Marcelo de Cerqueira Cesar.

O objetivo do projeto é promover uma atualização da literatura ligada ao tema hexoquinases. Essa enzima, que catalisa a primeira etapa da via glicolítica, possui muitas outras funções, além de estar presente em uma das principais vias metabólicas geradoras de energia. Atua no controle da apoptose, na sinalização celular e na regulação funcional. Participa ainda na ativação celular, bem como em mecanismos de envelhecimento e doenças degenerativas. Para entender essa multiplicidade de fenômenos, estamos empreendendo uma atualização do papel das isoformas da hexoquinase, seus aspectos estruturais, sua localização preferencial em diferentes órgãos. Estamos ainda empreendendo uma compreensão dos mais recentes desenvolvimentos no entendimento na relação estrutura ? função das hexoquinases.

Hexokinase catalyses the first regulatory step of the glycolytic pathway. We can say without any exaggeration that both hexokinase and glycolysis are involved in the control of brain cells life and death. To perform these pivotal roles, hexokinase occurs in four different isoforms in mammalian cells. Type I isozyme is best suited for energy generation, introducing glucose in glycolysis. In contrast, Type II and Type III isozymes produce Glc-6-P, which generates NADPH through the pentose phosphate pathway, utilized in biosynthetic processes. Nevertheless, hexokinase has another unique property to accomplish its multiple functions: the capacity of mitochondrial binding. Linked to its role in apoptosis control, upon binding, hexokinase inhibits the ability of Bax to induce the release of cytochrome c from the mitochondrial intermembrane space. Hexokinase II association with mitochondria is mediated by Akt. Overexpression of the phosphatase SHIP2 reduces Akt activity and enhances apoptosis, emphasizing hexokinase ´s role in cell death. Furhtermore, hexokinase also participates in cellular signaling and functional regulation. Adding complexity to this multidimensional enzyme´s attributes, glycolysis occurs in aerobic or anaerobic situations. Aerobic glycolysis participates in the regulation of cell excitability, in synapse formation and neurite growth. Here we provide an overview of the multiple roles of hexokinase and glycolysis in neuronal metabolic association with astrocytes, oligodendrocytes and microglia. We also provide an update on the role of hexokinase and glycolysis in astrocytes and microglia activation and in brain aging and neurodegenerative diseases.

A VDAC é a proteína mais abundante na membrana mitocondrial externa que exerce o controle da atividade desta organela através da regulação da troca de metabólitos e tem função crucial no mecanismo de apoptose. Os estudos dos complexos protéicos, das interações e das conseqüências dessas interações entre a VDAC e outras proteínas presentes no interior do neurônio auxiliam na manutenção das funções das organelas e da célula. O presente projeto determinará a estequiometria do complexo protéico Hexoquinase-VDAC-ANT presente em cérebro de rato, boi e ave, cujo objetivo é identificar se as expressões diferenciadas da VDAC1 e VDAC2 verificadas nos cérebros murino, aviar e bovino, estão associadas a diferenças nos interactomas dessas proteínas nesses cérebros. Estudos realizados nas espécies aviar, murina e bovina no Laboratório de Neurociência e Proteômica, revelaram que nas células de córtex cerebral de aves, ocorre maior expressão da VDAC 1 e menor expressão da VDAC 2, quando comparadas às demais. Além disso, estudaremos também se a expressão da VDAC 1 e VDAC 2 altera-se em neurônios aviares submetidos a estímulo apoptótico via estaurosporina. Um maior compreendimento do papel das VDACs na resposta neuronal é outro objetivo do presente estudo.

Trabalhos prévios do laboratório, indicaram a existência de dois sítios de ligação (A e B) da hexoquinase à mitocôndria de cérebros, baseando ? se na liberação da mesma com glicose ? 6 ? P (Cerqueira César & Wilson, 2002). Verificamos que a proporção de sítios A e B variava de acordo com a espécie animal estudada. Era de 90 % A : 10 % B no cérebro de rato, 60 % A : 40 % B no cérebro de coelho, e de 40 % A : 60 % B no caso do cérebro bovino. Posteriormente provamos que essa variação na proporção de sítios A e B de ligação da hexoquinase à mitocôndria espécie específica, era devida à diferenças na expressão relativa das isoformas de VDAC (voltage dependent anion channel). Provamos que com relação à VDAC 3, as isoformas de VDAC 1 e VDAC 2 eram mais expressas no cérebro bovino em comparação com o cérebro de rato (Cerqueira César & Wilson, 2004). Nossos resultados, juntamente com os de Shinohara et al. (2000) foram os primeiros a mostrar uma comparação quantitativa dos níveis de RNAm das diferentes isoformas de porina em tecidos de diferentes espécies animais.
	A teoria que pretendo comprovar no presente projeto é que existe uma correlação entre níveis de glicose circulante e expressão espécie ? específica diferenciada dos sítios A e B de ligação da hexoquinase à mitocôndria. Além de uma expressão diferenciada das isofomas de VDAC, hexoquinase e transportadores de glicose (GLUT). Os materiais biológicos para comprovar tal teoria serão tecidos cerebrais de bovino (57 mg/dl de glicose circulante), rato (73 mg/dl) e frango (168 mg/dl). Pretendemos apontar adaptações moleculares existentes para sobreviver com baixos níveis de glicose (cérebro de bovino) e altos níveis de glicose (aves) e apresentar possíveis sugestões para tratamentos moleculares de condições de hiperglicemia ou hipoglicemia em humanos e monogástricos. Acrescente ? se o fato que o entendimento da interação VDAC ? hexoquinase é de suma importância, pois trata ? se de um dos pontos estratégicos de controle da

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents. In the present study, chick muscle cell was used to verify the mode the cell death in an oxidative stress model induced by selenium and vitamin E dietary deficiencies. Antioxidants deficiency increased caspase-like activity assessed on the hydrolysis of quenched peptide substrates designed for these enzymes, but proteolytic activation was not accompanied by internucleosomal DNA fragmentation. Although the general caspase inhibitor Z-VAD-fmk did not block caspase-like activity, the hydrolyzed substrates presented the same cleavage profile in HPLC when incubated with a purified recombinant enzyme caspase-3. These preliminary data indicate that oxidative stress cause caspase-like activation in muscle cell and suggest that the cell death associated to exudative diathesis can follow the apoptotic program.

One-day-old chicks were reared using diets differing in their vitamin E and/or selenium content. In chicken depleted of both selenium and vitamin E, signs of exudative diathesis on superficial pectoralis muscle were observed. The purpose of this research was to determine the defective points of the antioxidant defense system, which turned this tissue highly susceptible to nutritionally induced oxidative stress. Vitamin E and in lower magnitude selenium were the factors, which strikingly affected the course of mitochondrial lipid peroxidation. Animals fed diets deficient in vitamin E and selenium displayed the lowest reduced glutathione level and glutathione peroxidase activity. The decreased levels of reduced glutathione were not due to one defective activity of glutathione reductase, which was increased in both mitochondria and cytosol. The absence of vitamin E was linked to one lowering of mitochondrial thiol levels. The Glutathione peroxidase/Cu,Zn Superoxide dismutase ratio was of 2.8 in animals fed selenium and vitamin E and decreased to 0.13 in animals deficient in both nutrients. This change was indicative of oxidant-induced damage mediated by hydrogen peroxide. Catalase activity increased in an attempt to counteract the decrease in glutathione peroxidase activity. The results obtained showed that  - tocopherol and Se deficiencies caused multiple alterations in the antioxidant system and adversely affected the redox state of chicken superficial pectoralis muscle.

Hexokinase bound to nonphosphorylating rat brain mitochondria, exhibits Michaelis-Menten kinetic behavior, with an apparent Km for ATP of 0.44 &#61617; 0.08 mM. After initiation of oxidative phosphorylation, a steady-state rate of Glc phosphorylation is maintained despite the fact that extramitochondrial [ATP] continues to increase but remains well below saturating levels (i.e. < 0.4 mM). This independence from extramitochondrial [ATP] is taken to indicate that hexokinase is not utilizing extramitochondrial ATP as substrate, but rather draws substrate ATP from an intramitochondrial compartment supplied by oxidative phosphorylation. The steady-state rate of Glc phosphorylation by hexokinase bound to phosphorylating mitochondria is not altered by increase in total rate of ATP production resulting from addition of hexokinase-depleted mitochondria to the system. In contrast, the steady-state rate of Glc phosphorylation by yeast hexokinase, which does not bind to mitochondria, is directly related to the total rate of ATP production in the system. These results are also consistent with the view that, during oxidative phosphorylation, mitochondrially bound hexokinase is selectively using intramitochondrially compartmented ATP; such substrate selectivity would be expected to require physical association of hexokinase with the mitochondria and be dependent solely on the oxidative phosphorylation activity of the hexokinase ? bearing organelles. The Km for Glc is only modestly affected by the binding of hexokinase to mitochondria and not further altered upon induction of active oxidative phosphorylation, suggesting that neither binding nor oxidative phosphorylation greatly affects the conformation of the Glc binding site. The reliance on intramitochondrial ATP is suggested to result from oxidative-phosphorylation-dependent changes in the interaction between the mitochondrial surface and the regions of the hexokinase molecule involved in binding ATP.