Mutations in the gene encoding Aprataxin are the second leading cause of an early onset hereditary ataxia termed ataxia-oculomotor apraxia. Early onset ataxias are progressive, neurological disorders, with the patients losing balance and motor coordination in their hands and legs, and suffering from other symptoms such as controlling ocular movements.
More seriously, patients usually have immune system abnormalities and are very sensitive to the effects of radiation treatments. In the United States, where antibiotics usually control recurrent infections typical of the disorder, patients are at high risk of developing and dying of cancer, particularly leukaemia's and lymphomas.
In this study, the researchers purified human Aprataxin and every disease-associated mutant form of Aprataxin and measured the ability of these proteins to function as AMP-lysine hydrolases.
Though the model substrate may not have all of the features Aprataxin is looking for in a substrate inside the cell, the authors showed that wild-type Aprataxin possessed AMP-lysine hydrolase activity that depends on its Hint active site and that all disease-associated mutant forms of Aprataxin reduced or eliminated this activity. The next step, according to Dr Seidle, is to identify the protein targets in vivo.
Dr Charles Brenner, associate professor of genetics and of biochemistry at Dartmouth Medical School recognised Aprataxin as having a protein domain related to "Hint," an enzyme they previously characterised.
A large number of proteins function by modifying the structures of other proteins. Hint is an AMP-lysine hydrolase, meaning that it has the ability to remove a nucleotide modification, typically AMP, from a lysine sidechain.
"As with many diseases for which genes were identified by positional cloning, one begins with insufficient information about the encoded protein that would allow one to formulate a disease hypothesis, let alone develop potential therapeutic strategies," said lead author Brenner.
"By identifying an enzymatic activity of Aprataxin, we were able to formulate the disease hypothesis that Aprataxin activity on protein substrates in the developing brain is required for normal neurological development," he added.
By establishing that Aprataxin has an enzymatic activity, Brenner said, researchers can focus attention on potential Aprataxin target proteins that might be regulated by this gene.
"Though we don't think we can reverse the disease by putting the Aprataxin gene back in, we think we might be able to improve the functions of target proteins once we understand their roles and the consequences of their regulation by Aprataxin," said Brenner.
"In this way, the enzymatic activity of Aprataxin takes us to Aprataxin target proteins and potential therapeutic strategies," he added.
In earlier work, Brenner and co-workers developed a synthetic chemical substrate that allowed Hint to produce a strong fluorescent signal when it did its job (AMP-lysine hydrolysis) on a model compound.
The study appears in the June 3, 2005 issue of the Journal of Biological Chemistry (JBC) as Paper of the Week.