Structural, Functional and Computational Studies of Proton-Coupled Eukaryotic Amino Acid Transporters

阳离子氨基酸转运蛋白（CATs）是一种跨细胞膜转运带正电荷氨基酸的膜蛋白。这个转运蛋白家族的成员参与免疫反应，但我们目前对精氨酸和赖氨酸-在免疫信号传导中发挥作用的氨基酸-如何被转运到细胞中的理解是有限的。CAT原核同源物（GkApcT）的结构模型最近得到解决，并显示是一个质子偶联转运蛋白。然而，由于炎性疾病随着寿命延长而变得越来越重要（例如类风湿性关节炎），因此需要用具有更高临床有效性的药剂来治疗这些病症。哺乳动物的真核结构模型的CAT是必需的，以告知基于结构的药物化学的抗炎药物设计的努力。为此，该项目的主要目标是解决哺乳动物CAT结构和功能注释的CAT家族成员与功能生物化学和模拟方法。特别是破译CAT 4是否像GkApcT一样是质子偶联的，以及它转运的氨基酸可能表明这种孤儿转运蛋白的底物。另外，已知亚型CAT 2-A/B对精氨酸具有不同的亲和力;然而，在机制上，仍然存在这样的问题：这是如何在两种转运蛋白具有如此高的序列相似性的情况下特异性地实现的（例如，差异是动力学（Vmax）驱动的还是动力学（KD）驱动的？）。脂质体重组纯化的转运蛋白将提供洞察这些问题沿着的就业分子动力学（MD）模拟和自由能方法从计算化学进一步询问动力学。

Cationic amino acid transporters (CATs) are membrane proteins tasked with the transport of positively charged amino acids across the cell membranes. Members of this family of transporters are involved in immune response, but our current understanding on how arginine and lysine - amino acids that play a role in immune signaling - are transported into the cell and total amounts is limited. A structural model of a CAT prokaryotic homolog (GkApcT) was recently solved and shown to be a proton-coupled transporter. However as inflammatory diseases are of increasing importance with lifespan elongation (e.g. rheumatoid arthritis) the need for therapeutic treatment of these conditions with agents of higher clinical effectiveness are needed. A mammalian eukaryotic structural model of CATs is required to inform structure-based medicinal chemistry efforts in anti-inflammatory drug design. To this end, the key goals of this project are solving a mammalian CAT structure and to functionally annotate members of the CAT family with functional biochemistry and simulation methods. Specifically deciphering whether CAT4 is proton-coupled like GkApcT and what amino acids it transports may suggest this orphan transporter's substrates. Additionally isoforms CAT2-A/B are known to have different affinities for arginine; however mechanistically the question remains of how this specifically is achieved with such high sequence similarity of the two transporters (e.g. is the difference kinetically (Vmax) or thermodynamically (KD) driven?). Liposomal reconstitution of the purified transporters will provide insight to these questions along with the employment of Molecular Dynamics (MD) simulations and free energy methods from computational chemistry to further interrogate dynamics.