Functional characterization of the alphavirus TF protein.

Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

Immune and inflammatory pathways are involved in inherent bone marrow ossification.

BACKGROUND: Bone marrow plays a key role in bone formation and healing. Although a subset of marrow explants ossifies in vitro without excipient osteoinductive factors, some explants do not undergo ossification. The disparity of outcome suggests a significant heterogeneity in marrow tissue in terms of its capacity to undergo osteogenesis. QUESTIONS/PURPOSES: We sought to identify: (1) proteins and signaling pathways associated with osteogenesis by contrasting the proteomes of ossified and poorly ossified marrow explants; and (2) temporal changes in proteome and signaling pathways of marrow ossification in the early and late phases of bone formation. METHODS: Explants of marrow were cultured. Media conditioned by ossified (n = 4) and poorly ossified (n = 4) subsets were collected and proteins unique to each group were identified by proteomic analysis. Proteomic data were processed to assess proteins specific to the early phase (Days 1-14) and late phase (Days 15-28) of the culture period. Pathways involved in bone marrow ossification were identified through bioinformatics. RESULTS: Twenty-eight proteins were unique to ossified samples and eight were unique to poorly ossified ones. Twelve proteins were expressed during the early phase and 15 proteins were specific to the late phase. Several identified pathways corroborated those reported for bone formation in the literature. Immune and inflammatory pathways were specific to ossified samples. CONCLUSIONS: The marrow explant model indicates the inflammatory and immune pathways to be an integral part of the osteogenesis process.

Increased expression of GAPDH protein is not indicative of nitrosative stress or apoptosis in liver of starved rainbow trout (Oncorhynchus mykiss).

Short-term starvation has been linked to in vivo protein degradation in liver of rainbow trout (Oncorhynchus mykiss). However, it is unclear whether this proposed increase in protein degradation is followed by programmed cell death (apoptosis) in liver of starved trout. A preliminary study in our laboratory revealed an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein that increased 4.5-fold in liver of starved trout. GAPDH is a glycolytic enzyme involved in other cellular functions, including apoptosis. Increased intracellular nitric oxide (NO) promotes nuclear translocation of GAPDH that is associated with increased apoptosis in mammals. If GAPDH protein is associated with apoptosis in rainbow trout, it could potentially be used as a biomarker of cellular stress in liver of teleost fish species. The purpose of this study was to determine whether increased GAPDH protein expression in liver of starved rainbow trout is associated with NO-induced apoptosis. Targeted proteomic analysis using multiple reaction monitoring (MRM) was used to determine the level of GAPDH in nuclear and cytoplasmic fractions and inducible nitric oxide synthase (iNOS) in cell lysates. Dot blot and DNA fragmentation analyses were conducted to evaluate protein S-nitrosylation and apoptosis, respectively. Results showed that cytoplasmic GAPDH was 3.4-fold higher in liver of starved versus fed rainbow trout but could not be detected in nuclear fractions. Starvation significantly reduced hepato-somatic index but had no effect on iNOS protein expression, protein S-nitrosylation, or apoptosis. Our results indicate that starvation promoted significant reduction in liver mass that was not associated with increased apoptosis or NO-induced stress and that greater GAPDH concentration in liver of starved rainbow trout was located primarily in the cytoplasm.

A large, consistent plasma proteomics data set from prospectively collected breast cancer patient and healthy volunteer samples.

BACKGROUND: Variability of plasma sample collection and of proteomics technology platforms has been detrimental to generation of large proteomic profile datasets from human biospecimens. METHODS: We carried out a clinical trial-like protocol to standardize collection of plasma from 204 healthy and 216 breast cancer patient volunteers. The breast cancer patients provided follow up samples at 3 month intervals. We generated proteomics profiles from these samples with a stable and reproducible platform for differential proteomics that employs a highly consistent nanofabricated ChipCube™ chromatography system for peptide detection and quantification with fast, single dimension mass spectrometry (LC-MS). Protein identification is achieved with subsequent LC-MS/MS analysis employing the same ChipCube™ chromatography system. RESULTS: With this consistent platform, over 800 LC-MS plasma proteomic profiles from prospectively collected samples of 420 individuals were obtained. Using a web-based data analysis pipeline for LC-MS profiling data, analyses of all peptide peaks from these plasma LC-MS profiles reveals an average coefficient of variability of less than 15%. Protein identification of peptide peaks of interest has been achieved with subsequent LC-MS/MS analyses and by referring to a spectral library created from about 150 discrete LC-MS/MS runs. Verification of peptide quantity and identity is demonstrated with several Multiple Reaction Monitoring analyses. These plasma proteomic profiles are publicly available through ProteomeCommons. CONCLUSION: From a large prospective cohort of healthy and breast cancer patient volunteers and using a nano-fabricated chromatography system, a consistent LC-MS proteomics dataset has been generated that includes more than 800 discrete human plasma profiles. This large proteomics dataset provides an important resource in support of breast cancer biomarker discovery and validation efforts.

Oxidative stress studies in yeast with a frataxin mutant: a proteomics perspective.

Cellular response of wild-type Saccharomyces cerevisiae and the Delta yfh1 mutant to oxidative stress (OS) was examined by stressing cells through the addition of H(2)O(2) to the growth medium. The Delta yfh1 mutant is unusual in that it accumulates iron in it is mitochondria. Wild-type growth was immediately arrested and recovered in 2 h following H(2)O(2) treatment. No change in viability was observed. Growth of the mutant, on the other hand, was similar to wild-type yeast for 4 h but then rapidly declined, eventually reaching zero. Levels of carbonyl groups and reactive oxygen species (ROS) reached their maximum at 3 h following exposure. The impact of OS on protein function was also evaluated by proteomic techniques targeting protein carbonylation. Oxidized proteins were selected by affinity chromatography, and following trypsin digestion, peptide fragments were identified by RPLC-MS/MS. A total of 53 proteins were identified in both wild-type and mutant cells, respectively.

Quantitation of Ubiquinone (Coenzyme Q10) in Serum/Plasma Using Liquid Chromatography Electrospray Tandem Mass Spectrometry (ESI-LC-MS/MS).

Dietary ubiquinone (Coenzyme Q10) is considered an essential co-factor in the mitochondrial respiratory chain responsible for oxidative phosphorylation. This oil-soluble vitamin-like substance is mobile in cellular membranes and plays a unique role in the electron transport chain (ETC). Coenzyme Q10 (CoQ10) is present in most eukaryotic cells and functions as an electron carrier and an antioxidant. Although the exact role of Coenzyme Q10 is often debated; there is a growing interest in the measurement of CoQ10 concentrations particularly in the area of cardiovascular disease, malignancies, exercise physiology, Parkinson's disease, and patients undergoing statin drug therapies. We describe a simple method for the quantitative measurement of the ammonium adduct of Coenzyme Q10 using a high-pressure liquid chromatography combined with positive electrospray ionization tandem mass spectroscopy (ESI-LC-MS/MS) utilizing a 3 µm PFP(2) 50 × 2.0 mm 100 Å column. A stable isotopic deuterated internal standard, in the form of Coenzyme Q10-[D9], is added to the patient serum. The extraneous proteins are precipitated from the sample with ethanol and isolation of the targeted compound is facilitated by the addition of hexane to aide in the cleanup and recovery. Quantitation occurs via a 6-point calibration that is linear from 0.16 to 6.0 µg with an observed error of 6.2 % across the analytical range.

Simultaneous Quantitation of Estradiol and Estrone in Serum Using Liquid Chromatography Mass Spectrometry.

Accurate measurement of the endogenous estrogens, estrone (E1) and estradiol (E2), is important in the clinical diagnosis and monitoring of multiple disorders. Typically, given the efficacy and low cost, radioimmunoassays (RIA) and enzyme-linked immunoassays (EIA) are used to quantify these hormones in biological samples. Unfortunately, at low levels these assays lack the necessary sensitivity and specificity for diagnosis of certain disorders in adult and pediatric endocrinology and oncology. In response to this need, we developed a fast and sensitive high performance liquid chromatography negative electrospray ionization tandem mass spectrometry (LC-MS/MS) method to measure serum estrone (E1) and estradiol (E2) without chemical derivatization. Samples are spiked with a stable isotopic carbon thirteen ((13)C) labeled internal standard and the estrogens are isolated by liquid-liquid extraction (LLE) with hexane:Methyl-tert-butyl ether (MTBE) (9:1). Following centrifugation and dry down samples are reconstituted with deionized water, and separated on a C18 reverse phase column. The analytes are quantified using a six point calibration curve with a linearity of 2.6-625 pg/ml and with a variability of less than 8 % across analytical range.

CNS neurotrophins are biologically active and expressed by multiple cell types.

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and -4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.

Systems-scale analysis reveals pathways involved in cellular response to methamphetamine.

BACKGROUND: Methamphetamine (METH), an abused illicit drug, disrupts many cellular processes, including energy metabolism, spermatogenesis, and maintenance of oxidative status. However, many components of the molecular underpinnings of METH toxicity have yet to be established. Network analyses of integrated proteomic, transcriptomic and metabolomic data are particularly well suited for identifying cellular responses to toxins, such as METH, which might otherwise be obscured by the numerous and dynamic changes that are induced. METHODOLOGY/RESULTS: We used network analyses of proteomic and transcriptomic data to evaluate pathways in Drosophila melanogaster that are affected by acute METH toxicity. METH exposure caused changes in the expression of genes involved with energy metabolism, suggesting a Warburg-like effect (aerobic glycolysis), which is normally associated with cancerous cells. Therefore, we tested the hypothesis that carbohydrate metabolism plays an important role in METH toxicity. In agreement with our hypothesis, we observed that increased dietary sugars partially alleviated the toxic effects of METH. Our systems analysis also showed that METH impacted genes and proteins known to be associated with muscular homeostasis/contraction, maintenance of oxidative status, oxidative phosphorylation, spermatogenesis, iron and calcium homeostasis. Our results also provide numerous candidate genes for the METH-induced dysfunction of spermatogenesis, which have not been previously characterized at the molecular level. CONCLUSION: Our results support our overall hypothesis that METH causes a toxic syndrome that is characterized by the altered carbohydrate metabolism, dysregulation of calcium and iron homeostasis, increased oxidative stress, and disruption of mitochondrial functions.

Dynamics of protein damage in yeast frataxin mutant exposed to oxidative stress.

Oxidative stress and protein carbonylation is implicated in aging and various diseases such as neurodegenerative disorders, diabetes, and cancer. Therefore, the accurate identification and quantification of protein carbonylation may lead to the discovery of new biomarkers. We have developed a new method that combines avidin affinity selection of carbonylated proteins with iTRAQ labeling and LC fractionation of intact proteins. This simple LC-based workflow is an effective technique to reduce sample complexity, minimize technical variation, and enable simultaneous quantification of four samples. This method was used to determine protein oxidation in an iron accumulating mutant of Saccharomyces cerevisiae exposed to oxidative stress. Overall, 31 proteins were identified with 99% peptide confidence, and of those, 27 proteins were quantified. Most of the identified proteins were associated with energy metabolism (32.3%), and cellular defense, transport, and folding (38.7%), suggesting a drop in energy production and reducing power of the cells due to the damage of glycolytic enzymes and decrease in activity of enzymes involved in protein protection and regeneration. In addition, the oxidation sites of seven proteins were identified and their estimated position also indicated a potential impact on the enzymatic activities. Predicted 3D structures of peroxiredoxin (TSA1) and thioredoxin II (TRX2) revealed close proximity of all oxidized amino acid residues to the protein active sites.

Multi-dimensional liquid chromatography in proteomics--a review.

Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. This still-emerging combination of technologies aims to describe and characterize all expressed proteins in a biological system. Because of upper limits on mass detection of mass spectrometers, proteins are usually digested into peptides and the peptides are then separated, identified and quantified from this complex enzymatic digest. The problem in digesting proteins first and then analyzing the peptide cleavage fragments by mass spectrometry is that huge numbers of peptides are generated that overwhelm direct mass spectral analyses. The objective in the liquid chromatography approach to proteomics is to fractionate peptide mixtures to enable and maximize identification and quantification of the component peptides by mass spectrometry. This review will focus on existing multidimensional liquid chromatographic (MDLC) platforms developed for proteomics and their application in combination with other techniques such as stable isotope labeling. We also provide some perspectives on likely future developments.

TIGR is upregulated in the chronic glial scar in response to central nervous system injury and inhibits neurite outgrowth.

Reactive astrocytes respond to central nervous system (CNS) injury and disease by elaborating a glial scar that is inhibitory to axonal regeneration. To identify genes that may be involved in the astrocytic response to injury, we used differential display polymerase chain reaction and an in vivo model of the CNS glial scar. Expression of the trabecular meshwork inducible glucocorticoid response (TIGR) gene was increased in gliotic tissue compared with the uninjured cerebral cortex. Increased TIGR expression by reactive astrocytes was confirmed by in situ hybridization, quantitative reverse transcriptase-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Although mutations of the TIGR gene have been implicated in glaucoma, a function for TIGR has not been reported. Since TIGR is secreted, we assessed a possible role in inhibition of neuronal regeneration with an in vitro bioassay and found that this protein is a potent inhibitor of neurite outgrowth. Thus, TIGR is a newly identified component of the CNS glial scar that is likely to contribute to neuronal regenerative failure characteristic of the mammalian CNS.