Structural insights into a HECT-type E3 ligase AREL1
Ontology highlight
ABSTRACT:
INSTRUMENT(S): TripleTOF 5600
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Sunil Singh
LAB HEAD: Prof. J. Sivaraman
PROVIDER: PXD016016 | Pride | 2019-11-25
REPOSITORIES: Pride
Publications
Structural insights into a HECT-type E3 ligase AREL1 and its ubiquitination activities <i>in vitro</i>.
Singh Sunil S Ng Joel J Nayak Digant D Sivaraman J J
The Journal of biological chemistry 20191115 52
The HECT E3 ligase family comprises three subfamilies: NEDD4 E3 ubiquitin protein ligase (NEDD4), HECT and RLD domain-containing E3 ubiquitin protein ligase (HERC), and "other." Most previous studies have focused on the NEDD4 subfamily. Apoptosis-resistant E3 ligase 1 (AREL1) belongs to "other" subfamily HECT that inhibits apoptosis by ubiquitinating and degrading proapoptotic proteins. Here, we report the crystal structure of the extended HECT domain of AREL1 (amino acids (aa) 436-823) at 2.4 Å ...[more]
Similar Datasets
Project description:Preparation of proteins from salt-gland-enriched tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant’s unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining sufficient proteins from salt gland-enriched tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Despite the lower protein yield obtained, one-dimensional protein gel profiles with greater resolution could be obtained, with more than twice the number of proteins detected when 1D-LC-MS/MS analyses were compared. Further analysis of proteins that were solely present in each extraction method favoured the phenol-based extraction. Phenol-based extracts contained nearly 10 times more solely-detected proteins than those were detectable in the extracts without using phenol. The protocol established could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent.
2015-07-17 | PXD001691 | Pride
Project description:Plant salt glands are nature’s desalination devices that harbour potentially useful information pertaining to salt and water transport during secretion. As part of the program toward deciphering secretion mechanisms in salt glands, we adopted a shotgun approach to look into the proteome of salt-gland enriched tissues of the mangrove tree species Avicennia officinalis. To achieve this, we isolated the adaxial epidermal peels (which harbour the salt glands), and separated them from the mesophyll tissues of the leaves. Three biological replicates were prepared and total proteins were extracted from these tissues. Proteins that were found to be unique in salt gland-enriched tissues were obtained by eliminating proteins found in the mesophyll tissues. The proteins that are uniquely present in salt gland-enriched tissues were then selected and categorized by GO analysis.
2015-07-22 | PXD000771 | Pride
Project description:We used tobramycin-affinity RNA-pulldown assays coupled with mass spectrometry to identify CK1α 5'-UTR-binding proteins.
2022-10-01 | PXD027124 | Pride
Project description:Plasmodium falciparum, the most pathogenic human malaria parasite, infects millions of human beings and causes a serious public health threat. Currently, the most potent anti-malarial drugs are artemisinin and its derivatives1,2. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge3. The activation of artemisinin requires the cleavage of the endoperoxide bridge in the presence of iron sources4. Once activated, artemisinins are converted into highly reactive carbon-centered radicals5,6 that attack macromolecules through alkylation and propagate a series of damages, eventually leading to parasite death7,8. Even though several parasite proteins have been reported as the targets of artemisinin9,10, the exact mechanism of artemisinin action is still controversial and its high potency and specificity against the malaria parasite could not be fully accounted. Here, we have developed an unbiased chemical proteomics approach to directly probe the mechanism of action of artemisinin in P. falciparum in situ. An alkyne-tagged artemisinin analogue coupled with biotin enables selective purification and identification of 124 artemisinin covalent-binding protein targets, many of which are involved in essential biological processes of the parasite. In vitro assays confirm the specific artemisinin binding and inhibition of selected targets. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using the alkyne-tagged artemisinin coupled with a fluorescent dye to monitor its protein binding, we showed that heme, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The extremely high level of heme released from the hemoglobin digestion by the parasite make artemisinin exceptionally potent against late-stage parasites (trophozoite and schizont stages) compared to parasites at early ring stage, which have low level of heme, mainly from endogenous synthesis. The ‘blood eating’ nature of the parasite with the release of large amounts of heme confers artemisinin with extremely high specificity against the parasites, with minimum side effects towards healthy red blood cells. Taken together, our results established a unifying model to explain the action and specificity of artemisinin in parasite killing. Our findings could facilitate the development of better alternative strategies to treat malaria in times of emerging artemisinin resistance11,12.
2015-12-17 | PXD002611 | Pride
Project description:Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some non-essential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation, the global protein synthesis is significantly reduced mainly due to suppression of mechanistic target of rapamycin (MTOR), there is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal non-canonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne bearing biotin and AHA’s bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 711 proteins were identified. The characterized functional profiles of these newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the pro-survival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy and cell death. Thus, the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.
2016-10-18 | PXD003546 | Pride
Project description:Alternative splicing (AS) has a significant potential to impact on the eukaryotic cell transcriptome and proteome. However, the extent of this influence as well as mechanisms, providing the tissue-specific pattern of AS are poorly studied. Here, we analyzed the AS of two life forms, protonema and gametophores, as well as the protoplasts, of a model organism, the Physcomitrella patens moss, using the data of transcriptome and proteome profiling. Altogether, 12,043 genes subjected to alternative splicing were identified. The alternative splicing patterns of 302 genes involved in stress response, growth, and development are changed considerably in the process of the gametophore development, whereas AS of 276 genes involved in transcription regulation, development, cell interactions, and cell response to biotic and abiotic stresses altered upon protoplastitation. Using mass spectrometry analysis, we studied the extent to which AS contributes to proteome diversity and identified 117 isoform-specific peptides for 36 genes with AS events. Our results indicate that AS event have a little impact on the proteome diversity and mostly result in the addition of extra amino acids rather than editing of existing proteins sequences. Among differentially expressed and spliced genes we found serine/arginine-rich (SR) genes, which are known to regulate AS in cells. We identified new isoforms of these genes and data evidencing their translation were obtained at transcriptome and proteome level. We also found that treatment with abscisic and jasmonic acids led to the isoform-specific response in protonema. Besides, we revealed the potential lncRNAmRNA and lncRNApre-mRNA interactions that can influence both the expression and alternative splicing of genes. It can point at an additional level of gene expression and AS regulation by non-coding transcripts in the Physcomitrella patens moss.
2018-01-15 | PXD005223 | Pride
Project description:Candida albicans, a major opportunistic fungal pathogen is frequently found together with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Previous studies have demonstrated that S. mutans and C. albicans synergizes virulence of plaque-biofilms in vivo. However, the nature of this bacterial-fungal relationship in this cross-kingdom biofilm remains largely uncharacterized. Using iTRAQ based quantitative proteomics, we found that proteins associated with carbohydrate metabolism such as alpha-1,4 glucan phosphorylase, Hexokinase-2, Isocitrate lyase and malate synthase were significantly upregulated in C. albicans in the mixed-species biofilms (P<0.05). C. albicans proteins associated with growth/morphogenesis such as pH-responsive protein-2, Fma1p and Hsp21 were also induced. Conversely, S. mutans proteins in the tricarboxylic acid cycle such as citrate synthase and in the pentose phosphate pathway such as Ribose-5-phosphate isomerase A as well as proteins associated with sugar transport systems were upregulated indicating enhanced carbohydrate metabolism. Interestingly mixed-species biofilm microenvironment had a lower pH than S. mutans single-species biofilms. This observation was supported by proteomics, wherein proteins associated with lactate and formate assimilation such as Glyoxalase and putative NADPH-dependent methylglyoxal reductase proteins were significantly upregulated in the mixed-species biofilms (P<0.05). Furthermore, we unexpectedly found that S. mutans derived glucosyltransferase B (GtfB), responsible for co-adhesion via glucans, can also contribute to C. albicans growth and carbohydrate metabolism by providing glucose and fructose from sucrose breakdown. These findings demonstrate synergistic bacterial-fungal interactions within mixed-species biofilms and a novel GtfB cross-feeding role. Taken together, quantitative proteomics provides new insights into this virulent cross-kingdom oral biofilm.
2019-07-30 | PXD010261 | Pride
Project description:iTRAQ-based comparative proteomic analysis on the critical node of viability loss in wheat seeds with obvious divergence in storability.
2021-09-25 | PXD025860 | Pride
Project description:In this study, we carried out a large-scale proteome profiling of human mitral valve tissues resected from patients with mitral valve prolapse.
2015-05-07 | PXD000774 | Pride
Project description:Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119 using an improved version of biotin ligase BioID2. The BioID2 tag was fused to either the N-terminus (3HA-BioID2-TbUnc119) or the C-terminus (TbUnc119-BioID2-HA) of TbUnc119 and expressed using a cumate-inducible expression system in the procyclic cells of T. brucei. We performed LC-MS/MS on the affinity-purified biotinylated samples followed by evaluation of emPAI score.
2020-06-25 | PXD019488 | Pride