bims-proteo Biomed News
on Proteostasis
Issue of 2020‒10‒04
forty-two papers selected by
Eric Chevet

  1. Autophagy. 2020 Sep 26.
    Broday L, Adir O, Bening-Abu-Shach U, Arbib S, Henis-Korenblit S.
      RNF5 is implicated in ERAD and in negative regulation of macroautophagy/autophagy. To better understand the function of RNF-5 under ER-stress conditions, we studied the ability of Caenorhabditis elegans rnf-5(tm794) mutant animals to cope with stress in the background of impaired UPR machinery. We demonstrate that downregulation of RNF-5 decreased sensitivity to tunicamycin both in wild type and in an ire-1 mutant. Double-mutant rnf-5;ire-1 animals showed increased starvation resistance and extended lifespan when compared to the ire-1 mutant. This partial rescue of ire-1 required functional autophagy. Downregulation of RNF-5 rescued ER maturation defects and protein secretion of a DAF-28::GFP intestinal reporter in the ire-1 background. Proteomics and functional studies revealed an increase in lysosomal protease levels, in the frequency of intestinal lysosomes, and in lysosomal protease activity in rnf-5(tm794) animals. Together, these data suggest that RNF-5 is a negative regulator of ER stress, and that inactivation of RNF-5 promotes IRE-1-independent elevation of ER capacity.
    Keywords:  IRE1; RMA1; RNF5; autophagy, C. elegans ; endoplasmic reticulum; lysosome
  2. Cell Death Dis. 2020 Sep 26. 11(9): 810
    Li R, Gu Z, Zhang X, Yu J, Feng J, Lou Y, Lv P, Chen Y.
      Autophagy is a highly conserved lysosome-dependent degradation system in eukaryotic cells. This process removes long-lived intracellular proteins, damaged organelles, and recycles biological material to maintain cellular homeostasis. Dysfunction of autophagy triggers a wide spectrum of human diseases, including cancer and neurodegenerative diseases. In the present study, we show that RNF115, an E3 ubiquitin ligase, regulates autophagosome-lysosome fusion and autophagic degradation under both nutrient-enriched and stress conditions. Depletion of the RNF115 gene caused the accumulation of autophagosomes by impairing fusion with lysosomes, which results in an accumulation of autophagic substrates. Further investigation suggests that RNF115 interacts with STX17 and enhances its stability, which is essential for autophagosome maturation. Importantly, we provide in vitro and in vivo evidence that RNF115 inactivation inhibits the tumorigenesis and metastasis of BGC823 gastric cancer cells. We additionally show that high expression levels of RNF115 mRNA correlate with poor prognosis in gastric cancer patients. These findings indicate that RNF115 may play an evolutionarily conserved role in the autophagy pathway, and may act to maintain protein homeostasis under physiological conditions. These data demonstrate the need to further evaluate the potential therapeutic implications of RNF115 in gastric cancer.
  3. Cell Res. 2020 Sep 28.
    Xu F, Du W, Zou Q, Wang Y, Zhang X, Xing X, Li Y, Zhang D, Wang H, Zhang W, Hu X, Liu X, Liu X, Zhang S, Yu J, Fang J, Li F, Zhou Y, Yue T, Mi N, Deng H, Zou P, Chen X, Yang X, Yu L.
      Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.
  4. New Phytol. 2020 Oct 02.
    Zhang CL, Wang GL, Zhang YL, Hu X, Zhou LJ, You CX, Li YY, Hao YJ.
      Post-translational modification of proteins mediated by SIZ1, a small ubiquitin-like modifier (SUMO) E3 ligase, regulates multiple biological processes in plants. However, its role in the regulation of lateral root formation remains unclear. Here, we demonstrate that the apple SUMO E3 ligase MdSIZ1 promotes lateral root formation. Using a yeast two-hybrid system, the auxin response factor MdARF8 was screened out as a protein-protein interaction partner of the SUMO-conjugating E2 enzyme MdSCE1, indicating that MdARF8 may be a substrate for MdSIZ1. The interaction between MdARF8 and MdSCE1 was confirmed by pull-down, Y2H, and Co-IP assays. MdSIZ1 enhanced the conjugating enzyme activity of MdSCE1 to form a MdSCE1-MdSIZ1-MdARF8 complex, thereby facilitating SUMO modification. We identified two arginine substitution mutations at K342 and K380 in MdARF8 that blocked MdSIZ1-mediated SUMOylation, indicating that K342 and K380 are the principal SUMOylation sites of the MdARF8 protein. Moreover, MdARF8 promoted lateral root formation in transgenic apple plants, and the phenotype of reduced lateral roots in the Arabidopsis siz1-2 mutant was restored in siz1-2/MdARF8 complementary plants. Our findings reveal an important role for sumoylation in the regulation of lateral root formation in plants.
    Keywords:  MdARF8; MdSIZ1; SUMOylation; apple; lateral root
  5. Dev Cell. 2020 Sep 24. pii: S1534-5807(20)30713-9. [Epub ahead of print]
    Milan M, Balestrieri C, Alfarano G, Polletti S, Prosperini E, Nicoli P, Spaggiari P, Zerbi A, Cirulli V, Diaferia GR, Natoli G.
      Many tumors of endodermal origin are composed of highly secretory cancer cells that must adapt endoplasmic reticulum (ER) activity to enable proper folding of secreted proteins and prevent ER stress. We found that pancreatic ductal adenocarcinomas (PDACs) overexpress the myelin regulatory factor (MYRF), an ER membrane-associated transcription factor (TF) released by self-cleavage. MYRF was expressed in the well-differentiated secretory cancer cells, but not in the poorly differentiated quasi-mesenchymal cells that coexist in the same tumor. MYRF expression was controlled by the epithelial identity TF HNF1B, and it acted to fine-tune the expression of genes encoding highly glycosylated, cysteine-rich secretory proteins, thus preventing ER overload. MYRF-deficient PDAC cells showed signs of ER stress, impaired proliferation, and an inability to form spheroids in vitro, while in vivo they generated highly secretory but poorly proliferating and hypocellular tumors. These data indicate a role of MYRF in the control of ER homeostasis in highly secretory PDAC cells.
    Keywords:  ER stress; MYRF; differentiation; pancreatic cancer; stress response; transcription; tumor heterogeneity; unfolded protein response
  6. Front Cell Dev Biol. 2020 ;8 802
    Zhang L, Afolabi LO, Wan X, Li Y, Chen L.
      Protein quality control (PQC) is pivotal for eukaryotic cells to eliminate misfolded proteins and maintain cellular homeostasis. A decreased or increased capacity of PQC is associated with various diseases, e.g., neurodegenerative diseases and cancers. Recently, increasing evidences have suggested that tripartite motif-containing family proteins (TRIMs) are the key players in PQC regulation. Most TRIMs are E3 ubiquitin ligases, such as TRIM11/19/25, which, through the ubiquitination modifications, can contribute to effectively remove the cellular misfolded proteins or protein aggregates via the UPS pathway. In this review, we summarized the participation of TRIM members in misfolded protein elimination through distinct pathways, including the ubiquitin-proteasome system (UPS), autophagy system, and ER-associated degradation (ERAD).
    Keywords:  ERAD; TRIMs; UPS; aggregates; autophagy; degradation; misfolded proteins
  7. J Org Chem. 2020 Sep 28.
    Izumi M, Araki H, Tominaga M, Okamoto R, Kajihara Y.
      Degradation of misfolded glycoproteins by the ubiquitin-proteasome system (UPS) is a very important process for protein homeostasis. To demonstrate the accessibility toward a ubiquitinated glycoprotein probe for the study of glycoprotein degradation by UPS, we synthesized ubiquitinated glycoprotein CC motif chemokine 1 (CCL1) bearing a high-mannose type N-glycan, starting from six peptide segments. A native isopeptide linkage was constructed using δ-thiolysine (thioLys)-mediated chemical ligation. CCL1 glycopeptide with a high-mannose-type N-glycan as well as a δ-thioLys residue was synthesized chemically. The chemical ligation between δ-thioLys-containing glycopeptide and ubiquitin-α-thioester successfully yielded a ubiquitinated glycopeptide with a native isopeptide bond after desulfurization, even in the presence of a large N-glycan. In vitro folding experiments under reduced and redox conditions gave the desired two types of ubiquitinated glycosylated CCL1s, consisting of unfolded CCL1 and folded ubiquitin, and folded form of both CCL1 as well as ubiquitin. We achieved the chemical synthesis of a complex protein molecule that contains not only the two major post-translational modifications, ubiquitination and glycosylation, but also controlled folding states of ubiquitin and CCL1. These chemical probes could have useful applications in the study of complex ubiquitin biology and glycobiology.
  8. PLoS Genet. 2020 Sep 28. 16(9): e1008704
    Guan L, Zhan Z, Yang Y, Miao Y, Huang X, Ding M.
      ER stress occurs in many physiological and pathological conditions. However, how chronic ER stress is alleviated in specific cells in an intact organism is an outstanding question. Here, overexpressing the gap junction protein UNC-9 (Uncoordinated) in C. elegans neurons triggers the Ire1-Xbp1-mediated stress response in an age-dependent and cell-autonomous manner. The p38 MAPK PMK-3 regulates the chronic stress through IRE-1 phosphorylation. Overexpressing gap junction protein also activates autophagy. The insulin pathway functions through autophagy, but not the transcription of genes encoding ER chaperones, to counteract the p38-Ire1-Xbp1-mediated stress response. Together, these results reveal an intricate cellular regulatory network in response to chronic stress in a subset of cells in multicellular organism.
  9. IBRO Rep. 2020 Dec;9 218-223
    Yshii LM, Manfiolli AO, Denadai-Souza A, Kinoshita PF, Gomes MD, Scavone C.
      Background: Parkinson's disease (PD) is a neurodegenerative disease characterized by intracellular inclusions named Lewy bodies (LB), and alpha-synuclein (asyn) is the major component of these protein aggregates. The precise physiological and pathological roles of asyn are not fully understood. Nevertheless, asyn present in LB is ubiquitinated but fails to reach the 26S proteasome. The mutation A30 P is related to an aggressive and early-onset form of PD. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase, and it interacts and ubiquitinates the asyn in atypical chains (lysine K6, K27, K29, and K33). Methods: Here, we investigated the role of TRAF6 interaction with asyn and the involvement of nuclear factor κB (NF-κB), a key transcription factor in pro-inflammatory signaling pathway activation.Results and Conclusion: We demonstrated that TRAF6 binds to both WT and the mutant form A30 P asyn in an SH-SY5Y cell model. Additionally, the interaction between TRAF6 and WT asyn induced an increase in the activation of NF-κB, leading to changes in TNF, IL-1β and IL-10 levels and culminating in reduced cell viability. Interestingly, the activation of NF-κB and gene regulation were not found in A30 P asyn. These data point to a novel role of TRAF6 in the pathophysiology of PD.
    Keywords:  CHIP, carboxyl terminus of Hsp70-interaction protein; EMSA, Electrophoretic Mobility Shift Assay; LB, Lewy bodies; NF-κB, nuclear factor κB; PD, Parkinson's disease; SIAH, seven in absentia homolog; TRAF6 and NF-κB; TRAF6, tumor necrosis factor receptor-associated factor 6; alpha-synuclein; asyn, alpha-synuclein; cell death; cytokines
  10. IBRO Rep. 2020 Dec;9 207-217
    Wu H, Wang J, Cao M, Liang J, Wu D, Gu X, Ke K.
      Intracerebral hemorrhage (ICH) is defined as bleeding into the brain parenchyma with a high mortality and morbidity rate. Unfortunately, it remains an unresolved medical problem. Therefore, it is necessary to find ways to reduce cellular apoptosis after ICH. Homocysteine-induced endoplasmic reticulum protein (HERP), a 54 kD transmembrane protein, is an early stress response protein encoded by ubiquitin-like domain member 1 (Herpud1) gene. In the present work, our group investigated the role of HERP after ICH and hemin stimulation, HERP expression was examined in mouse and primary cortical neurons after ICH and hemin stimulation by western blot and Immunofluorescent labeling. Using shRNA-HERP plasmid and recombinant adenovirus, we also investigated how HERP affected neuronal apoptosis after ICH and hemin stimulation. In addition, behavioral evaluation was used to ensure our models' success. In vivo and vitro studies, the expression of HERP was increased following ICH and hemin-exposed primary cortical neurons. HERP depletion activated the endoplasmic reticulum (ER) stress pathway and apoptosis in hemin-exposed primary cortical neurons, but inhibited autophagy in hemin-exposed primary cortical neurons. Overexpression of HERP inhibited the ER stress pathway and apoptosis, but activated autophagy in hemin-exposed primary cortical neurons. Consequently, we confirm that HERP plays a protective role in ICH model.
    Keywords:  Apoptosis; Autophagy; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation; Endoplasmic reticulum stress; HERP, homocysteine-induced endoplasmic reticulum protein; Herpud1, homocysteine-inducible and ER-stress-inducible, ubiquitin-like domain member 1; Homocysteine-induced endoplasmic reticulum protein; ICH, intracerebral hemorrhage; Intracerebral hemorrhage; Mice; TM, tunicamycin; TUDCA, tauroursodeoxycholic acid
  11. Expert Rev Hematol. 2020 Sep 29.
    Ninkovic S, Harrison SJ, Quach H.
      INTRODUCTION: Glucose-regulated protein 78 (GRP78) is a stress-inducible molecular chaperone expressed within the endoplasmic reticulum where it acts a master regulator of the unfolded protein response (UPR) pathway. At times of ER stress, activation of the UPR, a multimolecular pathway, limits proteotoxicity induced by misfolded proteins. In malignancies, including multiple myeloma which is characterised by an excessive accumulation of misfolded immunoglobulins, GRP78 expression is increased, with notable translocation of GRP78 to the cell surface. Studies suggest cell surface GRP78 (csGRP78) to be of prognostic significance with emerging evidence that it interacts with a myriad of co-ligands to activate signalling pathways which may promote cell proliferation and survival or apoptosis.AREAS COVERED: This review will focus on the role of ER and csGRP78 in physiology and oncogenesis in multiple myeloma, addressing the factors that shift the balance in GRP78 signalling from survival to apoptosis. The role of GRP78 as a potential prognostic biomarker is explored and current therapeutics in development aimed at targeting csGRP78 are addressed. We conducted a PubMed literature search using the key words "GRP78", "multiple myeloma" reviewing studies prior to 2020.
    EXPERT OPINION: Cell surface GRP78 expression is a potential novel prognostic biomarker in myeloma and targeting of csGRP78 is promising and requires further investigation.
    Keywords:  ER stress; biomarker; glucose-regulated protein 78 (GRP78); multiple myeloma; therapeutic target
  12. Fungal Genet Biol. 2020 Sep 26. pii: S1087-1845(20)30164-X. [Epub ahead of print] 103473
    Lim YJ, Lee YH.
      Ubiquitination, an important process in post-translational modification, regulates various mechanisms in eukaryotes including protein degradation and interaction, cell cycle, stress response, and pathogenicity. The Skp1/Cullin/F-box and the endoplasmic reticulum-associated degradation (ERAD) complexes, RING E3 ligase complexes, are involved in ubiquitin-mediated proteolysis and protein quality control. The F-box protein has FBXO (F-box only or others), FBXW (with WD40), and FBXL (with LRR) classes depending on which interaction domain is present on the C-terminus. The ubiquitin system component cue (CUE) protein is a key factor of ERAD. However, the biological roles of FBXO and CUE proteins are largely unknown in plant pathogenic fungi including Magnaporthe oryzae. To elucidate the roles of FBXO and CUE proteins in fungal development and pathogenicity, MoFBX15 and MoCUE1 were functionally characterized in M. oryzae. Two ubiquitination-associated genes were crucial for conidiation, alkaline stress tolerance, and pathogenicity in M. oryzae. In particular, MoCUE1 was important for ER stress response and localization and translocation of cytoplasmic effectors. Moreover, ubiquitination and SUMOylation levels were decreased and transcript levels of deSUMOylation-associated genes were increased in ΔMofbx15 and ΔMocue1. This study will provide not only comprehensive understanding of the role of ubiquitination but also new insights on crosstalk between ubiquitination and SUMOylation in rice blast fungus and other fungal pathogens.
    Keywords:  Magnaporthe oryzae; Pathogenicity; Post-translational modification; SUMOylation; Ubiquitination
  13. J Cell Mol Med. 2020 Sep 29.
    Chen Y, Guo Y, Li J, Chen YY, Liu Q, Tan L, Gao ZR, Zhang SH, Zhou YH, Feng YZ.
      Bone healing in tooth extraction sockets occurs in a complex environment containing saliva and many microorganisms and is affected by many factors. Endoplasmic reticulum (ER) stress affects bone metabolism, but the role of ER stress in bone healing after tooth extraction remains unclear. We utilized a rat tooth extraction model, in which we promoted wound healing by using salubrinal to regulate the ER stress response. Western blot analysis showed increased expression of p-eIF2α/eIF2α, Runx2 and alkaline phosphatase (ALP) in bone tissue, and histological assays showed irregularly arranged and new bone with more collagen fibres 14 days after tooth extraction and after modulating the degree of ER stress. Micro-CT showed that modulating ER stress to an appropriate degree increases bone filling in regards to the density in the bottom and the surrounding bone wall of the tooth extraction wounds. Transmission electron microscopy showed rough ER expansion and newly formed collagen fibrils in osteoblasts after modulating ER stress to an appropriate degree. We also used different concentrations of salubrinal to evaluate the resistance to tunicamycin-induced ER stress in an osteogenic induction environment. Salubrinal restored the tunicamycin-induced decrease in the viability of primary calvarial osteoblasts and increased the expression of Runx2 and ALP, and decreased p-eIF2α/eIF2α in a dose-dependent manner. Taken together, the results demonstrate that ER stress occurred after tooth extraction, and regulating the degree of ER stress can promote bone healing in tooth extraction sockets, providing clinical evidence for bone healing.
    Keywords:  bone remodelling; endoplasmic reticulum stress; p-eIF2α; primary calvarial osteoblasts; tooth extraction; unfolded protein response
  14. Immunol Lett. 2020 Sep 28. pii: S0165-2478(20)30385-0. [Epub ahead of print]
    Mukherjee D, Bercz LS, Torok MA, Mace TA.
      Activating transcription factor 4 (ATF4) is a DNA binding transcription factor belonging to the family of basic Leucine zipper proteins. ATF4 can be activated in response to multiple cellular stress signals including endoplasmic reticulum stress in the event of improper protein folding or oxidative stress because of mitochondrial dysfunction as well as hypoxia. There are multiple downstream targets of ATF4 that can coordinate the regulation between survival and apoptosis of a cell based on time and exposure to stress. ATF4, therefore, has a broad range of control that results in the modulation of immune cells of the innate and adaptive responses leading to regulation of the cellular immunity. Studies provide evidence that ATF4 can regulate immune cells such as macrophages, T cells, B cells, NK cells and dendritic cells contributing to progression of disease. Immune cells can be exposed to stressed environment in the event of a pathogen attack, infection, inflammation, or in the tumor microenvironment leading to increased ATF4 activity to regulate these responses. ATF4 can further control differentiation and maturation of different immune cell types becoming a determinant of effective immune regulation. Additionally, ATF4 has been heavily implicated in rendering effector immune cells dysfunctional that are used to target tumorigenesis. Therefore, there is a need to evaluate where the literature stands in understanding the overall role of ATF4 in regulating cellular immunity to identify therapeutic targets and generalized mechanisms for different disease progressions.
    Keywords:  ATF4; Cellular Immunity; ER stress; Unfolded protein response
  15. Front Physiol. 2020 ;11 1054
    Alam S, Abdullah CS, Aishwarya R, Morshed M, Bhuiyan MS.
      Mitochondria are the key to properly functioning energy generation in the metabolically demanding cardiomyocytes and thus essential to healthy heart contractility on a beat-to-beat basis. Mitochondria being the central organelle for cellular metabolism and signaling in the heart, its dysfunction leads to cardiovascular disease. The healthy mitochondrial functioning critical to maintaining cardiomyocyte viability and contractility is accomplished by adaptive changes in the dynamics, biogenesis, and degradation of the mitochondria to ensure cellular proteostasis. Recent compelling evidence suggests that the classical protein quality control system in cardiomyocytes is also under constant mitochondrial control, either directly or indirectly. Impairment of cytosolic protein quality control may affect the position of the mitochondria in relation to other organelles, as well as mitochondrial morphology and function, and could also activate mitochondrial proteostasis. Despite a growing interest in the mitochondrial quality control system, very little information is available about the molecular function of mitochondria in cardiac proteostasis. In this review, we bring together current understanding of the adaptations and role of the mitochondria in cardiac proteostasis and describe the adaptive/maladaptive changes observed in the mitochondrial network required to maintain proteomic integrity. We also highlight the key mitochondrial signaling pathways activated in response to proteotoxic stress as a cellular mechanism to protect the heart from proteotoxicity. A deeper understanding of the molecular mechanisms of mitochondrial adaptations and their role in cardiac proteostasis will help to develop future therapeutics to protect the heart from cardiovascular diseases.
    Keywords:  cardiac proteostasis; mitochondria; mitochondrial dysfunction; mitochondrial proteostasis; mitochondrial unfolded protein response; proteotoxicity
  16. JCI Insight. 2020 Oct 01. pii: 138530. [Epub ahead of print]
    Otterpohl KL, Busselman BW, Ratnayake I, Hart RG, Hart K, Evans C, Phillips CL, Beach JR, Ahrenkiel P, Molitoris B, Surendran K, Chandrasekar I.
      Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions including endocytosis and organelle transport pathways. Cell type-specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible, conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the GPI-anchored protein uromodulin and gradual loss of Na+ K+ 2Cl- cotransporter from the apical membrane of the thick ascending limb (TAL) epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules, activation of ER stress and unfolded protein response pathways in Myh9&10 cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress mediated progressive renal tubulointerstitial disease. These observations establish cell type-specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin..
    Keywords:  Cell Biology; Chronic kidney disease; Cytoskeleton; Molecular genetics; Nephrology
  17. J Transl Int Med. 2020 Jun;8(2): 71-79
    Qian H, Zhang Y, Wu B, Wu S, You S, Zhang N, Sun Y.
      Ubiquitination is a modification after protein transcription that plays a vital role in maintaining the homeostasis of the cellular environment. The Homologous to E6AP C-terminus (HECT) family E3 ubiquitin ligases are a kind of E3 ubiquitin ligases with a C-terminal HECT domain that mediates the binding of ubiquitin to substrate proteins and a variable-length N-terminal extension. HECT-ubiquitinated ligases can be divided into three categories: NEDD4 superfamily, HERC superfamily, and other HECT superfamilies. HECT ubiquitin ligase plays an essential role in the development of many human diseases. In this review, we focus on the physiological and pathological processes involved in oxidative stress and the role of E3 ubiquitin ligase of the HECT family.
    Keywords:  E3 ubiquitin ligase; oxidative stress; ubiquitination
  18. Int J Mol Sci. 2020 Sep 29. pii: E7181. [Epub ahead of print]21(19):
    Lenzi C, Ramazzina I, Russo I, Filippini A, Bettuzzi S, Rizzi F.
      Parkinson's Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn's aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aβ aggregation in Alzheimer's Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.
    Keywords:  chaperone; clusterin; gene expression; heat shock protein; neurodegeneration; protein aggregation; proteostasis; αSynuclein
  19. Front Oncol. 2020 ;10 1569
    Xia Q, Ali S, Liu L, Li Y, Liu X, Zhang L, Dong L.
      Glioblastoma (GB) is the most common and aggressive brain malignancy, characterized by heterogeneity and drug resistance. PTEN, a crucial tumor suppressor, exhibits phosphatase-dependent (PI3K-AKT-mTOR pathway)/independent (nucleus stability) activities to maintain the homeostatic regulation of numerous physiological processes. Premature and absolute loss of PTEN activity usually tends to cellular senescence. However, monoallelic loss of PTEN is frequently observed at tumor inception, and absolute loss of PTEN activity also occurs at the late stage of gliomagenesis. Consequently, aberrant PTEN homeostasis, mainly regulated at the post-translational level, renders cells susceptible to tumorigenesis and drug resistance. Ubiquitination-mediated degradation or deregulated intracellular localization of PTEN hijacks cell growth rheostat control for neoplastic remodeling. Functional inactivation of PTEN mediated by the overexpression of ubiquitin ligases (E3s) renders GB cells adaptive to PTEN loss, which confers resistance to EGFR tyrosine kinase inhibitors and immunotherapies. In this review, we discuss how glioma cells develop oncogenic addiction to the E3s-PTEN axis, promoting their growth and proliferation. Antitumor strategies involving PTEN-targeting E3 ligase inhibitors can restore the tumor-suppressive environment. E3 inhibitors collectively reactivate PTEN and may represent next-generation treatment against deadly malignancies such as GB.
    Keywords:  E3 ubiquitin ligases; drug resistance; glioblastoma; glioma; phosphatase and tensin homolog; ubiquitination
  20. PLoS Genet. 2020 Sep 28. 16(9): e1009053
    Mizuno T, Muroi K, Irie K.
      Autophagy is a fundamental process responsible for degradation and recycling of intracellular contents. In the budding yeast, non-selective macroautophagy and microautophagy of the endoplasmic reticulum (ER) are caused by ER stress, the circumstance where aberrant proteins accumulate in the ER. The more recent study showed that protein aggregation in the ER initiates ER-selective macroautophagy, referred to as ER-phagy; however, the mechanisms by which ER stress induces ER-phagy have not been fully elucidated. Here, we show that the expression levels of ATG39, encoding an autophagy receptor specific for ER-phagy, are significantly increased under ER-stressed conditions. ATG39 upregulation in ER stress response is mediated by activation of its promoter, which is positively regulated by Snf1 AMP-activated protein kinase (AMPK) and negatively by Mig1 and Mig2 transcriptional repressors. In response to ER stress, Snf1 promotes nuclear export of Mig1 and Mig2. Our results suggest that during ER stress response, Snf1 mediates activation of the ATG39 promoter and consequently facilitates ER-phagy by negatively regulating Mig1 and Mig2.
  21. Protein J. 2020 Oct 03.
    Lian Q, Gao Y, Li Q, He X, Jiang X, Pu Z, Xu G.
      Interleukin enhancer-binding factor 2 (ILF2) forms a heterodimer with interleukin enhancer-binding factor 3 (ILF3) via double-stranded RNA-binding motif and zinc finger associated domain and thus regulates gene expression and cancer cell growth. However, how ILF2 is degraded in cells remains elusive. In this work, using stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics, we find that ILF2 is downregulated in cells expressing cereblon (CRBN). Using affinity purification and immunoblotting analysis, we demonstrate that CRBN interacts with ILF2 and functions as a substrate receptor of the cullin-4 RING E3 ligase complex. Biochemical experiments disclose that CRBN expression reduces ILF2 protein level and this reduction is diminished when the proteasome is inhibited. Upon protein synthesis inhibition, the degradation of ILF2 is enhanced by CRBN. Moreover, CRBN promotes the ubiquitination of ILF2 and thus results in the ubiquitin-mediated proteasomal degradation. Analyses of previously identified post-translational modification sites and the crystal structure of ILF2 discover the potential ubiquitination sites on ILF2. Through mutagenesis and biochemical experiments, we further reveal that the K45R mutation completely abolishes the effect of CRBN on ILF2, suggesting that this is the key residue responsible for its ubiquitination. Taken together, we identify an E3 ligase that regulates ILF2 and uncover a molecular pathway for its degradation. This work might be helpful to elucidate the molecular mechanism by which CRBN regulates diverse cellular functions.
    Keywords:  Cereblon (CRBN); Degradation; Interleukin enhancer-binding factor 2 (ILF2); Quantitative proteomics; SILAC; Ubiquitination
  22. Cell Chem Biol. 2020 Sep 23. pii: S2451-9456(20)30377-9. [Epub ahead of print]
    Bushman JW, Donovan KA, Schauer NJ, Liu X, Hu W, Varca AC, Buhrlage SJ, Fischer ES.
      Deubiquitinating enzymes (DUBs) catalyze the removal of ubiquitin, thereby reversing the activity of E3 ubiquitin ligases and are central to the control of protein abundance and function. Despite the growing interest in DUBs as therapeutic targets, cellular functions for DUBs remain largely unknown and technical challenges often preclude the identification of DUB substrates in a comprehensive manner. Here, we demonstrate that treatment with potent DUB inhibitors coupled to mass spectrometry-based proteomics can identify DUB substrates at a proteome-wide scale. We applied this approach to USP7, a DUB widely investigated as a therapeutic target and identified many known substrates and additional targets. We demonstrate that USP7 substrates are enriched for DNA repair enzymes and E3 ubiquitin ligases. This work provides not only a comprehensive annotation of USP7 substrates, but a general protocol widely applicable to other DUBs, which is critical for translational development of DUB targeted agents.
    Keywords:  DUBs; chemical probe; deubiquitinating enzymes; drug discovery; proteomics
  23. Cell Death Differ. 2020 Sep 30.
    Kist M, Kőműves LG, Goncharov T, Dugger DL, Yu C, Roose-Girma M, Newton K, Webster JD, Vucic D.
      Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1K376R/K376R mice, Ripk1K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1K376R/cko mice was tolerated. However, adult Ripk1K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.
  24. Nat Cell Biol. 2020 Oct;22(10): 1170-1179
    Xu C, Wang L, Fozouni P, Evjen G, Chandra V, Jiang J, Lu C, Nicastri M, Bretz C, Winkler JD, Amaravadi R, Garcia BA, Adams PD, Ott M, Tong W, Johansen T, Dou Z, Berger SL.
      SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1-5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome-lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy-lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.
  25. Channels (Austin). 2020 Dec;14(1): 326-335
    Ma XE, Liu B, Zhao CX.
      Ca2+-induced Ca2+ release (CICR) from sarcoplasmic reticulum is a finely tuned process responsible for cardiac excitation and contraction. The ubiquitin-proteasome system (UPS) as a major degradative system plays a crucial role in the maintenance of Ca2+ homeostasis. The E3 component N-recognin (UBR) subfamily is a part of the UPS; however, the role of UBR in regulating cardiac CICR is unknown. In the present study, we found that among the UBR family, single knockdown of UBR3 or UBR6 significantly elevated the amplitude of sarcoplasmic reticulum Ca2+ release without affecting Ca2+ transient decay time in neonatal rat ventricular myocytes. The protein expression of alpha 1 C subunit of L-type voltage-dependent Ca2+ channel (Cav1.2) was increased after UBR3/6 knockdown, whereas the protein levels of RyR2, SERCA2a, and PLB remained unchanged. In line with the increase in Cav1.2 proteins, the UBR3/6 knockdown enhanced the current of Cav1.2 channels. Furthermore, the increase in Cav1.2 proteins caused by UBR3/6 reduction was not counteracted by a protein biosynthesis inhibitor, cycloheximide, suggesting a degradative regulation of UBR3/6 on Cav1.2 channels. Our results indicate that UBR3/6 modulates cardiac CICR via targeting Cav1.2 protein degradation.
    Keywords:  Ca2+-induced Ca2+ release; Cav1.2 channel; UBR
  26. Heliyon. 2020 Sep;6(9): e05000
    Moidu NA, A Rahman NS, Syafruddin SE, Low TY, Mohtar MA.
      Anterior gradient-2 (AGR2) protein mediates the formation, breakage and isomerization of disulphide bonds during protein maturation in the endoplasmic reticulum (ER) and contributes to the homoeostasis of the secretory pathway. AGR2 promotes tumour development and metastasis and its elevated expression is almost completely restricted to malignant tumours. Interestingly, this supposedly ER-resident protein can be localised to other compartments of cancer cells and can also be secreted into the extracellular milieu. There are emerging evidences that describe the gain-of-function activities of the extracellular AGR2, particularly in cancer development. Here, we reviewed studies detailing the expression, pathological and physiological roles associated with AGR2 and compared the duality of localization, intracellular and extracellular, with special emphasis on the later. We also discussed the possible mechanisms of AGR2 secretion as well as deliberating the functional impacts of AGR2 in cancer settings. Last, we deliberate the current therapeutic strategies and posit the potential use AGR2, as a prognosis and diagnosis marker in cancer.
    Keywords:  Biochemistry; Cancer; Cancer research; Cell biology; Enzymology; Molecular biology; Oncology; PDI; Protein folding; Protein secretion; Secretory pathway; UPR
  27. PLoS Pathog. 2020 Sep 30. 16(9): e1008918
    Mahmud SA, Qureshi MA, Sapkota M, Pellegrino MW.
      The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway promoting mitochondrial recovery and defense against infection. In C. elegans, the UPRmt is activated during infection with the pathogen Pseudomonas aeruginosa-but only transiently. As this may reflect a pathogenic strategy to target a pathway required for host survival, we conducted a P. aeruginosa genetic screen to uncover mechanisms associated with this temporary activation. Here, we find that loss of the P. aeruginosa acyl-CoA dehydrogenase FadE2 prolongs UPRmt activity and extends host survival. FadE2 shows substrate preferences for the coenzyme A intermediates produced during the breakdown of the branched-chain amino acids valine and leucine. Our data suggests that during infection, FadE2 restricts the supply of these catabolites to the host hindering host energy metabolism in addition to the UPRmt. Thus, a metabolic pathway in P. aeruginosa contributes to pathogenesis during infection through manipulation of host energy status and mitochondrial stress signaling potential.
  28. Cell Chem Biol. 2020 Sep 23. pii: S2451-9456(20)30344-5. [Epub ahead of print]
    Kowada T, Watanabe T, Amagai Y, Liu R, Yamada M, Takahashi H, Matsui T, Inaba K, Mizukami S.
      Fluorescent Zn2+ probes used for the quantitative analysis of labile Zn2+ concentration ([Zn2+]) in target organelles are crucial for understanding the role of Zn2+ in biological processes. Although several fluorescent Zn2+ probes have been developed to date, there is still a lack of consensus concerning the [Zn2+] in intracellular organelles. In this study, we describe the development of ZnDA-1H, a small-molecule fluorescent probe for Zn2+, which exhibits less pH sensitivity, high Zn2+ selectivity, and large fluorescence enhancement upon binding to Zn2+. Through protein labeling technology, ZnDA-1H was precisely targeted in various intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. ZnDA-1H exhibited a reversible fluorescence response toward labile Zn2+ in these organelles in live cells. Using this probe, the [Zn2+] in the Golgi apparatus was estimated to be 25 ± 1 nM, suggesting that labile Zn2+ plays a physiological role in the secretory pathway.
    Keywords:  Golgi apparatus; Zn(2+); fluorescence imaging; quantitative analysis
  29. Int J Mol Sci. 2020 Sep 25. pii: E7088. [Epub ahead of print]21(19):
    Voronin MV, Vakhitova YV, Seredenin SB.
      This review analyzes the current scientific literature on the role of the Sigma1R chaperone in the pathogenesis of depressive disorders and pharmacodynamics of antidepressants. As a result of ligand activation, Sigma1R is capable of intracellular translocation from the endoplasmic reticulum (ER) into the region of nuclear and cellular membranes, where it interacts with resident proteins. This unique property of Sigma1R provides regulation of various receptors, ion channels, enzymes, and transcriptional factors. The current review demonstrates the contribution of the Sigma1R chaperone to the regulation of molecular mechanisms involved in the antidepressant effect.
    Keywords:  BDNF; ER stress; NGF; Sigma1R chaperone; antidepressants; calcium signaling; depression; epigenetic regulation; unfolded protein response
  30. Front Cell Dev Biol. 2020 ;8 843
    Che X, Jian F, Wang Y, Zhang J, Shen J, Cheng Q, Wang X, Jia N, Feng W.
      F-box proteins, as substrates for S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes, mediate the degradation of a large number of regulatory proteins involved in cancer processes. In this study, we found that F-box only protein 2 (FBXO2) was up-regulated in 21 endometrial carcinoma (EC) samples compared with five normal endometrium samples based on our Fudan cohort RNA-sequencing. The increased FBXO2 expression was associated with tumor stage, tumor grade, and histologic tumor type, and poor prognosis based on The Cancer Genome Atlas (TCGA) database. FBXO2 knockdown inhibited EC cell proliferation, and FBXO2 overexpression promoted the parental cell phenotype in vivo and in vitro. Fibrillin1 (FBN1) was also identified as a substrate for FBXO2 using a ubiquitination-proteome approach. In addition, promotion of EC proliferation by FBXO2 was regulated by specific proteins of the cell cycle (CDK4, CyclinD1, CyclinD2, and CyclinA1) and the autophagy signaling pathway (ATG4A and ATG4D) based on RNA sequencing (RNA-seq). We concluded that FBXO2 acts as an E3 ligase that targets FBN1 for ubiquitin-dependent degradation, so as to promote EC proliferation by regulating the cell cycle and the autophagy signaling pathway. Targeting FBXO2 may represent a potential therapeutic target for EC.
    Keywords:  FBN1; FBXO2; autophagy; cell cycle; endometrial carcinoma; ubiquitination
  31. Clin Transl Med. 2020 Sep;10(5): e166
    Lu L, Ma J, Tang J, Liu Y, Zheng Q, Chen S, Gao E, Ren J, Yang L, Yang J.
      BACKGROUND: Myocardial ischemia/reperfusion (MI/R) injury imposes devastating cardiovascular sequelae in particular cardiac dysfunction as a result of restored blood flow. However, the mechanism behind MI/R injury remains elusive. Mitochondrial ubiquitin ligase (MITOL/MARCH5) is localized at the mitochondria-ER contact site and may be activated in response to a variety of pathophysiological processes, such as apoptosis, mitochondrial injury, ER stress, hypoxia, and reactive oxygen species (ROS) generation. Irisin as a cleaved product of fibronectin type III domain-containing protein 5 (FNDC5) displays cardioprotection in diverse cardiac diseases.METHODS: This study was designed to examine the role of irisin and MITOL in MI/R injury. Male C57BL/6J mice (8-10-week-old) were administered adenovirus MITOL shRNA through intracardiac injection followed by MI/R surgery through ligation and release the slipknot of cardiac left anterior descending coronary artery.
    RESULTS: Our results showed that irisin improved myocardial function in the face of MI/R injury as evidenced by reduced myocardial infarct size, apoptotic rate, serum lactate dehydrogenase (LDH), ROS generation, and malondialdehyde (MDA) levels as well as lessened ER stress injury. Moreover, our results indicated that protective role of irisin was mediated by upregulation of MITOL. Irisin also protected H9c2 cells against simulated I/R through negating ER stress, apoptosis, ROS and MDA levels, as well as facilitating superoxide dismutase (SOD) by way of elevated MITOL expression.
    CONCLUSIONS: To this end, our data favored that irisin pretreatment protects against MI/R injury, ER stress, ROS production, and mitochondrial homeostasis through upregulation of MITOL. These findings depicted the therapeutic potential of irisin and MITOL in the management of MI/R injury in patients with ST-segment elevation.
    Keywords:  apoptosis; endoplasmic reticulum stress; irisin (FNDC5); mitochondrial ubiquitin ligase (MITOL); myocardial ischemia/reperfusion (MI/R); reactive oxygen species (ROS)
  32. J Cell Biol. 2020 Nov 02. pii: e202007135. [Epub ahead of print]219(11):
    Stancheva VG, Li XH, Hutchings J, Gomez-Navarro N, Santhanam B, Babu MM, Zanetti G, Miller EA.
      Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which a cargo-bound inner coat layer recruits and is organized by an outer scaffolding layer to drive local assembly of a stable structure rigid enough to enforce membrane curvature. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces, including a newly defined charge-based interaction. These interfaces combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. The Sec31 disordered region could be replaced by evolutionarily distant sequences, suggesting plasticity in the binding interfaces. Such a multimodal assembly platform provides an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.
  33. PLoS Negl Trop Dis. 2020 Oct 02. 14(10): e0008709
    Förster B, Demangel C, Thye T.
      Mycobacterium ulcerans is a human pathogen that causes a necrotizing skin disease known as Buruli ulcer. Necrosis of infected skin is driven by bacterial production of mycolactone, a diffusible exotoxin targeting the host translocon (Sec61). By blocking Sec61, mycolactone prevents the transport of nascent secretory proteins into the endoplasmic reticulum of host cells. This triggers pro-apoptotic stress responses partially depending on activation of the ATF4 transcription factor. To gain further insight into the molecular pathways mediating the cytotoxic effects of mycolactone we conducted the first haploid genetic screen with the M. ulcerans toxin in KBM-7 cells. This approach allowed us to identify the histone methyltransferase SETD1B as a novel mediator of mycolactone-induced cell death. CRISPR/Cas9-based inactivation of SETD1B rendered cells resistant to lethal doses of the toxin, highlighting the critical importance of this gene's expression. To understand how SETD1B contributes to mycolactone cytotoxicity, we compared the transcriptomes of wild-type (WT) and SETD1B knockout KBM-7 cells upon exposure to the toxin. While ATF4 effectors were upregulated by mycolactone in both WT and SETD1B knockout cells, mycolactone selectively induced the expression of pro-apoptotic genes in WT cells. Among those genes we identified CHAC1, which codes for a major glutathione (GSH)-degrading enzyme, and whose strong upregulation in mycolactone-treated WT cells correlated with a marked reduction in GSH protein level. Moreover, GSH supplementation conferred cells with substantial protection against the toxic effects of mycolactone. Our data thus identify SETD1B/CHAC1/GSH as a novel, epigenetic mechanism connecting Sec61 blockade with apoptotic cell death. They suggest that GSH-based treatments might have the capacity to limit skin necrosis in Buruli ulcer.
  34. Mol Cell Biol. 2020 Sep 28. pii: MCB.00180-20. [Epub ahead of print]
    Lin Z, King R, Tang V, Myers G, Balbin-Cuesta G, Friedman A, McGee B, Desch K, Ozel AB, Siemieniak D, Reddy P, Emmer B, Khoriaty R.
      Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.
  35. PLoS Genet. 2020 Oct 02. 16(10): e1008821
    Urso SJ, Comly M, Hanover JA, Lamitina T.
      The conserved O-GlcNAc transferase OGT O-GlcNAcylates serine and threonine residues of intracellular proteins to regulate their function. OGT is required for viability in mammalian cells, but its specific roles in cellular physiology are poorly understood. Here we describe a conserved requirement for OGT in an essential aspect of cell physiology: the hypertonic stress response. Through a forward genetic screen in Caenorhabditis elegans, we discovered OGT is acutely required for osmoprotective protein expression and adaptation to hypertonic stress. Gene expression analysis shows that ogt-1 functions through a post-transcriptional mechanism. Human OGT partially rescues the C. elegans phenotypes, suggesting that the osmoregulatory functions of OGT are ancient. Intriguingly, expression of O-GlcNAcylation-deficient forms of human or worm OGT rescue the hypertonic stress response phenotype. However, expression of an OGT protein lacking the tetracopeptide repeat (TPR) domain does not rescue. Our findings are among the first to demonstrate a specific physiological role for OGT at the organismal level and demonstrate that OGT engages in important molecular functions outside of its well described roles in post-translational O-GlcNAcylation of intracellular proteins.
  36. Biochemistry. 2020 Oct 02.
    Jiang HK, Kurkute P, Li CL, Wang YH, Chen PJ, Lin SY, Wang YS.
      Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.
  37. Nat Cell Biol. 2020 Oct;22(10): 1252-1263
    Nakamura S, Shigeyama S, Minami S, Shima T, Akayama S, Matsuda T, Esposito A, Napolitano G, Kuma A, Namba-Hamano T, Nakamura J, Yamamoto K, Sasai M, Tokumura A, Miyamoto M, Oe Y, Fujita T, Terawaki S, Takahashi A, Hamasaki M, Yamamoto M, Okada Y, Komatsu M, Nagai T, Takabatake Y, Xu H, Isaka Y, Ballabio A, Yoshimori T.
      Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
  38. Front Genet. 2020 ;11 1023
    Kong Y, Qiao Z, Ren Y, Genchev GZ, Ge M, Xiao H, Zhao H, Lu H.
      Lung cancer is one of the most common human cancers both in incidence and mortality, with prognosis particularly poor in metastatic cases. Metastasis in lung cancer is a multifarious process driven by a complex regulatory landscape involving many mechanisms, genes, and proteins. Membrane proteins play a crucial role in the metastatic journey both inside tumor cells and the extra-cellular matrix and are a viable area of research focus with the potential to uncover biomarkers and drug targets. In this work we performed membrane proteome analysis of highly and poorly metastatic lung cells which integrated genomic, proteomic, and transcriptional data. A total of 1,762 membrane proteins were identified, and within this set, there were 163 proteins with significant changes between the two cell lines. We applied the Tied Diffusion through Interacting Events method to integrate the differentially expressed disease-related microRNAs and functionally dys-regulated membrane protein information to further explore the role of key membrane proteins and microRNAs in multi-omics context. Has-miR-137 was revealed as a key gene involved in the activity of membrane proteins by targeting MET and PXN, affecting membrane proteins through protein-protein interaction mechanism. Furthermore, we found that the membrane proteins CDH2, EGFR, ITGA3, ITGA5, ITGB1, and CALR may have significant effect on cancer prognosis and outcomes, which were further validated in vitro. Our study provides multi-omics-based network method of integrating microRNAs and membrane proteome information, and uncovers a differential molecular signatures of highly and poorly metastatic lung cancer cells; these molecules may serve as potential targets for giant-cell lung metastasis treatment and prognosis.
    Keywords:  lung cancer metastasis; membrane proteome; microRNA; multi-omics analysis; prognostic
  39. Trends Biochem Sci. 2020 Sep 29. pii: S0968-0004(20)30227-9. [Epub ahead of print]
    Jansen NS, Vertegaal ACO.
      Small ubiquitin-like modifiers (SUMOs) regulate virtually all nuclear processes. The fate of the target protein is determined by the architecture of the attached SUMO protein, which can be of polymeric nature. Here, we highlight the multifunctional aspects of dynamic signal transduction by SUMO polymers. The SUMO-targeted ubiquitin ligases (STUbLs) RING-finger protein 4 (RNF4) and RNF111 recognize SUMO polymers in a chain-architecture-dependent manner, leading to the formation of hybrid chains, which could enable proteasomal destruction of proteins. Recent publications have highlighted essential roles for SUMO chain disassembly by the mammalian SUMO proteases SENP6 and SENP7 and the yeast SUMO protease Ulp2. SENP6 is particularly important for centromere assembly. These recent findings demonstrate the diversity of SUMO polymer signal transduction for proteolytic and nonproteolytic purposes.
    Keywords:  SUMO; SUMO-specific protease; SUMO-targeted ubiquitin ligase; chains; small ubiquitin-like modifier; ubiquitin
  40. Proc Natl Acad Sci U S A. 2020 Sep 28. pii: 202007297. [Epub ahead of print]
    Debets MF, Tastan OY, Wisnovsky SP, Malaker SA, Angelis N, Moeckl LKR, Choi J, Flynn H, Wagner LJS, Bineva-Todd G, Antonopoulos A, Cioce A, Browne WM, Li Z, Briggs DC, Douglas HL, Hess GT, Agbay AJ, Roustan C, Kjaer S, Haslam SM, Snijders AP, Bassik MC, Moerner WE, Li VSW, Bertozzi CR, Schumann B.
      Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
    Keywords:  bioorthogonal; glycosylation; glycosyltransferase; mucin
  41. Sci Rep. 2020 Sep 28. 10(1): 15850
    Falconieri A, Minervini G, Bortolotto R, Piovesan D, Lopreiato R, Sartori G, Pennuto M, Tosatto SCE.
      Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoform-specific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases.
  42. Cells. 2020 Sep 28. pii: E2183. [Epub ahead of print]9(10):
    Sonninen TM, Goldsteins G, Laham-Karam N, Koistinaho J, Lehtonen Š.
      Protein homeostasis (proteostasis) disturbances and inflammation are evident in normal aging and some age-related neurodegenerative diseases. While the proteostasis network maintains the integrity of intracellular and extracellular functional proteins, inflammation is a biological response to harmful stimuli. Cellular stress conditions can cause protein damage, thus exacerbating protein misfolding and leading to an eventual overload of the degradation system. The regulation of proteostasis network is particularly important in postmitotic neurons due to their limited regenerative capacity. Therefore, maintaining balanced protein synthesis, handling unfolding, refolding, and degrading misfolded proteins are essential to preserve all cellular functions in the central nervous sysytem. Failing proteostasis may trigger inflammatory responses in glial cells, and the consequent release of inflammatory mediators may lead to disturbances in proteostasis. Here, we review the mechanisms of proteostasis and inflammatory response, emphasizing their role in the pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, we discuss the interplay between proteostatic stress and excessive immune response that activates inflammation and leads to dysfunctional proteostasis.
    Keywords:  ER stress; ROS; advanced glycation end-products; immunoproteosome; lipid peroxidation; neuroinflammation; pro-inflammatory cytokines; protein misfolding