Cellular effects of gliotoxin: Evaluation of a proteomic, isotope-based method to detect reactive cysteines

Cysteinyl residues in proteins are important for many cellular processes and unregulated modification of the cysteine thiol group can have negative effects on cell vitality and viability. In this thesis, the potential for use of the isotope coded affinity tag (ICAT) method for detection of cysteine modification has been investigated. ICAT reagents label free cysteine thiols. The aim of this study was to use HL-60 cells treated with gliotoxin, a fungal metabolite with a reactive disulfide bridge, as a system to evaluate the performance of ICAT for identification of cysteine modification in a whole cell proteome. Gliotoxin has antimicrobial, antitumor, immunosuppressive and cytotoxic properties that have been related to cysteine modification in proteins. Cellular assays including viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell cycle analysis, and measurement of reactive oxygen species using dichlorofluorescin diacetate were used to establish conditions for measuring the effects of gliotoxin on HL-60 cells prior to large-scale cellular damage. Cells exposed to gliotoxin and control cells were then labeled with ICAT reagents and analysed by offline reversed phase liquid chromatography followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. The pilot results identified tubulin, glyceraldehyde-3-phosphate dehydrogenase and peptidyl-prolyl cis-trans isomerase as putative targets of gliotoxin. Additionally, this study showed that ICAT can be used to detect modified cysteines from a highly complex sample, but further optimization is needed to unlock the full potential for detection of cysteine modification in complex samples

Cellular effects of gliotoxin: Evaluation of a proteomic, isotope-based method to detect reactive cysteines

Cysteinyl residues in proteins are important for many cellular processes and unregulated modification of the cysteine thiol group can have negative effects on cell vitality and viability. In this thesis, the potential for use of the isotope coded affinity tag (ICAT) method for detection of cysteine modification has been investigated. ICAT reagents label free cysteine thiols. The aim of this study was to use HL-60 cells treated with gliotoxin, a fungal metabolite with a reactive disulfide bridge, as a system to evaluate the performance of ICAT for identification of cysteine modification in a whole cell proteome. Gliotoxin has antimicrobial, antitumor, immunosuppressive and cytotoxic properties that have been related to cysteine modification in proteins. Cellular assays including viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell cycle analysis, and measurement of reactive oxygen species using dichlorofluorescin diacetate were used to establish conditions for measuring the effects of gliotoxin on HL-60 cells prior to large-scale cellular damage. Cells exposed to gliotoxin and control cells were then labeled with ICAT reagents and analysed by offline reversed phase liquid chromatography followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. The pilot results identified tubulin, glyceraldehyde-3-phosphate dehydrogenase and peptidyl-prolyl cis-trans isomerase as putative targets of gliotoxin. Additionally, this study showed that ICAT can be used to detect modified cysteines from a highly complex sample, but further optimization is needed to unlock the full potential for detection of cysteine modification in complex samples

The kappa opioid receptor agonist, nalfurafine, and its mechanism of action in promoting remyelination

Multiple sclerosis is a debilitating, neurodegenerative disorder which is characterized by the demyelinated lesion caused by immune cells infiltrating into the central nervous system and breaking down the myelin sheaths. Current treatments include immunosuppressives and immunomodulators to prevent or limit further neuronal damage but little is done to support the repair of the injured areas. Recently, kappa opioid receptors (KOR) were identified as a promising target to induce remyelination. Nalfurafine (Nalf) is a KOR agonist and thus far the only agonist approved for clinical use. It enhances the repair of myelin and reduces neuroinflammation in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). While the in volvement of KOR in nociception is well understood, the mechanism of how it influences remyelination is not. To gain a better understanding of the mechanism of action of Nalf in the context of remyelination, certain aspects, such as pharmakokinetics, neurotransmitter and lipid abundance, and gene expression changes were investigated. The pharmacokinetic study was preceded by the development and optimization of a liquid chromatography coupled mass spectrometry method. Due to the high potency of Nalf, only a low dose is required to achieve the desired effects. Therefore, a highly sensitive and high throughput detection method was developed, which allowed the detection of Nalf in plasma and brain tissue. For the pharmacokinetics, different parameters were evaluated such as drug delivery route, sex, and dose. KOR are known to influence the release of other neurotransmitters such as dopamine, γ-aminobutyric acid, and serotonin. Some of these have also been shown to regulate remyelination. This prompted the investigation of neurotransmitters by mass spectrometry imaging, a technique capable of detecting hundreds of molecules simultaneously without labeling. The distribution and abundance of neurotransmitter in the brain of EAE animals was investigated in response to Nalf treatment. Mass spectrometry imaging was also applied to explore alterations of lipid abundance in the spinal cord by Nalf. A certain lipid class, sulfatides, were the focus due to the recently proposed link between sulfatide fatty acid length and the developmental stage of oligodendrocyte precursor cells. Finally, the effects of Nalf on gene expression during EAE were investigated by RNA sequencing. This, taken together with the results from the pharmacokinetics the observed neurotransmitter and lipid changes, highlighted potential pathways of how Nalf could affect remyelination.</p

MOESM6 of Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease

Additional file 6: Summary of the proteins identified in FFPE PCa tissue that were selected for Western blot (WB) analysis

MOESM8 of Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease

Additional file 8: Summary of patient samples analysed

MOESM1 of Proteins from formalin-fixed paraffin-embedded prostate cancer sections that predict the risk of metastatic disease

Additional file 1: Two-dimensional electrophoresis of proteins extracted from FFPE tissue