Molecular mechanisms of protein crosslinking in the lens

晶状体蛋白在衰老过程中经历二硫交联和非二硫交联。这样的交联是

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.