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 synthesis of the complete genome of bacterium Mycoplasma genitalium and how two oncoproteins act together to promote tumorigenesis and cell proliferation. New contributors are always welcome. If you are interested please contact molbiosyst@rsc.org for more information, we’d like to hear from you.


Synthetic solutions

Reviewed by: Tim Meredith, Harvard Medical School, Boston, USA

 

H. O. Smith and co-workers of the J. Craig Venter Institute report the complete synthesis of the entire Mycoplasma genitalium genome through the assembly of chemically synthesized oligonucleotides. A number of technical challenges inherent to manipulating large DNA fragments were overcome to achieve this feat. At ∼583 kilobase pairs, the work represents the largest DNA fragment assembled to date, easily eclipsing the previous mark of 32 kilobases. A highly convergent synthetic strategy was employed, that first joined a set of 5–7 kb synthetic cassettes containing overlapping ends using an in vitro recombination method to generate a library of plasmids that could be propagated as bacterial artificial chromosomes (BAC) in Escherichia coli. Progressive rounds of assembly using these products produced BACs that contained up to one fourth of the M. genitalium genome. The BACs were ultimately assembled by transformation-associated recombination in the yeast Saccharomyces cerevisiae to complete the first entirely synthetic bacterial genome. As previous work from this group has shown that isolated genomic DNA can be transplanted into suitably prepared recipient cells to give rise to a recombinant organism, the technical advances achieved in the present study make the eventual realization of highly engineered organisms feasible. Whereas traditional approaches for genome engineering rely on tedious rounds of sequential genetic manipulations, the current strategy can conceivably incorporate a limitless number of genetic changes in a single assembly. Synthetic genomes hold potential for numerous applications, from defining the minimal essential gene set to the production of “designer” organisms for biofuels production.

Daniel G. Gibson, Gwynedd A. Benders, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Holly Baden-Tillson, Jayshree Zaveri, Timothy B. Stockwell, Anushka Brownley, David W. Thomas, Mikkel A. Algire, Chuck Merryman, Lei Young, Vladimir N. Noskov, John I. Glass, J. Craig Venter, Clyde A. Hutchison III and Hamilton O. Smith, Science, 2008, DOI: 10.1126/science.1151721


ERK and MDM2 promote tumorigenesis by targeting the proteasomal degradation of FOXO3a

Reviewed by: Sharon Aviram, UT Southwestern Medical Center, Dallas, Texas, USA

 

A recent work by Jen-Yen Yang and co-workers from M. D. Anderson Cancer Center in Houston reveals a novel pathway which explains how two bona fide oncoproteins ERK and MDM2 act together to promote tumorigenesis and cell proliferation by inactivation of the tumor suppressor FOXO3a. The transcription factor FOXO3a controls the expression of genes involved in apoptosis (Bim and fasL), cell cycle arrest (Cyclin D and p27kip1) and DNA damage repair (GADD45a).

The data presented in this paper suggest that the expression levels of FOXO3a are regulated post-translationally by the MEK/ERK signaling cascade. FOXO3a is shown to be specifically phosphorylated by ERK at three serine residues; 294, 344 and 425, with the phosphorylated product being primarily detected in the cytoplasm, preventing FOXO3a regulation of gene expression. Foxo3a is known to be negatively regulated by other kinases; AKT, IKKβ and GSK have been shown to trigger FOXO3a nuclear export and cytosolic sequestering. This is likely also occurring in the case of ERK phosphorylation.

Further, the authors identify the E3 ubiquitin ligase that targets FOXO3a proteasomal degradation as MDM2.

In an elegant experiment using a non-phosphorylated FOXO3a mutant (all three ERK specific serine residues were changed to alanine) and a phospho-mimic FOXO3a (all three specific serine residues were replaced with asparagine) the authors demonstrate that ERK specific phosphorylation of FOXO3a is required for its interaction with MDM2. This interaction is highly reduced with the non-phosphorylated mutant.

Finally the non-phosphorylated FOXO3a mutant is shown to inhibit colony formation of breast cancer cells at a higher percentage when compared to WT and phospho-mimic mutants, a conclusion backed up by an in vivo tumor model in mice. These data strongly suggest that phosphorylation of FOXO3a by ERK inhibits its tumor suppressor activity.

Identification of ERK target proteins and downstream events is critical to the basic understanding of tumorigenesis and designing more sophisticated therapeutic strategies. This paper threads pieces of previously reported data into a novel pathway that explains one of the mechanisms by which the MEK/ERK signaling cascade promotes tumor development. For example this paper clarifies one of the ways by which ERK controls Cyclin D and P27kip1 expression to promote the G1/S transition. In addition, while the oncogene MDM2 is known to be upregulated in response to MEK/ERK activation and FOXO3a is known to go through proteasomal degradation, how these enzymes cooperatively contribute to tumor development was not understood.

The finding that FOXO3a is downregulated by the MEK/ERK pathway in addition to other signaling pathways is an important milestone, as constitutive activation of either MEK/ERK or Akt is frequently observed in human cancers. These two pathways can be simultaneously activated by a single stimulus (such as EGF), and can operate either cooperatively as well as independently to promote tumorigenesis. Therapeutic targeting against one of these pathways may result in resistance due to mutations that activate the second pathway. In this case, a therapeutic approach targeting a common downstream substrate, such as FOXO3a could prove to be very valuable.

J.-Y. Yang, et al., Nat. Cell Biol., 2008, 10, 138–148.


Protein Translocation Followed by FRET

Reviewed by: Ljiljana Fruk, University of Dortmund, Germany

 

One of the important issues to tackle in order to expand the potential of protein based drugs is the delivery of proteins into the cell. Different strategies have been used up to now to achieve this resulting in the collection of a number of different delivery vectors. However, it is still difficult to determine if protein is really translocated through the membrane in its functional form or trapped inside endocytic vesicles or degraded.

Now researchers from Texas A&M University have used FRET methodology to investigate the translocation of a model protein into the cells. In their studies they utilized red fluorescent protein mCherry which was labeled with green fluorophore (fluorescein) via expressed protein ligation. Using this procedure they ended up with extra cysteine which can be used for site specific labeling (via disulfide bond formation) with construct containing quencher DABCYL and delivery vector HIV 1-TAT ( dabcyl-DV). Dabcyl-DV acts both as the green fluorescence quencher and the promoter of protein transduction. The authors reasoned that if protein is trapped in endosomes, where the disulfide linkage is stable, fluorescence would be quenched, but in cytosol dabcyl-DV would be cleaved by cytosolic glutathione leading to restoration of green fluorescence. If this is true, FRET could be used to distinguish between intact protein successfully delivered to cytosol (both green fluorescence and FRET present, disulfide bond cleaved by cytosolic glutathione), protein trapped in endocytosomes (no FRET, no green fluorescence because of DABCYL quenching) or protein degraded in cytosol (green fluorescence but no FRET since mCherry needs to be properly folded and linked to the fluorophore to observe FRET). This was indeed the case when this model was studied in HeLa cells by live-cell confocal microscopy. It could be seen that a large fraction of the protein is trapped in endocytic compartments after transduction indicating that TAT mediated translocation in the cytosol is not very efficient. Additionally, using the FRET system it was also estimated that only a small portion of intact protein is released into cytosol. Taking into account the amount of information that can be obtained from this model system it can be envisaged that the efficiency of known as well as the identification of novel delivery vectors could be performed.

Y. J. Lee, S. Datta and J. P. Pellois, Real Time Fluorescence Detection of Protein Transduction into Live Cells, J. Am. Chem. Soc., 2008, 130, 2398–2399.


Au-DNA Based Nanolens

Reviewed by: Ljiljana Fruk, University of Dortmund, Germany

 

Gold nanoparticle–DNA (Au–DNA) conjugates have been used in a number of applications ranging from development of biosensors, intracellular target delivery or recently, development of designer nanocrystals (Nature, 2008, 451, 553). Now scientists from the Netherlands and Spain have developed well defined Au structures with high local plasmon fields using DNA assembly. It is known that aggregates of noble metals lead to production of “hot spots” – areas of high local fields which can be used in optical readout of molecules placed in their vicinity by spectroscopic techniques like surface enhanced Raman scattering (SERS). However, it is very difficult to control the extent of aggregation as well as the number of such hot spots for a given sample. To achieve the spacing of a few nanometres between Au nanoparticles, which is needed for production of plasmons, the researchers used DNA as a scaffold to bring together Au nanoparticles of different sizes (i.e. 5 and 18 nm) creating a plasmon based nanolens. They demonstrated that by using simple methodology based on DNA hybridization, control of the nanoparticle assembly can be achieved with a predicted four orders of magnitude enhancement enabling SERS measurements. In addition, the DNA backbone can be used to immobilize the molecules of interest close to the plasmon hot spots leading to the development of sensors with single molecule sensitivity.

S. Bidault, F. J. Garcia de Abajo and A. Polman, Plasomn Based Nanolenses Assembled on a Well Defined DNA Template, J. Am. Chem. Soc., 2008, 130, 2750–2751.

Hot off the RSC press


Peptide coupling for potential cancer targeting

Reviewed by: David Parker, Royal Society of Chemistry, Cambridge, UK

 

Putting peptide coats on virus shells could lead to targeted medical imaging in cancer diagnostics, according to US chemists.

Matthew Francis, at the University of California, in Berkeley, and his colleagues have attached different peptides to the outer shell (capsid) of an icosahedrally-shaped virus particle called bacteriophage MS2. The group has previously used such capsids to house gadolinium complexes, which have applications in medical imaging. With the aim of eventually building targeted imaging agents, the group attached the capsids to peptides that target different human tissues, including a breast cancer cell line.


Peptides are attached to viral capsids through unnatural amino acids (left).
Fig. 1 Peptides are attached to viral capsids through unnatural amino acids (left).

Bacteriophage MS2 particles are formed by the self-assembly of 180 copies of a single protein monomer. Francis’s team used Escherichia coli bacteria to express mutants of this protein that included a single para-amino-L-phenylalanine group per monomer. From several mutants, the researchers chose the one that formed in the highest yield, which held the included groups on the capsid’s outer surface. The team then used oxidative bioconjugation reactions to couple each peptide in turn to these surface groups and found that the capsids remained intact.

According to Francis, peptide coupling to protein structures such as bacteriophage MS2 is ‘a particularly difficult challenge, because the reactions used for this purpose must proceed site-selectively in the presence of the full set of amino acid functional groups.’ Also, modifying the proteins after they are made means many more, non-biocompatible groups can be incorporated.

Looking to the future, Francis suggested that his peptide coupling method could find applications in both biochemical and materials science fields. His team is currently investigating the modified capsids’ abilities to target tumours in vivo.

Z. M. Carrico, D. W. Romanini, R. A. Mehl and M. B. Francis, Chem. Commun., 2008, 1205–1207


DNA detection with a twist

Reviewed by: Freya Mearns, Royal Society of Chemistry, Cambridge, UK

 

US scientists have set DNA detection in a spin by exploiting one of nature’s molecular motors.

Wayne Frasch and co-workers from Arizona State University, in Tempe, have used enzyme F1-ATPase as the engine of a new DNA detection device. ATPases catalyse adenosine triphosphate (ATP) decomposition to produce energy. F1-ATPase can use this released energy to spin—it can act as a rotary motor.


Dynamic connection: target DNA forms part of a bridge between molecular motor F1-ATPase (bottom left) and a gold nanorod.
Fig. 2 Dynamic connection: target DNA forms part of a bridge between molecular motor F1-ATPase (bottom left) and a gold nanorod.

Frasch’s device works by coupling gold nanorods with F1-ATPases bound to a surface. Two short, labelled DNA strands complementary to a target DNA sequence are added to a DNA sample. If the target DNA is present, the strands bind to it side-by-side, forming a stiff DNA bridge with labels at each end (see figure). The labels used are molecules of biotin (shown in blue), a vitamin that binds strongly to the glycoprotein avidin (shown in green), which is found in egg white. When a solution of the DNA is dropped onto a surface coated with avidin-modified F1-ATPases, the DNA bridges bind by one end to avidin units using one biotin. An avidin-coated gold nanorod is then bound to the other end of each bridge. The F1-ATPase is made to spin by adding ATP and the gold nanorods also spin, being attached through the bridges. This can be detected simply using microscopy.

The system’s detection limit is fewer than 600 DNA molecules in solutions of femtomolar concentrations. Conventional fluorescence-based DNA detection systems have detection limits of only about five picomolar; when fewer targets are present, either multiple fluorescent molecules must be used for each target or DNA amplification, typically using the polymerase chain reaction (PCR), is needed to generate a detectable fluorescent signal. The nanorod sensing method ‘avoids the problems inherent to PCR and is much faster than current assays,’ said Frasch.

Ulf Landegren, an expert on DNA detection from Uppsala University, Sweden, said, ‘the critical question is how the device performs under field or regular lab conditions, where PCR and its related variants have dominated so far.’ Frasch admits that he has reported the results from ‘clean systems’, but his team is now repeating experiments using real samples.

According to the US scientists, their system lends itself to an easy kit-based protocol. But the really exciting thing, said Frasch, is that it is the first practical nanodevice that employs a molecular motor that really works.

J. York, D. Spetzler, F. Xiong and W. D. Frasch, Lab Chip, 2008, 8, 415–419


This journal is © The Royal Society of Chemistry 2008
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