Early stage metabolic events associated with the establishment of Vitis vinifera - Plasmopara viticola compatible interaction.

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world, with 7.5 million of production area in 2017. The domesticated varieties of grapevine are highly susceptible to many fungal infections, of which downy mildew, caused by the biotrophic oomycete Plasmopara viticola (Berk. et Curt.) Berl. et de Toni is one of the most threatening. In V. vinifera, several studies have shown that a weak and transient activation of a defense mechanism occurs, but it is easily overcome by the pathogen leading to the establishment of a compatible interaction. Major transcript, protein and physiologic changes were shown to occur at later infection time-points, but comprehensive data on the first hours of interaction is scarce. In the present work, we investigated the major physiologic and metabolic changes that occur in the first 24 h of interaction between V. vinifera cultivar Trincadeira and P. viticola. Our results show that there was a negative modulation of several metabolic classes associated to pathogen defense such as flavonoids or phenylpropanoids as well as an alteration of carbohydrate content after inoculation with the pathogen. We also found an accumulation of hydrogen peroxide and increase of lipid peroxidation but to a low extent, that seems to be insufficient to restrain pathogen growth during the initial biotrophic phase of the interaction.

The role of fibrinogen glycation in ATTR: evidence for chaperone activity loss in disease.

Transthyretin amyloidosis (ATTR) belongs to a class of disorders caused by protein misfolding and aggregation. ATTR is a disabling disorder of autosomal dominant trait, where transthyretin (TTR) forms amyloid deposits in different organs, causing dysfunction of the peripheral nervous system. We previously discovered that amyloid fibrils from ATTR patients are glycated by methylglyoxal. Even though no consensus has been reached about the actual role of methylglyoxal-derived advanced glycation end-products in amyloid diseases, evidence collected so far points to a role for protein glycation in conformational abnormalities, being ubiquitously found in amyloid deposits in Alzheimer's disease, dialysis-related amyloidosis and Parkinson's diseases. Human fibrinogen, an extracellular chaperone, was reported to specifically interact with a wide spectrum of stressed proteins and suppress their aggregation, being an interacting protein with TTR. Fibrinogen is differentially glycated in ATTR, leading to its chaperone activity loss. Here we show the existence of a proteostasis imbalance in ATTR linked to fibrinogen glycation by methylglyoxal.

Metabolomics for undergraduates: Identification and pathway assignment of mitochondrial metabolites.

Metabolomics is a key discipline in systems biology, together with genomics, transcriptomics, and proteomics. In this omics cascade, the metabolome represents the biochemical products that arise from cellular processes and is often regarded as the final response of a biological system to environmental or genetic changes. The overall screening approach to identify all the metabolites in a given biological system is called metabolic fingerprinting. Using high-resolution and high-mass accuracy mass spectrometry, large metabolome coverage, sensitivity, and specificity can be attained. Although the theoretical concepts of this methodology are usually provided in life-science programs, hands-on laboratory experiments are not usually accessible to undergraduate students. Even if the instruments are available, there are not simple laboratory protocols created specifically for teaching metabolomics. We designed a straightforward hands-on laboratory experiment to introduce students to this methodology, relating it to biochemical knowledge through metabolic pathway mapping of the identified metabolites. This study focuses on mitochondrial metabolomics since mitochondria have a well-known, medium-sized cellular sub-metabolome. These features facilitate both data processing and pathway mapping. In this experiment, students isolate mitochondria from potatoes, extract the metabolites, and analyze them by high-resolution mass spectrometry (using an FT-ICR mass spectrometer). The resulting mass list is submitted to an online program for metabolite identification, and compounds associated with mitochondrial pathways can be highlighted in a metabolic network map.

Metabolite extraction for high-throughput FTICR-MS-based metabolomics of grapevine leaves.

In metabolomics there is an ever-growing need for faster and more comprehensive analysis methods to cope with the increase of biological studies. Direct infusion Fourier-transform ion cyclotron-resonance mass spectrometry (DI-FTICR-MS) is used in non-targeted metabolomics to obtain high-resolution snapshots of the metabolic state of a system. In any metabolic profiling study, the establishment of an effective metabolite extraction protocol is paramount. We developed an improved metabolite extraction method, compatible with DI-FTICR-MS-based metabolomics, using grapevine leaves. This extraction protocol allowed the extraction of polar and non-polar compounds, covering all major classes found in plants and increasing metabolome coverage.

Transthyretin Amyloidosis: Chaperone Concentration Changes and Increased Proteolysis in the Pathway to Disease.

Transthyretin amyloidosis is a conformational pathology characterized by the extracellular formation of amyloid deposits and the progressive impairment of the peripheral nervous system. Point mutations in this tetrameric plasma protein decrease its stability and are linked to disease onset and progression. Since non-mutated transthyretin also forms amyloid in systemic senile amyloidosis and some mutation bearers are asymptomatic throughout their lives, non-genetic factors must also be involved in transthyretin amyloidosis. We discovered, using a differential proteomics approach, that extracellular chaperones such as fibrinogen, clusterin, haptoglobin, alpha-1-anti-trypsin and 2-macroglobulin are overrepresented in transthyretin amyloidosis. Our data shows that a complex network of extracellular chaperones are over represented in human plasma and we speculate that they act synergistically to cope with amyloid prone proteins. Proteostasis may thus be as important as point mutations in transthyretin amyloidosis.

The proteome response to amyloid protein expression in vivo.

Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity.

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

The relative amounts of plasma transthyretin forms in familial transthyretin amyloidosis: a quantitative analysis by Fourier transform ion-cyclotron resonance mass spectrometry.

Familial transthyretin amyloidosis (ATTR) is a fatal autosomal dominant disease characterized by the formation of amyloid fibers, mainly composed of transthyretin (TTR). Protein aggregation and amyloid fiber formation are considered concentration dependent processes and since most ATTR patients are heterozygous it is crucial to determine the ratio between mutant and non-mutant TTR forms in human plasma. Using a high resolution mass spectrometry based approach we determined the ratio of TTR forms in ATTR patients, V30M mutation carriers, symptomatic and asymptomatic ones, as well as ATTR patients that received a wild type cadaveric liver transplant. Domino transplanted patients that received a liver from an ATTR patient were also investigated. We found that although wild type TTR is diminished in the plasma of non-transplanted ATTR patients comparatively to healthy subjects, the relationship with the V30M variant does not change with illness progression. Those who received a wild type liver showed no mutant protein while domino transplanted patients presented the same relative amount of V30M as found in asymptomatic and symptomatic individuals. The V30M to wild type TTR ratio in plasma is the same for all ATTR patients studied, showing no variation with disease clinical progression. Our results point to the involvement of additional non-genetic factors on the pathogenesis of this disease.

A non-invasive method based on saliva to characterize transthyretin in familial amyloidotic polyneuropathy patients using FT-ICR high-resolution MS.

PURPOSE: To identify, characterize and perform a relative quantification of human transthyretin (TTR) variants in human saliva. EXPERIMENTAL DESIGN: Serum and saliva samples were collected from healthy and familial amyloidotic polyneuropathy (FAP) patients, proteins separated by SDS-PAGE, TTR bands excised, in-gel digested and analyzed by MALDI-FTICR. RESULTS: We identified and performed a relative quantification of mutated and native TTR forms in human saliva, based on FTICR-MS. The results are quantitatively identical to the ones obtained with human serum. In FAP patients subjected to cadaveric liver transplant, the TTR mutant form is no longer detected in saliva, while in patients receiving a domino liver from a FAP donor the mutant form of TTR becomes detectable in saliva, thus demonstrating the serum origin of TTR in saliva. CONCLUSIONS AND CLINICAL RELEVANCE: Saliva TTR originates in serum and the ratio of mutant to native TTR is preserved. The method provides a non-invasive detection of mutated TTR and a relative quantification of TTR forms. Diagnostic and disease prognosis of FAP is crucial at early stages of the disease and after liver transplantation, the only curative therapy. A suitable non-invasive method was developed for monitoring the most important FAP biomarker in human saliva.

Protein glycation and methylglyoxal metabolism in yeast: finding peptide needles in protein haystacks.

Metabolism, the set of all chemical transformations inside a living cell, comprises nonenzymatic processes that generate toxic products such as reactive oxygen species and 2-oxoaldehydes. Methylglyoxal, a highly reactive 2-oxoaldehyde by-product of glycolysis, is able to react irreversibly and nonenzymatically with proteins, forming methylglyoxal advanced glycation end-products, which alter protein structure, stability and function. Therefore, protein glycation may influence cell metabolism and its physiology in a way beyond what can be predicted based on the implicit codification used in systems biology. Genome-wide approaches and transcriptomics, two mainstays of systems biology, are powerless to tackle the problems caused by nonenzymatic reactions that are part of cell metabolism and biochemistry. The effects of methylglyoxal-derived protein glycation and the cell's response to this unspecific posttranslational modification were investigated in Saccharomyces cerevisiae as a model organism. Specific protein glycation phenotypes were identified using yeast null-mutants for methylglyoxal catabolism and the existence of specific protein glycation targets by peptide mass fingerprint was discovered. Enolase, the major target, endures a glycation-dependent activity loss caused by dissociation of the active dimer upon glycation at a specific arginine residue, identified using the hidden information of peptide mass fingerprint. Once glycation occurs, a cellular response involving heat shock proteins from the refolding chaperone pathway is elicited and Hsp26p is activated by glycation.

Measuring intracellular enzyme concentrations: Assessing the effect of oxidative stress on the amount of glyoxalase I.

Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to in vivo conditions. The specificity constant, k(S) , is the ratio between k(cat) (the catalytic constant) and K(m) (Michaelis-Menten constant), and expresses the efficiency of an enzyme as a catalyst. This parameter is usually determined for purified enzymes, and in this work, we propose a classroom experiment for its determination in situ, in permeabilized yeast cells, based on a method of external enzyme addition, which was previously reported. Under these conditions, which resemble the in vivo state, enzyme concentrations and protein interactions are preserved. The students are presented with a novel approach in enzymology, based on the titration methods that allow the measurement of the enzyme amount, and thus the k(cat) and k(S) . The method will also be used to investigate the effect of exposure to oxidative stress conditions on yeast glyoxalase I.

Catalysis and structural properties of Leishmania infantum glyoxalase II: trypanothione specificity and phylogeny.

The glyoxalase pathway catalyzes the formation of d-lactate from methylglyoxal, a toxic byproduct of glycolysis. In trypanosomatids, trypanothione replaces glutathione in this pathway, making it a potential drug target, since its selective inhibition might increase methylglyoxal concentration in the parasites. Two glyoxalase II structures were solved. One with a bound spermidine molecule (1.8 A) and the other with d-lactate at the active site (1.9 A). The second structure was obtained by crystal soaking with the enzyme substrate (S)-d-lactoyltrypanothione. The overall structure of Leishmania infantum glyoxalase II is very similar to its human counterpart, with important differences at the substrate binding site. The crystal structure of L. infantum glyoxalase II is the first structure of this enzyme from trypanosomatids. The differential specificity of glyoxalase II toward glutathione and trypanothione moieties was revealed by differential substrate binding. Evolutionary analysis shows that trypanosomatid glyoxalases II diverged early from eukaryotic enzymes, being unrelated to prokaryotic proteins.

Teaching biochemistry at Lisbon University-Facing the challenge of the Bologna Declaration in the 25th anniversary of the biochemistry course.

The biochemistry degree has been taught at Lisbon University for 25 years. Since its creation, the curriculum is characterized for being widely eclectic and multidisciplinary. The adoption of the concepts proposed in Europe by the Declaration of Bologna and incorporation of these ideas at Lisbon University is discussed here for the biochemistry degree.

Yeast protein glycation in vivo by methylglyoxal. Molecular modification of glycolytic enzymes and heat shock proteins.

Protein glycation by methylglyoxal is a nonenzymatic post-translational modification whereby arginine and lysine side chains form a chemically heterogeneous group of advanced glycation end-products. Methylglyoxal-derived advanced glycation end-products are involved in pathologies such as diabetes and neurodegenerative diseases of the amyloid type. As methylglyoxal is produced nonenzymatically from dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate during glycolysis, its formation occurs in all living cells. Understanding methylglyoxal glycation in model systems will provide important clues regarding glycation prevention in higher organisms in the context of widespread human diseases. Using Saccharomyces cerevisiae cells with different glycation phenotypes and MALDI-TOF peptide mass fingerprints, we identified enolase 2 as the primary methylglyoxal glycation target in yeast. Two other glycolytic enzymes are also glycated, aldolase and phosphoglycerate mutase. Despite enolase's activity loss, in a glycation-dependent way, glycolytic flux and glycerol production remained unchanged. None of these enzymes has any effect on glycolytic flux, as evaluated by sensitivity analysis, showing that yeast glycolysis is a very robust metabolic pathway. Three heat shock proteins are also glycated, Hsp71/72 and Hsp26. For all glycated proteins, the nature and molecular location of some advanced glycation end-products were determined by MALDI-TOF. Yeast cells experienced selective pressure towards efficient use of d-glucose, with high methylglyoxal formation as a side effect. Glycation is a fact of life for these cells, and some glycolytic enzymes could be deployed to contain methylglyoxal that evades its enzymatic catabolism. Heat shock proteins may be involved in proteolytic processing (Hsp71/72) or protein salvaging (Hsp26).

Protein glycation in Saccharomyces cerevisiae. Argpyrimidine formation and methylglyoxal catabolism.

Methylglyoxal is the most important intracellular glycation agent, formed nonenzymatically from triose phosphates during glycolysis in eukaryotic cells. Methylglyoxal-derived advanced glycation end-products are involved in neurodegenerative disorders (Alzheimer's, Parkinson's and familial amyloidotic polyneurophathy) and in the clinical complications of diabetes. Research models for investigating protein glycation and its relationship to methylglyoxal metabolism are required to understand this process, its implications in cell biochemistry and their role in human diseases. We investigated methylglyoxal metabolism and protein glycation in Saccharomyces cerevisiae. Using a specific antibody against argpyrimidine, a marker of protein glycation by methylglyoxal, we found that yeast cells growing on d-glucose (100 mM) present several glycated proteins at the stationary phase of growth. Intracellular methylglyoxal concentration, determined by a specific HPLC based assay, is directly related to argpyrimidine formation. Moreover, exposing nongrowing yeast cells to a higher d-glucose concentration (250 mM) increases methylglyoxal formation rate and argpyrimidine modified proteins appear within 1 h. A kinetic model of methylglyoxal metabolism in yeast, comprising its nonenzymatic formation and enzymatic catabolism by the glutathione dependent glyoxalase pathway and aldose reductase, was used to probe the role of each system parameter on methylglyoxal steady-state concentration. Sensitivity analysis of methylglyoxal metabolism and studies with gene deletion mutant yeast strains showed that the glyoxalase pathway and aldose reductase are equally important for preventing protein glycation in Saccharomyces cerevisiae.

Analysis of methylglyoxal in water and biological matrices by capillary zone electrophoresis with diode array detection.

We describe a new method for the determination of methylglyoxal in water and biological matrices, using o-phenylenediamine as derivatizing agent and solid-phase extraction followed by capillary zone electrophoresis with diode array detection. 25 mM sodium phosphate running buffers at pH 2.2, 30 kV, and 25 degrees C allowed the best instrumental conditions for the optimum separation of methylglyoxal in a suitable analytical time (< 10 min), using an uncoated fused-silica capillary of 75 microm inner diameter and an effective length of 45.1 cm with an extended light path and the wavelength set to 200 nm. Under optimized instrumental conditions, good reproducibility of the migration time (< 1.1%), precision (< 5%), an excellent linear dynamic range from 0.1 to 3.6 mg/L (r(2) = 0.9997), and low limits of detection (7.2 microg/L) were obtained for methylglyoxal measurements, using the internal standard methodology. Assays on laboratory-spiked tap and ground water samples allowed a remarkable accuracy, presenting yields of 95.0 +/- 4.3 and 94.0 +/- 1.1%, respectively, and good performance to determine methylglyoxal in beer and yeast cells suspensions matrices was also obtained at trace level. The present methodology is a cost-effective alternative for routine quality control analysis, showing to be reliable, sensitive, and with a low sample volume requirement to monitor methylglyoxal in water and biological matrices.

Glucose-6-phosphate dehydrogenase deficiency in Portugal: biochemical and mutational profiles, heterogeneity, and haplotype association.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. This deficiency in erythrocytes has a prevalence of 0.51 +/- 0.109 in the Caucasoid male population of Portugal. The frequency for deficiency-conferring genes is 0.39% in the Portuguese population. In the herein study populations males from areas of Portugal presenting with the highest prevalence of G6PD deficiency (Castelo Branco, Setúbal, Faro, and Lisbon) as well as similar subjects located in the border Center/North area of the country (Viseu) have been analyzed for biochemical parameters and screened for mutations and haplotype-associated mutations commensurate with G6PD deficiency. Six intragenic restriction fragment length polymorphisms (RFLPs) were studied: exon 5, nt 376 A -->G, FokI; intron 5, nt 611 C--> G, PvuII; intron 8, nt 163 C--> T, BspHI; exon 10, nt 116 G --> A, PstI; exon 11, nt 1311 C--> T, BclI; and intron 11, nt 93 T -->C, NlaIII. New haplotypes were constructed with the inclusion of intron 11, nt 93 T--> C, NlaIII, and only 5 of 64 possible haplotypes were found to show a marked linkage disequilibrium for several RFLPs and also for mutations and specific haplotypes. The control population (n = 168 males) presented the G6PD B variant and corresponded to haplotypes I (- - + + - -), Ia (- - + + - +), and VIIa (- - + + + +), in 91.8, 2.3, and 5.9%, respectively. The PCR and sequencing analysis of extracted DNAs from the deficient G6PD group showed 48.6% (16/33) of individuals with the G6PD A- mutation, corresponding to haplotype VIa (+ + - + - +); 9% (3/33) with the Betica mutation and 18% (6/33) with the Santa Maria mutation, both of them associated with haplotype IVa (+ - - + \- +); 6.1% (2/33) with the Mediterranean mutation associated with haplotype VIIa; 12.3% (4/33) with the Seattle mutation, 3.0% (1/33) with Gaohe mutation; and a new mutation, 3.0% (1/33), which we designated by G6PD Flores, all of them associated with haplotype I.