Studies on molecular mechanisms underlying high pressure adaptation of alpha-actin from deep-sea fish

Deep-sea fish distribute to depths of several thousand meters and at these abyssal depths encounter pressures that shallower-living fish cannot tolerate. Tolerance to abyssal pressures by deep-sea fish is likely to depend at least in part on adaptive modifications of proteins. However, structural modifications that allow proteins to function at high pressures have not been well elucidated. The objective of this study is to disclose the mechanisms underlying adaptation of deep-sea fish to high pressures. First, in order to select sample fish for this study, the author constructed the molecular phylogenetic trees for the deep-sea fish Coryphaenoides using the nucleotide sequences of the mitochondrial 12S rRNA and COI genes. The trees showed new arrangements of seven Coryphaenoides species with distinct groups, abyssal and non-abyssal species, that differed from previous taxonomic studies. Using the mutation rate of mitochondrial genes, the divergence time between abyssal and nonabyssal Coryphaenoides was calculated to be 3.2-7.6 million years ago. The present study suggests that hydraulic pressures play an important role in the speciation process in the marine environment. Second, the author cloned cDNAs encoding alpha-actin. which was used as a model protein to elucidate the mechanisms involved in protein adaptation to high pressures, from two abyssal Coryphaenoides species. C. armatus and C. yaquinae. Consequently. the author identified three amino acid substitutions. V54A or L67P. Q137K and A155S. that distinguished these abyssal alpha-actins from orthologs from non-abyssal Coryphaenoides. Finally, the author examined by several biochemical analyses which of the three substitutions makes possible for alpha-actin of the deep-sea fish adapt to high hydrostatic pressures. It was found that the substitutions of Q137K and A155S prevent the dissociation reaction of ATP and Casup(2+) from being influenced by high pressures. In particular, the substitution of Q137K results in a much smaller change in the apparent volume for Casup(2+) dissociation reaction. The substitution of V54A or L67P reduced the volume change associated with actin polymerization and has a role in maintaining the DNasel activity of actin at high pressures. Taken together, these results indicate that a few amino acid substitutions in key functional positions can adaptively alter the pressure sensitivity of abyssal proteins.