Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase.

Area of Science:

• Biochemistry
• Steroid Metabolism
• Enzymology

Background:

• Pregnenolone (PREG) esterification occurs via plasma lecithin: cholesterol acyltransferase (LCAT) and other enzymes.
• Acyl-CoA:cholesterol acyltransferases (ACAT1 and ACAT2) esterify sterols, with activity enhanced by cholesterol.
• Pregnenolone is a sterol-like molecule with structural differences from cholesterol.

Purpose of the Study:

• To investigate the role of ACAT1 and ACAT2 in pregnenolone esterification.
• To elucidate the interaction of cholesterol with ACAT1 in pregnenolone esterification.
• To determine the contribution of LCAT and ACATs to pregnenolone ester levels in vivo.

Main Methods:

• Enzyme kinetics studies using purified ACAT1 with pregnenolone and cholesterol.
• Assessment of pregnenolone esterification in cells using ACAT inhibitors.
• Analysis of pregnenolone ester and adrenal steroid levels in ACAT1/2 and LCAT knockout mice.

Main Results:

• Cholesterol dramatically increases ACAT1 Vmax for pregnenolone esterification by 100-fold, acting as an activator.
• ACAT1 exhibits higher binding affinity for pregnenolone than cholesterol, with pregnenolone acting solely as a substrate.
• ACAT1/2 and LCAT play distinct roles in regulating pregnenolone ester content in adrenal tissues and serum.

Conclusions:

• ACAT1's substrate and activator sites involve different sterol-binding mechanisms.
• ACAT enzymes are crucial for pregnenolone esterification across various cell types.
• LCAT and ACATs collaboratively regulate pregnenolone ester levels in different tissues.