New Target to Enhance Cognitive Function

The ThinkerHistones are basic proteins that interact with negatively charged phosphate groups on DNA, compacting and protecting the DNA, and controlling gene expression. Histones are subject to a number of post-translational modifications, including methylation and acetylation. The balance between acetylation by histone acetyltransferases (HAT) and deacylation by histone deacetylases (HDAC) alters the strength of DNA interactions and plays a key role in regulating gene expression.

Collaborators led by scientists at the Picower Institute for Learning and Memory have identified a promising target that could enable the development of therapeutics to improve memory and learning in patients with neurodegenerative disorders such as Alzheimer’s. The team demonstrated that treatment with HDAC inhibitors enhanced memory and learning ability in normal mice and mouse models of neurodegeneration.

Histone and DNA are the major components of chromatin, the complex that packages genetic information into the chromosomes and inhibitors of the HDAC family have received much attention in recent years as potential treatments for various cancers. Chromatin modification, particularly via deacetylation, has also been implicated in memory formation.

Although HDACs are a family of 11 members, the team has shown that neuron-specific overexpression of HDAC2, but not HDAC1, in mice decreased synaptic plasticity, synapse number and memory formation. This effect was ameliorated by treatment with HDAC inhibitors. Conversely, Hdac2 deficient mice displayed enhancement of synapse number and memory facilitation.

The results, published in full in the journal Nature, suggest exploration of selective HDAC2 inhibitors for treatment of human neurodegenerative diseases involving memory impairment.

New Blood Pressure Target

heartAlthough normally clinically silent, persistent hypertension and atherosclerosis are leading risk factors for strokes, heart attacks, heart failure, aneurysms and chronic renal failure. How blood pressure is regulated is still not fully understood, but a team lead by scientists at the University of Pennsylvania School of Medicine has now suggested a role for prostaglandin F2α (PGF2α) in increasing blood pressure and accelerating atherosclerosis. Prostaglandins are a group of hormone-like substances that mediate many physiological and pathophysiological processes, and the team found that mice lacking the receptor for PGF2α had lower blood pressure and less atherosclerosis than wild-type mice. Knocking out the PGF2α receptor was found to suppress activity of the renin-angiotensin system which plays a key role in regulating blood pressure. When blood pressure is low, the liver secretes a protein, angiotensinogen, which is cleaved by renin to give angiotensin I. Further cleavage by angiotensin converting enzyme (ACE) produces angiotensin II which increases blood pressure by narrowing blood vessels and by stimulating release of aldosterone which leads in turn to retention of sodium and water.

If the results seen in mice translate to humans, blocking the PGF2α receptor may provide a novel strategy for controlling blood pressure and reducing atherosclerosis. The study is published in full in the Proceedings of the National Academy of Sciences.

Targeting the Riboswitch

Recent research has shown that gene expression can be regulated at the level of mRNA by riboswitches. A riboswitch is an aptamer region on an mRNA molecule that can specifically bind a small effector molecule, causing changes in the structure of the expression platform and so regulating the activity of the mRNA. Riboswitches most usually switch off the ability of mRNA to carry out protein synthesis but can also switch it on. A variety of riboswitch classes have been identified, with most of the examples being discovered in bacteria including E. coli and streptococcus as well as bacteria causing anthrax, gonorrhoea, meningitis and dysentry.

Crystal structure of preQ1 riboswitchResearchers at the University of Rochester Medical Center have now solved the crystal structure of the smallest known riboswitch, the preQ1 riboswitch. The preQ1 riboswitch controls the ability of bacteria to produce queuosine (Q), a molecule which enables accurate gene expression by overcoming an inbuilt defect in the mRNA-ribosome-tRNA system known as tRNA wobble, and which is essential for the survival of many important pathological bacteria. The preQ1 riboswitch ‘senses’ the level of preQ1, a precursor to Q. If too much preQ1 is present, genes responsible for producing preQ1, or for its transport, are shut down. The preQ1 precursor, known as preQ0, has the same effect in reducing production of Q. One gene that is regulated by the preQ1 riboswitch is that which codes for the enzyme queF, which converts preQ0 into preQ1.

The structure of the preQ1 riboswitch from Thermoanaerobacter tengcongensis complexed with preQ0 shows preQ0 bound in a buried pocket. The structure also reveals how the first base of the mRNA ribosome binding site binds to a loop of the riboswitch, and how the loop end of the preQ1 riboswitch aptamer domain binds to preQ0. Binding of the preQ1 aptamer loop to the first base in the ribosome binding site was found to be mediated by a standard G to C base pairing interaction. The preQ1 aptamer (34 nucleotides) is about 2.5-fold shorter than functionally related riboswitches that recognize similar metabolites.

An understanding of how bacterial species sequester their ribosome binding sites using divergent preQ1 riboswitch aptamers could lead to the design of a new class of antibiotics. There is evidence that some existing antibiotics act – in part at least – by targeting riboswitches and, since riboswitches have not yet been found in human cells, the hope is that antibiotics acting on riboswitches will have a low propensity for side effects. The study is published in the Journal of Biological Chemistry.

Keeping Malaria Locked Up

Animal parasites such as malaria have complex life cycles and, so far, most attempts to control infection have centred on preventing the parasite from entering host cells. Writing in the journal Science, a team led by Dr Doron Greenbaum at the University of Pennsylvania has now focussed on an alternative treatment approach – locking the parasites inside the host cell. The team found that Plasmodium falciparum, prison barsthe species responsible for the majority of human infections, and also the one that causes the most virulent form of malaria, uses a host protease to escape from cells. The protozoa replicate within a vacuole in infected cells and must escape to begin a new lytic cycle. The team used a variety of techniques to show that P falciparum makes use of host cell calpain proteases to facilitate escape.

The team were also interested to find out whether the distantly related parasite, Toxoplasma gondii adopts a similar strategy. Disease caused by T gondii infection is usually mild and self-limiting, but can be fatal to the unborn child if contracted during pregnancy. They found that in the absence of calpain, the parasites could not escape the infected cell, just as they had observed for malaria parasites.

Greenbaum plans to continue to explore the practicality of calpain as a target for anti-parasitic drugs. P falciparum has become increasingly resistant to anti-malarial drugs and targeting a host protein may afford less scope for the development of resistance. Calpains are a family of calcium-dependent cysteine proteases whose physiological roles are poorly understood.

Hitting Cancer with an Iron Fist?

The energy demands of rapidly proliferating cancer cells require high levels of nutrients, including iron. Since cells are sensitive to free iron, they utilise iron storage proteins to protect themselves. Scientists at the German Cancer Research Center (DFKZ) and University Medical Center Mannheim, studying Sézary’s disease, have now shown that manipulation of free intracellular iron can induce apoptosis in T-cell lymphomas.

iron-rich riverSézary’s disease is an aggressive and ultimately fatal type of cutaneous T-cell lymphoma that is resistant to currently available treatments. Apoptosis resistance in leukemias and lymphomas is mediated by aberrant signalling of the NF-κB pathway. The researchers have demonstrated that cell death of cutaneous T-cell lymphoma cell lines induced by inhibition of the NF-κB pathway is a result of increased free intracellular iron and reactive oxygen species (ROS). Using T-cells from Sézary patients they show that inhibition of constitutively active NF-κB causes down-regulation of ferritin heavy chain (FHC) that leads to an increase of free intracellular iron, which, in turn, induces massive generation of ROS. The involvement of FHC was confirmed by direct down-regulation using siRNA.

Importantly, T cells isolated from healthy donors do not display down-regulation of FHC and, therefore, do not show an increase in iron and cell death upon NF-κB inhibition.

The work, published in the journal Cancer Research, suggests FHC as a novel target for therapeutic intervention in lymphoma.

Target Identification by Numbers

Historically, most drugs were discovered either by identifying the active ingredient from traditional medicines or by observing the pharmacological effect of compounds in living animals and it wasn’t until the 1960s that an understanding of the relationship between chemical structure and biological activity began to develop. Since then, attempts have been made to link discrete molecular targets (usually proteins) to particular diseases and to identify small molecules which will interfere with the function of these targets.

Contemporary drug discovery is dominated by this molecular target-based paradigm but, with the advent of high throughput cell-based assays, scientists are now also able to test compounds for a desirable activity in whole cells. A disadvantage of this approach is that the precise protein target is not always easy to identify but, writing in the journal PNAS, Ong et. al. have now described the use of quantitative proteomics (SILAC, stable isotope labelling with amino acids in cell culture) together with affinity enrichment to identify the protein targets.
SILACSILAC is a technique based on mass-spectrometry which allows detection of differences in protein abundance between samples of cells. The growth medium of one cell population contains normal essential amino acids whilst that of the other cell population contains arginine or lysine labelled with stable heavy isotopes (13C or 15N). The growing cells incorporate these amino acids into their proteins, reaching full incorporation after 5 population doublings and producing a characteristic mass shift (6 Da with 13C6-Arg or 8 Da with 13C615N2-Lys). If the cells grown in the presence of the ‘heavy’ amino acids are also treated with a test compound, any difference in the ratio of peak intensities in the mass spectrum for protein pairs between treated and untreated cells indicates that the protein is, directly or indirectly, a target of the test compound. The authors describe the application of the method to the identification of targets for kinase inhibitors and immunophilin binders.