Retinal degeneration caused by alterations in protein O-sulfation

蛋白质 O-硫酸化改变引起的视网膜变性

• 批准号: 7843629
• 负责人: MUAYYAD R AL-UBAIDI
• 金额: $ 37.14万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7843629
• 关键词: 2 year old; Adult; Affect; Age; Age-Months; Animals; Antibodies; Behavior; Biochemical; Cell Nucleus; Column Chromatography; Crossbreeding; Data; Development; Electrodes; Electrons; Electroretinography; Embryo; Embryonic Development; Enzymes; Exhibits; Extracellular Matrix; Future; Genetic Transcription; Glycoproteins; Goals; Golgi Apparatus; Histologic; Human; Human Genome; Immunohistochemistry; Inorganic Sulfates; Isoenzymes; Knock-out; Knockout Mice; Light; Maintenance; Methods; Microscopic; Modification; Mus; Northern Blotting; Organ; Pattern; Phenotype; Phototransduction; Proteins; Research Design; Retina; Retinal; Retinal Cone; Retinal Defect; Retinal Degeneration; Retinal Diseases; Retinoids; Reverse Transcriptase Polymerase Chain Reaction; Role; Staging; Suction; TNFRSF5 gene; Techniques; Time; Tissues; Translations; Tyrosine; Unspecified or Sulfate Ion Sulfates; Western Blotting; aged; gel electrophoresis; insight; novel; protein expression; protein-tyrosine sulfotransferase; research study; response; retinal rods; sulfation; tyrosine O-sulfate; visual process; visual processing

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.

该提案的总体目标是研究蛋白质酪氨酰硫酸化的作用 在正常的视网膜功能中。中间目标包括鉴定由酪氨酰蛋白磺基转移酶硫酸化的视网膜蛋白，并确定缺乏这些酶中的一种（或两种）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-硫酸化。尚未在视网膜或其他眼组织中研究这些酶及其靶标。我们已经培育出了TPST-1和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 缺乏会导致功能发育迟缓。此外，这些小鼠表现出缓慢的视网膜变性，其特征是暗视和明视视网膜电图（ERG）逐渐丧失，在 4 个月大时变得非常明显（-30% 丧失）。 P120 Tpstrl-视网膜的组织学检查显示 ONL（P120 为 9 行，P50 为 11 行）和 INL（P120 为 4 行，P50 为 5-6 行）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 或其蛋白质产物在视网膜成熟中的直接作用。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 天至 2 岁时的正常小鼠视网膜中进行，使用对 TPST-1、TPST-2 和磺基酪氨酸具有特异性的抗体。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 TPST-1/2 修饰的蛋白质。在目标 2 中，我们将全面描述由于 TPST-1、TPST-2 或两者缺乏而导致的视网膜表型。将在光和电子显微镜水平上进行结构表征，通过暗视和明视 ERG 和抽吸电极记录进行功能表征，并通过 SOS-PAGE 分析视网膜蛋白的硫酸化和 TPST-1/2 靶标的表达进行生化表征。 这些研究将首次揭示 TPST-1 和 2 在视网膜蛋白质硫酸化中的作用。此外，它们将增加我们对视网膜发育和视网膜变性机制的了解。