Project description:An extended ChaFRADIC workflow was applied to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling, a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme were used, and only 1/3 of each ChaFRADIC-enriched fraction were analyzed by LC-MS. Furthermore, our goal was to gain insights of the Met-excision dogma where initiator Met residues are cleaved posstranslationally if the second residue is small, as well as the N-end rule degradation pathway (NERD) discriminating between stabilizing/destabilizing functions of N-terminal amino acid residues in Arabidopsis. We found bona fide NERD destabilizing residues underrepresented. The list of neo N-termini from wild type samples represents an extremely helpful resource for excluding pseudo-candidates of NERD.
Project description:The mitochondrial intramembrane rhomboid protease Parl plays essential roles in cell death but its physiological contribution remains unclear. In the present study we show that Parl ablation causes a dramatic necrotic neurodegeneration consistent with Leigh syndrome, a mitochondrial disease characterized by disrupted energy metabolism. Brain- but not liver or muscle- specific Parl deficient animals mimick Parl knock out animals. Deficiencies of the major substrates, Pink1, Pgam5, and of the complex III assembly factor Ttc19 are insufficient to modify or mimic the phenotype, suggesting that the mechanism involve a combination of Parl-/- induced effects. To investigate which mitochondrial protein changes could underlie the Parl-/- neurodegeneration we performed a quantitative mass spectrometry-based proteome analysis of brain mitochondria purified from three WT and three Parl-/- mice, leading to the quantification of 781 proteins annotated as mitochondrial resident in Swissprot. Statistical analysis revealed the accumulation or disappearance of Parl substrates, and following extensive validation, our data indicate that Pink1, Pgam5, Ttc19, Stard7, Diablo, and Clpb are genuine Parl substrates, and that Pink1, Pgam5, and Ttc19 are the most severely misprocessed substrates in brain. Together, alterations in the brain mitochondrial proteome of Parl-/- mice indicate defects in the ubiquinone pathway and complex III, which is confirmed by functional assays on neuronal mitochondria. Deficient processing of substrates by Parl leads to progressive loss of cristae structure, destabilization of electron transport, coenzyme Q deficiency, and mitochondrial calcium metabolism leading to Leigh syndrome.
Project description:Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid-like protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and cell death pathways, but its role in apoptosis remained enigmatic. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac/DIABLO as a PARL substrate. In apoptotic cells, Smac/DIABLO is released into the cytosol and promotes caspase activity by inhibiting IAPs. Intramembrane cleavage of Smac/DIABLO by PARL generates an amino terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac/DIABLO, which fails to bind XIAP. Smac/DIABLO peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac/DIABLO restores apoptosis in PARL-deficient cells. Our results discover a pro-apoptotic function of PARL and identify PARL-mediated Smac/DIABLO processing and cytochrome c release facilitated by OPA1 dependent cristae remodeling as two independent pro-apoptotic pathways in mitochondria. Please note that these data are overlapping with data uploaded under: PXD004914. Exclusively in this upload, you will find the S277A mutant immunoprecipitations.
Project description:MicroRNAs (miRNAs), a group of small non-coding RNAs, have recently become a subject of intense study. They are a class of post-transcriptional, negative regulators playing vital roles in plant development and growth. However, little is known about their regulatory roles in treeâs responses to the stressful environments incurred over its long-term growth. Here, we reported the cloning of small RNAs from abiotic stressed tissues of P. trichocarpa and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology. Among them, 9 families are novel, raising the number of the known ptc-miRNA families from 33 to 42. Total 346 targets are predicted for the cloned ptc-miRNAs using penalty scores of â¤2.5 for mismatched patterns in the miRNA:mRNA duplexes as the criterion. Six selected targets are experimentally validated. The expression of a majority of the novel miRNAs is altered in response to cold, heat, salt, dehydration, and mechanical stresses. Microarray analysis of known ptc-miRNAs identified 19 additional cold stress responsive ptc-miRNAs that originate from 14 miRNA gene families. Interestingly, we found that miRNAs of the same family respond differentially to the same stress. This suggests that members of a miRNA family may have different functions. These results reveal possible roles for miRNAs in the regulatory networks associated with development and stress response and therefore critical to plant long-term growth; and provide useful information for developing trees with a greater level of stress-resistance. Keywords: microRNA, cold stress, time course MicroRNA abundance of 21 samples from 7 cold stress time point were analyzed, each cold stress time point contain three replicates. 0 hour cold stress treatment were used as control.
Project description:The ppi2 mutant introgressed into the Columbia-0 ecotype as previously reported. Doublemutant ppi2xrpn8a was crossed with the above referenced genotypes using heterozygous ppi2 plants. After 2 days of stratification at 4 °C the plants were grown on half-strength Murashige and Skoog (M&S) medium supplemented with 0.8% (w/v) plant agar (Duchefa) and 0.8% (w/v) sucrose under short day conditions.
Project description:PeptideShaker is a user friendly tool for shotgun proteomic data interpretation and reprocessing. The present dataset is the example dataset and consists of a single run HeLa measurement.
Project description:Microtus fortis (M. fortis) is the only mammal in which the growth, development and maturation of schistosomes (Schistosoma japonicum) is prevented, resulting in the failure of the parasite to mature and complete its life cycle. MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs, has been found to introduce a whole new layer of gene regulation in eukaryotes. The anti-schistosomiasis mechanosm of M. fortis may require the participation of miRNA-mediated gene expression. In the present study, the difference pathological change of different tissue such as liver, spleen and lung of M. fortis were observed by using haematoxylin-eosin staining. Also, the miRNA expression in different tissue of M. fortis and mice before challenge and 10 days post-infection with schistosomes were first compared using microRNA microarray analysis. Histological analyses showed that S. japonicum infection in M. fortis resulted in more intensive inflammatory response and pathologic change than mice. The microarray investigations showed that 388 miRNAs detected common expressed in the two species, and 11 miRNAs in liver, 25 miRNAs in spleen and 28 miRNAs in lung differentially expressed in non-permissive M. fortis while increased, decreased or nearly fixed in mice. Further studies of the differentially expressed miRNAs demonstrated that many important signal pathway were triggered after the S. japonicum infection in M. fortis rather than the mouse, such as the metabolism of some nutrient material such as fatty-acid, cholesterol, lipid, insulin, and carbohydrate; immune response such as B and T cell differentiation, monocyte differentiation, the inflammation, NF-kappaB binding, even the in innate immune system; Cell differentiation and apoptosis such as erythrocytic differentiation and targeting proapoptotic and antiapoptotic proteins. These results may provide new insights into general mechanisms of regulation in non-permissive M. fortis, exploit the potential miRNA regulatory networks and the interaction between parasites and different hosts, which provide valuable new information on schistosome biology and valuable information for the better understanding of schistosome development and host-parasite interactions. We collected liver, spleen and lung from control and 10 days post-infection with schistosomes of M. fortis, mice and rat, respectively.
Project description:The major human pathogen Staphylococcus aureus possesses ten extracellular proteases that are central to the virulence process. While some host targets of these and other bacterial proteases are known, a major bottleneck to the study of their function is the low-throughout nature of substrate identification. Rapid identification of proteolytic cleavage events in complex substrates has, however, been rendered possible by recent developments in the proteomics sub discipline of N-terminomics. Thus, herein we perform the first study of bacterial protease-host interactions, combining the S. aureus V8 protease and the infection relevant substrate human serum. In so doing, we identified ~90 host-protein targets of V8, including previously known and novel targets. Amongst these were complement components (C1r, C1s, C3,C4b, C4BPA, C5, C6, C8G, C9, CfB, CfH, and ficolin), iron sequestration and transport proteins (ceruloplasmin, serotransferrin, hemopexin, haptoglobin), clotting cascade proteins (plasminogen, kallikrein proteins, prothrombin, factors X and XII), and multiple host protease inhibitors (α2M and members of the ITIH and SERPIN families). N-terminomics also provided insight into the disruption of host protein function by highlighting specific target regions of host proteins, where V8 cleaved within peptidase and sushi domains of complement proteins, and, in the case of host protease inhibitors, cleavage occurred predominantly outside their protease-trapping motifs. Collectively, our data highlight the potential for further application of N-terminomics for the discovery of bacterial protease substrates in other host niches, and provides unique insight into the role of the V8 protease in S. aureus pathogenesis.
Project description:Mass spectrometry-based proteomics data, obtained experimentally or via public repositories, remains challenging to analyze. This challenge is particularly felt when re-analysing or re-purposing public data sets, endeavours that otherwise hold great potential. PeptideShaker is an open source system that greatly simplifies the interpretation and dissemination of proteomics data, and that automates the re-analysis of public data, thus completing the proteomics data cycle (http://peptide-shaker.googlecode.com).