Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins

Sorting Signals, N-Terminal Modifications and Abundance of the Chloroplast Proteome

Characterization of the chloroplast proteome is needed to understand the essential contribution of the chloroplast to plant growth and development. Here we present a large scale analysis by nanoLC-Q-TOF and nanoLC-LTQ-Orbitrap mass spectrometry (MS) of ten independent chloroplast preparations from Arabidopsis thaliana which unambiguously identified 1325 proteins. Novel proteins include various kinases and putative nucleotide binding proteins. Based on repeated and independent MS based protein identifications requiring multiple matched peptide sequences, as well as literature, 916 nuclear-encoded proteins were assigned with high confidence to the plastid, of which 86% had a predicted chloroplast transit peptide (cTP). The protein abundance of soluble stromal proteins was calculated from normalized spectral counts from LTQ-Obitrap analysis and was found to cover four orders of magnitude. Comparison to gel-based quantification demonstrates that ‘spectral counting’ can provide large scale protein quantification for Arabidopsis. This quantitative information was used to determine possible biases for protein targeting prediction by TargetP and also to understand the significance of protein contaminants. The abundance data for 550 stromal proteins was used to understand abundance of metabolic pathways and chloroplast processes. We highlight the abundance of 48 stromal proteins involved in post-translational proteome homeostasis (including aminopeptidases, proteases, deformylases, chaperones, protein sorting components) and discuss the biological implications. N-terminal modifications were identified for a subset of nuclear- and chloroplast-encoded proteins and a novel N-terminal acetylation motif was discovered. Analysis of cTPs and their cleavage sites of Arabidopsis chloroplast proteins, as well as their predicted rice homologues, identified new species-dependent features, which will facilitate improved subcellular localization prediction. No evidence was found for suggested targeting via the secretory system. This study provides the most comprehensive chloroplast proteome analysis to date and an expanded Plant Proteome Database (PPDB) in which all MS data are projected on identified gene models

Tick-borne encephalitis virus in dogs - is this an issue?

The last review on Tick-borne encephalitis (TBE) in dogs was published almost ten years ago. Since then, this zoonotic tick-borne arbovirus has been geographically spreading and emerging in many regions in Eurasia and continues to do so. Dogs become readily infected with TBE virus but they are accidental hosts not capable to further spread the virus. They seroconvert upon infection but they seem to be much more resistant to the clinical disease than humans. Apart from their use as sentinels in endemic areas, however, an increasing number of case reports appeared during the last decade thus mirroring the rising public health concerns. Owing to the increased mobility of people travelling to endemic areas with their companion dogs, this consequently leads to problems in recognizing and diagnosing this severe infection in a yet non-endemic area, simply because the veterinarians are not considering TBE. This situation warrants an update on the epidemiology, clinical presentation and possible preventions of TBE in the dog

Genome-wide meta-analysis reveals shared new loci in systemic seropositive rheumatic diseases

OBJECTIVE: Immune-mediated inflammatory diseases (IMIDs) are heterogeneous and complex conditions with overlapping clinical symptoms and elevated familial aggregation, which suggests the existence of a shared genetic component. In order to identify this genetic background in a systematic fashion, we performed the first cross-disease genome-wide meta-analysis in systemic seropositive rheumatic diseases, namely, systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis and idiopathic inflammatory myopathies. METHODS: We meta-analysed ~6.5 million single nucleotide polymorphisms in 11 678 cases and 19 704 non-affected controls of European descent populations. The functional roles of the associated variants were interrogated using publicly available databases. RESULTS: Our analysis revealed five shared genome-wide significant independent loci that had not been previously associated with these diseases: NAB1, KPNA4-ARL14, DGQK, LIMK1 and PRR12. All of these loci are related with immune processes such as interferon and epidermal growth factor signalling, response to methotrexate, cytoskeleton dynamics and coagulation cascade. Remarkably, several of the associated loci are known key players in autoimmunity, which supports the validity of our results. All the associated variants showed significant functional enrichment in DNase hypersensitivity sites, chromatin states and histone marks in relevant immune cells, including shared expression quantitative trait loci. Additionally, our results were significantly enriched in drugs that are being tested for the treatment of the diseases under study. CONCLUSIONS: We have identified shared new risk loci with functional value across diseases and pinpoint new potential candidate loci that could be further investigated. Our results highlight the potential of drug repositioning among related systemic seropositive rheumatic IMIDs

Fractionation of tomato fruit chromoplasts

Chromoplast differentiation involves an active synthesis of carotenoids associated with the remodeling of the preexisting plastid membrane systems to form specialized structures involved in the sequestration and storage of the synthesized carotenoids. These subplastidial structures show remarkable morphological differences and seem to be adapted to the accumulation of particular carotenoids in some plant species and organs. At present, very little is known about chromoplast biogenesis and the role of the different suborganellar structures in the synthesis and storage of carotenoids. The combination of classical fractionation methods with the use of biochemical and -omics techniques represents an attractive approach to unravel novel aspects related with the biochemical and cellular mechanisms underlying the biogenesis of the structures involved in the biosynthesis and storage of carotenoids during chromoplast differentiation. Here we describe a combined protocol for the isolation, lysis and fractionation of tomato fruit chromoplast. The fractions obtained are suitable for metabolomics and proteomics analysis.We acknowledge the financial support of AGAUR-Generalitat de Catalunya (Grant 2017 SGR 710), the CERCA Programme of the Generalitat de Catalunya and the Severo Ochoa Programme for Centres of Excellence in R&D 2016–2019 to CRAG (SEV-2015-0533). AB is member of the Spanish Carotenoid Network (CaRed) funded by the Spanish Ministry of Economy and Competitiveness (Grants BIO2015-71703-REDT and BIO2017-90877-REDT).Peer reviewe

Chloroplast envelope membranes: a dynamic interface between plastids and the cytosol.

Chloroplasts are bounded by a pair of outer membranes, the envelope, that is the only permanent membrane structure of the different types of plastids. Chloroplasts have had a long and complex evolutionary past and integration of the envelope membranes in cellular functions is the result of this evolution. Plastid envelope membranes contain a wide diversity of lipids and terpenoid compounds serving numerous biochemical functions and the flexibility of their biosynthetic pathways allow plants to adapt to fluctuating environmental conditions (for instance phosphate deprivation). A large body of knowledge has been generated by proteomic studies targeted to envelope membranes, thus revealing an unexpected complexity of this membrane system. For instance, new transport systems for metabolites and ions have been identified in envelope membranes and new routes for the import of chloroplast-specific proteins have been identified. The picture emerging from our present understanding of plastid envelope membranes is that of a key player in plastid biogenesis and the co-ordinated gene expression of plastid-specific protein (owing to chlorophyll precursors), of a major hub for integration of metabolic and ionic networks in cell metabolism, of a flexible system that can divide, produce dynamic extensions and interact with other cell constituents. Envelope membranes are indeed one of the most complex and dynamic system within a plant cell. In this review, we present an overview of envelope constituents together with recent insights into the major functions fulfilled by envelope membranes and their dynamics within plant cells