Keywords
polymorphism - serotonin - tryptophan hydroxylase - SNPs - amino acid
Introduction
Human tryptophan hydroxylase 2 (hTPH-2) is one of the two isoforms of the tryptophan
hydroxylase (TPH) enzyme. One of them is found in a phosphorylated way in the digestive
system (hTPH-1). The other is present in the central and the peripheral nervous system.
The function of the hTPH-2 is the hydroxylation of tryptophan to 5-hydroxytrytophan,
a crucial step in the biosynthesis of the neurotransmitter serotonin.[1]
[2] The deficit or absence of the cofactor Fe (III) affects negatively the efficiency
of the production of 5-hydroxytrytophan. The hTPH-2 has three domains: the N-terminus
that evidences a regulatory function; the Catalytic α-helix domain, with the function
of tetramerization of tryptophan; and the C-terminus domain.[3]
Serotonin is the neurotransmitter associated in normal conditions with the regulation
of grades of pain, thermo-nociceptive response, intake, emotional behavior, and stress
response, among other functions.[4] A decrease in the serotonin production affects the muscle tissue of the vascular
system. For instance, this smooth muscle tissue evidences altered contractile responses
resulting on blood pressure disease due to a significant reduction of 5-hydroxytrytophan.[5]
Polymorphisms of hTHP-2 lead to changes in the amino acids and the structure of the
enzyme. These polimorphisms cause a declining serotonin outputs due non-functional
enzyme activity. Neuropsychiatric diseases such as depression, schizophrenia, and
autism are related with those changes.[6] Depression, for instance, is a common disease nowadays, evidenced by constant feelings
of sadness, loss of interest in self-care activities and self-esteem. The rs7305115
single nucleotide polymorphism (SNP) at the TPH2 may predispose to suicide attempts
in patients in the suicide and major depressive disorders (MMD) spectrum.[7] It is estimated that by 2020 depression will be the second most disabling disease
in the world.[8]
Thus, it is important to focus the research on neuropsychiatric and systemic serotonin-related
disorders, in order to implement therapeutic targets useful to improve treatments.[9] This in silico analysis corresponds to an initial modeling study of the reported
alteration on the enzyme structure. The results of this analysis serve as a background
of the SNPs associated to possible alterations in the function of the protein. The
purpose of this study is to analyze the influence on the stability and alterations
caused by changes in the associated SNPs of the hTHP-2 enzyme.
Materials and Methods
In this study, we applied bioinformatics techniques by using websites designed by
universities and organizations for the analysis of different SNPs. The code for TPH2
is Ensembl: ENSG00000139287.
The procedures used were:
-
Protein Data Bank (PDB)
We consulted the division of medicine found on this page to look for a protein according
to its function. The analyzed enzyme had to do with the production of serotonin, so
it was selected to determine the way in which an amino acid change could affect the
role of the treated protein. By using the PDB, we established the protein chain, primary
and secondary structures, as well as SNP. The primary structure was identified with
codes; in this case, the enzyme was labeled with the code 1MLW and contained 14 SNPs
on its structure. In this database, the files necessary to perform the following steps
in the investigation, such as those in Fast-All (FASTA) sequence and PDB format (3D
structure) were found.
-
Sorting Intolerant from Tolerant (SIFT)
This program indicated the probability of tolerance of the amino acids changed. Likewise,
the biochemical tolerance to the 3D structure of the chosen protein was determined.
To use this program, we started with the FASTA file that was downloaded from the PDB.
Then, the unique protein chain that constitutes the enzyme was selected from this
file. After adding the sequence of the enzyme in the space provided, we selected the
option of locating the position of the SNP and acquire the data for the analysis.
The fragment of protein sequence with the amino acid 341 (L), was as follows: GAGLLSSISELKHALSGHAKVKPF
-
Polymorphism Phenotyping v2 (PolyPhen-2)
By this software, it was demonstrated that making the change of an amino acid in the
protein sequence can lead to a pathogenic protein, based on biological characteristics
and protein evolution.
To obtain the results, the information of the protein chain and the position of SNP
to be treated were established. Then, the software evidenced the pathogenicity of
the amino acid change in the structure of the protein.
-
Non-synonymous single nucleotide polymorphism Analyzer (nsSNPAnalyzer):
To predict disease-associated non-synonymous single nucleotide polymorphism.
This tool provides additional information about the SNP investigated by reading and
interpreting the results in an easier way. With this software we used information
of multiple sequence alignment, and the information contained in the 3D structure
of the protein to make predictions through the analysis of biochemical and biological
information.
The procedure for data collection began with the use of the corresponding tryptophan
hydroxylase protein FASTA sequence. Following the procedure, the amino acid change
was placed in the protein sequence, and then the PDB file previously obtained was
used. The protein chain to be analyzed was introduced and the steps were repeated
with each of the SNPs.
-
Swiss-PDB Viewer 4.1.0
After the completion of the analysis with the described software, the 3D structure
of the studied protein was evidenced by Swiss-PDB. This 3D structure was obtained
through the option “file,” in which the “PDB file” option was selected. Then, the
structure of the protein was observed, and its protein chain for tryptophan hydroxylase
was used. The sequence of ordered protein chains was observed by using the bar called
control panel. In the control panel, the corresponding chain of interest was selected. We proceeded
to activate the Compute H-Bonds, near the command bar tools, to identify the hydrogen bonds that naturally possess the protein when it has normal
or abnormal changes on its structure.
For the analysis of the SNPs that were considered pathogens, we selected the amino
acid of interest in the control panel, and its location on the screen was presented.
First, the amino acid was located in the protein structure. Then, the amino acid change
or “mutation” was performed by selecting the corresponding option and finally, by
choosing the amino acid in the chain to make the exact change to the SNP.
A conformational change in the protein structure is evidenced when the SNP has a significant
effect on the protein function. This is one of the determinants in the protein to
be considered pathogenic. Another factor is given by the analysis through Force Field
(compute energy) in the Tools tab. After performing the last step, the Force Field analysis demonstrated the total
enthalpy of the protein and each amino acid in the chain.
Results
During evaluation and analysis of the protein 1MLW with its corresponding SNPs, it
was possible to demonstrate that only one SNP caused a pathogenic effect on the protein
structure. This polymorphism was classified by the four software as harmful/not tolerable.
The pathogenic SNP is the amino acid 341, which had a change of amino acid L to amino
acid F. Likewise, different results were found including the pathogenicity/tolerability
of SNPs. The first software, SIFT, performed a chemical assessment and determined
whether the protein is tolerable or not to the change. The second software, PolyPhen-2,
evaluated the biological characteristics of the protein, its evolution and whether
it is pathogenic or not. PolyPhen-2 labeled the SNPs as benign and possibly pathogenic.
[Table 1] summarizes the data obtained from the software for each SNP.
Table 1
Analysis of single-nucleotide polymorphisms according to tolerance, pathogenic effect
and enthalpy of the 3D structure of human tryptophan hydroxylase 2 (hTHP2)
|
SNPs
|
SIFT
|
PolyPhen-2
|
nsSNPAnalyzer
|
Enthalpy of the 3D structure
|
|
SNP 124 M-L
|
Tolerable
|
Benign 0.001
|
Neutral
|
12,296.042
|
|
SNP 142 R-C
|
Not tolerable
|
Possibly pathogen 1000
|
Disease
|
11,821.518
|
|
SNP 146 K-R
|
Tolerable
|
Possibly pathogen 0.471
|
Neutral
|
12,442.093
|
|
SNP 163 K-N
|
Tolerable
|
Benign 0.189
|
Neutral
|
12,441.610
|
|
SNP 177 V-I
|
Tolerable
|
Benign 0.015
|
Neutral
|
12,213.241
|
|
SNP 207 R-W
|
Tolerable
|
Possibly pathogen 0.999
|
Disease
|
12,033.135
|
|
SNP 224 L-I
|
Tolerable
|
Benign
|
Disease
|
12,030.536
|
|
SNP 274 L-I
|
Tolerable
|
Benign 0.375
|
Neutral
|
12,046.070
|
|
SNP 300 A-T
|
Not tolerable
|
Possibly pathogen 0.925
|
Neutral
|
12,020.028
|
|
SNP 304 A-V
|
Tolerable
|
Benign
|
Neutral
|
12,271.819
|
|
SNP 310 T-M
|
Not tolerable
|
Possibly pathogen 0.996
|
Disease
|
11,446.873
|
|
SNP 341 L-F
|
Not tolerable
|
Possibly pathogen 0.999
|
Disease
|
14,452.046
|
|
SNP 362 Q-R
|
Tolerable
|
Possibly pathogen 0.990
|
Neutral
|
12,400.587
|
Abbreviations: A, alanine; C, cysteine; F, phenylalanine; I, isoleucine; K, lysine;
L, leucine; M, methionine; nsSNPAnalyzer, non-synonymous single nucleotide polymorphism
analyzer; PolyPhen-2, polymorphism phenotyping v2; Q, glutamine; R, arginine; SIFT,
sorting intolerant from tolerant; SNP, single-nucleotide polymorphism; T, threonine;
V, valine; W, tryptophan.
PDB
-
In the PDB, we found the following SNPs:
-
rs201585879 change: K (lysine) - R (arginine)
-
rs147638867 change: V (valine) - I (isoleucine)
-
rs201751661 change: L (leucine) - F (phenylalanine)
-
rs200937558 change: R (arginine) - C (cysteine)
-
rs145855109 change: A (alanine) - T (threonine)
-
rs184973363 change: M (methionine) - L (leucine)
-
rs142170901 change: A (arginine) - V (valine)
-
rs41274348 change: T (threonine) - M (methionine)
-
rs139617975 change: R (arginine) - W (tryptophan)
-
rs147488937 change: E (glutamic acid) - K (lysine)
-
rs20222394 change: Q (glutamine) - R (arginine)
-
rs41274350 change: L (leucine) - I (isoleucine)
-
M.rs189455467 change: K (lysine) - N (asparagine)
-
rs145479597 change: R (arginine) - C (cysteine)
SIFT
The software evidenced that 9 of the 13 SNPs studied were tolerable; therefore, 4
SNPs were considered intolerable ([Table 1]).
PolyPhen-2
After analysis of the SNPs, this software revealed that 6 of the 13 SNPs were benign.
Thus, more than half of the SNPs were considered as possibly pathogenic. ([Table 1]).
Non-synonymous Single Nucleotide Polymorphism Analyzer (nsSNPAnalyzer)
This software showed that eight SNPs were neutral, while five might be disease-causing
([Table 1]).
Enthalpy of the 3D Structure
On the analysis of the enthalpy of the 3D structure of the protein, a change on its
primary structure was observed. The effect of the SNP that generated this outcome
was considered pathogenic. Likewise, the same SNP was evidenced as pathogenic in the
previous analysis performed through the software applications ([Table 1]). [Fig. 1A] shows the primary structure of the protein 1MLW without changes on its structure.
In contrast, [Fig. 1B] evidences the change on the 3D structure of the chain, due to the change on the
L341F, which generated an aromatic ring.
Fig. 1 (A) Normal protein. (B) Changes in the protein structure caused by mutation. The evident conformational
change is the formation of an aromatic ring (circle).
Discussion
This study evidenced an important change in the SNP 341 of the hTPH-2 enzyme that
caused a pathogenic effect. This negative impact resulted in the presence of an abnormal
aromatic ring in the structure of the protein ([Fig. 1B]). This structural disorder affects the polarity of the protein, causing loss of
the hydrogen bonds required for the stability so, leading to a functional alteration.
The changes on the other amino acids of the protein sequence mostly did not affect
it negatively; however, they caused minimal variations on the enthalpy. These outcomes
can be associated with abnormal expression in determined diseases that cause functional
defects. For instance, defects related with the decrease in the serotonin synthesis
that carries out symptoms evidenced in neurological diseases.
The change in a specific amino acid of the chain of this protein affects the serotonin
levels. This condition might lead to neuropsychiatric disorders such as depression,
schizophrenia, Parkinson's disease, aggressiveness, suicidal behavior, attention deficit
or hyperactivity, and autism.[8] In neuropsychiatric degenerative diseases, such as Parkinson's disease, according
to Ostrosky-Solis (2000),[10] the lack or deficiency in the synthesis of the serotonin is associated with depressive
and anxiety disorders in these patients.
Other systems, besides the nervous system, can be affected by the defects in the serotonin
synthesis. In the immune system, for example, evidence has been found that the synthesis
and transport of serotonin can be performed by T-lymphocytes. Researches performed
in psychiatric patients have shown a modification in the serotonergic system of the
T-lymphocytes cells when depression occurs.[11]
Our literature search evidenced very few articles that matched specifically the change
in the amino acid 341 of the structure of the hTPH-2, and/or the adverse effects caused
by the exchange of an amino acid, such as the one analyzed in this study.[11] Therefore, further research is needed to bring forth improvements in the treatment
for neuropsychiatric diseases.
Considering that a large percentage of people in the world suffer from depression
and other neuropsychiatric diseases, this study attempts to explore one of the multiple
possible causes of disorders in the synthesis of serotonin. This objective opens a
new window for research on the treatment of hTPH-2 enzyme deficiency and its effects
on patients.
Conclusion
While the causes of neurological and neuropsychiatric disorders are multiple, it is
very likely that polymorphisms in the different proteins involved in neurophysiological
processes have a significant influence on the development of these disorders. It is
suitable for future works to find a similar abnormal conformational tendency in other
enzymes of the nervous system. This allows researchers to establish a comparable association,
similar to the observed in hTPH-2 and its association with amino acids changes.
