192 results on '"Receptors, Autocrine Motility Factor"'
Search Results
2. Staphylococcal virulence factor HlgB targets the endoplasmic-reticulum-resident E3 ubiquitin ligase AMFR to promote pneumonia
- Author
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Lei Sun, Haibo Zhang, Huihui Zhang, Xinyi Lou, Zhiming Wang, Yaxian Wu, Xinyi Yang, Daijie Chen, Beining Guo, Ao Zhang, and Feng Qian
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Inflammation ,Microbiology (medical) ,Staphylococcus aureus ,Ubiquitin-Protein Ligases ,Immunology ,Pneumonia ,Cell Biology ,Endoplasmic Reticulum ,Endoplasmic Reticulum Stress ,Applied Microbiology and Biotechnology ,Microbiology ,Receptors, Autocrine Motility Factor ,Genetics ,Humans - Abstract
Staphylococcus aureus invades cells and persists intracellularly, causing persistent inflammation that is notoriously difficult to treat. Here we investigated host-pathogen interactions underlying intracellular S. aureus infection in macrophages and discovered that the endoplasmic reticulum (ER) is an important cellular compartment for intracellular S. aureus infection. Using CRISPR-Cas9 guide RNA library screening, we determined that the autocrine motility factor receptor (AMFR), an ER-resident E3 ubiquitin ligase, played an essential role in mediating intracellular S. aureus-induced inflammation. AMFR directly interacted with TAK1-binding protein 3 (TAB3) in the ER, inducing K27-linked polyubiquitination of TAB3 on lysine 649 and promoting TAK1 activation. Moreover, the virulence factor γ-haemolysin B (HIgB) of S. aureus bound to the AMFR and regulated TAB3. Our findings highlight an unknown role of AMFR in intracellular S. aureus infection-induced pneumonia and suggest that pharmacological interruption of AMFR-mediated TAB3 signalling cascades and HIgB targeting may prevent invasive staphylococci-mediated pneumonia.
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- 2023
3. Synergistic effects of autocrine motility factor and methyl jasmonate on human breast cancer cells
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Ae Lim Jo, Hee-Sung Park, and Nam Ho Jeoung
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0301 basic medicine ,Autocrine Motility Factor ,Biophysics ,Down-Regulation ,Estrogen receptor ,Breast Neoplasms ,Cyclopentanes ,Acetates ,Biochemistry ,Metastasis ,03 medical and health sciences ,0302 clinical medicine ,Breast cancer ,Cell Line, Tumor ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Humans ,Molecular Targeted Therapy ,Oxylipins ,Cloning, Molecular ,Autocrine signalling ,Molecular Biology ,Tumor Stem Cell Assay ,Cell Proliferation ,Chemistry ,fungi ,Glucose-6-Phosphate Isomerase ,Drug Synergism ,Cell Cycle Checkpoints ,Cell Biology ,medicine.disease ,Antineoplastic Agents, Phytogenic ,Recombinant Proteins ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Tumor progression ,030220 oncology & carcinogenesis ,Cancer cell ,MCF-7 Cells ,Cancer research ,Cytokines ,Female ,GPER ,Signal Transduction - Abstract
Autocrine motility factor (AMF) stimulates the motility of cancer cells via an autocrine route and has been implicated in tumor progression and metastasis. Overexpression of AMF is correlated with the aggressive nature of breast cancer and is negatively associated with clinical outcomes. In contrast, AMF also has the ability to suppress cancer cells. In this study, AMFs from different cancer cells were demonstrated to have suppressive activity against MCF-7 and MDA-MB-231 breast cancer cells. In a growth and colony formation assay, AMF from AsPC-1 pancreatic cancer cells (ASPC-1:AMF) was determined to be more suppressive compared to other AMFs. It was also demonstrated that AsPC-1:AMF could arrest breast cancer cells at the G0/G1 cell cycle phase. Quantified by Western blot analysis, AsPC-1:AMF lowered levels of the AMF receptor (AMFR) and G-protein-coupled estrogen receptor (GPER), concomitantly regulating the activation of the AKT and ERK signaling pathways. JAK/STAT activation was also decreased. These results were found in estrogen receptor (ER)-positive MCF-7 cells but not in triple-negative MDA-MB-231 cells, suggesting that AsPC-1:AMF could work through multiple pathways led to apoptosis. More importantly, AsPC-1:AMF and methyl jasmonate (MJ) cooperatively and synergistically acted against breast cancer cells. Thus, AMF alone or along with MJ may be a promising breast cancer treatment option.
- Published
- 2021
4. The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis
- Author
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Bing Cui, Fang Hua, Jiao-jiao Yu, Yang Xiao, Shuang Shang, Pingping Li, Xiaoxi Lv, Xiaowei Zhang, Zhi-guang Zhou, Yun-xuan Li, Zhuo-Wei Hu, Jun Yan, Jin-mei Yu, Shan-Shan Liu, Chang Liu, Feng Wang, and Ke Li
- Subjects
Pulmonary Fibrosis ,Immunology ,Inflammation ,CCL1 ,Lung injury ,CCR8 ,Biology ,Chemokine CCL1 ,Mice ,Chemokine receptor ,Pulmonary fibrosis ,medicine ,Animals ,Humans ,Immunology and Allergy ,Myofibroblasts ,Adaptor Proteins, Signal Transducing ,medicine.diagnostic_test ,Membrane Proteins ,Cell Differentiation ,Fibroblasts ,Phosphoproteins ,medicine.disease ,Receptors, Autocrine Motility Factor ,Bronchoalveolar lavage ,Infectious Diseases ,Cancer research ,medicine.symptom ,Signal Transduction - Abstract
Summary Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.
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- 2021
5. Autocrine motility factor receptor as a therapeutic target for asthma: comments on 'AMFR drives allergic asthma development by promoting alveolar macrophage-derived GM-CSF production'
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Huihui Zhang, Feng Qian, and Lei Sun
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Receptors, Autocrine Motility Factor ,Macrophages, Alveolar ,Genetics ,Granulocyte-Macrophage Colony-Stimulating Factor ,Humans ,Cell Biology ,General Medicine ,Molecular Biology ,Asthma - Published
- 2022
6. AMFR drives allergic asthma development by promoting alveolar macrophage–derived GM-CSF production
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Huihui Zhang, Ran Wei, Xinyi Yang, Lu Xu, Hongchao Jiang, Mengkai Li, Haixia Jiang, Haibo Zhang, Zhihong Chen, Feng Qian, and Lei Sun
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Inflammation ,Receptors, Autocrine Motility Factor ,Macrophages, Alveolar ,Immunology ,Granulocyte-Macrophage Colony-Stimulating Factor ,Humans ,Immunology and Allergy ,Asthma - Abstract
Alveolar macrophages (AMs) are specialized tissue-resident macrophages that orchestrate the immune response in allergic inflammation and asthma. However, what signals direct AMs to cross talk with other immune cells remains unclear. Here, we report that autocrine motility factor receptor (AMFR), an endoplasmic reticulum–resident E3 ubiquitin ligase, is upregulated in AMs of asthma and is critical for this condition. AMFR deficiency significantly decreased allergy-induced T helper 2 (Th2) and eosinophilic inflammation, with less granulocyte-macrophage colony-stimulating factor (GM-CSF) production in AMs. Mechanistically, following thymic stromal lymphopoietin (TSLP) stimulation, AMFR associated directly with cytokine-inducible SH2-containing protein (CIS), induced the ubiquitination of Lys48-linked polyubiquitination of CIS, and consequently blocked the inhibitory effect of CIS on signal transducer and activator of transcription 5 (STAT5) phosphorylation and the downstream pathway activation in AMs. In conclusion, our results demonstrate that AMFR serves a crucial role in promoting inflammation in asthma through regulating AM function, and may emerge as a new potential drug target for asthma therapy.
- Published
- 2022
7. Pharmacological induction of AMFR increases functional EAAT2 oligomer levels and reduces epileptic seizures in mice
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Longze Sha, Guanjun Li, Xiuneng Zhang, Yarong Lin, Yunjie Qiu, Yu Deng, Wanwan Zhu, and Qi Xu
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Receptors, Autocrine Motility Factor ,Mice ,Epilepsy ,Epilepsy, Temporal Lobe ,Excitatory Amino Acid Transporter 2 ,Seizures ,Animals ,Humans ,Anticonvulsants ,General Medicine ,Acetaminophen - Abstract
Dysregulation of excitatory amino acid transporter 2 (EAAT2) contributes to the development of temporal lobe epilepsy (TLE). Several strategies for increasing total EAAT2 levels have been proposed. However, the mechanism underlying the oligomeric assembly of EAAT2, impairment of which inhibits the formation of functional oligomers by EAAT2 monomers, is still poorly understood. In the present study, we identified E3 ubiquitin ligase AMFR as an EAAT2-interacting protein. AMFR specifically increased the level of EAAT2 oligomers rather than inducing protein degradation through K542-specific ubiquitination. By using tissues from humans with TLE and epilepsy model mice, we observed that AMFR and EAAT2 oligomer levels were simultaneously decreased in the hippocampus. Screening of 2386 FDA-approved drugs revealed that the most common analgesic/antipyretic medicine, acetaminophen (APAP), can induce AMFR transcriptional activation via transcription factor SP1. Administration of APAP protected against pentylenetetrazol-induced epileptogenesis. In mice with chronic epilepsy, APAP treatment partially reduced the occurrence of spontaneous seizures and greatly enhanced the antiepileptic effects of 17AAG, an Hsp90 inhibitor that upregulates total EAAT2 levels, when the 2 compounds were administered together. In summary, our studies reveal an essential role for AMFR in regulating the oligomeric state of EAAT2 and suggest that APAP can improve the efficacy of EAAT2-targeted antiepileptic treatments.
- Published
- 2022
8. RETREG1/FAM134B mediated autophagosomal degradation of AMFR/GP78 and OPA1 —a dual organellar turnover mechanism
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Rukmini Mukherjee, Subhrangshu Das, Swadhin Chandra Jana, Manindra Bera, Debdatto Mookherjee, Oishee Chakrabarti, and Saikat Chakrabarti
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0301 basic medicine ,Mitochondrion ,Biology ,Real-Time Polymerase Chain Reaction ,GTP Phosphohydrolases ,03 medical and health sciences ,Microscopy, Electron, Transmission ,Cell Line, Tumor ,Chlorocebus aethiops ,Organelle ,Animals ,Humans ,Molecular Biology ,Microscopy, Confocal ,030102 biochemistry & molecular biology ,Mechanism (biology) ,Endoplasmic reticulum ,Autophagy ,Autophagosomes ,Intracellular Signaling Peptides and Proteins ,Membrane Proteins ,Cell Biology ,DUAL (cognitive architecture) ,Cell biology ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Mitoplast ,Gene Knockdown Techniques ,COS Cells ,Degradation (geology) ,Lysosomes ,HeLa Cells ,Research Paper - Abstract
Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate “mitoplasts”. This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The “mitoplasts” generated are degraded by reticulo-mito-phagy – simultaneously affecting dual organelle turnover. Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.
- Published
- 2020
9. Achieving pure spin effects by artifact suppression in methyl adiabatic relaxation experiments
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R. Andrew Byrd, Fa An Chao, and Domarin Khago
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Models, Molecular ,0301 basic medicine ,Protein Folding ,Allosteric modulator ,Protein Conformation ,Recombinant Fusion Proteins ,Allosteric regulation ,010402 general chemistry ,Proton decoupling ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Allosteric Regulation ,Point Mutation ,Adiabatic process ,Anisotropy ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Binding Sites ,Chemistry ,Decoupling (cosmology) ,Peptide Fragments ,0104 chemical sciences ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Chemical physics ,Ubiquitin-Conjugating Enzymes ,Artifact suppression ,Thermodynamics ,Protons ,Artifacts - Abstract
Recent methyl adiabatic relaxation dispersion experiments provide examination of conformational dynamics across a very wide timescale (10(2) – 10(5) sec(−1)) and, particularly, provide insight into the hydrophobic core of proteins and allosteric effects associated with modulators. The experiments require efficient decoupling of (1)H and (13)C spin interactions, and some artifacts have been discovered, which are associated with the design of the proton decoupling scheme. The experimental data suggest that the original design is valid; however, pulse sequences with either no proton decoupling or proton decoupling with imperfect pulses can potentially exhibit complications in the experiments. Here, we demonstrate that pulse imperfections in the proton decoupling scheme can be dramatically alleviated by using a single composite π pulse and provide pure single-exponential relaxation data. It allows the opportunity to access high-quality methyl adiabatic relaxation dispersion data by removing the cross-correlation between dipole-dipole interaction and chemical shift anisotropy. The resulting high-quality data is illustrated with the binding of an allosteric modulator (G2BR) to the ubiquitin conjugating enzyme Ube2g2.
- Published
- 2020
10. Chemokine CCL1 as a therapeutic target for pulmonary fibrosis: comments on ‘The chemokine CCL1 triggers an AMFR‒SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis’
- Author
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Shanshan, Liu, Chang, Liu, Zhuowei, Hu, and Yang, Xiao
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Receptors, Autocrine Motility Factor ,Chemokine CCL1 ,Pulmonary Fibrosis ,Genetics ,Humans ,Membrane Proteins ,Cell Biology ,General Medicine ,Fibroblasts ,Myofibroblasts ,Phosphoproteins ,Lung ,Molecular Biology - Published
- 2021
11. iASPP suppresses Gp78-mediated TMCO1 degradation to maintain Ca
- Author
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Shanliang, Zheng, Dong, Zhao, Guixue, Hou, Song, Zhao, Wenxin, Zhang, Xingwen, Wang, Li, Li, Liang, Lin, Tie-Shan, Tang, and Ying, Hu
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Drug Resistance ,Intracellular Signaling Peptides and Proteins ,Mice, Nude ,Apoptosis ,Endoplasmic Reticulum-Associated Degradation ,Endoplasmic Reticulum ,HCT116 Cells ,Receptors, Autocrine Motility Factor ,Repressor Proteins ,Mice ,Cell Line, Tumor ,Neoplasms ,Animals ,Homeostasis ,Humans ,Calcium ,Calcium Channels ,HT29 Cells ,Cell Proliferation - Abstract
Ca
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- 2021
12. Correction to: MiR-139-5p inhibits migration and invasion of colorectal cancer by downregulating AMFR and NOTCH1
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Yaling Hu, Chao Quan, Leyuan Zhou, Yuan Yin, Zhaohui Huang, Dong Hua, Jiwei Zhang, Weili Wang, Mingxu Song, Bojian Fei, Binbin Zhang, Qifeng Wang, Zehua Bian, Xiang Du, and Shujuan Ni
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Male ,Colorectal cancer ,Down-Regulation ,Mice, Nude ,Biology ,Biochemistry ,Mir 139 5p ,Text mining ,Cell Movement ,Cell Line, Tumor ,Sequence Homology, Nucleic Acid ,Drug Discovery ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Receptor, Notch1 ,Mice, Inbred BALB C ,Base Sequence ,business.industry ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Correction ,Cell Biology ,Middle Aged ,medicine.disease ,HCT116 Cells ,Survival Analysis ,Xenograft Model Antitumor Assays ,Human genetics ,Gene Expression Regulation, Neoplastic ,Receptors, Autocrine Motility Factor ,MicroRNAs ,HEK293 Cells ,Cancer research ,Female ,RNA Interference ,Stem cell ,business ,Colorectal Neoplasms ,Developmental biology ,Biotechnology - Abstract
MicroRNAs (miRNAs) that exert function by posttranscriptional suppression have recently brought insight in our understanding of the role of non-protein-coding RNAs in carcinogenesis and metastasis. In this study, we described the function and molecular mechanism of miR-139-5p in colorectal cancer (CRC) and its potential clinical application in CRC. We found that miR-139-5p was significantly downregulated in 73.8% CRC samples compared with adjacent noncancerous tissues (NCTs), and decreased miR-139-5p was associated with poor prognosis. Functional analyses demonstrated that ectopic expression of miR-139-5p suppressed CRC cell migration and invasion in vitro and metastasis in vivo. Mechanistic investigations revealed that miR-139-5p suppress CRC cell invasion and metastasis by targeting AMFR and NOTCH1. Knockdown of the two genes phenocopied the inhibitory effect of miR-139-5p on CRC metastasis. Furthermore, the protein levels of the two genes were upregulated in CRC samples compared with NCTs, and inversely correlated with the miR-139-5p expression. Increased NOTCH1 protein expression was correlated with poor prognosis of CRC patients. Together, our data indicate that miR-139-5p is a potential tumor suppressor and prognostic factor for CRC, and targeting miR-139-5p may repress the metastasis of CRC and improve survival.
- Published
- 2021
13. Induction via Functional Protein Stabilization of Hepatic Cytochromes P450 upon gp78/Autocrine Motility Factor Receptor (AMFR) Ubiquitin E3-Ligase Genetic Ablation in Mice: Therapeutic and Toxicological Relevance
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Peyton Jacob, Yi Liu, Maria Almira Correia, Doyoung Kwon, and Sung-Mi Kim
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Male ,0301 basic medicine ,Inbred C57BL ,Transgenic ,Mice ,0302 clinical medicine ,Cytochrome P-450 Enzyme System ,Ubiquitin ,Receptors ,2.1 Biological and endogenous factors ,Pharmacology & Pharmacy ,Aetiology ,Receptor ,Cells, Cultured ,Cancer ,Mice, Knockout ,Cytochrome P-450 Enzyme Inducers ,0303 health sciences ,Cultured ,Autocrine Motility Factor ,biology ,Protein Stability ,Chemistry ,Liver Disease ,Articles ,Pharmacology and Pharmaceutical Sciences ,Prodrug ,3. Good health ,Ubiquitin ligase ,Liver ,5.1 Pharmaceuticals ,030220 oncology & carcinogenesis ,Enzyme Induction ,Molecular Medicine ,Development of treatments and therapeutic interventions ,Cells ,Knockout ,Ubiquitin-Protein Ligases ,Mice, Transgenic ,Endoplasmic-reticulum-associated protein degradation ,03 medical and health sciences ,Downregulation and upregulation ,Animals ,030304 developmental biology ,Pharmacology ,Aspirin ,CYP3A4 ,Neurosciences ,Protein ubiquitination ,Mice, Inbred C57BL ,Receptors, Autocrine Motility Factor ,Tamoxifen ,030104 developmental biology ,Hepatocytes ,biology.protein ,Cancer research ,Biochemistry and Cell Biology ,Digestive Diseases ,Gene Deletion ,030217 neurology & neurosurgery - Abstract
3.AbstractThe hepatic endoplasmic reticulum (ER)-anchored monotopic proteins, cytochromes P450 (P450s) are enzymes that metabolize endobiotics (physiologically active steroids and fatty acids) as well as xenobiotics including therapeutic/chemotherapeutic drugs, nutrients, carcinogens and toxins. Alterations of hepatic P450 content through synthesis, inactivation or proteolytic turnover influence their metabolic function. P450 proteolytic turnover occurs via ER-associated degradation (ERAD) involving ubiquitin (Ub)-dependent proteasomal degradation (UPD) as a major pathway. UPD critically involves P450 protein ubiquitination by E2/E3 Ub-ligase complexes. We have previously identified the ER-polytopic gp78/AMFR (autocrine motility factor receptor) as a relevant E3 in CYP3A4, CYP3A23 and CYP2E1 UPD. We now document that liver-conditional genetic ablation of gp78/AMFR in mice disrupts P450 ERAD, resulting in significant stabilization of Cyp2a5 and Cyps 2c, in addition to that of Cyps 3a and Cyp2e1. More importantly, we establish that such stabilization is of the functionally active P450 proteins, leading to corresponding significant enhancement of their drug metabolizing capacities. Our findings with clinically relevant therapeutic drugs (nicotine, coumarin, chlorzoxazone, and acetaminophen) and the prodrug (tamoxifen) as P450 substrates, reveal that P450 ERAD disruption could influence therapeutic drug response and/or toxicity, warranting serious consideration as a potential source of clinically significant drug-drug interactions (DDIs). Because gp78/AMFR is not only an E3 Ub-ligase, but also a cell-surface prometastatic oncogene that is upregulated in various malignant cancers, our finding that hepatic gp78/AMFR-knockout can enhance P450-dependent bioactivation of relevant cancer chemotherapeutic prodrugs is of therapeutic relevance and noteworthy in prospective drug design and development.4.Significance StatementThe cell surface and ER transmembrane protein gp78/AMFR, a receptor for the prometastatic autocrine motility factor (AMF), as well as an E3 ubiquitin-ligase involved in the ERAD of not only the tumor metastatic suppressor KAI1, but also of hepatic cytochromes P450, is upregulated in various human cancers, enhancing their invasiveness, metastatic potential and poor prognosis. Liver specific gp78/AMFR genetic ablation results in functional protein stabilization of several hepatic P450s and consequently enhanced drug and prodrug metabolism, a feature that could be therapeutically exploited in the bioactivation of chemotherapeutic prodrugs, through design and development of novel short-term gp78/AMFR chemical inhibitors.
- Published
- 2019
14. Gpnmb secreted from liver promotes lipogenesis in white adipose tissue and aggravates obesity and insulin resistance
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Chang Xie, Ting Xiao, Jiqiu Wang, Jie Hong, Wei Qi, Jian Wei, Yun-Feng Li, Xiongjie Shi, Xue-Min Gong, Bo-Liang Li, Ju-Qiong Wang, Guang Ning, Bao-Liang Song, and Jie Luo
- Subjects
medicine.medical_specialty ,Adipose Tissue, White ,Endocrinology, Diabetes and Metabolism ,Adipose tissue ,White adipose tissue ,Mice ,chemistry.chemical_compound ,Insulin resistance ,Downregulation and upregulation ,Physiology (medical) ,Internal medicine ,Internal Medicine ,medicine ,Animals ,Homeostasis ,Obesity ,Eye Proteins ,Fatty acid synthesis ,Mice, Knockout ,Membrane Glycoproteins ,GPNMB ,Lipogenesis ,Intracellular Signaling Peptides and Proteins ,Membrane Proteins ,Lipid metabolism ,Cell Biology ,Lipid Metabolism ,medicine.disease ,Up-Regulation ,Receptors, Autocrine Motility Factor ,Endocrinology ,Liver ,chemistry ,Insulin Resistance - Abstract
Metabolism in mammals is regulated by complex interplay among different organs. Fatty acid synthesis is increased in white adipose tissue (WAT) when it is inhibited in the liver. Here we identify glycoprotein non-metastatic melanoma protein B (Gpnmb) as one liver-WAT cross-talk factor involved in lipogenesis. Inhibition of the hepatic sterol regulatory element-binding protein pathway leads to increased transcription of Gpnmb and promotes processing of the membrane protein to a secreted form. Gpnmb stimulates lipogenesis in WAT and exacerbates diet-induced obesity and insulin resistance. In humans, Gpnmb is tightly associated with body mass index and is a strong risk factor for obesity. Gpnmb inhibition by a neutralizing antibody or liver-specific knockdown improves metabolic parameters, including weight gain reduction and increased insulin sensitivity, probably by promoting the beiging of WAT. These results suggest that Gpnmb is a liver-secreted factor regulating lipogenesis in WAT, and that Gpnmb inhibition may provide a therapeutic strategy in obesity and diabetes.
- Published
- 2019
15. Competitive oxidation and ubiquitylation on the evolutionarily conserved cysteine confer tissue-specific stabilization of Insig-2
- Author
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Xiongjie Shi, Zhangsen Zhou, Mei-Xin Li, Huabin Zhao, Wei Qi, Bao-Liang Song, Jie Liu, Liping Chu, Jie Luo, Jingrong Liu, Jing-Ming Xia, and Hengwu Jiao
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0301 basic medicine ,Male ,Muscle Fibers, Skeletal ,General Physics and Astronomy ,Biochemistry ,Myoblasts ,Mice ,Ubiquitin ,Sequence Analysis, Protein ,Myocyte ,lcsh:Science ,Mice, Knockout ,Sterol Regulatory Element Binding Proteins ,Multidisciplinary ,biology ,Myogenesis ,Chemistry ,Fishes ,Cell biology ,Liver ,Lipogenesis ,C2C12 ,Oxidation-Reduction ,Evolution ,Science ,Down-Regulation ,CHO Cells ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,Amphibians ,Evolution, Molecular ,03 medical and health sciences ,Cricetulus ,Lipid biosynthesis ,Animals ,Humans ,Cysteine ,Muscle Cells ,030102 biochemistry & molecular biology ,Ubiquitination ,Membrane Proteins ,Lipid metabolism ,General Chemistry ,Lipid Metabolism ,Sterol regulatory element-binding protein ,Mice, Inbred C57BL ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,biology.protein ,Hepatocytes ,lcsh:Q ,Reactive Oxygen Species ,Transcriptome - Abstract
Insig-2 is an ER membrane protein negatively controlling lipid biosynthesis. Here, we find that Insig-2 is increased in the tissues, including liver, but unaltered in the muscle of gp78-deficient mice. In hepatocytes and undifferentiated C2C12 myoblasts, Insig-2 is ubiquitylated on Cys215 by gp78 and degraded. However, the C215 residue is oxidized by elevated reactive oxygen species (ROS) during C2C12 myoblasts differentiating into myotubes, preventing Insig-2 from ubiquitylation and degradation. The stabilized Insig-2 downregulates lipogenesis through inhibiting the SREBP pathway, helping to channel the carbon flux to ATP generation and protecting myotubes from lipid over-accumulation. Evolutionary analysis shows that the YECK (in which C represents Cys215 in human Insig-2) tetrapeptide sequence in Insig-2 is highly conserved in amniotes but not in aquatic amphibians and fishes, suggesting it may have been shaped by differential selection. Together, this study suggests that competitive oxidation-ubiquitylation on Cys215 of Insig-2 senses ROS and prevents muscle cells from lipid accumulation., The protein Insig-2 negatively regulates lipid biosynthesis and is short-lived in liver cells but stable in muscle cells. Here, the authors show that in muscle cells producing reactive oxygen species, there is increased oxidation compared to ubiquitination of Insig-2, stabilising Insig-2 and reducing lipid biosynthesis.
- Published
- 2020
16. Calmodulin regulates MGRN1‐GP78 interaction mediated ubiquitin proteasomal degradation system
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Samita Basu, Oishee Chakrabarti, Saikat Chakrabarti, Abhishek Sau, Rukmini Mukherjee, and Anshu Bhattacharya
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0301 basic medicine ,Proteasome Endopeptidase Complex ,Cell biology ,Calmodulin ,Ubiquitin-Protein Ligases ,Protein degradation ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,Ubiquitin ,Mitophagy ,Genetics ,Animals ,Humans ,Calcium Signaling ,Molecular Biology ,Internal medicine ,Neurons ,biology ,Proteasome ,Chemistry ,Endoplasmic reticulum ,Signal transducing adaptor protein ,Ubiquitin ligase ,Molecular Docking Simulation ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Proteolysis ,biology.protein ,Calcium ,030217 neurology & neurosurgery ,HeLa Cells ,Biotechnology - Abstract
The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.
- Published
- 2018
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17. POLE mutations improve the prognosis of endometrial cancer via regulating cellular metabolism through AMF/AMFR signal transduction
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Yiran Li, Yiding Bian, Xiaoping Wan, and Kai Wang
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DNA Replication ,0301 basic medicine ,lcsh:Internal medicine ,lcsh:QH426-470 ,Somatic cell ,Biology ,Disease-Free Survival ,03 medical and health sciences ,0302 clinical medicine ,Endometrial cancer ,Genetics ,medicine ,Humans ,Poly-ADP-Ribose Binding Proteins ,lcsh:RC31-1245 ,Gene ,Genetics (clinical) ,Exome sequencing ,Survival analysis ,Glucose metabolism ,Glucose-6-Phosphate Isomerase ,Cancer ,DNA Polymerase II ,Middle Aged ,Prognosis ,medicine.disease ,Human genetics ,Endometrial Neoplasms ,Receptors, Autocrine Motility Factor ,lcsh:Genetics ,Glucose ,Polymerase ɛ (POLE) mutation ,030104 developmental biology ,Autocrine motility factor (AMF) ,030220 oncology & carcinogenesis ,Mutation ,Cancer research ,Female ,Cytokine secretion ,Research Article ,Signal Transduction - Abstract
Background The morbidity and mortality of endometrial tumors, a common type of malignant cancer in women, have increased in recent years. POLE encodes the DNA polymerase ε, which is responsible for the leading strand DNA replication. Somatic mutations of POLE have been acknowledged in numerous cancers, resulting in the accumulation of DNA errors, leading to ultra-mutated tumors. Mutations in the exonuclease domain of POLE have been reported to improve progression-free survival in endometrial cancer. However, the potential relationship and underlying mechanism between POLE mutations and the prognosis of endometrial cancer patients remains unclear. Methods The whole exome sequencing data, RNA sequencing data, and clinical information were obtained from the TCGA database and employed for the analyses in this study. The detailed mutational information was analyzed using whole exome sequencing data and the mutated genes were shown with OncoPlot. The survival curves and cox proportional hazards regression analysis were used to accessed patient prognosis, the association of clinical characteristics and prognosis. Differentially expressed genes were analyzed by the edgeR R/Bioconductor package, then the GSEA Pre-ranked tool was used for Gene Set Enrichment Analysis (GSEA) to estimate the function of genes. Expression values were clustered using hierarchical clustering with Euclidean distance and ward linkage by the dendextend R package. Results POLE mutational status was proven to be an independent prognostic factor for endometrial cancer patients. Patients with somatic POLE mutations presented a favorable prognosis. POLE mutations regulated glycolysis and cytokine secretion, affecting cell metabolism and immune response. Autocrine motility factor (AMF)/PGI and AMFR/gp78 exhibited higher expression levels in POLE mutant patients. The comprehensive high expressions of AMFR/gp78 and low expression of POLE were associated with the favorable prognosis of endometrial cancer patients. Conclusions This study showed the POLE mutations a vital factor in endometrial cancer patients, leading to a higher expression of AMF/PGI and AMFR/gp78. These results suggested comprehensive consideration of the POLE mutations, expression of AMF/PGI and AMFR/gp78 may provide a more feasible and effective approach for the treatment of endometrial cancer, which might improve the prognosis.
- Published
- 2019
18. A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum–associated degradation
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Zai-Rong Zhang, Jiqiang Li, Linhan Wang, Jia Ji, Xianyan Hu, Junfen Shi, and Yuan Guo
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Leupeptins ,Ubiquitin-Protein Ligases ,Human Immunodeficiency Virus Proteins ,macromolecular substances ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Humans ,Viral Regulatory and Accessory Proteins ,Molecular Biology ,Er associated degradation ,Protein Unfolding ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Endoplasmic reticulum ,Substrate (chemistry) ,Cell Biology ,Endoplasmic Reticulum-Associated Degradation ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Membrane ,HEK293 Cells ,Membrane protein ,biology.protein ,Biophysics ,Trimetrexate ,Protein folding - Abstract
The endoplasmic reticulum–associated degradation (ERAD) pathway mediates the endoplasmic reticulum–to–cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K–resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.
- Published
- 2019
19. Ubc7/Ube2g2 ortholog in Entamoeba histolytica: connection with the plasma membrane and phagocytosis
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Rinki Kumari, Preeti Gupta, and Swati Tiwari
- Subjects
0301 basic medicine ,Ubiquitin-Protein Ligases ,Protein Serine-Threonine Kinases ,Biology ,Ubiquitin-conjugating enzyme ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,03 medical and health sciences ,chemistry.chemical_compound ,Phagocytosis ,Endoribonucleases ,Animals ,Humans ,ERAD pathway ,Phagosome ,General Veterinary ,Ubiquitin ,Tunicamycin ,Endoplasmic reticulum ,Cell Membrane ,Entamoeba histolytica ,Endoplasmic Reticulum-Associated Degradation ,General Medicine ,Cell biology ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Infectious Diseases ,Secretory protein ,chemistry ,Insect Science ,Ubiquitin-Conjugating Enzymes ,Unfolded Protein Response ,Unfolded protein response ,Parasitology ,Calreticulin - Abstract
Endoplasmic reticulum (ER)-associated degradation (ERAD) and unfolded protein response (UPR) pathways are important for quality and quantity control of membrane and secretory proteins. We have identified orthologs of ER-associated ubiquitin conjugating enzymes (E2s) Ubc6/Ube2j2 and Ubc7/Ube2g2, ubiquitin ligases (E3) Hrd1 and GP78/AMFR, and sensor of UPR, Ire1 in E. histolytica that show conservation of important features of these proteins. Biochemical characterization of the ortholog of ERAD E2, Ubc7/Ube2g2 (termed as EhUbc7), was carried out. This E2 was transcriptionally upregulated several folds upon induction of UPR with tunicamycin. Ire1 ortholog was also upregulated upon UPR induction suggesting a linked UPR and ERAD pathway in this organism. EhUbc7 showed enzymatic activity and, similar to its orthologs in higher eukaryotes, formed polyubiquitin chains in vitro and localized to both cytoplasm and membranes. However, unlike its ortholog in higher eukaryotes, it also showed localization to the plasma membrane along with calreticulin. Inactivation of EhUbc7 significantly inhibited erythrophagocytosis, suggesting a novel function that has not been reported before for this E2. No change in growth, motility, or cell-surface expression of Gal/GalNAC lectin was observed due to inactivation of EhUbc7. The protein was present in the phagocytic cups but not in the phagosomes. A significant decrease in the number of phagocytic cups in inactive EhUbc7 expressing cells was observed, suggesting altered kinetics of phagocytosis. These findings have implications for evolutionary and mechanistic understanding of connection between phagocytosis and ER-associated proteins.
- Published
- 2018
20. Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductase
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Wei Jiang, Yan Ni Xiong, Na Tian, Jie Liu, Kai Yue Wu, Jian Wei, Jie Luo, Lu Yi Jiang, Bao-Liang Song, and Xiongjie Shi
- Subjects
0301 basic medicine ,CHO Cells ,Reductase ,Endoplasmic Reticulum ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Small hairpin RNA ,03 medical and health sciences ,Cricetulus ,Cricetinae ,Animals ,Humans ,Molecular Biology ,biology ,Ubiquitin ,Chemistry ,Endoplasmic reticulum ,Chinese hamster ovary cell ,Intracellular Signaling Peptides and Proteins ,Ubiquitination ,Membrane Proteins ,Cell Biology ,Lipids ,Sterol ,Ubiquitin ligase ,Cell biology ,Receptors, Autocrine Motility Factor ,RING finger domain ,Sterols ,030104 developmental biology ,Proteolysis ,HMG-CoA reductase ,biology.protein ,Hydroxymethylglutaryl CoA Reductases ,lipids (amino acids, peptides, and proteins) - Abstract
Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane–associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA–based screening targeting endoplasmic reticulum–localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.
- Published
- 2018
21. Role of HSPA1L as a cellular prion protein stabilizer in tumor progression via HIF-1α/GP78 axis
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Chul Won Yun, Dongjun Jeong, Sang-Cheol Lee, Hyog Young Kwon, SangMin Kim, Jun Hee Lee, Moo-Jun Baek, Hyunjoo Lee, Ho Jae Han, Sei-Jung Lee, Yeo Min Yoon, Yong-Seok Han, and Seungpil Yun
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Cancer Research ,Carcinogenesis ,animal diseases ,Cell Culture Techniques ,Biology ,Prion Proteins ,03 medical and health sciences ,Ubiquitin ,RNA interference ,mental disorders ,Genetics ,Humans ,HSP70 Heat-Shock Proteins ,Molecular Targeted Therapy ,HSPA1L ,RNA, Small Interfering ,Receptor ,Molecular Biology ,Gene knockdown ,Ubiquitination ,Flow Cytometry ,Hypoxia-Inducible Factor 1, alpha Subunit ,nervous system diseases ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Tumor progression ,Gene Knockdown Techniques ,Proteolysis ,Disease Progression ,biology.protein ,Cancer research ,RNA Interference ,Signal transduction ,Colorectal Neoplasms ,HT29 Cells ,Signal Transduction - Abstract
The cellular prion protein (PrPC) is associated with metastasis, tumor progression and recurrence; however, the precise mechanisms underlying its action is not well understood. Our study found that PrPC degradation decreased tumor progression in colorectal cancer (CRC). In a CRC cell line and human CRC tissue exposed to hypoxia, induced heat-shock 70-kDa protein-1-like (HSPA1L) expression stabilized hypoxia-inducible factor-1α (HIF-1α) protein and promoted PrPC accumulation and tumorigenicity in vivo. PrPC was degraded via the proteasome pathway mediated by the ubiquitin-protein E3 ligase glycoprotein 78 (GP78), which interacts directly with PrPC. However, hypoxia-induced HSPA1L interacted with GP78 and inhibited its functions. HSPA1L knockdown facilitated the interaction of GP78 and PrPC, thereby increasing PrPC ubiquitination. Thus, GP78 was identified as the ubiquitinase for PrPC, thereby revealing an essential mechanism that controls PrPC levels in CRC. Our results suggest that the HSPA1L/HIF-1α/GP78 axis has a crucial role in PrPC accumulation during tumor progression.
- Published
- 2017
22. Role of glycoprotein 78 and cidec in hepatic steatosis
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Jie Li, Zhiwen Zhang, Guocai Liu, Feng Zhang, Qing Li, and Yuqiao Xu
- Subjects
0301 basic medicine ,Cancer Research ,Cell ,Biology ,Biochemistry ,Cell Line ,cell death-inducing DFFA-like effector c ,lipids ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Lipid droplet ,Nonalcoholic fatty liver disease ,steatosis ,Genetics ,medicine ,Animals ,Oil Red O ,Molecular Biology ,glycoprotein 78 ,Proteins ,Articles ,Cell cycle ,medicine.disease ,Cell biology ,Ubiquitin ligase ,Fatty Liver ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,chemistry ,hepatic cell ,Cell culture ,Gene Knockdown Techniques ,Hepatocytes ,biology.protein ,Cancer research ,Molecular Medicine ,Steatosis - Abstract
Hepatic glycoprotein (gp78), a membrane-anchored E3 ubiquitin ligase, has been reported to be involved in regulating lipid and energy metabolism in animals, and cell death-inducing DFFA-like effector c (cidec) has emerged as an important regulator of metabolism, which has been implicated in the process of fat differentiation. Nonalcoholic fatty liver disease is a metabolic disorder associated with hepatic steatosis. In the present study, to investigate the role of gp78 and cidec in hepatic steatosis, an in vitro cell culture model of hepatic steatosis was established, using the AML12 mouse hepatocyte cell line to assess the protein expression of gp78. The results of Oil Red O staining, phase contrast microscopy and triglyceride content detection experiments indicated that the overexpression of gp78 induced lipid accumulation, whereas gp78-knockdown led to a reduction in lipid accumulation in the AML12 cells. The increased expression of gp78 was associated with steatosis. The expression of cidec was consistent with gp78, and the colocalization of gp78 and cidec was observed on the surface of lipid droplets using immunofluorescence analysis. Furthermore, an interaction between gp78 and cidec was detected using coimmunoprecipitation analysis, and this interaction promoted lipid accumulation. Based on these data, it was hypothesized that gp78 is a regulator of hepatic steatosis, and that it may be a putative molecular mediator in metabolic diseases.
- Published
- 2017
23. GP78 Cooperates with Dual-Specificity Phosphatase 1 To Stimulate Epidermal Growth Factor Receptor-Mediated Extracellular Signal-Regulated Kinase Signaling
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Madhumita Chatterjee, Andreas Vogt, Gen Sheng Wu, Dhong Hyo Kho, Avraham Raz, and Mohammed Hafiz Uddin
- Subjects
MAPK/ERK pathway ,Autocrine Motility Factor ,Carcinoma, Hepatocellular ,MAP Kinase Signaling System ,Biology ,Receptor tyrosine kinase ,03 medical and health sciences ,0302 clinical medicine ,Cell Movement ,Cell Line, Tumor ,Humans ,Neoplasm Invasiveness ,Epidermal growth factor receptor ,Phosphorylation ,Molecular Biology ,Cell Proliferation ,030304 developmental biology ,0303 health sciences ,Epidermal Growth Factor ,Kinase ,Liver Neoplasms ,Ubiquitination ,Dual Specificity Phosphatase 1 ,Cell Biology ,Ubiquitin ligase ,Cell biology ,ErbB Receptors ,Receptors, Autocrine Motility Factor ,HEK293 Cells ,030220 oncology & carcinogenesis ,Proteolysis ,biology.protein ,Signal transduction ,Research Article - Abstract
GP78 is an autocrine motility factor (AMF) receptor (AMFR) with E3 ubiquitin ligase activity that plays a significant role in tumor cell proliferation, motility, and metastasis. Aberrant extracellular signal-regulated kinase (ERK) activation via receptor tyrosine kinases promotes tumor proliferation and invasion. The activation of GP78 leads to ERK activation, but its underlying mechanism is not fully understood. Here, we show that GP78 is required for epidermal growth factor receptor (EGFR)-mediated ERK activation. On one hand, GP78 interacts with and promotes the ubiquitination and subsequent degradation of dual-specificity phosphatase 1 (DUSP1), an endogenous negative regulator of mitogen-activated protein kinases (MAPKs), resulting in ERK activation. On the other hand, GP78 maintains the activation status of EGFR, as evidenced by the fact that EGF fails to induce EGFR phosphorylation in GP78-deficient cells. By the regulation of both EGFR and ERK activation, GP78 promotes cell proliferation, motility, and invasion. Therefore, this study identifies a previously unknown signaling pathway by which GP78 stimulates ERK activation via DUSP1 degradation to mediate EGFR-dependent cancer cell proliferation and invasion.
- Published
- 2019
24. Autocrine motility factor promotes endometrial cancer progression by targeting GPER-1
- Author
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Kai Wang, Junjie Qu, Yuanhui Jia, Yiran Li, Xiaoping Wan, Huan Tong, and Yiding Bian
- Subjects
Autocrine Motility Factor ,Carcinogenesis ,Proliferation ,lcsh:Medicine ,G protein-coupled estrogen receptor 1 (GPER-1) ,Mice, Nude ,Context (language use) ,Apoptosis ,Biology ,Biochemistry ,Models, Biological ,Endometrial Cancer ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,PI3K signaling pathway ,Mice ,Phosphatidylinositol 3-Kinases ,Cell Line, Tumor ,Animals ,Humans ,Gene Silencing ,lcsh:QH573-671 ,Receptor ,Molecular Biology ,Cell Proliferation ,0303 health sciences ,Mice, Inbred BALB C ,Cell growth ,lcsh:Cytology ,Research ,030302 biochemistry & molecular biology ,lcsh:R ,fungi ,Glucose-6-Phosphate Isomerase ,Cell Biology ,Cell biology ,Endometrial Neoplasms ,Receptors, Autocrine Motility Factor ,Autocrine motility factor (AMF) ,Receptors, Estrogen ,Cell culture ,Disease Progression ,Female ,Signal transduction ,GPER ,Protein Binding ,Signal Transduction - Abstract
Background Autocrine motility factor (AMF) is a critical factor regulating aggressiveness of endometrial cancer (EC). Multiple pieces of evidence indicate that it is through G protein coupled estrogen receptor (GPER) signaling pathway that some growth factors promoted the migration and proliferation of tumor cells. The aim of this study is to explore the role of GPER-1 in AMF mediated regulatory mechanisms of EC recurrence and progression. Methods Real-Time Cell Analysis (RTCA) assays were performed to assess whether AMF depends on Autocrine motility factor recepter (AMFR) signaling in EC cells. A genome-wide expression microarray and Yeast Two-Hybrid assay were used to detect AMF and GPER-1 interaction in the context of AMFR depletion, and co-immunoprecipitation and immunofluorescence experiments were performed to confirm the physical interaction. Isobaric Tags for Relative and Absolute Quantification (iTRAQ) analysis was used for the identification of the target pathway activated by AMF-GPER-1 interaction. Cohorts of mice harboring xenografts derived from modified SPEC2 cell lines were treated with or without exogenous AMF to validate the results of previous experiments. Immunohistochemistry was performed to assess AMF and GPER-1 expression in endometrial cancer specimens and normal endometrium. Results Our data showed that GPER-1 binds to AMF and the formed complex translocates from the plasma membrane to the cytoplasm. Mechanistic investigations demonstrated that interaction between AMF and GPER-1 triggers phosphoinositide-3-kinase signaling and promotes EC cell growth. More importantly, through animal experiments and human tissue experiments, we found that AMF contributes to GPER-1-mediated EC progression, which is consistent with the above observations. Conclusions Our work not only delineated the regulatory mechanisms of endometrial cancer progression by AMF-GPER-1-AKT signaling cascade but also laid the foundation of targeting this pathway for treating endometrial cancer. Electronic supplementary material The online version of this article (10.1186/s12964-019-0336-4) contains supplementary material, which is available to authorized users.
- Published
- 2019
25. PINK1 Content in Mitochondria is Regulated by ER-Associated Degradation
- Author
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Serge Przedborski, Hediye Erdjument-Bromage, Yuhui Liu, Xuejun Jiang, Knut H. Lauritzen, Brittany Martin, Theresa C. Swayne, and Cristina Guardia-Laguarta
- Subjects
Proteasome Endopeptidase Complex ,Ubiquitin-Protein Ligases ,Cell ,PINK1 ,Endoplasmic-reticulum-associated protein degradation ,Mitochondrion ,Endoplasmic Reticulum ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Ubiquitin ,Valosin Containing Protein ,Cell Line, Tumor ,Mitophagy ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Research Articles ,030304 developmental biology ,Membrane potential ,0303 health sciences ,biology ,Chemistry ,General Neuroscience ,Intracellular Signaling Peptides and Proteins ,Ubiquitination ,Cell biology ,Mitochondria ,Receptors, Autocrine Motility Factor ,medicine.anatomical_structure ,HEK293 Cells ,Proteasome ,COS Cells ,Proteolysis ,biology.protein ,Protein Kinases ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
Maintaining a pool of functional mitochondria requires degradation of damaged ones within the cell. PINK1 is critical in this quality-control process: loss of mitochondrial membrane potential causes PINK1 to accumulate on the mitochondrial surface, triggering mitophagy. However, little is known about how PINK1 is regulated. Recently, we showed that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation of its mature form via a mechanism inconsistent with the proposed N-end rule process. Using both human female and monkey cell lines, we now demonstrate that once generated within the mitochondria, 52 kDa PINK1 adopts a mitochondrial topology most consistent with it being at the mitochondrial–endoplasmic reticulum (ER) interface. From this particular submitochondrial location, PINK1 interacts with components of the ER-associated degradation pathway, such as the E3 ligases gp78 and HRD1, which cooperate to catalyze PINK1 ubiquitination. The valosin-containing protein and its cofactor, UFD1, then target ubiquitinated PINK1 for proteasomal degradation. Our data show that PINK1 in healthy mitochondria is negatively regulated via an interplay between mitochondria and ER, and shed light on how this mitochondrial protein gains access to the proteasome. SIGNIFICANCE STATEMENT Regulation of mitochondrial content of PINK1, a contributor to mitophagy, is an important area of research. Recently, we found that PINK1 content is kept low in healthy mitochondria by continuous ubiquitination and proteasomal degradation. We now extend and refine this novel finding by showing that PINK1 localizes at the mitochondrial–endoplasmic reticulum (ER) interface, from where it interacts with the ER-associated degradation machinery, which catalyzes its ubiquitination and transfer to the proteasome. Thus, these data show that PINK1 in healthy mitochondria is negatively regulated via a mitochondria and ER interplay, and how this mitochondrial protein gains access to the proteasome.
- Published
- 2019
26. The ubiquitin specific protease USP34 protects the ubiquitin ligase gp78 from proteasomal degradation
- Author
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Dhonghyo Kho, Donghong Ju, Avraham Raz, Li Li, Hui Wang, Fei Sun, Youming Xie, and Huanjie Yang
- Subjects
0301 basic medicine ,Proteasome Endopeptidase Complex ,Biophysics ,Protein degradation ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Lipid droplet ,Cell Line, Tumor ,Humans ,RNA, Small Interfering ,Molecular Biology ,chemistry.chemical_classification ,DNA ligase ,biology ,Chemistry ,Endoplasmic reticulum ,Ubiquitination ,Cell Biology ,Endoplasmic Reticulum-Associated Degradation ,Lipid Droplets ,Lipid Metabolism ,Ubiquitin ligase ,Cell biology ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,HEK293 Cells ,030220 oncology & carcinogenesis ,Proteolysis ,biology.protein ,Hepatocytes ,Ubiquitin-Specific Proteases ,Biogenesis ,Protein Binding ,Signal Transduction - Abstract
The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.
- Published
- 2018
27. The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1
- Author
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Dick J. H. van den Boomen, James A. Nathan, Anna S Dickson, Richard T. Timms, Paul J. Lehner, Norbert Volkmar, Sam A. Menzies, Volkmar, Norbert [0000-0003-0766-5606], Nathan, James A [0000-0002-0248-1632], Lehner, Paul J [0000-0001-9383-1054], and Apollo - University of Cambridge Repository
- Subjects
RNF145 ,QH301-705.5 ,Ubiquitin-Protein Ligases ,Science ,Reductase ,Research Communication ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,gp78 ,0302 clinical medicine ,Ubiquitin ,cell biology ,Animals ,Humans ,human ,Biology (General) ,030304 developmental biology ,chemistry.chemical_classification ,HMGCR ,0303 health sciences ,DNA ligase ,biology ,Cholesterol ,Ubiquitination ,Membrane Proteins ,cholesterol ,Sterol ,3. Good health ,Cell biology ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,ER associated degradation ,Enzyme ,chemistry ,E3 ubiquitin ligase ,Proteolysis ,Ubiquitin-Conjugating Enzymes ,HMG-CoA reductase ,biology.protein ,Medicine ,Hydroxymethylglutaryl CoA Reductases ,lipids (amino acids, peptides, and proteins) ,CRISPR-Cas Systems ,030217 neurology & neurosurgery - Abstract
Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., eLife digest Cholesterol is a fatty molecule that is essential for our health; for example, it is a component of the outer membrane that surrounds every cell in our body. Yet, it also has a reputation for clogging arteries and causing heart attacks and strokes. Our organism can adjust the amount of cholesterol it creates through an enzyme called HMGCR, which is found in all cells. Switching off HMGCR, for instance by taking drugs called statins, reduces the amount of cholesterol made by cells. To regulate the activity of HMGCR, the body uses proteins known as E3 ubiquitin ligases, which can label the enzyme for destruction. However, the identity of the ligases that target HMGCR is a matter of intense debate. Here, Menzies, Volkmar et al. addressed this issue by using an approach called a genome-wide CRISPR forward genetic screen. First, HMGCR was marked inside the cells with a fluorescent tag to watch how its levels change in response to different amounts of cholesterol. Then, each gene in the cell was deleted, and the effects recorded. This allowed Menzies, Volkmar et al. to find the genes responsible for the rapid destruction of HMGCR. The experiments revealed that the E3 ubiquitin ligases RNF145 and gp78 are independently responsible for the degradation of the majority of HMGCR, with a third ligase, Hrd1, getting involved if the first two are absent. In particular, RNF145 builds up when a cell is starved of cholesterol, but it immediately marks HMGCR for destruction once cholesterol becomes more abundant. This ligase can therefore both sense and respond to the amount of cholesterol in a cell, making it a perfect candidate for regulating HMGCR based on what the body needs. Identifying the proteins that adjust the levels of HMGCR sheds light on how a cell controls the amount of cholesterol it creates. This knowledge could be relevant in the fight against the health problems associated with this molecule.
- Published
- 2018
28. Lunapark Is a Component of a Ubiquitin Ligase Complex Localized to the Endoplasmic Reticulum Three-way Junctions
- Author
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Yihong Ye, Huanhuan Huo, Yanfen Liu, Ting Zhang, and Yupeng Zhao
- Subjects
0301 basic medicine ,Atlastin ,Amino Acid Motifs ,Biology ,Endoplasmic Reticulum ,Biochemistry ,03 medical and health sciences ,Ubiquitin ,GTP-Binding Proteins ,Chlorocebus aethiops ,Animals ,Humans ,Small GTPase ,Molecular Biology ,Homeodomain Proteins ,chemistry.chemical_classification ,Endoplasmic reticulum ,Membrane Proteins ,Cell Biology ,Amino acid ,Cell biology ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,Protein Transport ,030104 developmental biology ,chemistry ,Protein Synthesis and Degradation ,Ubiquitin ligase complex ,COS Cells ,biology.protein ,Protein folding ,HeLa Cells - Abstract
The endoplasmic reticulum (ER) network comprises sheets and tubules that are connected by dynamic three-way junctions. Lunapark (Lnp) localizes to and stabilizes ER three-way junctions by antagonizing the small GTPase Atlastin, but how Lnp shapes the ER network is unclear. Here, we used an affinity purification approach and mass spectrometry to identify Lnp as an interacting partner of the ER protein quality control ubiquitin ligase gp78. Accordingly, Lnp purified from mammalian cells has a ubiquitin ligase activity in vitro. Intriguingly, biochemical analyses show that this activity can be attributed not only to associated ubiquitin ligase, but also to an intrinsic ubiquitin ligase activity borne by Lnp itself. This activity is contained in the N-terminal 45 amino acids of Lnp although this segment does not share homology to any known ubiquitin ligase motifs. Despite its interaction with gp78, Lnp does not seem to have a broad function in degradation of misfolded ER proteins. On the other hand, the N-terminal ubiquitin ligase-bearing motif is required for the ER three-way junction localization of Lnp. Our study identifies a new type of ubiquitin ligase and reveals a potential link between ubiquitin and ER morphology regulation.
- Published
- 2016
29. Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame
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YongQiang Wang, Sung-Mi Kim, Noushin Nabavi, Maria Almira Correia, and Yi Liu
- Subjects
p97 ,0301 basic medicine ,gp78 E3 ,0302 clinical medicine ,Cytochrome P-450 Enzyme System ,Ubiquitin ,Models ,Receptors ,Pharmacology (medical) ,Protein phosphorylation ,Pharmacology & Pharmacy ,General Pharmacology, Toxicology and Pharmaceutics ,26S proteasome ,Autocrine Motility Factor ,medicine.diagnostic_test ,CHIP ,Liver Disease ,p62 ,Endoplasmic Reticulum-Associated Degradation ,Pharmacology and Pharmaceutical Sciences ,K48 & K63 ubiquitination ,Cell biology ,Liver ,Biochemistry ,Generic Health Relevance ,030220 oncology & carcinogenesis ,Phosphorylation ,Cytochromes P450 ,autophagic lysosomal degradation ,phosphodegrons ,Ubiquitin-Protein Ligases ,Proteolysis ,macromolecular substances ,Endoplasmic-reticulum-associated protein degradation ,Biology ,Models, Biological ,Article ,03 medical and health sciences ,E2/E3 ubiquitin ligases ,medicine ,Animals ,Humans ,Endoplasmic reticulum ,Ubiquitination ,ERAD ,Biological ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Proteasome ,biology.protein ,Lysosomes ,Digestive Diseases - Abstract
The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or “conformational” phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural “LIR” motifs, and selective cellular “cargo receptors” as plausible P450-ALD determinants.
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- 2016
30. The linkage specificity determination of Ube2g2-gp78 mediated polyubiquitination
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Weixiao Liu, Wei Li, Yongliang Shang, and Fengyi Gao
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0301 basic medicine ,Biophysics ,macromolecular substances ,Plasma protein binding ,Computational biology ,Linkage (mechanical) ,Ubiquitin-conjugating enzyme ,environment and public health ,Biochemistry ,law.invention ,Structure-Activity Relationship ,03 medical and health sciences ,Enzyme activator ,Ubiquitin ,law ,Structure–activity relationship ,Binding site ,Polyubiquitin ,Molecular Biology ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,Cell Biology ,Enzyme Activation ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Amino Acid Substitution ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Proofreading ,Protein Binding - Abstract
Polyubiquitin chain linkage specificity or topology is essential for its role in diverse cellular processes. Previous studies pay more attentions to the linkage specificity of the first ubiquitin moieties, whereas, little is known about the editing mechanism of linkage specificity in longer polyubiquitin chains. gp78 and its cognate E2-Ube2g2 catalyze lysine48 (K48)-linked polyubiquitin chains to promote the degradation of targeted proteins. Here, we show that the linkage specificity of the entire polyubiquitin chain is determined by the conjugation manner of the first ubiquitin molecule but not the following ones. Further study discovered that the gp78 CUE domain works as a proofreading machine during the growth of K48-linked polyubiquitin chains to ensure the linkage specificity. Together, our studies uncover a novel mechanism underlying the linkage specificity determination of longer polyubiquitin chains.
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- 2016
31. Canalicular membrane MRP2/ABCC2 internalization is determined by Ezrin Thr567 phosphorylation in human obstructive cholestasis
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Sheng Chen, Wei Lian, Xiaochong He, Lei Chen, Huaizhi Wang, Liangjun Zhang, Ying Cheng, James L. Boyer, Jin Chai, Shi-Ying Cai, Rongquan Wang, Xiaocong Liu, Xinchan Feng, Wensheng Chen, and Yu He
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Adult ,Male ,Threonine ,media_common.quotation_subject ,Gallstones ,macromolecular substances ,Biology ,Models, Biological ,Ezrin ,Cholestasis ,Radixin ,medicine ,Humans ,RNA, Messenger ,Phosphorylation ,Internalization ,Protein Kinase C ,Protein kinase C ,media_common ,Hepatology ,Multidrug resistance-associated protein 2 ,Bile Canaliculi ,Membrane Proteins ,Hep G2 Cells ,Middle Aged ,Jaundice ,medicine.disease ,Molecular biology ,Multidrug Resistance-Associated Protein 2 ,Receptors, Autocrine Motility Factor ,Cytoskeletal Proteins ,Liver ,Case-Control Studies ,Gene Knockdown Techniques ,Female ,Multidrug Resistance-Associated Proteins ,medicine.symptom - Abstract
Background & Aims Multidrug resistance-associated protein 2 (MRP2) excretes conjugated organic anions including bilirubin and bile acids. Malfunction of MRP2 leads to jaundice in patients. Studies in rodents indicate that Radixin plays a critical role in determining Mrp2 canalicular membrane expression. However, it is not known how human hepatic MRP2 expression is regulated in cholestasis. Methods We assessed liver MRP2 expression in patients with obstructive cholestasis caused by gallstone blockage of bile ducts, and investigated the regulatory mechanism in HepG2 cells. Results Western blot detected that liver MRP2 protein expression in obstructive cholestatic patients (n=30) was significantly reduced to 25% of the non-cholestatic controls (n=23). Immunoprecipitation identified Ezrin but not Radixin associating with MRP2 in human livers, and the increased amount of phospho-Ezrin Thr567 was positively correlated with the amount of co-precipitated MRP2 in cholestatic livers, whereas Ezrin and Radixin total protein levels were unchanged in cholestasis. Further detailed studies indicate that Ezrin Thr567 phosphorylation plays an important role in MRP2 internalization in HepG2 cells. Since increased expression of PKCα, δ and e were detected in these cholestatic livers, we further confirmed that these PKCs stimulated Ezrin phosphorylation and reduced MRP2 membrane expression in HepG2 cells. Finally, we identified GP78 as the key ubiquitin ligase E3 involved in MRP2 proteasome degradation. Conclusions Activation of liver PKCs during cholestasis leads to Ezrin Thr567 phosphorylation resulting in MRP2 internalization and degradation where ubiquitin ligase E3 GP78 is involved. This process provides a mechanistic explanation for jaundice seen in patients with obstructive cholestasis.
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- 2015
32. gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum
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Yanfen Liu, Yihong Ye, Ting Zhang, and Yue Xu
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Protein Folding ,Ubiquitin-Protein Ligases ,macromolecular substances ,Plasma protein binding ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Ubiquitin ,Humans ,Molecular Biology ,biology ,Endoplasmic reticulum ,Ubiquitination ,Articles ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,Ubiquitin ligase ,Cell biology ,Receptors, Autocrine Motility Factor ,HEK293 Cells ,Biochemistry ,Membrane Trafficking ,Ubiquitin ligase complex ,Proteolysis ,biology.protein ,Chaperone complex ,Molecular Chaperones ,Protein Binding - Abstract
The functional relationship between mammalian ubiquitin ligase gp78 and Hrd1 was studied. Hrd1 is one of the essential retrotranslocation regulators conserved in yeast and mammalian cells, whereas gp78 serves an assisting role downstream of Hrd1 and possibly other ubiquitin ligases in mammalian cells., Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA–mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.
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- 2015
33. p38 MAP kinase–dependent phosphorylation of the Gp78 E3 ubiquitin ligase controls ER–mitochondria association and mitochondria motility
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Bharat H. Joshi, Leonard J. Foster, Ivan R. Nabi, Lei Li, Jay Shankar, and Guang Gao
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Consensus site ,Ubiquitin-Protein Ligases ,MFN2 ,Mitochondrion ,Endoplasmic Reticulum ,p38 Mitogen-Activated Protein Kinases ,Cell Line ,Mitochondrial Proteins ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Animals ,Humans ,Phosphorylation ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,Ubiquitination ,Articles ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,Molecular biology ,Mitochondria ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,mitochondrial fusion ,Membrane Trafficking ,030220 oncology & carcinogenesis ,biology.protein ,Mitochondrial fission - Abstract
Epitope mapping of the 3F3A mAb identified p38 MAPK phosphorylation of Ser-538 of the E3 ubiquitin ligase Gp78. p38 MAPK phosphorylation of Ser-538 prevents Gp78-dependent mitofusin degradation, mitochondrial fission, and ER–mitochondria association, defining a novel regulatory mechanism of Gp78 activity at the ER–mitochondria interface., Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8–amino acid motif (533–541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41–amino acid region containing 14-3-3– and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER–mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER–mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER–mitochondria association and mitochondria motility.
- Published
- 2015
34. The secreted glycolytic enzyme GPI/AMF stimulates glioblastoma cell migration and invasion in an autocrine fashion but can have anti-proliferative effects
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Katrin Lamszus, Keith L. Ligon, Annegret Kathagen-Buhmann, Manfred Westphal, Markus Glatzel, Cecile L. Maire, Mareike Holz, Alexander Schulte, Jakob Matschke, and Jonathan Weller
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0301 basic medicine ,Cancer Research ,Autocrine Motility Factor ,medicine.medical_treatment ,Apoptosis ,03 medical and health sciences ,Mice ,Cell Movement ,medicine ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Animals ,Humans ,Neoplasm Invasiveness ,Autocrine signalling ,Cell Proliferation ,Gene knockdown ,Cell growth ,Chemistry ,fungi ,Glucose-6-Phosphate Isomerase ,Cell migration ,Prognosis ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,Receptors, Autocrine Motility Factor ,Survival Rate ,Autocrine Communication ,030104 developmental biology ,Cytokine ,Oncology ,Cell culture ,Anaerobic glycolysis ,Basic and Translational Investigations ,Cancer research ,Cytokines ,lipids (amino acids, peptides, and proteins) ,Neurology (clinical) ,Glioblastoma - Abstract
Background Aerobic glycolysis confers several advantages to tumor cells, including shunting of metabolites into anabolic pathways. In glioblastoma cells, hypoxia induces a flux shift from the pentose phosphate pathway toward glycolysis and a switch from proliferation to migration. The mechanistic link between glycolysis and migration is poorly understood. Since glucose-6-phosphate isomerase (GPI) is identical to the secreted cytokine autocrine motility factor (AMF), we investigated whether GPI/AMF regulates glioblastoma cell invasion. Methods The expression and hypoxic regulation of GPI/AMF and its receptor AMFR were analyzed in glioblastoma tissue and cell lines. Functional effects were studied in vitro and in xenograft models. Results High GPI/AMF expression in glioblastomas was found to be associated with a worse patient prognosis, and levels were highest in hypoxic pseudopalisades. Hypoxia upregulated both GPI/AMF and AMFR expression as well as GPI/AMF secretion in vitro. GPI/AMF stimulated cell migration in an autocrine fashion, and GPI/AMF expression was upregulated in migratory cells but reduced in rapidly proliferating cells. Knockdown or inhibition of GPI/AMF reduced glioblastoma cell migration but in part stimulated proliferation. In a highly invasive orthotopic glioblastoma model, GPI/AMF knockdown reduced tumor cell invasion but did not prolong survival. In a highly proliferative model, knockdown tumors were even larger and more proliferative than controls; however, perivascular invasion, provoked by simultaneous bevacizumab treatment, was reduced. Conclusions GPI/AMF is a potent motogen for glioblastoma cells, explaining in part the association between glycolysis and migration. Targeting GPI/AMF is, however, problematic, since beneficial anti-invasive effects may be outweighed by unintended mitogenic effects. Key points 1.Increased glycolysis is linked with increased cell migration and invasion in glioblastoma cells. 2.The glycolysis enzyme GPI/AMF may serve as a target for antimetabolic and anti-invasive therapy. 3.Despite reducing tumor invasion, GPI/AMF targeting may have unwanted growth stimulatory effects.
- Published
- 2018
35. A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes
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James A. Olzmann, Daniel K. Nomura, Elizabeth A. Grossman, Milton To, Kirill Bersuker, Victoria Savikhin, Steffen J. Sahl, and Clark W.H. Peterson
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0301 basic medicine ,APEX2 ,Proteome ,lipid droplet ,Medical and Health Sciences ,0302 clinical medicine ,Lipid droplet ,Receptors ,biotinylation ,APEX ,biology ,Autocrine Motility Factor ,Membrane transport protein ,Peripheral membrane protein ,Endoplasmic Reticulum-Associated Degradation ,Biological Sciences ,endoplasmic reticulum ,Biotinylation ,proximity labeling ,Biotechnology ,endocrine system ,Computational biology ,Endoplasmic-reticulum-associated protein degradation ,complex mixtures ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,ubiquitin ,Humans ,Molecular Biology ,Staining and Labeling ,Endoplasmic reticulum ,technology, industry, and agriculture ,Membrane Proteins ,Membrane Transport Proteins ,Cell Biology ,Lipid Droplets ,ERAD ,eye diseases ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,proteasome ,Proteasome ,biology.protein ,ATPases Associated with Diverse Cellular Activities ,Generic health relevance ,Carrier Proteins ,030217 neurology & neurosurgery ,Biomarkers ,Developmental Biology - Abstract
Lipid droplet (LD) functions are regulated by a complement of integral and peripheral proteins that associate with the bounding LD phospholipid monolayer. Defining the composition of the LD proteome has remained a challenge due to the presence of contaminating proteins in LD-enriched buoyant fractions. To overcome this limitation, we developed a proximity labeling strategy that exploits LD-targeted APEX2 to biotinylate LD proteins in living cells. Application of this approach to two different cell types identified the vast majority of previously validated LD proteins, excluded common contaminating proteins, and revealed new LD proteins. Moreover, quantitative analysis of LD proteome dynamics uncovered a role forendoplasmic reticulum-associated degradation in controlling the composition of the LD proteome. These data provide an important resource for future LD studies and demonstrate the utility of proximity labeling to study the regulation of LD proteomes.
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- 2017
36. Gp78 involvement in cellular proliferation: Can act as a promising modulator for cell cycle regulatory proteins?
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Amit Mishra, Deepak Chhangani, Rajesh N. Sharan, Vibhuti Joshi, Ayeman Amanullah, and Arun Upadhyay
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0301 basic medicine ,Proteasome Endopeptidase Complex ,Physiology ,Ubiquitin-Protein Ligases ,Clinical Biochemistry ,Cell ,Cell Cycle Proteins ,03 medical and health sciences ,Ubiquitin ,Chlorocebus aethiops ,medicine ,Autophagy ,Animals ,Humans ,Cell Proliferation ,biology ,Chemistry ,Kinase ,Cell Biology ,Endoplasmic Reticulum Stress ,Ubiquitin ligase ,Cell biology ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,medicine.anatomical_structure ,Proteasome ,Gene Expression Regulation ,A549 Cells ,Gene Knockdown Techniques ,COS Cells ,Proteolysis ,biology.protein ,Unfolded protein response ,Intracellular ,Cyclin-Dependent Kinase Inhibitor p27 ,Metabolic Networks and Pathways - Abstract
In cells, protein synthesis and degradation are normal processes, which are tightly regulated by various cellular metabolic pathways. Cellular protein quality control (PQC) mechanisms always present a continuous and rigorous check over all intracellular proteins before they can participate in various cellular physiological processes with the help of PQC pathways like autophagy and ubiquitin proteasome system (UPS). The UPS employs few selective E3 ubiquitin ligases for the intracellular degradation of cyclin-dependent kinase inhibitor 1B (p27Kip1 ) that tightly controls cell cycle progression. But, the complex mechanistic interactions and the interplay between E3 ubiquitin ligases involved in the functional regulation as well as expression of p27 are not well known. Here, we demonstrate that cell surface glycoprotein Gp78, a putative E3 ubiquitin ligase, is involved in the stabilization of intracellular steady-state levels of p27. Transient overexpression of Gp78 increases the accumulation of p27 in cells in the form of massive inclusions like structures, which could be due to its cumulative increased stability in cells. We have also monitored how under stress condition, E3 ubiquitin ligase Gp78 regulates endogenous levels of p27 in cells. ER stress treatment generates a marginal increase in Gp78 endogenous levels, and this elevation effect was prominent for intracellular accumulation of p27 in cells. Taken together, our current findings suggest a valuable multifactorial regulatory mechanism and linkage of p27 with UPS pathway.
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- 2017
37. Conformational dynamics and allostery in E2:E3 interactions drive ubiquitination: gp78 and Ube2g2
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R. Andrew Byrd, Jess Li, Kalyan S. Chakrabarti, and Ranabir Das
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0301 basic medicine ,Stereochemistry ,Allosteric regulation ,Molecular Dynamics Simulation ,Ubiquitin-conjugating enzyme ,Article ,03 medical and health sciences ,Molecular dynamics ,Molecular recognition ,Allosteric Regulation ,Ubiquitin ,Structural Biology ,Humans ,Molecular Biology ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Ubiquitination ,Energy landscape ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Enzyme ,chemistry ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Allosteric Site - Abstract
Conformational dynamics plays a fundamental role in molecular recognition and activity in enzymes. The ubiquitin-conjugating enzyme (E2) Ube2g2 functions with the ubiquitin ligase (E3) gp78 to assemble poly-ubiquitin chains on target substrates. Two domains in gp78, RING and G2BR, bind to two distant regions of Ube2g2, and activate it for ubiquitin (Ub) transfer. G2BR increases the affinity between the RING and Ube2g2 by 50-fold, while the RING catalyzes the transfer of Ub from the Ube2g2 similar to Ub conjugate. How G2BR and RING activate Ube2g2 is unclear. In this work, conformational dynamics in Ube2g2 revealed a clear correlation of binding G2BR and RING with the sequential progression toward Ub transfer. The interrelationship of the existence and exchange between ground and excited states leads to a dynamic energy landscape model, in which redistribution of populations contributes to allostery and activation. These findings provide insight into gp78's modulation of conformational exchange in Ube2g2 to stimulate ubiquitination.
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- 2017
38. SVIP regulates Z variant alpha-1 antitrypsin retro-translocation by inhibiting ubiquitin ligase gp78
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Mariana E. Kirst, Chen Liu, George Marek, Karina Krotova, Farshid N. Rouhani, Nazli Khodayari, Mark L. Brantly, and Rejean liqun Wang
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0301 basic medicine ,lcsh:Medicine ,medicine.disease_cause ,Endoplasmic Reticulum ,Biochemistry ,Animal Cells ,Medicine and Health Sciences ,Small interfering RNAs ,lcsh:Science ,chemistry.chemical_classification ,Mutation ,Multidisciplinary ,Secretory Pathway ,biology ,Endoplasmic Reticulum Stress ,Cell biology ,Ubiquitin ligase ,Transport protein ,Amino acid ,Precipitation Techniques ,Nucleic acids ,Protein Transport ,Liver ,Cell Processes ,Hyperexpression Techniques ,Cellular Structures and Organelles ,Cellular Types ,Anatomy ,Research Article ,Context (language use) ,Endoplasmic-reticulum-associated protein degradation ,DNA construction ,Transfection ,Research and Analysis Methods ,Real-Time Polymerase Chain Reaction ,Cell Line ,03 medical and health sciences ,medicine ,Gene Expression and Vector Techniques ,Genetics ,Immunoprecipitation ,Humans ,Molecular Biology Techniques ,Non-coding RNA ,Molecular Biology ,Molecular Biology Assays and Analysis Techniques ,Endoplasmic reticulum ,lcsh:R ,Biology and Life Sciences ,Membrane Proteins ,Cell Biology ,Phosphate-Binding Proteins ,Gene regulation ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,chemistry ,Membrane protein ,alpha 1-Antitrypsin ,Plasmid Construction ,Vacuoles ,biology.protein ,Hepatocytes ,RNA ,lcsh:Q ,Gene expression ,Carrier Proteins - Abstract
Alpha-1 antitrypsin deficiency (AATD) is an inherited disorder characterized by early-onset emphysema and liver disease. The most common disease-causing mutation is a single amino acid substitution (Glu/Lys) at amino acid 342 of the mature protein, resulting in disruption of the 290-342 salt bridge (an electrophoretic abnormality defining the mutation [Z allele, or ZAAT]), protein misfolding, polymerization, and accumulation in the endoplasmic reticulum of hepatocytes and monocytes. The Z allele causes a toxic gain of function, and the E3 ubiquitin ligase gp78 promotes degradation and increased solubility of endogenous ZAAT. We hypothesized that the accumulation of ZAAT is influenced by modulation of gp78 E3 ligase and SVIP (small VCP-interacting protein) interaction with p97/VCP in ZAAT-expressing hepatocytes. We showed that the SVIP inhibitory effect on ERAD due to overexpression causes the accumulation of ZAAT in a human Z hepatocyte-like cell line (AT01). Overexpression of gp78, as well as SVIP suppression, induces gp78-VCP/p97 interaction in AT01 cells. This interaction leads to retro-translocation of ZAAT and reduction of the SVIP inhibitory role in ERAD. In this context, overexpression of gp78 or SVIP suppression may eliminate the toxic gain of function associated with polymerization of ZAAT, thus providing a potential new therapeutic approach to the treatment of AATD.
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- 2017
39. CDK5-Mediated Phosphorylation-Dependent Ubiquitination and Degradation of E3 Ubiquitin Ligases GP78 Accelerates Neuronal Death in Parkinson's Disease
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Xiang Peng, Hongwei Cai, Pei Zhang, Zheng Zhang, Qingzhi Wang, Ze-Xi Lv, Fengjuan Jiao, Feng He, Bo Tian, Jianguo Yan, and Qian Zhang
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0301 basic medicine ,Neuroscience (miscellaneous) ,Mitochondrion ,Protein degradation ,Cell Line ,Rats, Sprague-Dawley ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Ubiquitin ,Cyclin-dependent kinase ,medicine ,Animals ,Parkinson Disease, Secondary ,Phosphorylation ,Neurons ,biology ,Cell Death ,Cyclin-dependent kinase 5 ,MPTP ,Neurodegeneration ,Ubiquitination ,Cyclin-Dependent Kinase 5 ,medicine.disease ,Ubiquitin ligase ,Cell biology ,Rats ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,nervous system ,Neurology ,chemistry ,biology.protein ,Signal Transduction - Abstract
The molecular mechanisms responsible for the loss of dopaminergic neurons in Parkinson's disease (PD) remain obscure. Loss of function of E3 ubiquitin ligases is associated with mitochondria dysfunction, dysfunction of protein degradation, and α-synuclein aggregation, which are major contributors to neurodegeneration in PD. Recent research has thus focused on E3 ubiquitin ligase glycoprotein 78 (GP78); however, the role of GP78 in PD pathogenesis remains unclear. Notably, cyclin-dependent kinase 5 (CDK5) controls multiple cellular events in postmitotic neurons, and CDK5 activity has been implicated in the pathogenesis of PD. Thus, we addressed the relationship between CDK5 and GP78 in MPTP-based PD models. We found that GP78 expression is decreased in MPTP-based cellular and animal PD models, and CDK5 directly phosphorylated GP78 at Ser516, which promoted the ubiquitination and degradation of GP78. Importantly, overexpression of GP78 or interference of GP78 Ser516 phosphorylation protected neurons against MPP+-induced cell death. Thus, our research reveals that the CDK5-GP78 pathway is involved in the pathogenesis of PD and could be a novel candidate drug target for the treatment of PD.
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- 2017
40. Correlation between the GP78 Gene Polymorphism and Coronary Atherosclerotic Heart Disease
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Cheng-Dong Qiao, Long Wang, Jian Zhou, Zheng Zhang, Yong-Ming Liu, Bo Yang, and Hua-Cheng Li
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0301 basic medicine ,Adult ,Male ,lcsh:Diseases of the circulatory (Cardiovascular) system ,medicine.medical_specialty ,Heart disease ,Genotype ,Blotting, Western ,Blood lipids ,Locus (genetics) ,Enzyme-Linked Immunosorbent Assay ,Coronary Artery Disease ,030204 cardiovascular system & hematology ,Polymerase Chain Reaction ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,medicine ,Humans ,Gene ,Retrospective Studies ,Medicine(all) ,Polymorphism, Genetic ,business.industry ,DNA ,Middle Aged ,medicine.disease ,Atherosclerosis ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,Real-time polymerase chain reaction ,Endocrinology ,Blood pressure ,lcsh:RC666-701 ,Cardiology ,Female ,Gene polymorphism ,Restriction fragment length polymorphism ,Cardiology and Cardiovascular Medicine ,business ,Follow-Up Studies - Abstract
Objective: To study the correlation between the GP78 gene polymorphism and blood fat, blood glucose, blood pressure and coronary atherosclerotic heart disease. Methods: A total of 72 patients with coronary atherosclerotic heart disease were selected as the observation group, and 68 healthy participants were selected as the control group. The gp78 gene polymorphism of both groups was studied via polymerase chain reaction-restriction fragment length polymorphism (RFLP). At the same time, the multiple expression quantities of the GP78 gene in the tissues of both groups were tested via fluorogenic quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and Western-blotting assay. Furthermore, the blood fat, blood glucose and blood pressure of subjects in both groups were tested. Results: The percentages of the gp78 gene polymorphisms of Arg/Arg, Arg/Gly and Gly/Gly at the 145 locus of the study subjects in the observation group were 12.3%, 43.2% and 44.5%, respectively, while those in the control group were 74.3%, 11.2% and 14.5%, respectively, and there were significant differences between both groups. Based on the test results of the blood fat, blood glucose and blood pressure of the objects in the observation group and control group, significant differences were found between the two groups (P
- Published
- 2017
41. Endoplasmic reticulum stress–induced degradation of DNAJB12 stimulates BOK accumulation and primes cancer cells for apoptosis
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Douglas M. Cyr, Diane E. Grove, Hong Yu Ren, and Pattarawut Sopha
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0301 basic medicine ,Proteasome Endopeptidase Complex ,Carcinoma, Hepatocellular ,Recombinant Fusion Proteins ,Antineoplastic Agents ,Apoptosis ,Caspase 3 ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Cell Line, Tumor ,Chlorocebus aethiops ,Animals ,Humans ,Molecular Biology ,Caspase ,biology ,Protein Stability ,Endoplasmic reticulum ,Liver Neoplasms ,Cell Biology ,HSP40 Heat-Shock Proteins ,Endoplasmic Reticulum Stress ,Neoplasm Proteins ,Cell biology ,Receptors, Autocrine Motility Factor ,Thiazoles ,HEK293 Cells ,030104 developmental biology ,Proteostasis ,Amino Acid Substitution ,Proto-Oncogene Proteins c-bcl-2 ,Proteasome ,Protein Synthesis and Degradation ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Chaperone (protein) ,COS Cells ,Mutation ,Proteolysis ,Cancer cell ,biology.protein ,Unfolded protein response ,RNA Interference - Abstract
DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.
- Published
- 2017
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42. Regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmic-reticulum-associated degradation
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Sang Jun Lee, Suui Lee, Hyunju Kang, Yu-Ra Choi, M. Sun Kim, Ju Young Park, Hyun-Jun Lee, Jae-Hyung Song, Hyeongki Kim, Sunhong Kim, Seo Hyun Lee, Sungchan Cho, Ji-Ye Kim, Sung Hoon Back, Jong Heon Kim, Jeong-Eun Lee, Kwangman Choi, and So-Young Lee
- Subjects
Proteasome Endopeptidase Complex ,macromolecular substances ,Endoplasmic-reticulum-associated protein degradation ,Biochemistry ,Ubiquitin ,Cell Line, Tumor ,Lipid droplet ,Enzyme Stability ,Humans ,Diacylglycerol O-Acyltransferase ,Molecular Biology ,Messenger RNA ,biology ,Endoplasmic reticulum ,Ubiquitination ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,Lipid Metabolism ,Protein Structure, Tertiary ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,Transmembrane domain ,HEK293 Cells ,Proteasome ,Proteolysis ,Hepatocytes ,biology.protein - Abstract
Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by diacylglycerol acyltransferase (DGAT) in the endoplasmic reticulum (ER). DGAT2, one of the two DGAT enzymes, is barely detectable in cells, even though its mRNA transcripts are maintained at considerable levels. However, little is known about how DGAT2 expression is altered by protein stability. DGAT2 was highly unstable in cells and was rapidly degraded by proteasomes in an ubiquitin-dependent manner. Deletion mutation analysis identified transmembrane domain 1 (TMD1) as a protein degradation signal. TMD1 is also important for ER localization of DGAT2. Moreover, DGAT2 interacted with p97/VCP, a crucial component of the ER-associated degradation (ERAD) pathway, and polyubiquitinated DGAT2 accumulated following treatment with an ERAD inhibitor. Furthermore, gp78, an E3 ligase involved in ERAD, regulates the degradation of DGAT2 through direct interactions and ubiquitination. Consequently, the stabilization of DGAT2 increased the number of lipid droplets in hepatic cells. Therefore, DGAT2 is regulated by gp78-associated ERAD at the post-translational level.
- Published
- 2014
43. Regulation of Mitochondrial Antiviral Signaling (MAVS) Expression and Signaling by the Mitochondria-associated Endoplasmic Reticulum Membrane (MAM) Protein Gp78
- Author
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Jianzhong Zhu, Jana L. Jacobs, Saumendra N. Sarkar, and Carolyn B. Coyne
- Subjects
Immunology ,Regulator ,Mitochondrion ,Biology ,Endoplasmic-reticulum-associated protein degradation ,Biochemistry ,RNA interference ,Interferon ,Cell Line, Tumor ,medicine ,Humans ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Endoplasmic reticulum membrane ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,biology.organism_classification ,Protein Structure, Tertiary ,Cell biology ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,Gene Expression Regulation ,Vesicular stomatitis virus ,Interferon Type I ,biology.protein ,Signal Transduction ,medicine.drug - Abstract
In a previous study, we identified the E3 ubiquitin ligase Gp78 by RNAi high-throughput screening as a gene whose depletion restricted enterovirus infection. In the current study, we show that Gp78, which localizes to the ER-mitochondria interface, is a regulator of RIG-I-like receptor (RLR) antiviral signaling. We show that depletion of Gp78 results in a robust decrease of vesicular stomatitis virus (VSV) infection and a corresponding enhancement of type I interferon (IFN) signaling. Mechanistically, we show that Gp78 modulates type I IFN induction by altering both the expression and signaling of the mitochondria-localized RLR adaptor mitochondrial antiviral signaling (MAVS). Expression of mutants of Gp78 that abolish its E3 ubiquitin ligase and its participation in ER-associated degradation (ERAD) lost their ability to degrade MAVS, but surprisingly maintained their ability to repress RLR signaling. In contrast, Gp78 lacking its entire C terminus lost both its ability to degrade MAVS and repress RLR signaling. We show that Gp78 interacts with both the N- and C-terminal domains of MAVS via its C-terminal RING domain, and that this interaction is required to abrogate Gp78-mediated attenuation of MAVS signaling. Our data thus implicate two parallel pathways by which Gp78 regulates MAVS signaling; one pathway requires its E3 ubiquitin ligase and ERAD activity to directly degrade MAVS, whereas the other pathway occurs independently of these activities, but requires the Gp78 RING domain and occurs via a direct association between this region and MAVS.
- Published
- 2014
44. Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation
- Author
-
Shu-Yuan Guo, Yong-Jian Wang, Xu Lin, Ta-Yuan Chang, Catherine C.Y. Chang, Huiyong Yin, Qin Li, Bao-Liang Song, Ming Lu, Ying Xiong, Bo-Liang Li, Jie Luo, and Yan Bian
- Subjects
0301 basic medicine ,Male ,Time Factors ,Genotype ,CHO Cells ,Diet, High-Fat ,Transfection ,03 medical and health sciences ,chemistry.chemical_compound ,Residue (chemistry) ,0302 clinical medicine ,Insulin resistance ,Cricetulus ,Ubiquitin ,Oxidizing agent ,medicine ,Animals ,Homeostasis ,Humans ,Cysteine ,Mice, Knockout ,biology ,Cholesterol ,Fatty Acids ,Ubiquitination ,Cell Biology ,Hep G2 Cells ,medicine.disease ,Cell biology ,Mice, Inbred C57BL ,Receptors, Autocrine Motility Factor ,Disease Models, Animal ,030104 developmental biology ,Phenotype ,chemistry ,Biochemistry ,Lipotoxicity ,Liver ,030220 oncology & carcinogenesis ,Proteolysis ,biology.protein ,RNA Interference ,Cholesterol Esters ,Insulin Resistance ,Reactive Oxygen Species ,Oxidation-Reduction ,Sterol O-Acyltransferase - Abstract
Ubiquitin linkage to cysteine is an unconventional modification targeting protein for degradation. However, the physiological regulation of cysteine ubiquitylation is still mysterious. Here we found that ACAT2, a cellular enzyme converting cholesterol and fatty acid to cholesteryl esters, was ubiquitylated on Cys277 for degradation when the lipid level was low. gp78-Insigs catalysed Lys48-linked polyubiquitylation on this Cys277. A high concentration of cholesterol and fatty acid, however, induced cellular reactive oxygen species (ROS) that oxidized Cys277, resulting in ACAT2 stabilization and subsequently elevated cholesteryl esters. Furthermore, ACAT2 knockout mice were more susceptible to high-fat diet-associated insulin resistance. By contrast, expression of a constitutively stable form of ACAT2 (C277A) resulted in higher insulin sensitivity. Together, these data indicate that lipid-induced stabilization of ACAT2 ameliorates lipotoxicity from excessive cholesterol and fatty acid. This unconventional cysteine ubiquitylation of ACAT2 constitutes an important mechanism for sensing lipid-overload-induced ROS and fine-tuning lipid homeostasis.
- Published
- 2016
45. Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases
- Author
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Ying Wang, Seung Wook Ha, Avraham Raz, Dhong Hyo Kho, Youming Xie, and Tianpeng Zhang
- Subjects
TRIM25 ,Immunoprecipitation ,Ubiquitin-Protein Ligases ,Immunoblotting ,protein ubiquitylation ,Endoplasmic-reticulum-associated protein degradation ,ubiquitin ligase ,Tripartite Motif Proteins ,gp78 ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,AMF ,Humans ,Transcription factor ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,fungi ,Glucose-6-Phosphate Isomerase ,Ubiquitination ,3. Good health ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,HEK293 Cells ,Oncology ,030220 oncology & carcinogenesis ,biology.protein ,Research Paper ,Transcription Factors - Abstract
// Ying Wang 1,* , Seung-Wook Ha 1,* , Tianpeng Zhang 1,* , Dhong-Hyo Kho 1 , Avraham Raz 1 , and Youming Xie 1 1 Barbara Ann Karmanos Cancer Institute and Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA * These authors contributed equally to the work Correspondence: Avraham Raz, email: // Youming Xie, email: // Keywords : protein ubiquitylation, ubiquitin ligase, AMF, gp78, TRIM25 Received : October 8, 2013 Accepted : October 27, 2013 Published : October 28, 2013 Abstract gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.
- Published
- 2013
46. AMFR gene silencing inhibits the differentiation of porcine preadipocytes
- Author
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M.L. Chai, W.F. Lu, Yu Ding, Hao Jiang, Y.N. Zhu, Cheng-Zhen Chen, Lisheng Dai, L.J. Zhang, Jia-Bao Zhang, Si-Yuan Liu, Q.Y. Li, and Yanhang Gao
- Subjects
0301 basic medicine ,Swine ,Cellular differentiation ,Kruppel-Like Transcription Factors ,03 medical and health sciences ,chemistry.chemical_compound ,Western blot ,Adipocytes ,CCAAT-Enhancer-Binding Protein-alpha ,Genetics ,medicine ,Animals ,Gene silencing ,Oil Red O ,Gene Silencing ,Receptor ,Molecular Biology ,Cells, Cultured ,Messenger RNA ,medicine.diagnostic_test ,Cell Differentiation ,General Medicine ,Transfection ,Molecular biology ,PPAR gamma ,Receptors, Autocrine Motility Factor ,030104 developmental biology ,chemistry ,KLF2 ,Sterol Regulatory Element Binding Protein 1 ,Sterol Regulatory Element Binding Protein 2 - Abstract
Our study clarifies the role of the autocrine motility factor receptor (AMFR) gene in porcine preadipocyte differentiation. AMFR-siRNA was transfected into porcine preadipocytes and the preadipocytes were induced to differentiation. Subsequently, qRT-PCR was conducted to examine changes in mRNA expression of a series of genes in porcine preadipocytes, including AMFR, sterol-regulatory element-binding protein-1a (SREBP1a), SREBP2, insulin-induced gene 1 (Insig1), and Insig2. Expression changes in the mRNA of genes regulating adipocyte differentiation were also analyzed using qRT-PCR, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), and Kruppel-like factor 2 (KLF2). Western blot analysis was conducted to examine the changes in AMFR protein expression in porcine preadipocytes. Additionally, morphological changes in differentiated porcine preadipocytes were examined by oil red O staining, and changes in optical density (OD) values were measured using an ultraviolet spectrophotometer. At 24 h after transfection with AMFR-siRNA, AMFR mRNA expression significantly reduced (P < 0.01), and AMFR protein expression markedly decreased (P < 0.05). The mRNA expression of SREBP1a, SREBP2, Insig1, and C/EBPα was significantly reduced (P < 0.01), whereas the expression of KLF2 mRNA was significantly elevated (P < 0.01). After induction of preadipocyte differentiation, the number of lipid droplets decreased in the AMFR-silenced group, and the OD value markedly reduced (P < 0.05). In addition, the expression of C/EBPα mRNA significantly decreased (P < 0.05), whereas the expression of KLF2 mRNA considerably increased (P < 0.05). Taken together, silencing of the AMFR gene inhibits the differentiation of porcine preadipocytes.
- Published
- 2016
47. gp78: a Multifaceted Ubiquitin Ligase that Integrates a Unique Protein Degradation Pathway from the Endoplasmic Reticulum
- Author
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Zhiliang Chen, Shaojun Du, and Shengyun Fang
- Subjects
Molecular Sequence Data ,macromolecular substances ,Endoplasmic-reticulum-associated protein degradation ,Protein degradation ,Endoplasmic Reticulum ,Biochemistry ,Ubiquitin ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Secretory pathway ,biology ,Endoplasmic reticulum ,Ubiquitination ,Neurodegenerative Diseases ,Cell Biology ,General Medicine ,Ubiquitin ligase ,Cell biology ,Receptors, Autocrine Motility Factor ,Proteolysis ,biology.protein ,Protein folding ,Signal transduction ,Sequence Alignment ,Signal Transduction - Abstract
The endoplasmic reticulum (ER) is the site for maturation of proteins destined for the secretory pathway. Failure in maturation leads to production of misfolded proteins that are eliminated through the ER-associated degradation (ERAD) pathway. ERAD is a complex process that includes misfolded protein recognition, retrotranslocation to the cytosol, ubiquitination and proteasomal degradation. gp78 is an E3 ubiquitin ligase that integrates these ERAD steps by nucleating a unique degradation machine, which uses the p97/VCP-Npl4 complex for retrotranslocation instead of the wellknown p97/VCP-Ufd1-Npl4 complex. A growing list of substrates have been identified for gp78, which highlights the importance of gp78-mediated ERAD in essential physiological pathways and pathological processes.
- Published
- 2012
48. RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase
- Author
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Alaa El-Husseini, Ivan R. Nabi, Kun Huang, and Maria Fairbank
- Subjects
Gp78 ,Proteasome Endopeptidase Complex ,Amino Acid Motifs ,Palmitates ,Biophysics ,Palmitic Acids ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Biochemistry ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Palmitoylation ,Structural Biology ,Chlorocebus aethiops ,Genetics ,Ring finger ,medicine ,Animals ,Molecular Biology ,Degradation pathway ,030304 developmental biology ,0303 health sciences ,Trafficking ,Binding Sites ,biology ,Chemistry ,Endoplasmic reticulum ,technology, industry, and agriculture ,Cell Biology ,ERAD ,Protein Structure, Tertiary ,Cell biology ,Ubiquitin ligase ,Receptors, Autocrine Motility Factor ,Protein Transport ,S-palmitoylation ,medicine.anatomical_structure ,E3 ubiquitin ligase ,Gene Expression Regulation ,Microscopy, Fluorescence ,COS Cells ,Mutation ,Peripheral ER ,biology.protein ,lipids (amino acids, peptides, and proteins) ,030217 neurology & neurosurgery ,Cysteine - Abstract
Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER.
- Published
- 2012
49. ER stress response during the differentiation of H9 cells induced by retinoic acid
- Author
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Shengyun Fang, Chao Liu, Andria Apostolou, Yongwang Zhong, and Lihua Liu
- Subjects
medicine.medical_specialty ,Ubiquitin-Protein Ligases ,Cellular differentiation ,Eukaryotic Initiation Factor-2 ,Biophysics ,Retinoic acid ,Tretinoin ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Phosphorylation ,Endoplasmic Reticulum Chaperone BiP ,Molecular Biology ,Embryonic Stem Cells ,CD40 ,biology ,HEK 293 cells ,Cell Differentiation ,Cell Biology ,Endoplasmic Reticulum Stress ,Embryonic stem cell ,Cell biology ,Receptors, Autocrine Motility Factor ,HEK293 Cells ,Endocrinology ,P19 cell ,chemistry ,Cell culture ,biology.protein ,Unfolded protein response - Abstract
Endoplasmic reticulum (ER) stress occurs during early embryonic development. The aim of this study is to determine whether ER stress occurs during human embryonic stem cell differentiation induced by retinoic acid (RA). H9 human embryonic stem cells were subjected to RA treatment for up to 29days to induce differentiation. HEK293 cells were treated with RA as a control. The results demonstrate that several ER stress-responsive genes are differentially regulated in H9 and HEK293 cells in response to 5days of RA treatment. GRP78/Bip was upregulated in H9 cells but downregulated in HEK293 cells. eIF2α was downregulated in H9 cells but not in HEK293 cells. Phosphorylation of eIF2α was downregulated in H9 cells but upregulated in HEK293 cells. XBP-1 was downregulated immediately after RA treatment in H9 cells, but its downregulation was much slower in HEK293 cells. Additionally, two ER-resident E3 ubiquitin ligases, gp78 and Hrd1, were both upregulated in H9 cells following 5 days of exposure to RA. Moreover, the protein Bcl2 was undetectable in H9 cells and H9-derived cells but was expressed in HEK293 cells, and it expression in the two types of cells was unaltered by RA treatment. In H9 cells treated with RA for 29 days, GRP78/Bip, XBP-1 and Bcl2 were all upregulated. These results suggest that ER stress is involved in H9 cell differentiation induced by RA.
- Published
- 2012
50. Neuroleukin/Autocrine Motility Factor Receptor Pathway Promotes Proliferation of Articular Chondrocytes through Activation of AKT and Smad2/3
- Author
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Kang Tian, Han Liu, Jinrui Zhang, Weiguo Zhang, Pi-Xu Liu, Weiliang Zhong, and Xifu Zheng
- Subjects
Cartilage, Articular ,Type II collagen ,Gene Expression ,Smad2 Protein ,Biology ,Article ,Chondrocyte ,Chondrocytes ,medicine ,Animals ,Growth Plate ,Smad3 Protein ,Phosphorylation ,Autologous chondrocyte implantation ,Receptor ,Protein kinase B ,Cell Proliferation ,Multidisciplinary ,Cell growth ,Cartilage ,Glucose-6-Phosphate Isomerase ,Rats ,Receptors, Autocrine Motility Factor ,Phenotype ,medicine.anatomical_structure ,Cancer research ,Signal transduction ,Proto-Oncogene Proteins c-akt ,Signal Transduction - Abstract
Cartilage defect is an intractable clinical problem. Therapeutic strategies for cartilage repair are far from optimal due to poor proliferation capacity of chondrocytes. Autologous chondrocyte implantation is a cell based therapy that uses in vitro amplified healthy chondrocytes from the patient. However, chondrocyte dedifferentiation during in vitro culture limits its application. Neuroleukin (NLK) is a multifunctional protein that stimulates cell growth and migration, together with its receptor autocrine motility factor receptor (AMFR, also called gp78). We investigated expression of NLK and AMFR/gp78 during cartilage development in vivo and in cultured articular chondrocytes in vitro and found the pair associates with chondrocyte proliferation and differentiation. While applied to isolated articular chondrocytes, NLK promotes cell proliferation and secretion of type II collagen, a marker of proliferating chondrocytes. Further work demonstrates that NLK up regulates pAKT and pSmad2/3, but down regulates pSmad1/5. In animals, NLK treatment also promotes chondrocyte proliferation while inhibits terminal differentiation, leading to expanded proliferating zone but decreased prehypertrophic and hypertrophic zones in the growth plate region. NLK is therefore a candidate factor that can be applied in the treatment of cartilage defects.
- Published
- 2015
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