Identificación y cuantificación label-free de proteínas de suero humano dde media y baja abundancia con LC-LTQ-Orbitrap MS/MS tras inmunodeplecion y fraccionamiento SCX

Comunicaciones a congreso

Recognition of Host Proteins by Helicobacter Cysteine-Rich Protein C

Tetratricopeptide- and sel1-like repeat (SLR) proteins modulate various cellular activities, ranging from transcription regulation to cell-fate control. Helicobacter cysteine-rich proteins (Hcp) consist of several SLRs that are cross-linked by disulfide bridges and have been implicated in host/pathogen interactions. Using pull-down proteomics, several human proteins including Nek9, Hsp90, and Hsc71 have been identified as putative human interaction partners for HcpC. The interaction between the NimA-like protein kinase Nek9 and HcpC has been validated by ELISA and surface plasmon resonance. Recombinant Nek9 is recognized by HcpC with a dissociation constant in the lower micromolar range. This interaction is formed either directly between Nek9 and HcpC or via the formation of a complex with Hsc71. The HcpC homologue HcpA possesses no affinity for Nek9, suggesting that the reported interaction is rather specific for HcpC. These results are consistent with previous observations where Nek9 was targeted upon bacterial or viral invasion. However, further experiments will be required to show that the reported interactions also occur in viv

Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision-mass spectrometry

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision-mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N-hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrPC) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision-MS, with complete suppression of nonspecific bPrP bindin

Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function

Aims Reversible phosphorylation of mitochondrial proteins is essential in the regulation of respiratory function, energy metabolism, and mitochondrion-mediated cell death. We hypothesized that mitochondrial protein phosphorylation plays a critical role in cardioprotection during pre and postconditioning, two of the most efficient anti-ischaemic therapies. Methods and results Using phosphoproteomic approaches, we investigated the profiles of phosphorylated proteins in Wistar rat heart mitochondria protected by pharmacological pre and postconditioning elicited by isoflurane. Sixty-one spots were detected by two-dimensional blue-native gel electrophoresis-coupled Western blotting using a phospho-Ser/Thr/Tyr-specific antibody, and 45 of these spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven protein spots related to oxidative phosphorylation, energy metabolism, chaperone, and carrier functions exhibited significant changes in their phosphorylation state when protected mitochondria were compared with unprotected. Using a phosphopeptide enrichment protocol followed by liquid chromatography-MS/MS, 26 potential phosphorylation sites were identified in 19 proteins. Among these, a novel phosphorylation site was detected in adenine nucleotide translocator-1 (ANT1) at residue Tyr194. Changes in ANT phosphorylation between protected and unprotected mitochondria were confirmed by immunoprecipitation. The biological significance of ANT phosphorylation at Tyr194 was further tested with site-directed mutagenesis in yeast. Substitution of Tyr194 with Phe, mimicking the non-phosphorylated state, resulted in the inhibition of yeast growth on non-fermentable carbon sources, implying a critical role of phosphorylation at this residue in regulating ANT function and cellular respiration. Conclusions Our analysis emphasizes the regulatory functions of the phosphoproteome in heart mitochondria and reveals a novel, potential link between bioenergetics and cardioprotectio

Association of proteomic markers with nutritional risk and response to nutritional support: A secondary pilot study of the EFFORT trial using an untargeted proteomics approach

Background: By means of a structured nutritional support intervention, EFFORT showed a risk reduction for adverse events in medical in-patients. We were interested in the prognostic and therapeutic potential of an untargeted proteomics approach to understand response to nutritional support, risk of 30-day mortality, and distinct patterns in severity of malnutrition risk as assessed by the Nutritional Risk screening (NRS 2002), respectively. Methods: From 2,088 patients, we randomly took 120 blood samples drawn before treatment initiation on day 1 after hospital admission. Cases were selected by treatment allocation (nutritional support vs. usual nutrition), NRS 2002, and mortality at 30 days, but not on disease type. We measured proteins by untargeted liquid chromatography mass spectrometry (LC-MS/MS). Results: We found 242 distinct proteins in 120 patients of which 81 (67.5%) survived until day 30. Between group analysis revealed a slight difference between the treatment groups in patients with a NRS 3, but not in those with a higher NRS. C-statistic between non-survivors and survivors at day 30 ranged from 0.60 (95% confidence interval 0.34-0.78) for a combination of 3 proteins/predictors to 0.65 (95% CI 0.53-0.78) for a combination of 32 proteins/predictors. In nutritional support non-survivors, pathway analysis found significant enrichment in pathways for signal transduction, platelet function, immune system regulation, extracellular matrix organization, and integrin cell surface interactions compared to survivors. Conclusion: Within this pilot study using an untargeted proteomics approach, there was only little prognostic and therapeutic potential of proteomics for phenotyping the risk of malnutrition and response to nutritional therapy. The small sample size and high heterogeneity of our population regarding comorbidity burden calls for more targeted approaches in more homogenous populations to understand the true potential of proteomics for individualizing nutritional care. Trial registration: This is a pre-planned secondary analysis of the EFFORT trial (ClinicalTrials.gov NCT02517476)

Identification and characterization of chloroplast casein kinase II from Oryza sativa (rice)

Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identification and characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated rice chloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations of heparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolutio

Grape ASR-Silencing Sways Nuclear Proteome, Histone Marks and Interplay of Intrinsically Disordered Proteins

In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up- or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms

PARP1 ADP-ribosylates lysine residues of the core histone tails

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tail

Amnion Cells in Tailored Hydrogels Deposit Human Amnion Native Extracellular Matrix

Fetal therapies regularly result in iatrogenic preterm premature rupture of the fetal membranes (iPPROM), which is associated with preterm birth. Biomaterials that promote the healing of traumatized fetal membranes (FMs) may prevent iPPROM-associated preterm births, addressing this unmet clinical need. Here, a fully defined synthetic poly(ethylene glycol) (PEG) hydrogel is developed to study the healing functions of human amnion-derived mesenchymal stromal cells (hAMCs) in 3D cultures. A pipeline to analyze extracellular matrix (ECM) proteins deposited by hAMCs in PEG hydrogels is established involving label-free quantification of mass-spectrometry data. Owing to the contaminant-free PEG hydrogels and a short fetal bovine serum (FBS)-free culture period, 128 ECM proteins, of which 97 are present in the native amnion, are identified. Upon stimulation with platelet-derived growth factor BB (PDGF-BB), a cell proliferation and migration inducing factor, hAMCs remodel their surroundings and deposit ECM proteins pericellularly. Among the most abundantly deposited amnion proteins, transforming growth factor β-induced protein ig-h3 (TGFβi), a very distinctive amnion protein that is involved in the wound healing cascade, is identified. These data support the potential of PDGF-BB to promote the repair of traumatized FMs and encourage its use for the engineering of biomaterials for FM healing, to ultimately prevent iPPROM