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In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

Kalaiarasan P, Kumar B, Chopra R, Gupta V, Subbarao N, Bamezai RN - PLoS ONE (2015)

Bottom Line: SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important.The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2.We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India.

ABSTRACT
Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

No MeSH data available.


Related in: MedlinePlus

Prediction of Splice regulatory proteins to the PKM2 gene.(A) Splicing factor binding in presence of rs2856929_A (B) splicing factor binding in presence of rs2856929_C (C) splicing factor binding in presence of rs8192381_C (D) splicing factor binding in presence of rs8192381_U (E) splicing factor binding in presence of rs8192431_C (F) splicing factor binding in presence of rs8192431_U.
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pone.0120469.g007: Prediction of Splice regulatory proteins to the PKM2 gene.(A) Splicing factor binding in presence of rs2856929_A (B) splicing factor binding in presence of rs2856929_C (C) splicing factor binding in presence of rs8192381_C (D) splicing factor binding in presence of rs8192381_U (E) splicing factor binding in presence of rs8192431_C (F) splicing factor binding in presence of rs8192431_U.

Mentions: A population based case-control study carried out on 18 SNPs within PKM2 gene, including 15 intronic and 3 in the neighbouring UTR regions (2 close to 5’UTR and 1 close 3’UTR) in 205 sporadic breast cancer cases and 183 controls from northern India (Table 5), showed only 5 intronic SNPs (rs8192386, 0.029; rs1037680, 0.043; rs8192431, 0.053; rs2856929, 0.062; rs8192381, 0.088) in strong association with breast cancer (Table 5). Out of the 5 SNPs, 3 SNPs (rs8192386, rs1037680 and rs8192381) was located in intron 1; whereas other two SNPs (rs2856929 and rs8192431) were located in intron 8 and 10, respectively. To identify the role of these SNPs in silico, SpliceAid2 predicted the splicing factor binding to the minor and major alleles of these SNPs and its flanking sequences. Thus, multiple putative splice regulatory proteins binding sites were identified that could modulate PKM2 pre-mRNA splicing. Due to the single nucleotide difference in the sequences, representing minor and major alleles, 3 (rs8192431, rs2856929 and rs8192381) out of 5 SNPs, were predicted to influence the binding of different splicing factors (Fig. 7). SpliceAid2 identified rs2856929_A allele binding to hnRNP E1 and hnRNP E2 (Fig. 7A), whereas in case of rs2856929_G allele, the binding of these splicing factors was inhibited (Fig. 7B). Another SNP, rs8192381_C, allowed the binding of YB-1, SRp40, Nova-1 and Nova-2 (Fig. 7C); whereas rs8192381_U allele inhibited the binding of these four splicing factors, but allowed the binding of MBNL1 (Fig. 7D). SNP, rs8192431_C, inhibited the binding of SRp40 splicing factor (Fig. 7E), and in case of rs8192431_U the binding was possible (Fig. 7F).


In silico screening, genotyping, molecular dynamics simulation and activity studies of SNPs in pyruvate kinase M2.

Kalaiarasan P, Kumar B, Chopra R, Gupta V, Subbarao N, Bamezai RN - PLoS ONE (2015)

Prediction of Splice regulatory proteins to the PKM2 gene.(A) Splicing factor binding in presence of rs2856929_A (B) splicing factor binding in presence of rs2856929_C (C) splicing factor binding in presence of rs8192381_C (D) splicing factor binding in presence of rs8192381_U (E) splicing factor binding in presence of rs8192431_C (F) splicing factor binding in presence of rs8192431_U.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359060&req=5

pone.0120469.g007: Prediction of Splice regulatory proteins to the PKM2 gene.(A) Splicing factor binding in presence of rs2856929_A (B) splicing factor binding in presence of rs2856929_C (C) splicing factor binding in presence of rs8192381_C (D) splicing factor binding in presence of rs8192381_U (E) splicing factor binding in presence of rs8192431_C (F) splicing factor binding in presence of rs8192431_U.
Mentions: A population based case-control study carried out on 18 SNPs within PKM2 gene, including 15 intronic and 3 in the neighbouring UTR regions (2 close to 5’UTR and 1 close 3’UTR) in 205 sporadic breast cancer cases and 183 controls from northern India (Table 5), showed only 5 intronic SNPs (rs8192386, 0.029; rs1037680, 0.043; rs8192431, 0.053; rs2856929, 0.062; rs8192381, 0.088) in strong association with breast cancer (Table 5). Out of the 5 SNPs, 3 SNPs (rs8192386, rs1037680 and rs8192381) was located in intron 1; whereas other two SNPs (rs2856929 and rs8192431) were located in intron 8 and 10, respectively. To identify the role of these SNPs in silico, SpliceAid2 predicted the splicing factor binding to the minor and major alleles of these SNPs and its flanking sequences. Thus, multiple putative splice regulatory proteins binding sites were identified that could modulate PKM2 pre-mRNA splicing. Due to the single nucleotide difference in the sequences, representing minor and major alleles, 3 (rs8192431, rs2856929 and rs8192381) out of 5 SNPs, were predicted to influence the binding of different splicing factors (Fig. 7). SpliceAid2 identified rs2856929_A allele binding to hnRNP E1 and hnRNP E2 (Fig. 7A), whereas in case of rs2856929_G allele, the binding of these splicing factors was inhibited (Fig. 7B). Another SNP, rs8192381_C, allowed the binding of YB-1, SRp40, Nova-1 and Nova-2 (Fig. 7C); whereas rs8192381_U allele inhibited the binding of these four splicing factors, but allowed the binding of MBNL1 (Fig. 7D). SNP, rs8192431_C, inhibited the binding of SRp40 splicing factor (Fig. 7E), and in case of rs8192431_U the binding was possible (Fig. 7F).

Bottom Line: SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important.The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2.We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology, Shri Mata Vaishno Devi University, Katra, Jammu and Kashmir, India.

ABSTRACT
Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

No MeSH data available.


Related in: MedlinePlus