A survey of proteins encoded by non-synonymous single nucleotide polymorphisms reveals a significant fraction with altered stability and activity

Abstract

On average, each human gene has approximately four SNPs (single nucleotide polymorphisms) in the coding region, half of which are nsSNPs (non-synonymous SNPs) or missense SNPs. Current attention is focused on those that are known to perturb function and are strongly linked to disease. However, the vast majority of SNPs have not been investigated for the possibility of causing disease. We set out to assess the fraction of nsSNPs that encode proteins that have altered stability and activity, for this class of variants would be candidates to perturb cellular function. We tested the thermostability and, where possible, the catalytic activity for the most common variant (wild-type) and minor variants (total of 46 SNPs) for 16 human enzymes for which the three-dimensional structures were known. There were significant differences in the stability of almost half of the variants (48%) compared with their wild-type counterparts. The catalytic efficiency of approx. 14 variants was significantly altered, including several variants of human PKM2 (pyruvate kinase muscle 2). Two PKM2 variants, S437Y and E28K, also exhibited changes in their allosteric regulation compared with the wild-type enzyme. The high proportion of nsSNPs that affect protein stability and function, albeit subtly, underscores the need for experimental analysis of the diverse human proteome.

Direct Measurements of Oscillatory Glycolysis in Pancreatic Islet β-Cells Using Novel Fluorescence Resonance Energy Transfer (FRET) Biosensors for Pyruvate Kinase M2 Activity

“…First, we found that the dynamic range of PKAR was diminished by K433E, a mutant that blocks PKM2 binding to phosphotyrosine peptides that catalyze the release of FBP (43), as well as S437Y, which inhibits the ability of FBP to displace these peptides (48). Our data also suggest that the direct phosphorylation of PKM2 at Tyr-105 shifts PKM2 to its inactive state by disrupting FBP binding, as Y105F, which blocks this phosphorylation event (47), also diminished PKAR dynamic range.…”

“…To test this possibility we evaluated three PKM2 mutants that block 1) tyrosine phosphorylation of PKM2 (Y105F) (47), 2) PKM2 binding to endogenous phosphotyrosine peptides (K433E) (43), or 3) the ability of FBP to displace phosphotyrosine peptides (S437Y) (48). All three mutants decreased PKAR activation by glucose by Ն50% (Fig.…”

“…In this context the FRET method for moni- (47), K433E blocks phosphotyrosine peptide binding by PKM2 (43), and S437Y blocks the ability of PKM2 to displace phosphotyrosine peptides (48). Error bars in all panels represent the mean Ϯ S.E., n Ն 33 cells per treatment from Ն2 independent experiments.…”

Structural and Functional Impact of Cancer-Related Missense Somatic Mutations

“…A blind test of both methods against experimental data for a small set of germ line SNPs occurring in a set of enzymes also shows a high level of agreement. 32 …”

The Stress of Protein Misfolding: From Single Cells to Multicellular Organisms

“…A recent study examined 46 coding polymorphisms for 16 human enzymes with known three-dimensional structures and found that a high proportion (48%) of these natural variants results in altered thermal stability and, in some cases, catalytic efficiency or allosteric regulation (Allali-Hassani et al 2009). From the considerations discussed above, we speculate that altered thermostability leading to a phenotypic outcome may depend on factors such as the "strength" of the chaperone network in a given individual, influences by polymorphisms in chaperone or stress regulatory genes and imbalanced coexpression of chaperones and cochaperones, environmental influences and exposures to proteotoxic stresses, and the presence of other destabilized or misfolded proteins, particularly those acting in the same pathway or competing for the same chaperones (Fig.…”