O-GlcNAc modification of Sp1 inhibits the functional interaction between Sp1 and Oct1

AbstractSp1 is a ubiquitous transcription factor that is modified by multiple O-linked N-acetylglucosamines (O-GlcNAc). Previously, O-GlcNAcylation of a specific site of Sp1 was shown to inhibit Sp1 transcriptional activity. Yet, how O-GlcNAc on other modification sites affects Sp1 function and how O-GlcNAcylation of Sp1 affects the transcriptional regulation of a target gene remains unknown. Here we show that O-GlcNAc within the second serine/threonine-rich region of Sp1 interrupts a known interaction between Sp1 and Oct1, and inhibits the cooperative activation of the U2 snRNA gene by Sp1 and Oct1.Structured summaryMINT-6803452: Sp1 (uniprotkb-P08047) physically interacts (MI:0218) with Oct1 (uniprotkb:P14859) by anti tag coimmunoprecipitation (MI:0007)MINT-6803426, MINT-6803438: Oct1 (uniprotkb:P14859) binds (MI:0407) to Sp1 (uniprotkb:P08047) by pull down (MI:0096)MINT-6803470, MINT-6803484: Sp1 (uniprotkb:P08047) physically interacts (MI:0218) with Oct1 (uniprotkb:P14859) by anti bait coimmunoprecipitation (MI:0006

Cyanidin is an agonistic ligand for peroxisome proliferator-activated receptor-alpha reducing hepatic lipid

a b s t r a c t a r t i c l e i n f o To investigate the underlying mechanism of targets of cyanidin, a flavonoid, which exhibits potent anti-atherogenic activities in vitro and in vivo, a natural chemical library that identified potent agonistic activity between cyanidin and peroxisome proliferator-activated receptors (PPAR) was performed. Cyanidin induced transactivation activity in all three PPAR subtypes in a reporter gene assay and time-resolved fluorescence energy transfer analyses. Cyanidin also bound directly to all three subtypes, as assessed by surface plasmon resonance experiments, and showed the greatest affinity to PPARα. These effects were confirmed by measuring the expression of unique genes of each PPAR subtype. Cyanidin significantly reduced cellular lipid concentrations in lipid-loaded steatotic hepatocytes. In addition, transcriptome profiling in lipid-loaded primary hepatocytes revealed that the net effects of stimulation with cyanidin on lipid metabolic pathways were similar to those elicited by hypolipidemic drugs. Cyanidin likely acts as a physiological PPARα agonist and potentially for PPARβ/δ and γ, and reduces hepatic lipid concentrations by rewiring the expression of genes involved in lipid metabolic pathways

Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1

The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 10(3) transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts

SJ: The natural carotenoid astaxanthin, a PPAR-alpha agonist and PPAR-gamma antagonist, reduces hepatic lipid accumulation by rewiring the transcriptome in lipid-loaded hepatocytes. Mol Nutr Food Res 2012

Scope: A natural carotenoid abundant in seafood, astaxanthin (AX), has hypolipidemic activity, but its underlying mechanisms of action and protein targets are unknown. We investigated the molecular mechanism of action of AX in hepatic hyperlipidemia by measuring peroxisome proliferator-activated receptors (PPAR) activity. Methods and results: We examined the binding of AX to PPAR subtypes and its effects on hepatic lipid metabolism. AX binding activated PPAR-␣, but inhibited PPAR-␥ transactivation activity in reporter gene assay and time-resolved fluorescence energy transfer analyses. AX had no effect on PPAR␦/␤ transactivation. AX bound directly to PPAR-␣ and PPAR-␥ with moderate affinity, as assessed by surface plasmon resonance experiments. The differential effects of AX on PPARs were confirmed by measuring the expression of unique responsive genes for each PPAR subtype. AX significantly reduced cellular lipid accumulation in lipid-loaded hepatocytes. Transcriptome analysis revealed that the net effects of stimulation with AX (100 M) on lipid metabolic pathways were similar to those elicited by fenofibrate and lovastatin (10 M each), with AX rewiring the expression of genes involved in lipid metabolic pathways. Conclusion: AX is a PPAR-␣ agonist and PPAR-␥ antagonist, reduces hepatic lipid accumulation by rewiring the transcriptome in lipid-loaded hepatocytes