Human ras-converting enzyme (hRCE1) endoproteolytic activity on K-ras-derived peptides.
A human gene responsible for one of the steps in Ras post-translational modification and membrane localization, hRCE1, encodes a 35-kDa membrane-associated endoprotease. We examined hRCE1 activity using farnesylated 9 aa peptides with the core sequence, KSKTKC(farnesyl)VIM [(farnesyl) = (f)], from the C-terminus of K-Ras. We first demonstrated hRCE1 specificity in cleavage location and endoproteolysis. We then describe a direct fluorescent microtiter plate assay. We demonstrated that hRCE1 protease cleaved KSKTKC(f)VIM peptides between the C(f) and V positions, generating KSKTKC(f) and the corresponding tripeptides as products. We found that the sequence KSKTKC(f)VI was a better substrate for hRCE1 than KSKTKC(f)VIM. We also found that hRCE1 cleaved modified versions of KSKTKC(f)VIM that incorporated either MCA or ABZ fluorescent chromophores at the N-terminus, and quenching-group-containing amino acids at the V or M, but not the I, amino acid positions of VIM. The quenching-group-containing amino acids used were either Q(S) (dinitrophenyldiaminopropionic acid) or Q(L) (lysine epsilon-dinitrophenyl). Cleavage of KSKTKC(f)VIM and modified versions of this peptide by hRCE1 was initially evaluated by HPLC product resolution and quantitation. The hRCE1 cleavage of quenched peptides enabled us to directly monitor proteolytic activity in a 96-well microtiter fluorescent plate assay. The microtiter format assay was validated by its sensitivity to RPI, an inhibitor of prenyl protein protease. A direct fluorescent assay provides an effective tool for further characterization of this enzyme and also for detection of novel inhibitors.
Hollander
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I
Oncology Research, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York, 10965, USA.
Frommer
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E
Mallon
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R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D000596:Amino Acids; D010455:Peptides; D011994:Recombinant Proteins; D010450:Endopeptidases; C491326:RCE1 protein, human; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)