Small heat shock protein gene expression and function in Xenopus laevis

非洲爪蟾小热激蛋白基因的表达和功能

• 批准号: RGPIN-2014-04376
• 负责人: Heikkila, John
• 金额: $ 3.86万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629862
• 关键词: Small; heat; shock; protein; gene

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.

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。20多年来，我的实验室包括研究生和本科生，积极研究了应力诱导的sHsp基因家族Hsp30在非洲爪蟾中的表达和功能。在培养细胞中开始的研究可以扩展到早期胚胎。此外，卵子和胚胎的大尺寸使得蛋白质、核酸和其他分子的显微注射成为可能。此前，我们分离了2个功能性Hsp30基因。随后在鸟类、鱼类和其他蛙类中发现了类似hsp30的基因，但在哺乳动物中没有发现。虽然hsp30的氨基酸序列和表达模式与哺乳动物的sHsps不同，但它们都具有形成多聚体结构的能力，并作为分子伴侣，因为它们抑制应激诱导的蛋白质聚集。我们对非洲爪蟾培养细胞的研究表明，Hsp30可能参与应激时细胞骨架的调节，并可能抑制正常发育过程中的细胞凋亡。在培养的细胞中，各种应激如热休克、镉、亚砷酸盐和蛋白酶体抑制剂诱导Hsp30与f -肌动蛋白细胞骨架定位，这是细胞形状、运动和正常发育所必需的基本结构。事实上，应激诱导的Hsp30可能参与防止f -肌动蛋白在高温下的细胞骨架崩溃。蛋白酶体抑制剂和镉也诱导了含有Hsp30的大型结构的形成，这些结构被推定为聚集体（聚集蛋白的专门隔室）。在胚胎中，我们发现Hsp30基因在水泥腺中组成性表达，水泥腺是将发育中的尾芽胚胎固定在固体结构上的前器官，并被细胞凋亡消除。骨水泥腺中Hsp30的存在可能会抑制该器官的凋亡，直到蝌蚪期。本研究计划的长期目标是了解爪蟾sHsps，特别是Hsp30的表达和功能调控。本研究的具体目的是研究：1)Hsp30氨基末端结构域参与寡聚化和伴侣蛋白功能，2)应激诱导的Hsp30与f -肌动蛋白细胞骨架的关联，3)应激诱导的Hsp30参与细胞和胚胎中聚集体样结构的形成，4)Hsp30在尾芽胚胎中水泥腺的形成和凋亡消除中的作用。这个新颖的假设驱动的研究项目将更好地理解与sHsps的调控、结构和功能相关的机制。这一知识将提供对水生生物中sHsps在对抗环境应激中的作用的深入了解，并可能有助于确定sHsps在各种疾病状态中的作用。