The asymmetric cleavage of beta-carotene in mammalian embryonic development

 描述（由申请人提供）：维生素A是哺乳动物胚胎正常发育所必需的，因为其活性形式视黄酸（RA）的转录作用存在缺陷。在实验动物模型和人类中，母体维生素A摄入不足或过量会导致先天性畸形或胎儿死亡。值得注意的是，维生素A缺乏症是第三大最普遍的营养缺乏症，是一个压倒性的公共卫生问题，影响到发展中国家数亿人（主要是妇女和儿童）。世界上大多数人口，特别是在上述地理区域，依赖于维生素A前体β-胡萝卜素（BC）作为类维生素A（维生素A及其衍生物）的来源。在胚胎中，从母体饮食中获得的BC在通过对称BC裂解酶β，β-胡萝卜素-15，15 '-加氧酶CM 0 1转化为视黄醇后，可显著促进发育组织对类维生素A的需求。然后视黄醛被氧化为视黄酸，视黄酸是许多对胚胎发育至关重要的基因的主要调节因子。BC还被β，β-胡萝卜素-9 '，10'-加氧酶CMO 2不对称切割，产生C10 β-脱辅胡萝卜素醛（C10 apoAL）。虽然视黄醇也可以从C10 apoAL产生，但CMO 2及其反应产物在哺乳动物胚胎发育过程中的作用尚不清楚。我们的初步数据表明，众所周知的VAD对小鼠胚胎发生的不利影响加重时，CMO 2是无活性的和BC给药的母鼠，尽管CMO 1的表达。我们发现，在视黄醇结合蛋白（RBP）敲除背景下缺乏CMO 2的小鼠胚胎表型（一种已建立的VAD模型）是由于C10 apoAL水平低沿着类维生素A的可用性有限。补充C10 apoAL维生素A缺乏饮食的CMO 2-/-RBP-/-小鼠减少了先天性畸形。我们认为C10 apoAL是PKCδ的配体。这种PKC同工型向丙酮酸脱氢酶复合物（PDHC）发出信号，增加其活性，目的是协调柠檬酸循环的燃料流量与ATP生产的需求。PKCδ被氧化还原激活 该机制与结合激酶激活结构域的维生素A（视黄醇）的强制性催化作用有关。CMO 2定位于线粒体，C10 aopAL在结构上与视黄醇相似。我们发现，C10 apoAL与PKCδ的视黄醇结合域相互作用，并以PKCδ依赖的方式调节小鼠胚胎成纤维细胞的呼吸。总的来说，我们的数据表明，当C10 apoAL和维生素A限制时，PKC信号传导和线粒体功能的破坏可能是BC存在下缺乏CMO 2的小鼠表型恶化的根本原因。本研究旨在进一步了解CMO 2在哺乳动物胚胎发育过程中的功能和C10 apoAL的作用机制。具体而言，我们将测试C10 apoAL是否是VAD条件下胚胎存活所必需的（Aim 1），我们将定义在胚胎发育过程中CMO 2/β-apo胡萝卜素和PKCδ信号网络之间的相互作用（Aim 2），包括体外和离体（Aim 2A）和体内（Aim 2B）。独特的试剂，如纯合成的β-脱辅基类胡萝卜素化合物，和新的小鼠模型，与PKCδ和类胡萝卜素代谢途径的失活，将被用来解决上述问题。了解CMO 2/β-脱辅基类胡萝卜素在胚胎发生中的作用与人类健康有关，因为它将导致新的干预措施来改善VAD相关的先天性缺陷。

 DESCRIPTION (provided by applicant): Vitamin A is required for proper mammalian embryonic development, owing to defective transcriptional action of retinoic acid (RA), its active form. Deficient or excessive maternal vitamin A intake of this essential nutrient results in congenital abnormalities or fetal death in experimental animal models and humans. Notably, vitamin A deficiency (VAD) is the third most prevalent nutritional deficiency, and is an overwhelming public health issue, affecting hundreds of millions of people (predominantly women and children) in developing countries. The majority of the world population, especially in the above-mentioned geographic areas, relies on the vitamin A precursor β-carotene (BC) as a source of retinoids (vitamin A and its derivatives). In the embryo, BC obtained from the maternal diet can significantly contribute to the retinoid needs of the developing tissues upon conversion to retinaldehyde via the symmetric BC cleavage enzyme β,β-carotene-15,15'-oxygenase, CMO1. Retinaldehyde is then oxidized to retinoic acid, the master regulator of many genes that are crucial to embryogenesis. BC is also cleaved asymmetrically by β,β-carotene-9',10'-oxygenase, CMO2, generating C10 β-apocarotenal (C10 apoAL). While retinaldehyde could also be generated from C10 apoAL, the role of CMO2 and its reaction product during mammalian embryonic development is unknown. Our preliminary data indicate that the well-known detrimental effects of VAD on mouse embryogenesis are aggravated when CMO2 is inactive and BC is administered to the dams, despite expression of CMO1. We showed that the embryonic phenotype of mice lacking CMO2 on the retinol-binding protein (RBP) knockout background, an established model of VAD, was due to the low levels of C10 apoAL along with limited availability of retinoids. Supplementing CMO2-/-RBP-/- mice on a vitamin A deficient diet with C10 apoAL reduced congenital malformations. We propose that C10 apoAL serves as a ligand for PKCδ. This mitochondria-localized PKC isoform signals to the pyruvate dehydrogenase complex (PDHC), increasing its activity, with the purpose of coordinating the fuel flux to the citric acid cycle with the demands for ATP production. PKCδ is activated by redox mechanisms, with a mandatory catalytic role of vitamin A (retinol) that binds the activation domains of the kinase. CMO2 localizes to mitochondria and C10 aopAL is structurally similar to retinol. We showed that C10 apoAL interacts with the retinol-binding domain of PKCδ and modulates respiration in mouse embryonic fibroblasts, in a PKCδ-dependent manner. Overall, our data suggest that disruption of PKC signaling and mitochondrial functions when C10 apoAL and vitamin A are limiting could be the underlying cause of the exacerbated phenotypes of mice lacking CMO2 in the presence of BC. With this application we seek to further understand the function of CMO2 and the mechanisms of C10 apoAL action during mammalian embryogenesis. Specifically, we will test whether C10 apoAL is essential for embryonic survival under conditions of VAD (Aim 1) and we will define the interaction between CMO2/β- apocarotenoids and PKCδ signaling network during embryonic development (Aim 2), both in vitro and ex vivo (Aim 2A) and in vivo (Aim 2B). Unique reagents, such as pure synthetic β-apocarotenoid compounds, and new mouse models, with inactivation of the PKCδ and carotenoid metabolism pathways, will be used to address the above-mentioned questions. Understanding the role of CMO2/β-apocarotenoids in embryogenesis is relevant to human health, as it will lead to novel interventions to ameliorate VAD-associated congenital defects.