Structure and Function of Ocular Lacritin

描述(由申请人提供)： 我们的竞争性更新应用程序从我们发现的新的眼部特异型泪液因子“泪水”的角度来治疗干眼症。Lacritin从泪腺和眉板腺流入眼表，帮助促进眼表湿润。它也可能通过其基底层角膜上皮细胞的表达而促进角膜更新。泪液中催乳素的选择性下调与眼缘炎和隐形眼镜相关的干眼有关，这是迄今为止仅有的两种干眼综合征。在一到两年前摘除卵巢的正常兔子或干眼症雌性猴子的眼睛中添加催乳素，会触发增加和持续的泪水流量。在人类角膜上皮细胞培养中，lacritin比血清更有效地刺激MUC16蛋白的产生，我们最初的观察结果表明，lacritin促进培养的大鼠泪腺泡细胞释放泪液过氧化物酶，这不依赖于副交感神经(氨基甲胆碱)刺激途径。此外，人类角膜上皮细胞的亚融合培养以泪蛋白结构域和剂量特定的方式增殖。撕裂素是如何促进撕裂和再生的还不清楚。我们对Lacritin结构/功能和Lacritin信号的研究已经勾勒出了靶向细胞表面蛋白多糖syndecan-1的乙酰肝素酶依赖的机制。这可能导致G蛋白偶联受体通过G1i或G1o连接到PLC-PKC1/PLD/mTOR和PLC-PKC1/STIM1/Calcineurin/Ca~(2+)/NFATC1。新发现的剪接变异体lacritin-b或-c(怀疑不能分别结合gpr或syndecan-1)的差异上调与天然lacritin的下调或乙酰肝素酶的丢失(初步在干眼泪液中观察到)一致，是两种潜在的干眼刺激情况。我们的工作假设是，催乳素是眼表湿润和更新的调节剂。我们的直接目标是确定信号受体的特征，并了解其作用机制。我们的第一个目标是了解构成拉西丁/Syndecan-1/G蛋白偶联受体异源复合体的特定蛋白质-蛋白质相互作用。我们的第二个目标是研究上游撕裂蛋白信号是如何产生的。我们的第三个目标是询问下游的催乳素信号如何促进湿润和更新，从而促进全球对催乳素在眼表上的作用的认识。与公共卫生相关。干眼症很常见。这个项目将找出一种新的潜在的干眼疗法如何促进眼表湿润。这种疗法是基于一种天然的人类泪液蛋白。

DESCRIPTION (provided by applicant): Our competitive renewal application addresses dry eye from the perspective of the new ocular-specific tear factor 'lacritin' discovered by us. Lacritin flows onto the ocular surface from lacrimal and meibomian glands to help promote ocular surface wetting. It is also a presumed contributor to corneal renewal via its basal corneal epithelial expression. Selective downregulation of lacritin in tears is associated with blepharitis and contact lens-related dry eye, the only two dry eye syndromes proteomically examined to date. Adding lacritin to eyes of normal rabbits or dry eye female monkeys ovariectomized one to two years earlier triggers elevated and sustained tear flow. These observations are reinforced by human corneal epithelial cell culture in which lacritin stimulates MUC16 protein production more efficiently than serum and by our original observation that lacritin augments tear peroxidase release from cultured rat lacrimal acinar cells independent of the parasympathetic- (carbachol-) stimulation pathway. Also subconfluent cultures of human corneal epithelial cells proliferate in a lacritin domain- and dose-specific manner. How lacritin promotes tearing and renewal is not understood. Our lacritin structure/function and lacritin signaling studies have sketched out a heparanase-dependent mechanism for targeting cell surface proteoglycan syndecan-1. This leads to putative ligation of a G-protein coupled receptor for signaling through G1i or G1o to PLC-PKC1/PLD/mTOR and PLC- PKC1/STIM1/calcineurin/Ca2+/ NFATC1. Differential upregulation of newly identified splice variants lacritin-b or -c (suspected to be incapable of respectively binding the GPCR or syndecan-1) coincident with downregulation of native lacritin, or loss of heparanase (observed preliminarily in dry eye tears) are two potential dry eye-provoking scenarios. Our working hypothesis is that lacritin is a regulator of ocular surface wetting and renewal. Our immediate goal is to characterize the signaling receptor and understand its mechanism of action. Our first aim is to understand specific protein-protein interactions constituting the lacritin/syndecan-1/G-protein coupled receptor heterocomplex. Our second aim is to characterize how upstream lacritin signaling is generated. Our third aim asks how downstream lacritin signaling can promote wetting and renewal towards a global appreciation for lacritin's role on the ocular surface. PUBLIC HEALTH RELEVANCE. Dry eye is very common. This project will work out how a new potential therapeutic for dry eye promotes ocular surface wetting. The therapeutic is based on a natural human tear protein.