Pathogen and non-pathogen Spotted Fever Group Rickettsia trigger differential proteome signatures in macrophages
ABSTRACT: We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein profiled the host proteomic alterations resulted by the infection of THP-1 macrophages with R. conorii and R. montanensis using a high throughput quantitative proteomics approach (SWATH-MS). Our results revealed that these two members of SFG Rickettsia with distinct pathogenicity attributes for humans, trigger differential proteomic signatures in macrophage-like cells. Although infection by both rickettsial species resulted in a lower abundance of enzymes of glycolysis and pentose phosphate pathway, the pathogenic R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid -oxidation and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii towards a metabolic signature of an M2-like (anti-inflammatory) activation program. Moreover, our results revealed that several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. Remarkably, R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to compensate the accumulation of misfolded proteins by increasing the ER protein folding capacity and subsequently restore host cell homeostasis. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage.
Project description:This ArrayExpress record contains meta-data and results of quantitative analysis of cell lines from the NCI-60 panel using pressure cycling technology (PCT) and SWATH-mass spectrometry. Each cell line was analyzed in duplicate. Raw data files are available at the EMBL-EBI protemics data archive (PRIDE) at accession PXD003539 (http://www.ebi.ac.uk/pride/archive/projects/PXD003539). Since the record here does not include the raw data files and hence there is no need to explicitly link individual replicate to a raw file, each sample is only listed once in the ArrayExpress samples table for clarity.
Project description:Proteomic approaches are extremely valuable in many fields of research, where mass spectrometry methods have gained an increasing interest especially due to the ability to perform quantitative analysis. Nonetheless, sample preparation prior to mass spectrometry analysis is of the utmost importance. In this work two protein precipitation approaches, widely used for cleaning and concentrating protein samples, were tested and compared in a case of very diluted samples solubilized in a strong buffer (containing SDS). The amount of protein recovered after acetone and TCA/acetone precipitation was assessed, as well as the protein identification and relative quantification by SWATH-MS yields which were compared with the results from the same sample without precipitation. With this study it was possible to conclude that, in the case of diluted samples in denaturing buffers, the use of cold acetone as precipitation protocol is more favourable than the use of TCA/acetone in terms of reproducibility in protein recovery, number of identified and quantified proteins. Furthermore, the reproducibility in relative quantification of the proteins is even higher in samples precipitated with acetone when comparing to the original sample.
Project description:Pseudomonas aeruginosa is a Gram-negative, opportunistic bacterium and a major etiological agent in monogenic disease cystic fibrosis (CF). High density colonies of P. aeruginosa are often isolated from hypoxic mucus plugs in respiratory tract of CF patients, indicating high adaptive capacities of the bacterium. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to this hypoxic environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT labelled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2<1%) and aerobic conditions in media that mimics the nutrient components of the CF lung. 3,967 P. aeruginosa proteins were quantitated (FDR <1%) across all three strains, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640 and 364 proteins were altered by two-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2 and PASS3 respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.
Project description:Tau is a scaffolding protein which serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). We have recently shown that amyloid-, the second hallmark of AD, induces de novo protein synthesis of tau. Importantly, this activation was found to be tau-dependent, raising the question of whether FTD-tau by itself affects protein synthesis. To investigate this, we applied non-canonical amino acid labelling to visualise and identify newly synthesised proteins in the K369I tau transgenic K3 mouse model of FTD. This revealed that protein synthesis was massively decreased in neurons containing pathologically phosphorylated tau, a finding confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH-MS proteomics, we identified changes in 247 proteins of the de novo proteome of K3 mice. These included decreased synthesis of the ribosomal proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. Together, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.
Project description:The main objective of the present report was to unravel the Cx43-interaction network in the heart, and to establish the impact of heart ischemia and I/R upon these interactions. In order to characterize the cardiac Cx43 interactome under ischemia and I/R, a quantitative proteomic analysis was performed, through the combination of immunopurification of endogenous Cx43 (Cx43 IP) and identification of its binding partners with the SWATH-MS approach, using rat hearts maintained in a Langendorff apparatus. In the present approach 444 proteins were identified, including proteins identified based on a single peptide (supplementary table I). From these 444 proteins, 299 (approximately 67 % of the entire dataset) were quantified and compared between the various experimental conditions (including non-specific binding to control IP samples). These 299 proteins were further evaluated by a series of complementary analysis to deciphering the truly interactors of Cx43. According with this evaluation, 236 out of the 299 quantified proteins were considered as putative Cx43 interacting partners in the cardiac context presented The results obtained in this study demonstrate that Cx43 mainly interacts with proteins related with metabolism, signaling and trafficking, and that this interactome can be differentially modulated in diseased hearts. Our results shed new light upon the understanding of Cx43 functions in the heart, both in health and disease, which ultimately may lead to the establishment of new therapeutic targets to modulate cardiac homeostasis.
Project description:Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient specific. Ultrafiltration was used to fractionate hFF to High Molecular Weight (HMW) – proteome (>10kDa) and Low Molecular Weight (LMW) – peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, form which 59 were never reported before as hFF components. 55 (45 Never reported before) proteins were found by analyzing LMW fraction, 67 (14 never reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 18 proteins varied substantially among hFF samples from single donors and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.
Project description:Dioecy is an important sexual system wherein, male and female flowers are borne on separate unisexual plants. Knowledge of sex-related differences can enhance our understanding in molecular and developmental processes leading to unisexual flower development. Coccinia grandis is a dioecious species belonging to Cucurbitaceae, a family well-known for diverse sexual systems. Male and female plants of C. grandis have 22A+XY and 22A+XX chromosomes respectively. Previously, we have reported a gynomonoecious form (GyM) (22A+XX) of C. grandis bearing morphologically hermaphrodite flowers (GyM-H) and female flowers (GyM-F). Also, we showed that foliar spray of silver nitrate on female C. grandis plant induces development of morphologically hermaphrodite buds (Ag-H) despite the absence of Y chromosome. To identify sex-related differences, total protein from the flower buds of male, female, GyM-H and Ag-H of C. grandis at early and middle stages of development were analysed by a powerful label-free proteomics approach on ABSCIEX Triple TOF 5600 platform.
Project description:Glioblastoma (GBM), the most malignant primary brain tumor, is characterized by widespread heterogeneity, leading to poor and unpredictable clinical outcomes. Recent studies have provided evidences that the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. Here we investigate how GBM cells modulate glial cells, and how this modulation can influence back on the malignant phenotype of GBM cells. Primary mouse glial cultures were established and cultured in serum-free media (unprimed) or exposed to secretome derived from GL261 GBM cells (primed). Conditioned media (CM) from each glial culture were collected and a proteomic analysis was conducted. Glial cells CM (unprimed and primed) were also used in GL261 GBM cells to evaluate its impact in critical hallmarks of GBM cells, including viability, migration, and activation of tumor-related intracellular signaling pathways. The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells, consistent with a pattern of reactive astrogliosis. At the functional levels, CM derived from unprimed glial cells favored an increase in cell migration capacity, while CM from primed glial cells was more efficient in promoting GBM cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation. Together, our results demonstrate that glial cells can have a different impact on the progression of GBM tumors, suggesting that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells. Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.
Project description:For data-independent acquisition by means of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a reference library of data-dependent acquisition (DDA) runs is typically used to correlate the quantitative data from the fragment ion spectra with peptide identifications. The quality and coverage of such a reference library is therefore essential when processing SWATH data. In general, library sizes can be increased by reducing the impact of DDA precursor selection with replicate runs or fractionation. However, these strategies can affect the match between the library and SWATH measurement, and thus larger library sizes do not necessarily correspond to improved SWATH quantification. Here, three fractionation strategies to increase local library size were compared to standard library building using replicate DDA injection: protein SDS-PAGE fractionation, peptide high-pH RP-HPLC fractionation and MS-acquisition gas phase fractionation. The impact of these libraries on SWATH performance was evaluated in terms of the number of extracted peptides and proteins, the match quality of the peptides and the extraction reproducibility of the transitions. These analyses were conducted using the hydrophilic proteome of differentiating human embryonic stem cells.
Project description:Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation changes without considering the variation in the total levels of the proteins. However, the regulation of protein synthesis and protein degradation are also associated with many of the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in the Post Translational Modifications (PTMs) focused analyses, such as cysteine redox characterization. This aspect was only address in the cysTMTRAQ method, which combines two types of isobaric tags in the same experiment leading to a considerable increase in the costs of the analysis. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using non-isotopically labeled alkylating reagents (oxSWATH) is presented, thus integrating the information regarding relative cysteine oxidation with the comparative analysis of the total protein levels in a cost effective highthrouput approach. The proposed method was tested using a redox regulated protein and further applied to a comparative analysis of secretomes obtained under control or oxidative stress conditions to strengthen the importance of considering the changes in protein total levels. OxSWATH allowed to determine the relative proportion of reduced and reversible oxidized oxoforms of the proteins, and by considering total protein levels, to determine the total levels of each fraction, which are then used for comparative analysis. In this project it is presented the results from the validation of the method using the recombinant protein DJ-1.