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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Doutor em Genética e Bioquímica CHAPTER II: Ca+2/calmodulin complex interacts with and regulates various enzymes and target proteins, known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. Non-muscle myosin-IIB was shown in our study to be overexpressed in diabetic rat brains. In this study, we compared CaMBPs, purified by immobilized-calmodulin, affinity chromatography, isolated from non-diabetic and streptozotocin-induced diabetic rat brains. Proteins eluted with EGTA and urea were separated on a SDS-PAGE gel, digested and submitted to peptide mass fingerprinting analysis. There were fifteen and sixteen intense bands for non-diabetic and diabetic brains, respectively. Two proteins were exclusively in non-diabetic brains, four were found exclusively in diabetic brains and thirteen were found in both. Protein sequences were then analyzed for the presence of the calmodulin-binding sites. A large fraction of the eluted proteins (83%) had putative IQ motifs or calmodulin-binding sites. The myosin-IIB affinity chromatography elution in association with western blot, immunohistochemistry and RT-PCR analyses strongly suggest that myosin-IIB protein and mRNA are highly expressed levels in diabetic rat brains. This is the first study that identified and showed calmodulin-binding proteins differential expression in non-diabetic and diabetic rat brains through a comparative proteomic analysis, and it opens up a new field of study relating myosin-IIB expression in the brain, diabetes mellitus and intracellular calcium regulation. CHAPTER III: Diabetes mellitus is a chronic disease characterized by persistent hyperglycemia, which may lead to brain tissue damage due to oxidative stress. The enhancement of free radicals during hyperglycemia contributes to increased neuronal death and changes in synaptic transmission. This study evaluated the effect of oxidative stress and the use of antioxidants (calcium, zinc and vitamin E) supplementation on myosins expression levels in brain tissues of streptozotocin-induced diabetes experimental rat model. Lipid peroxidation, antioxidant enzymes activities and myosins IIB and Va expressions at transcriptional and protein levels were examined after 5 and 90 days post streptozotocin induction. The chronic effect of the streptozotocininduced diabetes led to upregulation of glutathione peroxidase (GPx) and malondialdehyde (MDA) levels, and downregulation of superoxide dismutase (SOD) and catalase (CAT) activities. These alterations were accompanied by increased myosin-IIB and decreased myosin-Va expressions in diabetic rat brains. The antioxidant supplementation protected against hyperglycemia damage by reducing CAT activity and increasing GPx and SOD activities, although it had no influence on MDA levels. Interestingly, only diabetic rats restored myosin-IIB expression at both transcriptional and protein levels in brain tissues after supplementation, and no differences were observed in myosin-Va expression in comparison to non-diabetic rat brains. The functional alterations of antioxidant enzymes and myosins IIB and Va in rat brains of experimentallyinduced diabetes were strongly associated with the disease and aging during acute and chronic stages, but only myosin-IIB expression could be reversed by antioxidants. These data highlight the supplementation (calcium, zinc and vitamin E) antioxidants effects on the oxidative stress and the involvement of myosins IIB and Va in the diabetes stages; however, their mechanisms of action on enzymes activities, lipid peroxidation and myosins expression remain to be elucidated.

The Ca 2+ /calmodulin complex interacts with and regulates various enzymes and target proteins known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. In this study, we show that non-muscle myosin-IIB is overexpressed in the brains of diabetic rats. We isolated CaMBPs from the brains of non-diabetic rats and rats with streptozotocin-induced diabetes and purified them by immobilized-calmodulin affinity chromatography. The proteins were eluted with EGTA and urea, separated by SDS-PAGE, digested and submitted to peptide mass fingerprinting analysis. Thirteen intense bands were found in both types of brains, two were found exclusively in non-diabetic brains and four were found exclusively in diabetic brains. A large fraction of the eluted proteins contained putative IQ motifs or calmodulin-binding sites. The results of the myosin-IIB affinity chromatography elution, western blot and RT-PCR analyses suggest that myosin-IIB protein and mRNA are expressed at high levels in diabetic brains. This is the first study that has demonstrated differential expression of CaMBPs in diabetic and non-diabetic brain tissue through a comparative proteomic analysis, and it opens up a new approach to studying the relationship between the expression of myosins in the brain, hyperglycemia and intracellular calcium regulation.