Department of Preventive Medicine Seminar
Tuesday November 22, 201610 to 11:00 a.m.
Stamler Conference Room
680 N. Lake Shore Drive, Suite 1400
Chicago, IL 60611
Charles L. Farnsworth, PhD | 12/15/15 | 10:15AM | Olson Pavilion 8-260
The ability to identify and quantify changes in protein modification state levels in cell lines or tissue samples is critical for gaining insight into the biology and chemistry of
biological systems. PTMScan® Proteomics Technologies, developed at Cell Signaling Technology, provide comprehensive analytical profiling of post-translational
modifications (PTMs), including Ser, Thr, and Tyr phosphorylation, as well as acetylation, ubiquitination, and methylation. Discovery-mode studies, carried out with
PTMScan® Discovery Technology, identify PTMs throughout the proteome in a variety of biological model systems and disease states. Alternatively, targeted-mode studies,
performed using PTMScan® Direct Technology, allow for targeted screening of known PTMs on protein targets within a defined group of signaling pathways in response to a
drug treatment or in the context of a specific disease state. Both technologies will be outlined with an emphasis on their direct application to drug discovery research.
Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid.
Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized. All compounds were assayed with nNOS, their IC50, KI, and kinact values were obtained, and their crystal structures were determined. The identification and characterization of the products formed during inactivation provide evidence for the details of the inactivation mechanism. On the basis of these studies, the most probable mechanism for the inactivation of nNOS involves oxidative demethylation with the resulting thiol coordinating to the cofactor heme iron. Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.
The lab of Northwestern University researcher Neil Kelleher has developed an on-line system for clean up of top-down proteomics samples following gel eluted liquid fraction entrapment electrophoresis (GELFrEE) fractionation.
The system, detailed in a paper published this month in the Journal of Proteome Research, uses asymmetrical flow field-flow fractionation (AF4) to address what is a key challenge in top-down proteomic workflows, and could make top-down approaches more widely shareable as well as improve their applicability to clinical work, Philip Compton, director of instrumentation at NorthWestern's Proteomics Center of Excellence and an author on the paper, told GenomeWeb.