Dichotomous effects of isomeric secondary amines containing an aromatic nitrile and nitro group on human aortic smooth muscle cells via inhibition of cystathionine-γ-lyase.

Excessive proliferation of vascular smooth muscle cells (SMC) is an important contributor to the progression of atherosclerosis. Inhibition of proliferation can be achieved by endogenously produced and exogenously supplied nitrogen monoxide, commonly known as nitric oxide (NO). We report herein the dichotomous effects of two isomeric families of secondary amines, precursors to the N-nitrosated NO-donors, on HASMC proliferation. The syntheses of these two families were carried out using two equivalents of homologous, aliphatic monoamines and 2,6-difluoro-3-nitrobenzonitrile (2,6-DFNBN, O family) or 2,4-difluoro-5-nitrobenzonitrile (2,4-DFNBN, P family). The secondary amines belonging to the P family inhibited HASMC proliferation at all concentrations, whereas the O family induced HASMC proliferation at low concentrations, and exhibited inhibitory properties at high concentrations. A probable explanation of these behaviors is proposed herein. l-homocysteine (HCY) is known to induce HASMC proliferation at low concentrations (<1 mM) and inhibit HASMC proliferation at higher concentrations (>2.5 mM). Our findings suggest that these two families of amines inhibit cystathionine-γ-lyase (CSE) to varying extents, which directly results in altered levels of intracellular HCY and consequent changes in HASMC proliferation.

Evidence for a proton-protein symport mechanism in the anthrax toxin channel.

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.

Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation.

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.

Cross-linked forms of the isolated N-terminal domain of the lethal factor are potent inhibitors of anthrax toxin.

The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF(N), the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF(N) retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LF(N)) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.

Identification of otubain 1 as a novel substrate for the Yersinia protein kinase using chemical genetics and mass spectrometry.

Yersinia encodes a protein kinase, YpkA, which disrupts the actin cytoskeleton. Using an approach termed chemical genetics, we identified a 36-kDa substrate for YpkA in both J774 lysates and bovine brain cytosol. Mass spectrometry analysis identified this substrate as FLJ20113, an open reading frame that corresponds to otubain 1, a deubiquitinating enzyme implicated in immune cell clonal anergy. We demonstrate that otubain 1 is phosphorylated by YpkA in vitro and interacts with YpkA and actin in vivo. Identification of otubain 1 as a YpkA substrate suggests that regulation of immune cell anergy may be a survival mechanism for Yersinia.

A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore.

The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen.

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.

Yersinia effectors target mammalian signalling pathways.

Animals have an immune system to fight off challenges from both viruses and bacteria. The first line of defence is innate immunity, which is composed of cells that engulf pathogens as well as cells that release potent signalling molecules to activate an inflammatory response and the adaptive immune system. Pathogenic bacteria have evolved a set of weapons, or effectors, to ensure survival in the host. Yersinia spp. use a type III secretion system to translocate these effector proteins, called Yops, into the host. This report outlines how Yops thwart the signalling machinery of the host immune system.