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In this study, we describe a new post-translational modification at position − 1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position − 1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position − 1.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais CHAPTER II: Myosin-IIB is a non-muscle isoform in the brain with increased expression in the brains of diabetic rats. Chronic hyperglycemia caused by diabetes can impair learning and memory. Oral hypoglycemic agents such as glibenclamide have been used to control hyperglycemia. We report changes in the expression and distribution of myosin-IIB in the frontal cortex and hippocampus of diabetic rats treated with glibenclamide. To establish this, brains were harvested after 43 days of treatment with glibenclamide (6 mg/kg bw orally), homogenized and analyzed by Western blotting, qRT-PCR and immunohistochemistry. As expected, myosin-IIB expression increased in the brains of diabetic rats. However, protein levels returned to normalcy after treatment with glibenclamide. In addition, MYH10 gene expression decreased in diabetic rats treated with glibenclamide. Moreover, we found weak myosin-IIB labeling in the hippocampus and frontal cortex of rats treated with glibenclamide. Therefore, the expression of myosin-IIB is affected by diabetes mellitus and may be modulated by glibenclamide treatment in rats. Structural changes in the hippocampus and prefrontal cortex are reversible, and glibenclamide treatment may reduce patho-physiological changes in the brain. Our findings suggest a possible correlation between glibenclamide effects and myosin-IIB function in the brain of diabetised rats. CHAPTER III: Introduction: Diabetes increases oxidative stress and causes several changes in protein transport and docking of synaptic vesicles. Plant extracts have long been used to treat hyperglycemia and diabetes. Aqueous extracts of Vochysia rufa, a species endemic to the Brazilian Cerrado ecossistem, have increasingly been prescribed and used as a complementary or alternative treatment. Objective: We examined the effects of an aqueous extract of Vochysia rufa on oxidative stress markers and on the expression and localization of transport proteins and synaptic vesicle docking in the diabetic rat brain. Design/Methods: Thirty-two male Wister rats were randomly divided into 4 groups: non-diabetic, diabetic, diabetic treated with glibenclamide (6 mg / kg bw orally) and diabetic treated with Vochysia rufa extract (500 mg/kg bw orally) for forty-three days. After the treatments, brains were collected and the following parameters were evaluated: enzymatic activity of glutathione peroxidase and glutathione S-transferase, superoxide dismutase concentration, total sulfhydryl, lipid peroxidation and reduced glutathione. The levels and localization of proteins such as CaMKII, myosin-Va, synapsin-1, SNAP-25 and GLUT4 were also analyzed. Results: Vochysia rufa extract decreased SOD, GSH, TBARS and total sulfhydryl levels and increased GST levels. Additionally, myosin-Va and synapsin-1 expression decreased and levels proteins such as CaMKII and SNAP-25 increased. These results were confirmed by immunolocalization. Conclusion: The results suggest that an aqueous extract of Vochysia rufa has a neuroprotective effect on diabetic rat brains by reducing oxidative stress and controlling changes in myosin-Va and synapsin-1 expression. O diabetes aumenta o estresse oxidativo e provoca várias alterações no transporte de proteínas e de ancoragem das vesículas sinápticas. Os extratos de plantas têm sido muito utilizados para tratar a hiperglicemia e diabetes. Extratos aquosos de Vochysia rufa, uma espécie do Cerrado brasileiro, têm sido prescritos e utilizados como tratamento complementar ou alternativo. Foram analisados os efeitos do extrato aquoso de Vochysia rufa sobre marcadores de estresse oxidativo e na expressão e localização de proteínas de transporte e ancoragem das vesículas sinápticas no cérebro de ratos diabéticos. Trinta e dois ratos Wistar machos foram divididos aleatoriamente em 4 grupos: não-diabéticos, diabéticos, diabéticos tratados com glibenclamida (6 mg/kg de peso corporal por via oral) e diabéticos tratados com extrato de Vochysia rufa (500 mg/kg de peso corporal por via oral) por 43 dias. Após os tratamentos, os cérebros foram coletadas e os seguintes parâmetros foram avaliados: atividade enzimática da glutationa peroxidase e glutationa S- transferase, a concentração de superóxido dismutase, sulfidrila total, a peroxidação lipídica e de glutationa reduzida. Os níveis e localização de proteínas, como CaMKII, miosina - Va, sinapsina -1, SNAP -25 e GLUT4 também foram analisados. O extrato de Vochysia rufa diminuiu SOD, GSH, TBARS e os níveis totais de sulfidrila e aumentou os níveis de GST. Além disso, a miosina -Va e sinapsina -1 apresentaram os níveis de expressão diminuídas, ao contrário de CaMKII e SNAP-25 que aumentaram. Estes resultados foram confirmados por imunohistoquímica. Os resultados sugerem que o extrato aquoso de Vochysia rufa tem um efeito neuroprotetor em cérebros de ratos diabéticos, reduzindo o estresse oxidativo e controlando as mudanças na miosina -Va e sinapsina -1. Mestre em Genética e Bioquímica

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.