Pisana Rawson

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1–infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.

Profiling the metabolic proteome of bovine mammary tissue

2‐DE and MALDI mass fingerprinting were used to analyse mammary tissue from lactating Friesian cows. The goal was detection of enzymes in metabolic pathways for synthesis of milk molecules including fatty acids and lactose. Of 418 protein spots analysed by PMF, 328 were matched to database sequences, resulting in 215 unique proteins. We detected 11 out of the 15 enzymes in the direct pathways for conversion of glucose to fatty acids, two of the pentose phosphate pathway enzymes and two of the enzymes for lactose synthesis from glucose. We did not detect enzymes that catalyse the first three reactions of glycolysis. Our results are typical of enzyme detection using 2‐DE of mammalian tissues. We therefore advocate caution when relating enzyme abundances measured by 2‐DE to metabolic output as not all relevant proteins are detected. 2‐D DIGE was used to measure interindividual variation in enzyme abundance from eight animals. We extracted relative protein abundances from 2‐D DIGE data and used a logratio transformation that is appropriate for compositional data of the kind represented in many proteomics experiments. Coefficients of variation for abundances of detected enzymes were 3–8%. We recommend use of this transformation for DIGE and other compositional data.

Metabolic proteomics of the liver and mammary gland during lactation

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by β-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and β-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than β-oxidation.

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have βI-tubulin mutations and altered expression of βII- and βIII-tubulin isotypes

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin–binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action. Mol Cancer Ther; 10(8); 1419–29. ©2011 AACR.

The Asia Oceania Human Proteome Organisation Membrane Proteomics Initiative. Preparation and characterisation of the carbonate‐washed membrane standard

The Asia Oceania Human Proteome Organisation (AOHUPO) has embarked on a Membrane Proteomics Initiative with goals of systematic comparison of strategies for analysis of membrane proteomes and discovery of membrane proteins. This multilaboratory project is based on the analysis of a subcellular fraction from mouse liver that contains endoplasmic reticulum and other organelles. In this study, we present the strategy used for the preparation and initial characterization of the membrane sample, including validation that the carbonate‐washing step enriches for integral and lipid‐anchored membrane proteins. Analysis of 17 independent data sets from five types of proteomic workflows is in progress.

Proteomic Analysis of Microsomes from Lactating Bovine Mammary Gland

Mammary gland has multiple metabolic potential including for large-scale synthesis of milk proteins, carbohydrate, and lipids including nutrient triacylglycerols. We have carried out a proteomic analysis of mammary tissue to discover proteins that affect lipid metabolism. Unfractionated microsomes from lactating bovine mammary tissue were analyzed using one-dimensional SDS-PAGE with RPLC-ESI-MS/MS. This approach gave 703 proteins including 160 predicted transmembrane proteins. Proteins were classified according to their subcellular localizations and biological functions. Over 50 proteins were associated with cellular uptake, metabolism, and secretion of lipids, including some enzymes that have been previously associated with breast cancer and potential therapeutic targets. This database develops a proteomic view of the metabolic potential of mammary gland that can be expected to contribute to a greater understanding of gene expression and tissue remodeling associated with lactation, and to further dissection of normal and pathological processes in mammary tissue.

Compatibility of toluidine blue with laser microdissection and saturation labeling DIGE

Tissue fixation and staining protocols for laser microdissection are frequently not fully compatible with subsequent proteomic analysis. We compared the effect of three common histological stains (toluidine blue (TB), hemotoxylin, and hematoxylin and eosin (HE)) on tissue visualization, protein recovery, the saturation labeling reaction, and 2‐D electrophoresis. TB provided the best visualization of colorectal tumor tissue during laser microdissection (LMD) and had a comparable effect on protein recovery and the saturation labeling reaction with hematoxylin, provided a modified 2‐D clean‐up protocol was used. Eosin inhibited both protein recovery and the saturation labeling reaction.

Identification of Cytokeratin 18 as a Biomarker of Mouse and Human Hepatosplenic Schistosomiasis

ABSTRACT Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.

Differential Patterns of Liver Proteins in Experimental Murine Hepatosplenic Schistosomiasis

ABSTRACT Schistosoma mansoni eggs produced by adult worms in the mesenteric vasculature become trapped in the liver, where they induce granulomatous lesions and strong immune responses. Infected individuals suffer from intestinal schistosomiasis (INT) in 90% of cases, whereas the remaining 10% present with severe hepatosplenic schistosomiasis (HS). The CBA/J mouse model mimics human disease, with 20% of infected mice developing hypersplenomegaly syndrome (HSS) that resembles HS and 80% developing moderate splenomegaly syndrome (MSS) similar to INT. We studied differential patterns of protein expression in livers of 20-week-infected CBA/J mice with MSS or HSS to understand the molecular changes that underlie these two disease forms. Using differential in-gel electrophoresis to identify differentially expressed protein spots, we found 80 protein spots significantly changed with infection and 35 changes specific to severe disease. In particular, the abundances of prohibitin 2, transferrin isoforms, and major urinary protein isoforms were significantly altered in HSS mice. Furthermore, annexin 5, glutathione S-transferase pi class, and S. mansoni phosphoenolpyruvate carboxykinase expression levels changed significantly with schistosome infection. Additionally, levels of major urinary protein decreased and levels of transferrin increased significantly in the sera of HSS mice compared to levels in sera of MSS or control mice, and these differences correlated to the degree of splenomegaly. These findings indicate that the liver protein abundances differ between MSS and HSS mice and may be used for the development of diagnostic markers for the early detection of hepatosplenic schistosomiasis.

Acquired Resistance to Peloruside A and Laulimalide is Associated with Downregulation of Vimentin in Human Ovarian Carcinoma Cells

ABSTRACTPurposeAcquired β-tubulin alterations in human ovarian carcinoma 1A9 cells were previously shown to confer resistance to the microtubule stabilizing agents peloruside A (PLA) and laulimalide (LAU). We examined the proteome of resistant cells to see what other protein changes occurred as a result of the acquired drug resistance.MethodsTwo-dimensional differential in-gel electrophoresis was performed to explore differentially expressed proteins in the resistant 1A9-R1 (R1) and 1A9-L4 (L4) cells. The proteins on the gels were identified by MALDI-TOF MS, and altered protein abundance was confirmed by Western blotting and immunocytochemistry. Vimentin expression was restored in vimentin-deficient L4 cells by transfecting a full-length human vimentin cDNA, and sensitivity to PLA and LAU were tested using an MTT cell proliferation assay.ResultsProteomic analysis identified several proteins that were significantly altered in the resistant cells relative to the parental 1A9 cells. Using Western blotting and immunocytochemistry, a decreased vimentin abundance in the L4 cells was validated. Vimentin levels were unchanged in PLA-resistant R1 cells and paclitaxel/epothilone-resistant derivatives of 1A9 cells. Vimentin cDNA transfection into L4 cells partially restored PLA and LAU sensitivity.ConclusionsDownregulation of vimentin contributes to the resistance of 1A9 cells to the microtubule stabilizing agents, PLA and LAU.