Gene Therapy and Nuclear Transport

For proteins to regulate gene expression, they must gain access to the nucleus. Proteins are targetted to the nucleus through the action of intrinsic nuclear targeting signals and a family of transport proteins known as importins. While the nuclear import process has been well characterised for a range of nuclear proteins, surprisingly little is known, especially in quantitative terms, regarding the pathways by which transcription factors localise in the nucleus. Since the transport mechanism by which proteins gain access to the nucleus relates integrally to the regulation of their function and thereby many cellular responses, the nuclear import pathway of transcription factors is of intrinsic interest and importance, and a focus of my research.

My research interests include the characterisation of the nuclear import pathways for a range of nuclear DNA-binding proteins including; the cAMP response element binding protein (CREB), AP-1 (activating protein-1), SRY (sex determining region Y), and TRF1 (the telomere release factor-1). Our research has so far demonstrated that these proteins are targetted to the nucleus by a distinct nuclear import pathway independent of importin alpha. These proteins interact directly with importin beta with nanomolar affinity, and thus bypassing possible regulatory interactions such as phosphorylation and nuclear localisation signal masking.

The ability of CREB, AP-1, SRY, and TRF1 to be used as gene transfer vehicles by binding DNA specifically and importing them to the nucleus is an import application of nuclear transport that we are exploring. We have shown already that CREB, AP-1 and TRF1, are suitable gene-transfer vehicles, while SRY binding to DNA and importin beta is mutually exclusive and therefore a poor choice for targetting DNA to the nucleus.

More recently, a research focus has been to understand the detailed mechanism by which nucleocytoplasmic transport is mediated. We have characterised the structures of various import molecules and complexes by x-ray crystallography and other protein-protein interaction techniques. These results are in the process of being published. Please contact me if you would like further details or to know future research projects in the lab.

Fatty Acid Metabolism

An important enzyme involved in fatty acid metabolism in humans is acyl-CoA thioesterase 7 (Acot7). This enzyme catalyzes the hydrolysis of fatty acyl-coenzyme A (CoA) ester molecules to CoA and free fatty acid and therefore able to modulate the cellular levels of activated fatty acids (acyl-CoAs), free fatty acids, and CoA.  and in turn regulate lipid metabolism and other intracellular processes that depend on such molecules. Acyl-CoAs regulate diverse proteins such as protein kinase C, ATP-dependent K+ channels, nuclear receptor HNF-4α, the PPARα and PPARγ families of nuclear receptors and transcription factors in E. coli.

Our latest research has determined the crystal structures of the N- and C-terminal thioesterase domains of mouse Acot7 and the structure of the full-length enzyme based on distance constraints obtained through chemical crosslinking. We show that both thioesterase domains are required for activity, but that only one of two active potential sites is functional, and identify the active site residues through mutagenesis. Importantly, we demonstrate that Acot7 has high specificity for arachidonoyl-CoA, an important precursor molecule for pro-inflammatory eicosanoids, and that Acot7 gene is highly expressed in macrophages and upregulated by pro-inflammatory factors. Furthermore, we show over-expression of Acot7 in macrophages alters the production of prostaglandins D2 and E2. These results suggest a role of Acot7 in eicosanoid synthesis and inflammation. This work has been protected by a patent due its exciting role in inflammation, and we are continuing to elucidate the function of Acot7. If you would like further information on these projects, please contact me.

Genetic Sex Reversal

In 355 BC, Aristotle suggested that the difference between the two sexes was due to the heat of semen during copulation: hot semen producing males, cold semen producing females. Our knowledge regarding the molecular basis of sex determination has come along way in the last 2000 years. We now know that in humans and most other mammals, sex determination is a genetically mediated process. For example, females possess the same sex chromosomes (XX), while males contain two distinct sex chromosomes (XY).

The sex chromosomes coordinate sex determination by allowing female development to progress unless diverted by genes located on the Y-chromosome. The single, most important gene located on the Y chromosome involved in testis development is SRY (sex determining region Y), whose presence triggers the bipotential gonad to enter the male pathway. This gene, located on Yp11.3, encodes a transcription factor that regulates downstream genes in the testicular differentiation cascade. Thus, SRY acts as a genetic switch at around six weeks in development to divert primordial gonads from the ovarian pathway toward male differentiation to form testes.

SRY is a transcription factor containing a single high mobility group (HMG) box located centrally within the protein. The HMG box, conserved across the HMG-1/HMG-2 family, consists of around 80 residues and binds DNA within the minor groove. Although HMG-1 and HMG-2 bind DNA with little or no sequence specificity, SRY has been reported to bind specifically to an eight-base pair recognition sequence within the MIS promoter. By binding DNA, SRY induces a large conformational change through helix unwinding, minor groove expansion, and DNA bending. This is believed to be the molecular mechanism that allows distantly bound proteins of the transcription machinery to attain close proximity, thereby permitting interaction in a way that can influence transcription.

For SRY to act as a genetic switch in sex determination, the protein needs to be specifically targeted to the nucleus where it can gain access to DNA. This localisation of proteins to the nucleus generally requires the presence of a nuclear localisation signal (NLS). NLSs are recognised by a family of transporters or cytosolic receptor proteins known as importins (or karyopherins), which work in concert with the guanine nucleotide-binding protein Ran and other regulatory proteins to transport proteins from the cytoplasm to the nucleus. For example, the NLS of many transcription factors are recognised by importin-beta, which mediates docking to the nuclear pore complex (NPC) and energy-dependent translocation into the nucleus. The process is finally terminated in the nucleus where RanGTP dissociates the NLS-Importin-alpha complex and importin-beta is recycled back to the cytoplasm for a further round of transport.

SRY contains two distinct nuclear localisation signals, while the majority of nuclear-localised proteins contain only a single NLS. These NLSs flank the HMG box and consist of a bipartite NLS (KRPMNAFIVWSRDQRRK77) at the N-terminus, and a monopartite NLS (RPRRKAK136) at the C-terminus. Both NLSs have shown to act independently and are able to target beta-galactosidase to the nucleus. While the precise functionality of containing dual NLSs within SRY remains unknown, effective delivery to the nucleus is dependent on both NLSs being operational: mutations in either NLS results in reduced nuclear import and genetic sex reversal (described below). Interestingly, the NLSs of SRY mediate nuclear import via distinct import pathways; the N-terminal NLS mediates translocation independent of importins and is regulated by calmodulin, while the C-terminal NLS mediates nuclear import by binding directly to importin-beta. While the precise cellular significance of employing two distinct nuclear import pathways for transporting SRY remains unclear, it possibly allows the levels of nuclear transport to be finely regulated.

The importance of SRY in sex determination has been most extensively documented in genetic reversal. For example, individuals with a male genotype (XY) but female phenotype contain either a deletion in the SRY gene, or specific point mutations that causes aberrations in function. Conversely, individuals containing a female genotype (XX) but develop as males contain a copy of SRY gene in one or both X chromosomes, moved there by chromosomal translocation. Moreover, genetically XX mice have been engineered to contain the SRY gene and these mice are phenotypically male. Projects focused on understanding the molecular basis of genetic sex reversal are currently on offer. Please contact me for details.