Protein Stability During Extreme Metabolic Rate Depression

克莱格如果环境中的分子氧被清除，大多数动物会在数小时内死亡。即使是适应良好的物种，如无根潮间带海洋无脊椎动物，也很少能在持续缺氧的情况下存活超过一个月。这项研究的重点是甲壳类动物阿耳特米亚·弗朗西亚纳（Artemia franciscana）的被囊胚胎，它们在缺氧时将新陈代谢带入可逆的停滞状态，有些人在这种状态下存活了6年以上，这是一种真正非凡的能力。这项研究的核心问题是它们蛋白质的稳定性：这些胚胎如何在多年的缺氧中避免可预测的蛋白质展开和聚集？由于在缺氧期间没有蛋白质合成或分解发生，因此不能发生有缺陷蛋白质的替换和/或去除。该项目扩展了先前对一种名为p26的小热休克/ α -结晶蛋白的研究。该蛋白的几个特征，包括其大量存在和在应激期间广泛的细胞内易位，导致p26代表这些胚胎适应性库的主要组成部分的假设，作为分子伴侣维持蛋白质完整性。为了进一步验证这一假设，我们将对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（在这些胚胎中大量存在的相容溶质）对蛋白质稳定性和p26伴侣活性的影响也将被检查，以及pH值的影响，已知pH值是Artemia胚胎中有氧-缺氧过渡期间代谢的主要调节因子。p26和模型靶点CS之间的物理关联将通过常规的凝胶过滤和电泳技术来寻找。除了伴侣活性外，p26的作用也将被探索，特别是它在好氧-缺氧过渡期间作为转录调节剂的潜在功能。为了进一步评估p26作为稳定适应性的重要性，我们将对转染p26基因（并表达该蛋白）的大肠杆菌对脱水应激和热休克的抗性进行研究。由于其他应激蛋白可能与这些胚胎忍受多年缺氧的能力有关，另外两个众所周知的热休克蛋白家族，热休克蛋白70和热休克蛋白90也将被检查，并将它们的行为与p26进行比较，特别是在应激诱导的细胞内易位方面，以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理解适应良好的动物的细胞如何在破坏绝大多数非适应动物的条件下存活下来。同样，这一知识可能会让我们深入了解细胞通常是如何抵御严重压力的。p26的丰度和独特性可能被证明在稳定具有商业重要性的蛋白质（如疫苗和具有生物活性但不稳定的蛋白质）方面具有价值。最后，Artemia是各种水产养殖企业非常重要的食物来源，其中一些企业严重依赖于使用这些胚胎和从中获得的幼虫。更多地了解它们的基本生物学知识，可以改善涉及它们使用的水产养殖做法。

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.