Hypoxia drives murine neutrophil protein scavenging to maintain central carbon metabolism

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues

A look behind the screens: Characterization of the HSP70 family during osmotic stress in a non-model crop

UNLABELLED: High-throughput molecular analysis is challenging in a non-model species. Proteomics has a great potential to characterize non-model species. We went beyond the level of simply 'identifying' the proteins of interest during osmotic stress experiments in an allopolyploid crop (in casu banana) and focus on an important stress family: the cytoplasmic HSP70s. HSP70s were already identified earlier in proteomics studies as an important player during stress but an insight into the different family members and their polymorphisms was lacking. One particular spot within a whole spot trail drew our attention: its abundance was significantly higher after osmotic stress. What distinguishes this spot from its brother and sister spots? To understand what was special about that particular spot, we characterized the whole spot family in roots and meristem cultures. Using an 2D-DIGE LC-MS/MS approach we were able to measure a proteotypic peptide for each paralog. From our data it is clear that the different paralogs have evolved over time and do not necessarily behave the same when subjected to a stress treatment. The presumable paralog that particularly reacted to the osmotic stress in roots and meristems is located on chromosome 2 and the promoter region contains a unique ABRE element. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first time that a proteomics approach has led to the exploration of a protein family at the paralog and allelic variant level in a crop. Moreover we identified a specific osmotic responsive cytoplasmic HSP70 isoform, the HSP70 paralog 2, at the protein level. We have shown that the availability of genomic resources as well as the technique used for proteomics analysis are crucial in being able to go beyond the 'usual suspects'.publisher: Elsevier articletitle: A look behind the screens: Characterization of the HSP70 family during osmotic stress in a non-model crop journaltitle: Journal of Proteomics articlelink: http://dx.doi.org/10.1016/j.jprot.2015.01.014 content_type: article copyright: Copyright © 2015 Elsevier B.V. All rights reserved.status: publishe

Screening the banana biodiversity for drought tolerance: can an in vitro growth model and proteomics be used as a tool to discover tolerant varieties and understand homeostasis

There is a great need for research aimed at understanding drought tolerance, screening for drought tolerant varieties and breeding crops with an improved water use efficiency. Bananas and plantains are a major staple food and export product with a worldwide production of over 135 million tonnes per year. Water however is the most limiting abiotic factor in banana production. A screening of the Musa biodiversity has not yet been performed. We at KU Leuven host the Musa International Germplasm collection with over 1200 accessions. To screen the Musa biodiversity for drought tolerant varieties, we developed a screening test for in vitro plants. Five varieties representing different genomic constitutions in banana (AAAh, AAA, AAB, AABp, and ABB) were selected and subjected to a mild osmotic stress. The ABB variety showed the smallest stress induced growth reduction. To get an insight into the acclimation and the accomplishment of homeostasis, the leaf proteome of this variety was characterized via 2D DIGE. After extraction of the leaf proteome of six control and six stressed plants, 2600 spots could be distinguished. A PCA analysis indicates that control and stressed plants can blindly be classified based on their proteome. One hundred and twelve proteins were significantly more abundant in the stressed plants and 18 proteins were significantly more abundant in control plants (FDR α 0.05). Twenty four differential proteins could be identified. The proteome analysis clearly shows that there is a new balance in the stressed plants and that the respiration, metabolism of ROS and several dehydrogenases involved in NAD/NADH homeostasis play an important role.status: publishe

MAIMS: a software tool for sensitive metabolic tracer analysis through the deconvolution of C-13 mass isotopologue profiles of large composite metabolites

© 2017, Springer Science+Business Media, LLC. Introduction: Metabolic tracer analysis (MTA) is a collection of principles, rules and tools for the interpretation of stable isotope incorporation patterns. One example is the GAIMS algorithm for the deconvolution of the UDP-GlcNAc 13C mass isotopologue profile. GAIMS has been presented as a powerful, yet currently unavailable, proof-of-concept-only technique. Objectives: We aimed to build a tool inspired by the original GAIMS algorithm, providing identical functionality and straightforward extensibility towards alternative composite metabolites. Methods: We implemented MAIMS by applying Multistart metaheuristics combined with an efficient hybrid stopping rule to solve the non-convex optimization underlying the deconvolution problem. By testing our tool on several theoretical datasets, we were able to confirm its robust and reproducible performance. Results: MAIMS is capable of finding the individual contributions of specifically labeled molecular subunits to large composite metabolites (such as UDP-GlcNAc and ATP) upon U-13C-glucose administration and thereby hinting on the activity of several metabolic pathway activities. Applied to proliferating endothelial cells (ECs), MAIMS led to several interesting metabolic insights and generally proved to be a sensitive way for relatively measuring specific pathway activities and for detecting compartmentalized pools of precursor metabolites. Conclusion: MAIMS is a powerful and extendible tool for isotopologue profile deconvolution tasks and is freely available on github as an open-source Python (Python 2.7 and 3.5 + compliant) script for command line usage. (http://github.com/savantas/MAIMS).status: publishe

Data for the characterization of the HSP70 family during osmotic stress in banana, a non-model crop

Here, we present the data from an in-depth analysis of the HSP70 family in the non-model banana during osmotic stress [1]. First, a manual curation of HSP70 sequences from the banana genome was performed and updated on the Musa hub http://banana-genome.cirad.fr/. These curated protein sequences were then introduced into our in-house Mascot database for an in-depth look at the HSP70 protein profiles in banana meristem cultures and roots during osmotic stress. A 2D-DIGE LC MS/MS approach was chosen to identify and quantify the different paralogs and allelic variants in the HSP70 spots

Develepment of a Comprehensive 2D-LC-MS method for the analysis and Identification of New Steviol Glycosides in Stevia Rebaudiana

A high efficient separation was setup for a commercial sample of steviol glycosides. Some 30 different compounds were found in theis samplestatus: publishe

A combination of polyunsaturated fatty acid, nonribosomal peptide and polyketide biosynthetic machinery is used to assemble the zeamine antibiotics

The zeamines are a unique group of antibiotics produced by Serratia plymuthica RVH1 that contain variable hybrid peptide–polyketide moieties connected to a common pentaamino-hydroxyalkyl chain. They exhibit potent activity against a broad spectrum of Gram-positive and Gram-negative bacteria. Here we report a combination of targeted gene deletions, high resolution LC-MS(/MS) analyses, in vitro biochemical assays and feeding studies that define the functions of several key zeamine biosynthetic enzymes. The pentaamino-hydroxyalkyl chain is assembled by an iterative multienzyme complex (Zmn10–13) that bears a close resemblance to polyunsaturated fatty acid synthases. Zmn14 was shown to function as an NADH-dependent thioester reductase and is proposed to release a tetraamino-hydroxyalkyl thioester from the acyl carrier protein domain of Zmn10 as an aldehyde. Despite the intrinsic ability of Zmn14 to catalyze further reduction of aldehydes to alcohols, the initially-formed aldehyde intermediate is proposed to undergo preferential transamination to produce zeamine II. In a parallel pathway, hexapeptide-monoketide and hexapeptide-diketide thioesters are generated by a hybrid nonribosomal peptide synthetase-polyketide synthase multienzyme complex (Zmn16–18) and subsequently conjugated to zeamine II by a stand-alone condensing enzyme (Zmn19). Structures for the resulting prezeamines were elucidated using a combination of high resolution LC-MS/MS and 1- and 2-D NMR spectroscopic analyses. The prezeamines are hypothesized to be precursors of the previously-identified zeamines, which are generated by the action of Zmn22, an acylpeptide hydrolase that specifically cleaves the N-terminal pentapeptide of the prezeamines in a post-assembly processing step. Thus, the zeamine antibiotics are assembled by a unique combination of nonribosomal peptide synthetase, type I modular polyketide synthase and polyunsaturated fatty acid synthase-like biosynthetic machinery.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 28 June 2014; Accepted 15 October 2014; Accepted Manuscript published 15 October 2014; Advance Article published 28 October 2014; Version of Record published 19 January 2015status: publishe

High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection

&lt;p&gt;Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomonas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology. However, a large fraction of the profound effect on host metabolism could not be attributed to the phage-encoded AMGs. We suggest a potentially crucial role for small, &amp;#39;non-enzymatic&amp;#39; peptides in metabolism take-over and hypothesize on potential biotechnical applications for such peptides. The highly phage-specific nature of the metabolic impact emphasizes the potential importance of the &amp;#39;phage diversity&amp;#39; parameter when studying metabolic interactions in complex communities.&lt;/p&gt;</p