Functional Validation of Hydrophobic Adaptation to Physiological Temperature in the Small Heat Shock Protein αA-crystallin

Abstract

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.

Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development

“…The recent development of methods for in vivo editing of the zebrafish genome, such as TALENS and the CRISPR-Cas system, will enhance the ability to manipulate lens crystallin expression and test hypotheses of crystallin function beyond early developmental stages (Chang et al, 2013; Hwang et al, 2013). We recently used evolutionary thermal adaptation of six fish αA-crystallins to identify specific amino acid changes that enhance anti-aggregation activity (Posner et al, 2012). Genome editing technologies and the confocal imaging of cataract demonstrated in this study will allow us to test the protective abilities of these and newly discovered modifications within the complex setting of a live lens.…”

A First Line of Stress Defense: Small Heat Shock Proteins and Their Function in Protein Homeostasis

“…Interestingly, the temperature range of activation reflects the physiological temperature of the respective organism. For example, mesophilic yeast Hsp26 is activated in a temperature range from 20°C to 43°C, Hsp16.5 from the hyperthermophile M. jannaschii is activated from 60°C to 95°C, human Hsp27 (HspB1) is activated from 37°C to 42°C, and sHsps from fish living below 15°C are activated at ∼10°C-25°C [35, 62, 101-103]. The shift from physiological to heat stress temperatures, which usually requires only a few degree increase, provides sufficient activation energy to shift the sHsp ensemble equilibrium towards enhanced dissociation.…”

Proteomic responses of fruits to environmental stresses

“…Several individual groups identified dehydrin protein in peach and citrus fruit stored at LT through traditional 2D-PAGE and difference gel electrophoresis (DIGE) (Nilo et al, 2010; Yun et al, 2010, 2012; Zhang et al, 2010). Other commonly identified proteins by these groups included heat shock protein and dehydrogenase which were well-known to play a key role in plant stress responses (Giannino et al, 2004; Posner et al, 2012). In nectarine fruits stored at LT, four differentially expressed proteins were characterized as allergens which can provide some form of protection to fruits during periods of stress (Pedreschi et al, 2007; Giraldo et al, 2012).…”