Biochemical Characterization of Hypothetical Proteins from Helicobacter pylori

The functional characterization of Open Reading Frames (ORFs) from sequenced genomes remains a bottleneck in our effort to understand microbial biology. In particular, the functional characterization of proteins with only remote sequence homology to known proteins can be challenging, as there may be few clues to guide initial experiments. Affinity enrichment of proteins from cell lysates, and a global perspective of protein function as provided by COMBREX, affords an approach to this problem. We present here the biochemical analysis of six proteins from Helicobacter pylori ATCC 26695, a focus organism in COMBREX. Initial hypotheses were based upon affinity capture of proteins from total cellular lysate using derivatized nano-particles, and subsequent identification by mass spectrometry. Candidate genes encoding these proteins were cloned and expressed in Escherichia coli, and the recombinant proteins were purified and characterized biochemically and their biochemical parameters compared with the native ones. These proteins include a guanosine triphosphate (GTP) cyclohydrolase (HP0959), an ATPase (HP1079), an adenosine deaminase (HP0267), a phosphodiesterase (HP1042), an aminopeptidase (HP1037), and new substrates were characterized for a peptidoglycan deacetylase (HP0310). Generally, characterized enzymes were active at acidic to neutral pH (4.0–7.5) with temperature optima ranging from 35 to 55°C, although some exhibited outstanding characteristics.     

Preferential recognition of undisruptable dimers of inducible nitric oxide synthase by a monoclonal antibody directed against an N-terminal epitope

Overproduction of NO by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer in which the subunits align in a head-to-head manner, with the N-terminal oxygenase domains forming the dimer interface and a zinc metal center stabilizing the dimer. Thus, dimerization represents a critical locus for therapeutic interventions for regulation of NO synthesis. We have recently shown that intracellular iNOS forms dimers that are "undisruptable (UD)" by heat, SDS, strong denaturants, and/or reducing agents. Our data further suggest that the zinc metal center plays a role in forming and/or stabilizing iNOS undisruptable dimers (UD-dimers). In this study, we show that a mAb directed against a unique epitope at the oxygenase domain of human iNOS preferentially recognizes UD-dimers. This observation has implications for the mechanism of formation and regulation of dimer formation of iNOS. Our data suggest that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, still maintain features of the quaternary structure of iNOS particularly at its N-terminal end and including head-to-head contact of the oxygenase domains.     

Power and sample size calculation of comparative diagnostic accuracy studies with multiple correlated test results

We consider the power and sample size calculation of diagnostic studies with normally distributed multiple correlated test results. We derive test statistics and obtain power and sample size formulas. The methods are illustrated using an example of comparison of CT and PET scanner for detecting extra-hepatic disease for colorectal cancer.     

Foraging strategies of black kites (<Emphasis Type="Italic">Milvus migrans govinda</Emphasis>) in urban garbage dumps

Large numbers of black kites (Milvus migrans govinda) forage with house crows (Corvus splendens) at garbage dumps in many Indian cities. Such aggregation of many individuals results in aggressiveness where adoption of a suitable behavioral approach is crucial. We studied foraging behavior of black kites in dumping sites adjoining two major corporation markets of Kolkata, India. Black kites used four different foraging tactics which varied and significantly influenced foraging attempts and their success rates. Kleptoparasitism was significantly higher than autonomous foraging events; interspecific kleptoparasitism was highest in occurrence with a low success rate, while ‘autonomous-ground’ was least adopted but had the highest success rate.     

Three-Dimensional Structure of Different Functional Forms of the Vibrio cholerae Hemolysin Oligomer: a Cryo-Electron Microscopic Study

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.Vibrio cholerae, a Gram-negative bacterium, is the causative agent of cholera in humans (9). The severe diarrhea of cholera is due primarily to the effects of cholera enterotoxin upon the small intestine epithelial cells (3). In addition to cholera toxin (9), most Vibrio cholerae strains secrete a membrane-damaging protein, designated cholera hemolysin (HlyA) (27) or cytolysin/hemolysin (VCC), with cytotoxic and cytolytic activity toward a wide spectrum of eukaryotic cells. VCC is an extracellular pore-forming toxin (PFT) (26) that exists in two stable forms: one is a water-soluble monomer with a molecular mass of 65 kDa (5, 6, 27, 31), and the other is a β-barrel amphipathic heptamer. The VCC monomer interacts with a cell surface receptor(s), self-assembles to an amphipathic β-barrel by circular oligomerization, and inserts into the membrane lipid bilayer (2, 7, 14, 21). Despite individual variations that depend on amino acid sequence, three-dimensional (3D) structure, and receptor specificity, PFTs have evolved a strikingly common strategy to destroy the permeability barrier of the target cell membrane (16). The soluble PFT monomer interacts with the target cell, self-assembles into an amphipathic β-barrel by circular oligomerization, and translocates from the cell surface or lipid-water interface to the core of the lipid bilayer, forming a transmembrane pore (5, 6).HlyA is expressed as an 82-kDa preprohemolysin protein, and following removal of the signal sequence, the protein is excreted during in vitro growth as the 79-kDa prohemolysin (proHlyA) (28). Proteolytic removal of 132 residues from the N-terminal region generates the mature 65-kDa HlyA, with a specific hemolytic activity of ∼100 pM toward rabbit erythrocytes (15). The mature toxin has a central cytolytic domain involved in oligomerization and anchoring to the hydrophobic core of the bilayer, followed by two lectin domains homologous to the carbohydrate-binding domains of the plant lectins ricin and jacalin (17). Proteolytic deletion of the 15-kDa β-prism lectin domain, which is apparently involved in specific interaction with β-galactosyl-terminated glycoconjugates (21), from the carboxy-terminal end of the 65-kDa mature hemolysin generates the truncated 50-kDa hemolysin (HlyA50) (8, 29). This truncated 50-kDa hemolysin (HlyA50) retains the oligomerization and membrane-anchoring domains (17, 18) and resembles the mature toxin in surface amphipathicity, nonspecific interaction with target biomembrane and lipid vesicles, and ability to undergo lipid-induced oligomerization (30). HlyA50 is about 1,000-fold less active than HlyA toward rabbit erythrocytes (29, 30), suggesting that the HlyA50 oligomer might be less capable of adopting an insertion-competent configuration.In the present study, we have attempted to determine the three-dimensional structures of the oligomers generated by the 65-kDa Vibrio cholerae hemolysin and the 50-kDa hemolysin, using cryo-electron microscopy and single-particle analysis techniques. Determination of the three-dimensional structure is important, as it might shed light on the shape of the transmembrane channel as well as on the anchoring and insertion processes of the two toxin forms.     

Trichomonas Transmembrane Cyclases Result from Massive Gene Duplication and Concomitant Development of Pseudogenes

Background

Trichomonas vaginalis has an unusually large genome (∼160 Mb) encoding ∼60,000 proteins. With the goal of beginning to understand why some Trichomonas genes are present in so many copies, we characterized here a family of ∼123 Trichomonas genes that encode transmembrane adenylyl cyclases (TMACs).

Methodology/Principal Findings

The large family of TMACs genes is the result of recent duplications of a small set of ancestral genes that appear to be unique to trichomonads. Duplicated TMAC genes are not closely associated with repetitive elements, and duplications of flanking sequences are rare. However, there is evidence for TMAC gene replacements by homologous recombination. A high percentage of TMAC genes (∼46%) are pseudogenes, as they contain stop codons and/or frame shifts, or the genes are truncated. Numerous stop codons present in the genome project G3 strain are not present in orthologous genes of two other Trichomonas strains (S1 and B7RC2). Each TMAC is composed of a series of N-terminal transmembrane helices and a single C-terminal cyclase domain that has adenylyl cyclase activity. Multiple TMAC genes are transcribed by Trichomonas cloned by limiting dilution.

Conclusions/Significance

We conclude that one reason for the unusually large genome of Trichomonas is the presence of unstable families of genes such as those encoding TMACs that are undergoing massive gene duplication and concomitant development of pseudogenes.     

Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase

The role of active site histidine residues in SCN^– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a second order rate constant of 0.26 M^–1 sec^–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6 indicating histidine derivatization. The UV difference spectrum of the modified versus native enzyme shows a peak at 242 nm indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with SCN^– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically detectable compound II formation is lost due to modification and is not evident after SCN^– protection. The data indicate that out of two histidines, one regulates compound I formation while the other one controls SCN^– binding. SCN^– protected enzyme is inactive due to loss of compound I formation. SCN^– binding studies by optical difference spectroscopy indicate that while the native enzyme binds SCN^– with the K_d of 15 mM, the modified enzyme shows very weak binding with the K_d of 660 mM. From the pH dependent binding of SCN^–, a plot of log K_d vs. pH shows a sigmoidal curve from which the involvement of an enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN^– binding. LGP has thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls SCN^– binding.