Helix Vol.

2:116-119 (2012)

A Novel Insilico Approach for Studying the Mutational affects of TPH2 Protein and its association with Suicidal behavior
Spoorthy Nirvani*, Dr. Amit Kumar2, P.Shalini3
Email Id: nirvana.spoorthy@gmail.com, 2amit.kumar@dnares.in, 3shalini@dnares.in

Received February 10, 2012; Accepted February 24, 2012; Published March 01, 2012

Abstract: Suicide is an important public health problem and a major issue of concern. Several reports suggest that the genetic factors play a prominent role in the predisposition to suicide. Tryptophan hydroxylase 2 (Tph2) is a neuronal enzyme involved in the synthesis of the neurotransmitter serotonin. Tph2 has its significance in suicide behavior which is less reported. Current work is emphasized on the mutational studies of TPH2 through Insilico approach and its association with suicide. From the literature and SNP data base, mutations which are responsible for the suicidal behavior are studied. A complete analysis of the protein with respect to the mutations was done to trace the significance of the mutations in functional and structural level. Analysis includes sequence, phylogenetic and structural analysis, domain, motif and binding site prediction analysis. The effect of these mutations in the sequence and structural level are studied. The mutational effect that leads to the diseased condition or neural polymorphism has been analyzed using insilico tools. Structure comparison of the normal and mutated domain was done to know the deviation of the structure and stability effect. The mutational study has been summarized by mentioning the mutational site significance, its effects on sequence and structural level, and the resultant condition due to the mutation. Keywords: Mutation, Tph2, Serotonin, Suicide. Introduction: Tryptophan hydroxylase is an enzyme critical for synthesis of serotonin (5-HT) in the brain, which plays a role in anxiety and depression related personality traits [1]. Brain serotonin (5-HT) a monoaminergic neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Some discoveries show that a second TPH isoform (TPH2)

in vertebrates, including man, which is predominantly, expressed in brain, while the previously known TPH isoform (TPH1) is primarily a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders (for ex: drug abuse, schizophrenia, suicide) [2]. TPH2 is encoded by two different genes, localized on Chromosome 12. As TPH2 is the brain-specific isoform of tryptophan hydroxylase, it has been the focus of genetic association studies addressing the relationship between the mutations in TPH2 and affective disorders [3]. TPH2 is a homo tetramer. Each monomer is catalytically active and comprises three functional regions: a regulatory N-terminal region, a catalytic domain and the tetramerization C-terminal domain. Three TPH2 mutations have been linked to serotonergic pathology: Pro206Ser (a mutation was manifested via stability and solubility effects, localized in the sixth exon of tph2 gene, SNP rs17110563), associated with BP disorder and Ala235Ser (a mutation localized in the sixth exon of tph2 gene, SNP rs120074176), associated with a hyperactivity disorder and Suicide behavior and Arg303Trp (a mutation had severely reduced solubility effects SNP rs4570625), also associated with a hyperactivity disorder [4]. The most concrete evidence for the connection between serotonin and depression is the decreased concentrations of serotonin metabolites in the cerebrospinal fluid and brain tissues of depressed people [5]. High levels of tryptophan in the brain directly influence increased serotonin production and new brain cell production begins to rise [6]. Suicide is a serious problem in Slovenia, where in the year 2002 of every 100.000 citizens, 44.4 men and 10.5 women committed suicide [World Health Organization, 2004]. Various published studies are seeking possible genetic markers that will be helpful for the early detection and prevention of suicidal behavior. One possible mechanism by which genetic factors might affect the risk for suicide is by the control of serotonergic neurotransmission [Stefulj et al., 2004a] [7].

116
Copyright 2012 Helix ISSN 2277 3495(Print)

Helix Vol. 2:116-119 (2012)

Material & Methodology: For the mutational studies of Tph2 through insilico approach SNP data analysis was done and found three mutated sites Pro206Ser, Arg303Trp, and Ala235Ser. The complete mutational analysis was done by studying the protein in detail with respect to mutational sites. The significance of these mutational sites on sequence and structural level was studied using various insilico tools. Sequence analysis is done by using BLAST [8], primary secondary and tertiary structure analysis is done by using ProtParam, SOPMA, CPHmodel, HHpred, and Phyre2 tools [9]. Domain analysis was done using SMART tool and Motif analysis using fingerprint scan [10], active sites were predicted using Q-site finder and CASTp [11], the disorderness of the sites are predicted using DisEMBL tool [12]. Using SYBYL software the domain was designed and optimized. Stability and Relative Solvent Accessible Areas for the mutated positions were calculated [13]. The effect of the mutation on the protein was analyzed. Results & Discussion: Tph2 sequence was retrieved for its further insilico analysis. Sequence analysis using BLAST was performed followed by the phylogenetic analysis using SDSC biology workbench. The fig: 1 shows the superimposition of normal tph2 with 206th mutated structure and RMSD value is calculated as 31.3086.

Figure2: Molegro superimposition of normal Tph2 structure With 235th mutated structure and RMSD value

Fig: 2 show the superimposition of normal tph2 with 235th mutated structure and the RMSD value is calculated as 0.136559.

Figure3: Molegro superimposition of normal Tph2 structure with 235th mutated structure and RMSD value

Fig: 3 shows the superimposition of normal tph2 with 303 mutated structure and RMSD value calculated as 0.297707.
Table: I-Mutant Result for Normal Tph2 sequence at the parameter sign DDG.

Table: I-Mutant Result for Normal Tph2 sequence at the Free Energy Change Value DDG Parameter

Figure1: Molegro superimposition of normal Tph2 structure with 206th mutated structure and RMSD value

116
Copyright 2012 Helix ISSN 2277 3495(Print)

Helix Vol. 2:116-119 (2012)

WT: Aminoacid in Wild-Type Protein NEW: New Aminoacid after Mutation RI: Reliability Index T: Temperature in Celsius degrees pH: -log [H+]
Table: I-Mutant Result for 1MLW structure at the Parameter Sign DDG

protein, aliphatic and hydrophilic in nature by analyzing its primary structure Structural analysis is performed using PROTPARAM, SOPMA. The contribution of the mutational sites in secondary structure formation was studied. Mutational sites are forming coil and helix conformations in the secondary structures. From tertiary structure analysis using CPH, HHpred, and Phyre, the protein ID similar to the tph2 protein was determined as 1MLW. Domains were analyzed using SMART an found that the mutational sites are lying in the domain Biopterin_H falls between 155-483 of the sequence. Disorder prediction was done using DisEMBL which includes the prediction of the disorderness and the disorder probability of the mutational sites. Fingerprint scan was used to predict the active site and the functional importance was traced. According to the Q-site finder results mutational site 257 is laying on 3rd binding site, 160th mutation is lying on 6th strong binding site, and CASTp results show that 189th mutational site is lying on 13th pocket. The structure comparison of the normal and mutated structure was performed. Structural comparison was done by superimposing the two structures and the change in the structure is defined by means of Root Mean Square Deviation (RMSD) value. Molegro virtual docker was used for structure superimposition. The effect of the mutational site was studied using I Mutant. The three mutations 206, 235 and 303 causes a decrease in the stability of the protein and the RSA (Relative Solvent Accessible Area) values for the mutations are calculated to be 64.8, 0.0, 5.7 respectively. The resultant condition due to the mutation was studied using PhD SNP [14]. All the three mutations lead to the disease related polymorphism condition. From the above analysis it can be hypothesized that the structural modifications caused due to mutation and its effect on the functionality of the protein could be the possible reason for the suicidal behavior. Conclusion: In this paper we predict the possibilities of the psychological disorder and their influence in suicide behavior due to the mutations in TPh2 protein. The alterations to the Tph2 protein at molecular level were studied using Insilico analysis. The mutational studies were performed and their effects on the structural and functional level were studied. The

Table: I-Mutant Result for 1MLW structure at the Parameter Free Energy Change Value DDG

RSA: Relative Solvent Accessible Area

PhD-SNP results
Table: PhD-SNP Result for 1MLW structure

WT: Amino acid in Wild-Type Protein NEW: New Amino acid after Mutation RI: Reliability Index Effect: Neutral: Neural Polymorphism Disease: Disease-related Polymorphism Through literature and genome analysis with the use of SNPdb, three variations has been identified, the 3 prominent mutations P206S, R303W, and A235S have been focused which have relation with the suicide which can be stated by considering their importance in major depression disorders. The mutation study was done on P206S, R303W, and A235S mutations. Complete mutational analysis was performed by studying the protein in detail. The significance of these mutational sites on sequence and structural level was studied using various insilico tools. The query protein is inferred as an unstable

117
Copyright 2012 Helix ISSN 2277 3495(Print)

Helix Vol. 2:116-119 (2012)

resultant effect is analyzed to be the disease related polymorphic condition thus emphazing its role in suicide behavior. Acknowledgment: We thank sincerely to Ms. P. Shalini for her guidance and suggestions, and thank to my parents for their encouragement. I also thank the BioAxis DNA Research Centre Pvt.Ltd. staff for their constant encouragement and support. References: [1]. Waider J, Araragi N et al. Tryptophan hydroxylase-2 (TPH2) in disorders of cognitive control and emotion regulation: a perspective. Department of Psychiatry. Psychosomatics, and Psychotherapy, University of Wuerzburg, Fuechsleinstrasse Germany. [2]. Maik Grohmann et al. Alternative Splicing and Extensive RNA Editing of Human TPH2 Transcripts [3]. Maria mernea et al, Pro206ser and arg441his mutations influence on human tryptophan hydroxylase 2 activity a molecular Modeling study, Academia romn, revue roumaine de chimie, Rev. Roum. Chim., 2011, 56(8), 833-841. 4]. McKinney J.A., Turel B., Winge I., Knappskog P.M., Haavik J. Functional properties of missense variants of human tryptophan hydroxylase 2. [5]. University of BRISTOL, Serotonin a molecule of happiness, http://www.chm.bris.ac.uk/motm/serotonin/depressio n.htm [6]. Integrative Psychiatry, Dont Just Treat the Symptoms: Treat the Cause, http://www.integrativepsychiatry.net/serotonin.html [7]. Alja Videtic,1 Galina Pungercic,1 Irena Zupanic Pajnic,2 Tomaz Zupanc,2 Joze Balazic,2 Brief Research Communication, Association Study of Seven Polymorphisms in Four Serotonin Receptor Genes on Suicide Victims. [8]. Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. [9]. Lavigne, R., Sun, W.D. and Volckaert, G. STORM towards protein function: Systematic Tailored ORF-data Retrieval and Managment. Applied Bioinformatics (2003) 2(3): 177-179 [10]. Subramaniam, S. (1998) The Biology Workbench--a seamless database and analysis environment for the biologist. Proteins, 32, 1-2. [11]. Sriram Sankararaman, Fei Sha, Jack F. Kirsch, Michael I. Jordan, Kimmen Sjolander,Active site prediction using evolutionary and structural information Bioinformatics, Vol. 26, No. 5. (1 March 2010), pp. 617-624., [12]. R. Linding, L.J. Jensen, F. Diella, P. Bork, T.J. Gibson and R.B. Russell, Protein disorder prediction: implications for structural proteomics Structure Vol 11, Issue 11, 4 November 2003. [13]. Matthew Clark, Richard D. Cramer, Nicole Van Opdenbosch,., Validation of the general purpose tripos 5.2 force field J. Comput. Chem., Vol. 10, No. 8. (7 September 1989), pp. 982-1012. [14]. Capriotti, E., Calabrese, R., Casadio, R. (2006) Predicting the insurgence of human genetic diseases associated to single point protein mutations with support vector machines and evolutionary information. Bioinformatics. *****

118
Copyright 2012 Helix ISSN 2277 3495(Print)