Shakhawat Bhuiyan

Characterization and ontogenic study of novel steroid-sulfating SULT3 sulfotransferases from zebrafish

In vertebrates, sulfation as catalyzed by members of the cytosolic sulfotransferase (SULT) family has been suggested to be involved in the homeostasis of steroids. To establish the zebrafish as a model for investigating how sulfation functions to regulate steroid metabolism during the developmental process, we have embarked on the identification of steroid-sulfating SULTs in zebrafish. By searching the GenBank database, we identified two putative cytosolic SULT sequences from zebrafish, designated SULT3 ST1 and ST2. The recombinant proteins of these two zebrafish SULT3 STs were expressed in and purified from BL21 (DE3) cells transformed with the pGEX-2TK expression vector harboring SULT3 ST1 or ST2 cDNA. Upon enzymatic characterization, purified SULT3 ST1 displayed the strongest sulfating activity toward 17beta-estradiol among the endogenous substrates tested, while SULT3 ST2 exhibited substrate specificity toward hydroxysteroids, particularly dehydroepiandrosterone (DHEA). The pH-dependence and kinetic constants of these two enzymes with 17beta-estradiol and DHEA were determined. A developmental expression study revealed distinct patterns of the expression of SULT3 ST1 and ST2 during embryonic development and throughout the larval stage onto maturity. Collectively, these results imply that these two steroid-sulfating SULT3 STs may play differential roles in the metabolism and regulation of steroids during zebrafish development and in adulthood.

Protein SRP68 of human signal recognition particle: Identification of the RNA and SRP72 binding domains

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro‐transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety‐four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68–SRP72 interaction remained stable at elevated salt concentrations and engaged ∼150 amino acids from the N‐terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)‐like motifs suggested to form a superhelical structure with a groove to accommodate the C‐terminal region of SRP68.

Identification and characterization of two novel cytosolic sulfotransferases, SULT1 ST7 and SULT1 ST8, from zebrafish

Cytosolic sulfotransferases (SULTs) constitute a family of Phase II detoxification enzymes that are involved in the protection against potentially harmful xenobiotics as well as the regulation and homeostasis of endogenous compounds. Compared with humans and rodents, the zebrafish serves as an excellent model for studying the role of SULTs in the detoxification of environmental pollutants including environmental estrogens. By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative SULTs were identified. Sequence analysis indicated that these two putative zebrafish SULTs belong to the SULT1 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT1 ST7 and SULT1 ST8, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) cells. Purified GST-fusion protein form of SULT1 ST7 and SULT1 ST8 exhibited strong sulfating activities toward environmental estrogens, particularly hydroxylated polychlorinated biphenyls (PCBs), among various endogenous and xenobiotic compounds tested as substrates. pH-dependence experiments showed that SULT1 ST7 and SULT1 ST8 displayed pH optima at 6.5 and 8.0, respectively. Kinetic parameters of the two enzymes in catalyzing the sulfation of catechin and chlorogenic acid as well as 3-chloro-4-biphenylol were determined. Developmental expression experiments revealed distinct patterns of expression of SULT1 ST7 and SULT1 ST8 during embryonic development and throughout the larval stage onto maturity.

A Novel Hydroxysteroid-Sulfating Cytosolic Sulfotransferase, SULT3 ST3, from Zebrafish: Identification, Characterization, and Ontogenic Study

To establish the zebrafish as a model for investigating the drug metabolism through sulfation, we had embarked on establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Based on the sequence analysis, this zebrafish SULT was found to belong to the SULT3 gene family. The recombinant protein of the zebrafish SULT, designated SULT3 ST3, was expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring SULT3 ST3 cDNA. Upon enzymatic characterization, purified SULT3 ST3 displayed sulfating activity toward hydroxysteroids, particularly pregnenolone and dehydroepiandrosterone (DHEA), as well as several drugs among various endogenous and xenobiotic compounds tested as substrates. The pH-dependence and kinetic constants of this enzyme with DHEA were determined. The regulatory effects of various divalent metal cations on the DHEA-sulfating activity of SULT3 ST3 were quantitatively evaluated. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. Collectively, these results suggest a possible involvement of the newly discovered SULT3 ST3 in the metabolism of hydroxysteroids and xenobiotics including drugs in zebrafish.

Identification and Characterization of Zebrafish SULT1 ST9, SULT3 ST4, and SULT3 ST5

By searching the GenBank database, we identified sequences encoding three new zebrafish cytosolic sulfotransferases (SULTs). These three new zebrafish SULTs, designated SULT1 ST9, SULT3 ST4, and SULT3 ST5, were cloned, expressed, purified, and characterized. SULT1 ST9 appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as β-naphthol, β-naphthylamine, caffeic acid and gallic acid. SULT3 ST4 showed strong activity toward endogenous compounds such as dehydroepiandrosterone (DHEA), pregnenolone, and 17β-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, as well as xenobiotics including mestranol, β-naphthylamine, β-naphthol, and butylated hydroxyl anisole (BHA). pH-dependency and kinetic constants of these three enzymes were determined with DHEA, β-naphthol, and 17β-estradiol as substrates. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the expression of these three new zebrafish SULTs at different developmental stages during embryogenesis, through larval development, and on to maturity.

Archaea Signal Recognition Particle Shows the Way

Archaea SRP is composed of an SRP RNA molecule and two bound proteins named SRP19 and SRP54. Regulated by the binding and hydrolysis of guanosine triphosphates, the RNA-bound SRP54 protein transiently associates not only with the hydrophobic signal sequence as it emerges from the ribosomal exit tunnel, but also interacts with the membrane-associated SRP receptor (FtsY). Comparative analyses of the archaea genomes and their SRP component sequences, combined with structural and biochemical data, support a prominent role of the SRP RNA in the assembly and function of the archaea SRP. The 5e motif, which in eukaryotes binds a 72 kilodalton protein, is preserved in most archaea SRP RNAs despite the lack of an archaea SRP72 homolog. The primary function of the 5e region may be to serve as a hinge, strategically positioned between the small and large SRP domain, allowing the elongated SRP to bind simultaneously to distant ribosomal sites. SRP19, required in eukaryotes for initiating SRP assembly, appears to play a subordinate role in the archaea SRP or may be defunct. The N-terminal A region and a novel C-terminal R region of the archaea SRP receptor (FtsY) are strikingly diverse or absent even among the members of a taxonomic subgroup.

A. fulgidus SRP54 M-domain

The signal recognition particle (SRP) is a ribonucleoprotein complex that binds ribosomes engaged in the synthesis of secretory proteins destined for cellular membranes. SRP associates with the nascent signal sequence and halts or delays translation of the secretory protein until the nascent protein–ribosome–SRP complex binds to the SRP receptor in the membrane. Following the hydrolysis of GTP, the SRP dissociates from the ribosome and is free to bind another emerging signal peptide (Doudna and Batey 2004). The SRP RNA and one molecule of protein SRP54, called fifty-four homolog (for Ffh) in bacteria, are at the core of the SRP in all organisms. SRP54 is composed of the N-terminal (N) domain, the GTPase (G) domain, and the C-terminal methionine-rich (M) domain. The M-domain binds to the highly conserved helix 8 of the SRP RNA and is in close proximity to the emerging signal sequence (High and Dobberstein 1991). SRP54 has been the object of intensive structural investigations owing to its role in signal peptide binding. Such studies have shown that the M-domain is all-helical and that it uses a helix-turn-helix motif to bind into the distorted minor groove of the SRP RNA (Batey et al. 2000; Kuglstatter et al. 2002; Rosendal et al. 2003). These structures have also provided insights as to possible mechanisms of signal peptide binding. One such mechanism, suggested by the crystal structure of Thermus aquaticus Ffh, is that the signal peptide binds within a wide and short groove formed by the ends of helices 1 and 2, the exposed face of helix 5, and the fingerloop, an extended segment bridging helices 1 and 2 (Supplementary Material Fig. 1a) (Keenan et al. 1998). A similar hydrophobic groove was observed in the crystal structure of Escherichia coli Ffh bound to its SRP RNA (Batey et al. 2000), although in this case the fingerloop was structurally disordered over most of its length (28 residues). In the crystal structure of the RNA-bound Sulfolobus solfataricus SRP54, the fingerloop was folded into the hydrophobic groove, suggestive of a possible ‘‘closed state’’ whereby the loop shielded the groove from solvent (Rosendal et al. 2003). Another possible mechanism of signal peptide binding was suggested by the crystal structure of the human SRP54 M-domain, which in both the free and RNA-bound forms, formed a domain-swapped dimer where the N-terminal a-helix ‘‘flipped out’’ of the helical core and was replaced by the N-terminal a-helix of a neighboring molecule (Clemons et al. 1999; Kuglstatter et al. 2002). Such dimeric structures led to the hypothesis that the N-terminal helices of the intertwined molecules mimicked the binding of the signal peptide in its a-helical state (Supplementary Material Fig. 1b) (Clemons et al. 1999). Electronic supplementary material The online version of this article (doi:10.1007/s10858-008-9252-4) contains supplementary material, which is available to authorized users.