ABSTRACT
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.
Subject(s)
Actins/metabolism , Endopeptidases/metabolism , Luciferases/metabolism , Proteomics/methods , Apoptosis , Autophagy , Endopeptidases/analysis , Recombinant Fusion ProteinsABSTRACT
The androgen receptor plays a major role in the development and function of normal and malignant prostate cells. Due to the relationship of the androgen receptor and prostatic growth, it has been proposed that polymorphisms within the androgen receptor may play a role in an individual's susceptibility to developing prostate cancer. An inverse relationship has been established between a highly polymorphic trinucleotide repeat located in the first exon of the androgen receptor and the transactivaton function of the receptor. Serum samples were collected from 131 patients with histologically confirmed adenocarcinoma of the prostate, DNA was isolated, and the polymorphic CAG repeat was amplified by PCR and sequenced. The CAG repeat lengths were then compared with age at diagnosis, age at time of study, baseline log(10) PSA, Gleason score, time from diagnosis to initiation of hormonal therapy, time to progression after androgen ablation, and overall survival time. No correlation was found between CAG length and time to progression or overall survival time, but a significant correlation was found between Gleason score and CAG length suggesting that shorter CAG lengths may predict a higher histological grade of prostate cancer.
