ABSTRACT
Viral infection induces numerous tripartite motif (TRIM) proteins to control antiviral immune signaling and viral replication. Particularly, SPRY-containing TRIM proteins are found only in vertebrates and they control target protein degradation by their RING-finger and SPRY domains, and proper cytoplasmic localization. To understand TRIM30 function, we analyzed its localization pattern and putative roles of its RING-finger and SPRY domains. We found that TRIM30 is located in actin-mediated cytoplasmic bodies and produces colocalized ubiquitin chains in SPRY domain- and RING-finger domain-dependent ways that are degraded by autophagy and the proteasome. These results suggest a TRIM protein-dependent degradation mechanism by cytoplasmic body formation with actin networks.
Subject(s)
Animals , Mice , Amino Acid Sequence , Autophagy , Cell Line , Inclusion Bodies/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Molecular Sequence Data , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Interaction Domains and Motifs , Protein Transport , Proteolysis , RING Finger DomainsABSTRACT
Parkinson's disease is a multifactorial disorder with several genes linked to the familial types of the disease. ATP13A2 is one of those genes and encode for a transmembrane protein localized in lysosomes and late endosomes. Previous studies suggested the roles of this protein in lysosomal functions and cellular ion homeostasis. Here, we set out to investigate the role of ATP13A2 in lysosomal function and in metabolism of alpha-synuclein, another PD-linked protein whose accumulation is implicated in the pathogenesis. We generated non-sense mutations in both copies of ATP13A2 gene in SH-SY5Y human neuroblastoma cells. We examined lysosomal function of ATP13A2-/- cells by measuring the accumulation of lysosomal substrate proteins, such as p62 and polyubiquitinated proteins, induction of acidic compartments, and degradation of ectopically introduced dextran. None of these measures were altered by ATP13A2 deficiency. The steady-state levels of alpha-synuclein in cells or secretion of this protein were unaltered either in ATP13A2-/- compared to the normal cells. Therefore, the proposed roles of ATP13A2 in lysosomal functions may not be generalized and may depend on the cellular context. The ATP13A2-/- cells generated in the current study may provide a useful control for studies on the roles of PD genes in lysosomal functions.
Subject(s)
Humans , alpha-Synuclein , Dextrans , Endosomes , Homeostasis , Lysosomes , Metabolism , Neuroblastoma , Parkinson Disease , PolyubiquitinABSTRACT
Retinoic acid-inducible gene I (RIG-I) is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS (also called IPS-1, CARDIF, or VISA). A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I. Recent studies have proposed that K63-linked ubiquitination of, or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling. Conversely phosphorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction. Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling. ATP and dsRNA binding triggered dimerization of RIG-I with conformational rearrangements of the tandem CARD domains. Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio. Compared with the previously reported crystal structures of RIG-I in inactive state, our electron microscopic structure of full length RIG-I in complex with blunt-ended dsRNA, for the first time, revealed an exposed active conformation of the CARD domains. Moreover, we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA, while S8E and T170E phosphorylation-mimicking mutants of RIG-I were defective in binding E3 ligase TRIM25, unanchored K63-linked polyubiquitin, and MAVS regardless of dsRNA. These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.
Subject(s)
Humans , Adaptor Proteins, Signal Transducing , Metabolism , Adenosine Triphosphate , Metabolism , DEAD Box Protein 58 , DEAD-box RNA Helicases , Chemistry , Genetics , Metabolism , Dimerization , Mutagenesis, Site-Directed , Phosphorylation , Polyubiquitin , Metabolism , Protein Binding , Protein Structure, Tertiary , RNA, Double-Stranded , Metabolism , Recombinant Proteins , Chemistry , Genetics , Signal Transduction , Transcription Factors , Metabolism , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Metabolism , UbiquitinationABSTRACT
<p><b>BACKGROUND</b>Wilms' tumor (nephroblastoma) is a cancer of the kidneys that occurs typically in children and rarely in adults. Early diagnosis is very important for the treatment and prognosis of the disease. The aim of our study was to discover and identify potential non-invasive and convenient biomarkers for the diagnosis of Wilms' tumor.</p><p><b>METHODS</b>Nude mice were used to construct a Wilms' tumor model by injecting nephroblastoma cells into their bilateral abdomen. We collected 94 serum samples from mice consisting of 45 samples with Wilms' tumor and 49 controls. The serum proteomic profiles of the samples were analyzed via surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. The candidate biomarkers were purified by high-performance liquid chromatography, identified by liquid chromatography-mass spectrometry, and validated using ProteinChip immunoassays.</p><p><b>RESULTS</b>We finally retrieved two differential proteins (m/z 4509.2; 6207.9), which were identified as apolipoprotein A-II and polyubiquitin, respectively. The expression of apolipoprotein A-II was higher in the Wilms' tumor group than in the control group (P < 0.01). By contrast, the expression of polyubiquitin was lower in the Wilms' tumor group than in the control group.</p><p><b>CONCLUSION</b>Apolipoprotein A-II and polyubiquitin may be used as potential biomarkers for nephroblastoma in children, and the analysis of apolipoprotein A-II may help diagnose and treat Wilms' tumor.</p>
Subject(s)
Animals , Mice , Apolipoprotein A-II , Blood , Biomarkers , Blood , Cell Line, Tumor , Mice, Nude , Polyubiquitin , Blood , Proteomics , Methods , Wilms Tumor , Blood , Metabolism , PathologyABSTRACT
Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.
Subject(s)
Animals , Humans , Mice , B-Lymphocytes , Allergy and Immunology , Metabolism , Cell Line , DEAD Box Protein 58 , DEAD-box RNA Helicases , Allergy and Immunology , Metabolism , Hep G2 Cells , Hepatitis B virus , Allergy and Immunology , Metabolism , I-kappa B Kinase , Allergy and Immunology , Metabolism , Immune Evasion , Immunity, Innate , Interferon Regulatory Factor-3 , Allergy and Immunology , Metabolism , Interferon Type I , Allergy and Immunology , Metabolism , Molecular Targeted Therapy , Polyubiquitin , Metabolism , Protein Binding , Allergy and Immunology , Sendai virus , Allergy and Immunology , Metabolism , Signal Transduction , Allergy and Immunology , TNF Receptor-Associated Factor 3 , Allergy and Immunology , Metabolism , Trans-Activators , Allergy and Immunology , MetabolismABSTRACT
BACKGROUND: A major pathway for protein degradation in eukaryocytes is ubiquitin-dependent. A novel species of plant and mammalian E2 homologous to yeast UBC4/UBC5 is involved in polyubiquitination and degradation of off and many other proteins as well. METHODS: By sequencing the Expressed Sequence Taqs(ESTs) of human dermal papilla cDNA library, we isolated a clone, named K183 which showed high homology to the yeast UBC4/UBC5. We designated this gene as UBC-dp. RESULTS: K183 clone is 1,222 nucleotides long, and has a coding region of 622 nucleotides and a 3' noncoding region of 538 nucleotides. The presumed open reading frame starting at the 5' terminus of UBC-dp encodes 207 amino acids. The amino acid sequence deduced from the open reading frame of UBC-dp shares 81%, 80% and 80% identities with that of HSUBCH5. yeast UBC4 and yeast UBC5, respectively. The transcripts were ubiquitously expressed in a variety of human tissues. The levels of transcript were relatively high in those tissues such as skeletal muscle, heart, testis and ovary. CONCLUSIONS: Homology search result suggests that K183 clone is human homolog of the UBC4 and UBC5 which are involved in p53 degradation so its function related with p53 should be studied.