PhD

dissertationNucleocytoplasmic trafficking of steroid hormone receptors is a conundrum that remains to be solved. The mechanism of nuclear import has been established, whereas the mode of nuclear export is unknown. Steroid receptors are vital to cell functioning and are involved in several signal transduction pathways. These receptors are also promising pharmacological targets as they are implicated in a number of diseases. Identifying the means of nucleocytoplasmic shuttling is important in understanding the molecular basis of functioning of these receptors and their potential use in therapeutics. The research presented here investigated the mechanism of nucleocytoplasmic shuttling of steroid hormone receptors. The knowledge of signal mediated transport was then applied to construct a regulatable protein switch with potential use in the field of gene therapy. The first half of the dissertation describes the search for the export mechanism. A model system using Enhanced Green Fluorescent Protein (EGFP) was established to examine classical nuclear export signals. The most well-known export pathway is mediated by a karyopherin called Crm1 and was the starting point of this investigation. The role of a protein called Calreticulin that binds to the DNA binding domain of these receptors was also tested. In addition, the involvement of heat shock proteins (HSPs) that bind to these steroid receptors at their ligand binding domain was investigated for potential in mediating export. Results could not rule out the possibility of Crm1 and Calreticulin being involved in the transport pathway but showed that the LBD determines the subcellular localization of receptors and also that HSPs may be involved in export. In the second half of this study, signal sequences, regulated by an external stimulus, were used to facilitate or block the localization of a delivered protein to its active compartment. A bidirectional on/off switch was constructed with nuclear export signals, import signals, and a truncated PRLBD, which could be regulated by an external ligand, mifepristone. This work describes a prospective direction for search of proteins responsible for steroid receptor nuclear transport and established a method to regulate protein function by altering its subcellular localization

Model system to study classical nuclear export signals

Signal-mediated protein transport through the nuclear pore complex is of considerable interest in the field of molecular pharmaceutics. Nuclear localization signals can be used to target genes/antisense delivery systems to the nucleus Studying nuclear export is useful in enhancing the expression and the efficiency of action, of these therapeutic agents. The mechanism of nuclear import has been well studied and most of the proteins participating in this mechanism have been identified. The subject of nuclear export is still in the initial stages and there is a considerable amount of uncertainty in this area. Two main export receptors identified so far are Exportin 1 (Crm1) and Calreticulin. Crm1 recognizes certain leucine-rich amino acid sequences in the proteins it exports called classical nuclear export signals. This paper describes a model system to study, identify, and establish these classical nuclear export signals using green fluorescent protein (GFP). Two putative export signals in the human progesterone receptor (PR) and the strongest nuclear export signal known (from mitogen activated protein kinase kinase [MAPKK]) were studied using this model system