Acyl-coenzyme A:cholesterol acyltransferase (ACAT)2 is a membrane-bound enzyme present in a variety of tissues and cells. It is mainly located at the endoplasmic reticulum, and catalyzes the biosynthesis of cholesteryl esters, using long-chain fatty acyl-coenzyme A and cholesterol as its substrates. ACAT plays important roles in cholesterol homeostasis. At the single cell level, it is a key enzyme that prevents excess free cholesterol from building up in the cell membranes. At the physiological level, it contributes cholesteryl esters as part of the neutral lipid cargo, to be packaged into the cores of very low density lipoproteins and chylomicrons. Under pathophysiological condition, in cholesterol-loaded macrophages, ACAT converts excess cholesterol into cholesteryl esters. This action reduces the amount of cholesterol available from the macrophage cell surface for efflux and converts the macrophages to foam cells, which are the hallmark of early lesions of the disease atherosclerosis (reviewed in Ref. 1). For these reasons, ACAT has been a drug target for pharmaceutical intervention of diseases, including atherosclerosis and hyperlipidemia. In mammals, two ACAT genes exist that encode for two similar but different proteins, ACAT1 and ACAT2. Available evidence suggests that ACAT1 and ACAT2 may function in distinct and complementary manners in various tissues (reviewed in Refs. 2 and 3). Unlike many other enzymes/proteins involved in cellular cholesterol metabolism, neither ACAT1 nor ACAT2 is regulated at the transcription level by the cholesterol-dependent SREBP (sterol regulatory element-binding protein) cleavage-activating protein (SCAP)/sterol regulatory element-binding protein pathway. Instead, available evidence suggests that ACAT1 may contain a distinct regulatory site that specifically recognizes cholesterol as its activator (4, 5). This mechanism allows ACAT1 to be up-regulated rapidly (within minutes) by cholesterol that builds up at the ER. The enzymological and biochemical characteristics of ACAT2 significantly diverge from those of ACAT1 in several ways; however, ACAT2 may also be allosterically regulated by cholesterol (5, 6).Molecular cloning of the human ACAT1 (hACAT1) gene (7) provided the opportunity to study its biochemical properties. The recombinant hACAT1 expressed in Chinese hamster ovary cells can be purified to homogeneity (8). However, due to the low quantities of protein derived from the purification process, current efforts in our laboratory focus on studies at the enzymological and cell biological levels, but not at the structural biology level. ACAT1 is homotetrameric in vitro and in intact cells (9). The region near the N-terminal contains a dimerization motif. Deleting the N-terminal region converts the enzyme into a homodimer; the dimeric enzyme is fully active catalytically, and remains to be allosterically regulated by cholesterol (10). ACAT1 contains multiple transmembrane domains (TMDs). To deduce its membrane topology, we had previously inserted the nine-amino acid HA tag a...

Liver‐expressed antimicrobial peptide 2 (LEAP2) is a highly conserved secretory peptide first isolated in 2003. However, its exact biological functions remained elusive until a recent study identified it as an endogenous antagonist for the growth hormone secretagogue receptor (GHSR1a), a G protein‐coupled receptor for which the gastric peptide ghrelin is the endogenous agonist. By tuning the ghrelin–GHSR1a system, LEAP2 has an important function in energy metabolism. In the present study, we first demonstrated that LEAP2 and ghrelin actually bound to GHSR1a in a competitive manner, rather than in a non‐competitive manner as previously reported, by binding assays and activation assays. Subsequently, we demonstrated that the antagonistic function of LEAP2 was drastically affected by the manner of its addition. LEAP2 primarily affected the maximal activation effect when added before ghrelin, whereas it primarily affected half‐maximal effective concentration when added at the same time as ghrelin. Thus, LEAP2 behaved as a competitive antagonist if added at the same time as the agonist and a non‐competitive antagonist if added before the agonist. This unusual property of LEAP2 might be caused by its slow dissociation from receptor GHSR1a. We also found that the N‐terminal fragment of LEAP2 was important for receptor binding. Our present study revealed an antagonistic mechanism for LEAP2, and will facilitate the design of novel antagonists for receptor GHSR1a in future studies.

Insulin-like peptide 5 (INSL5), a member of the insulin/relaxin superfamily, can activate the G-protein-coupled receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), but its precise biological functions are largely unknown. Recent studies suggest that INSL5/RXFP4 is involved in the control of food intake and glucose homoeostasis. We report in the present study that RXFP4 is present in the mouse insulinoma cell line MIN6 and INSL5 augments glucose-stimulated insulin secretion (GSIS) both in vitro and in vivo. RXFP4 is also expressed in the mouse intestinal L-cell line GLUTag and INSL5 is capable of potentiating glucose-dependent glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. We propose that the insulinotrophic effect of INSL5 is probably mediated through stimulation of insulin/GLP-1 secretion and the INSL5/RXFP4 system may be a potential therapeutic target for Type 2 diabetes.

Although insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar tertiary structure, weakly overlapped biological activity, and a common ancestor, the two highly homologous sequences encode different folding behavior: insulin folds into one unique stable tertiary structure while IGF-1 folds into two disulfide isomers with similar thermodynamic stability. To further elucidate the molecular mechanism of their different folding behavior, we prepared two single-chain hybrids of insulin and IGF-