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.

Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathies.

α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice.

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.

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.

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.

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.

The glyoxalase pathway: the first hundred years... and beyond.

The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time.

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains.

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h.

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.

The glyoxalase pathway in protozoan parasites.

The glyoxalase system is the main catabolic route for methylglyoxal, a non-enzymatic glycolytic byproduct with toxic and mutagenic effects. This pathway includes two enzymes, glyoxalase I and glyoxalase II, which convert methylglyoxal to d-lactate by using glutathione as a catalytic cofactor. In protozoan parasites the glyoxalase system shows marked deviations from this model.

α-Synuclein aggregation in the saliva of familial transthyretin amyloidosis: a potential biomarker.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the formation of transthyretin (TTR) amyloid deposits. This crippling and fatal disease is associated with point mutations in TTR, a protein mainly produced in the liver. Hence, liver transplantation is the only treatment capable of halting disease progression.

Optimization of time-course experiments for kinetic model discrimination.

Systems biology relies heavily on the construction of quantitative models of biochemical networks. These models must have predictive power to help unveiling the underlying molecular mechanisms of cellular physiology, but it is also paramount that they are consistent with the data resulting from key experiments. Often, it is possible to find several models that describe the data equally well, but provide significantly different quantitative predictions regarding particular variables of the network.

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.

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.

Enlightening the molecular basis of trypanothione specificity in trypanosomatids: mutagenesis of Leishmania infantum glyoxalase II.

Leishmania infantum glyoxalase II shows absolute specificity towards its trypanothione thioester substrate. In the previous work, we performed a comparative analysis of glyoxalase II structures determined by X-ray crystallography which revealed that Tyr291 and Cys294, absent in the human homologue, are essential for substrate binding. To validate this trypanothione specificity hypothesis we produced a mutant L.

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.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of glyoxalase I from Leishmania infantum.

Glyoxalase I (GLO1) is the first of the two glyoxalase-pathway enzymes. It catalyzes the formation of S-D-lactoyltrypanothione from the non-enzymatically formed hemithioacetal of methylglyoxal and reduced trypanothione. In order to understand its substrate binding and catalytic mechanism, GLO1 from Leishmania infantum was cloned, overexpressed in Escherichia coli, purified and crystallized.

Protein glycation in vivo: functional and structural effects on yeast enolase.

Protein glycation is involved in structure and stability changes that impair protein functionality, which is associated with several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer's disease, Parkinson's disease and Andrade's syndrome). To understand the relationship of protein glycation with protein dysfunction, unfolding and beta-fibre formation, numerous studies have been carried out in vitro. All of these previous experiments were conducted in non-physiological or pseudo-physiological conditions that bear little to no resemblance to what may happen in a living cell.

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.

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.

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.

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.

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production.

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.