Retinal degeneration caused by alterations in protein tyrosine O-sulfation

描述（由申请人提供）：对视网膜色素变性（RP）和年龄相关性黄斑变性（AMD）的研究大大提高了对其遗传原因的认识。虽然这些发现引发了许多治疗范例的探索，但我们仍然缺乏对视力丧失迟发性原因的理解，尽管突变蛋白从出生就存在。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。但是，为什么病人突然开始遇到视力困难，这是不清楚的？在这里，我们提出了一个额外的贡献假说的延迟发病的视力丧失的基础上两个相关的研究结果。第一个是CFH和fibulin 5作为硫酸化蛋白的鉴定。除了存在于玻璃疣中，两种蛋白质的突变都与AMD相关。第二个是，我们在两种经过充分研究的RP动物模型（VPP和rds+/-小鼠模型）中观察到视网膜变性期间酪氨酸硫酸化蛋白水平和类型的调节。由于硫酸化是一种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，当临界数量的ECM维持细胞丢失时，就会达到临界点，突然加速退化过程。我们提出了两个具体的目标来解决我们的假设。在目标1中，我们正在产生视网膜/RPE特异性条件敲除小鼠的两种酶负责蛋白质硫酸化，蛋白质酪氨酰磺基转移酶1和2。双敲除（DKO）小鼠将从出生至2岁期间在功能、结构和生化方面表征缺乏硫酸化的影响。为了确定ECM蛋白硫酸化的缺乏是否加剧了AMD表型，我们计划将这些小鼠回交成AMD转基因模型（cfhY 402 H）。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小鼠与两种充分研究的RP模型回交，这两种模型代表视紫红质基因（VPP）中的P23 H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fibulin 5功能中的作用。将鉴定的硫酸化酪氨酸突变为苯丙氨酸，并体外分析：1）未硫酸化CFH 结合CRP（C-反应蛋白），2）其自身二聚化的能力和3）其与聚阴离子的相互作用。对于fibulin 5，我们将测试未硫酸化的蛋白质与interins和ECM超氧化物歧化酶（SOD）3相互作用的能力。随后，我们将产生和表征视网膜/RPE特异性条件性敲入小鼠表达硫酸缺陷型CFH或fibulin 5。拟议研究的完成将为ECM在退行性过程中的作用打开一个新的窗口，并将注意力集中在开发新的治疗方法上，重点是保护ECM，以延长患者的视力，并扩大干预的窗口。这对于超过1000万患有RP和AMD的各个年龄段和种族的美国公民来说意义重大。

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.