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: 7525495
• 负责人: MUAYYAD R AL-UBAIDI
• 金额: $ 38.29万
• 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 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7525495
• 关键词: 2 year old; Adult; Age-Months; Aging; Animals; Antibodies; Behavior; Biochemical; Cell Nucleus; Column Chromatography; Crossbreeding; Data; Development; Electrodes; Electrons; Electroretinography; Embryo; Embryonic Development; Enzymes; Exhibits; Future; Genetic Transcription; Glycoproteins; Goals; Golgi Apparatus; Histologic; Human; Human Genome; Immunohistochemistry; Inorganic Sulfates; Isoenzymes; Knock-out; Knockout Mice; Light; Maintenance; Microscopic; Modification; Mus; Organ; Pattern; Phenotype; Proteins; Research Design; Retina; Retinal; Retinal Cone; Retinal Defect; Retinal Degeneration; Retinal Diseases; Reverse Transcriptase Polymerase Chain Reaction; Role; Suction; TNFRSF5 gene; Techniques; Time; Tissues; Translations; Tyrosine; Unspecified or Sulfate Ion Sulfates; Western Blotting; gel electrophoresis; insight; novel; protein expression; protein-tyrosine sulfotransferase; research study; sulfation; tyrosine O-sulfate

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.

本提案的总体目标是研究蛋白质酪氨酰硫酸化的作用 正常的视网膜功能。中间目标包括鉴定被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。在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的翻译后酪氨酸O-硫酸化。这些酶及其靶点在视网膜或其他眼组织中均未被研究。我们已经产生了TPST-1和2的敲除小鼠。这两种小鼠，以及通过杂交获得的双敲除小鼠，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的缺乏导致功能发育的延迟。此外，这些小鼠表现出缓慢的视网膜变性，其特征在于暗视和明视视网膜电图（ERG）的逐渐丧失，其在4月龄时变得非常明显（约30%丧失）。P120 Tpstrl-视网膜的组织学检查显示ONL（P120时9行对比P50时11行）和INL（P120时4行对比P50时5-6行）中的细胞核数量减少。与TpsWI -视网膜不同，TpstZI -视网膜从未完全发育至野生型水平。这非常清楚地证明了TPST-2或其蛋白产物在视网膜成熟中的直接作用。在目标1中，我们将表征发育过程中视网膜TPST-1和TPST-2的存在、定位和生化行为。这些实验将在胚胎第10天至2岁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和MS和MS/MS鉴定由TPST-1/2修饰的蛋白质。在目标2中，我们将充分表征由缺乏TPST-1、TPST-2或两者引起的视网膜表型。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和抽吸电极记录进行功能表征，通过SOS-PAGE分析视网膜蛋白的硫酸化和TPST-1/2靶点的表达进行生物化学表征。 这些研究将首次揭示TPST-1和2在视网膜蛋白硫酸化中的作用。此外，它们将增加我们对视网膜发育和视网膜变性所涉及的机制的理解。