JPST000115-1 Identification of ubiquitylation sites by K0-Ub [Reanalysis: JPST000115]
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ABSTRACT:
ORGANISM(S): Hek293t
SUBMITTER: Yasushi Ishihama
PROVIDER: RPXD034664 | JPOST Repository | Sat Mar 25 00:00:00 GMT 2023
REPOSITORIES: jPOST
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2022-01-09 | GSE124890 | GEO
Project description:Given our laboratory interest in IFITM3 S-fatty-acylation and antiviral activity, we sought to directly characterize fatty acids that are covalently attached to the Cys residues of IFITM3. IFITM3 comprises two S-fatty-acylation sites (C71 and C72) in proximity of a intramembrane domain, and another site adjacent to a transmembrane domain (C105). We directly identified the S-fatty-acylation sites of IFITM3 and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid.
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Project description:Given our laboratory interest in IFITM3 S-fatty-acylation and antiviral activity, we sought to directly characterize fatty acids that are covalently attached to the Cys residues of IFITM3. IFITM3 comprises two S-fatty-acylation sites (C71 and C72) in proximity of a intramembrane domain, and another site adjacent to a transmembrane domain (C105). We directly identified the S-fatty-acylation sites of IFITM3, and further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid.
2018-03-28 | PXD008401 | Pride
Project description:Pupylation is a posttranslational protein modification in bacteria resembling ubiquitination in eukaryotes. The prokaryotic ubiquitin-like protein Pup is covalently attached to other proteins via an isopeptide bond between its carboxyterminal glutamate residue and a lysine residue in the target. In mycobacteria, pupylation was shown to mark proteins for unfolding by the ATPase Mpa and subsequent degradation by the proteasome. However, the occurrence of pupylation in species without a proteasome like Corynebacterium glutamicum suggests that degradation may not be the only fate of pupylated proteins. The Îpup mutant senses a stronger iron limitation than the wild type. Among the 125 genes showing at least 2-fold changes in transcript levels in the Îpup mutant (p-value ⤠0.05) were 54% of all genes known to be regulated by the master regulator of iron homeostasis DtxR (Brune et al., 2006; Wennerhold and Bott, 2006). Except for ftn, which is activated by DtxR and showed a 2-fold decreased mRNA level, the other DtxR target genes showed increased mRNA levels in the Îpup strain. These included ripA, encoding a transcriptional regulator of iron proteins, which represses a number of prominent iron-containing proteins under iron limitation, such as aconitase or succinate dehydrogenase (Wennerhold et al., 2005). 79% of the known RipA target genes showed decreased mRNA levels in the Îpup strain. DNA microarray analyses were performed to compare the mRNA levels of the C. glutamicum Îpup mutant and its parent wild type under iron-limited conditions. The two strains precultivated in CGXII medium with 4% (w/v) glucose and 1 µM FeSO4 were inoculated into fresh medium to an OD600 of 1, cultured for 2 h, and harvested on ice by centrifugation (5 min at 4,000 g and 4°C). Please note that the GPL16989 array design comprises oligos for 4 different bacterial genomes. In the GPR-files in this study, all IDs/Names (oligonucleotides) which are not from the host C. glutamicum were replaced by the text EMPTY since only C. glutamicum expression was analyzed.
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Project description:The mitochondrial kinase PINK1 and the cytosolic ubiquitin ligase (E3) Parkin is involved in mitochondrial quality control. PINK1, which phosphorylates ubiquitin at serine 65 residue and the ubiquitin-like (Ubl) domain of Parkin at serine 65 residue, promotes the Parkin activation and translocation to damaged mitochondria. When Parkin is activated, the Ubl domain are ubiquitinated at lysine 27 (K27) and lysine 48 (K48) residues. It remains unclear whether K27/K48 ubiquitination contributes to Parkin activity. In this study, we blocked the ubiquitination of K27 and/or K48 residues to examine the effects of the Parkin Ubl ubiquitination in Drosophila. Parkin replaced K27 with arginine (K27R) rescued the lethality of flies by PINK1 and Parkin co-expression whereas K48 replacement with arginine did not. Parkin K27R exhibited less ability of mitochondrial fragmentation and mitochondrial arrest, which are mediated by the degradation of Parkin E3 substrates Mitofusin and Miro, respectively. The pathogenic K27N Parkin, unlike K27R Parkin, was found to be completely devoid of E3 activity, suggesting not only that K27N Parkin is not ubiquitin-modified at the K27 residue but also that the structure of the Ubl domain itself is largely affected. Ubiquitin attached to the K27 residue of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because K27R Parkin showed less ability of Parkin self-association upon the reduction of the mitochondrial membrane potential. Our study suggests a new Parkin activation mechanism where an activating complex for Parkin is formed through phospho-ubiquitin attached to the K27 residue of the Ubl domain.
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Project description:Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline residues on histone tails. This activity has been linked to the repression of gene transcription on a limited number of genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 cells. Results showed that PADI4 is enriched in the gene promoter regions near the transcription start sites (TSSs) and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. The expression of two well characterized Elk-1 target genes, c-fos and egr-1, was then found to be inhibited following treatment of MCF-7 cells with a PADI4 specific inhibitor. The inhibitor also significantly reduced levels of acetylation at H4 lysine 5 at these promoters suggesting that the activating function of PADI4 at these target genes is mediated, in part, by interplay between histone citrullination and HAT-mediated acetylation. These findings greatly expand our knowledge of the role of PADI4 in gene regulation by defining a new role for PADI4 catalyzed histone citrullination in mediating gene transactivation. Two PADI4 ChIP-chip biological replicates from MCF-7 human breast cancer cells are included.
2011-03-31 | E-GEOD-18755 | biostudies-arrayexpress
Project description:Hepatocyte nuclear factor 1α (HNF1α) play essential roles in controlling development and metabolism, its mutations are clearly linked to the occurrence of maturity-onset diabetes of the young (MODY3) in human. 117 lysine to glutamic acid (E117) mutation in the HNF1α gene was clinically associated with MOYD3, but no functional data on this variant is available. Here, we generated a knock-in mouse model in which the lysine 117 is replaced with glutamic acid in the endogenous murine HNF1α locus. The HNF1αE117/E117 mice reproduced HNF1-null phenotypes including dwarfism, hepatic dysfunction, renal Fanconi syndrome, diabetes and die from a progressive wasting syndrome, but no hyperphenylalaninemia. Moreover, HNF1αE117/E117 mice shared similar hepatic gene expression profiles with HNF1 deficient mice. In vitro experiments showed that HNF1 mutants in which the lysine 117 residue were replaced by glutamic acid, arginine or glutamine residues, respectively, had a significant decreased in overall transactivation and DNA binding capacity. K117 mutants had no effect on its protein stabilities and nuclear localization, but significantly blocked its own association. Our results clearly demonstrate that the lysine 117 residue is essential for the function of the HNF1a, and prove important to identify the molecular basis for diseases associated with defects in HNF1α We then performed gene expression profiling analysis using data obtained from RNA-seq of liver tissue of 3 WT mice and 3 KI mice.
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Project description:Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function. UHRF1 protein was depleted in HCT116 cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-regulated genes were identified by comparing the gene expression profiles of control and UHRF1-depleted HCT116 cells.
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