Functional consequences in Genitourinary (GU) development of human mutations in the genes of the ubiquitin ligase complex KCTD13-CUL3 E3.
Abhishek Seth, MD, Olga Medina-Martinez, PhD, Carolina J. Jorgez, PhD.
Baylor College of Medicine, Houston, TX, USA.
BACKGROUND:Copy number variation (CNV) are an important risk factor for congenital genitourinary defects. One of the most frequent CNV involved in congenital GU anomalies is the 16p11.2 locus. We identified KCTD13, as an important gene in this region for male lower GU tract defects. KCTD13 acts as a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin -protein ligase complex, which regulates the actin cytoskeleton to modulate cell migration and proliferation. The ubiquitin-proteasome machinery is mainly involved in protein degradation, but also regulate other cellular functions including cell signaling, protein trafficking, cell-cycle progression and cell death. Reduction of KCTD13 results in elevated RHOA, which positively regulates MAP3K1 and SOX9, two important regulators of sexual development. We generated a mouse model lacking Kctd13 that has cryptorchidism and micropenis with reduced levels of nuclear AR and SOX9 and increased levels of RHOA. We hypothesized that similar to the mouse model, KCTD13-CUL3 complex in boys may alters the masculinization axes by resulting in inefficient ubiquitination of the masculinization pathways leading to abnormal GU development, undervirilization and subfertility. METHODS: Using whole-exome sequencing (WES), we have identified single nucleotide variants (SNV) in KCTD13 that were generated in vitro using site-directed mutagenesis. We generated gain and loss (RNAi-mediated) of function studies of KCTD13 and CUL3 in GU cell lines. We generated a cell reconstitution ubiquitination assay.
RESULTS: Knock-down of KCTD13 in MLTC-1 cells (produces testosterone when activated by hCG since they carry LHr) showed a significant decrease in testosterone production compared to control cells with hCG stimulation. Knockdown of KCTD13 in TM4, Sertoli cell line, induced actin stress fiber formation. We identified two rare predictive damaging SNVs in KCTD13- E120K and P146L- in boys with cryptorchidism. We generated TM4 cell line with stably transfected pcDNA3.1-KCTD13 with the corresponding open reading frame or the missense mutations identified by WES of patients. Our preliminary data indicated these two SNV severely affect the formation of actin stress fibers. We are studying the effects of these SNV in testosterone production and KCTD13 CUL3 interaction and ubiquitination.
CONCLUSIONS: We have identified KCTD13 as an important promoter of AR transcriptional activity. Our data will provide insights into the mechanisms of KCTD13-CUL3 mediate the masculinization pathway in masculinization. It will also help to understand the functional consequences of each of the SNVs identified in patients and its association with GU development. The validation of these pathogenic mutations could generate potential clinical diagnostic test and targets for personalized therapeutic drugs.
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