Protein Kinase C Gamma in the Lens

描述(申请人提供)：在晶状体中，缝隙连接活性通过激活晶状体/神经特异性的PKC伽马亚型而降低。这种酶被生长和压力因素激活，如IGF-1、LEDGF和过氧化氢。一旦激活，PKC-γ转位到膜上，并磷酸化晶状体连接蛋白，如Cx43。这通过将缝隙连接斑块分解成含有小窝蛋白-1的质膜脂筏来抑制缝隙连接。这种PKC伽马功能可以通过分解缝隙连接和阻止损伤信号传递到邻近细胞来保护晶状体免受氧化应激，这一过程可能在缺血期间发生。我们的假设是，PKC伽马在晶状体中充当应力传感器。与其他经典的PKC亚型相比，该亚型对氧化和激活信号(如二酰甘油)更敏感。因此，当它存在于细胞中时，它是压力的主要PKC传感器。PKC伽马基因敲除模型不能减少过氧化氢引起的缝隙连接，该模型可能对导致白内障的氧化应激损伤更敏感。旨在测试PKC伽马作为透镜应力传感器的作用的具体目标是：1.识别结构 镜头的PKC伽马的特点，使这是一个应力传感器。这将包括鉴定对接蛋白和识别位点，这将使该酶保持不活跃状态。2.通过鉴定蛋白质上的相互作用位点，确定PKC-γ如何与脂筏中的Cx43和小窝蛋白-1相互作用。3.确定整个晶状体对过氧化氢胁迫的反应机制是通过激活PKCγ和降低Cx46和Cx50.4的功能来实现的。确定糖尿病期间链脲佐菌素大鼠对缝隙连接的PKC伽马控制是如何受到影响的。5.对PKC-γ基因敲除小鼠模型进行评估，以确定PKC-γ在整个晶状体应激反应中的作用。将设计能够从14-3-3对接蛋白中去除PKC伽马并激活晶状体中PKC伽马的多肽。这应该会导致缝隙连接的解体，并有助于防止氧化应激造成的损害。这些多肽也可能有助于防止缺血对视网膜的损害。

DESCRIPTION (provided by applicant): In the lens gap junction activity is decreased by activation of the lens/neural-specific PKC gamma isoform. This enzyme is activated by growth and stress factors such as IGF-1, LEDGF, and by hydrogen peroxide. Upon activation PKC gamma translocates to membranes and phosphorylates lens connexin proteins such as Cx43. This causes inhibiton of gap junctions through disassembly of gap junction plaques into caveolin-1-containing plasma membrane lipid rafts. This PKC gamma function could protect the lens from oxidative stress through disassembly of gap junctions and prevention of the passage of damaging signals to neighboring cells, a process which can occur during ischemia. Our hypothesis is that PKC gamma serves as a stress sensor in the lens. This isoform is more sensitive to oxidative and activation signals such as diacylglycerol, than other classical PKC isoforms. Thus, when present in cells it is the major PKC sensor of stress. The PKC gamma knock-out model is not able to decrease gap junctions in response to hydrogen peroxide and this model may be more sensitive to oxidative stress damage which causes cataracts. The specific aims, designed to test the role of PKC gamma as a lens stress sensor are: 1. Identify structural features of the lens PKC gamma which allow this to be a stress sensor. This will include identification of the docking protein and recognition sites, which maintain this enzyme in an inactive state. 2. Determine how PKC gamma interacts with Cx43 and caveolin-1 in lipid rafts by identification of interaction sites on the proteins. 3. Determine the mechanism by which whole lens responds to hydrogen peroxide stress through activation of PKC gamma and decreased function of Cx46 and Cx50.4. Determine how the PKC gamma control of gap junctions is compromised during diabetes in the streptozotocin rat. 5. Assess the PKC gamma knock-out mouse model to determine the role of PKC gamma in the whole lens stress response. Peptides will be designed which can remove the PKC gamma from the 14-3-3 docking protein and activate PKC gamma in lens. This should cause disassembly of gap junctions and help to prevent damage from oxidative stress. These peptides could also be useful to prevent damage to retina from ischemia.