The Role of Proline Metabolism During Osteoblast Differentiation and Bone Formation

项目总结： 脯氨酸是一种多功能亚氨基酸，在细胞中有多种用途。除了直接结合到蛋白质中外， 脯氨酸可以通过一个被称为脯氨酸循环的过程进行代谢。在这里，Pro被Pro氧化 氧化物酶(PROH)生成1-吡咯啉-5-羧酸盐(P5C)。PROSH是一种依赖于FAD的酶 将电子给予线粒体电子传输链的复合体II，从而将脯氨酸氧化偶联到 三磷酸腺苷合成。P5C被NADH依赖的酶-5-吡咯烷-5-羧酸盐重新转化为Pro 还原酶(PYCR)，为糖酵解和磷酸戊糖途径提供还原动力。它是 尚不清楚成骨细胞是如何获得Pro的，Pro摄取是如何被调节的，或者Pro循环是否以及何时被调节 在差异化过程中需要。成骨细胞表达多种膜系带氨基酸 转运蛋白以促进脯氨酸的摄取。我们已经鉴定了一个中性氨基酸转运蛋白系统 SNAT2(由Slc38a2编码)是成骨细胞中表达最高的可能的Pro转运蛋白。我们的 初步数据表明，WNT通过SNAT2刺激成骨细胞所必需的脯氨酸摄取 体外分化。此外，SLc38a2(Slc38a2-/-)零等位基因纯合的小鼠在 软骨内骨化。在这项提案中，我们将1)通过以下方式确定摄取脯氨酸的必要性 SLC38a2/SNAT2调节体内成骨细胞分化和骨形成，2)SNAT2如何决定 活性受WNT信号调节，3)阐明了通过Pro循环进行Pro代谢的必要性 在分化成骨细胞方面。我们的发现将对骨骼的发育、维持 骨量、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.