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RISS 인기검색어
- 검색키워드
- 저자 : (Robert R . Rando) (검색결과 3 건)
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Inhibitors of the Enzymes Related Post-Translational Modification
Lim, Young Hee,Chen, Yulong,Rando, Robert R 中央大學校 食糧資源硏究所 1997 食糧資源硏究所 論文集 Vol.4 No.-
The membrane-associated isoprenylated protein endoprotease and carboxymethy- transferase are of sunstantial interest because specific inhibitors of those block the processing and functioning of ras in vivo. Specific inhibitors of this type should allow for the identification and cloning of these enzymes, which are important for signal transduction. Potent and specific affinity labeling agent containing chloromethyl ketone have been developed. Irreversible chloromethyl ketone inhibitors, N-biotinyl-S-farnesyl-L-cystein chlorometyl ketone (Biotin- FCCMK) and N-t-Boc-S-farnesyl-L-cystein chloromehyl ketone(BFCCMK), and a reversible inhibitor,N-Boc-S-farnesyl-L-cysteinyl-y(CH2-NH)-valy-L-isoleucyl-L-methioni ne (RPI) have been designed and synthesized. These inhibitors inhibited the activity of a isoprenylated protein endoprotease. Furthermore, The biotin-tagging affinity labeling agent, Biotin-FCCMK was utilized to identify a polypeptide of 37 KDa which is believed for the endoprotease or as a catalytic domain of this enzyme. For isoprenylated protein carboxymethyltransferase, analogs of N-acetyl-S-farnesyl-L-cysteine (AFC) and S-farnesylthioacetic acid (FTA) were synthesized. These inhibitors inhibited the enzyme activity and inhibited the cell growth when applied to the cancer cells. The isoprenylation of proteins is an important hydrophobic posttranslational modification, which is thought to target the modified protein to a membrane(1). This modification is recognized to be critical for the fuction of the large class of proteins that it affect(2-4). Isoprenylated proteins are posttranslationally modified at the C-terminal CAAX box, or less frequently at either a CXC or a CC sequence, by either a geranylgeranyl or a farnesyl moiety(5-7). In the case of CAAX containing proteins, endoproteolysis occurs to remove AAX, followed by the reversible S-adenosyl-L- methione (AdoMet) dependent methylation of the newly formed isoprenylated cysteine C-terminus (Scheme I). These modifications allow isopreylated/methylated proteins to bind to membranes efficiently(8). Among these modification steps, farnesylation is believed to be essential for activity, since ras mutants unable to undergo farnesylation are inert(8). Much effort has been made on design and synthesis of farnesyl transferase inhibitors(9) because inhibitors of farnesylation step block the activity of ras in vivo and in vitro(2,10). Is endoproteolysis essential for the activity of isoprenylated proteins such as ras? Membrane binding studies of fully processed and partially processed ras suggest that proteolysis may be important in ras function(8). Although the membrane-bound isoprenylated protein methyltransferase has not been purified, aspects of its chemical biology have been explored. Recently, the enzymatic activity responsible for proteolysis in bovine liver and canine microsomal membranes(11), in yeast membranes(12), and later in rat liver microsomes(13) has been identified. Inhibition studies on isoprenylated protein endoprotease and carboxymethyltransferase strongly suggest that further characterization of these enzyme will be of substantial interest biologically and pharmacologically. Here we show that potent and specific inhibitors for these enzymes inhibited the activity of these enzymes. By using a biotin-tagging chloromethyl ketone inhibitor, Biotin-FCCMK, an 32 KDa protein is identified, which is strong candidate to be the isoprenyl protein endoprotease. The new irreversible inhibitors and the biotin-tagging affinity labeling agent reported here will facilitate the purifiction and cloning these enzymes in signal transduction and may be used as cancer theraphic agents.
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