Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein

Nitroxide spin labels were incorporated into selected sites within the β-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Cα carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.     

The Risk of Coronary Artery Disease Associated with Cigarette Smoking and Hypercholesterolemia Is Additionally Increased by the Presence of the AT 1 R Gene 1166C Allele

Cigarette smoking and hypercholesterolemia influence the renin-angiotensin system (RAS) functions, including increased RAS-mediated vasoconstriction, mitogenic signaling, and angiotensin II type 1 receptor (AT1R) expression. We have explored the interactions of the AT1R gene 1166 A>C polymorphism and traditional risk factors using an epidemiological approach. The study cohort included 341 subjects; 172 were patients with angiographically confirmed coronary artery disease (CAD) and 169 were blood donors. The 1166 A>C polymorphism was genotyped using the PCR-RFLP method. We found a synergy of the 1166C allele with cigarette smoking (synergy indices: SI = 1.41, SIM = 1.33), LDL cholesterol levels > or = 3 mmol/l (SI = 1.25, SIM = 1.19), and elevated total cholesterol (> or =5 mmol/l) levels (SI = 1.15, SIM = 1.13). In each case, the estimated CAD risk was greater than that predicted by assuming the additivity and multiplication of effects. We conclude that the 1166C allele increases the risk of CAD associated with the presence of cigarette smoking and hypercholesterolemia.     

Hsp90n - An accidental product of a fortuitous chromosomal translocation rather than a regular Hsp90 family member of human proteome

Human cells express two isoforms of the Hsp90 protein, called Hsp90alpha and Hsp90beta. Although existence of the third form called Hsp90alphaDeltaN, or Hsp90N was reported in 1998, our investigation, based on the sequence analysis and attempts to reproduce previous results, demonstrate that there is no evidence that Hsp90N gene is present in human genome and no homologs of such a protein are present in other known eukaryotic genomes. We propose that Hsp90N was created as an artifact of a cDNA synthesis or that it is a chimeric protein, being a result of the chromosomal rearrangement that occurred in a single cell line, after this line was established.     

The MLK Family Mediates c-Jun N-Terminal Kinase Activation in Neuronal Apoptosis

Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.     

Protective effects of resveratrol against oxidative/nitrative modifications of plasma proteins and lipids exposed to peroxynitrite

The protective effects of resveratrol (3, 4', 5-trihydroxystilbene; present naturally in different plants) against the oxidative/nitrative damage of human plasma proteins induced by peroxynitrite (ONOO-) were studied and compared with those of deferoxamine (DFO; a natural siderophore isolated from Streptomyces pilosus), which is a typical and well-known antioxidant. We also studied the effect of ONOO- on plasma lipid peroxidation and the role of tested antioxidants in this process. ONOO- at the used concentrations (0.01-1 mM) showed toxicity to human plasma components. Exposure of plasma to ONOO- (0.1 mM) resulted in an increase of the level of carbonyl groups and nitrotyrosine residues in plasma proteins (approximately 4-fold and 76-fold, respectively) and in a distinct augmentation of lipid peroxidation (approximately 2-fold). In the presence of 0.1-mM resveratrol, a distinct decrease of carbonyl group formation and tyrosine nitration in plasma proteins caused by 0.1-mM ONOO- was observed (by approximately 70% and 65%, respectively). Addition of 0.1-mM DFO to plasma also distinctly reduced the level of carbonyl groups and nitrotyrosines caused by 0.1-mM ONOO- (by approximately 50% and 60%, respectively). Moreover, these antioxidants also inhibited plasma lipid peroxidation induced by ONOO- (0.1 mM). The obtained results indicate that in vitro resveratrol, like well-known antioxidant DFO, has inhibitory effects on ONOO- -mediated oxidation of proteins and lipids in human plasma.     

Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae)

The systematic analysis of structure-activity relationships of insect kinins on two heterologous receptor-expressing systems is described. Previously, kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the dengue vector, the mosquito Aedes aegypti (L.), were functionally and stably expressed in CHO-K1 cells. In order to determine which kinin residues are critical for the peptide-receptor interaction, kinin core analogs were synthesized as an Ala-replacement series of the peptide FFSWGa and tested by a calcium bioluminescence plate assay. The amino acids Phe(1) and Trp(4) were essential for activity of the insect kinins in both receptors. It was confirmed that the pentapeptide kinin core is the minimum sequence required for activity and that the C-terminal amide is also essential. In contrast to the tick receptor, a large increase in efficacy is observed in the mosquito receptor when the C-terminal pentapeptide is N-terminally extended to a hexapeptide. The aminoisobutyric acid (Aib)-containing analog, FF[Aib]WGa, was as active as superagonist FFFSWGa on the mosquito receptor in contrast to the tick receptor where it was statistically more active than FFFSWGa by an order of magnitude. This restricted conformation Aib analog provides information on the conformation associated with the interaction of the insect kinins with these two receptors. Furthermore, the analog FF[Aib]WGa has been previously shown to resist degradation by the peptidases ACE and nephrilysin and represents an important lead in the development of biostable insect kinin analogs that ticks and mosquitoes cannot readily deactivate.     

A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation

The bootstrap-based method for calculation of the 3D variance in cryo-EM maps reconstructed from sets of their projections was applied to a dataset of functional ribosomal complexes containing the Escherichia coli 70S ribosome, tRNAs, and elongation factor G (EF-G). The variance map revealed regions of high variability in the intersubunit space of the ribosome: in the locations of tRNAs, in the putative location of EF-G, and in the vicinity of the L1 protein. This result indicated heterogeneity of the dataset. A method of focused classification was put forward in order to sort out the projection data into approximately homogenous subsets. The method is based on the identification and localization of a region of high variance that a subsequent classification step can be focused on by the use of a 3D spherical mask. After initial classification, template volumes are created and are subsequently refined using a multireference 3D projection alignment procedure. In the application to the ribosome dataset, the two resulting structures were interpreted as resulting from ribosomal complexes with bound EF-G and an empty A site, or, alternatively, from complexes that had no EF-G bound but had both A and P sites occupied by tRNA. The proposed method of focused classification proved to be a successful tool in the analysis of the heterogeneous cryo-EM dataset. The associated calculation of the correlations within the density map confirmed the conformational variability of the complex, which could be interpreted in terms of the ribosomal elongation cycle.     

Structure of the head of the Bartonella adhesin BadA

Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 A. Both domains are beta-prisms, the N-terminal one formed by interleaved, five-stranded beta-meanders parallel to the trimer axis and the C-terminal one by five-stranded beta-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens.     

Oxidative DNA damage in polymorphonuclear leukocytes of patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disorder characterized by recurrent, inflammatory self-limited episodes of fever and other symptoms. This disease is caused by more than 25 mutations in the gene MEFV. During fever attacks, there is a substantial influx of polymorphonuclear leukocytes into the affected tissues. Attack-free periods are accompanied by the up-regulation of neutrophil and monocyte phagocytic activity and oxidative burst. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in FMF patients. To test this hypothesis, we investigated oxidative DNA damage in polymorphonuclear leukocytes of FMF patients during the attack-free period in comparison with FMF-free control individuals. DNA was isolated from polymorphonuclear leukocytes of 17 FMF patients and 10 control individuals. DNA samples were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of DNA. We show, for the first time, that FMF patients accumulate statistically significant levels of these lesions in their DNA when compared to FMF-free control individuals. This work suggests that the persistent oxidative stress with excess production of free radicals in FMF patients may lead to accumulation of oxidative DNA damage. Defective DNA repair may also contribute to this phenomenon, perhaps due to mutations in the MEFV gene. The accumulation of mutagenic and cytotoxic DNA lesions may contribute to increased mutations and apoptosis in FMF patients, thus to worsening of the disease and well-being of the patients. Future research should deal with prevention of oxidative DNA damage and apoptosis in FMF patients, and also the elucidation of a possible role of DNA repair in this disease.