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 enhancing the efficacy of RNA interference, identification of targets of kinase inhibitors, analysis of low-abundance peptides, and secretion of recombinant proteins.

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Enhancing the efficacy of RNA interference; longer double-stranded RNAs are the better gene suppressor

Short interfering RNAs (siRNAs), double-stranded RNAs of 21 base-pair (bp) in length, have been developed as a powerful tool for silencing of gene expression in mammalian cells. Recently, two independent research groups reported that relatively long double-stranded RNAs (25–30 bp in length) have significantly higher inhibitory potency than the conventional 21 bp siRNAs.

John J. Rossi and colleagues at the Beckman Research Institute of the City of Hope, California, USA demonstrated enhanced efficacy of RNA interference using synthetic RNAs of 27 bp in length with blunt ends.

Gregory J. Hannon and colleagues at the Cold Spring Harbor Laboratory, New York, USA demonstrated that double-stranded RNAs of 29 bp with hairpin loop structure improved the inhibitory efficacy.

Since the longer double-stranded RNAs can be up to 100-fold more potent than conventional siRNAs, the double-stranded RNAs of 25–30 bp in length enable us to use synthetic RNAs at lower concentrations that can avoid non-specific gene silencing which has been problematic in laboratory experiments as well as in future clinical settings.

 

D. H. Kim, M. A. Behlke, S. D. Rose, M. S. Chang, S. Choi and J. J. Rossi, Nat. Biotechnol., 2005, 23, 222–226

D. Siolas, C. Lerner, J. Burchard, W. Ge, P. S. Linsley, P. J. Paddison, G. J. Hannon and M. A. Cleary, Nat. Biotechnol., 2005, 23, 227–231

Reviewed by: Kazunari Taira, Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Japan

Kinase inhibitor target identification

Kinase inhibitors are the second largest therapeutic agents in the market. However, most of the kinase inhibitors are poorly characterized regarding their target specificity. A chemical genetic strategy of sensitizing kinases to small molecule inhibition, by Kevan Shokat and co-workers, provides insight into identifying cellular targets of kinase inhibitors. Reference profiles of the cellular effects generated by small molecule inhibitor GW400426 in analog sensitive alleles of cyclin-dependent kinases CDK1 and PHO85 of yeast were compared to the whole genome transcriptional profile. While CDK1 has been identified as a target of GW400426 both in vitro and in vivo, PhO85 is an in vivo target. Multiplex inhibition of both kinases accurately explained the transcriptional effects of GW400426 treatment.

 

C. Kung, D. M. Kenski, S. H. Dickerson, R. W. Howson, L. F. Kuyper, H. D. Madhani and K. M. Shokat, Proc. Natl. Acad. Sci. USA, 2005, 102(10), 3587

Reviewed by: Mani Upreti, Department of Internal Medicine and the Center for Translational Research, University of Texas Southwestern Medical Center, USA

Chemical labeling to analyze low-abundance peptides

A recurring problem in proteomics research is to analyze low abundance peptides in the presence of a vast excess of other peptides. For example, it is often the case that one would like to isolate all phosphopeptides or all peptides that contain sites of ubiquitylation. Thus, there is great interest in chemical labeling methods that allow selective enrichment of these species from complex mixtures.

Eric Peters and co-workers provide proof-of-principle that fluorous tagging can be a powerful tool for this purpose. Molecules containing multiply fluorinated (fluorous) chains can be separated from non-fluorous molecules easily on a fluorocarbon chromatography matrix. To a first approximation, the structural features of the molecule have little effect on its ability to bind to a fluorous column; only the number of fluorine atoms is critical. The unique properties of the fluorous interaction have been used extensively for the purification of small molecules and for tagging small compound libraries. This paper reports the first application of this powerful technology to proteomics.

The authors show that the classical base-catalyzed elimination of phosphoserine and threonine groups followed by addition of a fluorous thiol to the resulting dehydroalanine intermediate allows the facile enrichment of the product from complex mixtures. Of even more interest, they show that branched chain peptides, such as those resulting from proteolysis of ubiquitylated proteins, can be highly enriched by capping the N-termini with the activated ester of a fluorous carboxylate. The branched peptides pick up two, rather than one, fluorous chains and can thus be separated from non-branched products. In many ways, this technology is akin to the addition of biotin to a peptide and subsequent enrichment on streptavidin-coupled resins. However, release of the desired products from a fluorous column is simple whereas disruption of the streptavidin–biotin complex is usually inefficient.

 

S. M. Brittain, S. B. Ficarro, A. Brock and E. C. Peters, Nat. Biotechnol., 2005, DOI: 10.1038/nbt1076

Reviewed by: Thomas Kodadek, Departments of Internal Medicine and Molecular Biology and the Center for Translational Research, University of Texas Southwestern Medical Center, USA

Secretion of recombinant proteins into growth media

Engineered Escherichia coli are the simplest, cheapest, and highest-yielding method of obtaining large quantities of heterologous protein for diagnostic, therapeutic, or biotechnology purposes. However, most high level E. coli expression systems produce recombinant protein in the cytoplasmic or periplasmic space, which limits the production of toxic proteins and requires efficient cell collection and lysis procedures. It would be advantageous to design efficient systems to secrete recombinant proteins into the growth media, which could allow for continuous high-level expression and facile purification. Westerlund-Wikström and colleagues have described such a system that makes use of a modified type III secretion system, which secretes flagellum components directly to the exterior of the cell, bypassing the periplasm. They accomplished this by expression from the 5′-UTR (including the promoter) of the filament protein flagellin fliC, in some cases with the 3′-UTR of fliC, and/or a small N-terminal fragment of fliC. Using this system, they were able to secrete three heterologous outer surface proteins, the D1–D3 repeats of the fibronectin-binding protein from Staphylococcus aureus, the mature form of the adhesion Peb1 from Campylobacter jejuni, and α-enolase from Streptococcus pneumoniae, as well as a solubilized form of GFP into the growth medium. In all cases, the proteins appeared to be folded correctly and retained biological function. Remarkably, all four of these proteins were secreted into the medium at 1–8 mg L⁻¹, one to two orders of magnitude higher than previous secretion systems. The scope of this system is being investigated, and the authors note that an additional application is to identify adhesion and invasion proteins by whole genome screening of pathogenic bacteria.

 

K. Majander, L. Anton, J. Antikainen, H. Lång, M. Brummer, T. K Korhonen and B. Westerlund-Wikström, Nat. Biotechnol., 2005, DOI:10.1038/nbt1077

Reviewed by: Benjamin Gross, Harvard Medical School, USA

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