Exploring PiTs: Functional and structural characterization of human SLC20 family members

SLC20家族的钠依赖的磷酸转运体的功能和转运机制还没有得到很好的描述。两个人类SLC20转运蛋白PiT1和PiT2广泛存在于人体内，它们的功能范围从PI内务的调节到适应性和遗传性钙化疾病的因子。它们的重要性是显而易见的，因为PiT1基因的敲除对小鼠是胚胎致命的，而人类PiT2的突变与严重的遗传病特发性基底节钙化有关。在缺乏人PiT1和PiT2的生产和纯化策略的情况下，功能研究大多局限于卵母细胞中小鼠同系物的电生理表征，并且只有一种细菌同源物的结构可用。现有的关于PIT转运蛋白的信息不足以理解其转运机制，也不允许对疾病相关突变体的病理生理功能做出任何结论。我想要阐明一些关于PIT转运蛋白功能的分子细节。我将实施人PIT转运蛋白的生产和纯化策略，建立体内和体外转运蛋白检测方法，并从冷冻-EM分析中获得这些二级活性转运蛋白的第一结构。我要探讨的四个关键方面是转运机制的分子细节，内源性脂类对转运蛋白活性的影响，二聚化对转运蛋白功能的影响，包括可能的协同效应和细胞内环的功能，这只在真核细胞的凹坑转运蛋白中存在。我以前在膜蛋白生产、纯化和处理方面的经验，再加上Pul Nissen教授团队和奥胡斯大学在二级活性转运蛋白和优秀的冷冻-EM基础设施方面的丰富知识，使我相信拟议的项目是合理和可行的。

The functional role and transport mechanism of sodium-dependent phosphate transporters of the SLC20 family are not well characterized. The two human SLC20 transporters PiT1 and PiT2 are omnipresent in the human body and their proposed functions range from regulators of Pi housekeeping to factors in adapted and genetic calcification disorders. Their importance is eminent because, a knockout of the PiT1 gene is embryonic lethal in mice and mutations of human PiT2 are linked to the severe genetic disease idiopathic basal ganglia calcification. In the absence of a production and purification strategy for human PiT1 and PiT2, functional studies are mostly limited to electrophysiological characterization of murine homologues in oocytes and only one structure from a bacterial homologue is available. The existing information on PiT transporters is not sufficient to understand their transport mechanism and does not allow any conclusion on the pathophysiological functions of disease-related mutants. I want to shed some light on the molecular details of PiT transporter function. I will implement a production and purification strategy for human PiT transporters, establish in vivo and vitro transport assays and obtain the first structures of these secondary active transporters from cryo-EM analysis. The four key aspects I will explore are the molecular details of the transport mechanism, the influence endogenous lipids have on transporter activity, the impact dimerization has on transporter function including possible cooperative effects and the function of the intracellular loop, which is only found in eukaryotic PiT transporters. My previous experience in membrane protein production, purification and handling in combination with the vast knowledge on secondary active transporters and excellent cryo-EM infrastructure present in the group of Prof. Poul Nissen and at Aarhus University make me confident that the proposed project is reasonable and feasible.