Wednesday, 28 October 2015

Molecule of the Month: November 2015 The Inflammasome

A recent article in the journal Science announced a discussion of the "Inflammasome" (see the diagram LHS, taken from the Invivogen web site) with the title "Assembling the Wheel of Death". It had to be read! The work was carried out by groups in he USA and China at the Universities of Harvard and Tsinghua. And after reading about this multi-protein complex, I thought it would make an excellent choice for my next Molecule of the Month! So before we look at the molecule itself, just what is the inflammasome and what is its biological role?

Immune defences are possibly amongst the most complex phenomena found in Nature; and from an evolutionary point of view, the sophistication of the immune response is quite different between organisms. In simple terms, the innate immune response is a quick-fire generic mechanism to fend off threats from pathogenic microbes and foreign bodies, such as asbestos fibres. The adaptive immune system, on the other hand, is confined to vertebrates, combats an attack and leaves us able to mount a defence against future attacks of the same kind. The inflammasome is a universal molecular assembly and is part of the innate immune response.

The recent articles in Science provide a structural basis to the triggering of the
assembly of the inflammasome and the image on the right gives you some idea of the overall shape of the complex. The pink "cap" is the NAIP2 protein and the pale blue colour are the NLRC4 proteins. The inflammasome has different components and stoichiometries in different organisms and the organisation shows interspecies variation, that relates to specific differences in the characteristics of the complex in each setting. However, the general features of the  inflammasome are shared by all inflammasomes, and I shall concentrate on these here. The first point to note is that the IF (fed up of typing inflammasome already!) is an intracellular complex that promotes the "activation" of proteases and the maturation of cytokines. The immune system is littered with "latent" or "pro" forms of proteins, that require some form of modification, such as the release of an N-terminal peptide, before they can perform their inflammatory-related role. Think of trypsinogen and chymotrypsinogen, pro-enzyme forms of the serine proteases, trypsin and chymotrypsin respectively. The conversion of an inactive form to an active one by limited proteolysis or ligand binding is a common thread in biochemical regulation: the latter is referred to as "allostery" (discussed in an earlier post on RNA Polymerase). 

The image on the left, illustrates the protease cleavage events associated with the activation of Caspase 7, an enzyme (an executioner enzyme, no less) that is intimately associated with programmed cell death (apoptosis). Using  cryo-electron microscopy (as an aside, you may have noticed how often this technique is appearing in the structural biology world: it seems to be an unstoppable force and is a major new focus at Sheffield) the authors derive a series of incredibly detailed molecular images. If you are looking for more information, the Wu lab home page contains links to publications that provide a comprehensive background to the molecules and their function. See here, for example.

Perhaps the most significant finding of this work (summarised schematically alongside related complexes on the left); notwithstanding the elegant microscopy, is the detail that has revealed how the IF is triggered by a single molecular recognition event from an invading bacterium (the pink blob in the image above), which in turn promotes the assembly of the intact and active IF. In addition, mutations in the NLRC4 protein (the pale blue wheel-shaped assembly attached to the pink cap, above) cause severe autoimmune inflammatory disease. The structure provides insight into how the IF fails to assemble properly in patients carrying these genetic lesions.The use of cryo-electron microscopy, drawing on X-ray crystallography has led to a molecular explanation for the triggering of the inflammatory response and moreover how mutations can throw a spanner in the works of macromolecular assembly, with such a devastating effect.

Saturday, 10 October 2015

The 2015 Nobel Prizes

I just posted this on my Sheffield Undergraduate Blog site, but I covered some of this material in my lecture on Thursday at the UTC. So I thought I would post here too. There is a power-point link on the RHS in the "Interesting Links" box, click Nobels 2015. It may be a little hard going for those not taking A level chemistry, but see what you think! 

The recent Nobel Prize in Chemistry awarded to Tomas Lindahl, Paul Modrich and Aziz Sancar (L to R opposite) provides a great example of just how powerful Biochemistry can be. It sits at the interface between Life Sciences and Chemistry, and can be instrumental in helping to elucidate the molecular basis of processes that are not only interesting in themselves, but also provide the foundations for future developments in medicine. 

The announcement made by the Nobel foundation last week, included the following phrase: “for mechanistic studies of DNA repair", which made me think immediately of the suggestion made to the Krebs Institute management board by one of our distinguished advisers, (Sir) Rich Roberts: "why don't you refresh the Krebs Institute mission..., how about mechanistic biology?". What a great concept, we all thought. The "strap line" was duly adopted, posted on the Institute web site,  incorporated into our grant applications and was bandied around at meetings just as much then, as it is now! Rich, a chemist by trade, obtained his PhD with David Ollis FRS, the head of Chemistry at Sheffield from 1963-1990. Rich always describes his project as simply being given a log from a Brazilian tree, and being told to find something interesting in it! Which he did pretty quickly! From every conversation I have had with Rich (and you can read the story he tells at these three blog posts: one, two and three), his understanding of Chemistry has always informed his Molecular Biology work: just take a look at Roberts RJ on PubMed

Getting back to DNA Repair, I can't think of many better examples of how the partnership of Chemistry and Biology, captured succinctly by the adjective "mechanistic",  has been so successful in explaining the underlying mechanisms of Darwinian evolution. In the absence of the advantages of the post Watson and Crick era, Darwin's ideas centred on a level of intrinsic  genetic change, on a "geological" timescale to explain the transitions in the fossil record. But mechanisms for change, or mutation, must be controlled in order that we don't stray too far from the "healthy" programme of reproduction and development. Without DNA repair mechanisms, we would succumb to diseases like cancer, much more frequently. Or as King Lear might have put it more eloquently:

Sans DNA repair... "O, that way madness lies"

What were the key pieces of work that led to the Nobel Committee's decision? As Tomas Lindahl commented when interviewed shortly after the announcement; there were perhaps a dozen scientists who contributed to our current understanding of the principles of DNA repair, so how did the committee pick out these three individuals? One way to find out is to take a look at their publication record: the time honoured way of assessing the "impact" of a scientist (you can read more about this here). Here are my own choices of a single paper from each of the three laureates. These papers exemplify the quality and strategies used by the laureates, and provide an insight into the quality of these worthy Prize winners.

In this paper Tomas Lindahl and colleagues at the Imperial Cancer Research Fund labs at Clare Hall (soon to be relocated with other London labs to the brand new Crick Institute), used a range of analytical techniques (protein purification, protease mapping and petide analysis by HPLC) to demonstrate that the adaptive response protein (ada) comprises two domains and that each domain can remove the alkylation damage conferred on guanines in DNA (a form of damage). In doing so, the alkyl group (eg a methyl) is transferred to a Cys residue in a conserved motif. The enzyme is subsequently inactivated and can no longer participate in repair functions (this is a suicide repair event, as far as the enzyme is concerned!). Interestingly, the ada protein has a second function: it can stimulate expression of the DNA sequence, encoding its own protein sequence! These incisive biochemical studies characterized the work from the Lindahl group over a period in excess of 30 years, of sustained, high impact science!  The image on the right was obtained by NMR later and both confirmed and added the molecular details to these landmark studies.

Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs.  Su SS, Modrich P. Proc Natl Acad Sci U S A. 1986 83:5057-61.

PDB 1oh6 EBI.jpgPaul Modrich's lab at Duke University  (North Carolina) unearthed the mechanism of mismatch repair catalysed by a complex of enzymes that are referred to by their genetic names: MutH, MutL, MutS, and MutU from E.coli. I have picked a paper relating to MutS. This early publication demonstrates the similar approach taken by the Modrich lab (as Lindahl) in purifying and characterising functions using a combination of protein and nucleic acid chemistries. The lovely example in the paper of DNAse footprinting reveals evidence for mismatch recognition by the MutS protein. The structure shown left , once again confirmed the elegant biochemistry from Modrich's lab.

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Object name is zpq0450641240005.jpgFinally, the Turkish born Aziz Sancar, now at the University of North Carolina has brought a rigorous approach to understanding DNA repair (and latterly a related set of proteins associated with  Circadian Rhythm and cell growth in plants and animals). In the above paper, a little more recent than the previous two, Sancar's lab tackle the relationship in activity terms between the cryptochrome and photolyase encoded genes referred to as Cry-DASH. This paper illustrates the systematic approach to addressing a controversial issue relating to a set of genes/proteins that share evolutionary links. Once again, the conclusions drawn are supported by associated structural work: the structure of a Cry-DASH protein interacting with damaged single-stranded DNA is shown opposite.

In conclusion, the elegant experimental work of Lindahl, Modrich and Sancar shares a lot in common. Their identification and characterisation of the proteins that recognise and fix a range of lesions from mismatches, chemical modifications through to light induced pyrimidine dimers provided a starting point. These authors and subsequently numerous other groups have confirmed and extended the work and we are now beginning to harness these results for therapeutic applications. The interdisciplinary nature of this work: chemistry, biochemistry, molecular cell biology and medicine, fell into the Chemistry Prize category, but it seems to me that it could equally have earned the Prize in Medicine or Physiology. Congratulations to all three winners!

Thursday, 1 October 2015

The fulminates, molecule(s) for the Nobel month of October

Mercury-fulminate-3D-vdW.pngOne dictionary definition of the verb to fulminate is "to explode violently". A second, more chemical definition would be "to react a metal with nitric acid and ethanol". The result of the latter, as discovered by Edward Charles Howard in 1824, is a highly explosive compound comprising a metal and a fulminate ion 
which is  also shown top left as mercury fulminate in a space filling format. In fact mercury and silver are the best known of the fulminates. The reason for discussing them here is partly because it is Nobel Prize month, and this time last year I discussed dynamite and nitroglycerin. This year I am attracted to these molecules because of what they can also teach us about electrons, bonding and energy. [If you didn't know, Alfred Nobel made his fortune from explosives!]. 

Chemistry at the turn of the 19th century was largely empirical. That is, most experiments were look-see, and relatively few scientists carried out systematic investigations. There were of course some notable exceptions: but in trying to harness the power of chemistry during Queen Victoria's reign in England, was not dissimilar to the attempts to harness the power of molecular biology in the early days of the Biotechnology industry. In other ways the knowledge base was inadequate. In fact Mendeleev's first Periodic Table was published in 1869, about 40 years after the start of the Industrial Revolution and the birth of the Chemical Industry! So when Edward Charles Howard (above) added mercury to nitric acid, followed by a dash of ethanol, mercury fulminate was "born". [Just think of the Health and Safety consequences for proposing this as a first year chemistry practical!]. Silver fulminate, prepared in the same way is even more unstable and can actually explode under its own weight, and under water!

So why are these fulminates so reactive? It is thought to be  the presence of the weak single nitrogen-oxygen bond which leads to its instability. Nitrogen easily forms a stable triple bond to another nitrogen atom, forming gaseous nitrogen. This "tension" generates the compound's intrinsic instability. Why is the silver salt more unstable than the mercury salt? I thought about this for a while and my conclusion is that mercury is a liquid at room temperature, owing to the unorthodox arrangement of its inner and outer electrons. It may be that this confers a minor stabilising effect that cannot be achieved by silver ions.  Silver has the electron distribution: 2,8,18,18,1. Any thoughts from inorganic chemistry experts?

There is another twist to the fulminate story. The chemists among you will have recognised that the formulae for silver cyanate (AgOCN) and and silver fulminate (AgCNO) are technically equivalent. In the 1820s, these observations led to a huge debate betwee one of history's most illustrious chemists, Justus Liebig, who discovered silver fulminate (Ag-CNO) and Friedrich Wšhler, who discovered silver cyanate (Ag-OCN). This was only resolved when  Jšns Jakob Berzelius came up with the concept of isomers. The tension in the fulminate molecule seemed to have rubbed off on Liebig and  Wšhler! It was also Alfred Nobel who had the last laugh: he patented the use of mercury fulminate in his explosives and therby generated even more wealth. Finally, although the fulminic acid molecule looks very simple, its structure was only determined about 10 years ago!  I think this is a suitable choice for the Nobel month of October and I wonder what explosions will emerge next week when the Nobel committee announce the results of their ruminations and deliberations.

End note. I came across a word I have never heard before in connection with the fulminates. Brisance: defined chemically, as the shattering capability of an explosive, measured by the state of a shell after an explosion. So, in summary, silver fulminate has a greater brisance than its mercury derivative: its "electron shells" directly impact on "ammunition shells!" Alfred Nobel was vilified in his lifetime for his contribution to death in warfare, but I believe his endowment of the Nobel Prize has had a major impact on the public awareness of some great Science!