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In Vitro Assay and Characterization of the Farnesylation-dependent Prelamin A Endoprotease

1997; Elsevier BV; Volume: 272; Issue: 8 Linguagem: Inglês

10.1074/jbc.272.8.5298

1083-351X

Fusun Kilic, Marguerite B. Dalton, Sarah K. Burrell, John P. Mayer, Scott D. Patterson, Michael Sinensky,

Cellular transport and secretion

The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S -farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S -farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro , with cleavage of the synthetic peptide at the expected site between Tyr 657 and Leu 658 . Endoproteolytic cleavage requires the S -prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N -Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.