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Molecular cellular proteomics [journal]
- Integrated omic analysis of oropharyngeal carcinomas reveals HPV-dependent regulation of the AP-1 pathway. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 30.
While HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcome relative to HPV-negative patients, the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n=27) and HPV-negative (n=26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis and immune response. The differential abundances of cortactin (CTTN) and methylthioadenosine phosphorylase (MTAP) were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p=0.023 and p=0.009, respectively). An additional 1,124 proteins were independently corroborated by comparison to a published proteomic dataset of OPC. Furthermore, utilizing The Cancer Genome Atlas (TCGA), we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundance to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs, potentially contributing to reduced survival in these patients via its established role in radiation resistance. Through interrogation of TCGA data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of AP-1 transcription factor target genes is a plausible consequence of elevated cortactin protein levels.
- Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 30.
Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in natural environment. This medium is of interest as a promising source of potential biomarkers and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of malignant ascites metabolome and proteome. To omit components that belong to systemic response to the ascites formation we compared malignant ascites with cirrhosis one. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively, 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrated that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest, the finding suggests that they may play a role in the communication between cancer cells. Besides, malignant ascites contains a high number of exosomes that are known to play an important role for signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.
- Quantitative phosphoproteomics of cytotoxic T cells to reveal Protein Kinase D 2 regulated networks. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 29.
The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3,505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription and translation. In other cell types PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages.
- The Proteomics of Networks and Pathways: A Movie is Worth a Thousand Pictures. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 24.
- A New In Vivo Cross-linking Mass Spectrometry Platform to Define Protein-Protein Interactions in Living Cells. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 24.
Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane permeable, enrichable and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells, and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach in studying in vivo PPIs, and provides a solid foundation for future studies towards the complete mapping of PPI networks in living systems.
- A transcriptional regulator Sll0794 regulates tolerance to biofuel ethanol in photosynthetic Synechocystis sp. PCC 6803. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 19.
To improve ethanol production directly from CO2 in photosynthetic cyanobacterial systems, one key issue that needs to be addressed is the low ethanol tolerance of cyanobacterial cells. Our previous proteomic and transcriptomic analyses found that several regulatory proteins were up regulated by exogenous ethanol in Synechocystis sp. PCC6803. In this study, through tolerance analysis of the gene disruption mutants of the up-regulated regulatory genes, we uncovered that one transcriptional regulator, Sll0794, was related directly to ethanol tolerance in Synechocystis. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible regulatory network of Sll0794. The proteomic analysis showed that in the ∆sll0794 mutant grown under ethanol stress a total of 54 and 87 unique proteins were down- and up-regulated, respectively. In addition, electrophoretic mobility shift assays (EMSAs) demonstrated that the Sll0794 transcriptional regulator was able to bind directly to the upstream regions of sll1514, slr1512 and slr1838, which encode a 16.6 kDa small heat shock protein, a putative sodium-dependent bicarbonate transporter and a carbon dioxide concentrating mechanism protein CcmK, respectively. The study provided a proteomic description of the putative ethanol-tolerance network regulated by the sll0794 gene, and revealed new insights on the ethanol-tolerance regulatory mechanism in Synechocystis. As the first regulatory protein discovered related to ethanol tolerance, the gene may serve as a valuable target for transcription machinery engineering to further improve ethanol tolerance in Synechocystis. All MS data have been deposited in the ProteomeXchange with identifier PXD001266 (http://proteomecentral.proteomexchange.org/dataset/ PXD001266).
- Identification of Novel Nuclear Targets of Human Thioredoxin 1. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 17.
The dysregulation of protein oxidative post-translational modifications (PTMs) has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine PTMs. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified due to low Trx1 abundance in the nucleus. Here, we report a large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild type Trx1 contains a conserved C32XXC35 motif, whereby the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, is sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed, which may include regulating transcription, RNA processing and nuclear pore function in human cells.
- Spatiotemporal proteomic analyses during pancreas cancer progression identifies STK4 as a novel candidate biomarker for early stage disease. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 15.
Pancreas cancer, or pancreatic ductal adenocarcinoma (PDA), is the deadliest of solid tumors with a 5-year survival rate of <5%. Detection of resectable disease improves survival rates but access to tissue and other biospecimens to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages when surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ~2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage, metastatic disease, respectively. A panel of 7 up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated and their levels assessed in tissues collected at preinvasive, early invasive and moribund stages of disease. Six of the 7 markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasias (PanIN) to PDA. Immunohistochemistry (IHC) of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. IHC of a panel of human pancreas cancers confirmed that STK4 levels are increased in tumor epithelia compared to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage including early (resectable), and therefore clinically meaningful, stages.
- A 'proteomic ruler' for protein copy number and concentration estimation without spike-in standards. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 15.
Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting and protein concentration measurements. Here we present a novel concept that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS-signal of histones can be used as a 'proteomic ruler' because is proportional to the amount of DNA in the sample, which in turn depends of the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows the estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived from the SILAC-PrEST method, which combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset - even in retrospect analysis - and we demonstrate its usefulness on a series of mouse organ proteomes.
- Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Sep 15.
Chagas disease is a tropical neglected disease endemic in Latin America and it is caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host-cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue culture-derived trypomastigotes in acidic DMEM. Here we use this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of the T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled and multiplexed. Subsequently, phosphopeptides were enriched using TiO2 matrix. Non-phosphorylated peptides were HILIC-fractionated prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification and phosphorylation site assignment. We could identify regulated proteins and pathways involved in coordinating amastigogenesis. We could also observe that a significant proportion of the regulated proteins are membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of the T. cruzi amastigogenesis and this data will provide trustworthy basis for future studies and possibly for new potential drug targets.