A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.
Abstract-Using a vaccine approach, we immunized New Zealand White rabbits with a peptide containing a region of cholesteryl ester transfer protein (CETP) known to be required for neutral lipid transfer function. These rabbits had significantly reduced plasma CETP activity and an altered lipoprotein profile. In a cholesterol-fed rabbit model of atherosclerosis, the fraction of plasma cholesterol in HDL was 42% higher and the fraction of plasma cholesterol in LDL was 24% lower in the CETP-vaccinated group than in the control-vaccinated group. Moreover, the percentage of the aorta surface exhibiting atherosclerotic lesion was 39.6% smaller in the CETP-vaccinated rabbits than in controls. The data reported here demonstrate that CETP activity can be reduced in vivo by vaccination with a peptide derived from CETP and support the concept that inhibition of CETP activity in vivo can be antiatherogenic. In addition, these studies suggest that vaccination against a self-antigen is a viable therapeutic strategy for disease management. (Arterioscler
The acceleration of atherosclerosis by polygenic (essential) hypertension is well-characterized in humans; however, the lack of an animal model that simulates human disease hinders the elucidation of pathogenic mechanisms. We report here a transgenic atherosclerosis-polygenic hypertension model in Dahl salt-sensitive hypertensive rats that overexpress the human cholesteryl ester transfer protein (Tg[hCETP]DS). Male Tg[hCETP]DS rats fed regular rat chow showed age-dependent severe combined hyperlipidemia, atherosclerotic lesions, myocardial infarctions and decreased survival. These findings differ from various mouse atherosclerosis models, demonstrating the necessity of complex disease modeling in different species. The data demonstrate that cholesteryl ester transfer protein can be proatherogenic. The interaction of polygenic hypertension and hyperlipidemia in the pathogenesis of atherosclerosis in Tg[hCETP]DS rats substantiates epidemiological observations in humans.
Bacteriophage T4 gene 32 encodes a singlestranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A+T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the X leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being derepressed at g32P concentrations that repress the wild-type gene 32 transcripts.The Mr 34,387 bacteriophage T4 gene 32 product (g32P) has served as a prototype for a class of proteins that bind tightly and cooperatively to single-stranded (ss) DNA and that in vitro stimulate the activity of their cognate DNA polymerase. In vivo, g32P has been shown to be required in stoichiometric amounts for DNA replication, repair, and recombination (for review see ref. 1). Physicochemical studies on g32P and its complexes with ss DNA are beginning to provide insight into general mechanisms for the binding of proteins to singlestranded nucleic acids. In the case of g32P, both nitration (2) and 1H-NMR (3, 4) studies indicate that five tyrosine side chains are involved in binding to ss DNA. Since one phenylalanine and three tyrosine residues have been implicated (5) in the binding of the bacteriophage fd gene 5 protein to ss DNA, it appears that hydrophobic interactions between the side chains of aromatic amino acids and the bases of a polynucleotide may represent a common binding mechanism for several ss DNA binding proteins (4).To identify which tyrosine residues in the known g32P sequence (6) are at the interface of the g32P-ss DNA complex, we have cloned gene 32 so that it could be subjected to in vitro oligodeoxynucleotide site-directed mutagenesis. Site-specific mutagenesis provides the opportunity to sequentially substitute nonaromatic amino acids for each tyrosine residue in g32P. 1H-NMR and nucleic acid binding experiments on the resulting mutant proteins can then be used to determine the contribution of each of the tyrosine residues that had been replaced or, for that matter, any other g32P amino acids to the overall free energy of binding of the protein to ss DNA.Previous attempts to clone gene 32 have failed presumably due to a deleterious effect of g32P on host cell viability (7). Even if this problem were avoided by placing gene 32 under the control of an inducible promoter, in vitro studies indicate that overexpression of g32P w...
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