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D.K. Shanks, D. Madgwick, M.E. Barnett, D. Lin, O. Prakash, L. Grauer, D. Takemoto; Lens PKC–C1B Domains Are Altered by Ataxia Mutations: A Modeling and NMR Study . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4092.
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© ARVO (1962-2015); The Authors (2016-present)
There are a number of human diseases which are linked to the failure of proper gap junction control. Mutations in PKCγ C1B domains are associated with Human Spinocerebellar Ataxia, SCA–14. The mutations lead to an impaired cellular response to extracellular signals and oxidative stress. The purpose of this research is to determine the structural influence of C1B domains mutations on the functional activation of PKCγ, a major lens PKC.
We used molecular dynamics simulations and the solved NMR structure to compare the energy–minimized structures of PKCγ WT and mutant proteins. We purified the C1B domain and several variants using a bacterial expression system and carried out multi–dimensional hetero–nuclear NMR. C1B domain mutations H101Y and S119P were introduced into a GST linked pGEX–6p vector and subsequently transfected into BLD–21 low competency E. coli cells. The fusion proteins were treated with lysate buffer and then clarified by centrifugation and filtration (.5µm). The GST–C1B proteins were eluted from a glutathione–sepharose 4B column to isolate the fusion products and then the tags were cleaved with a Proscission digestion enzyme. The purified C1B proteins were recovered after a second GST affinity column elution step. N15 and/or C13 labeling were applied for hetero–nuclear NMR experiments.
The results of our molecular modeling and NMR structural data indicate that different mutations within the C1B domain of PKCγ may affect the activation mechanism of this kinase. Although many of the residues in the C1B domain participate in binding to activators and responding to oxidative stress, our results indicate that each mutation may cause specific problems for the enzyme. For example, modeling conformational changes within the C1B domain, including a collapse in the DAG binding loop, predict that the enzyme’s functionality is altered by the H101Y mutation.
Mutations which are associated with Human Spinocerebellar Ataxia SCA14 cause structural and biochemical changes within the C1B domain of PKCγ that affect the mechanisms by which this enzyme becomes activated. This would alter the enzyme’s ability to inhibit lens gap junctions, Cx43 and/or Cx50.
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