Presentation Title

Evolution and natural variation of HSPA1A, the major stress inducible gene, in humans

Start Date

November 2016

End Date

November 2016

Location

HUB 302-35

Type of Presentation

Poster

Abstract

HSPA1A is a critical component of the cellular stress response and alterations in its function have been associated with several diseases, such as cancer. However, the extent of HSPA1A’s natural variation remains largely unknown. Here, we investigated the evolution of this gene in mammals. Our results reveal that in all mammals HSPA1A is actually two identical proteins coded by two tandemly arranged genes. Although this pattern can be explained by gene conversion, this mechanism alone cannot explain the presence of several non-amino acid altering (synonymous) mutations. Therefore, we conceptualized that both gene conversion and purifying selection act to conserve these genes. This notion is also supported by analyses of HSPA1A variation in humans, which revealed that all mutations have a very low frequency and that the majority is synonymous. The presence of purifying selection was further supported by experiments determining whether natural variants affect HSPA1A’s functions. First, proteins corresponding to the wild-type (WT) and five mutated variants were tested for their ability to hydrolyze ATP. These experiments revealed that the mutations did not significantly alter the rate of ATP hydrolysis. Second, Isothermal Titration Calorimetry was employed to test whether the mutations alter binding of HSPA1A to ATP, ADP, and protein substrates. These experiments showed that only one mutation increased the entropy of the reactions. Lastly, the effect of these mutations on protein stability was determined by calculating the protein’s melting temperature (Tm). This assay revealed that three mutations had significantly higher Tm as compared to the WT. These experiments suggest that although purifying selection acts to preserve the primary functions of HspA1A, other secondary functions of the molecule may be altered. Collectively, our results constitute an evolutionary paradigm in which gene conversion, purifying selection, and neofunctionalization act conversely to conserve a critical component of the heat shock response.

This project was supported by funds from NIH and CSUF to NN

keywords: natural variation, heat-shock proteins, biochemistry

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Evolution and natural variation of HSPA1A, the major stress inducible gene, in humans

HUB 302-35

HSPA1A is a critical component of the cellular stress response and alterations in its function have been associated with several diseases, such as cancer. However, the extent of HSPA1A’s natural variation remains largely unknown. Here, we investigated the evolution of this gene in mammals. Our results reveal that in all mammals HSPA1A is actually two identical proteins coded by two tandemly arranged genes. Although this pattern can be explained by gene conversion, this mechanism alone cannot explain the presence of several non-amino acid altering (synonymous) mutations. Therefore, we conceptualized that both gene conversion and purifying selection act to conserve these genes. This notion is also supported by analyses of HSPA1A variation in humans, which revealed that all mutations have a very low frequency and that the majority is synonymous. The presence of purifying selection was further supported by experiments determining whether natural variants affect HSPA1A’s functions. First, proteins corresponding to the wild-type (WT) and five mutated variants were tested for their ability to hydrolyze ATP. These experiments revealed that the mutations did not significantly alter the rate of ATP hydrolysis. Second, Isothermal Titration Calorimetry was employed to test whether the mutations alter binding of HSPA1A to ATP, ADP, and protein substrates. These experiments showed that only one mutation increased the entropy of the reactions. Lastly, the effect of these mutations on protein stability was determined by calculating the protein’s melting temperature (Tm). This assay revealed that three mutations had significantly higher Tm as compared to the WT. These experiments suggest that although purifying selection acts to preserve the primary functions of HspA1A, other secondary functions of the molecule may be altered. Collectively, our results constitute an evolutionary paradigm in which gene conversion, purifying selection, and neofunctionalization act conversely to conserve a critical component of the heat shock response.

This project was supported by funds from NIH and CSUF to NN

keywords: natural variation, heat-shock proteins, biochemistry