Hot off the Press

Hot off the Press highlights recently published work for the benefit of our readers. Our contributors this month have focused on the modulation of gene expression through varying the secondary structure of mRNA, a new fluorescent biosensor to monitor reversible redox cycles in cells and molecular imaging agents for angiogenesis. New contributors are always welcome. If you are interested please contact http://molbiosyst@rsc.org for more information, we'd like to hear from you.

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Altered mRNA secondary structure modulates human catechol-O-methyltransferase expression

Catechol-O-methyltransferase (COMT) catalyzes the transfer of a methyl group from S-adenosylmethionine to catecholamines, including the neurotransmitters dopamine, epinephrine and norepinephrine. This O-methylation is associated with pain perception, cognitive function and affects mood. Two types of human COMT are known, encoded by the same gene; soluble COMT (S-COMT) and membrane-bound COMT (MB-COMT). Both COMTs consist of a 221-amino-acid sequence encoded by the same exons, but MB-COMT has an additional 50 N-terminal residues, encoded in two 5′-exons, which function as a hydrophobic membrane anchor.

It has been shown that three combinations of four SNPs (one in the S-COMT promoter region and three in the COMT coding region) form three haplotypes associated with pain responsiveness (low pain sensitivity (LPS), average pain sensitivity (APS), and high pain sensitivity (HPS)). The mechanism(s) by which the pain sensitivity phenotypes are expressed have been poorly understood. Differences in COMT expression levels and activity are observed, yet the promoter SNP makes no contribution to the pain phenotype. As the mRNA levels in all three haplotypes are similar, it was hypothesized that differences in COMT expression are the result of altered mRNA secondary structure, which could affect mRNA stability or protein expression.

Nackley and colleagues used the Mfold program to predict mRNA secondary structure for the three haplotypes. HPS, which formed the most stable folded mRNA, had the lowest COMT expression level and activity, while LPS with the least stable folded structure was expressed with the highest activity. The authors also used mutagenesis to destroy the stable loop in the HPS mRNA without altering the encoded amino acid sequence. The new mRNA provided similar levels of COMT expression and activity to that provided by LPS mRNA. Although the mRNA degradation rates of the three haplotypes are different, the total mRNA levels are similar. Therefore, the different COMT expression levels are likely to be caused by the efficiency of translation of the mRNAs. In the case of APS, a Val¹⁵⁸Met mutation in the coding region may also contribute.

To summarize, this work demonstrates that one of the mechanisms of modulation of COMT gene expression is through variation in mRNA secondary structure. The work emphasizes the role of silent mutations, which can result in stronger effects on COMT gene function than changes in amino acid sequence.

 

Nackley A. G., Shabalina S. A., Tchivileva I. E., Satterfield K., Korchynskyi O., Makarov S. S., Maixner W., Diatchenko L., Science, 2006 Dec 22, 314(5807), 1930–3

Reviewed by: Deanpen Japrung and Amy Mason, University of Oxford, UK.

Imaging of Reversible Cell Cycles in Living Cells

Unregulated production of reactive oxygen species (ROS) in cellular processes results in oxidative stress, which in turn can lead to a damage of proteins and nucleic acids, vital components of a healthy cell. Recently, however, it has been shown that ROS intermediates play an important role in cellular signal transduction. Among them hydrogen peroxide, in particular, was shown to take part in this process by reversible oxidation and reduction of cystein thiols. Although understanding the signal transduction pathways is a key for understanding causes and development of human diseases, following all the processes is a difficult task. Fluorescence imaging with sensitive chemosensors played a major role in a research of oxidative stress. However, the number and effectiveness of available fluorescent probes is limited and they usually respond irreversibly to a single oxidation event.

Now, E. W. Miller and colleagues from University of California report on a successful synthesis of REDOXFLUOR-1 (RF1) fluorescent compound which can be used in the detection of reversible redox cycles in living cells. RF1 was synthesised in 3 steps and the final molecule contained disulfides, which are often, in biology, used as redox reservoirs. Fluorescent probe had a flourescein like characteristics when oxidised while the treatment with reducing agents generated non-fluorescent form. When reduced, the sensor can return to its fluorescent state by reoxidation by air or hydrogen peroxide and this cycle can be repeated at least 10 times without the loss of emission intensity. This was also tested for imagining of reversible redox cycle in living cell. To make it membrane permeable, RF1 dye was additionally modified to give acetoxymethyl ester, which was then injected into live cells. In the native reducing environment of the cells, only faint fluorescence is observed. However, the fluorescence is increased significantly when the conditions are changed ie. by addition of hydrogen peroxide, which induces oxidative stress. Additionally, the cells remained viable even after few cycles of reversible fluorescence responses, which showed great promise of RF1 fluorescence sensor to be used in in vivo studies of oxidative signal pathways in biological systems.

Fig. 1 Figure showing the fluorescent dye activated and reactivated after time by oxidation by hydrogen peroxide. Figure reproduced with permission from E. W. Miller, S. X. Bian, C. J. Chang, J. Am. Chem. Soc., 2007, 129, 3458–3459. Copyright 2007 American Chemical Society.

 

E. W. Miller, S. X. Bian, C. J. Chang, J. Am. Chem. Soc., 2007, 129, 3458–3459.

Reviewed by Ljiljana Fruk, University of Dortmund, Germany.

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

Angiogenesis has been proven as an important therapeutic target which is suitable for complex and personalized treatment regimens. Advancements in molecular imaging of angiogenic vasculature could be very useful in identification of tumors and in the process of drug development, as well as to follow tumor-targeted therapies. Vascular endothelial growth factor (VEGF) receptors that are over expressed in angiogenic endothelium are particularly attractive targets for VEGF-based molecular imaging agents. However, most of the VEGF-based imaging agents that have been reported are unsuitable for clinical development for many reasons, including the uncertainty of binding, damage caused by random radio labeling, unacceptably high liver uptake and complex probe design.

Joseph M. Backer and his colleagues recently reported a new approach for the labeling of targeted proteins, with development of a 15-amino-acid Cys-tag, which contains a unique cysteine for site-specific modification by thiol-directed chemistries. They developed novel molecular imaging agents based on this new, robust, single-chain Cys-tagged VEGF (scVEGF) fusion protein that combines two fragments (amino acids 3–112) of human VEGF121 cloned head to tail. They showed that these scVEGF-based imaging agents can be readily prepared for near-infrared fluorescence imaging (NIRF), single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of VEGF receptors in angiogenic vasculature.

They labeled Cys-tag in scVEGF with three agents namely, Cy5.5 dye for NIRF imaging, 99mTc chelator hydraziniumpyridine (HYNIC) for SPECT imaging and a de novo–synthesized 64Cu chelator, PEG-DOTA, for PET imaging. First, they demonstrated that these site specific modifications of Cys-tag do not affect the functional characteristics of scVEGF. Their results elaborate high specific focal uptake of these imaging agents into the vasculature of tumors and surrounding host tissue in vivo. By using fluorescence contrast agents with endothelial cell markers, they showed that internalization is mediated by the receptors as well as long-term persistence of the image. They ruled out induction of angiogenesis or secondary tumors by these VEGF based imaging agents by considering the tracer dosages used for imaging as well as by referring to the reported data. This novel class of molecular imaging agents would facilitate therapeutic developments in the angiogenesis area.

 

Marina V. Backer, Zoya Levashova, Vimalkumar Patel, Brian T. Jehning, Kevin Claffey, Francis G. Blankenberg & Joseph M Backer., Nature Medicine, 2007, 13, 504–509.

Reviewed by: Gomika Udugamasooriya, Division of Translational Research, University of Texas Southwestern Medical Center, Dallas, TX USA.

Hot off the RSC Press

Mixed-up inhibitors

The future for research into a key biological complex is a lot brighter thanks to researchers in the Netherlands. Herman Overkleeft and colleagues at Leiden University have developed a series of new inhibitors for the proteasome, a human protein complex, simply by swapping parts of well-known inhibitors.

The proteasome has many functions, one of which is to break down proteins into peptides. These peptides can interfere with the immune system, and so inhibiting the proteasome could find application in treating auto-immune diseases, such as type I diabetes.

Overkleeft and his colleagues have developed a panel of proteasome inhibitors by scrambling the structural elements of the best-known peptide-based examples. Each combined an ‘electrophilic trap' (to bind to the protein complex) with a peptide sequence (to mimic the proteasome's natural substrates). Edward Tate, a chemical geneticist at Imperial College London, UK, suggested that ‘the inhibitors represent the first in a new generation of powerful chemical tools to probe specific and multiple activities of the proteasome.'

One of the new inhibitors proved to be ‘one of the most potent proteasome inhibitors reported to date,' said Overkleeft. It contains a boronic ester as the trap, similar to a group present in the drug bortezomib, which is used to re-sensitise cancers of the immune system to chemotherapy. The other half of the molecule is a tetrapeptide sequence from the natural product epoxomicin, a known proteasome inhibitor.

Overkleeft said that the challenge now lies in assessing how good the inhibitors are at inhibiting specific functions of the proteasome, and then developing better inhibitors using different traps and peptides. This should lead to a ‘better understanding of the proteasome as a drug target, and maybe also to the development of new therapeutics,' said Overkleeft.

Fig. 2 Mix-and-match inhibitors could lead to a better understanding of the proteasome.

 

Martijn Verdoes, Bogdan I. Florea, Wouter A. van der Linden, Didier Renou, Adrianus M. C. H. van den Nieuwendijk, Gijs A. van der Marel and Herman S. Overkleeft, Org. Biomol. Chem., 2007, 5, 1416

Reviewed by: David Barden, Royal Society of Chemistry, Cambridge, UK.

Pores for thought

Researchers in Switzerland have made artificial membrane pores that can recognise nucleotides.

Pores in cell membranes are channel-like structures, made from proteins, which allow specific molecules to pass into and out of the cell. These pores can also act as sensors, such as those found in taste receptor cells in the human tongue. Natural sensors like these usually rely on ion-pairing interactions to recognise different molecules.

But the synthetic pore, made by Stefan Matile and colleagues at the University of Geneva, uses a different kind of interaction altogether. Matile's pore is made from rigid rods functionalised with pentapeptides, which self-assemble into a barrel structure, just like that of a natural pore. The pentapeptides are themselves functionalised with electron-poor naphthalenediimide molecules. A pair of these electron-poor molecules can act as a clamp, sandwiching an electron-rich analyte such as a nucleotide (a sub-unit of DNA).

‘The pores are bioinspired, but it is wonderful to achieve this small victory over nature in utilising this kind of interaction,' said Matile. ‘It may even give us access to new analytes.'

Jean-Marie Lehn, an expert in molecular recognition from Louis Pasteur University, Strasbourg, France, said, 'this work will be very important in understanding how natural pores work, and for designing new types of sensor based on this technology.'

Fig. 3 The rigid backbone (black) has electron-poor clamps (blue) attached, which can recognise electron-rich analytes (red).

Matile says there are many potential applications for this kind of system, which could lead to diagnostic sensors for cholesterol, inhibitor screens for drug discovery, or even a synthetic tongue that could 'taste' the difference between sweet, sour and umami. ‘The next step is to convince potential investors that this is a viable proposition,' he said.

The synthesis of the pore is not easy, but Matile believes the effort pays off; the high activity of these sensors means that hundreds of thousands of assays can be performed using only milligram quantities of pores. ‘Potentially, all that is required in order to realise new applications is the appropriate functionalisation of the pentapeptide with the right type of clamp,' said Matile.

 

Hiroyuki Tanaka, Guillaume Bollot, Jiri Mareda, Svetlana Litvinchuk, Duy-Hien Tran, Naomi Sakai and Stefan Matile, Org. Biomol. Chem., 2007, 5, 1369.

Reviewed by: Stephen Davey, Royal Society of Chemistry, Cambridge, UK.

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