Molecular cloning and biochemical characterization of Candida albicans acyl-CoA:sterol acyltransferase, a potential target of antifungal agents.
To determine whether Candida albicans acyl CoA:sterol acyltransferase (ASAT) can be a potential target enzyme for the protoberberine derivative (HWY-289), we have isolated a gene encoding Ca-ASAT and examined inhibitory effects of HWY-289 on the overexpressed Ca-ASAT. HWY-289 specifically inhibits Ca-ASAT in a non-competitive manner in vitro (IC(50) [9.2microM], K(i) [5.15microM]). The cloned CaARE2 gene (1830 nucleotides [nt]) encodes active Ca-ASAT protein that exhibits a calculated molecular mass of 71.3kDa. The amino acid sequence of CaAre2p is 33.4% and 35.1% identical to those of Saccharomyces cerevisiae ScAre1p and ScAre2p homologues, respectively. Recombinant and endogenous Ca-ASAT displayed identical patterns of inhibition upon exposure to HWY-289 and a preference for cholesterol and oleoyl-CoA as substrates. Northern blot analysis showed that CaARE2 was activated by HWY-289, but not by CI-976 (a human acyl-coenzyme A:cholesterol acyltransferase inhibitor), in a dose-dependent manner (up to 5mg/L), suggesting different selectivities of action between HWY-289 and CI-976 on Ca-ASAT activity.
Kim
Ki-Young
KY
Department of Biochemistry, Yonsei Proteome Research Center, and Biomedical Proteome Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul 120-749, Republic of Korea.
Shin
Yu-Kyong
YK
Park
Jong-Chul
JC
Kim
Jung-Ho
JH
Yang
Hongyuan
H
Han
Dong-Min
DM
Paik
Young-Ki
YK
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D000813:Anilides / Q000378:metabolism; D000935:Antifungal Agents / Q000378:metabolism; D001483:Base Sequence; D001599:Berberine / Q000031:analogs & derivatives* / Q000378:metabolism; D002176:Candida albicans / Q000201:enzymology*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000214:Acyl Coenzyme A; D000813:Anilides; D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C429324:HWY289; D011994:Recombinant Proteins; D001599:Berberine; C069304:PD 128042; D002785:Sterol O-Acyltransferase