Semi-synthesis of ubiquitinated androgen receptor peptides using an expressed protein ligation system Creator
|Department:||Department of Human Genetics|
|Keywords:||Molecular Biology - Genetics|
|Full text PDF:||http://digitool.Library.McGill.CA:80/R/-?func=dbin-jump-full&object_id=121439&silo_library=GEN01|
The 90-kb androgen receptor (AR) gene is located at locus Xq11-12 and exon 1 encodes the transactivational domain, exons 2 and 3 encode the DNA-binding domain, and exons 4 through 8 encode the ligand-binding domain. The AR protein, a nuclear receptor that belongs to the steroid/thyroid hormone receptor family, functions primarily as a DNA-binding transcription factor to regulate gene expression. In fact, cytoplasmic AR is activated by binding testosterone or dihydrotestosterone hormone before translocating to the nucleus. Spinal and Bulbar Muscular Atrophy (SBMA). The cause of SBMA is expansion of glutamine (Gln, Q), in Exon 1 of the AR gene. Therefore SBMA is considered to be a polyGln expansion disorder. The toxic gain of AR function that is characteristic of SBMA results in a slow progressive muscle weakness, atrophy of the tongue, fasciculations of bulbar, facial and limb muscles, and muscle cramping. A loss of AR function is related to breast enlargement and reduced fertility.The ubiquitin-proteasome system (UPS), which functions in protein degradation, plays an important role in neural cells from SBMA patients. UPS components are found in polyGln-expanded protein aggregates seen in these cells. Previous studies have suggested that the UPS is impaired in SBMA. Previous work suggests that 0QAR and 20QAR can be degraded via the UPS, however, the 50QAR appeared to inhibit the degradation of specific proteins by the UPS. It is also hypothesized that these SBMA-inducing mutant ARs can accumulate in the cell and cause disease by evading UPS degradation. The primary goal of this research was to develop a system for ubiquitinating AR by using an expressed protein ligation system. To prove this hypothesis, several intermediate goals have been achieved. PCR-amplified fragments of AR (0Q, 20Q, and 50Q) were cloned into the pTWIN2 vector and contructs were analyzed using restriction enzyme digests and gel electrophoresis.