| Section on Steroid Regulation | |||
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RESEARCH The
transformation of biomolecules by sulfonation is a fundamental process
of major importance. Sulfonation is essential for normal growth and
development, as well as maintenance of the internal milieu. It plays
a primary role in the post-translational modification of numerous
structural and membrane constituents that are necessary for normal
development. Sulfonated macromolecules We have isolated, cloned and biochemically characterized several members of the steroid sulfotransferase family including those that sulfonate phenolic as well as neutral steroids. Structure/function studies of both estrogen and hydroxysteroid sulfotransferases have revealed a number of previously unknown features including functionally unique isoforms and stereospecificity. One role of steroid sulfotransferases is to regulate the level of biologically active free steroid. However, steroid sulfoconjugates are not simply inactive metabolites and have well-characterized biological effects that are distinct from the well-known role of unconjugated steroids as ligands for nuclear receptors regulating gene expression. Most of these extranuclear effects of steroid sulfonates are exerted at cell membranes and involve such functions as regulation of phospholipases and ion channels. Several hydroxylated metabolites of cholesterol have been found to activate orphan nuclear hormone receptors that regulate genes involved in cholesterol homeostasis. Like the C21, C19, and C18 steroids, C27 oxysterols are subject to sulfonation, and we have recently characterized sulfotransferases that preferentially sulfonate cholesterol and oxysterols. These enzymes are highly expressed in skin, where cholesterol sulfonation is an important factor in the development of the skin barrier. The control of cholesterol sulfotransferase gene expression during keratinocyte differentiation is currently under investigation. Sulfonation requires the universal sulfonate donor molecule, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The biosynthesis of this essential biological molecule from inorganic sulfate and ATP is catalyzed by ATP sulfurylase and adenosine 5'-phosphosulfate kinase, activities that are intrinsic to a single polypeptide chain. We have cloned the bifunctional human PAPS synthase, localized it to chromosome 4 and identified its catalytic domains as well as unique ATP binding motifs in each domain. A second isozyme of human PAPS synthase was cloned and is located on chromosome 10. PAPS synthase 1 appears to be a housekeeping gene based on its ubiquitous expression, whereas PAPS synthase 2 appears to be regulated based on its tissue-specific expression. The PAPS synthase 2 gene was discovered during a search for the genetic basis of a developmental abnormality causing a form of spondyloepimetaphyseal dysplasia that presents with a skeletal phenotype involving the spine and long bones. This recessive dwarfing disorder is caused by a nonsense mutation located in the ATP sulfurylase domain of PAPS synthase 2. PAPS synthase 1, however, is the dominant isoform expressed in most tissues including cartilage. This apparent conundrum was resolved by examination of cartilage from guinea pigs as an animal model. Similar to humans, cartilage from mature animals predominantly expresses PAPS synthase 1. In contrast, however, expression of PAPS synthase 1 is barely detectable in the growth plate cartilage of guinea pig long bones, whereas PAPS synthase 2 is highly expressed. The final resolution of this paradox will require analysis of the manner in which the PAPS synthase genes are differentially regulated during growth and development, including identification of the crucial cis elements and transacting factors that regulate expression of the two genes. During initial studies the core promoters have been identified as well as downstream or proximal regulatory GC boxes, and we have determined that expression of both genes is at least partly under the influence of the Sp1 family of transcription factors. The search is now on for co-activators and/or repressors. SECTION CHIEF Charles
A. Strott, received his medical degree from the University of Pittsburgh
and did his internship and residency training in Internal Medicine
at the Presbyterian-University Hospital and Medical Center. After
completing his internship and post-doctoral training in Endocrinology
and Genetics at the University of Utah under Drs. Charles Nugen and
Frank Tyler, he pursued advanced studies in Steroid Biochemistry and
Metabolism at the National Cancer Institute under Dr. Mortimer Lipsett.
This was followed by a faculty appointment at Vanderbilt University
in the Department of Medicine under Dr. Grant Liddle, after which
he returned to the NICHD as a Medical Officer in the Endocrinology
and Reproduction Research Branch. He is currently head of the Section
on Steroid Regulation. Contact Information Charles
A. Strott, M.D. Telephone:
301-496-3025
PERSONNEL Personnel: ·
Young C. Lee, Ph.D., Bldg. 49; Rm. 6A-55; tel: 301-496-5909;
e-mail: younglee@box-y.nih.gov Collaborators: · Norman B. Javitt, M.D., Ph.D., Professor of Medicine and Pediatrics, Director Division of Hepatic Diseases, NYU Medical Center, New York, NY; e-mail: norman.javitt@med.nyu.edu
BIBLIOGRAPHY Shimizu C, Fuda H, Lee YC, Strott CA (2001) Transcriptional regulation of human 3'-phosphoadenosine synthase 1. Biochem Biophys Res Commun 234: 763-770. Javitt NB, Lee YC, Shimizu C, Fuda H, Strott CA. (2001) Cholesterol and hydroxycholesterol sulfotransferases: identification, distinction from dehydroepiandrosterone sulfotransferase and differential tissue expression. Endocrinology 142:2978-84. Park BC, Lee YC, Strott CA. (1999) Testosterone sulfotransferase: evidence in the guinea pig that this reaction is carried out by 3 alpha-hydroxysteroid sulfotransferase. Steroids. 64(8):510-7. Park BC, Lee YC, Strott CA. (1999) Identification by chimera formation and site-selected mutagenesis of a key amino acid residue involved in determining stereospecificity of guinea pig 3-hydroxysteroid sulfotransferase isoforms. J Biol Chem. 274(31):21562-8. Venkatachalam KV, Fuda H, Koonin EV, Strott CA. (1999) Site-selected mutagenesis of a conserved nucleotide binding HXGH motif located in the ATP sulfurylase domain of human bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase. J Biol Chem. 274(5):2601-4. Venkatachalam
KV, Akita H, Strott CA. (1998) Molecular
cloning, expression, and characterization of human bifunctional 3'-phosphoadenosine
5'-phosphosulfate synthase and its functional domains. J Biol
Chem. 273(30):19311-20. Luu NX, Driscoll WJ, Martin BM, Strott CA. (1995) Molecular cloning and expression of a guinea pig 3-hydroxysteroid sulfotransferase distinct from chiral-specific 3 alpha-hydroxysteroid sulfotransferase. Biochem Biophys Res Commun. 217(3): 1078-86. Driscoll WJ, Komatsu K, Strott CA. (1995) Proposed active site domain in estrogen sulfotransferase as determined by mutational analysis. Proc Natl Acad Sci U S A. 92(26):12328-32. Chiba H, Komatsu K, Lee YC, Tomizuka T, Strott CA. (1995) The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site. Proc Natl Acad Sci U S A. 92(18):8176-9. Lee YC, Park CS, Komatsu K, Kwack J, Strott CA. (1995) Adrenocortical pregnenolone binding activity resides with estrogen sulfotransferase. Endocrinology. 136(1):361-4. Lee YC, Komatsu K, Driscoll WJ, Strott CA. (1994) Structural and functional characterization of estrogen sulfotransferase isoforms: distinct catalytic and high affinity binding activities. Mol Endocrinol. 8(12):1627-35. Lee YC, Park CS, Strott CA.(1994) Molecular cloning of a chiral-specific 3 alpha-hydroxysteroid sulfotransferase. J Biol Chem. 269(22):15838-45. |
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