Protein Stability During Extreme Metabolic Rate Depression

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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.