Molecular BioSystems

doi: 10.1039/b614380fpmid: N/A

doi: 10.1039/b614381bpmid: N/A

doi: 10.1039/b613297apmid: N/A

In the section of members of the Editorial Board and their research groups highlight recent literature for the benefit of the community. This month the highlighted topics include high-throughput screening of mutant glycosyltransferases, the structure of a Mg channel/transporter and the use of t-RNA as a template for quantum dot synthesis and some items published recently in the RSC’s journals.

Jain, K. K.

doi: 10.1039/b611485gpmid: 17216033

The commercial potential of RNAi is assessed on the basis of successful translation of technology into applications in three areas: (1) drug discovery and research—currently the biggest segment; (2) potential therapeutic applications; and (3) the role of microRNA in molecular diagnostics. RNAi is an important method for analyzing gene function and identifying new drug targets that use dsRNA to knock down or silence specific genes. Sets of siRNAs focused on a specific gene class (siRNA libraries) have the capacity to greatly increase the pace of pathway analysis and functional genomics. RNAi plays an important role in drug discovery by facilitating target validation. The discovery of the role of microRNA (miRNAs) in various pathological processes opens up possible applications in molecular diagnostics, particularly that of cancer. The advantages of RNAi-based therapeutics over traditional pharmaceuticals include the capability for more specific therapies with small molecule siRNA. Drawbacks include the development of resistance in cancer and viral infections as well as the interferon effect. RNAi is closely related to gene therapy and the vectors developed for gene therapy are also being used for delivery of siRNAs. RNAi, along with other related technologies, will contribute to the development of personalised medicine. Although none of the RNAi-based drugs is in the market yet, some are in clinical trials. By the year 2010 the market for RNAi-based drugs is expected to be worth $3.5 billion and is expected to expand to $10.5 billion by the year 2015.

Corry, Ben

doi: 10.1039/b610062gpmid: 17216034

Ion channels play an essential role in the communication between and within cells. Here some of the different ion channel proteins and the roles they perform are introduced, before a discussion of the mechanisms by which they discriminate between different ion types and open and close to allow the passage of ions at the appropriate times.

Lim, Cheh Peng; Cao, Xinmin

doi: 10.1039/b606246fpmid: 17216035

The Signal Transducer and Activator of Transcription (STAT) family of proteins was first discovered in the 1990's as key proteins in cytokine signaling. Since then, the field has greatly advanced in the past 15 years, providing significant insight into the structure, function, and regulation of STATs. STATs are latent cytoplasmic transcription factors consisting of seven mammalian members. They are Tyr phosphorylated upon activation, a post-translational modification critical for dimerization, nuclear import, DNA binding, and transcriptional activation. In recent years, unphosphorylated STATs have also been observed to dimerize and drive transcription, albeit by yet an obscure mechanism. In addition, the function of cytoplasmic STATs is beginning to emerge. Here, we describe the structure, function, and regulation of both unphosphorylated and phosphorylated STATs. STAT isoforms from alternative splicing or proteolytic processing, and post-translational modifications affecting STAT activities are also discussed.

Ghosh, Indraneel; Stains, Cliff I.; Ooi, Aik T.; Segal, David J.

doi: 10.1039/b611169fpmid: 17216036

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.

Benjamin, Don; Colombi, Marco; Stoecklin, Georg; Moroni, Christoph

doi: 10.1039/b609448apmid: 17216037

Interleukin-3 (IL3) mRNA is intrinsically labile due to the presence of a destabilizing AU-rich element (ARE) that targets the transcript for rapid degradation. We review our experience with a sensitive reporter system where changes in IL3 mRNA stability are translated into increased/decreased green fluorescent protein (GFP) signals. A GFP reporter gene was fused to the full-length IL3 3′UTR containing the ARE motif that responds to regulatory signals that control transcript stability. The reporter system was tested against known IL3 mRNA stabilizing/destabilizing agents either through pharmacological treatment, siRNA knock-down of components of the decay machinery, mutation of the ARE motif, or in tumour lines harbouring stable IL3 mRNA. In all cases, the reporter transcript responds in an identical fashion to the endogenous IL3 message thereby verifying the fidelity of the system. This reporter system allows screening and identification of novel ARE-mRNA stabilizing compounds, or the isolation of mutants defective in ARE-mRNA turnover. We also report preliminary attempts to modify the system for high-throughput screening of an extensive compound library. The simplicity and effectiveness of this screen makes it ideal for screening of modulators of clinically important ARE-bearing transcripts such as TNFα, VEGF, the interferons and other cytokines.

Alluri, Prasanna; Liu, Bo; Yu, Peng; Xiao, Xiangshu; Kodadek, Thomas

doi: 10.1039/b608924kpmid: 17216038

Pharmacologic agents capable of activating the expression of specific genes would be valuable tools in biological research and could potentially be useful therapeutically. Efforts to develop a general solution to this problem have focused on the discovery of cell permeable mimics of native transcription factors comprised of linked DNA-binding and activation domain surrogates. Recently, we reported the isolation of a peptoid, called KBPo2, that binds a fragment of the mammalian coactivator CREB-binding protein (CBP). When delivered to a promoter-bound DNA-binding domain, this peptoid acted as a potent activation domain mimic in human cells. In this paper, we provide full details of the screening experiments and also report further characterization of this molecule as well as the other peptoids that came out of the screen. Of the three peptoids identified as putative CBP ligands, only KBPo2 demonstrated the necessary combination of binding affinity, specificity and cell permeability necessary to function as a potent activation domain mimic in cells. KBPo2 binds to CBP in a region different than that recognized by the native activation peptide from the transcription factor CREB.

Tomizaki, Kin-ya; Mihara, Hisakazu

doi: 10.1039/b609529apmid: 17216039

Protein kinases play important roles in signaling pathways that regulate many cellular biological processes, including apoptosis, cell growth, and differentiation in response to extracellular stimuli. Design of homogenous protein kinase assay platforms including design of potent protein kinase substrates is essential for exploration of the phosphoproteome. Here, we describe a unique chromism-based assay (CHROBA) technique for the direct measurement of protein kinase activities. The CHROBA is a novel chemosensor system that produces signals based on the photochromic and thermodynamic properties of a spiropyran derivative incorporated into peptide substrates. The CHROBA technique for detecting protein kinase activities involves the following five steps: (i) phosphorylation, (ii) photobleaching of the reaction mixture, (iii) addition of ionic polymer(s), (iv) incubation in the dark, and (v) signal readout. This simple ‘end-point’ assay method allows quantitative measurements of protein kinase A, Src protein tyrosine kinase, c-Abl protein tyrosine kinase, and protein kinase Cα activities even with excess ATP. Our results showed that spiropyran-containing peptide substrates with net charges between +2 and 0 are suitable for the present CHROBA method. This information should aid in the rational design of diverse protein kinase assay platforms. The present CHROBA technique can be adapted to a microplate format with both fluorometric and colorimetric readouts and would be useful for high-throughput drug discovery and analysis of the phosphoproteome.