Biophysical and Biochemical Studies of Alpha II Spectrin and Its Break-Down Products by Caspase-3

The classical view of spectrin has been that of a structural protein to maintain the morphology of erythrocytes. However, this view has shifted with the realization of spectrin’s diverse roles in non-erythroid cells. Spectrin isoforms are composed of several domains, which are very often referred to as repeats, suggesting that all spectrin structural domains are identical. However, this statement is far oversimplified. Each domain has its unique structural features that all together contribute to the overall functional properties of spectrins. We prepared spectrin model proteins containing the N-terminal partial domain region of alphaII (brain spectrin), and its binding partner, the C-terminal partial domain region of betaII to study the formation of functional spectrin tetramers. These regions are found to fold into coiled coil helices, and the tetramerization involves helical bundling of these helices. X-ray crystallography, isothermal titration calorimetry, and site directed mutagenesis were used to characterize the details in the structure of alphaII N-terminal region and the interactions between the alphaIIbetaII helices. Point mutations affecting structural flexibility and the tetramerization region conformation in alphaII, as well as inter-helix electrostatic interactions alter the equilibrium between spectrin dimers and spectrin tetramers and may lead to neurological disorders. It has been shown that spectrin-alphaII undergoes caspase-3 cleavage into several spectrin break-down products (SBDP) under various physiological and patho-physiological conditions. In particular two fragments are generated, SBDP of 150 kDa (SBDP150) and of 120 kDa (SBDP120). SBDP120 has recently been detected in patients with Sjögren’s Syndrome and in numerous brain related injuries and diseases, such as Alzheimer’s disease. Reactions leading to SBDP are physiologically relevant, well regulated, and complex. We have used gel electrophoresis and tryptophan fluorescence methods to monitor the cleavage of spectrin model proteins by caspase-3. Our results show that caspase-3 cleaves alphaII at two independent sites in the middle of alphaII with very different catalytic rates. Our findings suggest that SBDP150 and SBDP120, as well as a 37 kDa SBDP, may be involved in different cellular functions.