The Role of Proline Metabolism During Osteoblast Differentiation and Bone Formation

项目概要： 脯氨酸是一种多功能的亚氨基酸，在细胞中有无数的用途。除了直接掺入蛋白质外， 脯氨酸可通过称为脯氨酸循环的过程代谢。在这里，脯氨酸被脯氨酸氧化 氧化酶（PRODH）以形成D1-吡咯啉-5-羧酸酯（P5 C）。PRODH是FAD+依赖性酶， 给线粒体电子传递链的复合物II提供电子，从而偶联脯氨酸氧化， ATP合成。P5 C通过NADH依赖性酶吡咯啉-5-羧酸盐转化回脯氨酸 还原酶（PYCR）为糖酵解和戊糖磷酸途径提供还原能力。是 不知道成骨细胞如何获得脯氨酸，脯氨酸的摄取是如何调节的，或者脯氨酸循环是否以及何时被调节。 在分化过程中需要。成骨细胞表达多种膜系氨基酸 促进脯氨酸摄取的转运蛋白。我们已经确定了一个中性氨基酸转运蛋白系统 SNAT 2（由Slc 38 a2编码）是成骨细胞中最高表达的推定脯氨酸转运蛋白。我们 初步数据表明，WNT通过成骨细胞所必需的SNAT 2刺激脯氨酸摄取， 体外分化。此外，对于Slc 38 a2的无效等位基因纯合的小鼠（Slc 38 a2-/-）具有以下缺陷： 软骨内骨化在本提案中，我们将1）通过以下途径确定脯氨酸摄取的必要性： Slc 38 a2/SNAT 2在体内调节成骨细胞分化和骨形成，2）确定SNAT 2 活性受WNT信号传导调节，3）阐明通过脯氨酸循环进行脯氨酸代谢的必要性 分化成骨细胞。我们的研究结果将对骨骼发育、维持 骨质、骨骼修复和再生。

Project Summary: Proline is a multifunctional imino acid with myriad uses in the cell. Aside from direct incorporation into protein, proline can be metabolized via a process known as the proline cycle. Here, proline is oxidized by proline oxidase (PRODH) to form D1-pyrroline-5-carboxylate (P5C). PRODH is a FAD+ dependent enzyme that donates electrons to complex II of the mitorchondrial electron transport chain thus coupling proline oxidation to ATP synthesis. P5C is converted back into proline by the NADH dependent enzyme pyrroline-5-carboxylate reductase (PYCR) to provide reducing power for glycolysis and the pentose phosphate pathways. It is unknown how osteoblasts obtain proline, how proline uptake is regulated, or if and when the proline cycle is required during differentiation. Osteoblasts express a diverse array of membrane-tethered amino acid transporters to facilitate proline uptake. We have identified the system A neutral amino acid transporter SNAT2 (encoded by Slc38a2) as the most highly expressed putative proline transporter in osteoblasts. Our preliminary data indicates WNT stimulates proline uptake through SNAT2 that is necessary for osteoblast differentiation in vitro. Moreover, mice homozygous for a null allele of Slc38a2 (Slc38a2-/-) have defects in endochondral ossification. In this proposal, we will 1) establish the necessity of proline uptake through Slc38a2/SNAT2 to regulate osteoblast differentiation and bone formation in vivo, 2) determine how SNAT2 activity is regulated by WNT signaling and 3) elucidate the necessity of proline metabolism via the proline cycle in differentiating osteoblasts. Our findings will have broad implications in bone development, maintenance of bone mass, skeletal repair and regeneration.