Saturday, 29 November 2014

Prostaglandins: Molecule(s) of the month for December

I was driving to Liverpool a few days ago listening to the first part of the annual BBC Radio, Reith Lectures. In fact I was probably at the same point on Edge Lane, when I was struck by Grayson Perry's disruptive, irreverent and insightful take on the art world last year. This year, Dr. Atul Gawande is tackling the topic of contemporary medicine and in his first lecture, he posed the question: Why do doctors fail? In his moving description of his son's encounter with the medical profession, he mentioned the use of a class of drugs based on the prostaglandins. In the case of his son, Walker; an early tragedy was averted by the doctor's administration of Prostaglandin E1. This prevented closure of the patent ductus arteriosus in the newly born, Walker, had symptoms of a cyanotic (Gk dark blue colour) heart defect (the skin has a blue/purple colouration, owing to a deficiency in the supply of oxygenated blood, for which there are a number of causes). It made me think that prostaglandins (PGs) are often overlooked in mainstream Biochemistry courses, and yet they are a potent class of molecules. This is an attempt to whet your appetite for the prostaglandins!

Prostaglandins are derived enzymatically from essential fatty acids: they are 20 carbon molecules with a 5-membered ring, as shown on the RHS for Prostaglandin E1 (or Alprostadil). The enzyme Phospholipase A2, converts diacylglycerol to arachidonic acid, which in turn is converted to the prostaglandins by the enzyme called cyclooxygenase (or COX, for short). The main function of COX is to catalyse the formation of the "signature" ring, found in all prostaglandins, through ring closure and the addition of oxygen. You may have come across these enzymes, since they are the targets of one of the most
commonly taken drugs, aspirin. The mechanism of action of aspirin, lies in its irreversible acetylation of a key Serine side chain in the COX enzyme. Other pain killers/anti inflammatories, such as ibuprofen act on the same target, but are reversible (ie non-covalent) inhibitors. This work was recognised by the award of a Nobel Prize in 1982 to (Sir) John Vane, along with two others. The structure of aspirin (purple) is shown in the active site of a COX enzyme on the LHS. The Serine at position is acetylated as the aspirin molecule is hydrolysed in the active site. As a result, arachidonic acid is now prevented from entering the active site of the enzyme and consequently no PGs can be synthesised. 

There are around 10 receptors that specifically recognise and mediate the potent effects of PGs. These interactions are in turn "transduced" by G-protein coupled receptors (GPCRs, shown on the RHS) leading to reprogramming of normal hormonal responses, contraction and dilation of smooth muscle cells and a wide range of other physiological effects. We do not yet have a high resolution structure of the PG receptors, but I am sure it wont be long. The interest from my perspective, is in a compound class that is so potent. It reminds me of the story surrounding thalidomide, which you can read in an earlier Blog. The combination of the hydrophobicity, the structurally constraining ring and the oxygen atoms, give PGs their potency, when coupled to the GPCR pathways. But, as a simple biochemist, the challenges brought by working in the lab with such insoluble molecules, make me think I took the easy route early in my career by choosing amino acid dehydrogenases and DNA modifying enzymes, where everything can be carried out in the aqueous phase. Now, every time you take an aspirin, think of the molecular pharmacological events that you have triggered!

Saturday, 8 November 2014

Jack's project first steps: just a phase we are going through!

I thought it might be helpful to other students (and Jack and me!) to share some thoughts on the Y13 lab projects. I shall work through them all, but since it is on my mind, I thought I would say a few words about Jack Condron's project. Jack asked if he could develop a device for cleaning contaminated drinking water. His idea was to create a cheap and simple solution for helping solve one of the major challenges in developing countries: access to clean drinking water. A laudable ambition, and one that has and continues to occupy the minds of many scientists, engineers in both research organisations and commercial organisations. One patent image is shown on the LHS. The key to this project is simplicity and a solution that is independent of any power supply: i.e. purification might result from a shake or a hand operated mini-pump.

The first thing to point out is that Jack's initial thoughts, combined with his determination and drive was enough to get me behind him. We then started thinking about "proof of concept" experiments, the need for certain materials and reagents and in particular how we might "mimic" the system we are trying to develop. One of the great things about the UTC's Innovation Labs is the access we have to a 3D printer, and, more importantly, George Rule's ingenuity. So, Jack and George created a small "printed" chamber for testing the extraction of water from a "dirty" mixture. I have shown a competing design above, Jack's design is currently our in house design and will remain undisclosed for now. In simple terms, Jack's design comprises, two, 2ml compartments that can be connected, with a dialysis membrane providing an interface. This will allow us to establish whether our "chemistry" can achieve the desired result.

We sat down and thought about the model experimental system and our through our discussions it became clear that "phase transitions" and an understanding of solubility of molecules in water and organic solvents was only superficially taught at A level. Moreover, the use of a simple and safe polymer for creating phases, such as polyethylene glycol (PEG, for short), was also something for which a strong theoretical base was off curriculum. At this point Jack was beginning to wonder what had happened to his desire to save lives! (Me too!). I then thought, we need to "see" the movement of contaminating species between phases and across membranes. We needed a dye that was water insoluble, but reasonably soluble in ethanol and polyethylene glycol. Orcein, came to the rescue (top LHS).

We now have water, orcein, ethanol, PEG (at three mean molecular weight lengths), an experimental chamber and a water soluble dye (still under investigation). Jack also, threw in bleach tablets for good measure, thinking about the use of calcium hypochlorite (top RHS) for water purification, and as a compound that we might need to eliminate. So we now have to understand not only the physical chemistry of such mixtures, but also the reactivity of a strong oxidising agent as well. 

Jack has been exploring how these molecules behave in solution when mixed in a range of combinations. We are on a journey rich in chemistry and one that seems to be taking us farther and farther away from the end point, but (I like to think) deeper and deeper into fundamental chemistry. The visual demonstration of PEG:water phases (used to drive counter current distribution separation technology), the mixing of ethanol in water and the differential partitioning of dyes has been the first "learning" phase (sorry!). Jack has already noted that polymer length (PEG is shown top LHS) can also influence the absorbance maximum of the dye (purple to red shifts are reproducibly obtained). Dye precipitation at water PEG interfaces can be followed with time and then of course there is the observed "bleaching" of absorbance caused by the bleaching tablets. Does "bleach" affect all 8 component of the dye? Can we explain the bleaching of polyaromatic dyes and does the same phenomenon occur with similar water soluble polyaromatic dyes?

How does this help us with the initial aim? Well it helps us in many ways. First and foremost it is training a young enthusiastic scientist at the UTC, how challenging research can be, even when the problem seems (at face value) so simple. It is providing Jack with a terrific foundation in the relationship between pure and applied chemistry and empowering him as an investigative scientist. The fact that he is a Y13 student at the UTC, constantly amazes me! Jack is at an early stage in the project and I shall follow up at Christmas with project and he will write the third and final blog!

To follow, Kelly's project: fingerprints, identical twins, epigenetics and Alan Turing's legacy.

Thursday, 6 November 2014

How the BLAST rendered me speechless today. Molecule of the month November 2014 FOXP2

I started this Blog, intending to look at the properties of gunpowder, but remembered I had discussed dynamite in October, so two explosive molecules in the same number of months, seems a little excessive! Then I started writing about Cytochrome P450s, but although I was ready to go with the P450 story, I realised how nicely the FOXP2 protein structure, function and genetics fitted together in the BLAST sessions today. So, P450 for Christmas and FOXP2 for November. This is a story that might leave you speechless!

The human genome project has proved a treasure trove for evolutionary biologists, and a quick search of a well known gene encoding say haemoglobin or a histone protein, will inevitably identify a closely related sequence in chimpanzees, orang utans or another ape. So far not surprising. However, I then asked the question: what genes would you expect to point to differences between man and apes? I was delighted when someone gave the answer "communication" genes. So of course I suggested an analysis of the "language" gene, FOXP2. 

The FOXP2 protein is a transcription factor (here you can find a lovely summary with simple sketches of transcrition factors in higher organisms) found widely in the genomes of mammals, that is a regulator of neuronal plasticity, with a direct impact on speech and language development. The protein sequence that you all obtained, showed a dominant feature: a run of tens of Q (Glutamine) residues. These are located at the N terminal side of the protein sequence and are not uncommon in some DNA binding proteins. In addition FOXP2 contains a zinc finger and a leucine zipper, both of which are protein sequence motifs, often associated with DNA binding. From a bioinformatics perspective, the FOXP2 sequence is a dead give away! Transcription factors bind to DNA and RNA Polymerases and promote gene transcription (expression). In the case of FOXP2 it regulates the levels of expression of a set of genes involved in language development which are mapped to the brain. The FOXP2 gene, therefore encodes a "master regulator" and mutations to the sequence  of such genes can be disastrous, leading to negative effects on a significant range of "downstream " functions.

Full-size image (74 K)
The protein (shown left in complex with DNA from the work of an international collaboration published in the journal Structure) recognises a specific DNA sequence. There are two amino acid substitutions between the FOXP2 proteins between chimpanzees and man, which appear to interfere with the function of FOXP2 in such a way that the coordinated expression of a family of genes required for speech is not possible in apes. The story was made possible by pioneering work from communication scientists at McGill and London Universities, and geneticists at Oxford University and the UCL in London. From a family with a rare, inherited speech disorder, the region of the chromosome containing the mutation came the first clues. Shortly afterwards, the FOXP2 gene was isolated and, as with apes, the changes in function relate to subtle amino acid differences: you should attempt to rationalise these observations. You will find that FoxP2 from a number of species is highly conserved, but look closely at the features in the protein primary structure. Subtle differences can give rise to profound functional consequences, which makes a powerful case for understanding the details of chemistry, structure and reactivity of amino acid side chains in understanding biological phenomena: in this case language skills!

The FOXP2 gene expresses a protein that regulates the levels of expression of a subset of genes which in turn cascade down information that leads to coordination of brain function leading to controlled action of facial muscles and the larynx, thereby producing speech. It should also be remembered from your BLAST searching that by way of contrast some protein sequences can tolerate significant amino acid changes without loss of function. The critical evaluation of protein sequences is the key to a deep understanding of function, so always treat similarities with a healthy level of scepticism and try to validate ideas by experiment, where possible.