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One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of Tyrosine Phosphoproteome
No full textTyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance
Differentiation between Enamines and Tautomerizable Imines Oxidation Reaction Mechanism using Electron-Vibration-Vibration Two Dimensional Infrared Spectroscopy
No full textIntermediates lie at the center of chemical reaction mechanisms. However, detecting intermediates in an organic reaction and understanding its role in reaction mechanisms remains a big challenge. In this paper, we used the theoretical calculations to explore the potential of the electron-vibration-vibration two-dimensional infrared (EVV-2DIR) spectroscopy in detecting the intermediates in the oxidation reactions of enamines and tautomerizable imines with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). We show that while it is difficult to identify the intermediates from their infrared and Raman signals, the simulated EVV-2DIR spectra of these intermediates have well resolved spectral features, which are absent in the signals of reactants and products. These characteristic spectral signatures can, therefore, be used to reveal the reaction mechanism as well as monitor the reaction progress. Our work suggests the potential strength of EVV-2DIR technique in studying the molecular mechanism of organic reactions in general
Biochemical characterization of three new alpha-olefin-producing P450 fatty acid decarboxylases with a halophilic property
No full textBackground: The CYP152 family member OleT(JE) from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates.
Results: We biochemically characterized three new P450 fatty acid decarboxylases including OleT(JH), OleT(SQ) and OleT(SA), with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleT(JE)-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C-8-C-20) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleT(SA) revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C-8-C-12). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes.
Conclusion: The P450 fatty acid decarboxylases OleT(JE), OleT(JH), OleT(SQ) and OleT(SA) belong to a novel group of moderate halophilic P450 enzymes. OleT(JH) from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleT(JE). Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of alpha-olefin biohydrocarbons
