Small heat shock protein gene expression and function in Xenopus laevis

各种疾病或环境压力，如暴露于高温或重金属，可以产生对细胞有毒的受损蛋白质。细胞应急反应系统的一个组成部分是一类称为“小热休克蛋白”的应激诱导分子伴侣。在应激过程中，sHsps形成与未折叠蛋白结合的复合物，抑制有毒聚集体的形成，并在有利条件恢复后促进其重新折叠。细胞中大部分受损蛋白通过泛素-蛋白酶体系统（UPS）降解。SHsp的合成或突变与多种疾病有关，包括阿尔茨海默病、帕金森病、遗传性2型运动神经病变、夏尔克-玛丽-图斯病、癌症和末代相关肌病。因此，了解sHsps在正常和应激条件下是如何调节的至关重要。对于这项任务，非洲爪蟾（Xenopus laevis）是一种杰出的模式生物。

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.