.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-1357
  14. Cells. 2020 Sep 22. pii: E2138. [Epub ahead of print]9(9):
    Patel C, Saad H, Shenkman M, Lederkremer GZ.
      N-linked glycosylation and sugar chain processing, as well as disulfide bond formation, are among the most common post-translational protein modifications taking place in the endoplasmic reticulum (ER). They are essential modifications that are required for membrane and secretory proteins to achieve their correct folding and native structure. Several oxidoreductases responsible for disulfide bond formation, isomerization, and reduction have been shown to form stable, functional complexes with enzymes and chaperones that are involved in the initial addition of an N-glycan and in folding and quality control of the glycoproteins. Some of these oxidoreductases are selenoproteins. Recent studies also implicate glycan machinery-oxidoreductase complexes in the recognition and processing of misfolded glycoproteins and their reduction and targeting to ER-associated degradation. This review focuses on the intriguing cooperation between the glycoprotein-specific cell machineries and ER oxidoreductases, and highlights open questions regarding the functions of many members of this large family.
    Keywords:  ER quality control; ERAD; PDI; calnexin; mannosidase; oligosaccharyltransferase
    DOI:  https://doi.org/10.3390/cells9092138
  15. J Neurosci. 2020 Sep 24. pii: JN-RM-0957-20. [Epub ahead of print]
    Scapin C, Ferri C, Pettinato E, Bianchi F, Del Carro U, Feltri ML, Kaufman RJ, Wrabetz L, D'Antonio M.
      Myelin Protein Zero (MPZ/P0) is the most abundant glycoprotein of peripheral nerve myelin. P0 is synthesized by myelinating Schwann cells, processed in the endoplasmic reticulum (ER) and delivered to myelin via the secretory pathway. The mutant P0S63del, that causes Charcot-Marie-Tooth type 1B (CMT1B) neuropathy in humans and a similar demyelinating neuropathy in transgenic mice, is instead retained the ER where it activates an unfolded protein response (UPR). Under stress conditions, the ER-resident kinase PERK phosphorylates eIF2α to attenuate global translation, thus reducing the misfolded protein overload in the ER. Genetic and pharmacological inactivation of Gadd34, a subunit of the PP1 phosphatase complex that promotes the dephosphorylation of eIF2α, prolonged eIF2α phosphorylation and improved motor, neurophysiological and morphological deficits in S63del mice. However, PERK ablation in S63del Schwann cells ameliorated, rather than worsened S63del neuropathy, despite reduced levels of P-eIF2α. These contradictory findings prompted us to genetically explore the role of eIF2α-phosphorylation in P0S63del-CMT1B neuropathy through the generation of mice in which eIF2α cannot be phosphorylated specifically in Schwann cells. Morphological and electrophysiological analysis of male and female S63del mice showed a worsening of the neuropathy in the absence of eIF2α phosphorylation. However, we did not detect significant changes in ER-stress levels but, rather, a dramatic increase of the MEK/ERK/c-Jun pathway accompanied by reduction in myelin genes expression and a delay in Schwann cell differentiation. Our results support the hypothesis that eIF2α-phosphorylation is protective in CMT1B and unveil a possible crosstalk between eIF2α and the MEK/ERK pathway in neuropathic nerves.SIGNIFICANCE STATEMENT:In the P0S63del mouse model of CMT1B, the genetic and pharmacological inhibition of Gadd34 prolonged eIF2α phosphorylation leading to a proteostatic rebalance that significantly ameliorated the neuropathy. Yet, ablation of the kinase PERK also ameliorated the S63del neuropathy, despite reduced levels of P-eIF2α. In this study we provide genetic evidence that eIF2α-phosphorylation has a protective role in CMT1B Schwann cells by limiting ERK/c-Jun hyperactivation. Our data support the targeting of the P-eIF2α/Gadd34 complex as a therapeutic avenue in CMT1B and also suggest that PERK may hamper myelination via mechanisms outside its role in the UPR.
    DOI:  https://doi.org/10.1523/JNEUROSCI.0957-20.2020
  16. J Biol Chem. 2020 Sep 25. pii: jbc.RA120.013987. [Epub ahead of print]
    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, non-alcohol 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 (MOMP). 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:  endoplasmic reticulum stress (ER stress); endoplasmic-reticulum-associated protein degradation (ERAD); liver; mitochondrial disease; mitochondrial permeability transition (MPT)
    DOI:  https://doi.org/10.1074/jbc.RA120.013987
  17. Autophagy. 2020 Sep 23. 1-9
    Miller K, McGrath ME, Hu Z, Ariannejad S, Weston S, Frieman M, Jackson WT.
      The COVID-19 pandemic, caused by the SARS-CoV-2 virus, is the most recent example of an emergent coronavirus that poses a significant threat to human health. Virus-host interactions play a major role in the viral life cycle and disease pathogenesis, and cellular pathways such as macroautophagy/autophagy prove to be either detrimental or beneficial to viral replication and maturation. Here, we describe the literature over the past twenty years describing autophagy-coronavirus interactions. There is evidence that many coronaviruses induce autophagy, although some of these viruses halt the progression of the pathway prior to autophagic degradation. In contrast, other coronaviruses usurp components of the autophagy pathway in a non-canonical fashion. Cataloging these virus-host interactions is crucial for understanding disease pathogenesis, especially with the global challenge of SARS-CoV-2 and COVID-19. With the recognition of autophagy inhibitors, including the controversial drug chloroquine, as possible treatments for COVID-19, understanding how autophagy affects the virus will be critical going forward. Abbreviations: 3-MA: 3-methyladenine (autophagy inhibitor); AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; ATPase: adenosine triphosphatase; BMM: bone marrow macrophage; CGAS: cyclic GMP-AMP synthase; CHO: Chinese hamster ovary/cell line; CoV: coronaviruses; COVID-19: Coronavirus disease 2019; DMV: double-membrane vesicle; EAV: equine arteritis virus; EDEM1: ER degradation enhancing alpha-mannosidase like protein 1; ER: endoplasmic reticulum; ERAD: ER-associated degradation; GFP: green fluorescent protein; HCoV: human coronavirus; HIV: human immunodeficiency virus; HSV: herpes simplex virus; IBV: infectious bronchitis virus; IFN: interferon; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCoV: mouse coronavirus; MERS-CoV: Middle East respiratory syndrome coronavirus; MHV: mouse hepatitis virus; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2 (autophagy receptor that directs cargo to phagophores); nsp: non-structural protein; OS9: OS9 endoplasmic reticulum lectin; PEDV: porcine epidemic diarrhea virus; PtdIns3K: class III phosphatidylinositol 3-kinase; PLP: papain-like protease; pMEF: primary mouse embryonic fibroblasts; SARS-CoV: severe acute respiratory syndrome coronavirus; SKP2: S-phase kinase associated protein 2; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; ULK1: unc-51 like autophagy activating kinase 1; Vps: vacuolar protein sorting.
    Keywords:  Autophagy; COVID-19; ERAD; MERS; SARS-CoV-2; coronavirus
    DOI:  https://doi.org/10.1080/15548627.2020.1817280
  18. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31150-5. [Epub ahead of print]32(12): 108161
    Yang Y, Yue Y, Song N, Li C, Yuan Z, Wang Y, Ma Y, Li H, Zhang F, Wang W, Jia H, Li P, Li X, Wang Q, Ding Z, Dong H, Gu L, Li B.
      Sensing stressful conditions and adjusting the cellular metabolism to adapt to the environment are essential activities for bacteria to survive in variable situations. Here, we describe a stress-related protein, YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine-5'-monophosphate to a protein tyrosine/histidine residue, an unusual modification defined as UMPylation. Mn2+ serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn2+ binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. A detailed mechanism is proposed based on the crystal structures of Apo-YdiU and YdiU-AMPNPP-Mn2+ and on molecular dynamics simulation models of YdiU-UTP-Mn2+ and YdiU-UTP-peptide. In vivo data demonstrate that YdiU effectively protects Salmonella from stress-induced ATP depletion through UMPylation.
    Keywords:  AMPylation; UMPylation; bacterial stress resistence; chaperones; post-translational modification; the YdiU domain
    DOI:  https://doi.org/10.1016/j.celrep.2020.108161
  19. J Virol. 2020 Sep 23. pii: JVI.01229-20. [Epub ahead of print]
    Huang Y, Lin Q, Huo Z, Chen C, Zhou S, Ma X, Gao H, Lin Y, Li X, He J, Zhang P, Liu C.
      Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which has become a global epidemic threat due to its rapid spread and association with serious consequences of infection including neonatal microcephaly. Inositol requiring enzyme 1α (IRE1α) is an endoplasmic reticulum (ER)-related transmembrane protein that mediates unfolded protein response (UPR) pathway and has been indicated to play an important role in flavivirus replication. However, the mechanism of how IRE1α affects ZIKV replication remains unknown. Here, we explored the role of IRE1α in ZIKV infection in vitro and in vivo by using CRISPR/Cas9-based gene knockout and RNA interference-based gene knockdown techniques. Both knockout and knockdown of IRE1α dramatically reduced ZIKV replication levels, including viral RNA levels, protein expression, and titers in different human cell lines. Trans-complementation with IRE1α restored viral replication levels decreased by IRE1α depletion. Furthermore, the proviral effect of IRE1α was dependent on its kinase and ribonuclease activities. Importantly, we found that IRE1α promoted the replication of ZIKV through up-regulating the accumulation of monounsaturated fatty acids (MUFA) rate-limiting enzyme stearoyl-CoA desaturase 1 (SCD1), which further affected the production of oleic acid and lipid droplet. Finally, our data demonstrated that in the brain tissues of ZIKV-infected mice, the replication levels of ZIKV and viral related lesions were significantly suppressed by both the kinase and RNase inhibitors of IRE1α. Taken together, our work identified IRE1α as a ZIKV-dependency factor, which promotes viral replication through affecting SCD1 mediated lipid metabolism, potentially providing a novel molecular target for the development of anti-ZIKV agents.Importance Zika virus (ZIKV) has been linked to serious neurologic disorders and causes widespread concern in the field of global public health. Inositol requiring enzyme 1α (IRE1α) is an ER-related transmembrane protein that mediates unfolded protein response (UPR) pathway. Here, we revealed that IRE1α is a proviral factor for ZIKV replication both in culture cells and mice model, which relies on its kinase and RNase activities. Importantly, we further provided evidence that upon ZIKV infection, IRE1α is activated and splices XBP1 mRNA which enhances the expression of monounsaturated fatty acids rate-limiting enzyme stearoyl-CoA desaturase 1 (SCD1) and subsequent lipid droplet production. Our data uncover a novel mechanism of IRE1α proviral effect by modulating lipid metabolism, providing the first evidence of a close relationship between IRE1α-mediated UPR, lipid metabolism, and ZIKV replication, and indicating IRE1α inhibitors as potentially effective anti-ZIKV agents.
    DOI:  https://doi.org/10.1128/JVI.01229-20
  20. J Biol Chem. 2020 Sep 23. pii: jbc.RA120.015577. [Epub ahead of print]
    Guo XY, Liu YS, Gao XD, Kinoshita T, Fujita M.
      The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.
    Keywords:  ER quality control; endoplasmic reticulum (ER); glycobiology; glycosylphosphatidylinositol (GPI anchor); protein folding
    DOI:  https://doi.org/10.1074/jbc.RA120.015577
  21. Nature. 2020 Sep 23.
    Xu D, Zhao H, Jin M, Zhu H, Shan B, Geng J, Dziedzic SA, Amin P, Mifflin L, Naito MG, Najafov A, Xing J, Yan L, Liu J, Qin Y, Hu X, Wang H, Zhang M, Manuel VJ, Tan L, He Z, Sun ZJ, Lee VMY, Wagner G, Yuan J.
      Cell death in human diseases is often a consequence of disrupted cellular homeostasis. If cell death is prevented without restoring cellular homeostasis, it may lead to a persistent dysfunctional and pathological state. Although mechanisms of cell death have been thoroughly investigated1-3, it remains unclear how homeostasis can be restored after inhibition of cell death. Here we identify TRADD4-6, an adaptor protein, as a direct regulator of both cellular homeostasis and apoptosis. TRADD modulates cellular homeostasis by inhibiting K63-linked ubiquitination of beclin 1 mediated by TRAF2, cIAP1 and cIAP2, thereby reducing autophagy. TRADD deficiency inhibits RIPK1-dependent extrinsic apoptosis and proteasomal stress-induced intrinsic apoptosis. We also show that the small molecules ICCB-19 and Apt-1 bind to a pocket on the N-terminal TRAF2-binding domain of TRADD (TRADD-N), which interacts with the C-terminal domain (TRADD-C) and TRAF2 to modulate the ubiquitination of RIPK1 and beclin 1. Inhibition of TRADD by ICCB-19 or Apt-1 blocks apoptosis and restores cellular homeostasis by activating autophagy in cells with accumulated mutant tau, α-synuclein, or huntingtin. Treatment with Apt-1 restored proteostasis and inhibited cell death in a mouse model of proteinopathy induced by mutant tau(P301S). We conclude that pharmacological targeting of TRADD may represent a promising strategy for inhibiting cell death and restoring homeostasis to treat human diseases.
    DOI:  https://doi.org/10.1038/s41586-020-2757-z
  22. Int J Mol Sci. 2020 Sep 19. pii: E6884. [Epub ahead of print]21(18):
    Yang YL, Wang PW, Wang FS, Lin HY, Huang YH.
      MicroRNA-29a (miR-29a) has been shown to ameliorate hepatocellular damage, such as in the context of non-alcoholic fatty liver disease (NAFLD), steatohepatitis (NASH), and cholestatic injury. However, the mechanism mediating the hepatoprotective effect of miR-29a in diet-induced NASH remains elusive. In the present study, C57BL/6 mice of wild-type (WT) or miR-29a overexpression were fed with methionine-choline sufficient (MCS) or methionine-choline-deficient (MCD) diet for four weeks. The C57BL/6 mice harboring miR-29a overexpression presented reduced plasma AST, hepatic CD36, steatosis, and fibrosis induced by MCD. The TargetScan Release7.2-based bioinformatic analysis, KEGG pathway analysis, and luciferase reporter assay confirmed that miR-29a targets 3'UTR of glycogen synthase kinase 3 beta (Gsk3b) mRNA in the HepG2 hepatocyte cell line. Furthermore, miR-29a overexpression in the MCD-fed group resulted in inhibition of Gsk3b mRNA and GSK3β protein levels in the liver. GSK3β was notably expressed jointly with the extent of aggregated protein, which was then identified to be associated with mitochondrial unfolded protein response (UPRmt), but not with endoplasmic reticulum UPR (UPRER). Additionally, in silico analysis of protein-protein interaction, in vivo, and in vitro correlation analyses of protein expression demonstrated that GSK3β closely associated with sirtuin 1(SIRT1). Finally, the implication of SIRT1-mediated mitochondrial biogenesis in the perturbation of proteostasis was observed. We herein provide novel insight into a hepatoprotective pathway, whereby miR-29a inhibits GSK3β to repress SIRT1-mediated mitochondrial biogenesis, leading to alleviation of mitochondrial proteostatic stress and UPRmt in the context of NASH. miR-29a, GSK3β, and SIRT1 could thus serve as possible therapeutic targets to improve the treatment of NAFLD/NASH.
    Keywords:  GSK3β; NASH; SIRT1; liver fibrosis; microRNA-29a; mitochondrial unfolded protein response; proteostatic stress
    DOI:  https://doi.org/10.3390/ijms21186884
  23. Int J Mol Sci. 2020 Sep 17. pii: E6825. [Epub ahead of print]21(18):
    Kang JA, Jeon YJ.
      Eukaryotic proteomes are enormously sophisticated through versatile post-translational modifications (PTMs) of proteins. A large variety of code generated via PTMs of proteins by ubiquitin (ubiquitination) and ubiquitin-like proteins (Ubls), such as interferon (IFN)-stimulated gene 15 (ISG15), small ubiquitin-related modifier (SUMO) and neural precursor cell expressed, developmentally downregulated 8 (NEDD8), not only provides distinct signals but also orchestrates a plethora of biological processes, thereby underscoring the necessity for sophisticated and fine-tuned mechanisms of code regulation. Deubiquitinases (DUBs) play a pivotal role in the disassembly of the complex code and removal of the signal. Ubiquitin-specific protease 18 (USP18), originally referred to as UBP43, is a major DUB that reverses the PTM of target proteins by ISG15 (ISGylation). Intriguingly, USP18 is a multifaceted protein that not only removes ISG15 or ubiquitin from conjugated proteins in a deconjugating activity-dependent manner but also acts as a negative modulator of type I IFN signaling, irrespective of its catalytic activity. The function of USP18 has become gradually clear, but not yet been completely addressed. In this review, we summarize recent advances in our understanding of the multifaceted roles of USP18. We also highlight new insights into how USP18 is implicated not only in physiology but also in pathogenesis of various human diseases, involving infectious diseases, neurological disorders, and cancers. Eventually, we integrate a discussion of the potential of therapeutic interventions for targeting USP18 for disease treatment.
    Keywords:  ISG15; USP18; deubiquitinases; interferon signaling; post-translational modifications; ubiquitin
    DOI:  https://doi.org/10.3390/ijms21186825
  24. Adv Exp Med Biol. 2020 Sep 27.
    Chua NK, Brown AJ.
      Cholesterol synthesis is a fundamental process that contributes to cellular cholesterol homeostasis. Cells execute transcriptional and post-translational mechanisms to control the abundance of enzymes of the cholesterol synthesis pathway, consequently affecting cholesterol production. One such highly tuned enzyme is squalene monooxygenase (SM), which catalyzes a rate-limiting step in the pathway. A well-characterized mechanism is the cholesterol-mediated degradation of SM. Notably, lipids (cholesterol, plasmalogens, squalene, and unsaturated fatty acids) can act as cellular signals that either promote or reduce SM degradation. The N-terminal region of SM consists of the shortest known cholesterol-responsive degron, characterized by atypical membrane anchoring structures, namely a re-entrant loop and an amphipathic helix. SM also undergoes non-canonical ubiquitination on serine, a relatively uncommon attachment site for ubiquitination. The structure of the catalytic domain of SM has been solved, providing insights into the catalytic mechanisms and modes of inhibition by well-known SM inhibitors, some of which have been effective in lowering cholesterol levels in animal models. Certain human cancers have been linked to dysregulation of SM levels and activity, further emphasizing the relevance of SM in health and disease.
    Keywords:  Cholesterol synthesis; Degron; Endoplasmic reticulum-associated degradation (ERAD); Squalene; Squalene monooxygenase; Ubiquitin
    DOI:  https://doi.org/10.1007/5584_2020_583
  25. J Biomed Nanotechnol. 2020 Apr 01. 16(4): 432-445
    Sønstevold T, Engedal N, Mørch Ý, Iversen TG, Skotland T, Sandvig K, Torgersen ML.
      Nanoparticle drug carriers trigger a variety of cellular stress responses, including ER stress and antioxidant responses, but may also affect the intracellular degradative pathway autophagy. This can impose profound effects on drug delivery, cellular treatment responses, and nanoparticle cytotoxicity. We recently demonstrated that even small variations in the alkyl side chains of poly(alkylcyanoacrylate) (PACA) drug carrier nanoparticles, namely butyl (PBCA), ethylbutyl (PEBCA), or octyl (POCA), differentially induce ER stress and redox imbalance in human cell lines. Here, we systematically investigate how these PACA variants affect autophagy. Interestingly, treatment with PEBCA or POCA particles led to intracellular accumulation of the autophagosome marker LC3-II, but via different mechanisms. PEBCA induced an integrated stress response-and ATF4-mediated increase in LC3B mRNA, whereas POCA blocked autophagic degradation of LC3-II and long-lived proteins in bulk. PBCA also increased LC3B mRNA via the integrated stress response and ATF4, but unlike PEBCA, it inhibited LC3 lipidation and autophagic cargo degradation. Our data demonstrate that even subtle variations in NP structure can have profoundly different impacts on autophagy, and that careful monitoring of autophagic flux and cargo degradation is critical for drawing accurate conclusions. Our findings have important implications for the choice of PACA monomer in different therapeutic settings.
    DOI:  https://doi.org/10.1166/jbn.2020.2906
  26. Autophagy. 2020 Sep 25.
    Zhou C, Liang Y, Zhou L, Yan Y, Liu N, Zhang R, Huang Y, Wang M, Tang Y, Ali DW, Wang Y, Michalak M, Chen XZ, Tang J.
      Pancreatic cancer is one of the most aggressive tumors associated with a poor clinical prognosis, weakly effective therapeutic options. Therefore, there is a strong impetus to discover new therapeutic targets in pancreatic cancer. In the present study, we first demonstrated that TSPAN1 is upregulated in pancreatic cancer and that TSPAN1 depletion decreases pancreatic cancer cell proliferation in vitro and in vivo. TSPAN1 expression was correlated with poor overall survival of pancreatic cancer patients. Moreover, we demonstrated that TSPAN1 is a novel positive regulator of macroautophagy/autophagy characterized by decreased LC3-II and SQSTM1/p62 expressions, inhibited puncta formation of GFP-LC3 and autophagic vacuoles. We also demonstrated that tspan1 mutation impaired autophagy in the zebrafish model. Furthermore, we showed that TSPAN1 promoted autophagy maturation via direct binding to LC3 by two conserved LIR motifs. Mutations in the LIR motifs of TSPAN1 resulted in a loss of the ability to induce autophagy and promote pancreatic cancer proliferation. Second, we discovered two conservative TCF/LEF binding elements present in the promoter region of the TSPAN1 gene, which was further verified through luciferase activity and ChIP assays. Furthermore, TSPAN1 was upregulated by FAM83A through the canonical WNT-CTNNB1 signaling pathway. We further demonstrated that both TSPAN1 and FAM83A are both direct targets of MIR454 (microRNA 454). Additionally, we revealed the role of MIR454-FAM83A-TSPAN1 in the proliferation of pancreatic cancer cells in vitro and in vivo. Our findings suggest that components of the MIR454-FAM83A-TSPAN1 axis may be valuable prognosis markers or therapeutic targets for pancreatic cancer.
    Keywords:   MIR454 ; FAM83A; WNT-CTNNB1; autophagy; pancreatic cancer; tetraspanin 1
    DOI:  https://doi.org/10.1080/15548627.2020.1826689
  27. Mol Microbiol. 2020 Sep 21.
    Miwa T, Chadani Y, Taguchi H.
      Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the σ32 heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsps expression. Oligomeric IbpA self-represses the ibpA/ibpB translation, and mediates its own mRNA degradation, but the self-repression is relieved by sequestration of IbpA into the protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.
    Keywords:   E coli ; Bacterial; Gene Expression Regulation; IbpA protein; Molecular Chaperones; Protein Aggregates; Protein Biosynthesis; Small Heat-Shock Proteins
    DOI:  https://doi.org/10.1111/mmi.14606
  28. J Clin Invest. 2020 Sep 22. pii: 129374. [Epub ahead of print]
    Song K, Cai X, Dong Y, Wu H, Wei Y, Shankavaram U, Cui K, Lee Y, Zhu B, Bhattacharjee S, Wang B, Zhang K, Wen A, Wong S, Yu L, Xia L, Welm AL, Bielenberg DR, Camphausen K, Kang Y, Chen H.
      Estrogen receptor (ER)-negative breast cancer is thought to be more malignant and devastating than ER-positive breast cancer and exhibit elevated NF-κB activity. How abnormally high NF-κB activity is maintained in ER-negative breast cancer is poorly understood. The importance of linear ubiquitination, which is generated by the linear ubiquitin chain assembly complex (LUBAC), is increasingly appreciated in NF-κB signaling, which regulates cell activation and death. Here, we showed that epsin proteins, a family of ubiquitin-binding endocytic adaptors, interacted with LUBAC via its Ubiquitin-Interacting Motif (UIM) and bound LUBAC's bona fide substrate NEMO via its N-terminal homolog (ENTH) domain. Furthermore, epsins promoted NF-κB essential modulator (NEMO) linear ubiquitination and served as scaffolds for recruiting other components of the IκB kinase (IKK) complex; thereby, resulting in the heightened IKK activation and sustained NF-κB signaling essential for the development of ER-negative breast cancer. Heightened epsin levels in ER-negative human breast cancer are associated with poor, relapse-free survival. We showed that transgenic and pharmacological approaches eliminating epsins potently impeded breast cancer development in both spontaneous and patient-derived xenograft breast cancer mouse models. Our findings established the pivotal role epsins played in promoting breast cancer. Thus, targeting epsins may represent a strategy to restrain NF-κB signaling, and provide an important perspective into ER-negative breast cancer treatment.
    Keywords:  Adaptor proteins; Breast cancer; Cell Biology; NF-kappaB; Oncology
    DOI:  https://doi.org/10.1172/JCI129374
  29. J Integr Plant Biol. 2020 Sep 24.
    Yu J, Kang L, Li Y, Wu C, Zheng C, Liu P, Huang J.
      Mitogen activated protein kinase kinase kinase 18 (MAPKKK18) mediated signaling cascade plays important roles in Arabidopsis drought stress tolerance. However, the post-translational modulation patterns of MAPKKK18 are not characterized. In this study, we found that the protein level of MAPKKK18 was tightly controlled by the 26S proteasome. Ubiquitin ligases RGLG1 and RGLG2 ubiquitinated MAPKKK18 at lysine residue K32 and K154, and promoted its degradation. Deletion of RGLG1 and RGLG2 stabilized MAPKKK18 and further enhanced the drought stress tolerance of MAPKKK18-overexpression plants. Our data demonstrate that RGLG1 and RGLG2 negatively regulate MAPKKK18-mediated drought stress tolerance in Arabidopsis. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1111/jipb.13019
  30. Biochem J. 2020 Sep 30. 477(18): 3471-3497
    Zheng T, Yang Y, Castañeda CA.
      Cells rely on protein homeostasis to maintain proper biological functions. Dysregulation of protein homeostasis contributes to the pathogenesis of many neurodegenerative diseases and cancers. Ubiquilins (UBQLNs) are versatile proteins that engage with many components of protein quality control (PQC) machinery in cells. Disease-linked mutations of UBQLNs are most commonly associated with amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerative disorders. UBQLNs play well-established roles in PQC processes, including facilitating degradation of substrates through the ubiquitin-proteasome system (UPS), autophagy, and endoplasmic-reticulum-associated protein degradation (ERAD) pathways. In addition, UBQLNs engage with chaperones to sequester, degrade, or assist repair of misfolded client proteins. Furthermore, UBQLNs regulate DNA damage repair mechanisms, interact with RNA-binding proteins (RBPs), and engage with cytoskeletal elements to regulate cell differentiation and development. Important to the myriad functions of UBQLNs are its multidomain architecture and ability to self-associate. UBQLNs are linked to numerous types of cellular puncta, including stress-induced biomolecular condensates, autophagosomes, aggresomes, and aggregates. In this review, we focus on deciphering how UBQLNs function on a molecular level. We examine the properties of oligomerization-driven interactions among the structured and intrinsically disordered segments of UBQLNs. These interactions, together with the knowledge from studies of disease-linked mutations, provide significant insights to UBQLN structure, dynamics and function.
    Keywords:  autophagy; oligomerization; protein aggregation; protein quality control; ubiquilins (UBQLNs); ubiquitin proteasome system
    DOI:  https://doi.org/10.1042/BCJ20190497
  31. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 202011260. [Epub ahead of print]
    McWilliam HEG, Mak JYW, Awad W, Zorkau M, Cruz-Gomez S, Lim HJ, Yan Y, Wormald S, Dagley LF, Eckle SBG, Corbett AJ, Liu H, Li S, Reddiex SJJ, Mintern JD, Liu L, McCluskey J, Rossjohn J, Fairlie DP, Villadangos JA.
      The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.
    Keywords:  MAIT cells; MHC class I-related protein 1 (MR1); fluorescent probe; protein trafficking; vitamin B
    DOI:  https://doi.org/10.1073/pnas.2011260117
  32. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31151-7. [Epub ahead of print]32(12): 108162
    Fucci C, Resnati M, Riva E, Perini T, Ruggieri E, Orfanelli U, Paradiso F, Cremasco F, Raimondi A, Pasqualetto E, Nuvolone M, Rampoldi L, Cenci S, Milan E.
      FAM46C is a non-canonical poly(A) polymerase uniquely mutated in up to 20% of multiple myeloma (MM) patients, implying a tissue-specific tumor suppressor function. Here, we report that FAM46C selectively stabilizes mRNAs encoding endoplasmic reticulum (ER)-targeted proteins, thereby concertedly enhancing the expression of proteins that control ER protein import, folding, N-glycosylation, and trafficking and boosting protein secretion. This role requires the interaction with the ER membrane resident proteins FNDC3A and FNDC3B. In MM cells, FAM46C expression raises secretory capacity beyond sustainability, inducing ROS accumulation, ATP shortage, and cell death. FAM46C activity is regulated through rapid proteasomal degradation or the inhibitory interaction with the ZZ domain of the autophagic receptor p62 that hinders its association with FNDC3 proteins via sequestration in p62+ aggregates. Altogether, our data disclose a p62/FAM46C/FNDC3 circuit coordinating sustainable secretory activity and survival, providing an explanation for the MM-specific oncosuppressive role of FAM46C and uncovering potential therapeutic opportunities against cancer.
    Keywords:  FAM46C; FNDC3B; antibody; autophagy; bortezomib; endoplasmic reticulum; multiple myeloma; p62/SQSTM1; plasma cell; secretion
    DOI:  https://doi.org/10.1016/j.celrep.2020.108162
  33. Mol Biol Cell. 2020 Sep 23. mbcE20080541
    Singh A, Vashistha N, Heck J, Tang X, Wipf P, Brodsky JL, Hampton RY.
      Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone dependent ubiquitination by the action of E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70's role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of SSE1, and the action of J proteins are all needed for Ubr1 mediated quality control. To discern if chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage, in which efficiently folded proteins are spared, while slow- or non-folding proteins are iteratively tagged with ubiquitin for subsequent degradation.
    DOI:  https://doi.org/10.1091/mbc.E20-08-0541
  34. FEBS Open Bio. 2020 Sep 23.
    Düppre E, Schneider D.
      Hsp70 proteins and their Hsp40 co-chaperones are essential components of cellular chaperone networks in both prokaryotes and eukaryotes. Here, we performed a genetic analysis to define the protein domains required for the key functions of the major Hsp70/DnaJ protein Sll0897 of the cyanobacterium Synechocystis sp. PCC6803. Expression of the N-terminally located J- and G/F-domains is essential and sufficient for the proteins' fundamental in vivo functions, whereas presence of the full-length protein, containing the C-terminal substrate-binding domains, is crucial under stress conditions.
    Keywords:   Synechocystis ; DnaJ; Hsp40; chaperone; cyanobacteria; stress response
    DOI:  https://doi.org/10.1002/2211-5463.12980
  35. EMBO J. 2020 Sep 21. e105693
    Tüshaus J, Müller SA, Kataka ES, Zaucha J, Sebastian Monasor L, Su M, Güner G, Jocher G, Tahirovic S, Frishman D, Simons M, Lichtenthaler SF.
      To understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the "high-performance secretome protein enrichment with click sugars" (hiSPECS) method. To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons, and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion and brain function and to identify cell type-specific biomarkers for CNS diseases.
    Keywords:   CSF ; BACE1; brain cells; proteomics; secretomics
    DOI:  https://doi.org/10.15252/embj.2020105693
  36. FASEB J. 2020 Sep 22.
    López DM, Kählau L, Jungnickel KEJ, Löw C, Damme M.
      The two lysosomal integral membrane proteins MFSD1 and GLMP form a tight complex that confers protection of both interaction partners against lysosomal proteolysis. We here refined the molecular interaction of the two proteins and found that the luminal domain of GLMP alone, but not its transmembrane domain or its short cytosolic tail, conveys protection and mediates the interaction with MFSD1. Our data support the finding that the interaction is essential for the stabilization of the complex. These results are complemented by the observation that N-glycosylation of GLMP in general, but not the type of N-glycans (high-mannose-type or complex-type) or individual N-glycan chains, are essential for protection. We observed that the interaction of both proteins already starts in the endoplasmic reticulum, and quantitatively depends on each other. Both proteins can affect vice versa their intracellular trafficking to lysosomes in addition to the protection from proteolysis. Finally, we provide evidence that MFSD1 can form homodimers both in vitro and in vivo. Our data refine the complex interplay between an intimate couple of a lysosomal transporter and its accessory subunit.
    Keywords:  GLMP; MFSD1; accessory subunit; lysosomal; transporter
    DOI:  https://doi.org/10.1096/fj.202000912RR
  37. Elife. 2020 Sep 21. pii: e60742. [Epub ahead of print]9
    Johnson SL, Ranxhi B, Libohova K, Tsou WL, Todi SV.
      Spinocerebellar ataxia type 3 (SCA3) belongs to the family of polyglutamine neurodegenerations. Each disorder stems from the abnormal lengthening of a glutamine repeat in a different protein. Although caused by a similar mutation, polyglutamine disorders are distinct, implicating non-polyglutamine regions of disease proteins as regulators of pathogenesis. SCA3 is caused by polyglutamine expansion in ataxin-3. To determine the role of ataxin-3's non-polyglutamine domains in disease, we utilized a new, allelic series of Drosophila melanogaster. We found that ataxin-3 pathogenicity is saliently controlled by polyglutamine-adjacent ubiquitin-interacting motifs (UIMs) that enhance aggregation and toxicity. UIMs function by interacting with the heat shock protein, Hsc70-4, whose reduction diminishes ataxin-3 toxicity in a UIM-dependent manner. Hsc70-4 also enhances pathogenicity of other polyglutamine proteins. Our studies provide a unique insight into the impact of ataxin-3 domains in SCA3, identify Hsc70-4 as a SCA3 enhancer, and indicate pleiotropic effects from HSP70 chaperones, which are generally thought to suppress polyglutamine degeneration.
    Keywords:  D. melanogaster; aggregation; ataxia; cell biology; deubiquitinase; machado-joseph disease; neurodegeneration; neuroscience; proteostasis
    DOI:  https://doi.org/10.7554/eLife.60742
  38. Plant Physiol. 2020 Sep 21. pii: pp.01106.2020. [Epub ahead of print]
    Wang N, Liu W, Yu L, Guo Z, Chen Z, Jiang S, Xu H, Fang H, Wang Y, Zhang Z, Chen X.
      Drought is an important environmental factor affecting the growth and production of agricultural crops and fruits worldwide, including apple (Malus domestica). HEAT SHOCK FACTORs (HSFs) have well-documented functions in stress responses, but their roles in flavonoid synthesis and the flavonoid-mediated drought response mechanism remain elusive. In this study, we demonstrated that a drought-responsive HSF, designated MdHSFA8a, promotes the accumulation of flavonoids, scavenging of reactive oxygen species, and plant survival under drought conditions. A chaperone, HEAT SHOCK PROTEIN 90 (HSP90), interacted with MdHSFA8a to inhibit its binding activity and transcriptional activation. However, under drought stress, the MdHSP90-MdHSFA8a complex dissociated and the released MdHSFA8a further interacted with the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF)-family transcription factor RELATED TO AP2 12 (RAP2.12) to activate downstream gene activity. In addition, we demonstrated that MdHSFA8a participates in abscisic acid (ABA)-induced stomatal closure and promotes expression of ABA signaling-related genes. Collectively, these findings provide insight into the mechanism by which stress-inducible MdHSFA8a modulates flavonoid synthesis to regulate drought tolerance.
    DOI:  https://doi.org/10.1104/pp.20.01106
  39. EMBO Rep. 2020 Sep 24. e50905
    Catipovic MA, Rapoport TA.
      Bacterial secretory proteins are translocated post-translationally by the SecA ATPase through the protein-conducting SecY channel in the plasma membrane. During the ATP hydrolysis cycle, SecA undergoes large conformational changes of its two-helix finger and clamp domains, but how these changes result in polypeptide movement is unclear. Here, we use a reconstituted purified system and protease protection assays to show that ATP binding to SecA results in a segment of the translocation substrate being pushed into the channel. This motion is prevented by mutation of conserved residues at the finger's tip. Mutation of SecA's clamp causes backsliding of the substrate in the ATP-bound state. Together, these data support a power stroke model of translocation in which, upon ATP binding, the two-helix finger pushes the substrate into the channel, where it is held by the clamp until nucleotide hydrolysis has occurred.
    Keywords:   E. coli ; AAA ATPase; SecA; SecYEG; protein translocation
    DOI:  https://doi.org/10.15252/embr.202050905
  40. Proc Natl Acad Sci U S A. 2020 Sep 21. pii: 202005506. [Epub ahead of print]
    Li S, Wu Z, Tantray I, Li Y, Chen S, Dong J, Glynn S, Vogel H, Snyder M, Lu B.
      Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.
    Keywords:  C9-ALS/FTD; CAT-tailing; Notch; YME1L; ribosome-associated quality control
    DOI:  https://doi.org/10.1073/pnas.2005506117
  41. J Cell Biol. 2020 Oct 05. pii: e201912074. [Epub ahead of print]219(10):
    Wozniak AL, Adams A, King KE, Dunn W, Christenson LK, Hung WT, Weinman SA.
      Cells respond to inflammatory disease states by releasing exosomes containing highly specific protein and RNA cargos, but how inflammation alters cargo specificity and secretion of exosomes is unknown. We show that increases in exosome secretion induced by either viral infection or LPS/ATP exposure result from inflammasome activation and subsequent caspase-1-dependent cleavage of the trafficking adaptor protein RILP. This cleaved form of RILP promotes the movement of multivesicular bodies toward the cell periphery and induces selective exosomal miRNA cargo loading. We have identified a common short sequence motif present in miRNAs that are selectively loaded into exosomes after RILP cleavage. This motif binds the RNA binding protein FMR1 and directs miRNA loading into exosomes via interaction with components of the ESCRT (endosomal sorting complex required for transport) pathway. These results indicate that inflammasome-mediated RILP cleavage, and sequence-specific interactions between miRNAs and FMR1, play a significant role in exosome cargo loading and enhanced secretion during cellular inflammatory responses.
    DOI:  https://doi.org/10.1083/jcb.201912074
  42. Mol Cell Proteomics. 2020 Sep 21. pii: mcp.RA120.002290. [Epub ahead of print]
    Nie L, Wang C, Li N, Feng X, Lee N, Su D, Tang M, Yao F, Chen J.
      Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.
    Keywords:  Cancer Biology*; E3 ubiquitin ligase; Label-free quantification; Mass Spectrometry; Protein Degradation*; RNF146; Substrate identification; TNKS; Ubiquitin; Ubiquitinases
    DOI:  https://doi.org/10.1074/mcp.RA120.002290
  43. Cells. 2020 Sep 22. pii: E2140. [Epub ahead of print]9(9):
    Andrade-Tomaz M, de Souza I, Rocha CRR, Gomes LR.
      The cell cycle involves a network of proteins that modulate the sequence and timing of proliferation events. Unregulated proliferation is the most fundamental hallmark of cancer; thus, changes in cell cycle control are at the heart of malignant transformation processes. Several cellular processes can interfere with the cell cycle, including autophagy, the catabolic pathway involved in degradation of intracellular constituents in lysosomes. According to the mechanism used to deliver cargo to the lysosome, autophagy can be classified as macroautophagy (MA), microautophagy (MI), or chaperone-mediated autophagy (CMA). Distinct from other autophagy types, CMA substrates are selectively recognized by a cytosolic chaperone, one-by-one, and then addressed for degradation in lysosomes. The function of MA in cell cycle control, and its influence in cancer progression, are already well-established. However, regulation of the cell cycle by CMA, in the context of tumorigenesis, has not been fully addressed. This review aims to present and debate the molecular mechanisms by which CMA can interfere in the cell cycle, in the context of cancer. Thus, cell cycle modulators, such as MYC, hypoxia-inducible factor-1 subunit alpha (HIF-1α), and checkpoint kinase 1 (CHK1), regulated by CMA activity will be discussed. Finally, the review will focus on how CMA dysfunction may impact the cell cycle, and as consequence promote tumorigenesis.
    Keywords:  MYC; autophagy; cancer; chaperone-mediated autophagy (CMA), cell cycle; checkpoints; hypoxia-inducible factor-1 subunit alpha (HIF-1α), checkpoint kinase 1 (CHK1)
    DOI:  https://doi.org/10.3390/cells9092140
  44. Biophys J. 2020 Sep 11. pii: S0006-3495(20)30711-6. [Epub ahead of print]
    Kötter A, Mootz HD, Heuer A.
      Post-translational modification with one of the isoforms of the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome. The binding of SUMO to SUMO interacting motifs (SIMs) can translate the SUMOylation event into functional consequences. The E3 ubiquitin ligase RNF4 contains multiple SIMs and connects SUMOylation to the ubiquitin pathway. SIM2 and SIM3 of RNF4 were shown to be the most important motifs to recognize SUMO chains. However, the study of SIM-SUMO complexes is complicated by their typically low affinity and variable binding of the SIMs in parallel and antiparallel orientations. We investigated properties of complexes formed by SUMO3 with peptides containing either SIM2 or SIM3 using molecular dynamics simulations. The affinities of the complexes were determined using a state-of-the-art free energy protocol and were found to be in good agreement with experimental data, thus corroborating our method. Long unrestrained simulations allowed a new interpretation of experimental results regarding the structure of the SIM-SUMO interface. We show that both SIM2 and SIM3 bind SUMO3 in parallel and antiparallel orientations and identified main interaction sites for acidic residues flanking the SIM. We noticed unusual SIM-SUMO interfaces in a previously reported NMR structure (PDB: 2mp2) of a complex formed by a SUMO3 dimer with the bivalent SIM2-SIM3 peptide. Computational determination of the individual SIM-SUMO affinities based on these structural arrangements yielded significantly higher dissociation constants. To our knowledge, our approach adds new opportunities to characterize individual SIM-SUMO complexes and suggests that further studies will be necessary to understand these interactions when occurring in multivalent form.
    DOI:  https://doi.org/10.1016/j.bpj.2020.09.003
  45. Cancer Cell Int. 2020 ;20 459
    Shen T, Li Y, Liang S, Chen Z.
      Background: Centromere protein F (CENPF) is a key component of the kinetochore complex involved in mitosis, cell differentiation and cellular response to stresses. However, the alteration of CENPF in response to endoplasmic reticulum (ER) stress has not been well described. In the present study, we investigate CENPF regulation in response to ER stress.Methods: Quantitative real-time polymerase chain reaction and western blotting were used to determine CENPF expression under ER stress. Luciferase activity analysis was performed to investigate the promoter regions contributing to CENPF transcription in response to TG. Chromatin immunoprecipitation (ChIP) and ChIP Re-IP assays were used to determine if X-box binding protein 1 (XBP1) and/or activating transcription factor 6α (ATF6α) bind in the CENPF promoter region. Cell apoptosis and proliferation were analyzed using TUNEL, cell growth and clonogenic assays.
    Results: CENPF expression is dramatically reduced under ER stress induced by thapsigargin (TG), brefeldin A (BFA), or tunicamycin (TM) and this downregulation of CENPF expression was dependent on XBP1 and ATF6α. Luciferase activity analysis of the truncated CENPF promoter indicates that regions from bases - 679 to - 488 and from - 241 to - 78 in the CENPF promoter were sensitive to TG treatment. Additionally, ChIP and ChIP Re-IP assays reveal that XBP1 and ATF6α were assembled on the same regions of CENPF promoter. Notably, we identify two XBP1 binding sequences at positions - 567 and - 192, to which XBP1 binding was enhanced by TG. Finally, CENPF overexpression inhibits cell apoptosis and promotes cell proliferation in response to ER stress.
    Conclusion: In summary, these results demonstrate that ER stress plays a crucial role in CENPF expression, and XBP1 may up-regulate DNA-binding affinities after TG treatment to the promoter of CENPF. These findings may contribute to the understanding of the molecular mechanism of CENPF regulation.
    Keywords:  ATF6α; CENPF; ER stress; Expression regulation; XBP1
    DOI:  https://doi.org/10.1186/s12935-020-01553-9
  46. J Biol Chem. 2020 Sep 23. pii: jbc.RA120.014532. [Epub ahead of print]
    Kaur S, Chen Y, Shenoy SK.
      The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a 7-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, while a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By downregulating 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 (GPCR); deubiquitylation (deubiquitination); endocytosis; glucagon; receptor recycling
    DOI:  https://doi.org/10.1074/jbc.RA120.014532
  47. EMBO Rep. 2020 Sep 21. e50400
    Heim VJ, Dagley LF, Stafford CA, Hansen FM, Clayer E, Bankovacki A, Webb AI, Lucet IS, Silke J, Nachbur U.
      Signaling via the intracellular pathogen receptors nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2 requires receptor interacting kinase 2 (RIPK2), an adaptor kinase that can be targeted for the treatment of various inflammatory diseases. However, the molecular mechanisms of how RIPK2 contributes to NOD signaling are not completely understood. We generated FLAG-tagged RIPK2 knock-in mice using CRISPR/Cas9 technology to study NOD signaling mechanisms at the endogenous level. Using cells from these mice, we were able to generate a detailed map of post-translational modifications on RIPK2. Similar to other reports, we did not detect ubiquitination of RIPK2 lysine 209 during NOD2 signaling. However, using site-directed mutagenesis we identified a new regulatory region on RIPK2, which dictates the crucial interaction with the E3 ligase XIAP and downstream signaling outcomes.
    Keywords:   XIAP ; NOD signaling; RIPK2; inflammation; ubiquitin
    DOI:  https://doi.org/10.15252/embr.202050400
  48. Sci Adv. 2020 Sep;pii: eabb0205. [Epub ahead of print]6(39):
    Gambardella G, Staiano L, Moretti MN, De Cegli R, Fagnocchi L, Di Tullio G, Polletti S, Braccia C, Armirotti A, Zippo A, Ballabio A, De Matteis MA, di Bernardo D.
      Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.
    DOI:  https://doi.org/10.1126/sciadv.abb0205
  49. Biochem Biophys Res Commun. 2020 Sep 21. pii: S0006-291X(20)31784-8. [Epub ahead of print]
    Lee SW, Commisso C.
      The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.
    Keywords:  EGFR; Glutamine; Nutrient stress; Pak; Pancreatic; Rac
    DOI:  https://doi.org/10.1016/j.bbrc.2020.09.043
  50. Autophagy. 2020 Sep 22. 1-15
    Pantoom S, Konstantinidis G, Voss S, Han H, Hofnagel O, Li Z, Wu YW.
      Autophagosome formation is a fundamental process in macroautophagy/autophagy, a conserved self-eating mechanism in all eukaryotes, which requires the conjugating ATG (autophagy related) protein complex, ATG12-ATG5-ATG16L1 and lipidated MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). How the ATG12-ATG5-ATG16L1 complex is recruited to membranes is not fully understood. Here, we demonstrated that RAB33B plays a key role in recruiting the ATG16L1 complex to phagophores during starvation-induced autophagy. Crystal structures of RAB33B bound to the coiled-coil domain (CCD) of ATG16L1 revealed the recognition mechanism between RAB33B and ATG16L1. ATG16L1 is a novel RAB-binding protein (RBP) that can induce RAB proteins to adopt active conformation without nucleotide exchange. RAB33B and ATG16L1 mutually determined the localization of each other on phagophores. RAB33B-ATG16L1 interaction was required for LC3 lipidation and autophagosome formation. Upon starvation, a fraction of RAB33B translocated from the Golgi to phagophores and recruited the ATG16L1 complex. In this work, we reported a new mechanism for the recruitment of the ATG12-ATG5-ATG16L1 complex to phagophores by RAB33B, which is required for autophagosome formation. Abbreviations : ATG: autophagy-related; Cα: alpha carbon; CCD: coiled-coil domain; CLEM: correlative light and electron microscopy; DTE: dithioerythritol; EBSS: Earle's balanced salt solution; EDTA: ethylenediaminetetraacetic acid; EGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLIM: fluorescence lifetime imaging microscopy; FRET: Förster resonance energy transfer; GDP: guanosine diphosphate; GOLGA2/GM130: golgin A2; GppNHp: guanosine 5'-[β,γ-imido]triphosphate; GST: glutathione S-transferase; GTP: guanosine triphosphate; GTPγS: guanosine 5'-O-[gamma-thio]triphosphate; HA (tag): hemagglutinin (tag); HEK: human embryonic kidney; HeLa: Henrietta Lacks; HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); IgG: immunoglobulin G; Kd: dissociation constant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCF7: Michigan cancer foundation-7; MEF: mouse embryonic fibroblast; MEM: minimum essential medium Eagle; MST: microscale thermophoresis; NEAA: non-essential amino acids; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RAB: RAS-associated binding; RB1CC1/FIP200: RB1 inducible coiled-coil protein 1; RBP: RAB-binding protein; SD: standard deviation; SDS: sodium dodecyl sulfate; SQSTM1/p62: sequestosome 1; TBS-T: tris-buffered saline-tween 20; WD (repeat): tryptophan-aspartic acid (repeat); WIPI2B: WD repeat domain phosphoinositide interacting 2B; WT: wild type.
    Keywords:  ATG12–ATG5-ATG16L1 complex; ATG16L1; RAB33B; RAB33B-ATG16L1 complex; autophagosome formation; autophagy; crystal structure
    DOI:  https://doi.org/10.1080/15548627.2020.1822629
  51. J Hepatol. 2020 Sep 17. pii: S0168-8278(20)33622-9. [Epub ahead of print]
    Lee-Law PY, Olaizola P, Caballero-Camino FJ, Izquierdo-Sánchez L, Rodrigues PM, Santos-Laso A, Azkargorta M, Elortza F, Martinez-Chantar ML, Perugorria MJ, Aspichueta P, Marzioni M, LaRusso NF, Bujanda L, Drenth JPH, Banales JM.
      BACKGROUND & AIMS: polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple fluid-filled biliary cysts. Most PLD-causative genes participate in protein biogenesis and/or transport. Post-translational modifications (PTMs) are implicated in protein stability, localization and activity, contributing to human pathobiology; however, their role in PLD is unknown. Here, we aimed to unveil the role of protein SUMOylation in PLD and its potential therapeutic targeting.METHODS: levels and functional effects of SUMOylation, along with response to S-adenosylmethionine (SAMe, inhibitor of the SUMOylation enzyme UBC9) and/or short-hairpin RNAs (shRNAs) against UBE2I (UBC9), were evaluated in vitro, in vivo and/or in patients with PLD. SUMOylated proteins were determined by immunoprecipitation and proteomic analyses by mass spectrometry.
    RESULTS: most SUMOylation-related genes were found overexpressed (mRNA) in polycystic human and rat liver tissue, as well as in cystic cholangiocytes in culture compared to controls. Increased SUMOylated protein levels were also observed in cystic human cholangiocytes in culture, which decreased after SAMe administration. Chronic treatment of polycystic (PCK: Pkdh1-mut) rats with SAMe halted hepatic cystogenesis and fibrosis, and reduced liver/body weight ratio and liver volume. In vitro, both SAMe and shRNA-mediated UBE2I knockdown increased apoptosis and reduced cell proliferation of cystic cholangiocytes. High-throughput proteomic analysis of SUMO1-immunoprecipitated proteins in cystic cholangiocytes identified candidates involved in protein biogenesis, ciliogenesis and proteasome degradation. Accordingly, SAMe hampered the proteasome hyperactivity in cystic cholangiocytes, leading to the activation the unfolded protein response (UPR) and stress-related apoptosis.
    CONCLUSIONS: cystic cholangiocytes exhibit increased SUMOylation of proteins involved in cell survival and proliferation, thus promoting hepatic cystogenesis. Inhibition of protein SUMOylation with SAMe halts PLD, representing a novel therapeutic strategy.
    Keywords:  Hepatic cystogenesis; S-adenosylmethionine (SAMe); SUMOylation; post-translational modifications; therapy
    DOI:  https://doi.org/10.1016/j.jhep.2020.09.010
  52. Cell Rep. 2020 Sep 22. pii: S2211-1247(20)31165-7. [Epub ahead of print]32(12): 108176
    Buch-Larsen SC, Hendriks IA, Lodge JM, Rykær M, Furtwängler B, Shishkova E, Westphall MS, Coon JJ, Nielsen ML.
      ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of activated ion electron transfer dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profile 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress and identify 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and electron-transfer higher-energy collisional dissociation (EThcD), respectively. Under physiological conditions, AI-ETD identifies 450 ADPr sites on low-abundant proteins, including in vivo cysteine modifications on poly(ADP-ribosyl)polymerase (PARP) 8 and tyrosine modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provide insights into the physiological regulation of ADPr.
    Keywords:  ADP-ribosylation; AI-ETD; Golgi apparatus; PARP14; PARP8; endoplasmic reticulum; glycosylation; mass spectrometry; physiological ADPr; proteomics
    DOI:  https://doi.org/10.1016/j.celrep.2020.108176
  53. Oncogene. 2020 Sep 24.
    Qian W, Li Q, Wu X, Li W, Li Q, Zhang J, Li M, Zhang D, Zhao H, Zou X, Jia H, Zhang L, Yang XD, Hou Z.
      Snail is a master inducer of epithelial-mesenchymal transition (EMT) and metastasis, however, Snail protein is labile and is quickly degraded through the predominate ubiquitination-mediated proteasome pathway. Deubiquitinases (DUBs) can counteract the Snail degradation process to maintain high level of Snail protein in cancer cells. In this study, we screened a cDNA library containing 79 DUBs, and discovered that a panel of DUBs consisting of USP13, USP28, USP29, USP37, OTUD6A, and DUB3 can markedly stabilize Snail protein, with USP29 displaying the strongest activity to prevent Snail degradation. Mechanistically, USP29 enhances the interaction of Snail and SCP1, resulting in simultaneous dephosphorylation and deubiquitination of Snail and thereafter cooperative prevention of Snail degradation. Biologically, ectopic expression of USP29 promotes gastric cancer cell migration, and depletion of Snail abolishes USP29-mediated cell migration; and USP29 can be induced by major EMT and metastatic inducing factors such as TGFβ, TNFα, and hypoxia. More importantly, high expression levels of Snail, USP29, and SCP1 are associated with poor survival and prognosis. Collectively, these data indicate that Snail is a crucial substrate for USP29 to promote cell migration and USP29/SCP1 complex may be new therapeutic targets to treat metastatic cancer.
    DOI:  https://doi.org/10.1038/s41388-020-01471-0