Stephen E. Leonard

Bio: Stephen E. Leonard is an academic researcher from University of Washington. The author has contributed to research in topics: Sulfenic acid & Proto-oncogene tyrosine-protein kinase Src. The author has an hindex of 12, co-authored 13 publications receiving 1359 citations. Previous affiliations of Stephen E. Leonard include University of Michigan & Indiana Wesleyan University.

Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity

Abstract: A sensitive probe that detects protein sulfenylation in cells reveals that sulfenylation of the active site cysteine in EGFR enhances its kinase activity. Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor–mediated signaling results in H2O2 production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H2O2 at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.

Mining the Thiol Proteome for Sulfenic Acid Modifications Reveals New Targets for Oxidation in Cells

Abstract: Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox- mediated signal transduction; however, the identity of modified proteins remains largely unknown. We recently reported DAz-1, a cell-permeable chemical probe ca- pable of detecting sulfenic acid modified proteins directly in living cells. Here we describe DAz-2, an analogue of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfe- nome in a tumor cell line identifies most known sulfenic acid modified proteins: 14 in total, plus more than 175 new candidates, with further testing confirming oxida- tion in several candidates. The newly identified proteins have roles in signal trans- duction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Cross-comparison of these re- sults with those from disulfide, S-glutathionylation, and S-nitrosylation proteomes reveals moderate overlap, suggesting fundamental differences in the chemical and biological basis for target specificity. The combination of selective chemical enrichment and live-cell compatibility makes DAz-2 a powerful new tool with the potential to reveal new regulatory mechanisms in signaling pathways and identify new therapeutic targets.

A Periplasmic Reducing System Protects Single Cysteine Residues from Oxidation

Abstract: The thiol group of the amino acid cysteine can be modified to regulate protein activity. The Escherichia coli periplasm is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, many periplasmic proteins contain single cysteine residues, which are vulnerable to oxidation to sulfenic acids and then irreversibly modified to sulfinic and sulfonic acids. We discovered that DsbG and DsbC, two thioredoxin-related proteins, control the global sulfenic acid content of the periplasm and protect single cysteine residues from oxidation. DsbG interacts with the YbiS protein and, along with DsbC, regulates oxidation of its catalytic cysteine residue. Thus, a potentially widespread mechanism controls sulfenic acid modification in the cellular environment.

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Abstract: Oxidation of the thiol functional group in cysteine (Cys–SH) to sulfenic (Cys–SOH), sulfinic (Cys–SO2H) and sulfonic acids (Cys–SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting proteinsulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteineresidues.

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Cellular mechanisms and physiological consequences of redox-dependent signalling

Abstract: Reactive oxygen species (ROS), which were originally characterized in terms of their harmful effects on cells and invading microorganisms, are increasingly implicated in various cell fate decisions and signal transduction pathways. The mechanism involved in ROS-dependent signalling involves the reversible oxidation and reduction of specific amino acids, with crucial reactive Cys residues being the most frequent target. In this Review, we discuss the sources of ROS within cells and what is known regarding how intracellular oxidant levels are regulated. We further discuss the recent observations that reduction-oxidation (redox)-dependent regulation has a crucial role in an ever-widening range of biological activities - from immune function to stem cell self-renewal, and from tumorigenesis to ageing.

Chemistry and biology of reactive oxygen species in signaling or stress responses

Abstract: Reactive oxygen species (ROS) are a family of molecules that are continuously generated, transformed and consumed in all living organisms as a consequence of aerobic life. The traditional view of these reactive oxygen metabolites is one of oxidative stress and damage that leads to decline of tissue and organ systems in aging and disease. However, emerging data show that ROS produced in certain situations can also contribute to physiology and increased fitness. This Perspective provides a focused discussion on what factors lead ROS molecules to become signal and/or stress agents, highlighting how increasing knowledge of the underlying chemistry of ROS can lead to advances in understanding their disparate contributions to biology. An important facet of this emerging area at the chemistry-biology interface is the development of new tools to study these small molecules and their reactivity in complex biological systems.

Quantitative reactivity profiling predicts functional cysteines in proteomes

Abstract: Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs

Computational protein–ligand docking and virtual drug screening with the AutoDock suite

Abstract: Computational docking can be used to predict bound conformations and free energies of binding for small-molecule ligands to macromolecular targets. Docking is widely used for the study of biomolecular interactions and mechanisms, and it is applied to structure-based drug design. The methods are fast enough to allow virtual screening of ligand libraries containing tens of thousands of compounds. This protocol covers the docking and virtual screening methods provided by the AutoDock suite of programs, including a basic docking of a drug molecule with an anticancer target, a virtual screen of this target with a small ligand library, docking with selective receptor flexibility, active site prediction and docking with explicit hydration. The entire protocol will require ∼5 h.