Structure and chemistry of lysinoalanine crosslinking in the spirochaete flagella hook.

The flagellar hook protein FlgE from spirochaete bacteria self-catalyzes the formation of an unusual inter-subunit lysinoalanine (Lal) crosslink that is critical for cell motility. Unlike other known examples of Lal biosynthesis, conserved cysteine and lysine residues in FlgE spontaneously react to form Lal without the involvement of additional enzymes. Oligomerization of FlgE via its D0 and Dc domains drives assembly of the crosslinking site at the D1-D2 domain interface. Structures of the FlgED2 domain, dehydroalanine (DHA) intermediate and Lal crosslinked FlgE subunits reveal successive snapshots of the reaction. Cys178 flips from a buried configuration to release hydrogen sulfide (H2S/HS-) and produce DHA. Interface residues provide hydrogen bonds to anchor the active site, facilitate ß-elimination of Cys178 and polarize the peptide backbone to activate DHA for reaction with Lys165. Cysteine-reactive molecules accelerate DHA formation, whereas nucleophiles can intercept the DHA intermediate, thereby indicating a potential for Lal crosslink inhibitors to combat spirochaetal diseases.

Functional comparision between truncated MTT1 and truncated MTT2 from Tetrahyemna thermophila.

Metallothioneins (MTs) are low-molecular-weight proteins with high Cys content and high metal-chelating ability. CdMT and CuMT subfamilies present different characteristics in Tetrahymena. To explore the effect of the cysteine arrangement and sequence length of MTs for binding different metal ions, MTT1, truncated MTT1 (TM1), MTT2, and truncated MTT2 (TM2) were expressed in E. coli. The half-maximal inhibiting concentrations (IC50) of Cd2+ and Cu+ for the recombinant strains were different. Furthermore, E. coli cells expressing MTT1 and TM1 exhibited higher accumulating ability for Cd2+ than cells expressing MTT2 and TM2. However, the opposite is true for Cu+. The binding ability of the different recombinant proteins to Cd2+ and Cu+ were also different. MTT1 and truncated mutant TM1 were the preference for Cd2+, whereas MTT2 and truncated mutant TM2 were the preference for Cu+ coordination. These results showed that metal ion tolerance and accumulation ability not only depended on cysteine arrangement pattern but also on sequence length of MT in Tetrahymena.

The therapeutic value of protein (de)nitrosylation in experimental septic shock.

Cardiovascular dysfunction and organ damage are hallmarks of sepsis and septic shock. Protein S-nitrosylation by nitric oxide has been described as an important modifier of protein function. We studied whether protein nitrosylation/denitrosylation would impact positively in hemodynamic parameters of septic rats. Polymicrobial sepsis was induced by cecal ligation and puncture. Female Wistar rats were treated with increasing doses of DTNB [5,5'-dithio-bis-(2-nitrobenzoic acid)] 30min before or 4 or 12h after sepsis induction. Twenty-four hours after surgery the following data was obtained: aorta response to phenylephrine, mean arterial pressure, vascular reactivity to phenylephrine, biochemical markers of organ damage, survival and aorta protein nitrosylation profile. Sepsis substantially decreases blood pressure and the response of aorta rings and of blood pressure to phenylephrine, as well as increased plasma levels of organ damage markers, mortality of 60% and S-nitrosylation of aorta proteins increased during sepsis. Treatment with DTNB 12h after septic shock induction reversed the loss of response of aorta rings and blood pressure to vasoconstrictors, reduced organ damage and protein nitrosylation and increased survival to 80%. Increases in protein S-nitrosylation are related to cardiovascular dysfunction and multiple organ injury during sepsis. Treatment of rats with DTNB reduced the excessive protein S-nitrosylation, including that in calcium-dependent potassium channels (BKCa), reversed the cardiovascular dysfunction, improved markers of organ dysfunction and glycemic profile and substantially reduced mortality. Since all these beneficial consequences were attained even if DTNB was administered after septic shock onset, protein (de)nitrosylation may be a suitable target for sepsis treatment.

Effect of Redox-Modifying Agents on the Activity of Channelrhodopsin-2.

BACKGROUND: The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. AIM: In this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. METHODS: Whole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. RESULTS: We show that the reducing agent dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current-voltage relationship by 23 ± 4.31 mV, suggesting a change in ion selectivity. CONCLUSION: Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.

Gamma-interferon-inducible, lysosome/endosome-localized thiolreductase, GILT, has anti-retroviral activity and its expression is counteracted by HIV-1.

The mechanism by which type II interferon (IFN) inhibits virus replications remains to be identified. Murine leukemia virus (MLV) replication was significantly restricted by γ-IFN, but not human immunodeficiency virus type 1 (HIV-1) replication. Because MLV enters host cells via endosomes, we speculated that certain cellular factors among γ-IFN-induced, endosome-localized proteins inhibit MLV replication. We found that γ-IFN-inducible lysosomal thiolreductase (GILT) significantly restricts HIV-1 replication as well as MLV replication by its thiolreductase activity. GILT silencing enhanced replication-defective HIV-1 vector infection and virion production in γ-IFN-treated cells, although γ-IFN did not inhibit HIV-1 replication. This result showed that GILT is required for the anti-viral activity of γ-IFN. Interestingly, GILT protein level was increased by γ-IFN in uninfected cells and env-deleted HIV-1-infected cells, but not in full-length HIV-1-infected cells. γ-IFN-induced transcription from the γ-IFN-activation sequence was attenuated by the HIV-1 Env protein. These results suggested that the γ-IFN cannot restrict HIV-1 replication due to the inhibition of γ-IFN signaling by HIV-1 Env. Finally, we found that 4,4'-dithiodipyridine (4-PDS), which inhibits S-S bond formation at acidic pH, significantly suppresses HIV-1 vector infection and virion production, like GILT. In conclusion, this study showed that GILT functions as a host restriction factor against the retroviruses, and a GILT mimic, 4-PDS, is the leading compound for the development of novel concept of anti-viral agents.

Thioredoxin (Trx1) regulates CD4 membrane domain localization and is required for efficient CD4-dependent HIV-1 entry.

BACKGROUND: CD4 is a glycoprotein expressed on the surfaces of certain immune cells. On lymphocytes, an important function of CD4 is to co-engage Major Histocompatibility Complex (MHC) molecules with the T Cell Receptor (TCR), a process that is essential for antigen-specific activation of T cells. CD4 localizes dynamically into distinct membrane microdomains, an important feature of its immunoregulatory function that has also been shown to influence the efficiency of HIV replication. However, the mechanism by which CD4 localization is regulated and the biological significance of this is incompletely understood. METHODS: In this study, we used confocal microscopy, density-gradient centrifugation and flow cytometry to analyze dynamic redox-dependent effects on CD4 membrane domain localization. RESULTS: Blocking cell surface redox exchanges with both a membrane-impermeable sulfhydryl blocker (DTNB) and specific antibody inhibitors of Thioredoxin-1 (Trx1) induces translocation of CD4 into detergent-resistant membrane domains (DRM). In contrast, Trx1 inactivation does not change the localization of the chemokine receptor CCR5, suggesting that this effect is targeted. Moreover, DTNB treatment and Trx1 depletion coincide with strong inhibition of CD4-dependent HIV entry, but only moderate reductions in the infectivity of a CD4-independent HIV pseudovirion. CONCLUSIONS: Changes in the extracellular redox environment, potentially mediated by allosteric consequences of functional disulfide bond oxidoreduction, may represent a signal for translocation of CD4 into DRM clusters, and this sequestration, another potential mechanism by which the anti-HIV effects of cell surface oxidoreductase inhibition are exerted. GENERAL SIGNIFICANCE: Extracellular redox conditions may regulate CD4 function by potentiating changes in its membrane domain localization.

Evidence for redox sensing by a human cardiac calcium channel.

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca(2+) channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.

Activation of paraoxonase 1 is associated with HDL remodeling ex vivo.

BACKGROUND: We hypothesize that during high density lipoprotein (HDL) remodeling PON1 reaches an optimal distribution in HDL subclasses by which it achieves maximum activity. We conducted this study to gain insight on PON1 fate and activation during short-term HDL remodeling ex vivo. METHODS: Serum from 8 healthy volunteers was either frozen at -80°C (time 0) or incubated under sterile conditions for up to 48h at 37°C or at 4°C. Aliquots were taken at 3, 6, 9, 24 and 48 h and immediately frozen at -80°C. PON1 activities were measured, as well as PON1 and apolipoprotein distributions in HDL subclasses by gradient gel electrophoresis. RESULTS: The first novel finding in our study is the evidence provided for a significant activation of both lactonase and arylesterase activities of PON1 that ensues in a very short time frame of incubation of serum ex vivo at 37°C. All subjects studied displayed these changes, the activation was apparent in <3h, peaked at 6h and amounted to >20%. This is associated with a temperature and time-dependent redistribution of PON1 activity in HDL subclasses, with an increase in activity in both very large HDL2 and small HDL3 in the first phase (3-9h), followed by a progressive transfer of PON1 to very large HDL2 as the particles mature. These changes are paralleled by the appearance of weak, but apparent PON1 activity at subspecies that correspond to sdLDL. During the first phase of PON1 activation and shifts, a parallel shift of apoE can be evidenced: at 3-9h, apoE increases in sdLDL, after that time it is lost from HDL and also from sdLDL and stays in VLDL at the origin of the run. ApoA-I shifts towards larger particles, which parallels the change in PON1. As HDL matures there is a progressive shift of apoA-II towards larger HDL. Low levels of apoA-IV at the initiation of the incubation are followed by time dependent quick disappearance of apoA-IV in HDL which parallels the changes in PON1, apoE and A-II. CONCLUSION: Short, ex vivo incubation of serum leads to quick activation of PON1 associated with transfers to HDL3c, large HDL and sdLDL. The process is blocked by CETP and LCAT inhibitors. The data suggest that HDL maturation optimizes PON1 activity. These findings may be of interest for future studies aimed at modulating PON-1 activity for its cardioprotective effects and suggest a new mechanism whereby CETP inhibitors failed in clinical trials.

Hypoxia limits inhibitory effects of Zn2+ on spreading depolarizations.

Spreading depolarizations (SDs) are coordinated depolarizations of brain tissue that have been well-characterized in animal models and more recently implicated in the progression of stroke injury. We previously showed that extracellular Zn(2+) accumulation can inhibit the propagation of SD events. In that prior work, Zn(2+) was tested in normoxic conditions, where SD was generated by localized KCl pulses in oxygenated tissue. The current study examined the extent to which Zn(2+) effects are modified by hypoxia, to assess potential implications for stroke studies. The present studies examined SD generated in brain slices acutely prepared from mice, and recordings were made from the hippocampal CA1 region. SDs were generated by either local potassium injection (K-SD), exposure to the Na(+)/K(+)-ATPase inhibitor ouabain (ouabain-SD) or superfusion with modified ACSF with reduced oxygen and glucose concentrations (oxygen glucose deprivation: OGD-SD). Extracellular Zn(2+) exposures (100 µM ZnCl2) effectively decreased SD propagation rates and significantly increased the initiation threshold for K-SD generated in oxygenated ACSF (95% O2). In contrast, ZnCl2 did not inhibit propagation of OGD-SD or ouabain-SD generated in hypoxic conditions. Zn(2+) sensitivity in 0% O2 was restored by exposure to the protein oxidizer DTNB, suggesting that redox modulation may contribute to resistance to Zn(2+) in hypoxic conditions. DTNB pretreatment also significantly potentiated the inhibitory effects of competitive (D-AP5) or allosteric (Ro25-6981) NMDA receptor antagonists on OGD-SD. Finally, Zn(2+) inhibition of isolated NMDAR currents was potentiated by DTNB. Together, these results suggest that hypoxia-induced redox modulation can influence the sensitivity of SD to Zn(2+) as well as to other NMDAR antagonists. Such a mechanism may limit inhibitory effects of endogenous Zn(2+) accumulation in hypoxic regions close to ischemic infarcts.

A conformational intermediate in glutamate receptor activation.

Ionotropic glutamate receptors (iGluRs) transduce the chemical signal of neurotransmitter release into membrane depolarization at excitatory synapses in the brain. The opening of the transmembrane ion channel of these ligand-gated receptors is driven by conformational transitions that are induced by the association of glutamate molecules to the ligand-binding domains (LBDs). Here, we describe the crystal structure of a GluA2 LBD tetramer in a configuration that involves an ∼30° rotation of the LBD dimers relative to the crystal structure of the full-length receptor. The configuration is stabilized by an engineered disulfide crosslink. Biochemical and electrophysiological studies on full-length receptors incorporating either this crosslink or an engineered metal bridge show that this LBD configuration corresponds to an intermediate state of receptor activation. GluA2 activation therefore involves a combination of both intra-LBD (cleft closure) and inter-LBD dimer conformational transitions. Overall, these results provide a comprehensive structural characterization of an iGluR intermediate state.

Antidepressant-like responses in the forced swimming test elicited by glutathione and redox modulation.

Glutathione (GSH) displays a broad range of functions, among them a role as a neuromodulator with some neuroprotective properties. Taking into account that oxidative stress has been associated with depressive disorders, this study investigated the possibility that GSH, a major cell antioxidant, elicits an antidepressant-like effect in mice. Thus, GSH was administered by i.c.v. route to mice that were tested in the forced swimming test and in the tail suspension test, two predictive tests for antidepressant drug activity. In addition, GSH metabolism and the redox environment were modulated in order to study the possible mechanisms underlying the effects of GSH in the forced swimming test. The administration of GSH decreased the immobility time in the forced swimming test (300-3000nmol/site) and tail suspension test (100-1000nmol/site), consistent with an antidepressant-like effect. GSH depletion elicited by l-buthionine sulfoximine (3.2µmol/site, i.c.v.) did not alter the antidepressant-like effect of GSH, whereas the inhibition of extracellular GSH catabolism by acivicin (100nmol/site, i.c.v.) prevented the antidepressant-like effect of GSH. Moreover, a sub-effective dose (0.01nmol/site, i.c.v.) of the oxidizing agent DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) potentiated the effect of GSH (100nmol/site, i.c.v.), while the pretreatment (25-100mg/kg, i.p.) with the reducing agent DTT (dl-dithiothreitol) prevented the antidepressant-like effect of GSH (300nmol/site, i.c.v.). DTNB (0.1nmol/site, i.c.v.), produced an antidepressant-like effect, per se, which was abolished by DTT (25mg/kg, i.p.). The results show, for the first time, that centrally administered GSH produces an antidepressant-like effect in mice, which can be modulated by the GSH metabolism and the thiol/disulfide reagents. The redox environment may constitute a new venue for future antidepressant-drug development.

Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

BACKGROUND: The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. RESULTS: We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. CONCLUSIONS: Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences among previously published studies. More importantly, from an in vivo perspective, the preferential utilization of PDI may be relevant to the HIV-1 entry and establishment of virus reservoirs in resting CD4+ cells, while the elevated levels of Trx reported in the chronic stages of HIV-1 infection may facilitate the virus entry in macrophages and help to sustain high viremia during the decline of T lymphocytes.

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase.

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases.

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase and endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids.

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ∼threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.

Inhibiting rotavirus infection by membrane-impermeant thiol/disulfide exchange blockers and antibodies against protein disulfide isomerase.

OBJECTIVES: Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. METHODS: Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. RESULTS: Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 mM DTNB, respectively; infectivity became reduced by 50% by 20 mM bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 mM) or bacitracin (20 mM) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. CONCLUSIONS: It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.

Partial characterization of an atypical family I inorganic pyrophosphatase from cattle tick Rhipicephalus (Boophilus) microplus.

The present paper presents the partial characterization of a family I inorganic pyrophosphatase from the hard tick Rhipicephalus (Boophilus) microplus (BmPPase). The BmPPase gene was cloned from the tick embryo and sequenced. The deduced amino acid sequence shared high similarity with other eukaryotic PPases, on the other hand, BmPPase presented some cysteine residues non-conserved in other groups. This pyrophosphatase is inhibited by Ca(2+), and the inhibition is antagonized by Mg(2+), suggesting that the balance between free Ca(2+) and free Mg(2+) in the eggs could be involved in BmPPase activity control. We observed that the BmPPase transcripts are present in the fat body, midgut and ovary of ticks, in two developmental stages (partially and fully engorged females). However, higher transcription amounts were found in ovary from fully engorged females. BmPPase activity was considerably abolished by the thiol reagent dithionitrobenzoic acid (DTNB), suggesting that cysteine residues are exposed in its structure. Therefore, these cysteine residues play a critical role in the structural stability of BmPPase. Molecular dynamics simulation analysis indicates that BmPPase is the first Family I PPase that could promote disulfide bonds between cysteine residues 138-339 and 167-295. Finally, we believe that these cysteine residues exposed in the BmPPase structure can play an important controlling role regarding enzyme activity, which would be an interesting mechanism of redox control. The results presented here also indicate that this enzyme can be involved in embryogenesis of this arthropod, and may be useful as a target in the development of new tick control strategies.

Hydrogen peroxide targets the cysteine at the active site and irreversibly inactivates creatine kinase.

In our study, we showed that at a relatively low concentration, H(2)O(2) can irreversibly inactivate the human brain type of creatine kinase (HBCK) and that HBCK is inactivated in an H(2)O(2) concentration-dependent manner. HBCK is completely inactivated when incubated with 2mM H(2)O(2) for 1h (pH 8.0, 25°C). Inactivation of HBCK is a two-stage process with a fast stage (k(1)=0.050 ± 0.002 min(-1)) and a slow (k(2)=0.022 ± 0.003 min(-1)) stage. HBCK inactivation by H(2)O(2) was affected by pH and therefore we determined the pH profile of HBCK inactivation by H(2)O(2). H(2)O(2)-induced inactivation could not be recovered by reducing agents such as dl-dithiothreitol, N-acetyl-L-cysteine, and l-glutathione reduced. When HBCK was treated with DTNB, an enzyme substrate that reacts specifically with active site cysteines, the enzyme became resistant to H(2)O(2). HBCK binding to Mg(2+)ATP and creatine can also prevent H(2)O(2) inactivation. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence data showed no tertiary structure changes after H(2)O(2) treatment. The thiol group content of H(2)O(2)-treated HBCK was reduced by 13% (approximately 1 thiol group per HBCK dimer, theoretically). For further insight, we performed a simulation of HBCK and H(2)O(2) docking that suggested the CYS283 residue could interact with H(2)O(2). Considering these results and the asymmetrical structure of HBCK, we propose that H(2)O(2) specifically targets the active site cysteine of HBCK to inactivate HBCK, but that substrate-bound HBCK is resistant to H(2)O(2). Our findings suggest the existence of a previously unknown negative form of regulation of HBCK via reactive oxygen species.

Antiviral propierties of 5,5'-dithiobis-2-nitrobenzoic acid and bacitracin against T-tropic human immunodeficiency virus type 1.

Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are agents known to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI). Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-ß-gal (CD4) and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env) indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

A nonproton ligand sensor in the acid-sensing ion channel.

Acid-sensing ion channels (ASICs) have long been considered as extracellular proton (H(+))-gated cation channels, and peripheral ASIC3 channels seem to be a natural sensor of acidic pain. Here, we report the identification of a nonproton sensor on ASIC3. We show first that 2-guanidine-4-methylquinazoline (GMQ) causes persistent ASIC3 channel activation at the normal pH. Using GMQ as a probe and combining mutagenesis and covalent modification analysis, we then uncovered a ligand sensor lined by residues around E423 and E79 of the extracellular "palm" domain of the ASIC3 channel that is crucial for activation by nonproton activators. Furthermore, we show that GMQ activates sensory neurons and causes pain-related behaviors in an ASIC3-dependent manner, indicating the functional significance of ASIC activation by nonproton ligands. Thus, natural ligands beyond protons may activate ASICs under physiological and pathological conditions through the nonproton ligand sensor, serving for channel activation independent of abrupt and marked acidosis.