<em>Zea mays</em> Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

<div><p><em>Zea mays</em> (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as <em>in vivo</em> assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.</p> </div

ZmTaxilin can repress the transcriptional activity of ZmO2.

<p>(<b>A</b>) Structure of effecter and reporters. (<b>B</b>) GUS/luciferase values of different reporter and effecter combinations. The values are the averages with SD of three independent experiments, after normalisation to the internal control. Statistical significance between YFP-O2 and YFP-O2+CFP-Taxilin was calculated using a two-tailed T-test. *p≤0.05.</p

ZmTaxilin and ZmO2 interact in a GST pull-down assay.

<p>(<b>A</b>) Western blot detection of the GST pull-down sample with a GST antibody. The kernel sample was a pool of equal amounts of RNA from different developmental stages between 3 and 36 days after pollination (DAP). (<b>B</b>) Western blot detection of the GST pull-down sample with the ZmO2 antibody. Lane 1 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing the GST-Taxilin protein and the maize seed protein containing ZmO2. Lane 2 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing GST and maize seed protein containing ZmO2. The expected molecular weight of the GST-Taxilin fusion protein, the GST tag and ZmO2 are 75.397, 27.895 and 47.075 kDa, respectively. The apparent molecular weight of the ZmO2 protein was approximately 68–72 kDa.</p

<i>ZmTaxilin</i> clones identified by yeast two-hybrid.

<p><i>ZmTaxilin</i> clones identified by yeast two-hybrid.</p

Computational analyses of obesity associated loci generated by genome-wide association studies

<div><p>Objectives</p><p>Genome-wide association studies (GWASs) have discovered associations of numerous SNPs and genes with obesity. However, the underlying molecular mechanisms through which these SNPs and genes affect the predisposition to obesity remain not fully understood. Aims of our study are to comprehensively characterize obesity GWAS SNPs and genes through computational approaches.</p><p>Methods</p><p>For obesity GWAS identified SNPs, functional annotation, effects on miRNAs binding and impact on protein phosphorylation were performed via RegulomeDB and 3DSNP, miRNASNP, and the PhosSNP 1.0 database, respectively. For obesity associated genes, protein-protein interaction network construction, gene ontology and pathway enrichment analyses were performed by STRING, PANTHER and STRING, respectively.</p><p>Results</p><p>A total of 445 SNPs are significantly associated with obesity related phenotypes at threshold <i>P</i> < 5×10⁻⁸. A number of SNPs were eQTLs for obesity associated genes, some SNPs located at binding sites of obesity related transcription factors. SNPs that might affect miRNAs binding and protein phosphorylation were identified. Protein-protein interaction network analysis identified the highly-interconnected “hub” genes. Obesity associated genes mainly involved in metabolic process and catalytic activity, and significantly enriched in 15 signal pathways.</p><p>Conclusions</p><p>Our results provided the targets for follow-up experimental testing and further shed new light on obesity pathophysiology.</p></div

Effect of obesity lead SNPs and proxy SNPs on protein phosphorylation.

<p>Effect of obesity lead SNPs and proxy SNPs on protein phosphorylation.</p

The workflow of obesity associated loci derived from GWAS.

<p>Data were obtained from the NCBI Association Results Browser (<a href="http://www.ncbi.nlm.nih.gov/projects/gapplusprev/sgap_plus.htm" target="_blank">http://www.ncbi.nlm.nih.gov/projects/gapplusprev/sgap_plus.htm</a>) and HuGE Navigator (<a href="http://64.29.163.162:8080/HuGENavigator/startPagePhenoPedia.do" target="_blank">http://64.29.163.162:8080/HuGENavigator/startPagePhenoPedia.do</a>) and analyzed by many tools.</p

SNPs located in the binding sites of obesity related transcription factors.

<p>SNPs located in the binding sites of obesity related transcription factors.</p

Protein-protein interaction network of obesity GWAS associated genes.

<p>The nodes and edges represent the proteins (genes) and their interactions, respectively. Colored nodes represent query proteins and first shell of interactors, white nodes represent second shell of interactors, empty nodes represent proteins of unknown 3D structure, filled nodes represent some 3D structure is known or predicted. Purple edges (experimentally determined) and light blue edges (from curated databases) represent known interactions, green edges (gene neighborhood), red edges (gene fusions) and dark blue edges (gene co-occurrence) represent predicted interactions, yellow-green edges (textmining), black edges (co-expression) and light blue edges (protein homology) represent others.</p