Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms

Methionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H(2)O(2) and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging.     

Receptor actions of synaptically released glutamate: the role of transporters on the scale from nanometers to microns

Actions of the excitatory neurotransmitter glutamate inside and outside the synaptic cleft determine the activity of neural circuits in the brain. However, to what degree local glutamate transporters affect these actions on a submicron scale remains poorly understood. Here we focus on hippocampal area CA1, a common subject of synaptic physiology studies. First, we use a two-photon excitation technique to obtain an estimate of the apparent (macroscopic) extracellular diffusion coefficient for glutamate, ∼0.32 μm²/ms. Second, we incorporate this measurement into a Monte Carlo model of the typical excitatory synapse and examine the influence of distributed glutamate transporter molecules on signal transmission. Combined with the results of whole-cell recordings, such simulations argue that, although glutamate transporters have little effect on the activation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, this does not rule out the occurrence of up to several dozens of transporters inside the cleft. We further evaluate how the expression pattern of transporter molecules (on the 10-100 nm scale) affects the activation of N-methyl-D-aspartic acid or metabotropic glutamate receptors in the synaptic vicinity. Finally, we extend our simulations to the macroscopic scale, estimating that synaptic activity sufficient to excite principal neurons could intermittently raise extracellular glutamate to ∼1 μM only at sparse (microns apart) hotspots. Greater rises of glutamate occur only when <5% of transporters are available (for instance, when an astrocyte fails). The results provide a quantitative framework for a better understanding of the relationship between glutamate transporters and glutamate receptor signaling.     

Engineering the NK1 fragment of hepatocyte growth factor/scatter factor as a MET receptor antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1-MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.     

Estrogen upregulates renal angiotensin II AT1 and AT2 receptors in the rat

We studied renal AT1 and AT2 receptors in male, female, ovariectomized and ovariectomized-estrogen-treated Wistar-Hanover and Wistar-Kyoto rats. AT1 receptors and AT1A receptor mRNA predominated, with no significant differences between males and females. AT2 receptor expression was restricted in female rats to the capsule, the transition zone between outer and inner medulla, the endothelium lining the papilla, and arcuate arteries and veins. There were no AT2 receptors in male rats, while male mice express substantial numbers of estrogen-dependent AT2 receptors. Arcuate arteries and veins expressed AT1B mRNA in males and females, and AT2 mRNA in females only. AT1 receptor and AT2 receptor expression were estrogen-dependent, with increases in AT1 and AT2 receptor expression after estrogen treatment in ovariectomized rats. Estrogen treatment increased prostaglandin E2 (PGE2) and cGMP concentrations in the renal medulla, and eNOS expression in cortical arteries. In rodents, expression of renal Angiotensin II receptor types is estrogen-dependent, with significant species, strain and area differences. Our results support an important role for AT2 receptors in the regulation of renal function and in the protective effects of estrogen in the kidney.     

Simulation of Effects of Soils,Climate and Management on N<Subscript>2</Subscript>O Emission from Grasslands

Nitrous oxide (N₂O) is a potent greenhouse gas with a high contribution from agricultural soils and emissions that depend on soil type, climate, crops and management practices. The N₂O emissions therefore need to be included as an integral part of environmental assessments of agricultural production systems. An algorithm for N₂O production and emission from agricultural soils was developed and included in the FASSET whole-farm model. The model simulated carbon and nitrogen (N) turnover on a daily basis. Both nitrification and denitrification was included in the model as sources for N₂O production, and the N₂O emissions depended on soil microbial and physical conditions. The model was tested on experimental data of N₂O emissions from grasslands in UK, Finland and Denmark, differing in climatic conditions, soil properties and management. The model simulated the general time course of N₂O emissions and captured the observed effects of fertiliser and manure management on emissions. Scenario analyses for grazed and cut grasslands were conducted to evaluate the effects of soil texture, climatic conditions, grassland management and N fertilisation on N₂O emissions. The soils varied from coarse sand to sandy loam and the climatic variation was taken to represent the climatic variation within Denmark. N fertiliser rates were varied from 0 to 500 kg N ha⁻¹. The simulated N₂O emissions showed a non-linear response to increasing N rates with increasing emission factors at higher N rates. The simulated emissions increased with increasing soil clay contents. N₂O emissions were slightly increased at higher temperatures, whereas increasing annual rainfall generally lead to decreasing emissions. Emissions were slightly higher from grazed grasslands compared with cut grasslands at similar rates of total N input (fertiliser and animal excreta). The results indicate higher emission factors and thus higher potentials for reducing N₂O emissions for intensively grazed grasslands on fine textured soils than for extensive cut-based grasslands on sandy soils.     

Computer-aided rodent carcinogenicity prediction

The potential of the computer program PASS (Prediction Activity Spectra for Substances) to predict rodent carcinogenicity for chemical compounds was studied. PASS predicts carcinogenicity of chemical compounds on the basis of their structural formula and of structure-activity relationship analysis of known carcinogens and non-carcinogens. The data on structures and experimental results of 2-year carcinogenicity assays for 412 chemicals from the NTP (National Toxicological Program) and 1190 chemicals from the CPDB (Carcinogenic Potency Database) were used in our study. The predictions take into consideration information about species and sex of animals. For evaluation of the predictive accuracy we used two procedures: leave-one-out cross-validation (LOO CV) and leave-20%-out cross-validation. In the last case we randomly divided the studied data set 20 times into two subsets. The data from the first subset, containing 80% of the compounds, were added to the PASS training set (which includes about 46,000 compounds with about 1500 biological activity types collected during the last 20 years to predict biological activity spectra), the second subset with 20% of the compounds was used as an evaluation set. The mean accuracy of prediction calculated by LOO CV is about 73% for NTP compounds in the 'equivocal' category of carcinogenic activity and 80% for NTP compounds in the 'evidence' category of carcinogenicity. The mean accuracy of prediction for the CPDB database is 89.9% calculated by LOO CV and 63.4% calculated by leave-20%-out cross-validation. Influence of incorporation of species and sex data on the accuracy of carcinogenicity prediction was also investigated. It was shown that the accuracy was increased only for data on male animals.     

1,3-Dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines as potent caspase-3 inhibitors

Synthesis, biological evaluation and structure-activity relationships for a series of novel nonpeptide small molecule inhibitors of caspase-3 are described. Among the studied compounds, 8-sulfamide derivatives of 1,3-dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines have been identified as potent inhibitors of caspases-3. The most active compound within this series (8f) inhibited caspase-3 with IC(50)=4 nM.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits an important intracellular cleavage function and causes chromosome instability

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in non-malignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation.