Joe Magliocca

The AIDS epidemic has been a pervasive health crisis over the past thirty years.  While the rate of death from the disease has declined due to advances in antiviral drugs, reducing the spread of the virus is still an important goal for researchers. There are many routes for this research, but one of the more unusual directions has involved an attempt to “reawaken” a lost protein coded by the human genome.  The protein is called retrocylin. This is a circular defensin protein with antimicrobial properties.

Defensins are small proteins that were known to have antimicrobial and antiviral properties. The first discovered defensins were linear proteins, chains of amino acids with two ends, an N-terminus and a C-terminus. They are active against many bacteria and some viruses, one of which is HIV. The main weakness that defensins have is that they are inactivated when they are exposed to high salt concentrations. Retrocyclin shares the abilities of the linear defensins. Unfortunately, humans have lost the ability to synthesize this protein because of a nonsense mutation, a mutation that causes protein synthesis to stop before a mature protein is made.  Recent research has led not only to the discovery of retrocyclin, but also to a method for “reawakening” it in some human cells.

How do we even know about retrocyclin if no human cell can produce it?  Our knowledge comes from a similar protein accidently discovered in a close human relative, the rhesus monkey. In the late 1990s a team of researchers from UC Irvine was looking for undiscovered defensins in rhesus white blood cells using HPLC, and they found an unrecognized protein. Normally, when a new protein is discovered, the first thing that is done is sequencing. However, Edman degradation, the most commonly used method for protein sequencing did not work for this new protein. In Edman degradation, a protein sequence is obtained by removing each amino acid individually from the N-terminus. Because this did not work, they concluded that the protein was circular and therefore had no distinct termini. In order to sequence the protein, the team of scientists cut the protein with a low concentration of proteases, enzymes that hydrolyze the peptide bond between amino acids. A low concentration was necessary because they did not want to break down the protein completely, so the procedure resulted in breaking down the protein into fragments that could be sequenced.

After the protein was sequenced, they decided to call it a “theta defensin.”  Its structure looks similar to the Greek letter theta, hence the name. Because this first theta defensin was found in rhesus monkeys, the newly discovered protein was named “rhesus theta defensin one” or RTD-1. RTD-1 was found to have similar antimicrobial properties to other defensins, but it does not lose its activity at higher salt concentration.

A capuchin monkey-(S)he’s sad because as a new world monkey, (s)he has no theta defensin. 

After the discovery of RTD-1, researchers looked for homologous proteins in other primates. Similar proteins were found in other old world monkeys (the monkeys without prehensile tails from Africa, south and southeast Asia, and southern Spain) and in Orangutans. In contrast, no homologue was found in new world monkeys (the monkeys with prehensile tails from Central and South America), gorillas, chimpanzees, or humans.  Because humans are so closely related to old world monkeys, a group from UCLA decided to look for a pseudogene that was similar to RTD-1, and they found the sequence for one (retrocyclin) by looking in a cDNA library from human leukocytes.  Although an early stop codon prevents retrocyclin from being produced naturally, the team was able to learn the sequence, which is similar to that of RTD-1. With the sequence knowledge, the group was able to make a synthetic version of retrocyclin as well as a protein that is identical to retrocyclin with one amino acid substituted, so the protein could be radioactively labeled. The altered version is called RC-101.

A rhesus monkey-(S)he’s happy because as an old world monkey, (s)he has a theta defensin.

The group tested the effects of retrocyclin and RC-101 on bacterial growth and found effects similar to those of RTD-1. After this, because linear defensins are known to also reduce the growth of some viruses, they tested retrocylin’s effects on HIV-growth in two different types of cells that can be infected with HIV. By measuring the amount of an antigen produced, they found that exposure to retrocylin or RC-101 reduced HIV production. While they couldn’t determine the complete mechanism of RC-101 for blocking HIV, the data showed that retrocyclin collected on the cell membrane, so they hypothesized that the protein somehow prevents HIV viruses from entering the cells.

A summary of the hypothesized mechanism of the synthesis of RTD-1 from its linear precursors. From Tang Y. et al. 1999.

Seven years later, the same group from UCLA and their collaborators from the University of Central Florida developed a plan to make human cells produce retrocyclin. They first had to determine whether human cells still have the machinery to synthesize a circular protein. To do this, they transfected DNA for a gene that coded for RC-101 into human cells. After they confirmed that these cells produced active RC-101, they tried to make human cervical cells express retrocyclin without adding a gene. Because a stop codon prevents the expression of retrocylin, a group of drugs that are known to cause protein synthesis beyond a stop codon were used. The drugs used were aminoglycosides, which are a group of antibiotics that, at very high concentrations, are known to cause “read-through” mutations in animal cells. One aminoglycoside, tobramycin, caused detectable retrocyclin to be formed in the cervicovaginal cells without killing the cell.

Comparison of RTD-1 and Retrocyclin (RC-100): Panel C shows amino acid sequence of RC-100, and panel D shows that of RTD-1. Unlabeled amino acids are cysteines. Cole, A. et al. 2002

Because retrocyclin reduces the production of HIV, the authors thought that HIV positive women could apply tobramycin to the vagina. This would not cure the disease, but it could help reduce the spread of HIV. The antibiotic tobramycin would have to applied topically to “reawaken” retrocyclin because systemic tobramycin at concentrations high enough to cause read-through mutations would have disastrous consequences. Most stop codons are important! Unfortunately, the interior of men’s genitals are less accessible than women’s, so the topical method would be less useful, even though HIV spreads much more frequently from men than from women.

References:

Tang, Y. A Cyclic Antimicrobial Peptide Produced in Primate Leukocytes by the Ligation of Two Truncated -Defensins, Science, 286 (5439) 502. DOI: 10.1126/science.286.5439.498

Cole, A.M. Retrocyclin: A primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1, Proceedings of the National Academy of Sciences, 99 (4) 1818. DOI: 10.1073/pnas.052706399

Venkataraman N, Cole AL, Ruchala P, Waring AJ, Lehrer RI, et al. (2009) Reawakening Retrocyclins: Ancestral Human Defensins Active Against HIV-1. PLoS Biol 7(4): e1000095. DOI: 10.1371/journal.pbio.1000095

Cascales, L. & Craik, D.J. (2010). Naturally occurring circular proteins: distribution, biosynthesis and evolution, Organic & Biomolecular Chemistry, 8 (22) DOI: 10.1039/c0ob00139b

Photo credits:

1.  http://www.flickr.com/photos/31406414@N04/4365830523/

2. http://www.flickr.com/photos/alexpenev/6921920751/