Limits...
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

Enzyme activity of wild and variants.Under optimal conditions, the activity assay of purified PK-WT and variant proteins showed ~18% and 55% reduction in C31F and S437Y nsSVPs activities respectively. However, Q310P nsSVP was catalytically dead. Binding of allosteric activator FBP increased the activity up-to 27% and 35% in PKWT and C31F nsSVP, however, showed no increase in activity in S43Y nsSVP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359060&req=5

pone.0120469.g006: Enzyme activity of wild and variants.Under optimal conditions, the activity assay of purified PK-WT and variant proteins showed ~18% and 55% reduction in C31F and S437Y nsSVPs activities respectively. However, Q310P nsSVP was catalytically dead. Binding of allosteric activator FBP increased the activity up-to 27% and 35% in PKWT and C31F nsSVP, however, showed no increase in activity in S43Y nsSVP.

Mentions: The purified recombinant wild type human-PKM2 protein (PKWT) and its nsSVPs expressed in E. coli was used for PKM2 activity assay. PKWT showed ~41.1 U/mg of PKM2 activity while C31F and S437Y variants showed 29.9 and 18.82 U/mg activity, respectively (Fig. 6). Interestingly, another non-synonymous variant, Q310P, was catalytically dead (Fig. 2). Since PKM2 allosteric activator FBP is known to increase its affinity for substrate PEP with a net increase in activity [6], in order to investigate the effect of non-synonymous variations on FBP dependent change in activity, we incubated proteins with 2mM FBP and measured the activity under the similar optimal condition as assessed in the absence of FBP. As expected, FBP increased the activity by 27% in the case of wild PKM2. In C31F variant, a 35% increase in activity in the presence of FBP was significantly higher than wild type which is not expected. The S437Y variant did not show any change in activity, while Q310P variant remained catalytically dead relatively. All these results indicated the possible change in the structures of variants which potentially led to modulations in binding of either substrate or other ligands like FBP, ADP to the enzyme.


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)

Enzyme activity of wild and variants.Under optimal conditions, the activity assay of purified PK-WT and variant proteins showed ~18% and 55% reduction in C31F and S437Y nsSVPs activities respectively. However, Q310P nsSVP was catalytically dead. Binding of allosteric activator FBP increased the activity up-to 27% and 35% in PKWT and C31F nsSVP, however, showed no increase in activity in S43Y nsSVP.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120469.g006: Enzyme activity of wild and variants.Under optimal conditions, the activity assay of purified PK-WT and variant proteins showed ~18% and 55% reduction in C31F and S437Y nsSVPs activities respectively. However, Q310P nsSVP was catalytically dead. Binding of allosteric activator FBP increased the activity up-to 27% and 35% in PKWT and C31F nsSVP, however, showed no increase in activity in S43Y nsSVP.
Mentions: The purified recombinant wild type human-PKM2 protein (PKWT) and its nsSVPs expressed in E. coli was used for PKM2 activity assay. PKWT showed ~41.1 U/mg of PKM2 activity while C31F and S437Y variants showed 29.9 and 18.82 U/mg activity, respectively (Fig. 6). Interestingly, another non-synonymous variant, Q310P, was catalytically dead (Fig. 2). Since PKM2 allosteric activator FBP is known to increase its affinity for substrate PEP with a net increase in activity [6], in order to investigate the effect of non-synonymous variations on FBP dependent change in activity, we incubated proteins with 2mM FBP and measured the activity under the similar optimal condition as assessed in the absence of FBP. As expected, FBP increased the activity by 27% in the case of wild PKM2. In C31F variant, a 35% increase in activity in the presence of FBP was significantly higher than wild type which is not expected. The S437Y variant did not show any change in activity, while Q310P variant remained catalytically dead relatively. All these results indicated the possible change in the structures of variants which potentially led to modulations in binding of either substrate or other ligands like FBP, ADP to the enzyme.

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