Biochemistry and Molecular Biology
Presenter(s): Abbigael Eli
Advisor(s): Dr. Marco Bisoffi
Prostate cancer (PCa) is one of the most frequent cancers in the world’s male population. The androgen receptor (AR), which responds to the binding of androgens (for example, testosterone), is a major oncogenic driver in cancer cells. Androgens bind the AR in the cytoplasm and initiate its translocation to the nucleus, where it acts as a transcription factor for genes that promote growth and survival. In cancerous cells, AR signaling is upregulated and constitutive, leading to uncontrolled cell growth. The diarylpentanoid ca27, an analog of the natural product curcumin, has been shown to downregulate AR expression in PCa cells, but its mechanism of action remains unknown. This study explores the possibility of ca27 interfering with AR nuclear translocation, thereby leading to its degradation and reduced expression. Androgen-dependent human LNCaP prostate cancer cells were treated with ca27, curcumin, and dimethyl sulfoxide (DMSO) vehicle control, followed by the generation of cytoplasmic and nuclear protein lysates. The protein lysates were size-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunodetection of AR, histone H3 (nuclear protein), and beta-tubulin (cytoplasmic protein) by chemiluminescent Western blotting (WB). AR expression levels were determined by AR band densitometric analysis of digitalized images using ImageJ software. In this project, we aim at testing the potential effect of ca27 on AR translocation by determining the ratio of cytoplasmic-to-nuclear AR band intensity between ca27-, curcumin-, and DMSO-treated protein lysates. We report here on our first data set and provide an experimental plan moving forward to elucidate the effect of ca27 on AR translocation and expression as a potential mechanism of action.
Engineering a Prenyltransferase Enzyme for Biocatalytic Diversification of Bioactive Compounds
Presenter(s): Ahmad Alrusayes
Advisor(s): Dr. Sherif Elshahawi
Enzymes serve an important role in any biological system as the biocatalysis of reactions is essential for the survival of the living systems. Under biologically relevant conditions, uncatalyzed reactions tend to be extremely slow and the presence of enzymes catalyze these reactions in a specific and selective manner rapidly. Many chemical reactions require toxic petroleum-derived solvents and extreme reaction conditions which complicate process development and oftentimes create safety hazards to exclude even traces of water from their reaction media. Indeed, enzymes circumvent these problems by providing a specific environment within which a given reaction can occur more rapidly. This inspired chemists to manipulate these natural enzymes to catalyze difficult reactions under favorable reactions of pH, temperature and green conditions. Furthermore, engineering the active binding sites of enzymes to become more efficient is a hot research area and can lead to the catalysis of challenging chemical reactions. Prenyltransferases (PTs) are important enzymes that catalyze the transfer of a prenyl moiety to diverse compounds naturally. By transferring a prenyl moiety to a compound, the bioactivity of the substance might increase because the hydrophobicity of the substance will increase. This lowers the substance interaction with water in the system and, therefore, the reaction will have higher yield and reproducibility. By engineering PTs to have higher promiscuity, it is possible to use them as unique tools to generate more active compounds. In this project, we will study the X-ray crystal structure of one of these PTs and use mutagenesis to make it more proficient and promiscuous. The enzymes are then screened for their ability to diversify drugs which their counterparts wild type do not perform. A combination of different mutagenesis approaches will be used to enhance the activity of PTs towards nonnative substrates.