Small heat shock protein gene expression and function in Xenopus laevis

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Various diseases or environmental stresses, such as exposure to high temperature or heavy metals, can produce damaged proteins that are toxic to cells. One component of the cell’s emergency response system is a class of stress-inducible molecular chaperones called “small heat shock proteins” (sHsps). During stress, sHsps form complexes that bind to unfolded protein, inhibit the formation of toxic aggregates and facilitate their refolding once favorable conditions have returned. Most of the damaged protein in the cell is degraded by means of the ubiquitin-proteasome system (UPS). SHsp synthesis or mutation is associated with a variety of diseases including Alzheimer's, Parkinson’s, hereditary motor neuropathy type 2, Charcot-Marie-Tooth, cancer, and desmin-related myopathy. Therefore, it is critical to understand how sHsps are regulated during normal and stress conditions. For this task, the frog, Xenopus laevis, is an outstanding model organism. For more than 2 decades, my laboratory, which includes graduate and undergraduate students, has actively researched the expression and function of the stress-inducible sHsp gene family, Hsp30, in Xenopus. Research initiated in cultured cells can be extended to early embryos. Furthermore, the large size of the eggs and embryos permits the microinjection of proteins, nucleic acids and other molecules. Previously, we isolated 2 functional Hsp30 genes. Hsp30-like genes were subsequently found in birds, fish and other frogs, but not in mammals. While the amino acid sequences and expression patterns of the Hsp30s were different from mammalian sHsps, they shared the ability to form multimeric structures and act as molecular chaperones since they inhibited stress-induced protein aggregation. Our research with Xenopus cultured cells revealed that Hsp30 may be involved in the regulation of the cytoskeleton during stress and possibly inhibits apoptosis during normal developmental processes. In cultured cells, various stresses such as heat shock, cadmium, arsenite and proteasomal inhibitors induced localization of Hsp30 with the F-actin cytoskeleton, an essential structure necessary for cell shape, motility, and normal development. In fact, stress-induced Hsp30 may be involved in preventing the collapse of the F-actin cytoskeletal collapse at high temperatures. Proteasomal inhibitors and cadmium also induced the formation of large Hsp30 containing structures that were putatively identified as aggresomes (specialized compartments of aggregated protein). In embryos, we found that Hsp30 genes were expressed constitutively in the cement gland, an anterior organ that anchors the developing tailbud embryo to solid structures and is eliminated by apoptosis. It is possible that the presence of Hsp30 in the cement gland may inhibit apoptosis of this organ until the tadpole stage. The long-term goals of this research program are to understand the regulation of expression and function of Xenopus sHsps, particularly Hsp30. The specific aims of this proposal are to investigate the: 1) involvement of the Hsp30 amino terminal domain in oligomerization and chaperone function, 2) association of stress-induced Hsp30 with the F-actin cytoskeleton, 3) involvement of stress-induced Hsp30 in the formation of aggresome-like structures in cells and embryos and 4) the role of Hsp30 in the formation and apoptotic elimination of the cement gland in tailbud embryos. This novel and hypothesis-driven research program will provide a better understanding of the mechanisms associated with regulation, structure and function of sHsps. This knowledge will provide insight into the role of sHsps in counteracting environmental stress in aquatic organisms, and may aid in defining the role of sHsps in various disease states.