María Elena Chánez-Cárdenas

Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model

BackgroundAged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells.ResultsWe found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC.ConclusionsObtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions.

The Aggregation of Huntingtin and α-Synuclein.

Huntingtons and Parkinsons diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and α-synuclein resp.), are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.

The conserved salt bridge linking two C‐terminal β/α units in homodimeric triosephosphate isomerase determines the folding rate of the monomer

Triosephosphate isomerase (TIM), whose structure is archetypal of dimeric (β/α)8 barrels, has a conserved salt bridge (Arg189–Asp225 in yeast TIM) that connects the two C‐terminal β/α segments to rest of the monomer. We constructed the mutant D225Q, and studied its catalysis and stability in comparison with those of the wild‐type enzyme. Replacement of Asp225 by Gln caused minor drops in kcat and KM, but the catalytic efficiency (kcat/KM) was practically unaffected. Temperature‐induced unfolding–refolding of both TIM samples displayed hysteresis cycles, indicative of processes far from equilibrium. Kinetic studies showed that the rate constant for unfolding was about three‐fold larger in the mutant than in wild‐type TIM. However, more drastic changes were found in the kinetics of refolding: upon mutation, the rate‐limiting step changed from a second‐order (at submicromolar concentrations) to a first‐order reaction. These results thus indicate that renaturation of yTIM occurs through a uni–bimolecular mechanism in which refolding of the monomer most likely begins at the C‐terminal half of its polypeptide chain. From the temperature dependence of the refolding rate, we determined the change in heat capacity for the formation of the transition state from unfolded monomers. The value for the D225Q mutant, which is about 40% of the corresponding value for yTIM, would implicate the folding of only three quarters of a monomer chain in the transition state. Proteins 2008.

The folding pathway of triosephosphate isomerase.

Publisher Summary This chapter discusses the folding pathway of triosephosphate isomerase (TIM), which is a widely studied enzyme. The ubiquity, efficient catalytic activity, and (β/α)8 barrel three-dimensional conformation of this enzyme makes it an excellent model to perform almost any kind of research. TIM is a glycolytic enzyme that catalyzes the interconversion between glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) in the fifth reaction of glycolysis. This reaction is catalyzed with high efficiency, and it is a diffusive process, which is limited only by the rate at which GAP encounters or departs from the active site. TIM is active only in the oligomeric form, and the structure of the native dimer suggests that the presence of both subunits is important for stabilizing the active-site residues. Each monomer contains catalytic residues and the loops at the carboxyl termini of the barrel form the interface among subunits. Protein denaturation is a natural and artificial process that involves the disruption of the three-dimensional organization of protein molecules, that is, the secondary, tertiary, and quaternary structures.

Different catalytic properties of two highly homologous triosephosphate isomerase monomers

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.

The Unfolding and Refolding Reactions of Triosephosphate Isomerase from Trypanosoma Cruzi Follow Similar Pathways. Guanidinium Hydrochloride Studies

The unfolding and refolding reactions of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was studied under equilibrium conditions at increasing guanidinium hydrochloride concentrations. The changes in activity intrinsic fluorescence and far‐ultraviolet circular dichroism as a function of denaturant were used as a quaternary, tertiary and secondary structural probes respectively. The change in extrinsic ANS fluorescence intensity was also investigated. The results show that the transition between the homodimeric native enzyme to the unfolded monomers (unfolding), and its inverse reaction (refolding) are described by similar pathways and two equilibrium intermediates were detected in both reactions. The mild denaturant concentrations intermediate is active and contains significant amount of secondary and tertiary structures. The medium denaturant concentrations intermediate is inactive and able to bind the fluorescent dye. This intermediates are maybe related with those observed in the denaturation patt...

Evaluation of Aged Garlic Extract Neuroprotective Effect in a Focal Model of Cerebral Ischemia

The oxidant species generated in cerebral ischemia have been implicated as important mediators of neuronal injury through damage to lipids, DNA, and proteins. Since ischemia as well as reperfusion insults generate oxidative stress, the administration of antioxidants may limit oxidative damage and ameliorate disease progression. The present work shows the transitory neuroprotective effect of the aged garlic extract (AGE) administration (a proposed antioxidant compound) in a middle cerebral artery occlusion (MCAO) model in rats and established its therapeutic window. To determine the optimal time of administration, animal received AGE (1.2 mL/kg) intraperitoneally 30 min before onset of reperfusion (−0.5 R), at the beginning of reperfusion (0R), or 1 h after onset of reperfusion (1R). Additional doses were administrated after 1, 2, or 3 h after onset of reperfusion. To establish the therapeutic window of AGE, the infarct area was determined for each treatment after different times of reperfusion. Results sh...