Endothelial Hyaluronan Synthase 3 Augments Postischemic Arteriogenesis Through CD44/eNOS Signaling.

Objective: The dominant driver of arteriogenesis is elevated shear stress sensed by the endothelial glycocalyx thereby promoting arterial outward remodeling. Hyaluronan, a critical component of the endothelial glycocalyx, is synthesized by 3 HAS isoenzymes (hyaluronan synthases 1-3) at the plasma membrane. Considering further the importance of HAS3 for smooth muscle cell and immune cell functions we aimed to evaluate its role in collateral artery growth. Approach and Results: Male Has3-deficient (Has3-KO) mice were subjected to hindlimb ischemia. Blood perfusion was monitored by laser Doppler perfusion imaging and endothelial function was assessed by measurement of flow-mediated dilation in vivo. Collateral remodeling was monitored by high resolution magnetic resonance angiography. A neutralizing antibody against CD44 (clone KM201) was injected intraperitoneally to analyze hyaluronan signaling in vivo. After hindlimb ischemia, Has3-KO mice showed a reduced arteriogenic response with decreased collateral remodeling and impaired perfusion recovery. While postischemic leukocyte infiltration was unaffected, a diminished flow-mediated dilation pointed towards an impaired endothelial cell function. Indeed, endothelial AKT (protein kinase B)-dependent eNOS (endothelial nitric oxide synthase) phosphorylation at Ser1177 was substantially reduced in Has3-KO thigh muscles. Endothelial-specific Has3-KO mice mimicked the hindlimb ischemia-induced phenotype of impaired perfusion recovery as observed in global Has3-deficiency. Mechanistically, blocking selectively the hyaluronan binding site of CD44 reduced flow-mediated dilation, thereby suggesting hyaluronan signaling through CD44 as the underlying signaling pathway. Conclusions: In summary, HAS3 contributes to arteriogenesis in hindlimb ischemia by hyaluronan/CD44-mediated stimulation of eNOS phosphorylation at Ser1177. Thus, strategies augmenting endothelial HAS3 or CD44 could be envisioned to enhance vascularization under pathological conditions.

Cellular Plasticity in Response to Suppression of Storage Proteins in the Brassica napus Embryo.

The tradeoff between protein and oil storage in oilseed crops has been tested here in oilseed rape (Brassica napus) by analyzing the effect of suppressing key genes encoding protein storage products (napin and cruciferin). The phenotypic outcomes were assessed using NMR and mass spectrometry imaging, microscopy, transcriptomics, proteomics, metabolomics, lipidomics, immunological assays, and flux balance analysis. Surprisingly, the profile of storage products was only moderately changed in RNA interference transgenics. However, embryonic cells had undergone remarkable architectural rearrangements. The suppression of storage proteins led to the elaboration of membrane stacks enriched with oleosin (sixfold higher protein abundance) and novel endoplasmic reticulum morphology. Protein rebalancing and amino acid metabolism were focal points of the metabolic adjustments to maintain embryonic carbon/nitrogen homeostasis. Flux balance analysis indicated a rather minor additional demand for cofactors (ATP and NADPH). Thus, cellular plasticity in seeds protects against perturbations to its storage capabilities and, hence, contributes materially to homeostasis. This study provides mechanistic insights into the intriguing link between lipid and protein storage, which have implications for biotechnological strategies directed at improving oilseed crops.

A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.

The Role of Persulfide Metabolism During Arabidopsis Seed Development Under Light and Dark Conditions.

The sulfur dioxygenase ETHE1 oxidizes persulfides in the mitochondrial matrix and is involved in the degradation of L-cysteine and hydrogen sulfide. ETHE1 has an essential but as yet undefined function in early embryo development of Arabidopsis thaliana. In leaves, ETHE1 is strongly induced by extended darkness and participates in the use of amino acids as alternative respiratory substrates during carbohydrate starvation. Thus, we tested the effect of darkness on seed development in an ETHE1 deficient mutant in comparison to the wild type. Since ETHE1 knock-out is embryo lethal, the knock-down line ethe1-1 with about 1% residual sulfur dioxygenase activity was used for this study. We performed phenotypic analysis, metabolite profiling and comparative proteomics in order to investigate the general effect of extended darkness on seed metabolism and further define the specific function of the mitochondrial sulfur dioxygenase ETHE1 in seeds. Shading of the siliques had no morphological effect on embryogenesis in wild type plants. However, the developmental delay that was already visible in ethe1-1 seeds under control conditions was further enhanced in the darkness. Dark conditions strongly affected seed quality parameters of both wild type and mutant plants. The effect of ETHE1 knock-down on amino acid profiles was clearly different from that found in leaves indicating that in seeds persulfide oxidation interacts with alanine and glycine rather than branched-chain amino acid metabolism. Sulfur dioxygenase deficiency led to defects in endosperm development possibly due to alterations in the cellularization process. In addition, we provide evidence for a potential role of persulfide metabolism in abscisic acid (ABA) signal transduction in seeds. We conclude that the knock-down of ETHE1 causes metabolic re-arrangements in seeds that differ from those in leaves. Putative mechanisms that cause the aberrant endosperm and embryo development are discussed.

Characterization of Naïve and Vitamin C-Treated Mouse Schwann Cell Line MSC80: Induction of the Antioxidative Thioredoxin Related Transmembrane Protein 1.

Schwann cells (SCs) are essential in the production of the axon-wrapping myelin sheath and provide trophic function and repair mechanisms in the peripheral nerves. Consequently, well-characterized SC in vitro models are needed to perform preclinical studies including the investigation of the complex biochemical adaptations occurring in the peripheral nervous system (PNS) under different (patho)physiological conditions. MSC80 cells represent a murine SC line used as an in vitro system for neuropathological studies. Here, we introduce the most abundant 9532 proteins identified via mass spectrometry-based protein analytics, and thus provide the most comprehensive SC protein catalogue published thus far. We cover proteins causative for inherited neuropathies and demonstrate that in addition to cytoplasmic, nuclear and mitochondrial proteins and others belonging to the protein processing machinery are very well covered. Moreover, we address the suitability of MSC80 to examine the molecular effect of a drug-treatment by analyzing the proteomic signature of Vitamin C-treated cells. Proteomic findings, immunocytochemistry, immunoblotting and functional experiments support the concept of a beneficial role of Vitamin C on oxidative stress and identified TMX1 as an oxidative stress protective factor, which might represent a promising avenue for therapeutic intervention of PNS-disorders with oxidative stress burden such as diabetic neuropathy.

Platelet proteomics: from discovery to diagnosis.

INTRODUCTION: Platelets are the smallest cells within the circulating blood with key roles in physiological hemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signaling cascades. Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications. Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signaling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.

Quantification of Cardiovascular Disease Biomarkers in Human Platelets by Targeted Mass Spectrometry.

Platelets are known to be key players in thrombosis and hemostasis, contributing to the genesis and progression of cardiovascular diseases. Due to their pivotal role in human physiology and pathology, platelet function is regulated tightly by numerous factors which have either stimulatory or inhibitory effects. A variety of factors, e.g., collagen, fibrinogen, ADP, vWF, thrombin, and thromboxane promote platelet adhesion and aggregation by utilizing multiple intracellular signal cascades. To quantify platelet proteins for this work, a targeted proteomics workflow was applied. In detail, platelets are isolated and lyzed, followed by a tryptic protein digest. Subsequently, a mix of stable isotope-labeled peptides of interesting biomarker proteins in concentrations ranging from 0.1 to 100 fmol is added to 3 µg digest. These peptides are used as an internal calibration curve to accurately quantify endogenous peptides and corresponding proteins in a pooled platelet reference sample by nanoLC-MS/MS with parallel reaction monitoring. In order to assure a valid quantification, limit of detection (LOD) and limit of quantification (LOQ), as well as linear range, were determined. This quantification of platelet activation and proteins by targeted mass spectrometry may enable novel diagnostic strategies in the detection and prevention of cardiovascular diseases.

Cardiolipin Supports Respiratory Enzymes in Plants in Different Ways.

In eukaryotes the presence of the dimeric phospholipid cardiolipin (CL) is limited to the mitochondrial membranes. It resides predominantly in the inner membrane where it interacts with components of the mitochondrial electron transfer chain. CL deficiency has previously been shown to affect abundances of the plant NADH-dehydrogenase complex and its association with dimeric cyctochrome c reductase. Using an Arabidopsis thaliana knock-out mutant for the final enzyme of CL biosynthesis we here extend current knowledge on the dependence of plant respiration on CL. By correlating respiratory enzyme abundances with enzymatic capacities in mitochondria isolated from wild type, CL deficient and CL complemented heterotrophic cell culture lines a new picture of the participation of CL in plant respiration is emerging. Data indicate a loss of a general reduction of respiratory capacity in CL deficient mitochondria which cannot solely be attributed to decreased abundances or capacities of mitochondrial electron transfer protein complexes and supercomplexes. Instead, it most likely is the result of a loss of the mobile electron carrier cytochrome c. Furthermore, enzymes of the tricarboxylic acid cycle are found to have lower maximum activities in the mutant, including the succinate dehydrogenase complex. Interestingly, abundance of the latter is not altered, indicative of a direct impact of CL deficiency on the enzymatic capacity of this electron transfer chain protein complex.

Differential proteomics analysis of Frankliniella occidentalis immune response after infection with Tomato spotted wilt virus (Tospovirus).

Tomato spotted wilt virus (TSWV) is mainly vectored by Frankliniella occidentalis Pergande, and it potentially activates the vector's immune response. However, molecular background of the altered immune response is not clearly understood. Therefore, using a proteomic approach, we investigated the immune pathways that are activated in F. occidentalis larvae after 24 h exposure to TSWV. Two-dimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-IEF/SDS/PAGE) combined with mass spectrometry (MS), were used to identify proteins that were differentially expressed upon viral infection. High numbers of proteins were abundantly expressed in F. occidentalis exposed to TSWV (73%) compared to the non-exposed (27%), with the majority functionally linked to the innate immune system such as: signaling, stress response, defense response, translation, cellular lipids and nucleotide metabolism. Key proteins included: 70 kDa heat shock proteins, Ubiquitin and Dermcidin, among others, indicative of a responsive pattern of the vector's innate immune system to viral infection.

Dealing with the sulfur part of cysteine: four enzymatic steps degrade l-cysteine to pyruvate and thiosulfate in Arabidopsis mitochondria.

Amino acid catabolism is essential for adjusting pool sizes of free amino acids and takes part in energy production as well as nutrient remobilization. The carbon skeletons are generally converted to precursors or intermediates of the tricarboxylic acid cycle. In the case of cysteine, the reduced sulfur derived from the thiol group also has to be oxidized in order to prevent accumulation to toxic concentrations. Here we present a mitochondrial sulfur catabolic pathway catalyzing the complete oxidation of l-cysteine to pyruvate and thiosulfate. After transamination to 3-mercaptopyruvate, the sulfhydryl group from l-cysteine is transferred to glutathione by sulfurtransferase 1 and oxidized to sulfite by the sulfur dioxygenase ETHE1. Sulfite is then converted to thiosulfate by addition of a second persulfide group by sulfurtransferase 1. This pathway is most relevant during early embryo development and for vegetative growth under light-limiting conditions. Characterization of a double mutant produced from Arabidopsis thaliana T-DNA insertion lines for ETHE1 and sulfurtransferase 1 revealed that an intermediate of the ETHE1 dependent pathway, most likely a persulfide, interferes with amino acid catabolism and induces early senescence.

Depletion of the "gamma-type carbonic anhydrase-like" subunits of complex I affects central mitochondrial metabolism in Arabidopsis thaliana.

"Gamma-type carbonic anhydrase-like" (CAL) proteins form part of complex I in plants. Together with "gamma carbonic anhydrase" (CA) proteins they form an extra domain which is attached to the membrane arm of complex I on its matrix exposed side. In Arabidopsis two CAL and three CA proteins are present, termed CAL1, CAL2, CA1, CA2 and CA3. It has been proposed that the carbonic anhydrase domain of complex I is involved in a process mediating efficient recycling of mitochondrial CO2 for photosynthetic carbon fixation which is especially important during growth conditions causing increased photorespiration. Depletion of CAL proteins has been shown to significantly affect plant development and photomorphogenesis. To better understand CAL function in plants we here investigated effects of CAL depletion on the mitochondrial compartment. In mutant lines and cell cultures complex I amount was reduced by 90-95% but levels of complexes III and V were unchanged. At the same time, some of the CA transcripts were less abundant. Proteome analysis of CAL depleted cells revealed significant reduction of complex I subunits as well as proteins associated with photorespiration, but increased amounts of proteins participating in amino acid catabolism and stress response reactions. Developmental delay of the mutants was slightly alleviated if plants were cultivated at high CO2. Profiling of selected metabolites revealed defined changes in intermediates of the citric acid cycle and amino acid catabolism. It is concluded that CAL proteins are essential for complex I assembly and that CAL depletion specifically affects central mitochondrial metabolism.

Brassica napus seed endosperm - metabolism and signaling in a dead end tissue.

Oilseeds are an important element of human nutrition and of increasing significance for the production of industrial materials. The development of the seeds is based on a coordinated interplay of the embryo and its surrounding tissue, the endosperm. This study aims to give insights into the physiological role of endosperm for seed development in the oilseed crop Brassica napus. Using protein separation by two-dimensional (2D) isoelectric focusing (IEF)/SDS polyacrylamide gel electrophoresis (PAGE) and protein identification by mass spectrometry three proteome projects were carried out: (i) establishment of an endosperm proteome reference map, (ii) proteomic characterization of endosperm development and (iii) comparison of endosperm and embryo proteomes. The endosperm proteome reference map comprises 930 distinct proteins, including enzymes involved in genetic information processing, carbohydrate metabolism, environmental information processing, energy metabolism, cellular processes and amino acid metabolism. To investigate dynamic changes in protein abundance during seed development, total soluble proteins were extracted from embryo and endosperm fractions at defined time points. Proteins involved in sugar converting and recycling processes, ascorbate metabolism, amino acid biosynthesis and redox balancing were found to be of special importance for seed development in B. napus. Implications for the seed filling process and the function of the endosperm for seed development are discussed. BIOLOGICAL SIGNIFICANCE: The endosperm is of key importance for embryo development during seed formation in plants. We present a broad study for characterizing endosperm proteins in the oilseed plant B. napus. Furthermore, a project on the biochemical interplay between the embryo and the endosperm during seed development is presented. We provide evidence that the endosperm includes a complete set of enzymes necessary for plant primary metabolism. Combination of our results with metabolome data will further improve systems-level understanding of the seed filling process and provide rational strategies for plant bioengineering.