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IScience Mar 2021Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being...
Ubiquitination is a major post-translational modification of ribosomal proteins. The role of ubiquitination in the regulation of ribosome functions is still being elucidated. However, the importance of ribosome deubiquitination remains unclear. Here, we show that the cycle of ubiquitination and deubiquitination of the 40S ribosome subunit eS7 is important for efficient translation. eS7 ubiquitination at lysine 83 is required for efficient protein translation. We identified Otu2 and Ubp3 as the deubiquitinating enzymes for eS7. An ΔΔ mutation caused a defect in protein synthesis. Ubp3 inhibited polyubiquitination of eS7 in polysomes to keep eS7 in a mono-ubiquitinated form, whereas Otu2 was specifically bound to the free 40S ribosome and promoted the dissociation of mRNAs from 40S ribosomes in the recycling step. Our results provide clues for understanding the molecular mechanism of the translation system via a ubiquitination-deubiquitination cycle.
PubMed: 33665564
DOI: 10.1016/j.isci.2021.102145 -
TRABID targets DDB2 for deubiquitination to promote proliferation of hepatocellular carcinoma cells.Biochemical and Biophysical Research... Oct 2022TRAF-binding domain-containing protein (TRABID), a member of the OTU deubiquitinase family, has an important role in regulating cellular functions via deubiquitinating...
TRAF-binding domain-containing protein (TRABID), a member of the OTU deubiquitinase family, has an important role in regulating cellular functions via deubiquitinating substrate proteins such as EZH2 and Jmjd2d. However, the mechanism of its role in the proliferation of hepatocellular carcinoma (HCC) cells has not been fully elucidated. Here, we analyzed the interactome of TRABID in HepG2 cells through mass spectrometry-based proteomics and found that TRABID is associated with damaged DNA-binding protein2 (DDB2). Immunoprecipitation assay showed that the interaction of TRABID and DDB2 is mediated by their OTU domain and N-terminal region, respectively. Furthermore, TRABID deubiquitinates DDB2, and this deubiquitination effect of TRABID depends on its active site. Functionally, we showed that TRABID-mediated hepatocellular carcinoma cell proliferation is attenuated by DDB2 knockdown. Thus, our data revealed a critical role of the TRABID-DDB2 axis in the proliferation of hepatocellular carcinoma cells.
Topics: Carcinoma, Hepatocellular; Cell Line; Cell Proliferation; DNA-Binding Proteins; Humans; Immunoprecipitation; Liver Neoplasms
PubMed: 35944360
DOI: 10.1016/j.bbrc.2022.07.109 -
Placenta Feb 2024The dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse...
INTRODUCTION
The dysregulation of deubiquitination has been shown to affect the development of pre-eclampsia (PE). A disintegrin and metalloprotease 9 (ADAM9) plays roles in diverse physiological contexts, including PE. Here, this study aimed to investigate whether ADAM9 regulated trophoblast cell dysfunction through ubiquitin-specific protease 22 (USP22) deubiquitinase-mediated deubiquitination during PE.
METHODS
Levels of genes and proteins were tested via qRT-PCR and western blotting assays. Cell proliferation, migration, and invasion were detected using cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell and wound healing assays, respectively. Epithelial-mesenchymal transition related markers were assayed using western blotting. Proteins between USP22 and ADAM9 were identified by co-immunoprecipitation assay.
RESULTS
ADAM9 was highly expressed in PE patients, functionally, ADAM9 overexpression weakened the proliferation, migration, invasion, and EMT progression in trophoblast cells. Mechanistically, the deubiquitinase USP22 removed ubiquitination on ADAM9 and maintained its stability. Forced expression of USP22 also suppressed the proliferation and mobility in trophoblast cells. Moreover, the regulatory effects of USP22 on trophoblast cells were reversed by ADAM9 silencing. In addition, USP22 interacted with ADAM9 to regulate the activation of Wnt/β-catenin pathway.
DISCUSSION
ADAM9 was deubiquitinated and stabilized by USP22 and then suppressed the proliferation, migration, invasion, and EMT progression in trophoblast cells, indicating a new pathway of USP10/RUNX1 axis in PE process.
Topics: Pregnancy; Female; Humans; Pre-Eclampsia; Trophoblasts; Wnt Signaling Pathway; Epithelial-Mesenchymal Transition; Cell Proliferation; Cell Movement; MicroRNAs; Ubiquitin Thiolesterase; Membrane Proteins; ADAM Proteins
PubMed: 38176298
DOI: 10.1016/j.placenta.2023.12.008 -
Biochemical and Biophysical Research... Jul 2021Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes...
Conjugation of K48-linked ubiquitin chains to intracellular proteins mainly functions as a signal for proteasomal degradation. The conjugating enzyme E2-25K synthesizes not only canonical (noncyclic) but also cyclic K48-linked ubiquitin chains. Although the cyclic conformation is expected to repress molecular recognition by ubiquitin binding proteins due to restricting the flexibility of the ubiquitin subunits in a chain, multiple proteins are reported to associate with cyclic ubiquitin chains similar to noncyclic chains. However, the molecular mechanism of how cyclic ubiquitin chains are recognized remains unclear. Here we investigated the effect of cyclization on ubiquitin-chain cleavage and molecular recognition by a K48-linkage specific deubiquitinating enzyme OTUB1 for cyclic diubiquitin by NMR spectroscopic analyses. Compared to noncyclic diubiquitin, we observed slow but unambiguously detectable cleavage of cyclic diubiquitin to monoubiquitin by OTUB1. Intriguingly, upon ubiquitin chain cleavage, cyclic diubiquitin appeared to alter its "autoinhibited" conformation to an incompletely but partially accessible conformation, induced by interaction with OTUB1 via the ubiquitin-subunit specific recognition patches and adjacent surfaces. These data imply that cyclic ubiquitin chains may exist stably in cells in spite of the presence of deubiquitinating enzymes and that these chains can be recognized by intracellular proteins in a manner distinct from that of noncyclic ubiquitin chains.
Topics: Cyclization; Deubiquitinating Enzymes; Humans; Kinetics; Lysine; Nitrogen Isotopes; Protein Conformation; Proton Magnetic Resonance Spectroscopy; Ubiquitin; Ubiquitination
PubMed: 34049206
DOI: 10.1016/j.bbrc.2021.05.031 -
Pathology, Research and Practice Oct 2019Breast cancer is the most common malignant tumor among women in China, which seriously threatens women's physical and mental health. Tumorigenesis is closely related to...
Breast cancer is the most common malignant tumor among women in China, which seriously threatens women's physical and mental health. Tumorigenesis is closely related to the dysregulation of cell cycle. The cell cycle progression includes interphase and mitotic phase (M phase). Cyclin B1 is a key protein in regulating M phase, which is essential for the whole cell cycle progression. CyclinB1 can be degraded through ubiquitination mediated by the anaphase promoting complex/cyclosome (APC/C). However, the mechanism of how CyclinB1 is deubiquitinated in breast cancer still remains unclear. In this study, we discovered that CyclinB1 interacted with ubiquitin-specific peptidase 14 (USP14). Based on the deubiquitinating function of USP14, we detected the effect of USP14 on the ubiquitination of CyclinB1. Inhibiting the activity of USP14 or USP14 knockdown significantly increased the ubiquitination of CyclinB1. In accordance with this, knocking down USP14 arrested cell cycle at G2/M phase. Knocking down USP14 with siRNAs significantly inhibited the proliferation and migration of breast cancer cells. In conclusion, our study demonstrated that USP14 regulated the cell cycle of breast cancer cells by regulating the ubiquitination of CyclinB1, which will provide a solid theoretical basis for the development of anti-cancer drugs targeting USP14.
Topics: Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cyclin B1; Female; Humans; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 31474315
DOI: 10.1016/j.prp.2019.152592 -
Cell Cycle (Georgetown, Tex.) Jan 2020Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by...
Cockayne syndrome group B (CSB) protein participates in transcription-coupled nucleotide excision repair. The stability of CSB is known to be regulated by ubiquitin-specific protease 7 (USP7). Yet, whether USP7 acts as a deubiquitinating enzyme for CSB is not clear. Here, we demonstrate that USP7 deubiquitinates CSB to maintain its levels after ultraviolet (UV)-induced DNA damage. While both CSB and UV-stimulated scaffold protein A (UVSSA) exhibit a biphasic decrease and recovery upon UV irradiation, only CSB recovery depends on USP7, which physically interacts with and deubiquitinates CSB. Meanwhile, CSB overexpression stabilizes UVSSA, but decrease UVSSA's presence in nuclease-releasable/soluble chromatin, and increase the presence of ubiquitinated UVSSA in insoluble chromatin alongside CSB-ubiquitin conjugates. Remarkably, CSB overexpression also decreases CSB association with USP7 and UVSSA in soluble chromatin. UVSSA exists in several ubiquitinated forms, of which mono-ubiquitinated form and other ubiquitinated UVSSA forms are detectable upon 6xHistidine tag-based purification. The ubiquitinated UVSSA forms, however, are not cleavable by USP7 . Furthermore, USP7 disruption does not affect RNA synthesis but decreases the recovery of RNA synthesis following UV exposure. These results reveal a role of USP7 as a CSB deubiquitinating enzyme for fine-tuning the process of TC-NER in human cells.
Topics: Carrier Proteins; Chromatin; DNA Damage; DNA Helicases; DNA Repair Enzymes; HCT116 Cells; HeLa Cells; Humans; Poly-ADP-Ribose Binding Proteins; Polyubiquitin; Protein Binding; RNA; Ubiquitin-Specific Peptidase 7; Ubiquitination; Ultraviolet Rays; Valosin Containing Protein
PubMed: 31775559
DOI: 10.1080/15384101.2019.1695996 -
The EMBO Journal Jan 2020Psychiatric diseases are often accompanied by circadian disruptions, but the molecular underpinnings remain largely unclear. To address this, we screened genes that have...
Psychiatric diseases are often accompanied by circadian disruptions, but the molecular underpinnings remain largely unclear. To address this, we screened genes that have been previously reported to be associated with psychiatric diseases and found that TRRAP, a gene associated with schizophrenia, is involved in circadian rhythm regulation. Knocking down Nipped-A, the Drosophila homolog of human TRRAP, leads to lengthened period of locomotor rhythms in flies. Molecular analysis demonstrates that NIPPED-A sets the pace of the clock by increasing the mRNA and protein levels of core clock genes timeless (tim) and Par domain protein 1ε (Pdp1ε). Furthermore, we found that NIPPED-A promotes the transcription of tim and Pdp1ε possibly by facilitating deubiquitination of histone H2B via the deubiquitination module of the transcription co-activator Spt-Ada-Gcn5 acetyltransferase complex. Taken together, these findings reveal a novel role for NIPPED-A in epigenetic regulation of the clock.
Topics: Animals; Basic-Leucine Zipper Transcription Factors; Circadian Clocks; Deubiquitinating Enzymes; Drosophila Proteins; Drosophila melanogaster; Epigenesis, Genetic; Histones; Male; Protein Processing, Post-Translational; Transcription Factors; Ubiquitination
PubMed: 31538360
DOI: 10.15252/embj.2018101259 -
European Journal of Immunology Jan 2019Retinoic acid-inducible gene I (RIG-I) is a critical RNA virus sensor that initiates antiviral immune response through K63-linked ubiquitination. In this study, we...
Retinoic acid-inducible gene I (RIG-I) is a critical RNA virus sensor that initiates antiviral immune response through K63-linked ubiquitination. In this study, we demonstrated USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by directly deubiquitinating K63-linked RIG-I. USP14 knockdown significantly enhanced RIG-I-triggered type I IFN signaling and inhibited vesicular stomatitis virus (VSV) replication both in mouse peritoneal macrophages and THP1 cells. USP14 overexpression in HeLa cells attenuated RIG-I-triggered IFN-β expression and promoted VSV replication. Besides, USP14-specific inhibitor, IU1, increased RIG-I-mediated type I IFN production and antiviral responses in vitro and in vivo. In addition, USP14 could interact with RIG-I and remove RIG-I K63-linked polyubiquitination chains. This article is the first to report that USP14 acts as a negative regulator in antiviral response through deubiquitinating K63-linked RIG-I. These findings provide insights into a potential new therapy targeting USP14 for RNA virus-related diseases.
Topics: Animals; DEAD Box Protein 58; Female; HeLa Cells; Humans; Interferon Type I; Macrophages; Mice; Mice, Inbred C57BL; RNA, Small Interfering; Receptors, Immunologic; Rhabdoviridae Infections; Signal Transduction; THP-1 Cells; Ubiquitin Thiolesterase; Ubiquitination; Vesiculovirus; Virus Replication
PubMed: 30466171
DOI: 10.1002/eji.201847603 -
Molecular Cell Jan 2013NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various...
NLRP3 is an important pattern recognition receptor involved in mediating inflammasome activation in response to viral and bacterial infections as well as various proinflammatory stimuli associated with tissue damage or malfunction. Upon activation, NLRP3 assembles a multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the activation of caspase-1. Although NLRP3 expression is induced by the NF-κB pathway, the posttranscriptional molecular mechanism controlling the activation of NLRP3 remains elusive. Using both pharmacological and molecular approaches, we show that the activation of NLRP3 inflammasome is regulated by a deubiquitination mechanism. We further identify the deubiquitinating enzyme, BRCC3, as a critical regulator of NLRP3 activity by promoting its deubiquitination and characterizing NLRP3 as a substrate for the cytosolic BRCC3-containing BRISC complex. Our results elucidate a regulatory mechanism involving BRCC3-dependent NLRP3 regulation and highlight NLRP3 ubiquitination as a potential therapeutic target for inflammatory diseases.
Topics: Animals; Carrier Proteins; Caspase 1; Deubiquitinating Enzymes; Endopeptidases; Gene Knockdown Techniques; HEK293 Cells; Humans; Inflammasomes; Interleukin-1beta; Lipopolysaccharides; Macrophages, Peritoneal; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Peptide Mapping; Protein Processing, Post-Translational; Protein Structure, Tertiary; Pyrans; RNA, Small Interfering; Sulfhydryl Compounds; Toll-Like Receptor 4; Ubiquitination
PubMed: 23246432
DOI: 10.1016/j.molcel.2012.11.009 -
Structure (London, England : 1993) Mar 2008
Topics: Deubiquitinating Enzyme CYLD; Humans; Lysine; Models, Molecular; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination
PubMed: 18334208
DOI: 10.1016/j.str.2008.02.001