An investigation of the PsbS protein isolated from spinach chloroplast membranes.

Dissipation of excess light energy in plant photosynthetic membranes plays an important role in the response of plants to the environment, providing short-term balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid zeaxanthin and the photosystem Il subunit PsbS play vital roles in this process, but the mechanism of their action is largely unexplained. This thesis reports a novel procedure for the extraction of the PsbS protein from spinach thylakoids, including a detailed account of the developmental process and characterisation of the isolated protein. The ability of the PsbS protein to bind xanthophyll cycle carotenoids in vitro was assessed, leading to the observation that the isolated protein was able to bind exogenous zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and the appearance of characteristic features in the resonance Raman spectrum and a distinct circular dichroism spectrum, indicating pigment-protein, as well as specific pigmentpigment, interaction. A strong shift in the absorption spectrum of PsbS phenylalanine residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding to PsbS is the origin of the well known energy dissipation-related 535-nm absorption change. The ability of this PsbS-zeaxanthin complex to affect the rate of chlorophyll fluorescence quenching of the major LHcn antenna protein is detailed, revealing an increase in the rate of quenching, whilst the magnitude of quenching remained constant. The altered properties of zeaxanthin and PsbS after in vitro reconstitution and their subsequent effect on LHCnb provide the first direct indication about how they regulate energy dissipation

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

This protocol describes a highly reproducible antibody-based method that provides protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV-activated linking chemistry to detect changes in phosphorylation status. As an example application, we describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adaptor protein ​CrkL, a major substrate of the oncogenic tyrosine kinase ​BCR-​ABL in chronic myeloid leukemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5 pg/nl limit of protein detection (<0.2% of a stem cell sample containing <104 cells). Additional assays are described for phosphorylated tyrosine 207 (pTyr207)-​CrkL and the protein tyrosine phosphatase ​PTPRC/​CD45; these assays were developed using this protocol and applied to CML patient samples. This method is of high throughput, and it can act as a screen for in vitro cancer stem cell response to drugs and novel agents

ERK and AKT phosphorylation status in lung cancer and emphysema using nanocapillary isoelectric focusing.

BACKGROUND: Emphysema is an independent risk factor for the development of lung cancer in smokers. Activation of oncogenic signalling proteins AKT and ERK by phosphorylation has an established role in the development of lung cancer and has also been implicated in the pathogenesis of emphysema. The aim of this study was to compare the protein level and phosphorylation status of AKT and ERK in paired lung cancer and emphysema tissue using a highly sensitive phosphoprotein analysis approach. METHODS: An antibody-based, nanocapillary isoelectric focusing (cIEF) assay was used to determine the relative quantities and phosphorylation status of AKT and ERK in tumour and matched lung tissue from patients, with or without evidence of emphysema, undergoing curative resection for non-small cell lung cancer. RESULTS: 20 patients with adenocarcinoma (n=9) or squamous cell carcinoma (n=11) of the lung were included (mean age 67.3 years (SD 7.5, range 47–80 years)), 12 were men and all were current (n=10) or former smokers (n=10). Paired macroscopically normal lung tissue was either histologically normal (n=7) or showed emphysema (n=13). Total and phosphorylated AKT levels were fourfold (p=0.0001) and fivefold (p=0.001) higher in tumour compared with matched lung, respectively. There was no correlation with tumour histology, stage or differentiation; however, total AKT signal in tumour was significantly correlated with fluorodeoxyglucose avidity on positron emission tomography-CT scan (r=0.53, p=0.035). Total ERK was not differentially expressed, but doubly phosphorylated (activated) ERK was threefold higher in emphysema (23.5%, SD 9.2) than either matched tumour (8.8%, SD 8.6) or normal lung tissue (8.3%, SD 9.0) and correlated with the histological severity of emphysema (p=0.005). CONCLUSIONS: cIEF offers opportunities for quantifying subtle shifts in the phosphorylation status of oncoproteins in nanogram amounts of lung tissue. ERK activation is a feature of emphysema

Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases

A specific PTPRC/CD45 phosphorylation event governed by stem cell chemokine CXCL12 regulates primitive hematopoietic cell motility

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells