Re-awakening ancestral genes…

Research bloggingHUMANS, as we know, are the product of tens of thousands of genes, but hidden elsewhere in your DNA are genes that are no longer functional; these vestigial genes are known as pseudogenes, and they are ancestral remnants from an earlier point in our evolution. In many cases they are simply inactivated duplicates of a current functional gene. In other cases they are genes that have been cut out, reversed and stitched back in; in this position, some believe they may act to regulate the correctly oriented ‘functional’ version of the gene. Alternatively, they may be ancestral genes encoding functions that have become inactive beacuse they are ultimately not necessary for survival. Now, what if we could turn on one of these ancestral genes? One that could actually help protect us from a modern day infection?

In the recent edition of PLoS Biology is an interesting study that describes the re-activation of just such a dormant human pseudogene, retrocyclin, and its potential use as a defensive barrier against infection with HIV-1 (a strain of the Human Immunodeficiency Virus that causes AIDS). Retrocyclin is theta-defensin, which are naturally produced, circular chains of 18 amino acids (a peptide). I have previously research blogged about the application of other such antimicrobial peptides.

Active, functional theta-defensins have only so far been identified in the old world monkeys: the Rhesus Macaque and Olive Baboon; in Humans and other primates, they exist as pseudogenes. At some point in evolutionary history, our ancestors started inheriting a genetic mutation, all be it one that exists at 100%. The Human version of the gene, retrocyclin, is inactive in Humans because of a premature ‘stop’ signal, which makes the cell abandon the production of the peptide too early.

Retrocyclin can be synthesised chemically in a lab, and in this manner that the authors of this paper (from laboratories at the University of Central Florida and UCLA) have previously shown that it is capable of inactivating HIV-1, thereby preventing its entry into cells; in fact, they have also shown that it can similarly prevent entry of Herpes Simplex Virus type I (responsible for coldsores) and type II (responsible for genital warts).

The study focussed on two objectives:

Firstly, whilst the group has shown that synthetic retrocyclin can inhibit HIV-1 entry into test cells, they wanted to demonstrate that a retrocyclin gene that had been ‘corrected’,  by removal of the early ‘stop’ signal, could be produced and processed in human cells. As the gene has been inactive for so long, it may not be enough to just introduce a corrected gene; human cells may not have retained the ability to process the peptide properly once it is produced.

So, the authors inserted various corrected forms of the retrocyclin into a human cell line, which is a standard means of growing a studying human cells – outside of the body and in a controlled environment. After some growth, any peptides produced were extracted and this extract used to see if it effected the ability of HIV-1 to infect a different human cell line. The extract from one of the corrected retrocyclins did in fact show a significant decrease in the number of cells containing virus, compared with a control extract from cells that never received the corrected retrocyclin. Importantly, they performed additional rigorous experiments to ensure that the expected retrocyclin peptide was in fact present in the extracts that had shown defensive activity against HIV-1, concluding that correctly folded retrocyclin peptides can be expressed by human cells.

Secondly, the authors look at means to get human cells to produce their own retrocyclin naturally, without the addition of corrected DNA. The authors turned to the interesting phenomenon wherein a particular class of antibiotics called aminoglycosides, typically used against bacteria, has been shown to suppress premature ‘stop’ signals in human cells; the same problem effecting retrocyclin.

[In fact, a study was reported some years ago in the New England Journal of Medicine, which described the use of one such aminoglycoside, gentamicin, to treat cystic fibrosis. In 10% of sufferers, the mutation is caused by a premature ‘stop’ signal. Treatment with gentamicin was effective on a sub-set of patients, who fulfilled certain genetic criteria, correcting the abnormalities caused by the disease.]

The authors test three antibiotics from the aminoglycoside class to see if they overcame the premature ‘stop’ signal. Now, it is not enough to just add the antibiotic to the cells and see what happens, as it’s necessary to make sure that the antibiotic is doing what it is expected to do, namely, allow the production of the peptide. In order to visualise this, you can fuse your gene of interest, i.e. the retrocyclin, with another gene that acts as a ‘reporter protein’. The reporter protein is typically something that is easy to detect; in this case it was a protein called luciferase, an aptly named protein that produces bioilluminescence (responsible for Firefly glowing). Along side your gene of study, you use a control gene that you know is produced well, giving a strong ‘glowing’ result. Using the antibiotic tobramycin, a 26-fold increase in ‘reading’ of the retrocyclin was possible.

The next step is to see if the optimised tobramycin concentration works on the native retrocyclin pseudogene, as found in human cells, restoring anti-HIV-1 activity. The authors found that treated cells inhibited HIV-1 infection, compared to untreated cells. They then tested the tobramycin on a more complex 3D tissue model, called an organotypic model, which is a more medically relevant system of looking at responses of tissues to treatments, though not as good as doing it humans themselves. This model consisted of cervicovaginal skin cells, an obvious target for looking at preventing HIV-1 infection.

Application of tobramycin to this tissue resulted in the production of retrocyclin and, usefully, was not toxic, nor did it adversely effect the metabolism of the cells. HIV-1 invasion studies of the tissue were not addressed, but the concentration of retrocyclin indicated that it would be sufficient to represent a defensive mechanism against HIV-1.

Caveats

It is anticipated that a treatment of this manner would be a topical treatment, such as a cream, but I would raises several questions of application. How, and under what circumstances, would one expect to use such a treatment? Would it be an addition to the coating on contraceptives such as condoms for example? The use of antibiotics is well known to adversely effect the vaginal bacterial flora, which is necessary to maintain the health of the tissue. Fungal infections such as Candida albicans (Thrush) are common following treatment with antibiotics.

Furthermore, persistent exposure of bacteria to antibiotics does no one any favours, expect for perhaps bacteria. It is been shown that low concentrations of aminoglycosides actually stimulate bacteria in many ways, increasing their mutational frequency, increasing production of other bacterial genes, in other words, giving them a metabolic kick up the arse [nice review about this here].

Indeed, one could raise the question of why such an apparently useful defensin became inactive in Human ancestry. On an evolutionary basis, which works along the lines of ‘just good enough‘, clearly such a defensin would only continue to be maintained in its functional state if a mutation of the gene resulted in individuals who did not reach a sufficient age to procreate and raise their young. However, perhaps the gene is genetically linked with another gene, or genes, that resulted in a fitness cost to individuals, and it was the selection against such gene(s) that drove retrocyclin into pseudogenery.

As ever, such scientific studies raise more questions than they answer, but it is none the less an interesting study. Indeed, it may certainly be useful to identify other pseudogenes that may be effected by aminoglycoside antibiotics. There is a lot of further work to do, and as the authors state, the safety and effectiveness of this approach in actual humans has yet to be assessed.

Venkataraman, N., Cole, A., Ruchala, P., Waring, A., Lehrer, R., Stuchlik, O., Pohl, J., & Cole, A. (2009). Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1 PLoS Biology, 7 (4) DOI: 10.1371/journal.pbio.1000095

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