Whitney Hoopes, Brigham Young University
Microbiology and Molecular Biology
HspB2 is one of eleven known small Heat Shock Proteins (sHSP) that is expressed in human heart and skeletal muscle. In response to cellular stress, heat shock proteins play a vital role to help misfolded proteins and proteins susceptible to denaturation maintain their structure. Two members of the sHSP family, CryAB and HspB2, are both required for normal heart function and cardiac muscle integrity. CryAB-deficient mice have defects in cardiac muscle structure whereas HspB2-deficient mice display energy deficits (Rajasekaran et al. 2007). The contrasting phenotypes of CryAB and HspB2 suggest differential roles for these molecular chaperones in the heart. HspB2 has been found to localize with the mitochondria in several different cell lines and overexpression of this sHSP has been shown to support survival of cells against heat stress (Nakagawa, 2001). To understand the role and mechanism of HspB2 in cardiac muscle energy regulation, we have used a yeast two-hybrid (Y2H) system to uncover the novel protein binding partners specific to HspB2. From screening a human heart cDNA library, HspB2 interacted with approximately 10,000 out of 20 million plasmids. We have sequenced more than 1000 of these putative interactors and have identified over 100 unique proteins. Over 40% of these protein partners are involved in mitochondrial energy production and another 25% in cardiac muscle structure maintenance. In addition, we have identified an interaction between HspB2 and the related sHSP CryAB. We then compared this data with mitochondrial HspB2 binding partners identified by mass spectroscopy (MS) through a large-scale bioinformatics analysis and constructed a protein-protein network. Y2H dependency tests were conducted to verify interactions identified by both Y2H and MS. Following yeast verification, a subset of the interactions were confirmed in C9H2 cardiac cells through coimmunopurification. Our research describes the first protein-protein interaction network for any sHSP, supports a role for HspB2 in mitochondrial energy production and suggests a link between mitochondrial energy production/redox stasis and stressed cardiac muscle maintenance.