- Welcome Message
- Latest News
- Contact Us
- Child Health Research PhD Programme
- Teaching and Learning at ICH
- Vacation studentships
- Bill Marshall Fellowships
- Statistical courses
- ICH Intranet
- ICH News
The detamination of the anabolism and catabolism of ceramide trihexoside in Fabry Disease: The search for the elusive deacylase
Supervisors: Dr Wendy Heywood, Dr Simon Eaton, Professor Simon Heales and Dr Kevin Mills
The lysosome is the recycling organelle of the cell, whereby larger macro-molecules are broken down by a pathway of specific and sequential enzymes and then recycled to make new compounds. Fabry Disease is a lysosomal storage disease (LSD) and is caused by a deficiency of the lysosomal enzyme-galactosidase. This enzyme removes a single galactose residue from the molecule ceramide trihexoside (CTH). However, a deficiency of this enzyme leads to a build-up of CTH in the lysosome because it cannot be degraded, the lysosome begins to swell and CTH starts to ‘leak’ out into the circulation. This build-up of CTH in the body results in patients dying of stroke, cardiac arrest or kidney failure in the fourth decade of life (1).
However, in addition to CTH, there is also a build-up of a truncated or deacylated form of CTH called lyso-CTH. Lyso-CTH has also been shown to correlate better with disease progression and treatment than CTH levels. In addition, it has also been demonstrated to be significantly more potent in the damage of organ systems than CTH. However, scientists have no idea where lyso-CTH originates from, how it causes the cellular damage and why lyso-CTH only affects certain organs. We aim to use cutting edge proteomic and metabolomic technology to study this phenomenon in the hope of identifying the disease mechanism and the elusive enzyme responsible for creating lyso-CTH molecule. This would allow the development of compounds to target the enzyme responsible thereby creating an additional therapy for Fabry disease.
Aims and methods:
Where does lyso-CTH originate from?
Our laboratory have synthesised deuterated CTH molecules which are exactly the same as the endogenous CTH found in the body but ‘weigh’ more in the mass spectrometer . Using this difference in molecular weight between the synthetic and endogenous compounds, we can use the deuterated CTH as a metabolic tracer to see how CTH is metabolised in the body. Using patient fibroblast cell lines and/or cardiac/kidney/endothelial cell culture models of Fabry disease, we will incubate these cells with deuterated-CTH and observe how CTH is metabolised in the hope of identifying or detecting the elusive deacylase. Although these experiments will monitor the catabolism of CTH there is increasing evidence that the salvage mechanism of sphingolipid metabolism may also be involved in the production of lyso-CTH . Therefore, we will also incubate cell lines with deuterated fatty acid molecules of various lengths and monitor their incorporation into CTH, thus monitoring both the anabolism and catabolism of CTH in the cell. Using a combination of classical and state-of-the-art techniques such as cellular models, subcellular fractionation of organelles, enzymology and mass spectrometry, the student will gain full training and transferable skills during the course of this research.
Why and how do high levels CTH and lyso-CTH lead to stroke, cardiac disease and kidney failure?
The targeted and hypothesis driven analyses described above will also be augmented with hypothesis generating omic analyses carried out in the same experiment and even on the same sample. The purification of the subcellular organelles from the kidney, cardiomyocyte and endothelium cell culture experiments will not only allow us to monitor the metabolism of CTH, but also allow us to monitor which of these organelles are more susceptible to CTH incorporation / uptake. In addition, these metabolomic analyses will require precipitation of the protein content of the organelles. These protein fractions can then be used for proteomic analyses such as the mass spectrometry based method of label- free quantitation to study what effect that high levels of CTH/lyso-CTH have on protein expression and hence giving us insights into the disease mechanisms of Fabry disease which are still not understood.
1) Zarate YA, Hopkin RJ. Fabry's disease. Lancet 2008 Oct 18;372(9647):1427-35.
2) Mills K, Johnson A, Winchester B. Synthesis of novel internal standards for the quantitative determination of plasma ceramide trihexoside in Fabry disease by tandem mass spectrometry. FEBS
3) Mullen TD, Hannun YA, Obeid LM. Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J 2012 Feb 1;441(3):789-802.