THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3.

Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes.

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex.

Chemical and pathway proteomics: powerful tools for oncology drug discovery and personalized health care.

In recent years mass spectrometry-based proteomics has moved beyond a mere quantitative description of protein expression levels and their possible correlation with disease or drug action. Impressive progress in LC-MS instrumentation together with the availability of new enabling tools and methods for quantitative proteome analysis and for identification of posttranslational modifications has triggered a surge of chemical and functional proteomics studies dissecting mechanisms of action of cancer drugs and molecular mechanisms that modulate signal transduction pathways. Despite the tremendous progress that has been made in the field, major challenges, relating to sensitivity, dynamic range, and throughput of the described methods, remain.

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors.

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells.

Partial oncogenic transformation of chicken embryo fibroblasts by Jun dimerization protein 2, a negative regulator of TRE- and CRE-dependent transcription.

Jun dimerization protein 2 (JDP2) was identified as a bZIP protein that forms dimers with Jun proteins. JDP2 represses transcriptional activation of reporter constructs containing 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) or cyclic AMP responsive elements (CRE). JDP2, overexpressed by the avian retroviral vector RCAS, induces partial oncogenic transformation of chicken embryo fibroblasts.

Genomic organization, splice products and mouse chromosomal localization of genes for transcription factor Nuclear Factor One.

Transcription factor Nuclear Factor One (NFI) proteins are derived from a small family of four vertebrate genes (NFIA, B, C and X), all of which produce a fair number of protein variants by alternative splicing. In order to ultimately locate RNA signal sequences around exon/intron borders for the production of regulated splice variants, we have determined the exon structure of the chicken NFIB gene as the last of the four vertebrate genes for which the gene structure was not yet elucidated. This made it possible to compile nine newly isolated and sequenced mouse NFI cDNA sequences together with all previously available ones and to deduce corresponding splicing patterns for the orthologous vertebrate genes of all four paralogous gene types.