Browsing by Author "Goldstein, Joseph L."
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Item Identification and Biochemical Characterization of Ghrelin O-Acyltransferase (GOAT)(2009-06-19) Yang, Jing; Goldstein, Joseph L.Ghrelin is a 28-amino acid, appetite-stimulating hormone secreted by the food-deprived stomach. Ser-3 of ghrelin is acylated with an eight-carbon fatty acid, octanoate, which is critically required for its endocrine actions. However, the octanoylating enzyme had remained elusive for nearly a decade. By expression cloning, I have identified GOAT (Ghrelin O-Acyltransferase), an enzyme belonging to a family of 16 polytopic membrane-bound O-acyltransferases. GOAT activity requires catalytic Asn and His residues, which are conserved through vertebrates. Consistent with its function, GOAT mRNA is largely restricted to stomach and intestine, the major ghrelin-secreting tissues. To further characterize GOAT function biochemically, I have developed a robust in vitro assay using membranes from insect cells infected with baculovirus encoding recombinant mouse GOAT. GOAT-containing membranes catalyze the transfer of [3H]octanoyl from [3H]octanoyl CoA to recombinant proghrelin in vitro. 50 microM palmitoyl CoA is necessary in the assays to prevent the deacylation of [3H]octanoyl CoA by crude membrane preparations. Maximal GOAT activity is observed at pH 7.0, and there is no apparent requirement for metals as determined by a lack of inhibition by 1 mM EDTA. The apparent Km for proghrelin is 6 microM and for [3H]octanoyl CoA is 0.6 microM. The octanoylation reaction strictly depends on the GOAT recognition site comprising three of the four N-terminal amino acids of proghrelin: Gly-1, Ser-3, and Phe-4. A pentapeptide containing only the N-terminal five amino acids of ghrelin is octanoylated by the enzyme. Moreover, I have demonstrated that the activity of GOAT is subjected to end-product inhibition. Together, the insights provided by my research may facilitate the design of useful inhibitors of GOAT.Item Role of Insig-Mediated Cholesterol Homeostasis in the Development of the Palate and Hair Follicles in Mice(2012-08-15) Goldstein, Joseph L.; Brown, Michael S.Proper cholesterol homeostasis is crucial in mammalian development. Currently, there are 8 known human malformation syndromes due to genetic defects in cholesterol biosynthesis. Patients with these syndromes present with a constellation of developmental defects, ranging from skeletal and craniofacial dysmorphologies to skin and cardiovascular anomalies. In each of these diseases, there is a block at a specific enzymatic step leading to a deficiency in cholesterol and a concomitant buildup in sterol intermediates proximal to the block; however, it is unclear whether cholesterol deficiency or sterol intermediate accumulation causes the observed abnormalities. In this work, we generated mice lacking Insig-1 and Insig-2. These proteins regulate cholesterol biosynthesis by both inhibiting the proteolytic activation of SREBPs, transcription factors for cholesterol biosynthetic genes, and enhancing the degradation of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis. We first created Insig-double-knockout (Insig-DKO) mice homozygous for null mutations in Insig-1 and Insig-2. Insig-DKO mice exhibited defects in midline facial fusion, ranging from cleft palate to complete cleft face, and overproduced sterols, creating a significant buildup of cholesterol and sterol intermediates. When Insig-DKO mice were treated with lovastatin, an HMG-CoA reductase inhibitor, sterol intermediate levels were significantly reduced and the craniofacial defects were ameliorated. Next, we generated mice lacking Insigs in the epidermis (Epi-Insig-DKO). All Epi-Insig-DKO mice failed to grow body hair, had decreased body weights, and died 6 weeks after birth. Early hair follicle development was normal while later development was disrupted and led to hair cycle arrest in these mice. Skin from these mutant mice showed a significant buildup of both cholesterol and sterol intermediates. Topical treatment of Epi-Insig-DKO mice with the HMG-CoA reductase inhibitor simvastatin during the first two postnatal weeks lowered sterol intermediate levels in the skin and completely corrected the hairless and low body weight phenotypes. We conclude that the accumulation of sterol intermediates, not a deficiency of cholesterol, interferes with both the fusion of facial structures and the proper development of hair follicles in mice. Our findings have implications for the pathogenesis of the palate and skin/hair follicle defects observed in human malformation syndromes due to aberrant cholesterol biosynthesis.