Jordi Villà-Freixa

Amyloid-dependent triosephosphate isomerase nitrotyrosination induces glycation and tau fibrillation

Alzheimers disease neuropathology is characterized by neuronal death, amyloid beta-peptide deposits and neurofibrillary tangles composed of paired helical filaments of tau protein. Although crucial for our understanding of the pathogenesis of Alzheimers disease, the molecular mechanisms linking amyloid beta-peptide and paired helical filaments remain unknown. Here, we show that amyloid beta-peptide-induced nitro-oxidative damage promotes the nitrotyrosination of the glycolytic enzyme triosephosphate isomerase in human neuroblastoma cells. Consequently, nitro-triosephosphate isomerase was found to be present in brain slides from double transgenic mice overexpressing human amyloid precursor protein and presenilin 1, and in Alzheimers disease patients. Higher levels of nitro-triosephosphate isomerase (P < 0.05) were detected, by Western blot, in immunoprecipitates from hippocampus (9 individuals) and frontal cortex (13 individuals) of Alzheimers disease patients, compared with healthy subjects (4 and 9 individuals, respectively). Triosephosphate isomerase nitrotyrosination decreases the glycolytic flow. Moreover, during its isomerase activity, it triggers the production of the highly neurotoxic methylglyoxal (n = 4; P < 0.05). The bioinformatics simulation of the nitration of tyrosines 164 and 208, close to the catalytic centre, fits with a reduced isomerase activity. Human embryonic kidney (HEK) cells overexpressing double mutant triosephosphate isomerase (Tyr164 and 208 by Phe164 and 208) showed high methylglyoxal production. This finding correlates with the widespread glycation immunostaining in Alzheimers disease cortex and hippocampus from double transgenic mice overexpressing amyloid precursor protein and presenilin 1. Furthermore, nitro-triosephosphate isomerase formed large beta-sheet aggregates in vitro and in vivo, as demonstrated by turbidometric analysis and electron microscopy. Transmission electron microscopy (TEM) and atomic force microscopy studies have demonstrated that nitro-triosephosphate isomerase binds tau monomers and induces tau aggregation to form paired helical filaments, the characteristic intracellular hallmark of Alzheimers disease brains. Our results link oxidative stress, the main etiopathogenic mechanism in sporadic Alzheimers disease, via the production of peroxynitrite and nitrotyrosination of triosephosphate isomerase, to amyloid beta-peptide-induced toxicity and tau pathology.

Knowledge management for systems biology a general and visually driven framework applied to translational medicine

BackgroundTo enhance our understanding of complex biological systems like diseases we need to put all of the available data into context and use this to detect relations, pattern and rules which allow predictive hypotheses to be defined. Life science has become a data rich science with information about the behaviour of millions of entities like genes, chemical compounds, diseases, cell types and organs, which are organised in many different databases and/or spread throughout the literature. Existing knowledge such as genotype - phenotype relations or signal transduction pathways must be semantically integrated and dynamically organised into structured networks that are connected with clinical and experimental data. Different approaches to this challenge exist but so far none has proven entirely satisfactory.ResultsTo address this challenge we previously developed a generic knowledge management framework, BioXM™, which allows the dynamic, graphic generation of domain specific knowledge representation models based on specific objects and their relations supporting annotations and ontologies. Here we demonstrate the utility of BioXM for knowledge management in systems biology as part of the EU FP6 BioBridge project on translational approaches to chronic diseases. From clinical and experimental data, text-mining results and public databases we generate a chronic obstructive pulmonary disease (COPD) knowledge base and demonstrate its use by mining specific molecular networks together with integrated clinical and experimental data.ConclusionsWe generate the first semantically integrated COPD specific public knowledge base and find that for the integration of clinical and experimental data with pre-existing knowledge the configuration based set-up enabled by BioXM reduced implementation time and effort for the knowledge base compared to similar systems implemented as classical software development projects. The knowledgebase enables the retrieval of sub-networks including protein-protein interaction, pathway, gene - disease and gene - compound data which are used for subsequent data analysis, modelling and simulation. Pre-structured queries and reports enhance usability; establishing their use in everyday clinical settings requires further simplification with a browser based interface which is currently under development.

On the generation of catalytic antibodies by transition state analogues.

The effective design of catalytic antibodies represents a major conceptual and practical challenge. It is implicitly assumed that a proper transition state analogue (TSA) can elicit a catalytic antibody (CA) that will catalyze the given reaction in a similar way to an enzyme that would evolve (or was evolved) to catalyze this reaction. However, in most cases it was found that the TSA used produced CAs with relatively low rate enhancement as compared to the corresponding enzymes, when these exist. The present work explores the origin of this problem, by developing two approaches that examine the similarity of the TSA and the corresponding transition state (TS). These analyses are used to assess the proficiency of the CA generated by the given TSA. Both approaches focus on electrostatic effects that have been found to play a major role in enzymatic reactions. The first method uses molecular interaction potentials to look for the similarity between the TSA and the TS and, in principle, to help in designing new haptens by using 3D quantitative struture–activity relationships. The second and more quantitative approach generates a grid of Langevin dipoles, which are polarized by the TSA, and then uses the grid to bind the TS. Comparison of the resulting binding energy with the binding energy of the TS to the grid that was polarized by the TS provides an estimate of the proficiency of the given CA. Our methods are used in examining the origin of the difference between the catalytic power of the 1F7 CA and chorismate mutase. It is demonstrated that the relatively small changes in charge and structure between the TS and TSA are sufficient to account for the difference in proficiency between the CA and the enzyme. Apparently the environment that was preorganized to stabilize the TSA charge distribution does not provide a sufficient stabilization to the TS. The general implications of our findings and the difficulties in designing a perfect TSA are discussed. Finally, the possible use of our approach in screening for an optimal TSA is pointed out.

Chromatin-Bound IκBα Regulates a Subset of Polycomb Target Genes in Differentiation and Cancer

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation.

Teaching structural bioinformatics at the undergraduate level

Understanding the basic principles of structural biology is becoming a major subject of study in most undergraduate level programs in biology. In the genomic and proteomic age, it is becoming indispensable for biology students to master concepts related to the sequence and structure of proteins in order to develop skills that may be useful in a wide range of applications. Within this context, this article shows a scheme for teaching structural biology based on hands‐on computer approaches, including computational genomics, and structural bioinformatics tools. The students learn to use most of the leading computer programs available for the complete path that goes from sequence to structure and eventually function. During the course, they are taught to build models of proteins based on sequence and structure information. The students are also provided with a critical point of view on automatic procedures and learn to discern between likely and unlikely structures of their final models.

Energetics of K+ permeability through Gramicidin A by forward‐reverse steered molecular dynamics

The estimation of ion channel permeability poses a considerable challenge for computer simulations because of the significant free energy barriers involved, but also offers valuable molecular information on the ion permeation process not directly available from experiments. In this article we determine the equilibrium free energy barrier for potassium ion permeability in Gramicidin A in an efficient way by atomistic forward‐reverse non‐equilibrium steered molecular dynamics simulations, opening the way for its use in more complex biochemical systems. Our results indicate that the tent‐shaped energetics of translocation of K+ ions in Gramicidin A is dictated by the different polarization responses to the ion of the external bulk water and the less polar environment of the membrane. Proteins 2008.

Simulation methods with extended stability for stiff biochemical kinetics

BackgroundWith increasing computer power, simulating the dynamics of complex systems in chemistry and biology is becoming increasingly routine. The modelling of individual reactions in (bio)chemical systems involves a large number of random events that can be simulated by the stochastic simulation algorithm (SSA). The key quantity is the step size, or waiting time, τ, whose value inversely depends on the size of the propensities of the different channel reactions and which needs to be re-evaluated after every firing event. Such a discrete event simulation may be extremely expensive, in particular for stiff systems where τ can be very short due to the fast kinetics of some of the channel reactions. Several alternative methods have been put forward to increase the integration step size. The so-called τ-leap approach takes a larger step size by allowing all the reactions to fire, from a Poisson or Binomial distribution, within that step. Although the expected value for the different species in the reactive system is maintained with respect to more precise methods, the variance at steady state can suffer from large errors as τ grows.ResultsIn this paper we extend Poisson τ-leap methods to a general class of Runge-Kutta (RK) τ-leap methods. We show that with the proper selection of the coefficients, the variance of the extended τ-leap can be well-behaved, leading to significantly larger step sizes.ConclusionsThe benefit of adapting the extended method to the use of RK frameworks is clear in terms of speed of calculation, as the number of evaluations of the Poisson distribution is still one set per time step, as in the original τ-leap method. The approach paves the way to explore new multiscale methods to simulate (bio)chemical systems.

Computational study on the carboligation reaction of acetohidroxyacid synthase: New approach on the role of the HEThDP - intermediate

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl‐thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP–). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP– from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP– has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP–) and a second pyruvate molecule. Our results suggest that additional acid–base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid–base properties of the HEThDP– intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP– intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. Proteins 2010.

The role of residue stability in transient protein-protein interactions involved in enzymatic phosphate hydrolysis. A computational study.

Finding why protein–protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so‐called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S‐LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate “hot spots” for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper “energetic” orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above‐mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient PPIs. Proteins 2006.

Methylglyoxal Produced by Amyloid-β Peptide-Induced Nitrotyrosination of Triosephosphate Isomerase Triggers Neuronal Death in Alzheimer's Disease

Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimers disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.