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. Because protein synthesis occurs out of the nucleus in the cytoplasm, proteins that 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 (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).
We have more recently been examining the nuclear import of proteins from the causative agent of Dengue fever (Dengue virus), upper respiratory tract diseases (Respiratory Syncytial virus and Rhinovirus) and Rabies (Rabies Virus). We have found that certain viral proteins localise in the nucleus as part of the viral infectious cycle, and that they do so through both importin-dependent and 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.
Dr Leon Caly
Pathogenic Mechanisms of Respiratory Viruses
Viruses often appropriate and/or disrupt regulatory mechanisms of the infected host cell to ensure the appropriate localization of viral proteins and mislocalization of cellular proteins, with consequent increased virus replication/pathogenesis and inhibited host antiviral response. We are examining two respiratory viruses of medical importance, respiratory syncytial virus (RSV) and rhinovirus (RV) as models to study the importance of regulated protein subcellular localization in pathogenesis. The ultimate aim is to exploit this basic information to identify new targets for development of urgently needed antivirals or to assist in generating attenuated virus for vaccines.
|RSV budding out of an infected cell. Vero cells were infected with RSV, fixed 24h later, probed for RSV proteins by immunofluorescence followed by confocal microscopy.||RV replication complexes in an infected primary lung cell. Primary cells were infected with RV, fixed 6h later, probed for dsRNA by immunofluorescence and confocal microscopy|
Respiratory syncytial virus (RSV) is the chief cause of viral pneumonia in infants worldwide and an important lower respiratory pathogen in the elderly. Through its ability to shuttle into and out of the nucleus, the matrix (M) protein is believed to play a role in pathogenesis. M's role in the cytoplasm is to facilitate virus assembly, whereas its role in the nucleus may be to inhibit host transcription; localisation is regulated by phosphorylation and interactions with the cytoskeleton. Current research is aimed at understanding the importance of regulated nucleocytoplasmic transport of M in virus infection and disease.
Rhinovirus (RV) is responsible for 70% of virus-induced asthma exacerbations, accounting for up to 16 deaths/week in Australia alone, where as many as 30% of Australians are believed to suffer asthma. RV infection leads to degradation of key components of the cellular nuclear transport machinery, transcription factors and regulators of apoptosis. We have shown that RV 3C protease, essential for RV replication, is localised to the nuclei of infected cells, resulting in degradation of components of the nuclear pore complex the key structure regulating transport into and out of the nucleus. Current research is aimed at investigating the nucleocytoplasmic transport of 3C protease with a view to understanding its role in RV induced asthma exacerbations.
Dr Kylie Wagstaff
Drug/DNA delivery to the nucleus
Gene therapy, the expression in cells of therapeutic DNA to replace faulty genes, has long been awaited to treat numerous debilitating inherited diseases including many cancers. The reality, however has been somewhat disappointing, with most gene therapy delivery agents (vectors) relying on viruses to deliver the therapeutic DNA, resulting in numerous safety complications including insertional mutagenesis and unwanted immunological responses. Non-viral based vectors, while safer are very inefficient due to the poor ability to deliver DNA into the nucleus of the cell. In fact <1% of DNA taken up by a cell ever reaches the nucleus to be expressed. Transport into and out of the nucleus depends on facilitated passage through the nuclear pores within the double membrane (nuclear envelope) that defines the nuclear compartment. We have begun developing a range of non-viral modular DNA carriers that mimic viruses in their ability to enter cells, transport through the cytoplasm and deliver DNA to the nucleus. These modular DNA carriers can be used for safe non-viral gene therapy approaches, which is of even greater importance due to the recent suspension of viral based gene therapy trials in the US due to fatalities in trials using viral vectors. Current projects use novel modular protein molecules that combine novel cell entry sequences with modules mediating DNA association and nuclear import to enhance nuclear delivery of therapeutic agents, such as drugs/DNA, for application in gene therapy and anti-cancer therapy.
In this context, we have recently identified a novel tumor-enhanced nuclear targeting signal (tNTS) from a chicken anemia virus protein, which localizes strongly in the nucleus of tumour cells but not in the nucleus in non-tumour cells. Apart from examining the detailed mechanisms behind the function of the tNTS, current projects aim to derive novel tumour specific nuclear targeting signals and to exploit both these and the tNTS to enhance the delivery of nuclear acting drugs/DNA by incorporating them into our modular DNA delivery systems, along with other tumour-enhanced modules with an aim towards the development of truly tumour specific anti-cancer therapies.
To aid in the development of these systems, novel technology platforms are being developed to complement/enhance the ongoing work in these fields, including high-throughput protein-protein interaction assays, multi-colour FACS based analysis techniques and microarray based systems for analyzing the efficiency of DNA/drug delivery to cells.
Regulation of Nuclear Import
Negative regulators of nuclear import (NRNIs) are novel molecules we have recently identified that inhibit IMP-dependent nuclear import, and thereby influence processes such as oncogenesis/apoptosis in addition to development. The BRCA1 binding-protein, BRAP2, is one example, being highly expressed in the testis and able to bind NLS-containing proteins to inhibit their nuclear import. An exciting project intends to examine the role of BRAP2 and other NRNIs in spermatogenesis, in particular looking for their binding partners in the testis, some of which have already been identified. Understanding how NRNIs regulate the process of spermatogenesis may enable the programming/reprogramming of germ cells in vitro, through modulation of the balance of IMPs/NRNIs and hence TF nuclear entry.
- Wagstaff, K.M., Glover, D.J., Tremethick, D.J. and Jans, D.A. (2007) Histone mediated transduction as an efficient means for gene delivery. Molecular Therapy 15 (4), 721-731
- Wagstaff, K.M., Fan, J.Y., De Jesus, M.A., Tremethick, D.J. and Jans, D.A. Chromatin reconstituted with engineered histones as an efficient means for gene delivery. FASEB Journal 22(7), 2232-2242
- Glover DJ, Ng SM, Mechler A, Martin LL, Jans DA. FASEB J. 2009 Sep;23(9):2996-3006
Techniques for the above projects include confocal laser scanning microscopy (CLSM), fluorescence recovery after photobleaching, protein engineering, site-directed mutagenesis, in vitro nuclear transport assays, protein-protein interaction assays, FACS analysis, virological/immunochemical techniques, gene reporter assays, pull down assays and mass spectrophotometry (MS), and the yeast 2-hybrid system.
Jans Laboratory Members
Head of Laboratory Professor David A. Jans
|Hong-Mei Li||Leon Caly||Johanna Dean|