Silent metabolites talk

Many microbial biosynthetic gene clusters that encode putative secondary metabolites are 'silent' under laboratory conditions, which prevents the isolation and characterization of the small molecules. Through genome mining in Aspergillus nidulans, Bergmann et al. found a gene cluster that seemed to encode a rare fungal hybrid polyketide synthase–nonribosomal peptide synthase; however no PKS-NRPS hybrid metabolite could be detected. By taking advantage of a putative gene activator found within the gene cluster, the authors induced production of the silent gene cluster and characterized new pyridone-containing metabolites, aspyridones A and B. This new strategy is likely to be generally applicable to inducing cryptic fungal gene clusters, and the identification of these metabolites provides new insights into A. nidulans metabolic pathways. [Letters, p. 213 ] JK

ATP and thiamine connect

Thiamine diphosphate, a form of vitamin B1, is an essential enzymatic cofactor. Recent biosynthetic investigations have highlighted that thiamine metabolites may have additional functions. A study in the current issue by Bettendorff et al. now reports the isolation and characterization of a new cofactor metabolite, adenosine thiamine triphosphate (AThTP). The authors had observed the accumulation of a new thiamine metabolite in Escherichia coli under carbon starvation conditions and posited that it was derived from thiamine diphosphate and ATP. After isolating the compound from bacteria, the authors determined its structure by spectroscopic methods and confirmed its identity by independent synthesis. The authors further demonstrated that AThTP formation is not restricted to prokaryotes, but is also found in yeast, higher plants and diverse animal cells. Although the molecular effectors and biological functions of AThTP remain to be explored, the discovery of a nucleotide metabolite of vitamin B1 suggests that there are aspects of vitamin metabolism that are still uncharted. [Brief Communications, p. 211 ; News & Views, p. 202 ] TLS

Kinases spill their secrets

Kinases are key regulators of cellular function, and their inhibitors can be useful for investigating kinase signaling pathways. However, the high degree of homology of the ATP binding sites within kinases makes exclusive inhibition of a single kinase very challenging. Blair et al. now report a new method for assessing kinase inhibition in vivo. Using insights from structural biology, the authors engineered kinase variants by mutating a bulky 'gatekeeper' residue and introducing a reactive cysteine residue as a site for covalent labeling. The resulting kinases were irreversibly and selectively modified with an engineered class of quinazoline kinase inhibitors. The authors used the approach to assess how the level of kinase function in epidermal growth factor receptor affects downstream kinase signaling. The general approach provides a new method for determining the effect of kinase inhibition in vivo, thus offering a way to dissect complex kinase signaling cascades. [Articles, p. 229 ] CG

Groovy peptides

During an immune response, major histocompatibility complex (MHC) proteins bind to and present antigenic peptides on the cell surface to induce a T-cell response. Studies of antigen presentation have been hampered by the lack of methods for visualizing the process in real time. Venkatraman et al. have developed a new fluorescence-based method to monitor peptide binding to MHC. Synthetic peptides were designed that incorporated environmentally sensitive fluorophores that bound hydrophobic pockets in the MHC binding groove. Binding of the modified peptides to MHC induced a 1,000-fold increase in fluorescence, which could be monitored in living cells by flow cytometry. Using this approach, the authors demonstrated that immature dendritic cells can efficiently present peptides. These probes could be used to investigate the dynamic regulation of peptide loading activity in living cells, and environmentally sensitive fluorophores can be applied to the study of other protein-protein interactions. [Letters, p. 222 ; News & Views, p. 201 ] JK

Less perplexed by G-quadruplexes

Guanine-rich nucleic acids can form diverse secondary structures in vitro, includingG-quadruplexes—four-stranded structures stabilized by planar arrays in which each of four guanines pairs with two neighbors by Hoogsteen bonding. G-quadruplexes are established elements in functionally important DNAs such as telomeres and ribosomal DNA, though they have yet to be ascribed a biological function. It is thought that in vivo, G-quadruplexes are more likely to be found in RNA than in DNA because of RNA's single-stranded nature and thermodynamic stability. Kumari et al. performed a computational search and found a new RNA G-quadruplex in the 5´ UTR of the NRAS proto-oncogene mRNA. This G-quadruplex folds into a parallel conformation and has remarkable thermodynamic stability under near-physiological conditions. In a reticulocyte lysate reporter system this G-quadruplex inhibits the translation of the proto-oncogene transcript. From bioinformatics scans of the human genome, the authors found over 3,000 RNA G-quadruplexes in other 5´ UTRs (including those of many proto-oncogenes) that may similarly modulate translation. [Letters, p. 218 ; News & Views, p. 203 ] MB

In This Issue written by Mirella Bucci, Catherine Goodman, Joanne Kotz and Terry L. Sheppard