Biomedical subjects

Charles A Strott

Publications and source records attributed to Charles A Strott.

7 recordsLinked to original sources

PubMed2003

Crystal structure of human cholesterol sulfotransferase (SULT2B1b) in the presence of pregnenolone and 3'-phosphoadenosine 5'-phosphate. Rationale for specificity differences between prototypical SULT2A1 and the SULT2BG1 isoforms.

The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket.

Adenosine Diphosphate↗

PubMed2003

Cholesterol sulfate in human physiology: what's it all about?

Cholesterol sulfate is quantitatively the most important known sterol sulfate in human plasma, where it is present in a concentration that overlaps that of the other abundant circulating steroid sulfate, dehydroepiandrosterone (DHEA) sulfate. Although these sulfolipids have similar production and metabolic clearance rates, they arise from distinct sources and are metabolized by different pathways. While the function of DHEA sulfate remains an enigma, cholesterol sulfate has emerged as an important regulatory molecule. Cholesterol sulfate is a component of cell membranes where it has a stabilizing role, e.g., protecting erythrocytes from osmotic lysis and regulating sperm capacitation. It is present in platelet membranes where it supports platelet adhesion. Cholesterol sulfate can regulate the activity of serine proteases, e.g., those involved in blood clotting, fibrinolysis, and epidermal cell adhesion. As a result of its ability to regulate the activity of selective protein kinase C isoforms and modulate the specificity of phosphatidylinositol 3-kinase, cholesterol sulfate is involved in signal transduction. Cholesterol sulfate functions in keratinocyte differentiation, inducing genes that encode for key components involved in development of the barrier. The accumulating evidence demonstrating a regulatory function for cholesterol sulfate appears solid; the challenge now is to work out the molecular mechanisms whereby this interesting molecule carries out its various roles.

Amino Acid Sequence↗

PubMed2003

Conservation of the hydroxysteroid sulfotransferase SULT2B1 gene structure in the mouse: pre- and postnatal expression, kinetic analysis of isoforms, and comparison with prototypical SULT2A1.

A novel mouse hydroxysteroid sulfotransferase cDNA has been cloned, and organization of its gene structure has been determined. The new mouse sulfotransferase, SULT2B1a, and its closely related isoform, SULT2B1b, are derived from a single SULT2B1 gene as a result of an alternative exon I and differential splicing. Thus, the only structural distinction between the two SULT2B1 isoforms is at their amino-terminal ends. Importantly, in contrast to the prototypical mouse hydroxysteroid sulfotransferase SULT2A1, the SULT2B1 isoforms have a predilection for cholesterol. Real-time RT-PCR reveals that the SULT2B1a isoform is most abundantly expressed in the brain and spinal cord, whereas SULT2B1b and SULT2A1 are weakly, if at all, expressed in the central nervous system. On the other hand, the SULT2B1b isoform is the most prominent hydroxysteroid sulfotransferase expressed in skin, whereas SULT2A1 is strikingly expressed in the liver. The substrate specificities and differential expression patterns of the three SULT2 isozymes strongly suggest that they have distinct biologic roles to play. Of further interest, the mouse SULT2B1 and SULT2A1 genes are differentially expressed during embryonic development, with the former being expressed at all stages from E8.5-E19, whereas the latter is not expressed until E19. It is speculated that, during embryonic development, SULT2B1b is required for production of cholesterol sulfate essential for normal skin development, whereas SULT2B1a produces pregnenolone sulfate, an essential neurosteroid during development of the central nervous system.

Amino Acid Sequence↗

PubMed2002

Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase, whereas exon 1A yields pregnenolone sulfotransferase.

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.

Amino Acid Sequence↗

PubMed2002

Characterization and expression of human bifunctional 3'-phosphoadenosine 5'-phosphosulphate synthase isoforms.

Sulphonation, a fundamental process essential for normal growth and development, requires the sulphonate donor molecule 3'-phosphoadenosine 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the bifunctional enzyme PAPS synthase. In humans, two genes encode isoenzymes that are 77% identical at the amino acid level, and alternative splicing creates two subtypes of PAPS synthase 2. The question as to whether distinctions in amino acid composition are reflected in differences in activity has been examined. The specific activity of the PAPS synthase 2 subtypes is 10- to 15-fold higher than that for PAPS synthase 1. The greater catalytic efficiency of the PAPS synthase 2 subtypes is demonstrated further by the 3- to 6-fold higher k(cat)/K(m) ratios for ATP and inorganic sulphate as compared with the ratios for PAPS synthase 1. In humans, PAPS synthase 1 is expressed ubiquitously, and is the dominant isoform in most tissues, whereas expression of the PAPS synthase 2 subtypes is variable and tissue-specific. It is noteworthy that, similar to other human tissues, PAPS synthase 1 also appears to be the dominant isoform expressed in cartilage. The latter finding initially created a conundrum, since there is a specific human dwarfing disorder that is known to be caused by a mutation in the PAPS synthase 2 gene. This apparent enigma would seem to be 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, expression of PAPS synthase 1 is relatively low in the cartilage of immature guinea-pigs, including the growth plate of long bones, whereas PAPS synthase 2 is the highly expressed isoenzyme.

Amino Acid Sequence↗

PubMed2002

Transcriptional regulation of human 3'-phosphoadenosine 5'-phosphosulphate synthase 2.

Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3'-phosphate 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5'-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp -84 and bp -124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.

Base Sequence↗

PubMed2002

Sulfonation and molecular action.

The sulfonation of endogenous molecules is a pervasive biological phenomenon that is not always easily understood, and although it is increasingly recognized as a function of fundamental importance, there remain areas in which significant cognizance is still lacking or at most minimal. This is particularly true in the field of endocrinology, in which the sulfoconjugation of hormones is a widespread occurrence that is only partially, if at all, appreciated. In the realm of steroid/sterol sulfoconjugation, the discovery of a novel gene that utilizes an alternative exon 1 to encode for two sulfotransferase isoforms, one of which sulfonates cholesterol and the other pregnenolone, has been an important advance. This is significant because cholesterol sulfate plays a crucial role in physiological systems such as keratinocyte differentiation and development of the skin barrier, and pregnenolone sulfate is now acknowledged as an important neurosteroid. The sulfonation of thyroglobulin and thyroid hormones has been extensively investigated and, although this transformation is better understood, there remain areas of incomplete comprehension. The sulfonation of catecholamines is a prevalent modification that has been extensively studied but, unfortunately, remains poorly understood. The sulfonation of pituitary glycoprotein hormones, especially LH and TSH, does not affect binding to their cognate receptors; however, sulfonation does play an important role in their plasma clearance, which indirectly has a significant effect on biological activity. On the other hand, the sulfonation of distinct neuroendocrine peptides does have a profound influence on receptor binding and, thus, a direct effect on biological activity. The sulfonation of specific extracellular structures plays an essential role in the binding and signaling of a large family of extracellular growth factors. In summary, sulfonation is a ubiquitous posttranslational modification of hormones and extracellular components that can lead to dramatic structural changes in affected molecules, the biological significance of which is now beginning to be appreciated.

Amino Acid Sequence↗