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 (IC50 [9.2μM], K i [5.15μM]). 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.
|Number of pages||9|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 2004 Jul 2|
Bibliographical noteFunding Information:
We thank Drs. Catherine Chang and Ta Yuan Chang at Dartmouth College for their generosity in supplying CI-976, an ACAT inhibitor. We also thank Dr. Stephen L. Sturley (Columbia, NY, USA) for provision of sterol ester deficient yeast and Dr. Chery A. Gale (Minesota, USA) for provision of pMG1602. This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (03-PJ10-PG6-GP01-002 toY.K.P.), Seoul, Korea.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology