Department of Pharmacology

Cell Biology ~ Proteomics & Calcium Biology

Personnel

1. Our Biology
2. Our Research Technologies
3. Independent Publications

 

1. Our Biology

Cell Signalling

The mechanisms used to relay messages within cells are of intense biomedical interest. Complex signalling pathways underlie normal development and health of cells. Many diseases are associated with cell-signalling anomalies. Numerous drugs target the principal signalling effectors, calcium and protein phosphorylation.
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Calcium biology

Calcium has many roles inside and outside cells necessitating strict regulation at different concentrations in various locations. Calcium signals are transmitted through cells as transient increases of calcium which normally is kept at a very low concentration in the cytosol (cellular fluid). Cellular toxicity arises if these calcium elevations are too large or frequent (calcium cytotoxicity). It is clear that excess calcium can lead to cell death but disease-related disruptions of calcium signalling are not well understood.
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Dental enamel cells

We initiated investigations of calcium handling in enamel cells questioning how they make such a highly-calcified product (tooth enamel is 40% calcium) without suffering the cytotoxic effects of excess intracellular calcium. Of broader biomedical value, this research model comprises epithelial cells that have an informatively elongate morphology and undergo functionally-distinct phases of development linked to production of a hypermineralized extracellular matrix. Dentally, these cells are also central to the understanding of enamel mineralization and associated developmental defects; more information

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Current research focus

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2. Our Research Technologies

 

Molecular and cellular biology

We are using a broad range of experimental approaches from the DNA level (eg cDNA cloning, qPCR) through protein (eg 2-D gels, recombinant protein engineering), cellular (eg confocal and transmission electron microscopy) and physiological levels (eg knockout mouse characterisation).

 

Proteomics and protein biochemistry

Our speciality is microscale protein biochemistry, a challenging area necessitated by the scarcity of enamel cells. Approaches include mass spectrometry, Edman analysis, amino acid composition, gel electrophoresis and the purification, biophysical and functional analysis of proteins.

An online database (ToothPrint) of dental proteins in rat has been established.

 

Hard tissue microanalysis

We phenotypically characterise mouse and rat teeth using a variety of approaches including polarised light and scanning electron microscopy, histology, micro-CT and microradiography, microhardness and quantitative fracture analysis.

 

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3. Independent Publications

A. Enamel cell biology – how is bulk calcium handled safely?

Calcium plays numerous key roles in cells from their birth through to their death. Consequently there is much medical interest in manipulating calcium-dependent activities, for example to help keep damaged cells alive in neurodegenerative diseases or hastening the death of renegade cells in cancer. Enamel-forming cells hold interest in this regard as a calcium-savvy cell type that handles a lot of calcium (for mineralisation of dental enamel) without succumbing to the potentially cytotoxic effects of excessive intracellular calcium. To learn how enamel cells survive such a calcium onslaught, we developed microscale proteomic approaches and characterised enamel epithelial cells from developing teeth in neonatal rats and mice. This information was used to investigate the mechanistic basis of calcium transport across enamel cells. Our findings contradicted the classical "calcium ferry" dogma and led to development of a new paradigm for transcellular calcium transport that we've named "calcium transcytosis". Increasingly it appears this organelle-based mechanism could be more generally applicable across biology.

B. Enamel defects - can they be prevented?

Developmental defects of enamel are costly to patients and society. Many of these dental defects may become preventable if a better understanding of their causes and pathologies can be gained. Recently we assembled a multidisciplinary team to investigate a common dental defect, termed Molar Hypomineralization, which manifests as soft, porous (chalky) enamel – this congenital defect affects 'six-year-old molars' in over 10% of otherwise healthy children, causing life-long risk of pain, dental caries and perhaps tooth loss in severe cases. Our initial proteomics investigation provided intriguing insights to the nature and possible cause of Molar Hypomineralization, and so suggests novel avenues for basic research and clinical development.

C. Calbindins - what do they do?

Our cells contain numerous proteins thought to bind calcium as their primary role. Many of these calcium-binding proteins play pivotal roles in cell signalling, regulation and structure, making them attractive targets for therapeutic intervention in multiple diseases including cancer and neurodegeneration. However, for such applications to be fully effective, better understanding is needed about the functions of these calcium-binding proteins at individual and collective levels. Calbindins comprise three types of calcium-binding protein (calbindin-28kDa, calbindin-9kDa, calretinin) that have long been regarded as mobile calcium buffers in the cytosol and consequently are widely investigated as potential targets in calcium transport and neurodegeneration. Our investigations have contradicted this view and instead pointed to calbindins having a role that involves interactions with other proteins. These findings, which lead us to contemplate an alternative role in cell signalling, hold fundamental significance for medical targeting of calbindins.

D. ERp29 – what does it do?

The Endoplasmic Reticulum (ER) plays several critical roles in cell biology including the production of secretory proteins and the safe storage of calcium inside cells. Numerous proteins reside in the ER and so hold medical importance as potential therapeutic targets for the many diseases associated with ER dysfunction (e.g. cystic fibrosis, type 2 diabetes, cancer). However, much needs to be learned about these ER residents both in terms of their individual functions and how they work together as the 'ER machinery'. We discovered ERp29 during proteomic analysis of rat enamel cells, leading to the naming and first description of this ubiquitous ER resident in 1997. The challenge since has been to figure out the functional role of ERp29, bioinformatics having offered only limited insight. In a series of pioneering studies, we've gathered a variety of clues that collectively point to a novel "housekeeping" role of general importance, probably as a new type of chaperone. Now linked to a broad array of physiological processes and diseases including cancer, ERp29 holds broad potential as a medical target.

E. Other reports and articles

 

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