Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones.
The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.
Porter
Stephen B
SB
Department of Biochemistry and Molecular Biology, The University of Georgia, 120 Green Street, Athens, GA 30602, USA.
Hildebrandt
Emily R
ER
Breevoort
Sarah R
SR
Mokry
David Z
DZ
Dore
Timothy M
TM
Schmidt
Walter K
WK
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D002384:Catalysis; D025141:Drug Resistance, Fungal / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor; D010450:Endopeptidases / Q000378:metabolism; D007659:Ketones / Q000737:chemistry / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017368:Protein Prenylation / Q000187:drug effects*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity
D000970:Antineoplastic Agents; D007659:Ketones; D008565:Membrane Proteins; D010455:Peptides; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D011496:Protein Methyltransferases; C498863:Ste14 protein, S cerevisiae; D010450:Endopeptidases; D043484:Proprotein Convertases; C105193:RCE1 protein, S cerevisiae; D008666:Metalloendopeptidases; C491251:STE24 protein, S cerevisiae