Interaction of the Brain-Selective Sulfotransferase SULT4A1 with Other Cytosolic Sulfotransferases: Effects on Protein Expression and Function.

Sulfotransferase (SULT) 4A1 is a brain-selective sulfotransferase-like protein that has recently been shown to be essential for normal neuronal development in mice. In the present study, SULT4A1 was found to colocalize with SULT1A1/3 in human brain neurons. Using immunoprecipitation, SULT4A1 was shown to interact with both SULT1A1 and SULT1A3 when expressed in human cells. Mutation of the conserved dimerization motif located in the C terminus of the sulfotransferases prevented this interaction. Both ectopically expressed and endogenous SULT4A1 decreased SULT1A1/3 protein levels in neuronal cells, and this was also prevented by mutation of the dimerization motif. During differentiation of neuronal SH-SY5Y cells, there was a loss in SULT1A1/3 protein but an increase in SULT4A1 protein. This resulted in an increase in the toxicity of dopamine, a substrate for SULT1A3. Inhibition of SULT4A1 using small interference RNA abrogated the loss in SULT1A1/3 and reversed dopamine toxicity. These results show a reciprocal relationship between SULT4A1 and the other sulfotransferases, suggesting that it may act as a chaperone to control the expression of SULT1A1/3 in neuronal cells. SIGNIFICANCE STATEMENT: The catalytically inactive sulfotransferase (SULT) 4A1 may regulate the function of other SULTs by interacting with them via a conserved dimerization motif. In neuron-like cells, SULT4A1 is able to modulate dopamine toxicity by interacting with SULT1A3, potentially decreasing the metabolism of dopamine.

Expression of the orphan cytosolic sulfotransferase SULT4A1 and its major splice variant in human tissues and cells: dimerization, degradation and polyubiquitination.

The cytosolic sulfotransferase SULT4A1 is highly conserved between mammalian species but its function remains unknown. Polymorphisms in the SULT4A1 gene have been linked to susceptibility to schizophrenia. There are 2 major SULT4A1 transcripts in humans, one that encodes full length protein (wild-type) and one that encodes a truncated protein (variant). Here, we investigated the expression of SULT4A1 in human tissues by RT-PCR and found the wild-type mRNA to be expressed mainly in the brain, gastrointestinal tract and prostate while the splice variant was more widely expressed. In human cell-lines, the wild-type transcript was found in neuronal cells, but the variant transcript was expressed in nearly all other lines examined. Western blot analysis only identified SULT4A1 protein in cells that expressed the wild-type mRNA. No variant protein was detected in cells that expressed the variant mRNA. Ectopically expressed full length SULT4A1 protein was stable while the truncated protein was not, having a half-life of approximately 3 hr. SULT4A1 was also shown to homodimerize, consistent with other SULTs that contain the consensus dimerization motif. Mutation of the dimerization motif resulted in a monomeric form of SULT4A1 that was rapidly degraded by polyubiquitination on the lysine located within the dimerization motif. These results show that SULT4A1 is widely expressed in human tissues, but mostly as a splice variant that produces a rapidly degraded protein. Dimerization protects the protein from degradation. Since many other cytosolic sulfotransferases possess the conserved lysine within the dimerization motif, homodimerization may serve, in part, to stabilize these enzymes in vivo.

Phosphorylation/dephosphorylation of human SULT4A1: role of Erk1 and PP2A.

SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr(11) residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr(11) by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bß was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.

Regulation of mouse brain-selective sulfotransferase sult4a1 by cAMP response element-binding protein and activating transcription factor-2.

Sulfotransferase 4A1 (SULT4A1) is a novel cytosolic sulfotransferase that is primarily expressed in the brain. To date, no significant enzyme activity or biological function for the protein has been identified, although it is highly conserved between species. Mutations in the SULT4A1 gene have been linked to schizophrenia susceptibility, and recently, its stability was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. In this study, we investigated the transcriptional regulation of mouse Sult4a1. Using a series of promoter deletion constructs, we identified three cAMP-responsive elements (CREs) that were required for maximal promoter activity. The CREs are located within 100 base pairs of the major transcription start site and are also present in the same region of the human SULT4A1 promoter. Electrophoretic mobility shift assays (EMSAs) identified two specific complexes that formed on each of the CREs. One complex contained cAMP response element-binding protein (CREB), and the other contained activating transcription factor-2 (ATF-2) and c-Jun. Overexpression of CREB or ATF-2 increased not only reporter promoter activity but also endogenous Sult4a1 mRNA levels in Neuro2a cells. Moreover, [d-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) treatment increased both reporter promoter activity and Sult4a1 levels in mu-opioid receptor expressing Neuro2a/mu-opioid receptor cells, and EMSAs showed this to be due to increased binding of CREB and ATF-2 to the Sult4a1 promoter. We also show that DAMGO treatment increases Sult4a1 mRNA and protein levels in primary mouse neurons. These results suggest that Sult4a1 is a target gene for the mu-opioid receptor signaling pathway and other pathways involving activation of CREB and ATF-2.

Cytosolic Aryl sulfotransferase 4A1 interacts with the peptidyl prolyl cis-trans isomerase Pin1.

Sulfonation by cytosolic sulfotransferases plays an important role in the metabolism of both endogenous and exogenous compounds. Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase found primarily in neurons in the brain. It is highly conserved between species, but no substantial enzyme activity has been identified for the protein. Consequently, little is known about the role of this enzyme in the brain. We performed a yeast two-hybrid screen of a human brain library to isolate potential SULT4A1-interacting proteins that might identify the role or regulation of the sulfotransferase in humans. The screen isolated the peptidyl-prolyl cis-trans isomerase Pin1. Its interaction with SULT4A1 was confirmed by coimmunoprecipitation studies in HeLa cells and by in vitro pull-down of expressed proteins. Moreover, Pin1 binding was dependent on phosphorylation of the SULT4A1 protein. Pin1 destabilized SULT4A1, decreasing its half-life from more than 8 h to approximately 4.5 h. This effect was dependent on the isomerase activity of Pin1 and was inhibited by okadaic acid, suggesting a role for the phosphatase PP2A. Pin1-mediated SULT4A1 degradation did not involve the proteosomes or macroautophagy, but it was inhibited by the calpain antagonists N-acetyl-Leu-Leu-Nle-CHO and Z-Val-Phe-CHO. Finally, Pin1 binding was mapped to two threonine-proline motifs (Thr(8) and Thr(11)) that are not present in any of the other human cytosolic sulfotransferases. Our findings suggest that SULT4A1 is subject to post-translational modification that alters its stability in the cell. These modifications may also be important for enzyme activity, which explains why specific substrates for SULT4A1 have not yet been identified.

Cellular uptake of self-assembled cationic peptide-DNA complexes: multifunctional role of the enhancer chloroquine.

A small library of carriers consisting of various combinations of the cell penetrating peptide TAT, the SV40 Large T protein nuclear localisation signal (NLS) and a cationic dendrimer of 7 lysine residues (DEN) was synthesised and each member was tested for its ability to deliver exogenous DNA to human HeLa cells. We found that the TAT peptide was essential, but not sufficient for efficient uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake and expression with the most active carrier consisting of the TAT, NLS and DEN peptides. For those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required incubation with a fusogenic agent such as chloroquine before gene expression was observed. However, our data suggest that chloroquine did not enhance expression solely by promoting endosomal release since a fusogenic peptide derived from the influenza virus haemagglutinin protein did not improve gene expression. Chloroquine was found to protect DNA from degradation and enhance transcription of DNA bound to the respective carriers. Our results demonstrate that multi-component peptide-based gene carriers can be designed to deliver exogenous DNA. The actions of lysosomotropic agents such as chloroquine reveal the multifactorial properties required for carriers used in non-viral gene delivery.

Sulfotransferase 4A1.

In this review, we highlight the physical and enzymatic properties of the novel human sulfotransferase, SULT4A1. The gene is most highly expressed in selective regions of the brain, although work to date has failed to identify any specific endogenous substrate for the enzyme. SULT4A1 shares low homology with other human sulfotransferases. Nevertheless, it is highly conserved between species. Despite the low homology, it is structurally very similar to other cytosolic sulfotransferases with a conserved substrate binding domain, dimerization site and partial cofactor binding sites. However, the catalytic cavity is much smaller, and it has been suggested that the cofactor may not be accommodated within it. A recent link between variability in the 5'UTR of the SULT4A1 gene and schizophrenia has heightened interest in the endogenous function of the enzyme and its possible role in human disease.

The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1.

Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4(114-135)) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.

P450 2C18 catalyzes the metabolic bioactivation of phenytoin.

The safe clinical use of phenytoin (PHT) is compromised by a drug hypersensitivity reaction, hypothesized to be due to bioactivation of the drug to a protein-reactive metabolite. Previous studies have shown PHT is metabolized to the primary phenol metabolite, HPPH, then converted to a catechol which then autoxidizes to produce reactive quinone. PHT is known to be metabolized to HPPH by cytochromes P450 (P450s) 2C9 and 2C19 and then to the catechol by P450s 2C9, 2C19, 3A4, 3A5, and 3A7. However, the role of many poorly expressed or extrahepatic P450s in the metabolism and/or bioactivation of PHT is not known. The aim of this study was to assess the ability of other human P450s to catalyze PHT metabolism. P450 2C18 catalyzed the primary hydroxylation of PHT with a kcat (2.46 +/- 0.09 min-1) more than an order of magnitude higher than that of P450 2C9 (0.051 +/- 0.004 min-1) and P450 2C19 (0.054 +/- 0.002 min-1) and Km (45 +/- 5 microM) slightly greater than those of P450 2C9 (12 +/- 4 microM) and P450 2C19 (29 +/- 4 microM). P450 2C18 also efficiently catalyzed the secondary hydroxylation of PHT as well as covalent drug-protein adduct formation from both PHT and HPPH in vitro. While P450 2C18 is expressed poorly in the liver, significant expression has been reported in the skin. Thus, P450 2C18 may be important for the extrahepatic tissue-specific bioactivation of PHT in vivo.

Characterization of a spontaneously polarizing HT-29 cell line, HT-29/cl.f8.

A cloned cell line that spontaneously polarizes in standard glucose-containing media was derived from a single cell of the adenocarcinoma cell line HT-29. The cloned line, designated HT-29/cl.f8, has remained stable over 2 yr in culture, maintained high transepithelial resistance (300 ohm cm(2) or higher), and correctly sorted influenza virus and vesicular stomatitis virus to apical or basolateral domains, respectively. The newly cloned cells also displayed apical microvilli, tight junctions, and desmosomes, the morphological characteristics of mature epithelia. The cloned HT-29/cl.f8 cells function as epithelial enterocytes as shown by the apical expression of intestinal alkaline phosphatase, the expression of vimentin and cytokeratin, and lack of expression of mucin. We propose that the newly cloned HT-29/cl.f8 cells offer a viable alternative for studies of enterocyte function that will readily yield interpretable data not complicated by cell alterations due to the presence of drugs or chemicals that induce differentiation.