Dataset Information

Proteomic analysis of ovarian cancer cells reveals dynamic processes of protein secretion and shedding of extra-cellular domains.

ABSTRACT:

Background

Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid.

Methodology and findings

To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [(13)C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions.

Conclusions and significance

Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by in-depth proteomic profiling of ovarian cancer cells may provide new targets for diagnosis and therapy.

SUBMITTER: Faca VM

PROVIDER: S-EPMC2409963 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of Trypanosoma brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes.T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varying milieus. One mechanism by which all cells respond to changing environments is through posttranslational modification of proteins. Arginine methylation is one such modification that can dramatically impact protein-protein and protein-nucleic acid interactions and subcellular localization of proteins. To define the breadth of arginine methylation in trypanosomes and identify target proteins, we performed a global proteomic analysis of arginine methylated proteins in insect stage T. brucei. We identified 1332 methylarginines in 676 proteins, generating the largest compilation of methylarginine containing proteins in any organism to date. Numerous arginine methylated proteins function in RNA and DNA related processes, suggesting this modification can impact T. brucei genome integrity and gene regulation at numerous points. Other processes that appear to be strongly influenced by arginine methylation are intracellular protein trafficking, signaling, protein folding and degradation, and flagellar function. The widespread nature of arginine methylation in trypanosomes highlights its potential to greatly affect parasite biology and pathogenesis.

Project description:INTRODUCTION: We have previously linked HER4 expression with increased survival in breast cancer. However, other reports have associated HER4 with adverse prognostic significance. One possible explanation for the conflicting reports may be that these results are antibody dependent. The HER4 protein is enzymatically cleaved, which may alter the function of its intracellular domain (ICD). We have therefore compared the staining patterns of antibodies against its intracellular and extracellular domains using tissue microarray technology. METHODS: Immunohistochemistry was performed and evaluated on tumours from 402 tamoxifen treated oestrogen receptor positive patients. The HFR1 antibody recognises the ICD of HER4 and thus recognises both the intact receptor and the cleaved ICD. The H4.77.16 clone recognises an extracellular domain of HER4 and thus detects the full length receptor only. RESULTS: Both antibodies demonstrated nuclear, cytoplasmic and membranous staining. Concordance between the membrane staining patterns was high (88.44%, kappa 0.426). The HFR1 antibody, however, demonstrated generally higher levels of cytoplasmic staining (concordance 74.77%, kappa 0.351). The antibodies demonstrated very different patterns of nuclear staining. Over 60% of patients stained with the H4.77.16 had no nuclear staining whereas the vast majority showed staining with the HFR1 antibody (concordance 40.12%, kappa 0.051). Neither antibody demonstrated relationships between membranous or cytoplasmic HER4 staining and survival, although associations were seen with known poor prognostic markers. Cases with H4.77.16-determined nuclear staining had significantly poorer survival outcomes. CONCLUSION: The difference in antigen site may explain the different staining patterns we have seen with respect to location; with each antibody appearing to select for distinct compartments. Thus, HFR1 may select for cytoplasmic and nuclear HER4 ICD, whilst H4.77.16 selects for membranous HER4 and/or HER4 being recycled in cytoplasm or nucleus. This ability to distinguish between site and function of HER4 and its fragments is particularly important, with recent evidence highlighting the different functions of nuclear and mitochondrial HER4.