What was the most lethal epidemic of infectious disease in modern times? AIDS perhaps? You might think so, but you would be wrong. Between the beginning of the epidemic and the end of 2007, AIDS killed people 25 million; the influenza epidemic of 1918 killed 40 million. This vast figure is also about double the number who died in the First World War, which ended the same year. Furthermore, a high proportion of those deaths were of healthy individuals in the prime of life. Yet most of the time we think of influenza as little more than a very nasty nuisance...
However, there has recently been a renewed interest in past influenza epidemics as a result of the result of the virulent strain of influenza currently sweeping through populations of wild and domestic birds worldwide. The influenza virus is endemic in birds, and strains tend to spread periodically from them to mammalian hosts: pigs as well as humans. Mapping genetic changes in the influenza virus, and how these affect its spread, is an important research area. Richard Goldstein of the National Institute for Medical Research, based in Mill Hill, London, gave a fascinating Monday seminar on this topic yesterday, looking into the past to see how genetic changes could have led to the lethal 1918 epidemic.
Influenza viruses contain two proteins on their spherical surfaces: a neuraminidase and a hemagglutinin. These proteins come in various forms: 16 different hemagglutinins are known, and 9 different neuraminidases. Any influenza virus can be characterised by these variants - for example, the most common type of influenza currently afflicting humans is H3N2, and the feared bird flu H5N1. Recent flu epidemics appear to have been caused by reassortment events, where the genomes of different viral subtypes combine to form an entirely new one that will not be recognised by human immune systems. An epidemic in 1957, for instance, coincided with a shift from influenza H1N1 (which had been circulating since 1918) to H2N2.
Influenza virus hemagglutinin and neuraminidase are both mainly-beta proteins, and their structures are described further on this page of PPS section 5.
So, what happened in, or before, 1918 to cause the epidemic of H1N1 flu? Molecular geneticists, such as Goldstein, study this by reconstructing phylogenetic trees showing the evolutionary distance between viral isolates taken in different places at different times. Yet most of these calculations can only show evolutionary distance, not the direction of change - in the jargon of phylogeny, they produce unrooted, rather than rooted, trees (there is no known "top"). The research was at rather an impasse until postdoc Mario dos Reis (a Birkbeck Ph.D.) noticed that the GC content of viruses infecting humans, but not of those infecting birds, decreased over time. This enabled the group to add an evolutionary "clock" to the phylogenetic tree for each of the influenza virus' 11 genes. This showed that some of the genes had entered the human population at different times, indicating that the variant that caused the 1918 flu had arisen from several recombination events. Interestingly, only one gene (neither H nor N) could have made the jump in 1918; most viral proteins were present in the human population in their 1918 forms well before that year. M1, like some other proteins, appears to have made the shift in 1899.
So, what did happen in 1918? There do appear to have been changes to the H gene then. But it may also be possible that the world population was so debilitated, and susceptible, after four years of war that a variant that had already been around for a few years was, unusually, able to cause such an epidemic...
Tuesday, 19 February 2008
Monday, 11 February 2008
Function of vFLIP - a protein from the virus associated with Kaposi's sarcoma
Today's Monday seminar was given by Professor Mary Collins from the Division of Infection and Immunity at University College London's medical school. She described some of her studies of the function and mechanism of vFLIP, a protein from the human herpesvirus 8 (HHV8). This is a cancer-causing (oncogenic) virus and is responsible for the AIDS-associated tumour, Kaposi's sarcoma.
While very many people - about 10-15% of blood donors in London, for example - have been exposed to, and have antibodies for, this virus, it only causes problems in people with defective immune systems. In these people, however, it can cause lymphoma or multicentric Castleman's disease as well as Kaposi's sarcoma. This, however, is now - thanks to HIV and AIDS - one of the commonest cancers worldwide, in fact the commonest in some African countries. AIDS-related Kaposi's sarcoma is fairly rapidly fatal if untreated; however, it can now be controlled very well (although not completely cured) with anti-retroviral therapy.
HHV8 is a large (by viral standards!) double-stranded DNA virus. The protein vFLIP is one of a cluster of proteins expressed while the virus is latent. It binds to a protein called i-kappa kinase gamma in virus-infected cells. This causes the kinase to phosphorylate inhibitors of the cytokine NF-kappa B, leading to the release of NF-kappa B from inhibitor complexes and ultimately the degradation of the inhibitors. This leads to a cascade of gene expression that is essential for the prevention of apoptosis; in contrast, knockdown of vFLIP levels by siRNA will lead to induction of apoptosis. vFLIP is therefore implicated in the survival of virally infected cells.
Endothelial cells that have been infected by HHV8 adopt a characteristic "spindle cell" phenotype. Microarrays have shown that the virus induces complex re-programming of gene transcription in these cells, which is likely to induce this dramatic change in their morphology. Future work will elucidate the precise role of the signal transduction cascade induced by vFLIP in this "morphological reprogramming".
There is no structure available for vFLIP, but there are a large number of kinase structures known. From the UniProt database, the closest to i-kappa kinase is the intracellular Ser/Thr protein kinase domain of Mycobacterium tuberculosis PknB (PDB entry 1MRU).
While very many people - about 10-15% of blood donors in London, for example - have been exposed to, and have antibodies for, this virus, it only causes problems in people with defective immune systems. In these people, however, it can cause lymphoma or multicentric Castleman's disease as well as Kaposi's sarcoma. This, however, is now - thanks to HIV and AIDS - one of the commonest cancers worldwide, in fact the commonest in some African countries. AIDS-related Kaposi's sarcoma is fairly rapidly fatal if untreated; however, it can now be controlled very well (although not completely cured) with anti-retroviral therapy.
HHV8 is a large (by viral standards!) double-stranded DNA virus. The protein vFLIP is one of a cluster of proteins expressed while the virus is latent. It binds to a protein called i-kappa kinase gamma in virus-infected cells. This causes the kinase to phosphorylate inhibitors of the cytokine NF-kappa B, leading to the release of NF-kappa B from inhibitor complexes and ultimately the degradation of the inhibitors. This leads to a cascade of gene expression that is essential for the prevention of apoptosis; in contrast, knockdown of vFLIP levels by siRNA will lead to induction of apoptosis. vFLIP is therefore implicated in the survival of virally infected cells.
Endothelial cells that have been infected by HHV8 adopt a characteristic "spindle cell" phenotype. Microarrays have shown that the virus induces complex re-programming of gene transcription in these cells, which is likely to induce this dramatic change in their morphology. Future work will elucidate the precise role of the signal transduction cascade induced by vFLIP in this "morphological reprogramming".
There is no structure available for vFLIP, but there are a large number of kinase structures known. From the UniProt database, the closest to i-kappa kinase is the intracellular Ser/Thr protein kinase domain of Mycobacterium tuberculosis PknB (PDB entry 1MRU).
Tuesday, 5 February 2008
Seminar by Prof Armitage, Oxford
This weeks Monday seminar was by Prof Judy Armitage from Oxford (http://www.bioch.ox.ac.uk/aspsite/research/brochure/Armitage/) . Do go to her website as it will help you to see some of the pictures. She was interesting to us as a biologist (microbial physiologist) heading up a Systems Biology centre. Systems Biology is a trendy phrase but has almost as many meanings as there are centres. Systems Biology is really putting biology on a quantitative basis by developing mathematical models that accurately predict experiments. This can be at various scales, whole organisms, individual organs, whole cells or just a particular pathway. Prof Armitage has been studying the photosynthetic bacteria Rhodobacter sphaeroides for many years. It has now become more significant as Craig Ventner has found that a close relation is the most abundant organism in sea water and therefore central to photosynthesis in the sea.
However what Judy is currently studying is the chemotactic response where these bacteria swim towards their food source. In contrast to the well studied E.coli system where there is only one set of proteins, there are 4 operons in R.spheroides. Although in vitro the proteins were able to cross react and phosphorylate, they did not compensate for each other when deleted.
Her group showed by attaching fluorescent proteins (GFP/CFP) etc to the various components that unlike E.coli where the chemotactic proteins were all at the pole of the cell, there were two systems in R.spheroides, one at the pole but the other in the mid cell. One to sense the internal "happiness" of the cell- ie was it well fed and the other to go and find new food sources when it was not. She pointed out the importance of a number of their experimental details. Firstly they add the fluorescent protein in frame with the protein of interest in the genome so as to get natural abundance not overexpression. Secondly they only pay attention to those mutants where the pathway still functions as the fusions can sometimes disrupt the true localisation and give a false result, but when this happens the pathway does not work. The group has also done work on the flagellae that drive the bacteria and using a special fluorescent microscope were able to count the numbers of the subunits that drive the motor- 22 per complex.
Her take home message was that you cannot assume that all things work the same as E.coli!
However what Judy is currently studying is the chemotactic response where these bacteria swim towards their food source. In contrast to the well studied E.coli system where there is only one set of proteins, there are 4 operons in R.spheroides. Although in vitro the proteins were able to cross react and phosphorylate, they did not compensate for each other when deleted.
Her group showed by attaching fluorescent proteins (GFP/CFP) etc to the various components that unlike E.coli where the chemotactic proteins were all at the pole of the cell, there were two systems in R.spheroides, one at the pole but the other in the mid cell. One to sense the internal "happiness" of the cell- ie was it well fed and the other to go and find new food sources when it was not. She pointed out the importance of a number of their experimental details. Firstly they add the fluorescent protein in frame with the protein of interest in the genome so as to get natural abundance not overexpression. Secondly they only pay attention to those mutants where the pathway still functions as the fusions can sometimes disrupt the true localisation and give a false result, but when this happens the pathway does not work. The group has also done work on the flagellae that drive the bacteria and using a special fluorescent microscope were able to count the numbers of the subunits that drive the motor- 22 per complex.
Her take home message was that you cannot assume that all things work the same as E.coli!
Saturday, 2 February 2008
Monday, 21 January 2008
Structure and mechanism of Hsp90, a heat shock protein
Heat shock proteins are over-expressed when cells are exposed to heat or stress. Their function is to help other proteins to fold "correctly" into their mature, functional forms and, as such, they are classified as "chaperones". The structures and functions of these proteins will be described in much more detail in section 8 of the PPS course ("The Protein Lifecycle").
Today (21 January), in the School of Crystallography's Monday seminar programme, Maruf Ali from the Institute of Cancer Research in London spoke about his research on the structure of the heat shock protein Hsp90. This protein is found in all kingdoms except for the Archaea; it interacts with many other proteins (known as "client proteins" to help them enter their mature, active structural forms. Hsp90 client proteins include kinases that are important targets for anti-cancer drugs - hence the ICR's interest.
Hsp90 is a three-domain protein. The N-terminal domain binds ATP, which is necessary for the protein's activity; the middle domain binds client proteins; and the C-terminal one is involved in dimerisation. Structures of each domain separately were already known when Maruf started his post-doc a few years ago; each of these domains has a fold in Scop's alpha+beta class. Maruf's work involved solving the structure of a mutated form of the intact protein bound to a co-chaperone, (Ali et al. (2006), Nature 440, 1013-1019; PDB code 2CG9). This structure gives a clear picture of a complex structure, showing how a "lid" of structure closes to enabl the client protein to bind, and supports a previously proposed model in which the N-terminal domain is also involved in dimerisation.
Try downloading the structure, loading it into Jmol or a similar program, and seeing if you can identify the three domains.
Today (21 January), in the School of Crystallography's Monday seminar programme, Maruf Ali from the Institute of Cancer Research in London spoke about his research on the structure of the heat shock protein Hsp90. This protein is found in all kingdoms except for the Archaea; it interacts with many other proteins (known as "client proteins" to help them enter their mature, active structural forms. Hsp90 client proteins include kinases that are important targets for anti-cancer drugs - hence the ICR's interest.
Hsp90 is a three-domain protein. The N-terminal domain binds ATP, which is necessary for the protein's activity; the middle domain binds client proteins; and the C-terminal one is involved in dimerisation. Structures of each domain separately were already known when Maruf started his post-doc a few years ago; each of these domains has a fold in Scop's alpha+beta class. Maruf's work involved solving the structure of a mutated form of the intact protein bound to a co-chaperone, (Ali et al. (2006), Nature 440, 1013-1019; PDB code 2CG9). This structure gives a clear picture of a complex structure, showing how a "lid" of structure closes to enabl the client protein to bind, and supports a previously proposed model in which the N-terminal domain is also involved in dimerisation.
Try downloading the structure, loading it into Jmol or a similar program, and seeing if you can identify the three domains.
Tuesday, 15 January 2008
CCP4 2008 Study Weekend
CCP4 (Collaborative computing Project 4) is responsible for one of the main X-ray crystallography computer packages. It holds a study weekend the first weekend in January each year attended by 3-500 delegates. It is the social event of the UK protein crystallographers calendar as well as being the best methods meeting each year certainly in Europe. This year the topic was "Low Resolution Structure Determination and Validation" inspired by the retraction in Dec 2006 of five high profile structures of membrane proteins, which had been wrongly determined due to the author using an incorrect piece of software (not CCP4!) which meant that his maps were inverted ie his helices were all left handed rather than right handed. Because the resolution was low, as membrane protein crystals often are, this mistake was not immediately obvious from the maps and he forced through the refinement of right handed helices into left handed density by some dubious methods. Less was said about another structure published in Nature which essentially does not have the copies of the protein touching in the crystal as this is still under investigation by the American University from where this structure originates. Analysing these mistakes should help the community to avoid them themselves and possibly spot them as referees- all these papers had got past expert referees.
On a more positive note the first example of an ab initio (ie without a sequence homologue) prediction of a protein structure that was good enough to solve a crystal structure was presented. The main conclusion of the meeting is that the resolution of the structure is pretty much the only thing that determines the quality of the structure. Big structures at the same resolution are just as good as small structures. However big structures tend not to be at as high a resolution so that on average big structures are less well determined than small structures, but this is because they are lower resolution. Structural genomics groups are no better or worse than targetted labs at determining structures.
From a PPS point of view probably the most interesting talk was by Chris Tate from MRC in Cambridge who said that the retracted structure had to be wrong because it was incompatible with the biochemical data. Although structure is powerful, it has to be compatible with the biology. The system he worked on (EmrE) has another peculiarity in that half the protein inserts into the membrane in one direction (N terminus in) and half in the other (N terminal out) and the active molecule consists of one of each of these protein chains.
On a more positive note the first example of an ab initio (ie without a sequence homologue) prediction of a protein structure that was good enough to solve a crystal structure was presented. The main conclusion of the meeting is that the resolution of the structure is pretty much the only thing that determines the quality of the structure. Big structures at the same resolution are just as good as small structures. However big structures tend not to be at as high a resolution so that on average big structures are less well determined than small structures, but this is because they are lower resolution. Structural genomics groups are no better or worse than targetted labs at determining structures.
From a PPS point of view probably the most interesting talk was by Chris Tate from MRC in Cambridge who said that the retracted structure had to be wrong because it was incompatible with the biochemical data. Although structure is powerful, it has to be compatible with the biology. The system he worked on (EmrE) has another peculiarity in that half the protein inserts into the membrane in one direction (N terminus in) and half in the other (N terminal out) and the active molecule consists of one of each of these protein chains.
Friday, 11 January 2008
Tim Hunt's Lecture
The Institute of Structural Molecular Biology, based at Birkbeck and University College, hosted a star performer as its first seminar speaker of 2008: Tim Hunt of the Cancer Research UK London Research Institute. Tim was awarded the Nobel Prize for Physiology or Medicine in 2001, with Leland Hartwell and Paul Nurse, for his discovery of cyclins - proteins that control the expression of the cyclin-dependent kinases (CDKs) which control a cell's passage through the cell cycle. So cyclins can be described as regulators of the regulators of the cell cycle.
Maybe only a Nobel Laureate could do it. Tim started his lecture with a quick tour through several hundred years of Physics, inspired by his small daughter's (unanswered) question "Why is the sky opaque?" He introduced (or re-introduced) his audience to Schrodinger's equation and Maxwell's idea of a "field" before confessing that he didn't understand quantum mechanics: no one should ever be ashamed of admitting as much.
The cell cycle, and its control, is, like quantum mechanics, "very interesting and very complicated". Tim's critical observation, which he made studying frog oocytes, was that cell division is controlled by the concentrations of the proteins that we now know as cyclins. They were given this name because their concentration in cells goes up and down according to where those cells are in the cell cycle - whether they are growing, replicating their DNA, undergoing mitosis...
Cyclins control the progress of cells through cell division by regulating the function of cyclin dependent kinases (CDKs). By binding to CDKs, cyclins control their activation state, and active CDKs drive the cells through the cell cycle. The press release for the 2001 Nobel Prize succinctly described CDKs as the cells' "motors" and cyclins as the gear boxes that control whether cells will be in idle or overdrive.
Cyclins are all-alpha proteins (link is to the PPS material) with 5-helical cores. PDB entry 1H1S shows human cyclin A bound to CDK2.
It is impossible to do justice to such a complex topic, and lecture, in a few paragraphs. To learn more, try the resources on the Nobel website would be a good first port of call.
Maybe only a Nobel Laureate could do it. Tim started his lecture with a quick tour through several hundred years of Physics, inspired by his small daughter's (unanswered) question "Why is the sky opaque?" He introduced (or re-introduced) his audience to Schrodinger's equation and Maxwell's idea of a "field" before confessing that he didn't understand quantum mechanics: no one should ever be ashamed of admitting as much.
The cell cycle, and its control, is, like quantum mechanics, "very interesting and very complicated". Tim's critical observation, which he made studying frog oocytes, was that cell division is controlled by the concentrations of the proteins that we now know as cyclins. They were given this name because their concentration in cells goes up and down according to where those cells are in the cell cycle - whether they are growing, replicating their DNA, undergoing mitosis...
Cyclins control the progress of cells through cell division by regulating the function of cyclin dependent kinases (CDKs). By binding to CDKs, cyclins control their activation state, and active CDKs drive the cells through the cell cycle. The press release for the 2001 Nobel Prize succinctly described CDKs as the cells' "motors" and cyclins as the gear boxes that control whether cells will be in idle or overdrive.
Cyclins are all-alpha proteins (link is to the PPS material) with 5-helical cores. PDB entry 1H1S shows human cyclin A bound to CDK2.
It is impossible to do justice to such a complex topic, and lecture, in a few paragraphs. To learn more, try the resources on the Nobel website would be a good first port of call.
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