HspB2 is a small heat shock protein encoded on human chromosome 11. Less than 1000 base pairs away from HSPB2 and situated in a head-to-head orientation lies the gene encoding another small heat shock protein, CRYAB. Because they are uncommonly close to one another they share regulatory elements. In addition, they share protein homology as sHSPs, suggesting that they perhaps perform aimilar functions. SHSPs such as HspB2 and CryAB are traditionally thought to provide protective effects to cells in response to a variety of stress inducers. In response to stress they form complexes around misfolded proteins or proteins in danger of denaturation. HspB2 has been shown to exhibit protective effects during cellular stress and to localize to the Z-line of skeletal muscle. It has also been implicated in cardiac energetics, specifically in the production of ATP, however little is known about its molecular targets. Here I report the use of yeast two-hybrid screening to uncover the molecular targets of HspB2. I also detail the process by which the screens are performed as well as the verification steps, including co-precipitation experiments in mammalian cells. Through these studies we identify many novelbinding partners of HspB2, including CryAB as well as multiple muscle and mitochondrial proteins. Proteins discovered to bind to HspB2 include such proteins as actin and myosin, enzymes catalyzing various steps of glycolysis and the electron transport chain, as well as redox-, small heat shock protein-, kinase-, and electrolyte-related proteins, among others. Studies of the binding partners of HspB2 in cardiac tissue will provide important information clarifying the involvement of HspB2 in cardiac muscle maintenance and metabolism.

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It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.