The Role of Human HHRH Mutations in the Function of NaPi-IIc

描述(申请人提供)：这个项目将解决的问题是，当钠-磷酸共转运体Napi-IIc在非洲爪哇卵母细胞和哺乳动物细胞系中表达时，是形成二聚体还是多聚体，以及人类napi-IIc突变是否通过形成非功能性异二聚体来干扰钠-磷酸共转运体的运输。 遗传性低磷血症高钙尿症(HHRH)是由SLC34A3/napi-IIc纯合子或复合杂合性错义或缺失突变引起的。HHRH是一种持续终生的疾病，受HHRH影响的个体的杂合子亲属通常会有高钙尿和肾脏钙化，这是由于肾脏磷酸盐丢失导致1，25(OH)2D水平升高所致。相反，缺乏SLC34A3/napi-IIc的小鼠由于1，25(OH)2D水平升高，在早期只表现出轻微的高钙血症和吸收性高钙尿，但这些动物不会出现低磷血症，杂合子动物是正常的。虽然单倍体功能不全可以解释在杂合的napi-IIc突变携带者中观察到的高钙尿，因此一些突变，特别是那些与更严重的表型相关的突变，如肾结石，可能会导致对野生型napi-IIc或其他钠磷共转运体的显性负面影响。在我的KO8课程开始的过去两年里，我已经建立了对SLC34A3/Napi-IIc突变进行功能分析所需的所有技术，这些突变是在遗传性低磷性软骨病伴高钙尿症(HHRH)和相关疾病的转介患者中发现的。突变体T137M和V446Stop在非洲爪哇卵母细胞中共表达时，对野生型Napi-IIc功能的影响为显性-负性，而Napi-IIa功能不受影响。利用交联和免疫共沉淀技术，我已经展示了野生型Napi-IIc在非洲爪哇卵母细胞中过度表达后的二聚化。这些实验对非特异性蛋白质聚集效应进行了仔细的控制。我的计划是在OK、HEK293和LLCPK-1细胞中重复这些研究，以表明二聚化也发生在哺乳动物近端肾小管细胞系中。我还计划建立FRET技术，它不需要将二聚体的交联和纯化作为一种独立的方法来表明发生了二聚化。我将进一步使用野生型和突变型Napi-IIc的腺病毒转导，以显示在OK，HEK293和LLCPK-1细胞中的显性负效应。拟议的体外研究将提供第一个证据，证明Napi-IIc的二聚化对其功能至关重要。如果成功，这些数据将支持在代谢病房研究中对人类T137M和V446Stop杂合携带者的系统评估，并使用转基因或敲入小鼠模型来测试假设，即这些突变是否会在体内产生显性负面影响并导致肾结石形成和/或肾钙质沉着症。 公共卫生相关性：SLC34A3/NAPI-IIc是一种肾脏钠-磷联合转运蛋白，有助于从尿液中回收磷以提高血磷水平。在人类中，napi-IIc纯合子突变会导致遗传性低磷软骨病伴高钙尿症(HHRH)，但尚不清楚为什么napi-IIc突变杂合携带者有时也会有相对严重的表型，如肾结石。这一提议将检验这样一种假设，即某些突变可能通过形成非功能性异二聚体来干扰野生型共转运蛋白，从而解释杂合子携带者相对严重的表型。

DESCRIPTION (provided by applicant): This project is going to address the question whether the sodium-phosphate co-transporter NaPi-IIc forms dimers or multimers when expressed in Xenopus oocytes and mammalian cell lines and whether human mutations in NaPi-IIc interfere with sodium-phosphate co-transport by the formation of non-functional heterodimers. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by homozygous or compound heterozygous missense or deletion mutations in SLC34A3/NaPi-IIc. HHRH is a disorder that persists life-long and heterozygous relatives of individuals affected by HHRH often have hypercalciuria and renal calcifications due to elevated 1,25(OH)2D levels as a result of renal phosphate losses. In contrast, mice lacking SLC34A3/NaPi-IIc show only mild hypercalcemia and absorptive hypercalciuria at an early age due to increased 1,25(OH)2D levels, but these animals do not develop hypophosphatemia, and heterozygous animals are normal. While haploinsufficiency could explain the hypercalciuria observed in heterozygous human carriers of NaPi-IIc mutations, it is therefore possible that some mutations, particularly those associated with a more severe phenotype such as renal stones, lead to dominant negative effects on wild-type NaPi-IIc or other sodium-phosphate co-transporters. In the past two years since the start of my KO8 "Role of the renal sodium-phosphate co-transporter NaPi-IIc in phosphate homeostasis" I have established all techniques required for functional analysis of SLC34A3/NaPi-IIc mutations, which were discovered in patients referred with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and related disorders. When co-expressed in Xenopus oocytes, the mutants T137M and V446Stop impair wild-type NaPi-IIc function in a dominant-negative fashion, while NaPi-IIa function was unaffected. Using cross-linking and co-immunoprecipitation techniques, I have shown dimerization of wild-type NaPi-IIc after over-expression in Xenopus oocytes. These experiments were carefully controlled for non-specific protein aggregation effects. My plan is to repeat these studies in OK, HEK293 and LLCPK-1 cells to show that dimerization also occurs in mammalian proximal tubular cell lines. I also plan to establish the FRET technology, which does not require cross-linking and purification of dimers as an independent method to show that dimerization occurs. I will furthermore use adenoviral transduction of mutant and wild-type NaPi-IIc to show the dominant negative effect in OK, HEK293 and LLCPK-1 cells. The proposed in vitro studies will provide first proof that dimerization of NaPi-IIc is important for it's function. If successful, these data will support a systematic evaluation of the human heterozygous carriers of T137M and V446Stop in metabolic ward studies, and the use of transgenic or knock-in mouse models to test the hypothesis whether these mutations can have a dominant negative effect and lead to kidney stone formation and/or nephrocalcinosis in vivo. PUBLIC HEALTH RELEVANCE: SLC34A3/NaPi-IIc is a renal sodium-phosphate co-transporter, which helps reclaiming phosphate from the urine to increase blood phosphate levels. Homozygous mutations in NaPi-IIc cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in humans, but it is unclear why also heterozygous carriers of NaPi-IIc mutations sometimes have a relatively severe phenotype such as renal stones. This proposal is going to test the hypothesis that some mutations may interfere with wild-type co-transporters by the formation of non-functional heterodimers thereby explaining the relatively severe phenotype of heterozygous carriers.