No abstract

The intravenous administration of heparin to patients before open heart surgery reduced ristocetin cofactor activity by 58% (P < 0.01, t test), and this impairment of von Willebrand factordependent platelet function was closely related to plasma heparin levels (r2 = 0.9), but not to plasma von

StarD4 protein is a member of the StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins that includes StarD5 and StarD6, proteins whose functions remain poorly defined. The objective of this study was to isolate and characterize StarD4ʼs sterol binding and to determine in a hepatocyte culture model its sterol transport capabilities. Cholesterol is a structural component of mammalian cell membranes and also serves as a precursor to bile acids (in the liver), steroid hormones (in the adrenal, testis, and ovaries), and vitamin D. Homeostasis of cholesterol within the body is maintained through the coordinated regulation of its cell-mediated uptake, transport/trafficking, sorting, biosynthesis, storage (i.e., esterification), secretion, and catabolism to bile acids (1). More specifically, the steroidogenic acute regulatory-related lipid transfer (START) domain superfamily of proteins has been shown to be involved in several pathways of intracellular trafficking of cholesterol (2-4). It has been predicted that all proteins with a START domain contain a similar binding pocket with modifications in that pocket that determine ligand binding specificity and function (5). The START domains are 200-210 amino acid motifs that appear in a wide range of proteins and have been implicated in several cellular functions, including lipid transport and metabolism, signal transduction, and transcriptional regulation (3, 4, 6). The START-related lipid transfer protein 4 (StarD4) belongs to the StarD4 subfamily, a START subfamily that also contains the proteins StarD5 and StarD6. The proteins in the StarD4 subfamily have been shown to contain 205-233 amino acid residues, sharing 26-32% identity with each other (7). Within the StarD4 subfamily, StarD5 has been shown in vitro to bind cholesterol and 25-hydroxycholesterol (4). StarD1 and MLN64/StarD3 (the nearest related proteins to the StarD4 subfamily) have been shown to bind only cholesterol (4,8). Furthermore, full-length StarD1, StarD4, StarD5 and truncated MLN64/StarD3 have been reported to increase steroidogenesis and/or increase free cholesterol in the microsomes after overexpression in cell culture, representative evidence of their ability to transfer cholesterol (4, 9-12).The first START domain protein crystal structure reported was the C-terminal portion of human MLN64/ StarD3 (8), followed subsequently by the structure of mouse StarD4 (13). The protein structures of MLN64/ StarD3 and StarD4 revealed similar secondary structural elements and a hydrophobic tunnel with a size consistent with the binding of one cholesterol molecule (8, 13). Less

There are two major pathways of bile acid synthesis: the "neutral" pathway, initiated by highly regulated microsomal cholesterol 7 ␣ -hydroxylase (CYP7A1), and an "alternative" pathway, initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In hepatocyte cultures, overexpression of CYP7A1 increases bile acid synthesis by Ͼ 8-fold. However, overexpression of CYP27A1 in hepatocytes only increases it by 1.5-fold, suggesting that additional rate-limiting steps must be involved in the regulation of this pathway. The effects of intracellular cholesterol transport proteins on bile acid synthesis have been investigated in the current study. Under culture conditions in which the neutral pathway was inactive, selective overexpression of the gene encoding steroidogenic acute regulatory protein (StAR), MLN64 (StAR homolog protein), and sterol carrier protein-2 (SCP-2) led to 5.7-, 1.2-, and 1.7-fold increases, respectively, in the rates of bile acid synthesis in primary rat hepatocytes. Surprisingly, co-overexpression of MLN64 with StAR, SCP-2, or CYP7A1 blunted the upregulated bile acid synthesis by 48, 47, and 45%, respectively.These results suggest that MLN64, in its full-length form, is not responsible for the transport of cholesterol to the mitochondria or the endoplasmic reticulum, where CYP27A1 or CYP7A1 is located, respectively. -Ren, S., P. Hylemon, D. Marques, E. Hall, K. Redford, G. Gil, and W. M. Pandak. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis.

This study reports the discovery of a novel sulfonated oxysterol found at high levels in the mitochondria and nuclei of primary rat hepatocytes after overexpression of the gene encoding steroidogenic acute regulatory protein (StarD1). Forty-eight hours after infection of primary rat hepatocytes with recombinant adenovirus encoding StarD1, rates of bile acid synthesis increased by 4-fold. Concurrently, [ 14 C]cholesterol metabolites (oxysterols) were increased dramatically in both the mitochondria and nuclei of StarD1-overexpressing cells, but not in culture medium. A watersoluble [ 14 C]oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Enzymatic digestion, HPLC, and tandem mass spectrometry analysis identified the water-soluble oxysterol as 5-cholesten-3b,25-diol 3-sulfonate. Further experiments detected this cholesterol metabolite in the nuclei of normal human liver tissues. Based upon these observations, we hypothesized a new pathway by which cholesterol is metabolized in the mitochondrion.-Ren, S