The mapping and reconstitution of a conformational discontinuous B-cell epitope of HIV-1

A method for the discovery of the structure of conformational discontinuous epitopes of monoclonal antibodies (mAbs) is described. The mAb is used to select specific phages from combinatorial phage-display peptide libraries that in turn are used as an epitope-defining database that is applied via a novel computer algorithm to analyze the crystalline structure of the original antigen. The algorithm is based on the following: (1) Most contacts between a mAb and an antigen are through side-chain atoms of the residues. (2) In the three-dimensional structure of a protein, amino acid residues remote in linear sequence can juxtapose to one another through folding. (3) Tandem amino acid residues of the selected phage-displayed peptides can represent pairs of juxtaposed amino acid residues of the antigen. (4) Contact residues of the epitope are accessible to the antigen surface. (5) The most frequent tandem pairs of amino acid residues in the selected phage-displayed peptides can reflect pairs of juxtaposed amino acid residues of the epitope. Application of the algorithm enabled prediction of epitopes. On the basis of these predictions, segments of an antigen were used to reconstitute an antigenic epitope mimetic that was recognized by its original mAb.     

Multifunctional xylooligosaccharide/cephalosporin C deacetylase revealed by the hexameric structure of the Bacillus subtilis enzyme at 1.9A resolution

Esterases and deacetylases active on carbohydrate ligands have been classified into 14 families based upon amino acid sequence similarities. Enzymes from carbohydrate esterase family seven (CE-7) are unusual in that they display activity towards both acetylated xylooligosaccharides and the antibiotic, cephalosporin C. The 1.9A structure of the multifunctional CE-7 esterase (hereinafter CAH) from Bacillus subtilis 168 reveals a classical alpha/beta hydrolase fold encased within a 32 hexamer. This is the first example of a hexameric alpha/beta hydrolase and is further evidence of the versatility of this particular fold, which is used in a wide variety of biological contexts. A narrow entrance tunnel leads to the centre of the molecule, where the six active-centre catalytic triads point towards the tunnel interior and thus are sequestered away from cytoplasmic contents. By analogy to self-compartmentalising proteases, the tunnel entrance may function to hinder access of large substrates to the poly-specific active centre. This would explain the observation that the enzyme is active on a variety of small, acetylated molecules. The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues. Protein residues involved in catalysis are tethered by interactions with protein excursions from the canonical alpha/beta hydrolase fold. These excursions also mediate quaternary structure maintenance, so it would appear that catalytic competence is only achieved on protein multimerisation. We suggest that the acetyl xylan esterase (EC 3.1.1.72) and cephalosporin C deacetylase (EC 3.1.1.41) enzymes of the CE-7 family represent a single class of proteins with a multifunctional deacetylase activity against a range of small substrates.     

In CCR2-/- mice monocyte recruitment and egress of LDL cholesterol in vivo is impaired

Recruitment of macrophages plays an important role in initiation of atheroma, but their involvement in cholesterol clearance during regression is unknown. We developed a mouse model to quantitate cholesterol clearance from a depot of cationized LDL injected into a leg muscle, which evokes a sterile inflammatory reaction. In the CCR2(-/-) mice, cholesterol clearance was significantly slower than in C57BL controls because of decrease in cholesteryl ester (CE) hydrolysis, which is mandatory prior to cholesterol efflux. In CCR2(-/-) mice, macrophage recruitment to the injected site, identified by immunohistochemistry, was markedly delayed. CE hydrolysis was also significantly reduced in thioglycollate elicited peritoneal exudate cells of CCR2(-/-) mice, related to paucity of macrophages in the cell differential. The present study provides definite evidence that recruitment of macrophages is required for LDL cholesterol clearance, which plays a prominent role in regression of an atheroma.     

Cellvibrio japonicus alpha-L-arabinanase 43A has a novel five-blade beta-propeller fold

Cellvibrio japonicus arabinanase Arb43A hydrolyzes the alpha-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans. The three-dimensional structure of Arb43A, determined at 1.9 A resolution, reveals a five-bladed beta-propeller fold. Arb43A is the first enzyme known to display this topology. A long V-shaped surface groove, partially enclosed at one end, forms a single extended substrate-binding surface across the face of the propeller. Three carboxylates deep in the active site groove provide the general acid and base components for glycosidic bond hydrolysis with inversion of anomeric configuration.     

Sequence-specific interaction of U1 snRNA with the SMN complex

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.     

Immuno-detection of aluminium and aluminium induced conformational changes in calmodulin - implications in Alzheimer's disease

Studies were conducted to ascertain any involvement of free radical mediated prooxidative processes in different brain regions following diazopam administration. A significant decrease in TBA reactive substance formation was observed in cerebral cortex, cerebellum and brain stem regions after single doses of 1.5, 3 and 6 mg/kg b.wt. For further studies rats were given diazepam (i.p.) at 3 mg/kg body weight dose and sacrificed after 1 h to follow changes in the pro/antioxidant status. An enhancement in the TBARS formation was found in the mitochondrial fractions from cerebral cortex and brain stem. This effect was highest in brain stem being 107% as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a decrease in activity which was region dependent. Even though, total thiols were not significantly altered, free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Glutathione reductase activity was also decreased in cerebellum and brain stem. The results indicate that a single dose of diazepam causes free radical mediated changes and the modulatory response of antioxidant defences appears to be region specific.     

Planktonic functional diversity changes in synchrony with lake ecosystem state

Managing ecosystems to effectively preserve function and services requires reliable tools that can infer changes in the stability and dynamics of a system. Conceptually, functional diversity (FD) appears as a sensitive and viable monitoring metric stemming from suggestions that FD is a universally important measure of biodiversity and has a mechanistic influence on ecological processes. It is however unclear whether changes in FD consistently occur prior to state responses or vice versa, with no current work on the temporal relationship between FD and state to support a transition towards trait-based indicators. There is consequently a knowledge gap regarding when functioning changes relative to biodiversity change and where FD change falls in that sequence. We therefore examine the lagged relationship between planktonic FD and abundance-based metrics of system state (e.g. biomass) across five highly monitored lake communities using both correlation and cutting edge non-linear empirical dynamic modelling approaches. Overall, phytoplankton and zooplankton FD display synchrony with lake state but each lake is idiosyncratic in the strength of relationship. It is therefore unlikely that changes in plankton FD are identifiable before changes in more easily collected abundance metrics. These results highlight the power of empirical dynamic modelling in disentangling time lagged relationships in complex multivariate ecosystems, but suggest that FD cannot be generically viable as an early indicator. Individual lakes therefore require consideration of their specific context and any interpretation of FD across systems requires caution. However, FD still retains value as an alternative state measure or a trait representation of biodiversity when considered at the system level.