Blog Archives

The Right Tool for the Job?

tools

Image: Flickr - Janne Moren

Tool compounds are used to explore the role of a specific protein in a biological context and – it goes without saying – that to obtain meaningful results in a complex situation, the tool compound should have appropriate potency and selectivity. The family of phospholipase C (PLC) enzymes play important regulatory roles and a small molecule inhibitor, U73122, has been extensively used to provide evidence for the involvement of PLCs in many cellular pathways. Recent reports, however, have questioned the selectivity of U73122 and scientists at the University of North Carolina and GlaxoSmithKline have now discovered that, even in its interaction with PLCs, U73122 may not be quite what it seems. When the team explored the effects of U73122 on human PLCs in cell-free micellar systems, they found that the compound actually increased the enzymatic activity of a number of isoforms in a concentration- and time-dependent manner. At micromolar concentrations, U713122 increased the activity of PLCβ3 by up to eight-fold, that of PLCγ1 by more than ten-fold, and that of PLCβ2 by around two-fold; PLCδ1 was neither activated nor inhibited.

U73122 structure

U73122

Activation of PLCβ3 was attenuated by competing nucleophiles, suggesting that activation involves covalent modification of the protein by the reactive maleimide group of U73122; the analogous succinimide, U73343, was not effective as an activator. Involvement of specific cysteine residues in the protein was demonstrated by LC/MS/MS experiments. Although N-ethyl maleimide (NEM) itself did not activate PLCβ3, excess NEM attenuated the U73122-mediated activation in a concentration-dependent manner. The authors propose an activation model in which U73122 irreversibly binds to multiple cysteine residues on PLCβ3 and acts as either a lipid anchor or interfacial recognition site for the enzyme, facilitating adsorption to the substrate interface (i.e. the micelle surface). The protein-linked U73122 increases the rate of lipase activity by keeping the enzyme in close proximity to substrate which is held in the membrane.

The study, which is published in the Journal of Biological Chemistry, provides strong evidence that U73122 activates PLC enzymes in cell-free systems, in contrast to its ‘established’ role as a specific inhibitor of this family. The authors suggest that U73122 may have opposing effects on cytosolic and membrane-bound enzymes and/or may modify other cellular nucleophiles and advise great caution when forming hypotheses based on the observed effects of U73122 in cellular systems.


Study Casts New Light on Prozac Mechanism

Moonlight

Image: Flickr - Cindy See

Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac®) have been used to treat depression for more than three decades but researchers from INSERM and Hoffmann-La Roche have now shed new light on their mechanism of action. SSRIs are believed to act by inhibiting uptake of serotonin into presynaptic cells, thereby increasing the amount available in the synapse to bind to postsynaptic receptors. Typically, an ‘adaptation phase’ of several weeks is needed before the antidepressant effects are fully manifest and the new study helps to explain this latency. The study identified a key role for microRNA-16 (miR-16) in regulating expression of the serotonin transporter (SERT) which is responsible for the recapture of serotonin. Under normal conditions, SERT is present in serotonergic neurons where levels of miR-16 are low, but expression is silenced in noradrenergic cells by higher levels of miR-16; a reduction of miR-16 in noradrenergic cells causes de novo SERT synthesis.

In mice, chronic treatment with fluoxetine was shown to increase levels of miR-16 in serotonergic cells, leading to reduced SERT expression. The cells also released the neurotrophic factor S100β, which decreased miR-16 in noradrenergic cells, resulting in cells with a mixed phenotype that produced both noradrenaline and serotonin and which were sensitive to fluoxetine. Treatment with fluoxetine thus increases serotonin levels both by preventing reuptake by serotonergic neurons and by stimulating production by noradrenergic neurons through reduction of miR-16.

The study is published in the journal Science.


Stomach Bugs Need Vitamin B6

H Pylori electron micrograph

Electron micrograph of H. pylori showing multiple flagella. Image: Wikimedia Commons - Yutaka Tsutsumi, M.D. Professor Department of Pathology Fujita Health University School of Medicine

Approximately half of the world’s population is infected with Helicobacter pylori, the bacterium that causes peptic ulcers and some forms of stomach cancer. Although ‘triple therapy’ with a proton pump inhibitor and two antibiotics – selected from a very limited number – can eradicate H.pylori, an increasing number of people are found to be infected with antibiotic-resistant bacteria. Scientists in Australia, New Zealand and France have now shown that H.pylori needs vitamin B6 to establish and maintain chronic infection, and have identified two genes in the vitamin B6 biosynthesis pathway as potential targets for new antibiotics.

The team used an established technique known as in vitro attenuation to create variants of a mouse-colonising strain of H.pylori with low infectivity and then compared the gene expression profiles of the attenuated bacteria with the original highly virulent strain. The most significant changes were found to be in the genes that encode homologues of the Escherichia coli vitamin B6 biosynthesis enzymes, PdxA and PdxJ, which catalyse sequential steps in the pathway. In vitro, H. pylori PdxA mutants could only be recovered when pyridoxal-5’-phosphate, the bioactive form of vitamin B6, was added to the growth medium whereas it was not possible to produce viable bacteria with mutated PdxJ. PdxA was also shown to be necessary for H. pylori to establish a chronic infection in mice.

Further studies showed that, in addition to its well known metabolic roles, vitamin B6 is needed for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. The study, which is published in the new open access journal mBio, suggests that Pdx enzymes, which are present in a number of human pathogens, but not in mammalian cells, may present attractive targets for new antibiotic medicines.


Common Pathway for Neurodegenerative Diseases

pathway

Image: Flickr - A_of_DooM

Nitrosylation of proteins is emerging as a key post-translational modification important in both normal physiology and a wide spectrum of diseases, including neurodegenerative diseases. Physiological levels of nitric oxide (NO) can be neuroprotective, in part at least, by inhibiting caspase activity, but excess NO production leads to activation of cell death signalling cascades involved in many neurodegenerative disorders. Neuronal cell injury and death, which are prominent features of disorders such as Alzheimer’s, Huntington’s, and Parkinson’s diseases, are often mediated by the caspase family of cysteine proteases. Caspase activity is inhibited by S-nitrosylation and is also regulated by inhibitors of apoptosis such as X-linked inhibitor of apoptosis (XIAP) which associates with active caspases and represses their catalytic activity. XIAP also functions as an E3 ubiquitin ligase, targeting caspases for degradation by the proteasome.

A team of scientists led by Sanford-Burnham researchers have now discovered a new twist in caspase regulation. They showed that S-nitrosylation of XIAP (forming SNO-XIAP) inhibits the protein’s E3 ligase and antiapoptotic activity and also found that XIAP can be transnitrosylated by SNO-caspase but not vice versa. They found significant amounts of SNO-XIAP, but not SNO-caspase, in the brains of individuals with neurodegenerative diseases, suggesting that SNO-XIAP contributes to neuronal injury or death. The team hope that their study, which is published in the journal Molecular Cell, might lead to better biomarkers and earlier diagnosis for neurodegenerative diseases.


PPARγ– A New Twist in the Tale

Twists

Image: Flickr - alexdecarvalho

Obesity and related disorders such as diabetes have reached epidemic proportions. Although the anti-diabetic thiazolidinediones (glitazones) are effective insulin sensitizers, some members of the class have been withdrawn or had their use restricted because of safety concerns. Increased responsiveness to insulin is believed to be mediated by activation of the nuclear receptor, PPARγ but differences in clinically important side effects suggest subtle differences in pharmacology, even amongst full agonists.

Researchers at the Scripps Research Institute and the Dana-Farber Cancer Institute at Harvard University have now shown that cyclin-dependent kinase 5 (Cdk5) in adipose tissue is activated in obese mice fed a high-fat diet, resulting in phosphorylation of PPARγ. This has no effect on the adipogenic capacity of PPARγ but does alter the expression of a large number of obesity-related genes, including a reduction in expression of the insulin-sensitizing adipokine, adiponectin. Phosphorylation of PPARγ by Cdk5 was blocked both in vitro and in vivo by the full agonist, rosiglitazone, and by the partial agonist, MRL-24, leading to increased adiponectin production. The anti-diabetic effect of rosiglitazone in obese patients was also found to be closely associated with inhibition of PPARγ phosphorylation, suggesting that this may be a mechanism of insulin resistance. The authors of the study, which is published in the journal Nature, suggest that drugs that inhibit PPARγ phosphorylation by Cdk5, without necessarily activating the receptor, may provide an improved generation of anti-diabetic drugs.


Saying it with Flowers

flowers

Image: Flickr - graftedno1

The process of cell competition is believed to provide a mechanism to optimise tissue ‘fitness’ during development by eliminating weaker cells from the overall cell population. First described in Drosophila, a number of genes have been linked to cell competition but the precise details of the process are poorly understood. A new study conducted by scientists at the Spanish National Cancer Centre (CNIO), however, has furthered our understanding.

Using a combination of genomic analysis and functional assays, the team investigated how cells of Drosophila wing imaginal discs distinguished ‘winner’ and ‘loser’ cells. They found that six genes were upregulated early in loser cells and five of these encoded cell membrane proteins, suggesting that cell-cell communication is critical in the initial stages of cell competition. One of these membrane proteins, Flower (Fwe), was examined in detail.

Fwe is conserved in multicellular organisms and in the Drosophila study was found to be required and sufficient to label cells as winners or losers. The win/lose decision is mediated by three differentially expressed forms of fwe (fweubi, fweLoseA and fweLoseB) and cells are identified as losers when relative differences in fweubi and fweLose levels are detected – stress conditions that uniformly affect the entire population result in cell survival. Although further work is necessary to elucidate the detail, the team proposes that, in outcompeted cells, the fwe transcript is alternatively spliced and fweLose isoforms are induced at the expense of fweubi. It is likely that downregulation of fweubi and upregulation of fweLose both contribute to establish the lose/win decision.

The cellular tagging by Flower isoforms may have biomedical implications beyond cell competition since imbalances in cell fitness also occur during ageing, cancer formation and metastasis.

The study is published in Developmental Cell.


How ApoE4 May Increase Alzheimer’s Risk

neurons

Image: Flickr - fbobolas

ApoE is a lipid transport protein with roles in transport of dietary lipids, regulation of plasma cholesterol, and protection from atherosclerosis. In humans, there are three variants of ApoE (ApoE2, ApoE3 and ApoE4) and one of these, ApoE4, has been linked to earlier onset of Alzheimer’s disease. The mechanisms underlying the increased risk of Alzheimer’s disease remain unclear but researchers at UT Southwestern Medical Center have now shown that the ApoE4 variant reduces surface expression of receptors involved in synaptic plasticity by sequestering the receptors inside the cell.

ApoE interacts with members of the LDL receptor family and one of the receptors for ApoE, Apoer2, also acts as a signalling receptor for reelin, a protein that is important in the developing brain but also enhances NMDA receptor activity and increases long-term potentiation (LTP) in the adult brain. ApoE4 was found to reduce surface expression of NMDA and AMPA receptors as well as Apoer2 receptors, thereby impairing glutamatergic neurotransmission. β-Amyloid peptide, a hallmark of Alzheimer’s disease, suppresses LTP and the ability of reelin to counter the effects of β-amyloid peptide was almost completely abolished in mice expressing human ApoE4. The team are now trying to understand whether it is possible to build on their findings to develop new treatments for Alzheimer’s disease.

The study is published in the Proceedings of the National Academy of Sciences.


Dual-receptor T-cells may Trigger MS

mushrooms

Image: Flickr - didier.bier

Multiple sclerosis (MS) is an autoimmune disorder in which T-cells attack and damage the fatty myelin sheaths around the axons of the brain and spinal cord, disrupting the conduction of electrical signals along the nerve fibres. Although both genetic factors and viral infections have been suggested to contribute to the development of MS, no single virus has been conclusively linked to the disease and other mechanisms could also play a role. Animal models induced by CD8+ T-cells show similarities to human MS, and researchers at the University of Washington investigating the causes of MS have engineered mice that over-express CD8+ T-cells that recognise myelin basic protein (MBP), a candidate autoantigen in MS.

When infected with vaccinia virus engineered to produce MBP, the infection should activate the CD8+ T-cells to attack virally infected cells and also other cells that produce MBP. As expected, mice infected with the engineered virus developed MS-like disease but, surprisingly, symptoms were also triggered by infection with wild-type virus. This suggested that the engineered CD8+ T cells expressed a second receptor that recognised wild-type virus and subsequent cross-breeding experiments confirmed that some of the CD8+ T cells did indeed have receptors for both MBP and wild-type virus. Once activated by the virus, the dual-receptor CD8+ T cells were than able to attack cells producing MBP.

The study suggests a role for dual-receptor cells in autoimmune diseases and could explain how infection with a common virus triggers MS in genetically predisposed people, whilst having no lasting effects in most of the population. In the ‘dual-receptor model’, autoimmune activation could be triggered by a chance event leading to T-cells that recognise both MBP and a viral antigen. The prevalence of dual-receptor T cells is presently unclear and the team plan to assess whether they are more common in MS patients.

The study is published in Nature Immunology.


Adenosine may Mediate Effects of Acupuncture

acupuncture chart

Ming dynasty acupuncture chart - Wikimedia Commons

The origins of acupuncture are lost in antiquity and, today, the effectiveness of such treatment remains controversial. Despite the sometimes extravagant claims of proponents, there is little scientific basis to explain how acupuncture works and many researchers believe that the effects are attributable to a placebo effect. In a study published in Nature Neuroscience, researchers led by a team at the University of Rochester Medical Center have now suggested a physiological mechanism that may explain the analgesic effects of acupuncture.

The team found that adenosine was released during acupuncture in mice and that the analgesic effect of acupuncture could be replicated by direct injection of an adenosine A1 receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA). Adenosine, which is released in response to injury or inflammation, has known pain-relieving properties and acupuncture was found to be ineffective in A1 receptor knock-out mice. 2′-Deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase, was found to boost the effects of acupuncture, increasing the accumulation of adenosine in tissue as well as the duration of analgesia.

It will be interesting to see whether similar effects are observed in human subjects and also whether adenosine is also released during ‘sham’ acupuncture treatment in which needles are pressed against the skin without puncturing it – lack of differentiation from sham treatment is one of the main reasons that detractors cite for the placebo effect of acupuncture.


The Mysteries of Arsenic

arsenolite

Arsenolite

Image: Wikimedia Commons

For many people, the word ‘arsenic’ conjures up thoughts of murder mysteries and, in fact, arsenic has been a popular murder weapon since the middle ages. In the Victorian era, arsenic trioxide found favour as a cosmetic and it has also been used in both Chinese and Western medicine. Most recently, arsenic trioxide has been used to treat acute promyelocytic leukaemia (APL) that is unresponsive to first line therapies.

Arsenic trioxide is able to induce complete remission in patients with relapsed or refractory APL and is generally well-tolerated with minimal chemotherapy-related side effects. How arsenic trioxide kills cancer cells is not clear but scientists in China and France believe they have made a key step towards solving the mystery. APL cells are characterised by the occurrence of chromosomal translocations involving the retinoic acid receptor α gene (RARα)and the promyelocytic leukaemia gene (PML). These translocations lead to production of a fusion protein, PML-RORα that has altered functions and protects the cells from apoptosis. Arsenic trioxide triggers small ubiquitin-like modifier (SUMO) proteins to tag PML-RORα as part of a degradation mechanism. The new study has shown that the arsenic binds directly to cysteine residues in zinc fingers located within RBCC (N-terminal RING finger/B-box/coiled coil) domains of PML causing cross-linking and oligomerisation. The aggregated protein then undergoes SUMO modification and degradation. The identification of PML as a direct target of arsenic trioxide provides new insights into how the drug is able to treat APL and may lead to new treatment options.

The study is published in the journal Science.