Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2)

Abstract

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (N⌬150 and N⌬150/ C⌬24) using isopropyl ␤-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH 2 -terminal regulatory domain. The solubility of hTPH2, N⌬150, and N⌬150/ C⌬24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t1 ⁄ 2 at 37°C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, N⌬150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH 2 -terminal domain and the COOHterminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH 2 -terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V max values. These data identify the NH 2 -terminal regulatory domain as the source of hTPH2 instability and reduced solubility.Tryptophan hydroxylase (TPH 2 ; EC 1.14.16.4), a tetrahydropterin (BH 4 )-dependent amino acid hydroxylase, is the key regulator of serotonin (5-hydroxytryptamine) biosynthesis. Utilizing BH 4 and O 2 as co-substrates and Fe 2ϩ as a cofactor, TPH hydroxylates tryptophan to 5-hydroxytryptophan ( Fig. 1). Subsequent decarboxylation of 5-hydroxytryptophan by amino acid decarboxylase generates serotonin (1-3). This essential monoamine has been found in a variety of tissues and implicated in a wide range of physiological functions. In the central nervous system, serotonin is synthesized primarily in the dorsal raphe nucleus and acts as a neurotransmitter; in the pineal gland, it serves as a precursor for melatonin biosynthesis (4). In the periphery, serotonin constricts large blood vessels and regulates platelet adhesion (5-7). Serotonin, produced by the enterochromaffin cells of the intestinal system, initiates peristaltic and secretory reflexes (8). Altered activity of serotonin is associated with various disorders such as depression, impulsive behaviors, aggression, suicide, drug abuse and alcoholism, sleep disorders, gastrointestinal diseases (such as irritable bowel syndrome), and cardiovascular dysfunction leading to heart failure (9).Recently, Walther et al. (10) discovered the gene responsible for nervous system TPH (TPH2). Unlike TPH1 now known to be the peripheral enzyme (11), TPH2 is neuron-specific and expressed predominantly in serotonergic neurons of the raphe nuclei (10, 12) and in the peripheral myenteric neurons in the gut (13). Human TPH1 and TPH2 display 72% sequence h...

Functional properties of missense variants of human tryptophan hydroxylase 2

“…Similar to the cell free system, all TPH2 variants appeared to be more soluble in HEK 293 cells than in E. coli: 73-83% of the total amount of TPH2 was observed in the supernatant fraction after centrifugation at 12,000 Â g for 10 min (data not shown), except for p.Arg303Trp which was mainly recovered in the insoluble fraction as shown previously . These solubility estimates are higher than previously reported for WT, p.Pro206-Ser, and p.Arg441His [Carkaci-Salli et al, 2006;Cichon et al, 2008;Winge et al, 2007]. The difference is explained by the use of a more effective cell lysis procedure and avoidance of repeated freeze/thaw cycles of the cells.…”

“…2 and 3). It has been reported that the N-terminal domain of TPH2 is responsible for the lower solubility and stability of TPH2 in comparison to TH and PAH [Carkaci-Salli et al, 2006;Thorolfsson et al, 2002]. However, for all three enzymes, phosphorylation of serines in the N-terminal appears to stabilize the protein [Miranda et al, 2002;Moy and Tsai, 2004;Royo et al, 2005;Winge et al, 2008).…”

“…Except for p.Pro206Ser, which was cleaved at levels similar to the WT, mutants with reduced solubility also had relatively more uncleaved fusion protein, that is, 100%, 24%, 36%, and 22% for p.Arg303Trp, p.Ala328Val, p.Arg441His, and p.Asp479Glu, respectively. In a study where a similar method was used, less than 50% cleavage was reported for 6xHis-TPH2 WT using the same protease [Carkaci-Salli et al, 2006], indicating that 6xHisMBP-TPH2 is not an optimal substrate for this protease.…”

A Regulatory Domain in the N Terminus of Tryptophan Hydroxylase 2 Controls Enzyme Expression

“…1). The regulatory domains of other members of the family, such as TPH1 and TH, have been shown to modulate enzyme activity and thermostability through various mechanisms, including phosphorylation by protein kinases (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). In line with this, the extended N terminus of TPH2 contains a serine at position 19, which is a candidate for PKA phosphorylation (32) and has been recently shown to interact with 14-3-3 proteins in a phosphorylationdependent manner to increase protein stability (33).…”

A functional alternative splicing mutation in human tryptophan hydroxylase-2

“…34 As the truncated TPH2-TR retains the entire regulatory domain and a small portion of catalytic domain (Figure 1a), its dominant-negative activity may lie in its interference with the normal function of the regulatory domain in the full-length protein. 35,36 A number of genetic analyses have indicated association between the TPH2 gene and central nervous system disorders like depression, 14 bipolar disorder 37,38 and attention-deficit/hyperactivity disorder. 39 A functional characterization was carried out for a rare loss-of-function R441H mutation in an elderly depression cohort whose patients experienced life-long depression, and were resistant to treatment.…”