Molecular mechanisms of protein crosslinking in the lens

晶状体中蛋白质交联的分子机制

• 批准号: 10320416
• 负责人: Ram H Nagaraj
• 金额: $ 33.94万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320416
• 关键词: Advanced Glycosylation End Products; Age; Aging; Aldehydes; Apoptotic; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Blindness; Cataract; Cataract Extraction; Cells; Chemicals; Client; Complex; Crystallins; Data; Developing Countries; Development; Disulfides; Electrophoresis; Environment; Excision; Exhibits; Fluorescence Resonance Energy Transfer; Funding; Glucose; Heat shock proteins; Human; Individual; Innovative Therapy; Intraocular lens implant device; Ketones; Lead; Life; Light; Mediating; Modification; Molecular; Molecular Chaperones; Molecular Sieve Chromatography; Molecular Weight; Mus; Nature; Operative Surgical Procedures; Organ; Oxidative Stress; Permeability; Physiological; Post-Translational Protein Processing; Presbyopia; Proteins; Pyruvaldehyde; Solubility; Stress; Testing; Time; Transplantation; Western Blotting; Work; aged; alpha-Crystallins; amino group; ascorbate; base; cost; crosslink; dehydroascorbate; experimental study; flexibility; gamma-Crystallins; glycation; inhibitor; innovation; lens; lens transparency; light scattering; novel; preservation; prevent; protein aggregation; protein complex; protein crosslink; resilience; small molecule inhibitor; therapy development; thermal stress; water solubility

Lens proteins undergo disulfide and non-disulfide crosslinking during aging. Such crosslinkings are associated with protein aggregation, insolubilization, light scattering and loss of lens accommodation. While disulfide crosslinking is well studied, the biochemical nature and the mechanism of formation of non-disulfide crosslinking are poorly understood. We propose to test a novel hypothesis that non-disulfide covalent crosslinking of proteins in the lens could arise from glycation-mediated crosslinking of the complexes that are formed between α-crystallin and its chaperoned proteins, which leads to the formation of high molecular weight proteins and protein insolubilization during lens aging. Our preliminary studies strongly support this hypothesis. In the proposed project, we will systematically investigate this hypothesis via three specific aims. In Aim 1, we will perform experiments to establish the long-term stability of α-crystallin-client protein complexes under the conditions of the lens by employing fluorescence resonance energy transfer (FRET)- based assays. We will then determine whether α-crystallin-client protein complexes undergo more covalent crosslinking by glycation than their individual protein components by quantifying protein-crosslinking advanced glycation end products (AGEs). In Aim 2, we will extend our studies to intact human and mouse lenses to determine whether oxidative or thermal stress (to promote α-crystallin-client protein complex formation) would promote glycation-mediated protein crosslinking in the lens. We will then determine the collective effects of stress and glycation on light transmittance and stiffness (resilience) in lenses. In Aim 3, we will first determine whether crosslinking by α-crystallin-client protein glycation has a direct relationship with non-disulfide crosslinked high-molecular-weight proteins in aging lenses; we will then use a novel inhibitor that we developed during the previous funding period to inhibit protein crosslinking in human lenses. Finally, we will determine whether the inhibitor prevents losses in light transmittance and losses in resilience due to AGE-mediated protein crosslinking. Together, the three aims will test an innovative concept of protein crosslinking in the lens and test a novel chemical inhibitor against such crosslinking. The findings in this study could lead to innovative therapies against presbyopia and cataracts.

晶状体蛋白在老化过程中发生二硫键和非二硫键交联。 与蛋白质聚集、不溶解、光散射和晶状体调节损失有关。 二硫键交联已得到充分研究，非二硫键的生化性质和形成机制 我们对交联知之甚少，建议测试一个新的假设，即非二硫键共价键。 晶状体中蛋白质的交联可能是由糖基化介导的复合物交联引起的 α-晶状体蛋白与其陪伴蛋白之间形成，从而形成高分子 我们的初步研究有力地支持了这一点。 在拟议的项目中，我们最终将通过三个具体目标来研究该假设。 在目标 1 中，我们将进行实验以确定 α-晶状体蛋白客户蛋白的长期稳定性 通过采用荧光共振能量转移（FRET）在透镜条件下形成复合物- 然后我们将确定 α-晶状体蛋白-客户蛋白复合物是否经历更多共价作用。 通过定量蛋白质交联，通过糖化交联比其单独的蛋白质成分进行交联 在目标 2 中，我们将研究范围扩展到完整的人类和小鼠。 镜片以确定氧化应激或热应激（促进 α-晶状体蛋白-客户蛋白复合物 形成）将促进晶状体中糖化介导的蛋白质交联，然后我们将确定 在目标 3 中，应力和糖化对镜片透光率和硬度（弹性）的共同影响。 我们将首先确定 α-晶状体蛋白-客户蛋白糖化的交联是否与 然后我们将在老化镜片中使用非二硫键交联的高分子量蛋白质； 我们在上一个资助期间开发的用于抑制人类晶状体中的蛋白质交联的最后， 我们将确定抑制剂是否可以防止由于光透射率损失和弹性损失 AGE 介导的蛋白质交联将共同测试蛋白质的创新概念。 这项研究的结果是在镜片中发生交联，并测试了一种新型化学抑制剂来对抗这种交联。 可能会导致针对老花眼和白内障的创新疗法。

项目成果

Transient elevation of temperature promotes cross-linking of α-crystallin-client proteins through formation of advanced glycation endproducts: A potential role in presbyopia and cataracts.

温度的瞬时升高通过高级糖基化终产物的形成促进α-晶状体蛋白-客户蛋白的交联：在老花眼和白内障中的潜在作用。

• 发表时间: 2020
• 影响因子: 3.1
• 作者: Nandi, Sandip K;Rankenberg, Johanna;Glomb, Marcus A;Nagaraj, Ram H
• 通讯作者: Nagaraj, Ram H

Advanced oxidation protein products activate vascular endothelial cells via a RAGE-mediated signaling pathway.

高级氧化蛋白产品通过 RAGE 介导的信号通路激活血管内皮细胞。

• DOI: 10.1089/ars.2007.1999
• 发表时间: 2008-08-15
• 期刊: Antioxidants & redox signaling
• 影响因子: 6.6
• 作者: Zhi;Hong;F. Hou;Lu Zhang;Ning Fu;R. Nagai;Xiao Lu;Baihui Chen;Y. Shan
• 通讯作者: Y. Shan

Effects of photobleaching on selected advanced glycation end products in the human lens.

光漂白对人类晶状体中选定的高级糖基化终产物的影响。

• 发表时间: 2015-01-16
• 影响因子: 1.8
• 作者: Holm, Thomas;Raghavan, Cibin T;Nahomi, Rooban;Nagaraj, Ram H;Kessel, Line
• 通讯作者: Kessel, Line

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

糖化介导的蛋白质间交联由α-晶状体蛋白的伴侣-客户复合物促进：对晶状体老化和老花眼的影响。

• 发表时间: 2020-04-24
• 作者: Nandi, Sandip K;Nahomi, Rooban B;Rankenberg, Johanna;Glomb, Marcus A;Nagaraj, Ram H
• 通讯作者: Nagaraj, Ram H

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

体外，羧西丁可抑制小鼠晶状体中糖化介导的蛋白质交联和硬化。

• 发表时间: 2021
• 影响因子: 3
• 作者: Nandi, Sandip K;Rankenberg, Johanna;Rakete, Stefan;Nahomi, Rooban B;Glomb, Marcus A;Linetsky, Mikhail D;Nagaraj, Ram H
• 通讯作者: Nagaraj, Ram H