Hot off the Press

In the Hot off the Press section of Molecular BioSystems members of the Editorial Board and their research groups highlight recent literature for the benefit of the community. This month the highlighted topics include the use of small molecules to alter cellular functions, using quantum dots to identify leukemia cells, and a quantitative proteomic strategy for investigating yeast pheromone signalling.

---

Altering cellular functions with small molecules—novel use of ruthenium complexes

The search for novel drugs requires constant update of design strategies and novel and economic synthetic routes. Researchers at the University of Pennsylvania recently designed a small molecule probe utilising the complexing properties of metals and used it as a potent inhibitor of glycogen synthase kinase 3 (GSK3).

Eric Meggers and his colleagues based their principle design on naturally occurring staurosporine; the bidendate ligand functionality present in staurosporine was conserved in a synthetic variant. Their compound is shown to be a potent GSK3 inhibitor with a higher inhibiting activity than that of staurosporine.

Since GSK3 is a negative regulator of the wnt signaling pathway (wnt proteins form a family of signaling molecules that regulate cell-to-cell interactions during embryogenesis), their complex was tested as a pathway switch. The level of β-catenin was used as the activity indicator; when GSK3 is active, unstable phosphorylated β-catenin is produced, GSK inhibition leads to stabilisation of β-catenin, therefore its cellular level can be used as an indicator of GSK inhibition.

The switching of the wnt pathway was demonstrated in vivo using Xenopus laevis embryos whose phenotype is linked to the level of β-catenin. All of the embryos injected with the ruthenium compound demonstrated the expected phenotype proving that in vivo inhibition of GSK3 is possible and very effective. The significant finding was also that the new compound did not show any signs of cytotoxicity when tested on human embryonic kidney cells. The authors suggest that inert metal centers can be used as rigid structural scaffolds in the inhibitor's design thus leading to the production of highly potent molecular probes and drugs.

 

Douglas S. Williams, G. Ekin Atilla, Howard Bregman, Arpine Arzoumanian, Peter S. Klein, Eric Meggers, Angew. Chem. Int. Ed., 2005, 44, 1984

Reviewed by: Ljiljana Fruk, Universität Dortmund, Germany

Top-down approach to kinase specific target analysis

Advances in mass spectrometry have vastly increased the capacity to detect the multitude of phosphorylated proteins within biological samples. Facile identification of the kinase responsible for an individual phosphorylation event, however, remains a challenge. A recent communication from Kevan Shokat and colleagues at the University of California presents a methodology to address this challenge in a top-down approach.

The researchers showed that an engineered mutant allele of CDK1 (cyclin dependent kinase 1) possesses the ability to utilize the unnatural nucleotide A*TPγS and thereby introduce a thiophosphate on target proteins, i.e. “thiophosphorylation”. When exposed to the alkylating agent, PNBM (p-nitrobenzylmesylate), the enzymatically introduced thiophosphate group, along with other nucleophiles in the protein extract, becomes alkylated.

A key advance in the communication was the creation of an antibody, α-3-IgY, that specifically recognizes the thiophosphate alkylation product distinct from all other alkylation products in the extract. As a result this antibody can be used to isolate only those proteins or peptides that were “thiophosphorylated” by the mutant kinase, providing a pool of kinase targets that can be subsequently identified by mass spectrometry.

In short, this creative suite of reagents allows for kinase specific target analysis in the midst of a complex phospho-proteome.

 

Jasmina J. Allen, Scott E. Lazerwith, and Kevan M. Shokat, J. Am. Chem. Soc., 2005, 127(15), 5288

Reviewed by: Lyle Burdine, Center for Translational Research, University of Texas Southwestern Medical Center, USA

Identification of leukemia cells—a novel application of quantum dots

In recent years quantum dots (QDs) have been applied to many different systems. Particularly interesting is a new class of water soluble QDs which are also functionalized with groups that enable the coupling of biologically important molecules. Among the advantages of using these novel fluorescent markers as compared to organic fluorophores are higher brightness, higher stability against photobleaching and single excitation source needed for different colors.

Now Hideki Ohba and Rumiana Bakalova and their colleagues at the Single Molecule Bioanalysis Laboratory and Nagoya University, Japan used biologically compatible QDs for the identification of leukemia cells. Their approach is based on the ability of lectin molecules to interact with specific target oligosaccharides on the cancer cell surface. Using simple carboiimide chemistry they linked 3 different plant-derived lectins to water-soluble QDs. The QD–lectin conjugates were allowed to interact with leukemia cells from different lines and labeled cells were monitored using fluorescence confocal microscopy. When compared to the commercially available fluorophore (FITC), the QD–lectin conjugates showed higher fluorescence intensity and the stability of the signal was stable over a longer time (30 min compared to 5 min for FITC). The authors also suggest that they can calculate the percentage of leukemia cells in a mixture with normal lymphocytes when flow cytometry is used in detection. The worry about a possible negative effect of the biological activity of lectin was diminished by controlling the size of QD (3–5 nm in diameter, smaller than the usual 20–30 nm). The small size enabled 1 : 1, QD : lectin, conjugation therefore decreasing the possibility of cross-linking between the cells but still preserving the specificity of lectin cytoagglutination activity.

The simplicity of the QD–lectin conjugate preparation and two-step purification procedure demonstrates the viability of QD–lectins, in the words of the authors: “as appropriate and stable florescent markers for identification of several leukemia cell lines”. This is yet another valuable addition to biological uses of quantum dots.

 

Zhivko Zhelev, Hideki Ohba, Rumiana Bakalova, Rajan Jose, Satoshi Fukuoka, Toshimi Nagase, Mitsuru Ishikawa and Yoshinobu Baba, Chem. Commun., 2005, 1980

Reviewed by: Ljiljana Fruk, Universität Dortmund, Germany

Designing novel DNA bases—purine-like Ni(II) base pair

Christopher Switzer and colleagues from the University of California have designed a novel base that can be incorporated into oligonucleotides and could be used in the future to suppress or activate enzymatic activity. All of this was possible with a little help from Ni²⁺. By minimal modification of adenine (replacement of a 6-amino group with a pyridyl group) a novel base named Pur^P was obtained and 12mer oligonucleotides containing the base were then synthesized using standard triester methodology. The denaturation profiles of those oligonucleotides with different metals were obtained.

Ni²⁺ was the most stabilizing and therefore it was used in further studies. It was shown that Pur^P·Ni²⁺·Pur^P base pairs resemble normal base pairs dimensionally, but show greater stability than the C–G pair. Additionally, none of the natural bases make stable pairs with Pur^P·Ni²⁺. In fact, mismatches between Pur^P·Ni²⁺ and natural bases rival even the most severe natural mismatch such as C–A. Thus, the authors argue, metallo-base pairs could be incorporated into oligonucleotides and serve as ion activated switches for enzymatic activity.

 

Christopher Switzer, Surajit Sinha, Paul H. Kim, Benjamin D. Heuberger, Angew. Chem. Int. Ed, 2005, 44, 1529

Reviewed by: Ljiljana Fruk, Universität Dortmund, Germany

Investigation of yeast pheromone signaling by quantitative phosphoproteomics

Many techniques for determining the phosphorylation state of single, isolated proteins have been developed and successfully applied. In contrast, the analysis, particularly if quantitative, of protein phosphorylation on a proteome-wide scale poses significant challenges with respect to the selective isolation of phosphopeptides, their mass spectrometric analysis and the interpretation of the generated data.

Ole Jensen and co-workers present a proteomic strategy for the quantitative investigation of pheromone induced phosphorylation signalling in yeast. For the enrichment of phosphopeptides, a combination of strong cationic exchange and immobilized metal affinity chromatography was applied. Quantitation relied on stable isotope labelling by amino acids in cell culture and mass spectrometry. For an improved certainty of peptide identification and phosphosite mapping the high mass accuracy of a hybrid linear ion trap-Fourier Transform ion cyclotron resonance mass spectrometer in combination with three stages of mass spectrometry were used.

Overall 724 phosphopeptides (from 503 proteins) were identified and quantified in response to the α-factor pheromone. Of these, 139 were differentially regulated at least two-fold. Remarkably, in spite of the high complexity of the sample enriched in phosphopeptides, many proteins known to be involved in the pheromone signalling pathway were found.

 

Albrecht Gruhler, Jesper V. Olsen, Shabaz Mohammed, Peter Mortensen, Nils J. Færgeman, Matthias Mann and Ole N. Jensen, Mol. Cell Proteomics, 2005, 4, 310–327

Reviewed by: Bernd Bodenmiller, Institute for Molecular Systems Biology, ETH Hönggerberg, Switzerland

Function of the BRCA1/BARD1 heterodimer in DNA repair

The ability of a cell to respond to DNA damage is essential for its survival. Transcription-coupled repair (TCR), a process where DNA damage is repaired more rapidly on the actively transcribed strand, transiently inhibits mRNA transcription. This inhibition is thought to result from both a decrease in processing precursor mRNAs and the ubiquitylation and degradation of the elongating form of RNA polymerase II (RNAP II) by the proteasome. The heterodimer BRCA1/BARD1 is thought to play a role in the cell's response to DNA damage, but until now its function has remained elusive.

James Manley and colleagues have provided evidence for the mechanism of BRCA1/BARD1 inhibition of mRNA transcription in response to DNA damage. They show that the BRCA1/BARD1 heterodimer is responsible for ubiquitylation of the hyperphosphorylated, elongating form of RNAP II in vitro. In the presence of DNA damage knockdown of BRCA1/BARD1 protein levels with small interfering RNAs stabilized RNAP II levels and prevented inhibition of mRNA processing. These results provide an explanation of the function of BRCA1/BARD1 in the cellular response to DNA damage.

 

F. E. Kleinman, F. Wu-Baer, D. Fonseca, S. Kaneko, R. Baer, and J. L. Manley, Genes & Dev., 2005, 19, 1227–1237

Reviewed by: Chase Archer, Center for Translational Research, University of Texas Southwestern Medical Center, USA

Unexpected plasticity of the proteasome

The 26S proteasome is the large multi-protein complex responsible for most of the non-lysosomal proteolysis in eukaryotic cells. The proteasome is comprised of two major subunits. A barrel-like 20S core particle that contains internal proteolytic sites and a 19S regulatory particle that caps both ends of the 20S core. The traditional view of the proteasome catalytic cycle is that poly-ubiquitylated proteins are bound by the 19S cap after which six ATPase components both unwind the protein and feed the corresponding unstructured chain into the interior of the 26S barrel where they are degraded. An interesting issue is how the peptide products get out of the barrel.

Dorota Skowyra and colleagues recently presented evidence for a novel model for the catalytic cycle of the proteasome in which disassembly of the 26S is inextricably linked to the proteolytic event as well as ATP hydrolysis. They also provide evidence that this disassembly may require proteasome-associated proteins that co-immunoprecipitate with the 26S complex. Combined with earlier reports of non-proteolytic functions of the proteasomal ATPases, this report helps build the case that the 26S proteasome is far from a stable, monolithic species in cells, but rather is a highly dynamic assembly.

 

S. Babbitt, A. Kiss, A. E. Deffenbaugh, Y.-H. Chang, E. Bailly, H. Erdjument-Bromage, P. Tempst, T. Buranda, L. A. Sklar, J. Baumler, E. Gogol and D. Skowyra, Cell, 2005, 121, 553–565

Reviewed by: Thomas Kodadek, Center for Translational Research, University of Texas Southwestern Medical Center, USA

---