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Targeting Proteins to the Nucleus: Development, Viral Disease and Drug Delivery

JANS LABORATORY

Link to Prof David A Jans' Personal Webpage

Animal and plant cells differ from bacteria in that they are compartmentalised. Rather than being a simple "bag of enzymes" where chemical reactions take place rather haphazardly, animal and plant cells are partitioned into highly specialised membrane-bound structures called organelles, such as the nucleus or mitochondrion, which carry out specific functions largely in isolation from the rest of the cell. The cell requires specific "address systems" to target the specific molecules that are required in these organelles to their correct site, and this involves targeting signals and transport systems that recognise them.

We are interested in the nucleus because it is where the cellular DNA is located, and where the very important process of copying DNA into mRNA or transcription takes place (see schematic of illustration). Because protein synthesis occurs out of the nucleus in the cytoplasm, proteins which are required in the nucleus such as those regulating transcription, need to be specifically transported from the cytoplasm into the nucleus. Generally speaking, these proteins require specific targeting signals called nuclear localisation sequences (NLSs) in order to be able to interact with the cellular nuclear transport machinery, and subsequently localise in the nucleus. Specific proteins, the importins or karyopherins (the NLS "receptors"), recognise the NLSs, and mediate "docking" at the nuclear pore followed by interaction with other cellular factors to effect energy-dependent translocation through the pore and into the nucleus. The regulation of nuclear import of proteins such as those controlling transcription (transcription factors - TFs) or growth (eg. cancer-related proteins or "oncogene" products) is central to important cellular processes such as differentiation and oncogenesis (cancer).

Among other techniques, we use a high resolution digital imaging approach called confocal laser scanning microscopy to analyse transport at the level of single cells. The importance of nuclear import to eukaryotic ( cells with nuclei) cell function has led us to attempt to examine the mechanisms by which nuclear protein import is regulated. We have demonstrated that nuclear transport is not only dependent on targeting signals (i.e. NLSs), but also can be regulated by phosphorylation (the chemical bonding of phosphate groups to proteins, carried out by protein kinase enzymes in the cell).

We and others have shown that specific phosphorylation sites can enhance or inhibit NLS-dependent nuclear transport of TFs. Hormonal/growth factor/cytokine signals modulate gene expression through regulating phosphorylation at such sites, thereby specifically controlling the nuclear entry of TFs or other signalling molecules such as protein kinases. The real challenge is to demonstrate how this may work in the context of the whole cell, which harbours a myriad of other nuclear transport substrates and pathways competing with one another for access to the nucleus. Clearly in this context, a mechanism to effect high affinity interaction with the nuclear import machinery may be critical in the face of the imposing volume of proteins and other molecules required in large amounts in the nucleus such as ribosomal subunits, histones, and RNA binding proteins.

We have more recently been examining the nuclear import of proteins from the causative agents of auto-immune deficiency syndrome (AIDS) - the HIV-1 virus - and Dengue fever (Dengue virus), which is of significance in tropical Australia. We have found that certain viral proteins localise in the nucleus as part of the viral infectious cycle, and that they do so through importin-independent pathways that are quite distinct from those used by normal cellular proteins i.e. viruses may use additional mechanisms to access the nucleus. If our observations prove correct, and we are able to understand how these viral proteins localise in the nucleus, we should be able to devise new therapeutic strategies to block the viral nuclear import pathways, and thereby block viral infection.

Understanding of the mechanisms regulating nuclear protein import enables their application in targeting therapeutic molecules to the nucleus. In the latter case, efficient and tightly regulated nuclear uptake of DNA will be very useful in gene therapy applications or alternatively, toxins can be efficiently targeted to the nucleus of tumour cells in cancer therapy applications. We are currently developing strategies using modular conjugate molecules containing modified NLSs with these applications in mind.

An AIDS-virus protein accumulating in the nucleus or at the nuclear envelope of hepatic cells. The HIV-1 Vpr protein (red) accumulates in the nucleus of cells under normal conditions (A), but in the absence of nuclear transport components provided by the cell only accumulates at the nuclear envelope (B). The cell nucleus, nucleolus (nuclear subcompartment from which Vpr is excluded), cytoplasm, and nuclear envelope are indicated. On the left is shown a schematic of a cell showing the nucleus containing the DNA and site of transcription, and some other subcompartments.