Retinal degeneration caused by alterations in protein tyrosine O-sulfation

描述（申请人提供）：对色素性视网膜炎（RP）和年龄相关性黄斑变性（AMD）的研究已显著提高了对其遗传原因的认识。尽管这些发现引发了许多治疗范例的探索，但尽管突变蛋白从出生起就存在，但我们对迟发性视力丧失的原因的理解仍然不足。通常认为这些细胞的存活是由于神经保护因子的作用，这些因子显著延长了患病细胞的寿命。然而，目前还不清楚为什么病人突然开始遇到视觉障碍。在这里，我们提出了一个额外的贡献假设延迟发作的视力丧失基于两个相关的发现。首先是鉴定CFH和纤维蛋白5为硫酸化蛋白。除了存在于drusen中，这两种蛋白的突变都与AMD有关。第二，我们在RP和rds+/-小鼠两种动物模型中观察到视网膜变性过程中酪氨酸磺酸蛋白水平和类型的调节。由于硫酸化是发生在分泌和膜相关细胞外基质（ECM）蛋白上的翻译后修饰，我们假设细胞的缓慢死亡将导致ECM的逐渐恶化。然而，当维持ecm的细胞数量达到临界数量时，就会达到一个临界点，突然加速退化过程。我们提出两个具体目标来解决我们的假设。在Aim 1中，我们正在培养视网膜/ rpe特异性条件敲除小鼠，用于两种负责蛋白质硫酸化的酶，蛋白质酪氨酸硫转移酶1和2。双敲除（DKO）小鼠将从出生到2岁的功能、结构和生化特征来表征缺乏硫酸化的影响。为了确定缺乏ECM蛋白硫酸化是否会加剧AMD表型，我们计划将这些小鼠回交到AMD转基因模型（cfhY402H）中。为了解决ECM蛋白缺乏硫酸化对RP表型的影响，我们计划将DKO小鼠回交到两种已经得到充分研究的RP模型，分别代表视紫红质基因（VPP）的P23H突变和视网膜变性缓慢（rds+/-）蛋白的单倍不足。在目标2中，我们将评估硫酸化在CFH和纤维蛋白5功能中的作用。鉴定的磺化酪氨酸将突变为苯丙氨酸和1)非磺化CFH能力的体外分析

DESCRIPTION (provided by applicant): Research on retinitis pigmentosa (RP) and age related macular degeneration (AMD) has significantly advanced knowledge into their genetic causes. Although such findings triggered exploration of numerous therapeutic paradigms, we still come short in our understanding of the cause of the late onset of visual loss although the mutant protein is present from birth. It is often proposed that these cells survive due to effects f neuroprotective factors that significantly prolong the life of the sick cell. However, it is not clar as to why the patient starts to encounter visual difficulties all of a sudden? Here we propose an additional contributing hypothesis for the delayed onset of visual loss based upon two related findings. The first is the identification of CFH and fibulin 5 as sulfated proteins. Besides being present in drusen, mutations in both proteins associate with AMD. Our second is that we observe modulations in levels and types of tyrosine-sulfated proteins during retinal degeneration in two well-studied animal models of RP, the VPP and the rds+/- mouse models. Since sulfation is a post-translational modification that occurs on secreted and membrane-associated extracellular matrix (ECM) proteins, we hypothesize that the slow death of cells will lead to gradual deterioration of ECM. However, when a critical number of ECM-maintaining cells are lost, a breaking point is reached, suddenly accelerating the degenerative process. We propose two specific aims to address our hypothesis. In Aim 1, we are generating retina/RPE-specific conditional knockout mice for both of the enzymes responsible for protein sulfation, protein tyrosyl sulfotransferases 1 and 2. The double knockout (DKO) mice will be characterized functionally, structurally and biochemically from birth to 2 years of age for effects of lack of sulfation. To determine if the lack of sulfation of ECM proteins exacerbate the AMD phenotype, we plan to backcross these mice into an AMD transgenic model (cfhY402H). To address the effect of lack of sulfation of ECM proteins on the RP phenotype, we plan to backcross the DKO mice to two well-studied RP models representing the P23H mutation in the rhodopsin gene (VPP) and the haploinsufficiency of the retinal degeneration slow (rds+/-) protein. In Aim 2, we will evaluate the role of sulfation in the function of CFH and fibulin 5. The identified sulfated tyrosine(s) will be mutated to phenylalanines and in vitro analyses of 1) ability of unsulfated CFH to bind CRP (C-reactive protein), 2) its ability to self-dimerize and 3) its interactions with polyanions. For fibulin 5, we will test the ability of the unsulfated protein to interact with interins and ECM super oxide dismutase (SOD) 3. Subsequently, we will generate and characterize retina/RPE-specific conditional knock-in mice expressing either sulfation-deficient CFH or fibulin 5. Accomplishment of the proposed studies will open a new window on the role of ECM in degenerative process and direct attention to development of new therapeutic approaches focusing on protection of the ECM to prolong vision in patients and widen the window for interventions. This is of significance to over 10 million US Citizens of all ages and races that suffer from RP and AMD.