Oxford University Department of Biochemistry
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James Allen
Unusual mitochondrial protein assembly and biochemistry in the African sleeping sickness parasite

Co-workers: Katharine Sam, Elena Nomerotskaia

The parasites Trypanosoma brucei (causative agent of African sleeping sickness), Trypanosoma cruzi (Chagas disease) and pathogenic Leishmania species are a major source of tropical diseases. We are interested in the unusual mitochondrial biochemistry of these organisms.

C-type cytochromes are essential proteins for respiration in most organisms. They contain the iron cofactor heme, covalently bound to the protein in a post-translational modification that requires dedicated accessory proteins. Remarkably, the three known heme-attachment systems are all absent from trypanosomes. Furthermore, the trypanosome mitochondrial c-type cytochromes are structurally unique – the heme is attached to a single cysteine residue (an XXXCH motif), rather than to two cysteines (CXXCH) as in all other organisms. Together, this is persuasive evidence that biogenesis of the trypanosome c-type cytochromes requires a novel heme-attachment apparatus. Our key research questions are, what is this trypanosome cytochrome c biogenesis system and how does it work?

Disulfide bond oxidizing and reducing proteins are essential for protein import and folding in the mitochondrial intermembrane space (IMS) of animals and yeast, but is that the case in trypanosomes? In yeast, small cysteine rich proteins enter the IMS and are oxidized to form intramolecular disulfide bonds by the disulfide isomerase protein Mia40; this traps the imported protein in the IMS. Mia40 is reoxidised by the sulphydryl oxidase Erv1, which in turn passes electrons into the respiratory chain. However, in trypanosomes there is no homologue of Mia40, and trypanosomes do not always have a functional respiratory chain. We are therefore investigating how cysteine rich proteins are imported into the IMS of trypanosomes.

Together, our observations about cytochrome c and Mia40 suggest that the redox environment of the trypanosomatid mitochondrial IMS may be distinct from that in model eukaryotes such as yeast and animals.

 

 


Publications

Research Images


Figure 1: Trypanosomes require a classical mitochondrial respiratory chain for at least part of their life cycles. We are interested in the biogenesis of cytochromes c and c1 (shown in red), which are essential components of the respiratory chain. In particular, we are investigating the mechanism by which the heme cofactor becomes covalently attached to the polypeptide chain, a mechanism that is clearly novel in, and probably unique to, trypanosomes


Figure 2: Detail showing covalent attachment of heme to a typical mitochondrial cytochrome c. The attachment is through two thioether bonds between cysteines in the protein (yellow) and the heme (red). The cysteines occur in a characteristic Cys-Xxx-Xxx-Cys-His motif, where the histidine (green) is a ligand to the heme iron atom (brown). Uniquely, trypanosomatid cytochromes c lack the first cysteine of the CXXCH motif and heme is attached through only one thioether bond to an XXXCH motif


Figure 3: The structure of a typical mitochondrial cytochrome c


Figure 4: Cytochromes are brightly coloured and absorption spectroscopy provides a powerful and sensitive method for investigating heme binding and attachment to the polypeptide. This figure shows the spectrum of a typical mitochondrial cytochrome c (blue), where the heme is attached through two thioether bonds to the polypeptide, in comparison with the spectrum of a trypanosomatid cytochrome c (red) that has single cysteine attachment. The inset shows the pyridine hemochrome spectrum of the same proteins – this analytical method provides, through the positions of the peak maxima, diagnostic information on the nature of the heme attachment

Contact: james.allen@bioch.ox.ac.uk

Graduate Student and Postdoctoral Positions: Enquiries with CV welcome


DPhil studentship projects in my lab

A DPhil student would be integrated quickly into one of the research projects above, and would play a major role in the progression of this laboratory’s work. Enquiries from potential students are welcome. The techniques we use include microbiological culture, protein expression and purification, bacterial and eukaryotic molecular biology, protein chemistry, mass spectrometry, absorption and other spectroscopies, bioinformatics, crystallography and enzymology.

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