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CURATOR
A pinboard by
Marisa Duong

PhD Candidate, The University of Western Australia

PINBOARD SUMMARY

My research aims to develop analytical technique to pinpoint oxidised proteins in muscle tissues.

My field is proteomics, which is the study of proteins. Proteins are little machines in the cells of our body. They have fascinating functions such as forming the skeleton of our cells, catalysing our life chemistry, transporting substances into and out of the cells and around our body and so on. In many diseases, however, these proteins may be oxidised (by chemicals known as ROS) and thus rendered dysfunctional. In my research, I am developing biochemical methods that help measure the oxidation levels of thousands of proteins in the disease Duchenne Muscular Dystrophy. I hope to apply my analytical techniques to other disease models as well in the future.

4 ITEMS PINNED

In vivo ROS production and use of oxidative stress-derived biomarkers to detect the onset of diseases such as Alzheimer's disease, Parkinson's disease, and diabetes.

Abstract: Breakthroughs in biochemistry have furthered our understanding of the onset and progression of various diseases, and have advanced the development of new therapeutics. Oxidative stress and reactive oxygen species (ROS) are ubiquitous in biological systems. ROS can be formed non-enzymatically by chemical, photochemical and electron transfer reactions, or as the byproducts of endogenous enzymatic reactions, phagocytosis, and inflammation. Imbalances in ROS homeostasis, caused by impairments in antioxidant enzymes or non-enzymatic antioxidant networks, increase oxidative stress, leading to the deleterious oxidation and chemical modification of biomacromolecules such as lipids, DNA, and proteins. While many ROS are intracellular signaling messengers and most products of oxidative metabolisms are beneficial for normal cellular function, the elevation of ROS levels by light, hyperglycemia, peroxisomes, and certain enzymes causes oxidative stress-sensitive signaling, toxicity, oncogenesis, neurodegenerative diseases, and diabetes. Although the underlying mechanisms of these diseases are manifold, oxidative stress caused by ROS is a major contributing factor in their onset. This review summarizes the relationship between ROS and oxidative stress, with special reference to recent advancements in the detection of biomarkers related to oxidative stress. Further, we will introduce biomarkers for the early detection of neurodegenerative diseases and diabetes, with a focus on our recent work.

Pub.: 05 Apr '17, Pinned: 28 Jul '17

Levels of inflammation and oxidative stress, and a role for taurine in dystropathology of the Golden Retriever Muscular Dystrophy dog model for Duchenne Muscular Dystrophy.

Abstract: Duchenne Muscular Dystrophy (DMD) is a fatal skeletal muscle wasting disease presenting with excessive myofibre necrosis and increased inflammation and oxidative stress. In the mdx mouse model of DMD, homeostasis of the amino acid taurine is altered, and taurine administration drastically decreases muscle necrosis, dystropathology, inflammation and protein thiol oxidation. Since the severe pathology of the Golden Retriever Muscular Dystrophy (GRMD) dog model more closely resembles the human DMD condition, we aimed to assess the generation of oxidants by inflammatory cells and taurine metabolism in this species. In muscles of 8 month GRMD dogs there was an increase in the content of neutrophils and macrophages, and an associated increase in elevated myeloperoxidase, a protein secreted by neutrophils that catalyses production of the highly reactive hypochlorous acid (HOCl). There was also increased chlorination of tyrosines, a marker of HOCl generation, increased thiol oxidation of many proteins and irreversible oxidative protein damage. Taurine, which functions as an antioxidant by trapping HOCl, was reduced in GRMD plasma; however taurine was increased in GRMD muscle tissue, potentially due to increased muscle taurine transport and synthesis. These data indicate a role for HOCl generated by neutrophils in the severe dystropathology of GRMD dogs, which may be exacerbated by decreased availability of taurine in the blood. These novel data support continued research into the precise roles of oxidative stress and taurine in DMD and emphasise the value of the GRMD dogs as a suitable pre-clinical model for testing taurine as a therapeutic intervention for DMD boys.

Pub.: 10 Sep '16, Pinned: 28 Jul '17

Detection of ROS Induced Proteomic Signatures by Mass Spectrometry.

Abstract: Reversible and irreversible post-translational modifications (PTMs) induced by endogenously generated reactive oxygen species (ROS) in regulatory enzymes and proteins plays an essential role in cellular signaling. Almost all cellular processes including metabolism, transcription, translation and degradation have been identified as containing redox regulated proteins. Specific redox modifications of key amino acids generated by ROS offers a dynamic and versatile means to rapidly alter the activity or functional structure of proteins in response to biochemical, environmental, genetic and pathological perturbations. How the proteome responds to these stimuli is of critical importance in oxidant physiology, as it can regulate the cell stress response by reversible and irreversible PTMs, affecting protein activity and protein-protein interactions. Due to the highly labile nature of many ROS species, applying redox proteomics can provide a signature footprint of the ROS species generated. Ideally redox proteomic approaches would allow; (1) the identification of the specific PTM, (2) identification of the amino acid residue that is modified and (3) the percentage of the protein containing the PTM. New developments in MS offer the opportunity of a more sensitive targeted proteomic approach and retrospective data analysis. Subsequent bioinformatics analysis can provide an insight into the biochemical and physiological pathways or cell signaling cascades that are affected by ROS generation. This mini-review will detail current redox proteomic approaches to identify and quantify ROS induced PTMs and the subsequent effects on cellular signaling.

Pub.: 25 Jul '17, Pinned: 28 Jul '17

Limiting the Hydrolysis and Oxidation of Maleimide-Peptide Adducts Improves Detection of Protein Thiol Oxidation.

Abstract: Oxidative stress, caused by reactive oxygen and nitrogen species (RONS), is important in the pathophysiology of many diseases. A key target of RONS is the thiol group of protein cysteine residues. As thiol oxidation can affect protein function, mechanistic information about how oxidative stress affects tissue function can be ascertained by identifying oxidized proteins. The probes used must be specific and sensitive, such as maleimides for the alkylation of reduced cysteine thiols. However, we find that maleimide-alkylated peptides (MAPs) are oxidized and hydrolysed under sample preparation conditions common for proteomic studies. This can result in up to 90% of the MAP signal being converted to oxidized and/or hydrolysed MAPs, decreasing the sensitivity of the analysis. A substantial portion of these modifications were accounted for by Coomassie "blue silver" staining (~ 14%) of gels and proteolytic digestion buffers (~ 20%). More than 40% of the MAP signal can be retained with the use of thioglycolic acid during gel electrophoresis, trichloroethanol - UV protein visualization in gels, and proteolytic digestion buffer of pH 7.0 TRIS. This work demonstrates that is possible to decrease modifications to MAPs through changes to the sample preparation workflow, enhancing the potential usefulness of maleimide in identifying oxidised peptides.

Pub.: 30 Mar '17, Pinned: 28 Jul '17