zfDHHC16介导的蛋白质棕榈酰化修饰对小鼠眼睛发育和视网膜功能的影响

Protein palmitoylation is an important form of post-translational modification which affects the localization and activity of targeted proteins. Based on previous research, we generated a knockout mouse model of zfDHHC16, one of the palmitol transferase genes. Both homozygous and heterozygous knockout mice showed abnormal eye development. The homozygous knockout mouse was born with small eye or no eye and died within one day after birth. The heterozygous knockout mouse showed normal growth. However, further study revealed increased apoptosis of retinal cells while the rest of ocular tissues appeared to be normal in these mice under standard housing. They were also more susceptible to light-exposure induced retinal degeneration than control. These preliminary observations showed that zfDHHC16 protein plays an irreplaceable role in mouse eye development and retinal cell metabolism. Furthermore, our pilot study showed that BMP receptor IA and II are palmitoylated and the palmitoylation was decreased in cells of homozygous mouse, with subsequent decrease of Smad protein activation. Based on these results, we hypothesize that zfDHHC16 has different protein substrates during embryonic eye development and adulthood in mouse, which affect ocular morphogenesis and retinal apoptosis, respectively. While BMPR appear to be a rightful candidate for the embryonic stage, other protein(s) may also be its target(s). To test this hypothesis, we will first evaluate the developmental and functional abnormalities in homo and heterozygous knockout mice. Subsequently, we are going to identify the substrates of zfDHHC16 in embryo and adult mouse eyes and determine the mechanisms which lead to the respective abnormalities. Through these experiments, we will evaluate the values of these two models as models of embryonic eye development and retinal degereration, respectively. We believe that this is the first animal model of palmitol transferase knockout mouse which showed extensive eye abnormalities. Our study will provide new mechanisms and systems to the regulation of embryonic eye development and retinal cell function.

蛋白质棕榈酰化是调控蛋白质分布和活性的重要机制。本课题组自主建立了棕榈酰化酶zfDHHC16基因敲除小鼠模型。初步分析发现zfDHHC16-/-小鼠眼胚胎发育异常，新生鼠无眼或小眼，zfDHHC16+/-成年鼠在正常饲养下视网膜各类细胞凋亡高于正常，光照后凋亡细胞愈发增加。本模型证实zfDHHC16基因对眼胚胎发育和视网膜细胞代谢有极其重要作用。初步机理探究显示BMP受体具棕榈酰化修饰，在zfDHHC16-/-细胞中减少，且Smad1/5/8磷酸化减弱，提示BMP信号通路可能异常。本课题将在全面阐述zfDHHC16-/-和+/-小鼠眼睛异常基础上，通过各种实验手段确定zfDHHC16在眼内的底物及其作用机制，进而评估其分别作为眼发育及视网膜退化模型的价值。本模型可能是世上唯一的棕榈酰化引起眼睛异常的模型，将为进一步了解及调控眼睛发育及视网膜功能提供崭新的线索和系统。

蛋白质棕榈酰化是细胞内蛋白质翻译后修饰的一种，由棕榈酸乙酰转移酶介导。由于这些酶都含有一个天冬酰胺-组氨酸-组氨酸-半胱氨酸（DHHC）片段，因此也被统称为zDHHC或zfDHHC蛋白。蛋白质棕榈酰化通过影响被修饰蛋白在细胞内的定位，转运以及稳定性，从而影响蛋白的活性和功能。本课题基于zDHHC16全身性基因敲除小鼠胚胎致死同时伴有小眼或无眼现象，推测其对小鼠发育有重要作用。我们首先揭示了zDHHC16-/-胚胎致死的原因是由于其在心脏的作用底物，肌质网磷酸受钙蛋白（PLN）缺乏棕榈酰化修饰，从而导致其低磷酸化，继而引起心脏发育异常，小鼠胚胎死亡。而zDHHC16-/-小鼠眼睛发育缺陷的原因可能与BMP受体BMPR1A和BMPRII棕榈酰化缺失有关。在zDHHC16-/-小鼠胚胎成纤维细胞中Smad1/5/8蛋白表达水平低于正常小鼠细胞，BMP2诱导的Smad1/5/8磷酸化稳定时间较短。BMP在视杯和晶状体发育中有关键性作用，BMP7基因敲除小鼠也伴有眼睛发育异常。接着我们在细胞水平揭示了蛋白质棕榈酰化对细胞DNA损伤反应的影响，我们发现在棕榈酰化修饰受抑制的情况下，细胞DNA损伤反应诱导的Atm磷酸化，p53磷酸化以及p53下游蛋白表达减少，DNA损伤灶的形成和解散异常，而zDHHC16在介导这一过程中起重要作用。由于视网膜色素上皮细胞（RPE）在正常视网膜功能和一系列眼底致盲性疾病发病中的重要作用，我们对其进行了棕榈酰化蛋白组学的研究，通过CLICK以及ABE化学法结合液相色谱和串联质谱分析发现了51个从未被报道的存在棕榈酰化修饰的蛋白，其中包括一些与RPE特异功能相关的蛋白。我们对其中一个胆固醇转运蛋白-NPC1做了深入分析，发现其799和800位半胱氨酸的棕榈酰化为其转运胆固醇功能所必须。本课题同时也建立了zDHHC16条件性敲除小鼠模型，克服了由于全身敲除导致胚胎死亡带来的研究的不便，为进一步更加准确地了解这个棕榈酰化酶在眼组织发育和视功能中的作用提供了动物模型。本课题的研究使得我们第一次对zDHHC16的功能有了突破性的了解，目前已发表相关SCI论文3篇，总计影响因子超过15分，培养硕博学生各一名。

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