Molecular Mechanisms in Retinal Degeneration

描述（由申请人提供）：本提案的长期目标是表征与人类视网膜变性有关的基因，并找到阻止或最终治愈这些致盲疾病的可能方法。具体来说，我们将研究锥体中存在的两种蛋白质，15A15和视黄裂素，它们是由我们实验室之前分离的基因编码的，它们的功能可能是相互关联的。我们已经发现15A15蛋白结合到几个包含激素反应元件核心共识序列的DNA片段上，并且它具有核激素受体（nhr）的共激活因子/共抑制因子的几个特征。在我们的第一个特异性目标中，我们将：证实15A15发挥这种作用，通过GST下拉试验研究其蛋白质-蛋白质相互作用；研究15A15与nhr的相互作用是否依赖于激素，并增强内源性nhr的交互激活；绘制这些相互作用所需的15A15区域；确定15A15是否直接结合视网膜分裂素启动子和其他锥体中表达的基因中的应答元件，调节其转录，以及15A15模块的突变是否会消除nhr的转录增强。染色质免疫沉淀试验将显示15A15是否作为与其他因子的复合体的一部分存在于体内。我们还将筛选影响视锥细胞的视网膜变性患者的DNA，寻找可能导致疾病的15A15突变。在我们的第二个特定目标中，我们将继续研究视黄裂素，一种参与细胞相互作用的分泌蛋白，当突变导致x连锁幼年性视网膜裂（XLRS）。我们发现cGMP， Ca2+和可能的g蛋白可能参与调节视网膜裂素从光感受器内节段分泌。在这些数据的基础上，我们将对控制这一过程的机制进行系统的研究，调查膜和可溶性鸟苷酸环化酶（GC）的参与，GC激活蛋白，一氧化氮和一氧化碳的作用；杆状、锥体和Ca2+/钙调素依赖性pde的参与，以及鸟嘌呤脱氨酶将cGMP转化为cXMP的作用。我们还将确定l型电压门控Ca2+通道和细胞内Ca2+储存，以及gβ - γ和mGluR8是否影响视黄裂素的分泌。对于所研究的每个光感受器成分，我们将从药理学方法开始，以确定其参与作用，然后使用shrna将其从系统中去除，以证实其作用。总的来说，我们的研究将增加对视锥细胞中两个重要蛋白15A15和retinoschisin相互作用和功能的理解。通过了解正常生理条件下视黄鳞素分泌调节的生化途径，这可能对XLRS的潜在药理治疗具有重要意义，我们希望为探索目前无法治愈的疾病挽救视力的可能途径开辟新的途径。

DESCRIPTION (provided by applicant): The long-term objectives of this proposal are to characterize genes involved in human retinal degenerations and to find possible ways to halt or eventually cure these blinding diseases. Specifically, we will study two proteins present in cones, 15A15 and retinoschisin, encoded by genes isolated previously in our laboratory, that may be interrelated in their function. We have found that the 15A15 protein binds to several DNA fragments containing the core consensus sequences for hormone response elements and that it has several features characteristic of coactivators/ co-repressors of nuclear hormone receptors (NHRs). In our first Specific Aim we will: corroborate that 15A15 plays this role studying its protein-protein interactions with GST pull-down assays; investigate if 15A15 interactions with NHRs are hormone-dependent and enhance the transactivation of endogenous NHRs; map the region of 15A15 necessary for these interactions; determine if 15A15 binds directly to response elements present in the promoter of retinoschisin and other genes expressed in cones, regulating their transcription, and if mutations in 15A15 modules abolish transcriptional enhancement of NHRs. Chromatin immunoprecipitation assays will show if 15A15 exists as part of a complex with other factors, in vivo. We will also screen the DNA of patients with retinal degenerations affecting cones for mutations in 15A15 that may result in disease. In our second Specific Aim, we will continue studying retinoschisin, the secreted protein involved in cell-cell interactions that when mutated causes X-linked juvenile retinoschisis (XLRS). We have found that cGMP, Ca2+ and possibly G-proteins may participate in regulation of secretion of retinoschisin from the photoreceptor inner segments. On the basis of these data, we will carry out a systematic study on the mechanisms that control this process, investigating the involvement of membrane and soluble guanylate cyclases (GC), the effects of GC-activating proteins, nitric oxide and carbon monoxide; the participation of rod, cone and Ca2+/calmodulin-dependent PDEs, and the role of guanine deaminase, which converts cGMP into cXMP. We will also determine whether L-type voltage gated Ca2+ channels and intracellular Ca2+ stores, as well as Gbeta-gamma and mGluR8 influence the secretion of retinoschisin. For each photoreceptor component studied, we will start with a pharmacological approach to establish its involvement, followed by its removal from the system with the use of shRNAs to corroborate its effect. Overall, our research will increase the understanding of the interaction and function of 15A15 and retinoschisin, two important proteins in cones. By learning about the biochemical pathways involved in the regulation of retinoschisin secretion in normal physiological conditions, that may be important for potential pharmacological treatment of XLRS, we hope to open new avenues to explore possible ways to rescue vision for diseases for which there are no current cures.