The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrixassisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3-and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines. Molecular & Cellular Proteomics 1:91-98, 2002.The ability to determine statistically significant alterations in protein expression that correlate with disease or occur consequent to experimentally induced changes in cells is fundamental to the exploitation of proteomics. Previous studies from our laboratory have used two-dimensional (2D) 1 gel analysis of immunomagnetic affinity-sorted primary human breast cells to establish the protein expression profiles of luminal and myoepithelial cells (1). These differential expression studies were carried out using replicate 2D gels of each sample and post-staining with a fluorescent protein dye. The subsequent detailed curation and correlation of images was used to derive data sets for statistical analysis. This approach was attractive, because the fluorescent protein stain has a wide linear range of detection, thus giving improved quantification of both high and low abundance proteins. However, in these experiments, many pairs of gels were required to establish statistically significant differences in protein expression as each gel contains inherent experimental variations that limit image superimposition.The introduction of fluorescent 2D differential gel electrophoresis (DIGE) by Unlu et al. (2) has now made it possible to detect and quantitate differences between experimental pairs of samples resolved on the same 2D gel. The basis of the technique is the use of two mass-and charge-matched Nhydroxy succinimidyl ester derivatives of the fluorescent cyanine dyes Cy3 and Cy5, which possess distinct excitation and emission spectra. These are used to differentially label lysine residues of two protein samples for comparative analysis of the mixed sample on one gel. The ab...

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85␣ subunit of phosphatidylinositol 3-kinase, phospholipase C␥1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90B at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action. [Cancer Res 2007;67(7):3239-53]

In all, 31 differentially regulated proteins were identified by mass spectrometry analysis, several of which are involved in chaperone-assisted protein folding in the endoplasmic reticulum (ER) or in regulated protein degradation. Our results suggest that the exclusion of proteins by the ER quality control system, crosstalk between the EGF-and INF-induced signalling pathways and the regulation of INF-inducible genes are all altered in the prostate cancer cells. The combination of upregulated activity in the growth-promoting PI3K/Akt pathway, suppression of Nmi and overexpression of hnRNP-K and c-myc proteins may explain why the prostate cancer cells were found to be more resistant to the growth inhibitory effects of INFc.