Apoptotic marker expression in the absence of cell death in staurosporine-treated Leishmania donovani.

The protozoan parasite Leishmania donovani undergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility of L. donovani promastigotes in vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition of L. donovani protein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development.

Probing the dynamic nature of signalling pathways by IMAC and SELDI-tof MS.

One major obstacle to the analysis of signalling pathways is the dynamic nature of signalling response to environmental stimuli. To overcome this limitation we applied immobilized metal affinity chromatography (IMAC) in combination with SELDI-tof MS to investigate the temporal variation of protein phosphorylation. We analysed the phospho-proteome variations in our model organism, Leishmania donovani, in response to changes in pH and temperature, which induce differentiation from promastigotes to amastigotes. Investigation of total cell extracts did not allow promastigote and amastigote life cycle stages to be distinguished. However, using IMAC enriched samples, the pattern and intensity of phospho-proteins analysed distinguished both stages reproducibly. Approximately 61% of the phospho-proteins analysed were significantly different in abundance (p<0.02). Of these 61%, 73% showed an increased phosphorylation in promastigotes while 27% showed an increase phosphorylation in amastigotes. The workflow developed is currently being applied to the temporal analysis of environmental stimuli.

Analysis of stage-specific expression of basic proteins in Leishmania infantum.

Prior analyses of the proteome of the protozoan parasite Leishmania have underrepresented basic proteins. Here, we applied protein fractionation by isoelectric point (pI) using free-flow electrophoresis (FFE) to study stage-specific expression of basic proteins in this pathogen. Overall, we resolved 2469 protein spots in both the flagellated promastigote and the nonmotile amastigote forms in the basic range by two-dimensional gel electrophoresis (2-DE). Highly basic proteins were enriched by FFE fractionation, allowing many to be identified and characterized for the first time by proteomics analysis. Among proteins upregulated in the promastigote stage, we found glycolytic enzymes and flagellar proteins. Proteins upregulated in the amastigote stage included enzymes involved in gluconeogenesis and fatty acid beta-oxidation. In both life stages, many proteins were found in multiple spots or as proteolytic fragments, suggesting that extensive post-translational modification and processing occur. Interestingly, evidence was obtained suggesting that some of these processes may be stage-specific.

Application of free flow electrophoresis to the analysis of the urine proteome.

Urine is a complex fluid, which is thought to contain valuable diagnostic information regarding general health. In particular, there is great diagnostic potential in the peptide and/or protein content of urine, but the information is present in low abundance. Most traditional proteomic techniques lack sufficient sensitivity/dynamic range, especially for dilute and/or complex samples. However, orthogonal separation methods can be applied prior to protein/peptide analysis to increase the success rate of urine proteomic studies and access this potentially valuable information. In this chapter, we describe isoelectric focusing (IEF) of intact urine proteins, via free flow electrophoresis (FFE), prior to typical peptide-based mass spectrometry analysis, facilitating the deep analysis of urine protein detection and identification, for biomarker discovery. Our work demonstrates that such an approach can be used as a preprocessing step and can be integrated into a workflow for the successful identification of protein components (biomarkers) from urine.

Resolution of adiponectin oligomers in human plasma using free flow electrophoresis.

Adiponectin is an important adipocytokine hormone which circulates in blood as homo-oligomers (trimer, hexamer and high molecular weight (HMW) forms) as well as a truncated form corresponding to the globular domain. Free flow electrophoresis (FFE) used in zone electrophoresis mode revealed the presence of isoforms within these oligomeric forms in plasma. HMW adiponectin oligomer showed two isoforms which carry different charge density at pH 4.7, only one of which is susceptible to dissociation by SDS. The adiponectin hexamer was shown to consist of a doublet and also shown to have at least two isoforms. A truncated form of adiponectin was identified as the main constituent of adiponectin in plasma and appeared to circulate bound to a basic protein, potentially one of the chemokines reported to bind to the globular domain. Analysis of the monomer composition of the oligomers revealed differences in monomeric isoforms used to build up the oligomers.

Analysis of human plasma proteins: a focus on sample collection and separation using free-flow electrophoresis.

Due to ease of accessibility, plasma has become the sample of choice for proteomics studies directed towards biomarker discovery intended for use in diagnostics, prognostics and even in theranostics. The result of these extensive efforts is a long list of potential biomarkers, very few of which have led to clinical utility. Why have so many potential biomarkers failed validation? Herein, we address certain issues encountered, which complicate biomarker discovery efforts originating from plasma. The advantages of stabilizing the sample at collection by the addition of protease inhibitors are discussed. The principles of free-flow electrophoresis (FFE) separation are provided together with examples applying to various studies. Finally, particular attention is given to plasma or serum analysis using multidimensional separation strategies into which the FFE is incorporated. The advantages of using FFE separation in these workflows are discussed.

Prefractionation by digitonin extraction increases representation of the cytosolic and intracellular proteome of Leishmania infantum.

Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.

A proteomic analysis of arsenical drug resistance in Trypanosoma brucei.

We have undertaken 2-DE and MS to identify proteins associated with arsenical drug resistance in Trypanosoma brucei. This parasite causes sleeping sickness in humans, and arsenical drug resistance is a significant potential problem. Comparative analysis of approximately 2000 spots resolved by 2-DE in the soluble proteomes of drug-sensitive and drug-resistant isogenic lines of T. brucei identified a protein spot whose absence associated with resistance to the arsenical drug, Cymelarsan. MS matched this protein to an identical pair of tandem genes Tb09.211.0120 and 0130 that encode a putative nascent polypeptide associated complex subunit. This protein also occurs as an isoform located in both resistant and sensitive lines at a similar molecular weight, but different pI. The difference between isogenic lines was confirmed by Western blot using an antibody against recombinant protein. Both genes were identical in sequence between drug-sensitive and drug-resistant lines and both were transcribed as determined by RT-PCR. We postulate that the missing protein isoform arose due to the lack of a PTM.