Abstract
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.
Highlights
From the ‡Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892, §Gene Logic Inc., Gaithersburg, Maryland 20878, and the ¶Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, Campusvej 55, 5230 Odense M, Denmark
A-sterol regulatory element-binding protein (SREBP)-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region
Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF
Summary
The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequencespecific DNA to regulate the expression of genes involved in lipid metabolism. Under conditions of low sterol concentrations, SREBP cleavage-activating protein escorts SREBP from the endoplasmic reticulum to the Golgi apparatus, where Site-1 protease and Site-2 protease proteolytically cleave the inactive SREBP This releases the N-terminal fragment containing the B-HLH-ZIP domain that enters the nucleus and binds to its cognate DNA binding site as a dimer [2]. SREBPs bind to a 10-bp sequence (5Ј-ATCACCCCAC-3Ј) known as the sterolregulatory element (SRE) [5] This dual DNA binding specificity is due to the presence of a tyrosine in place of an arginine found in other B-HLH-ZIP transcription factors [6]. We show that A-SREBP-1 is effective and specific in co-transfection assays at inhibiting the activity of either SREBP-1 or SREBP-2
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
