Small Heat Shock Proteins (sHSP) are critical molecular chaperones that function to maintain protein homeostasis (proteostasis) and prevent the aggregation of other proteins during cellular stress. Any disruption in the process of proteostasis can lead to prevalent diseases ranging from cancer and cataract to cardiovascular and Alzheimer's disease. CryAB (αB-crystallin, HspB5) is one of ten known human sHSP that is abundant in the lens, skeletal, and cardiac muscle. This protein is required for cardiac function and muscle cell integrity. When the cell experiences physiological stress, including heat shock, CryAB moves to the cytoskeleton to act as a chaperone and prevent aggregation of its protein clientele. This research is designed to investigate the molecular role of CryAB in cell proteostasis through the identification of putative protein clientele and chaperone activity analysis. We have identified over twenty CryAB-binding partners through combined yeast two-hybrid (Y2H) and co-purification approaches, including interactions with myofibril proteins. Previously reported disease-associated CryAB missense variants were analyzed in comparison to wild type CryAB through Y2H binding assays. The characterization of the similarities and differences in binding specificities of these variants provide a foundation to better understand the chaperone pathways of CryAB and how these changes in molecular function result in the development of disparate diseases such as cataract, cancer, and various myopathies.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity. As such, mutations in this family of proteins result in a wide variety of diseases from cancers to cardiomyopathies. The sHSPs Beta-2 (HspB2) and alpha-beta crystalline (CryAB) are two of the ten human sHSPs and are both expressed in cardiac and skeletal muscle cells. A heart that cannot properly recover or defend against stressors such as extreme heat or cold, oxidative/reductive stress, and heavy metal-induced stress will constantly struggle to maintain efficient function. Accordingly, CryAB is required for myofibril recovery from ischemia/reperfusion (I/R) and HspB2 is required I/R recovery as well as efficient cardiac ATP production. Despite these critical roles, little is known about the molecular function of these chaperones. We have identified over two hundred HspB2-binding partners through both yeast two-hybrid and copurification approaches, including interactions with myofibril and mitochondrial proteins. There is remarkable overlap between the two approaches (80%) suggesting a high confidence level in our findings. The sHSP, CryAB, only binds a subset of the HspB2 interactome, showing that the HspB2 interactome is specific to HspB2 and supporting non-redundant roles for sHSPs. We have confirmed a subset of these binding partners as HspB2 clients via in vitro chaperone activity assays. In addition, comparing the binding patterns and activity of sHSP variants in comparison to wild type can help to elucidate how variants participate in causing disease. Accordingly, we have used Y2H and in vitro chaperone activity assays to compare the disease-associated human variants R120GCryAB and A177PHspB2 to wild type and have identified differences in binding and chaperone function. These results not only provide the first molecular evidence for non-redundancy of the sHSPs, but provides a useful resource for the study of sHSPs in mitochondrial and myofibril function.