Protein Stability During Extreme Metabolic Rate Depression

如果氧气分子从它们的环境中消失，大多数动物在几小时内就会死亡。 即使是适应性很好的物种，如固着的潮间带海洋无脊椎动物，也很少能在连续缺氧中存活超过一个月。 这项研究的重点是甲壳类动物卤虫的包囊胚胎，它们在缺氧期间使新陈代谢可逆地停止，有些人以这种方式存活了6年以上，这是一种真正了不起的能力。 这项研究的核心问题是它们蛋白质的稳定性：这些胚胎如何避免在多年缺氧期间蛋白质的可预测的展开和聚集？ 由于在缺氧期间不发生蛋白质合成或分解，因此不能发生缺陷蛋白质的替换和/或去除。 该项目扩展了先前对称为p26的小热休克/α-晶状体蛋白的研究。 这种蛋白质的几个特征，包括其大量存在和在应激过程中广泛的细胞内易位，导致了这样的假设，即p26代表了这些胚胎的适应性库中的主要成分，作为分子伴侣来维持蛋白质的完整性。 为了进一步检验这一假设，将对p26在体外陪伴模型靶蛋白柠檬酸合酶（CS）的能力进行实验。 海藻糖和甘油（这些胚胎中存在的大浓度相容性溶质）对蛋白质稳定性和p26分子伴侣活性的影响也将进行检查，pH值的影响也将进行检查，已知pH值是卤虫胚胎有氧-缺氧过渡期间代谢的主要调节剂。 p26和模型目标，CS之间的物理关联，将寻求使用传统的凝胶过滤和电泳技术。 p26除了分子伴侣活性的作用也将被探讨，特别是其潜在的功能作为一个调节器的转录在有氧缺氧过渡。 为了进一步评价p26作为稳定适应的重要性，将对用p26基因转染的大肠杆菌（并表达该蛋白质）的脱水应激和热休克抗性进行研究。 由于其他应激蛋白可能与这些胚胎耐受多年缺氧的能力有关，因此还将检查另外两个众所周知的热休克蛋白家族，Hsp-70和Hsp-90，并将它们的行为与p26的行为进行比较，特别是关于应激诱导的细胞内易位，为了评估这些应激蛋白可能作为“伴侣机器”协同作用的可能性，这项研究的一个重要成果将是更好地了解适应性良好的动物的细胞在摧毁绝大多数非适应性细胞的条件下生存的方式。 类似地，这些知识可能会让我们深入了解细胞一般是如何被保护免受严重压力的。 p26的丰富性和独特性可能被证明在稳定具有商业重要性的蛋白质如疫苗和生物活性但不稳定的蛋白质方面具有价值。 最后，卤虫是各种水产养殖企业的一个非常重要的食物来源，其中一些严重依赖于使用这些胚胎和从它们获得的幼虫。 更多地了解它们的基本生物学可以改善涉及它们使用的水产养殖实践。

CleggMost animals die within hours if molecular oxygen is removed from their environment. Even well-adapted species, such as sessile intertidal marine invertebrates, rarely survive more than a month of continuous anoxia. This research focuses on encysted embryos of the crustacean, Artemia franciscana, who bring their metabolism to a reversible standstill during anoxia, some surviving that way for over 6 years, a truly remarkable ability. The central question of this research concerns the stability of their proteins: how do these embryos avoid the predictable unfolding and aggregation of proteins during years of anoxia? Since no protein synthesis or breakdown take place during anoxia the replacement and/or removal of defective proteins can not occur. This project extends previous study of a small heat shock/alpha-crystallin protein called p26. Several characteristics of this protein, including its presence in massive amounts and its extensive intracellular translocation during stress, led to the hypothesis that p26 represents a major component in the adaptive repertoire of these embryos, acting as a molecular chaperone to maintain protein integrity. To further test this hypothesis experiments will be performed on the ability of p26 to chaperone a model target protein, citrate synthase (CS) in vitro. The effects of trehalose and glycerol (compatible solutes present in large concentration in these embryos) on protein stability and on p26 chaperone activity will also be examined, as will the effects of pH, known to be a major regulator of metabolism during aerobic-anoxic transitions in Artemia embryos. Physical associations between p26 and the model target, CS, will be sought using conventional gel filtration and electrophoretic techniques. Roles of p26 in addition to chaperone activity will also be explored, particularly its potential function as a regulator of transcription during aerobic-anoxic transitions. To further evaluate the importance of p26 as a stabilizing adaptation, a study will be carried out on the resistance to dehydration stress and heat shock in the bacterium Escherichia coli transfected with the gene for p26 (and expressing this protein). Since other stress proteins may be involved in the ability of these embryos to endure years of anoxia two other well known heat shock protein families, Hsp-70 and Hsp-90 will also be examined and their behavior compared to that of p26, particularly with regard to stress-induced intracellular translocations, to evaluate the possibility that these stress proteins may be acting in concert as "chaperone machines".A significant outcome of this research will be a better understanding of the means by which cells in well-adapted animals survive conditions that destroy the vast majority of non-adapted ones. Similarly, this knowledge may provide insight into how cells, in general, might be protected against severe stress. It is possible that the abundance and uniqueness of p26 may prove to be of value in stabilizing proteins of commercial importance such as vaccines and biologically-active, but unstable proteins. Finally, Artemia is a very important food source for a wide variety of aquaculture ventures, some of which depend heavily on the use of these embryos and the larvae obtained from them. Knowing more about their basic biology could result in improved aquacultural practices involving their use.