Ligand binding promotes CDK-dependent phosphorylation of ER-alpha on hinge serine 294 but inhibits ligand-independent phosphorylation of serine 305.

Phosphorylation of estrogen receptor-α (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ERα-positive breast cancers. Recent attention has turned to the poorly understood ERα hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ERα immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ERα and not by growth factor stimulation (EGF, insulin, heregulin-β).

Systematic mapping of posttranslational modifications in human estrogen receptor-alpha with emphasis on novel phosphorylation sites.

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory.

A novel serine phosphorylation site detected in the N-terminal domain of estrogen receptor isolated from human breast cancer cells.

Activated estrogen receptor (ERalpha) plays a critical role in breast cancer development and is a major target for drug treatment. Serine phosphorylation within the N-terminal domain (NTD) contributes to ERalpha activation and may also cause drug resistance. Previous biochemical identification of phosphorylated ERalpha residues was limited to protein artificially overexpressed in transfected cell lines.

Quantification of cysteine oxidation in human estrogen receptor by mass spectrometry.

Redox-dependent modifications of sulfhydryl groups within the two Cys4 zinc fingers of the estrogen receptor DNA-binding domain (ER-DBD) result in structural damage and loss of ER DNA-binding function, which parallels the situation observed in many ER-positive breast cancers. Quantitation of the redox status of cysteinyl thiols within ER-DBD employed cysteine-specific oxidants to induce varying degrees of oxidation in recombinant ER, followed by differential alkylation with the stable isotopic labeling reagents [12C2]-iodoacetic acid and [13C2]-bromoacetic acid. Subsequent proteolysis with LysC/Asp-N generated diagnostic peptides of which the C-terminal peptide of the second zinc finger is most strongly detected by mass spectrometry (MS) and serves as a suitable marker of ER-DBD redox status.

Novel pathways associated with quinone-induced stress in breast cancer cells.

Hormone-dependent breast cancers that overexpress the ligand-binding nuclear transcription factor, estrogen receptor (ER), represent the most common form of breast epithelial malignancy. Exposure of breast epithelial cells to a redox-cycling and arylating quinone induces mitogen-activated protein kinase phosphorylation of the cytoskeletal filament protein, cytokeratin-8, along with thiol arylation of H3 nuclear histones. Exogenous or endogenous quinones can also induce ligand-independent nuclear translocation and phosphorylation of ER; with excess exposure, these quinones can arylate ER zinc fingers, impairing ER DNA-binding and altering ER-inducible gene expression.

Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin.

The Clostridial neurotoxins, botulinum and tetanus, gain entry into motor neurons by binding to the sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides and specific protein receptors attached to the cell's surface. While the C-fragment of tetanus toxin (TetC) has been identified to be the ganglioside binding domain, remarkably little is known about how this domain discriminates between the structural features of different gangliosides. We have used electrospray ionization mass spectrometry (ESI-MS) to examine the formation of complexes between TetC and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding.

Analysis of glycosylphosphatidylinositol protein anchors: the prion protein.

Membrane proteins constitute a substantial fraction of the human proteome. A small subgroup associates with membranes through the presence of a C-terminal lipid anchor that is joined to the protein via a phosphoglycan. The prion protein (PrP), an abnormally folded form that causes fatal neurodegeneration, is one example of a glycosylphosphatidylinositol (GPI)-anchored protein.

Bioinformatic methods to exploit mass spectrometric data for proteomic applications.

The new technologies in mass spectrometric analysis of peptides and proteins necessary to accommodate proteomics-scale analyses require, in turn, concomitant development of informatics technologies suitable for very large-scale data handling and analysis. This chapter focuses on the data analysis tools available to the community for analysis of mass spectrometric proteomics data. Different database searching strategies are discussed for peptide and protein identification, and approaches and tools available for comparative quantitative analysis of samples are outlined.

Mass spectrometers for the analysis of biomolecules.

Mass spectrometry (MS) has become a vital enabling technology in the life sciences. This chapter summarizes the fundamental aspects of MS, with reference to topics such as isotopic abundance and accurate mass and resolution. A broad and comprehensive overview of the instrumentation, techniques, and methods required for the analysis of biomolecules is presented.

Salvage nasopharyngectomy for radiation recurrences.

Background: Salvage nasopharyngectomy has proven to be worthwhile in the management of persistent or recurrent nasopharyngeal cancer after radiotherapy failure; however, surgical complications are common and the indications for surgery and the choice of operation remain controversial.

Methods: Over a 17-year period from 1985 to 2001 salvage nasopharyngectomy was undertaken on 11 patients. In six patients an anterolateral disassembly approach was employed and five patients were treated with the more limited maxillary swing approach.

Reactivity of zinc finger cysteines: chemical modifications within labile zinc fingers in estrogen receptor.

Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane.

Vitamin K3 (menadione)-induced oncosis associated with keratin 8 phosphorylation and histone H3 arylation.

The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 microM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis.

Experience with mucosal melanoma of the nose and paranasal sinuses.

Purpose: Sinonasal mucosal melanoma is a rare and aggressive disease and its incidence does not mimic that of its cutaneous counterpart in the Australian population. The present study examines one unit's experience with the disease and proposes a treatment strategy. The significance of macroscopic widespread mucosal melanosis and histological melanoma in situ is considered in the present study to be crucial in overall survival and the main cause of local failure and is specifically addressed.

pH-dependent Interactions of the carboxyl-terminal helix of steroidogenic acute regulatory protein with synthetic membranes.

Steroidogenic acute regulatory (StAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM.

Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin.

Essential cysteine-alkylation strategies to monitor structurally altered estrogen receptor as found in oxidant-stressed breast cancers.

Oxidant-induced structural modifications within the cysteine-rich DNA-binding domain (DBD) of the overexpressed estrogen receptor (ER) likely contribute to its loss of DNA-binding function and altered transcriptional activity during human breast cancer development. Using recombinant ER protein as a model, procedures to detect such endogenously produced structural changes in the two Cys(4)-type zinc fingers within the DBD of ER extracted from breast cancer cells are being developed. Unfortunately, ex vivo oxidation of these ER-DBD cysteine residues can occur during routine ER purification and preparation procedures.

Differential inhibition of prion propagation by enantiomers of quinacrine.

Prion diseases are fatal neurologic disorders caused by accumulation of a pathogenic isoform (PrP(Sc)) of the prion protein (PrP). The recent discovery of the inhibitory action of quinacrine on PrP(Sc) formation in scrapie-infected neuroblastoma (ScN2a) cells raised the possibility of a treatment for patients with prion disease. To investigate the efficacy of quinacrine enantiomers, we measured the inhibitory effect of these isomers on PrP(Sc) formation in ScN2a cells.

Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography.

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography.

Attomole detection of in vivo protein targets of benzene in mice: evidence for a highly reactive metabolite.

Modified proteins were detected in liver and bone marrow of mice following treatment with [(14)C]benzene. Stained sections were excised from one-dimensional and two-dimensional gels and converted to graphite to enable (14)C/(13)C ratios to be measured by accelerator mass spectrometry. Protein adducts of benzene or its metabolites were indicated by elevated levels of (14)C.

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization.

Pathway complexity of prion protein assembly into amyloid.

In vivo under pathological conditions, the normal cellular form of the prion protein, PrP(C) (residues 23-231), misfolds to the pathogenic isoform PrP(Sc), a beta-rich aggregated pathogenic multimer. Proteinase K digestion of PrP(Sc) leads to a proteolytically resistant core, PrP 27-30 (residues 90-231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP(Sc) and found that it can adopt two non-native abnormal isoforms, a beta-oligomer and an amyloid fibril.