SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative.
The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.
Rishi
Vikas
V
Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Gal
Jozsef
J
Krylov
Dmitry
D
Fridriksson
Jakob
J
Boysen
Maria Sandberg
MS
Mandrup
Susanne
S
Vinson
Charles
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D018257:Helix-Loop-Helix Motifs; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D051780:Sterol Regulatory Element Binding Protein 1; D013816:Thermodynamics; D014157:Transcription Factors; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors