FAT10 a candidate gene for epithelial apoptosis in HIV-associated nephropathy

描述(申请人提供)：HIV相关肾病(HIVAN)是HIV血清阳性患者慢性肾功能衰竭的最常见原因，是由HIV-1感染肾上皮细胞引起的，HIVAN的易感性受到遗传因素的强烈影响。利用寡核苷酸芯片，我们已经确定泛素样蛋白FAT10是感染HIV-1后肾小管上皮细胞(RTECs)中表达最高的基因之一。我们发现FAT10在HIVAN活检标本中表达上调，并在体外诱导RTECs凋亡和细胞周期紊乱。此外，我们还发现FAT10基因的多态与发生HIVAN的易感性增加有关。这些研究的目的是确定FAT10在HIV诱导RTEC凋亡中的作用，并确定FAT10基因中的非同义单核苷酸多态(SNPs)如何改变蛋白质的分子功能，从而使个体患肾脏疾病的风险增加。在第一个特定目标中，我们将定位导致FAT10上调的HIV-1基因，并确定FAT10的表达是否对HIV-1诱导的肾小管上皮细胞凋亡是必需的。HIV-1多基因突变病毒构建物将用于体外转导肾小管上皮细胞，并定位导致FAT10表达增加的HIV-1基因(S)。慢病毒小发夹RNA(ShRNA)构建也将用于抑制人肾小管上皮细胞中FAT10的表达。用这些构建体稳定转导的细胞将感染HIV-1，并将通过流式细胞仪对细胞凋亡进行量化。在第二个具体目标中，我们将确定FAT10中的非同义SNPs如何影响其分子功能。我们已经确定了FAT10编码序列的四种不同的单倍型。一种单倍型，称为HPT-1，在FAT10的第二个外显子上包含四个非同义SNP，与HIVAN发病风险增加有关。我们将确定FAT10蛋白的四个变体在它们的亚细胞定位以及它们诱导细胞周期失调和凋亡的能力上的不同之处。我们还将确定这些SNPs是否改变了FAT10与有丝分裂纺锤体检查点蛋白MAD2结合的能力。这些研究将促进我们对HIVAN发病机制的理解，并可能为HIVAN和其他肾脏疾病的治疗提供新的治疗靶点。

DESCRIPTION (provided by applicant): HIV-associated nephropathy (HIVAN), the most common cause of chronic renal failure in HIV-seropositive patients, is caused by HIV-1 infection of renal epithelial cells and susceptiblity to HIVAN is strongly influenced by genetic factors. Using oligonucleotide microarrays, we have determined that the ubiquitin-like protein FAT10 is one of the most highly upregulated genes in renal tubular epithelial cells (RTECs) after infection by HIV-1. We have shown that FAT10 is upregulated in HIVAN biopsy specimens and that FAT10 expression induces apoptosis and cell cycle dysregulatin of RTECs in vitro. Moreover, we have found that polymorphisms in the FAT10 gene are associated with enhanced susceptibility to developing HIVAN. The goals of these studies are to determine the role of FAT10 in HIV-induced RTEC apoptosis and to determine how non-synonymous single nucleotide polymorphisms (SNPs) in the FAT10 gene alter the molecular function of the protein, thereby predisposing individuals to increased risk of developing renal disease. In the first Specific Aim we will map the HIV-1 genes that are responsible for upregulation of FAT10 and determine if FAT10 expression is necessary for HIV-1 induced apoptosis of renal tubular epithelial cells. HIV-1 multigenic mutant viral constructs will be used to transduce renal tubular epithelial cells in vitro and map the HIV-1 gene(s) responsible for increasing expression of FAT10. Lentiviral small hairpin RNA (shRNA) constructs will also be used to inhibit expression of FAT10 in human renal tubular epithelial cells. Cells that have been stably transduced with these constructs will be infected with HIV-1 and apoptosis will be quantified by flow cytometry. In the second Specific Aim, we will determine how non-synonymous SNPs in FAT10 affect its molecular function. We have identified four distinct haplotypes of the FAT10 coding sequence. One haplotype, referred to as HPT-1, contains four non-synonymous SNPs in the second exon of FAT10 and is associated with increased risk of developing HIVAN. We will determine how the four variants of the FAT10 protein differ in their subcellular localization and their ability to induce cell cycle dysregulation and apoptosis. We will also determine whether these SNPs alter the ability of FAT10 to associate with the mitotic spindle checkpoint protein MAD2. These studies will advance our understanding of HIVAN pathogenesis and may yield a novel therapeutic target for the treatment of HIVAN and other renal diseases.