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

[unreadable] 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. [unreadable] [unreadable]