Identification of an extended half‐site motif required for the function of peroxisome proliferator‐activated receptor α

Osada, Shiho; Tsukamoto, Tomoko; Takao, Masaki; Mori, Masataka; Osumi, Takashi

doi:10.1046/j.1365-2443.1997.1220319.x

Cited by 45 publications

(47 citation statements)

References 55 publications

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“…Plasmids-The expression vector of full-length PPAR␣ was pNCM-VPPAR␣ (11), constructed by replacing the lacZ gene in the cytomegalovirus promoter-driven vector pCMV␤ (24) with mouse PPAR␣ cDNA. A deletion construct lacking the A/B region was created by inserting a double-strand linker composed of 5Ј-TGTCGAATATGTGGGGA-CAAGG-3Ј and 5Ј-CCTTGTCCCCACATATTCGACACATG-3Ј between the BstXI site encompassing the initiation codon and the StuI site in the early portion of the C domain-encoding region.…”

Section: Methodsmentioning

confidence: 99%

“…For assaying the transcriptional activation by PPAR constructs containing the C region, a reporter vector, pAOXPPREluc (11), containing the PPAR-binding site and the basal promoter derived from the rat acyl-CoA oxidase gene was used (25). For assaying the activation by Gal4 fusion constructs, tk-GALpx3-luc, containing three copies of the Gal4-binding sites (upstream activation site (UAS)) and the herpes simplex virus thymidine kinase promoter, was mostly used.…”

Section: Methodsmentioning

confidence: 99%

“…PPAR recognizes the 5Ј-half-site, whereas the retinoid X receptor recognizes the 3Ј-half-site (11)(12)(13)(14). Moreover, for optimal binding, PPAR requires an extended half-site constituted by the AGGTCA hexanucleotide motif and four extra residues on the 5Ј-side (11,13,14).…”

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confidence: 99%

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Characterization of the Amino-terminal Activation Domain of Peroxisome Proliferator-activated Receptor α

Hi¹,

Osada²,

Yumoto³

et al. 1999

Journal of Biological Chemistry

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The transactivating function of the A/B region of mouse peroxisome proliferator-activated receptor ␣ (PPAR␣; NR1C1) was characterized. The truncated version of PPAR␣ lacking the A/B region had 60 -70% lower transactivating function than full-length PPAR␣ in both the presence and absence of the peroxisome proliferator ciprofibrate. When tethered to the yeast Gal4 DNA-binding domain, the A/B region exhibited the significant ligand-independent transactivating function, AF-1 activity. The first 44 amino acid residues were necessary for maximal transactivation, and the minimally essential region was further delimited to amino acids 15-44. This region is highly enriched with acidic residues, but mutational analyses showed that the protein structure, rather than the negative charge itself, was important for the AF-1 activity. An ␣-helical configuration was predicted for this region, and a CD spectrum analysis of the synthetic peptides showed that mutant sequences with higher AF-1 activity have higher helical contents and vice versa. The most active mutant, in which Met 31 was replaced with Leu, was ϳ5-fold more potent than the wild-type A/B region. These findings indicate that the AF-1 region of PPAR␣ is an acidic activation domain and that the helix-forming property is implicated in the transactivating function.Peroxisome proliferator-activated receptors (PPARs) 1 constitute a subfamily (NR1C, according to the unified nomenclature (1)) of nuclear hormone receptors. Since the first cloning of PPAR␣ (NR1C1) from mouse (2), three isoforms (␣, ␤ (or ␦), and ␥) have been identified in many organisms, including human, rat, and Xenopus (for review, see Ref.3). Many studies suggest that PPAR␣ regulates the fatty acid metabolism by controlling the expression of the genes involved in fatty acid oxidation as well as lipoprotein subunits, positively or negatively (3). On the other hand, PPAR␥ (NR1C3) seems to govern more versatile physiological processes such as adipocyte differentiation (4), inflammatory response (5, 6), and maturation of macrophages into foam cells (7,8). Very recently, evidence showing the involvement of PPAR␦ (NR1C2) in embryo implantation was presented (9).PPAR recognizes direct repeat motifs consisting of two AG-GTCA or related half-sites separated by a single nucleotide (DR-1) through heterodimerization with another nuclear hormone receptor, the retinoid X receptor (NR2B) (10). PPAR recognizes the 5Ј-half-site, whereas the retinoid X receptor recognizes the 3Ј-half-site (11-14). Moreover, for optimal binding, PPAR requires an extended half-site constituted by the AGGTCA hexanucleotide motif and four extra residues on the 5Ј-side (11,13,14). This is in contrast to other nuclear receptors forming heterodimers with the retinoid X receptor, e.g. the retinoic acid receptor (NR1B), thyroid hormone receptor (NR1A), and vitamin D receptor (NR1I1) (15).Many compounds have been identified as ligands of PPAR (3). The fibrate class of peroxisome proliferators and leukotriene B 4 are relatively selective for PPAR␣, whe...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of the Amino-terminal Activation Domain of Peroxisome Proliferator-activated Receptor α

Hi¹,

Osada²,

Yumoto³

et al. 1999

Journal of Biological Chemistry

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“…For the detection of endogenous CGI-58, differentiated 3T3-L1 cells (day 3) were double immunostained with antiperilipin and affinity-purified anti-CGI-58, followed by appropriate Cy3-or fluorescein isothiocyanate-labeled secondary antibodies (Jackson ImmunoResearch). CHO-K1 cells were transfected by a calcium phosphate method as described previously (29). Cells were fixed with cold methanol for 2 min and then treated as described above.…”

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confidence: 99%

CGI-58 Interacts with Perilipin and Is Localized to Lipid Droplets

Yamaguchi

Omatsu

Matsushita

et al. 2004

Journal of Biological Chemistry

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236

110

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Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. In this study, we used a yeast two-hybrid system to examine the potential function of perilipin. We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP.

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“…Studies on PPARα binding to nuclear receptor half-sites have established a precedent for such a model [35]. Gel retardation assays using the mouse G6pc promoter sequence between −62 and −29, which includes both the MUT 3 and MUT 1 regions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Sequence variation between the mouse and human glucose-6-phosphatase catalytic subunit gene promoters results in differential activation by peroxisome proliferator activated receptor gamma coactivator-1α

et al. 2008

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Identification of an extended half‐site motif required for the function of peroxisome proliferator‐activated receptor α

Cited by 45 publications

References 55 publications

Characterization of the Amino-terminal Activation Domain of Peroxisome Proliferator-activated Receptor α

Characterization of the Amino-terminal Activation Domain of Peroxisome Proliferator-activated Receptor α

CGI-58 Interacts with Perilipin and Is Localized to Lipid Droplets

Sequence variation between the mouse and human glucose-6-phosphatase catalytic subunit gene promoters results in differential activation by peroxisome proliferator activated receptor gamma coactivator-1α

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