Soft-shell clam (Mya arenaria) p53: a structural and functional comparison to human p53.

The tumor suppressor p53 regulates genes involved in progression through the cell cycle, DNA repair, senescence or apoptosis in response to cell stress. Dysregulation of p53 can result in uncontrolled cellular proliferation. Invertebrate homologues to human p53 (Hsp53) have been identified, including a putative p53 gene (Map53) from the soft-shell clam (Mya arenaria). Predicted sequences for human and clam p53 proteins exhibit conservation in key domains. In light of this similarity, and the apparent dysregulation of Map53 under morphologically aberrant/pathologic conditions, we tested the hypothesis that the two proteins function in a similar manner. Plasmids expressing either Hsp53 or Map53 were introduced by transient transfection into the p53-null H1299 cell line. Functionality was assessed by monitoring the p53/mdm2 feedback loop and expression of p53-mediated downstream markers of growth arrest and apoptosis under non-stressed conditions. Hsp53 spontaneously induced markers of growth arrest, while Map53 expression induced neither cell arrest nor apoptosis. The difference in downstream activation is not likely the result of cytosolic sequestration since Map53, like Hsp53, localized almost exclusively to the nucleus. Functional similarity was observed in regulation by human MDM2, suggesting that the clam may have an mdm2 homologue. Protein modeling identified an apparent MDM2 binding site in Map53, supporting the observation of a potential Map53/MDM2 interaction. Significant amino acid differences present in the Map53 tetramerization domain may potentially affect p53 protein/protein interactions. Taken together, these data suggest that the Map53 shares some functional similarity with human p53 as well as with other invertebrates, positioning the mollusk at a critical juncture in evolution of this gene family.

Detecting the active conformation of calpain with calpastatin-based reagents.

The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.

Structural and kinetic characterization of myoglobins from eurythermal and stenothermal fish species.

Teleost myoglobin (Mb) proteins from four fish species inhabiting different temperature environments were used to investigate the relationship between protein function and thermal stability. Mb was isolated from yellowfin tuna (homeothermal warm), mackerel (eurythermal warm), and the Antarctic teleost Notothenia coriiceps (stenothermal cold). Zebrafish (stenothermal tropical) myoglobin was expressed from cloned cDNA. These proteins differed in oxygen affinity, as measured by O2 dissociation rates and P50 values, and thermal stability as measured by autooxidation rates. Mackerel Mb had the highest P50 value at 25 degrees C (3.7 mmHg), corresponding to the lowest O2 affinity, followed by zebrafish (1.0 mmHg), yellowfin tuna (1.0 mmHg), and N. coriiceps (0.6 mmHg). Oxygen dissociation rates and Arrhenius plots were similar between all teleost species in this study, with the exception of mackerel myoglobin, which was two-fold faster at all temperatures tested. Myoglobin from the Antarctic teleost had the highest autooxidation rate (0.44 h(-1)), followed by mackerel (0.26 h(-1)), zebrafish (0.22 h(-1)), and yellowfin tuna (0.088 h(-1)). Primary structural analysis revealed residue differences distributed throughout the polypeptide sequences, making it difficult to identify, which, if any, residues contribute to structural flexibility. However, analysis of molecular dynamics trajectories indicates that Mb from the eurythermal mackerel is predicted to be the most flexible protein within the D loop and FG turn. At the same time, it has the lowest O2 affinity and the highest O2 dissociation rates when compared to myoglobins from teleosts that appear to be less flexible in our dynamics simulations.

Isolation, characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus.

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K(m) at 0.5 degrees C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K(m) values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6-7.7 at 0.5 degrees C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 degrees C. The apparent V(max) of C. aceratus MMCK at 0.5 degrees C is 94+/-4 S.D. (n=9) micromol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 degrees C and 8-fold greater than rabbit MMCK measured at 0.5 degrees C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.

Hemoglobin in Frankia, a nitrogen-fixing actinomycete.

Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s(-1), and the oxygen association rate constant was 206 microM(-1) s(-1).

Hemoglobin in five genetically diverse Frankia strains.

Five strains of Frankia were selected to represent a wide range of genetic diversity and examined for presence of hemoglobin. All five strains produced hemoglobin when grown on media without (-N) or with (+N) combined nitrogen. This indicates that hemoglobin is common in Frankia and is not directly associated with nitrogen fixation. Frankia strain EAN1(pec) was examined in more detail. It showed greater hemoglobin concentration when grown at 2% O2 than at 20% O2 in the -N treatment but no effect of oxygen on hemoglobin concentration in the +N treatment. At both oxygen levels, it produced substantially more biomass in +N than in -N culture. It also produced significantly more biomass when the medium contained 0.2% CO2 than in the absence of CO2. The molecular mass of the hemoglobin as determined by size exclusion chromatography was 13.4 +/- 0.2 kDa (mean +/- SE, n = 3) and is consistent with that of a truncated hemoglobin. The hemoglobin had absorption spectra that were typical of a hemoglobin. The oxygen dissociation rate constants for the hemoglobin were 131.2 +/- 5.8 s(-1) for -N culture and 166 +/- 8.2 s(-1) for +N culture. These rapid rates are consistent with a function in facilitated diffusion of oxygen.