Computational Screening and Analysis of Lung Cancer Related Non-Synonymous Single Nucleotide Polymorphisms on the Human Kirsten Rat Sarcoma Gene

The human KRAS (Kirsten rat sarcoma) is an oncogene, involved in the regulation of cell growth and division. The mutations in the KRAS gene have the potential to cause normal cells to become cancerous in human lungs. In the present study, we focus on non-synonymous single nucleotide polymorphisms (nsSNPs), which are point mutations in the DNA sequence leading to the amino acid variants in the encoded protein. To begin with, we developed a pipeline to utilize a set of computational tools in order to obtain the most deleterious nsSNPs (Q22K, Q61P, and Q61R) associated with lung cancer in the human KRAS gene. Furthermore, molecular dynamics simulation and structural analyses of the 3D structures of native and mutant proteins confirmed the impact of these nsSNPs on the stability of the protein. Finally, the experimental results demonstrated that the structural stability of the mutant proteins was worse than that of the native protein. This study provides significant guidance for narrowing down the number of KRAS mutations to be screened as potential diagnostic biomarkers and to better understand the structural and functional mechanisms of the KRAS protein.

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Nonsynonymous single nucleotide polymorphisms of NHE3 differentially decrease NHE3 transporter activity

AJP Cell Physiology ◽

10.1152/ajpcell.00421.2014 ◽

2015 ◽

Vol 308 (9) ◽

pp. C758-C766 ◽

Cited By ~ 7

Author(s):

Xinjun Cindy Zhu ◽

Rafiquel Sarker ◽

John R. Horton ◽

Molee Chakraborty ◽

Tian-E Chen ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Large Fraction ◽

Regulatory Protein ◽

Nucleotide Polymorphisms ◽

Genetic Determinants ◽

Single Nucleotide ◽

Mutant Proteins ◽

Homologous Protein ◽

Genetic Abnormalities ◽

The Impact

Genetic determinants appear to play a role in susceptibility to chronic diarrhea, but the genetic abnormalities involved have only been identified in a few conditions. The Na+/H+ exchanger 3 (NHE3) accounts for a large fraction of physiologic intestinal Na+ absorption. It is highly regulated through effects on its intracellular COOH-terminal regulatory domain. The impact of genetic variation in the NHE3 gene, such as single nucleotide polymorphisms (SNPs), on transporter activity remains unexplored. From a total of 458 SNPs identified in the entire NHE3 gene, we identified three nonsynonymous mutations (R474Q, V567M, and R799C), which were all in the protein's intracellular COOH-terminal domain. Here we evaluated whether these SNPs affect NHE3 activity by expressing them in a mammalian cell line that is null for all plasma membrane NHEs. These variants significantly reduced basal NHE3 transporter activity through a reduction in intrinsic NHE3 function in variant R474Q, abnormal trafficking in variant V567M, or defects in both intrinsic NHE3 function and trafficking in variant R799C. In addition, variants NHE3 R474Q and R799C failed to respond to acute dexamethasone stimulation, suggesting cells with these mutant proteins might be defective in NHE3 function during postprandial stimulation and perhaps under stressful conditions. Finally, variant R474Q was shown to exhibit an aberrant interaction with calcineurin B homologous protein (CHP), an NHE3 regulatory protein required for basal NHE3 activity. Taken together, these results demonstrate decreased transport activity in three SNPs of NHE3 and provide mechanistic insight into how these SNPs impact NHE3 function.

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