Retinal degeneration caused by alterations in protein O-sulfation

这项提案的总体目标是研究蛋白质酪氨酰硫化的作用。 在正常的视网膜功能中。中间目标包括鉴定被酪氨酸蛋白磺基转移酶硫化的视网膜蛋白，并确定缺乏其中一种(或两种)这些酶如何影响视网膜功能。大多数组织中存在两种不同的TPST同工酶。这两种酶负责细胞蛋白质的翻译后酪氨酸O-硫化。这些酶或它们的靶标在视网膜或其他眼睛组织中都没有被研究过。我们已经培育出了针对TPST-1和TPST-2的基因敲除小鼠。这两只小鼠以及通过杂交获得的双基因敲除小鼠，在视网膜发育、功能和衰老方面表现出不同的视网膜缺陷。TPST-1的缺失导致功能发育迟缓。此外，这些小鼠表现出缓慢的视网膜变性，特征是暗视和明视视网膜电图(ERG)逐渐消失，在4个月大时变得非常明显(-30%消失)。组织学检查显示ONL(P120时9行，P50时11行)和INL(P120时4行，P50时5-6行)的细胞核数量减少。与TpsWI-视网膜不同，TpstZI-视网膜永远不会完全发育到野生型水平。这非常清楚地证明了TPST-2或其蛋白产物在视网膜成熟过程中的直接作用。在目标1中，我们将描述视网膜TPST-1和TPST-2在发育过程中的存在、定位和生化行为。这些实验将在胚胎10至2岁的正常小鼠视网膜上进行，使用针对TPST-1、TPST-2和硫代酪氨酸的抗体。作为这一目标的一部分，我们将使用经典的免疫亲和纯化技术以及MS和MS/MS来鉴定被TPST-1/2修饰的蛋白质。在目标2中，我们将充分表征由于缺乏TPST-1和/或TPST-2而导致的视网膜表型。将在光学和电子显微镜水平上进行结构表征，通过暗视和明视ERG和吸入电极记录进行功能表征，并通过SOS-PAGE分析视网膜蛋白质的硫酸盐化和TPST-1/2靶标的表达来进行生化表征。 这些研究将首次阐明TPST-1和2在视网膜蛋白质硫酸盐化中的作用。此外，它们将增加我们对视网膜发育和视网膜变性涉及的机制(S)的了解。

The overall goal of this proposal is to investigate the role of protein tyrosyl-sulfation in normal retinal function. The intermediate aims include the identification of retinal proteins that are sulfated by tyrosylprotein sulfotransferases and determine how lack of either (or both) of these enzymes influence retinal function. There are two distinct TPST isoenzymes present in most tissues. These two enzymes are responsible for the post-translational tyrosine O-sulfation of cellular proteins. Neither these enzymes nor their targets have been studied in the retina or other ocular tissues. We have generated knockout mice for TPST-1 & 2. These two mice, and the double knockout obtained by crossbreeding, exhibit different retinal defects in retinal development, function and aging. Lack of TPST-1 results in the retardation of functional development. Moreover, these mice demonstrate a slow retinal degeneration characterized by a gradual loss of scotopic and photopic electroretinography (ERGs) that become very apparent at 4 months of age (-30% loss). Histologic examination of P120 Tpstrl- retinas revealed a reduced number of nuclei in ONL (9 rows at P120 versus 11 rows at P50) and INL (4 rows at P120 versus 5-6 at P50). Unlike TpsWI - retinas, TpstZI - retinas never fully develop to the wild-type level. This very clearly demonstrates a direct role for TPST-2, or its protein products, in retinal maturity. In Aim 1, we will characterize the presence, localization and biochemical behavior of retinal TPST-1 and TPST-2 during development. These experiments will be performed in normal mouse retinas from embryonic day 10 to 2 years of age utilizing antibodies that are specific for TPST-1, TPST-2, and to sulfotyrosine. As part of this aim, we will identify the proteins modified by TPST-1/2 using classic immunoaffinity purification techniques and MS and MS/MS. In Aim 2, we will fully characterize retinal phenotypes caused by lack of TPST-1, TPST-2 or both. Characterization will be performed structurally at the light and electron microscopic levels, functionally by scotopic and photopic ERGs and suction electrode recordings, and biochemically by analysis of the sulfation of retinal proteins and expression of the targets of TPST-1/2 by SOS-PAGE. These studies will, for the first time, shed light on the role of TPST-1 and 2 in protein sulfation in the retina. Further, they will increase our understanding of retinal development and the mechanism(s) involved in retinal degeneration.