Calcium Signalling and Related Signal Transduction Systems in Normal and Cancer Cells: Structure to Function
Multicellular communication is governed by numerous and complex signalling systems. In mammals, tissue formation and maintenance requires strict controls on cell division, differentiation and growth: all processes which depend upon signal transduction. In cancer, cells escape from these normal controls, and proceed along a path of uncontrolled growth and migration.
Our laboratory is interested in determining how the molecular network governing signal transduction processes behave and interact in living systems, and exactly what happens, at an atomic/molecular level when signalling molecules are mutated or damaged.
To this end, we have been investigating structure-function relationships of signalling proteins by means of biochemical and biophysical methods, including nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and fluorescence resonance energy transfer (FRET) imaging microscopy.
More specifically, these methods enable us to determine the three-dimensional atomic structures of proteins and protein complexes (NMR, X-ray) as well as their dynamic behaviors in living cells (FRET). Currently our research focus has been on proteins involved in Ca2+ signalling, cell adhesion, transcriptional regulation, and bacterial signal transduction. Such molecular studies will contribute to our understanding of human afflictions including cancer, neurological disorders, heart disorders and infectious diseases. Some specific studies of this laboratory are outlined below.
Ca2+ signalling (supported by CIHR and HSF):
Signalling systems translate external signals such as hormones, growth factors and neurotransmitters into intracellular second messengers. These messengers can then serve a number of functions by turning on or off specific pathways. Two important second messengers are Ca2+ and inositol 1,4,5-trisphosphate (IP3).
The concentration of Ca2+ in the cell is tightly regulated by various ion transporters. To understand how the Ca2+ signal is created, we need to investigate the mechanism of Ca2+ transportation in the cell. We are currently studying two major Ca2+ transporters, IP3 receptors and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). The former has been implicated in neurological disorders, such as cerebellar ataxia and epileptic seizures, and the latter is implicated in heart diseases.
While IP3 receptors act as a Ca2+ release channel on internal stores within the endoplasmic reticulum (ER), SERCA is a Ca2+ pump which translocates cytoplasmic Ca2+ into the internal stores. Our current work focuses on the structural determination of the cytoplasmic ligand binding portions of IP3 receptors and SERCA by X-ray crystallography or NMR spectroscopy.
In response to a Ca2+ increase in the cell, calmodulin (CaM), a central member of the Ca2+ signalling network, binds Ca2+ and undergoes a conformational change. CaM acts as an intracellular sensor for the second messenger Ca2+, turning on or off specific pathways by interacting with numerous downstream proteins including serine/threonine protein kinases (CaM kinases I, II, IV, CaM kinase kinase, myosin light chain kinases), protein phosphatases (calcineurin and RDGC), nitric oxide synthetase, NMDA receptor, IP3 receptors, Ca2+ channels, and many others. We are investigating how this ubiquitous Ca2+ sensor protein recognizes these diverse proteins and how exactly CaM regulates the biological activity of the targets. In order to organize and compare numerous sequence and structural information in the calcium research-related literature, we have established the Cellular Calcium Information Server (http://calcium.uhnres.utoronto.ca
), which currently features databases for calmodulin target proteins, the cadherin superfamily and the EF-hand protein superfamily.
Cell adhesion and cytoskeletal regulation (supported by NCIC):
The ability of cells to adhere to each other is fundamental to normal development and morphogenesis of multicellular organisms. Cancer cells often lead to a loss of the adhesion function, resulting in cell spreading from the primary cancer site to other locations in the body. The behaviour of cell adhesion proteins also influences the physiology of the cell, as the cell adhesion system is coupled with intracellular signalling systems and cytoskeletal assembly. These cellular processes together control the shape and motility of cells in a dynamic manner.
In order to understand the normal cell adhesion process and defects which may occur in cancer cells, we are investigating one of the essential cell-cell adhesion molecules, cadherin. We are interested in determining the structural basis of the cadherin-mediated cell-cell interaction and the role of Ca2+ in cell adhesion. The cadherin superfamily is also one of our favourite targets of bioinformatics studies.
Our ultimate goal in these studies is to fully understand the mechanisms underlying normal cell adhesion and cancer metastasis, ultimately leading to the design of therapeutics to eliminate the spread of cancer throughout the body.
Transcriptional regulation (supported by CIHR):
Cancer and many other diseases have a genetic link. Genetic information is encoded in DNA, which is transcribed and translated by complex molecular machineries. Cancer viruses directly target the transcriptional machinery of the host cells.
In human and other eukaryotes, gene transcription is controlled by an array of general transcription factors (GTFs) and RNA polymerases. TFIIB and TFIID are highly ubiquitous from yeast to humans and play crucial roles in transcription initiation and activation. We are interested in how these TGFs mediate transcription and participate in the activation of various genes.
We are also interested in the internal regulatory mechanism of TFIID, involving the TATA-box binding protein (TBP) and TBP-associated factors (TAFs), as well as the TFIIB-mediated transcriptional activation. The adenoviral oncoprotein, E1A, and the herpes simplex virus protein, VP16, both directly interact with TBP or TFIIB to regulate gene transcription activities of various genes. We are investigating the structure and dynamics/kinetics of the interaction of these viral transcription regulators with the GTFs. We hope to understand the molecular actions of such foreign gene products in host cells and to prevent such unwanted invasions.
Bacterial signal transduction (supported by CIHR):
The aforementioned signal transduction and gene expression systems are intimately coupled in bacterial signal transduction. Bacteria provide a simple but elegant means to study the relationship between the two cellular systems. The His-Asp phosphorelay system, or two-component system, for example, contains a sensor histidine kinase and a response regulator acting as a transcription factor. This signal transduction system is central in many prokaryotic organisms playing an essential role in some pathogenic bacterial strains, such as salmonella. For these reasons, the His-Asp phosphorelay system is a good target for antibiotic development.
We are also currently investigating the structure and mechanism of the EnvZ-OmpR two component system which severs as an osmosensor in E. coli. The histidine autophosphorylation of EnvZ and the phosphotransfer from EnvZ to an aspartate in OmpR is presently being studied by NMR and X-ray in combination with various biochemical techniques.
Fluorescence Resonance Energy Transfer (FRET) imaging(supported by CRS):
More recently, our laboratory has been involved in protein engineering and in vivo fluorescence imaging studies. Taking advantage of the existing 3D structural knowledge of various proteins, we have engineered the green fluorescent protein-based calcium indicator cameleons (GFP) and applied fluorescence resonance energy transfer (FRET) imaging microscopy to monitor dynamic changes in concentration and localization of cellular Ca2+.
In addition, we have designed a new cameleon molecule that possesses preferred fluorescence properties in terms of in vivo imaging, based on our structure determination of calmodulin (CaM) in complex with CaM-dependent kinase kinase.
We have further applied FRET imaging microscopy to monitor protein-protein interactions of various signalling systems in vivo. The 3D structural information of proteins can also provide useful clues in designing new therapeutic compounds to specifically inhibit proteins activities. An example of such an effort is the development of new antibiotics targeted for bacteria-specific signal transduction systems involving histidine kinases.
In coming years, we hope to determine 3D structures of more protein-protein complexes by NMR or X-ray in order to visualize, via FRET imaging microscopy, specific protein-protein interactions in various cell types, including tumour cells.