Role for Tau in ‘Microtentacles’ and Metastasis

Image: Flickr – quinn.anya
Tau proteins interact with tubulin to stabilise microtubules and are abundant in neurons but less common in non-neuronal cells. Recently, tau proteins have received a bad press because of the association between tangles of hyperphosphorylated tau and Alzheimer’s disease and now researchers at the University of Maryland have reported that tau proteins may also play a role in tumour metastasis. Tau was found to promote formation of extensions of the plasma membrane or ‘microtentacles’ on breast cancer cells which break away from the primary tumour and circulate in the bloodstream. The microtentacles then increase the ability of the cells to attach to the walls of capillaries in the lung and seed new tumours. Tau protein has previously been associated with chemotherapy-resistant breast cancers and a poor prognosis but this is the first time that it has been implicated in metastasis. In the present study involving 102 breast cancer patients, 52% showed tau expression in metastases and 26% showed significantly increased tau expression as the disease progressed.

The team hope that drugs might be identified which will prevent the growth of microtentacles and inhibit tumour metastasis. Although current breast cancer treatments are often effective in treating the primary tumour, they are less effective in treating metastatic cancer which can develop years after the primary tumour is discovered and is the leading cause of death in cancer patients.

The study is published in the journal Oncogene.

Keeping out Newcomers Helps Virus Spread

Beach Huts
Image: Flickr - S John Davey
For more than 50 years, it has been supposed that the rate of spread of viruses is limited by replication kinetics in an iterative process of infection, replication and release, but scientists at Imperial College London have now challenged this view. Using live video microscopy, vaccinia virus was found to spread four times more quickly than should have been possible, based on the rate at which it can replicate. The videos showed that the virus spreads by surfing from cell to cell, using a mechanism that allows it to bounce past cells that are already infected and reach uninfected cells as quickly as possible. Soon after vaccinia infects a cell, two viral proteins, A33 and A36, are expressed at the cell surface, marking the cell as infected. When new viruses approach the infected cell, these proteins trigger the host cell to project actin ‘tails’ which physically repel approaching viruses. In this way, the viruses are propelled from cell to cell until they find one that is not already infected.

HSV-1 also spreads at a faster rate than should be possible given its replication rate and may use a similar spreading mechanism. If the ability to signal that a cell is already infected proves to be a common feature of pathogenic viruses, the discovery could eventually lead to new antiviral drugs that exploit this mechanism.

The study is published in the journal Science.

Opposing Roles for Brain Macrophages

Personality and the mask
Image: Flickr - Vagamundos
Systemic infection and inflammation lead to release of cytokines, such as IL-1, which activate the brain’s stress response mechanisms, producing typical symptoms such as lethargy, fever, and lack of appetite. In response to inflammation or infection, the hypothalamus releases corticotropin-releasing factor which, in turn, stimulates the pituitary gland to secrete adrenocorticotropic hormone. This then causes the adrenal glands to increase production of glucocorticoids, which both mobilise energy reserves to cope with the inflammatory insult and also act as powerful immunosuppressants, preventing excessive cytokine production and immune cell proliferation. Since cytokines are not able to freely cross the blood-brain barrier, exactly how they initiate this cascade of events has not been clear but researchers at the Salk Institute for Biological Studies have now begun to unravel the process.

It had been suggested that cytokines might interact with epithelial cells in the brain’s vasculature to produce prostanoids which act as secondary messengers transmitting the signal onwards. Epithelial cells are ideally positioned to receive inflammatory signals from circulating blood but need a very strong signal to become activated. In contrast, perivascular macrophages, a subset of brain-resident macrophages, are much more sensitive to cytokines but are not in direct contact with the bloodstream. To clarify the roles of both cell types, liposomes containing clodronate, which specifically deplete macrophages, were injected into the lateral cerebral ventricles of rats. This procedure abolished responses to IL-1 which activates prostanoid synthesis only in perivascular cells, but enhanced responses to LPS which stimulates prostanoid synthesis by both perivascular cells and endothelial cells. Resident macrophages lined up along the blood-brain barrier thus play opposing roles in the transmission of immune signals to the brain depending on the nature of the stimulus.

As well as clarifying the cellular mechanisms of CNS responses to inflammatory insults, the team hope that a better understanding of how immune signals are transmitted across the blood-brain barrier may also lead ultimately to new treatments for chronic neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Parkinson’s disease, Alzheimer’s disease and prion diseases, in which inflammation is believed to play an important role.

The study is published in the January 14th issue of Neuron.

More ‘Moonlighting’ Proteins – This Time in Yeast

Coenzyme A (CoA) - Wikipedia
Coenzyme A (CoA) - Wikipedia
Coenzyme A (CoA) is an indispensable cofactor in all living organisms, operating as an acyl carrier and carbonyl-activating group in a variety of biochemical transformations, including fatty acid metabolism. Many bacteria as well as plants and yeast are capable of de novo CoA biosynthesis from aspartate and ketovalerate via pantothenic acid. In contrast, animals and some pathogenic microbes lack a de novo route, and they are totally dependent on scavenging exogenous pantothenic acid (vitamin B5). Biosynthesis of CoA from pantothenic acid is an essential, universal pathway in prokaryotes and eukaryotes, comprising five steps. The third step is the decarboxylation of phosphopantothenoylcysteine to 4′-phosphopantetheine by phosphopantothenoylcysteine decarboxylase (PPCDC).

The gene involved in the formation of PPCDC has previously been identified in plants and humans, where the functional enzyme is a homotrimeric complex. Until now, however, the nature of the enzyme in the yeast Saccharomyces cerevisiae has been unclear. Researchers at Universitat Autònoma de Barcelona (UAB), Spain, in collaboration with the University of Stellenbosch, South Africa, have now unravelled the mystery.

S. cerevisiae appears to contain three genes capable of coding a PPCDC (HAL3, VHS3 and YKL088w), but none of them have been associated with this function. In recent years the UAB group has discovered that the genes HAL3 and VHS3 regulate the activity of a protein phosphatase involved in saline tolerance and in the cell cycle, but the new research has demonstrated that the proteins encoded by these genes have additional functionality. Unlike the plant and human counterparts, the S. cerevisiae PPCDC exists as a heterotrimer. One of these proteins is necessarily coded by the YKL088w gene and the others can be two molecules coded by either HAL3 or VHS3, or one of each. The active site in this case is made up of amino acids from two different proteins: the one coded by YKL088w, which provides a catalytic cysteine, and the one coded by HAL3 or VHS3, which provides a histidine, also essential for the catalysis. So, in S. cerevisiae, HAL3 and VHS3 have apparent multiple functions.

The research, published in Nature Chemical Biology, demonstrates that the heterotrimeric structure of PPCDC can exist in a wide group of yeasts from the Ascomycetes family. This group not only includes yeasts which are used in biotechnology and industry, such as S. cerevisiae and Pichia pastoris, but also potential pathogens such as Candida albicans. The difference between the PPCDC structure in these organisms and that of the human enzyme, together with its essential nature, makes it a potential target for antifungal therapy.

Extracurricular Role for ERK2 as a Transcription Factor

spiral structure
Image: Flickr - Andrew Stawarz
Scientists at Johns Hopkins School of Medicine set out to systematically map protein-DNA interactions across the human genome using a combination of bioinformatics and a protein-microarray strategy. Their exploration of the protein-DNA interactome identified over 17,000 interactions between 460 DNA sequences predicted to regulate transcription and 4191 proteins of varied functional classes. As well as finding known transcription factors (TFs), the work uncovered a large number of previously uncharacterised TFs. Of the unconventional DNA-binding proteins, over 300 exhibited sequence-specific DNA-binding.

One example of the sequence-specific DNA binders identified was ERK2 (MAP kinase-1), a serine/threonine kinase involved in cellular signalling. The Johns Hopkins team found that ERK2 is a transcriptional repressor of interferon-γ inducible genes and that this function is independent of its catalytic kinase activity. Whilst ERK2 has been extensively studied because of its importance in regulation of cellular proliferation, this unexpected additional role of ERK2 adds another level of complexity. Knowledge of the transcriptional repressor function may shed new light on knock-out experiments with ERK2.

The study, published in the journal Cell, examined only a fraction of the human proteome, raising the possibility that there may be thousands of proteins that also function as transcription factors. Certainly something else to consider in drug discovery when your enzyme inhibitor doesn’t do the same as the knockout!

Amitriptyline Directly Stimulates Trk Receptors

TrkA crystal structureNeurotrophins – a family of proteins essential for the development, survival and function of neurons – exert their actions through two classes of receptor, Trk tyrosine kinase receptors and p75NTR. There has been considerable interest in using neurotrophins such as nerve growth factor (NGF) for the treatment of neuronal damage as well as for conditions such as stress and depression, but the use of NGF itself has been limited by poor CNS penetration and side effects such as hyperalgesia. This has led to a search for stable small molecules with neurotrophic activity and specificity for TrkA or TrkB receptors although, so far, none of the mimetics can fully reproduce the effects of NGF in animals.

Writing in the journal Chemistry & Biology, researchers at Emory University School of Medicine have now shown that the tricyclic antidepressant, amitriptyline, which had been thought to act predominantly by blocking serotonin and noradrenaline transporters, interacts directly with the extracellular domain of both TrkA and TrkB receptors. Amitriptyline induced TrkA and TrkB homo- and heterodimerization and activation in mouse brain, but heterodimerization was found not to be required for Trk receptor activation. Truncation of the amitriptyline binding motif on TrkA, but not the corresponding region on TrkB, abolished the receptor homo- and heterodimerization. Amitriptyline, but not other tricyclic antidepressants or selective serotonin reuptake inhibitors, promoted amitriptyline and imipramine structure TrkA autophosphorylation in primary neurons and induced neurite outgrowth in PC12 cells. In mice, amitriptyline was further shown to suppress neuronal apoptosis caused by the neuroexitotoxin, kainic acid, in a TrkA-dependent manner. Inhibition of TrkA, but not TrkB, abolished the neuroprotective effect of amitriptyline without affecting its antidepressant activity.

Amitriptyline was found to bind to a motif in the first leucine-rich motif of the extracellular domain of the TrkA receptor with a Kd of 3 µM, which approximates to the brain concentration achieved when used to treat depression or neuropathic pain. Although significantly lower than the affinity of NGF for TrkA, it suggests that amitriptyline affinity for TrkA might be sufficient to explain at least some of its biological activity.

When Two Heads are not Better than One

Schistosomiasis is caused by infection with one of several species of parasitic worms and, although not usually fatal, is considered to be the second only to malaria in terms of human impact. Infection with the parasite typically occurs when wading, swimming or washing in water containing fresh water snails, the intermediate host for the parasite.

2-headed wormAlthough many countries are working to eradicate the disease by drug treatment, snail control, education and improved sanitation, the disease remains endemic in more than 70 countries and is estimated to affect around 200 million people worldwide. Schistosomiasis is readily treated with praziquantel but, despite being the mainstay of treatment for several decades, the mechanism of action of praziquantel is still debated. Writing in the journal PLoS Neglected Tropical Diseases, researchers at University of Minnesota Medical School have now shown how praziquantel kills a species of free-living flatworm that is often used as a model organism. These worms have remarkable regenerative properties and are able to reform a complete body from even a small fragment. Praziquantel was found to cause aberrant regeneration, producing two-headed organisms with duplicated, integrated central nervous systems and organs. The team further showed that voltage-operated calcium channel (VOCC) β subunits are important for the activity of praziquantel, supporting an earlier hypothesis about its mechanism of action.

The authors hope that elucidation of the mechanism of praziquantel toxicity – albeit in a free-living flatworm species – will help in the rational design of new antischistosomal drugs.

S1P1 Signalling Maintained Despite Receptor Internalisation

The immunomodulator, FTY720 (fingolimod) is currently undergoing phase III clinical trials for the treatment of relapsing-remitting multiple sclerosis. FTY720 is a prodrug that, once phosphorylated to FTY720-P, is believed to act primarily by targeting sphingosine-1-phosphate (S1P) receptors on lymphocytes and endothelial cells. This leads to retention of lymphocytes in lymph nodes that, in turn, prevents attacks on myelin sheaths.

FTY720-PAlthough FTY720-P is a potent agonist of several S1P receptors, its beneficial effects in multiple sclerosis are believed to be mediated primarily through the S1P1 receptor. Because specific knockout of the S1P1 receptor on haematopoietic cells in mice and treatment with FTY720 show similar effects on lymphocyte recirculation, the efficacy of FTY720-P has been attributed to ‘functional antagonism’ leading to complete internalisation and desensitisation of receptors. However, writing in the journal Nature Chemical Biology, scientists at Novartis now provide evidence that, despite internalisation, signalling by S1P1 receptors bound to FTY720-P persists for hours. Although calcium signalling – which depends on cell surface localisation of the receptor – was inhibited by treatment with FTY720, other signalling pathways remained activated. In both stably transfected and primary cell lines, persistent activation of S1P1 receptors by FTY720 led to prolonged inhibition of adenylate cyclase and increased ERK phosphorylation. Similar effects were not observed with the endogenous agonist, S1P, and by exploring analogues of FTY-720, the length of the aliphatic side chain was found to be crucially important for persistent signalling and receptor internalisation.

Shortening the lipophilic side chain by one methylene group decreased the effect and shortening by two methylene groups abolished the effect completely, despite all three compounds having similar intrinsic potencies. The ability of the S1P1 antagonist, WN146 – which does not itself induce lymphocyte sequestration – to inhibit prolonged S1P1 signalling caused by treatment with FTY720-P suggests that direct agonism rather than functional antagonism may be the predominant mechanism of action of FTY720-P.

New Insight into Action of Curcumin

turmericAs well as being used as a spice to add flavour and colour to a variety of dishes, turmeric has long been valued in parts of Asia for its medicinal properties. Although turmeric has been used for centuries as an antiseptic and has been claimed to be effective in a wide range of conditions including autoimmune diseases, heart disease, Alzheimer’s disease and cancer, it is only recently that scientists have begun to explore its properties in detail.

curcuminCurcumin, a molecule with a broad spectrum of antioxidant and anti-inflammatory properties, has been identified as the main active ingredient of turmeric and scientists at the University of Michigan have now described how curcumin acts in the body. Instead of interacting directly with numerous unrelated membrane proteins, the team found that curcumin regulates the action of membrane proteins indirectly by changing the physical properties of the bilayer.

Writing in the Journal of the American Chemical Society, the researchers describe in detail how curcumin inserts deep into the membrane in a transbilayer orientation, anchored by hydrogen bonds to the phosphate groups of lipids. The insertion into the membrane is similar to that seen with cholesterol and, like cholesterol, curcumin induces segmental ordering in the membrane. Using a combination of solid-state NMR and differential scanning calorimetry experiments, the team showed that curcumin has a strong effect on membrane structure even at low concentrations. The team plan to use similar experiments to explore the action of other drugs, such as capsaicin, which also interact with membranes.

MTAN Inhibitors Disrupt Bacterial Quorum Sensing

E coli O157Bacterial resistance – often arising as a result of over, or inappropriate, use of antibiotics – is a major obstacle to the treatment of many bacterial infections. Recently, interference with quorum sensing has emerged as a strategy for the development of new antibiotics which minimises the evolution of drug-resistant strains. Quorum sensing is a process used by bacteria to coordinate gene expression according to local population densities. The bacteria secrete signalling molecules, known as autoinducers, and have receptors that specifically recognize the signalling molecules released by other bacteria of the same or different species. Bacterial cell density and concentration of autoinducers control factors such as expression of virulence factors, pathogenicity and biofilm formation.

BuT-DADMe-Immucillin-AWriting in the journal Nature Chemical Biology, researchers from Albert Einstein College of Medicine of Yeshiva University have recently described the effectiveness of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) inhibitors against Vibrio cholera and Escherichia coli O157:H7. MTAN plays a key role in the synthesis of autoinducers essential for bacterial quorum sensing and the absence of the nucleosidase in mammals suggests that it is likely to be an attractive target for antimicrobial drug design. Three transition state analogue inhibitors of MTAN were found to be highly potent at blocking quorum sensing, bacterial virulence and biofilm formation. Importantly, the effect persisted for several generations.

Crystal structure of MTAN complexed with BuT-DADMe-Immucillin-A

See also this earlier article on quorum sensing.