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Disulfide bond formation in vivo is linked to many essential intracellular processes; protein regulation and signaling, chemical transformations, and oxidative protein folding. Oxidative protein folding is an enzyme catalyzed process which is controlled by dedicated protein thiol oxidoreductases. In this work the oxidative protein folding within the mammalian endoplasmic reticulum (ER) is examined from an enzymological perspective. Evidence for the rapid reduction of PDI by reduced glutathione is presented in the context of PDI-first pathways. Next, strategies and challenges for the determination of the concentrations of reduced and oxidized glutathione and of the ratios of PDIred:PDIox is discussed. After a discussion of the use of natively encoded fluorescent probes to report the glutathione redox poise of the ER, a complementary strategy to discontinuously survey the redox state of as many redox-active disulfides as can be identified by ratiometric LC–MS–MS methods in order to better understand redox linked species. Next, we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Mia40 is found to be effective oxidant of non-cognate substrates, but is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40’s ability to bind amphipathic peptides tested by the insulin reductase assay. The consequences of these studies, mitochondrial oxidative protein folding, and the transit of polypeptides is discussed. Finally, the development of disulfide linked genetically encoded fluorescent probes for analyte-specific imaging are demonstrated. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here, novel probes were designed by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide hydrogenase, were successfully developed into thioredoxin and NAD+/NADH specific probes respectively and their performance was evaluated in vitro and in vivo. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in living cells.
Methodology and applications of redox proteomics The relatively new and rapidly changing field of redox proteomics has the potential to revolutionize how we diagnose disease, assess risks, determine prognoses, and target therapeutic strategies for people with inflammatory and aging-associated diseases. This collection brings together, in one comprehensive volume, a broad array of information and insights into normal and altered physiology, molecular mechanisms of disease states, and new applications of the rapidly evolving techniques of proteomics. Written by some of the finest investigators in this area, Redox Proteomics: From Protein Modifications to Cellular Dysfunction and Diseases examines the key topics of redox proteomics and redox control of cellular function, including: * The role of oxidized proteins in various disorders * Pioneering studies on the development of redox proteomics * Analytical methodologies for identification and structural characterization of proteins affected by oxidative/nitrosative modifications * The response and regulation of protein oxidation in different cell types * The pathological implications of protein oxidation for conditions, including asthma, cardiovascular disease, diabetes, preeclampsia, and Alzheimer's disease Distinguished by its in-depth discussions, balanced methodological approach, and emphasis on medical applications and diagnosis development, Redox Proteomics is a rich resource for all professionals with an interest in proteomics, cellular physiology and its alterations in disease states, and related fields.
This detailed volume presents a wide range of techniques for plant mitochondrial analysis, ranging from tried-and-tested work horse techniques to the latest innovations. Within its pages, it explores subjects such as affinity-based isolation of mitochondria with magnetic beads, mitochondrial quality assessment protocols, measurement of uptake and release of specific metabolites, mitochondrial protein identification and visualization, as well as gene splicing and editing, and much more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Plant Mitochondria: Methods and Protocols provides a highly useful set of methodologies for the plant mitochondrial community to help discover more interesting aspects of plant mitochondria in the years to come.
This volume compiles a broad range of step-by-step protocols, complementary to the ones published in the first edition of this book, to study various aspects of mitochondrial structure and function in different model organisms, both in vitro and in vivo. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Mitochondria: Practical Protocols, Second Edition aims to be useful for beginners as well as for experienced researchers in the field.
This report considers the biological and behavioral mechanisms that may underlie the pathogenicity of tobacco smoke. Many Surgeon General's reports have considered research findings on mechanisms in assessing the biological plausibility of associations observed in epidemiologic studies. Mechanisms of disease are important because they may provide plausibility, which is one of the guideline criteria for assessing evidence on causation. This report specifically reviews the evidence on the potential mechanisms by which smoking causes diseases and considers whether a mechanism is likely to be operative in the production of human disease by tobacco smoke. This evidence is relevant to understanding how smoking causes disease, to identifying those who may be particularly susceptible, and to assessing the potential risks of tobacco products.
Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.