Cytophysiologic effects and molecular inhibition of a functional actin specific ADP ribosyltransferase CDT from clostridium difficile 4

Chapter Peptide antibiotic and actin-binding protein as mixed-type inhibitors of Clostridium difficile CDT toxin activities 5.1. Introduction Aside from ATP phosphorylation and GTP-protein binding, ADP-ribosylation is a posttranslational, protein-modification process involved in many cellular functions (Obara et al., 1991; Sooki-Toth et al., 1987). Eukaryotes produce nuclear poly (ADP-ribosyl) synthetase which mediates substrate linkage of several ADP-ribose moieties and mono (ADP-ribosyl) transferase which is likewise produced by prokaryotes. The catalytic process involves N-glycosidic bond hydrolysis of NAD+ resulting in the release of nicotinamide and attachment of adenosinediphosphoribose group to an acceptor protein (Moss and Vaughan, 1988). The bulky ADP- ribose group is attached to arginine-177 of actin subdomain III forming bulk hindrance that prevents actin polymerization (Vandekerckhove et al., 1988). The acceptor amino acid is embedded in the filamentous actin form, thus explaining F-actin role as a poor CDT substrate. A variety of acceptor molecules has been identified such as histones, enzymes, regulatory and structural proteins (Ding and Smulson, 1994; Nakajima et al., 2004; Obara et al., 1991). Having demonstrated the actin-specific ADPRT activity of CDT, the abundance and equimolar concentration of G- and F-actin must be protected against modifying agents through the development of effective inhibitors. Moreso, there has been an increase in the isolation of cdt-encoding pathogenic C. difficile, an organism implicated in mammalian enterotoxemia, antibiotic-associated diarrhea and colitis (Borriello and Carman, 1983; Goncalves et al., 2004; Hatheway, 1990). Thus, the urgency of discovering potential molecules which can neutralize the activities of the toxin. Previous studies have focused largely on compounds against eukaryotic poly (ADPribosyl) synthetases whose inhibitory actions were found to be highly specific (Banasik et al., 1992; Rankin et al., 1989). In fact, differences in inhibitory capability yielded a broad range of 114 ordered efficacy. While novobiocin, vitamin K1 and vitamin K3 were reported most potent for eukaryotic mono (ADP-ribosyl) transferase from hen heterophil, preventive efficacy of the vitamin derivative nicotinamide has differed depending on ADPRT source (Banasik et al., 1990; Rankin et al., 1989). Here we report in vitro effects of several groups of natural and synthetic compounds such as metal ions, nucleotides, antimicrobials, vitamins and actin-binding proteins (ABPs) on bacterial mono ADPRT activities. The most potent inhibitors were α-actinin and striated muscle thin filament constituents, antagonistic to specific CDT actions. 5.2. Results 5.2.1. Nucleotide binding in CDT On testing direct CDTa action on actin, ADP-ribosylation proceeded in a time-dependent manner having peaked at h mark (197.32) (Fig. 5.1A). Proportional rise in CDTa dosage and actin labeling (Fig. 5.1B) supported substrate specificity of CDT action. NAD binding to actin was then determined via UV cross-linking. Photoinsertion into actin was proportional to [32P]NAD concentration with saturation effects observed at 46 µM (Fig. 5.1C). Competition for binding by increasing unlabeled parent nucleotide resulted in corresponding decrease in radiolabeling (Fig. 5.1D) with half maximal prevention obtained at 42.7 µM. These demonstrate specificity of [32P]NAD interaction with actin nucleotide binding site. Since attachment of NAD to CDT catalytic site was requisite in ADP-ribosylation, we next compared prevention of [32P]NAD photoinsertion into CDT by increasing NAD and ATP. Both nucleotides prevented photoinsertion with ATP causing 16% while NAD had 29% prevention (Fig. 5.1E), indicating higher NAD affinity to CDT nucleotide binding site. For control reactions, non-irradiated reaction mixtures and those with CDT or actin alone showed no detectable band indicating absence of autologous phosphorylation and endogenous or contaminant radiolabels. 115 Figure 5.1. Kinetics of recombinant CDT. A, time-course analysis of actin-directed ADP-ribosylation arrested at indicated times. Purified SK1203 (0.5 µg) was substituted for CDTa in the (-) lane. B, ADP-ribosylation with increasing CDTa in increasing concentrations (ng/ml). C, saturation of actin nucleotide binding site by photolabeling with increasing [32P]NAD concentrations (mCi/mmol). No actin was added for the (-) lane. D, prevention of [32P]NAD photolabeling of actin with increasing NAD concentrations (µM). E. comparative prevention of [32P]NAD photoinsertion into CDT by NAD and ATP at increasing concentrations (µM). 116 5.2.2. Divalent metal ions enhanced CDT-NAD binding We then quantified the effects of ionic compounds on NAD photolabeling of CDT to detect cofactors which could hasten or inhibit biochemical reactions. Relative to MgCl2 (maximal activity) with mean pixel value of 62, ZnCl2 at 112 and MnCl2 at 79 units enhanced photoinsertion by 81% and 27 % respectively, while Ca2+ and other monovalent ions weakened reactions by 2-7% indicating inability to substitute for Mg2+ (Fig. 5.2A-C). This was reflected in phosphorimages showing distinctly stronger intensities for Mn2+ and Zn2+ ions particularly at 100 µM and still evident at higher concentration. When chelators were added, differential reduction in labeling was observed (Fig. 5.2D), implicating the influence of metal ions in nucleotide binding. At 50 µM concentrations, the effect of ZnCl2 was lowered upon EGTA (62%), EDTA (43%) and calmodulin (37%) preincubation. MgCl2 and MnCl2 effects were diminished except by calmodulin which enhanced photoinsertion by 14-27%. EDTA lowered Zn2+, Mn2+ and Mg2+ effects by 43%, 38% and 34% respectively. EGTA caused 49% reduction on Mg2+ and only 26% on Mn2+ effects. Such divergent influence signifies a pattern of hierarchy for functional specificity of ionic species on NAD attachment to CDT. Overall, Zn2+ and Mn2+ appeared to have fulfilled the metal-ion requirement more efficiently than Mg2+. 5.2.3. Effects of various compounds on CDT activities Several groups of compounds proved inhibitory to CDT actions (Table 5.1). The more potent among them were ATP and nitrogenous compounds. Consistent with its direct competition with the labeled counterpart (Fig. 5.1D,E), NAD expectedly posted a high significant reduction of 36% and 58% on ARTase and NADse activities respectively (p vancomycin > cloxacillin > penicillin G and bacitracin while the rest exhibited either low to moderate action or even stimulatory effects (Table 5.1) (Fig. 5.2E,F). On NADse activity, polymyxin B > cephradine and cefotaxime > bacitracin > cyclosporin A > penicillin G proved most inhibitory. To illustrate the magnitude of prevention, polymyxin B posed 21% more ARTase inhibition compared to nystatin’s -18% at 0% control value without inhibitor. In addition, polymyxin concentration for half maximal inhibition is 21fold lower than the highest positive value for streptomycin. While vancomycin and cloxacillin worked against ARTase and not NADse activity indicating interference on CDT-ADP-ribose+actin interaction, cephradine and cefotaxime showed more effective NADse prevention (Table 121 5.1)(Fig. 5.2E,F) suggesting specific disruption of CDT-NAD interaction. Of the vitamins and derivatives tested, synthetic vitamin K3 had strong inhibition on both activities particularly ARTase (p[...]... induction by CDT via caspase-3 and ATF-2 activation CDT via the stress MAPKs may have activated ATF2 to form Jun:ATF2 and ATF2:ATF2 dimers which possess anti-apoptotic actions that promote cell proliferation, survival and transformation Therefore, we propose that CDT- imposed actin restructuring and cell shape alteration can trigger cellular responses against stress Previous reports have likewise illustrated... levels of such factors through immunoassay and found that CDT is an inducer of stress-related MAP kinase pathway and 1 34 apoptosis The sustained presence of CDT hindered the development of integrin aggregates into more contractile adhesions which briefly delayed Rho activation to allow actin nucleation/polymerization associated with Rac and Cdc42 activities This is reflective of the fact that immediate... Thus, ADP- ribosylation can be considered as an innate process whereby cells maintain the optimal G -actin pool, an event similar to G -actin engagement and regulation by actin- destabilizing or severing actin binding proteins (ABPs) like profilin, cofilin or thymosin 4 (Safer et al., 1991) to maintain isoform equilibrium We have shown the lethality of CDT action involving actin ADP- ribosylation, actin. .. of transduction Thus, it is becoming clear that cell death was not a manifestation of direct CDTa action on actin exclusively but of combined effects with CDTstimulated signal transduction and apoptosis Whether signaling was triggered as prompt as CDTb attachment on the membrane or after CDTa-induced cytoskeletal collapse (whereby increased G -actin relayed a stress signal to nuclear factors via MAPKs)... and CDTb and their effects on cellular physiology CDTa exhibited modification of actin monomer that caused dramatic F -actin depolymerization We have followed and quantified the dynamics of actin rearrangement through flow cytometry using actin- specific fluorescent reagents Other toxins like jasplakinolide and cytochalasin whose actions against actin are established were used in synchrony or as controls... towards foreign antigens will be downplayed if not entirely evaded Endogenous ABPs would be much tolerated In terms of beneficial applications, CDTa as an actin- freezing molecule and inducer of cAMP, MAPKs and other components of signal transduction pathways, is a promising pharmacologic agent that can be utilized for physiological research at the cellular to organ level It may also be tapped as an anti-tumor... establishment of focal adhesion and spreading was prioritized under stress conditions over that of network connections which occurs later in the developmental process Upregulation of talin and cAMP gave preliminary indication that CDT attachment to receptor may be linked to a transductional process promoting actin nucleation and polymerization CDT may have likewise intensified cAMP/PKA-enhanced actin. .. residues Asp1, Glu2, Asp3, Glu4, Asp 24, Asp25, Arg28, Glu93, Arg95 and Lys336 (Kabsch et al., 1990) and subdomain 3 residues Glu99, Glu100, Ala 144 , Ile 341 , Ile 345 , Leu 349 , Phe352, Pro332-333 and Trp79-Asn92 (Schroder et al., 1993) These regions overlaid by tropomyosin in the absence of Ca+ and S1 (tropomyosin off position) are in juxtaposition with Arg177 and may present steric impedance on ADP- ribosylation... withdrawal of electron from NN7 causing cleavage of the N-glycoside bond; the stabilization of the resulting oxocarbenium cation via charge-charge chelation of the positive NC1 of ADP- ribose by negative carboxylate group of Glu380 and Ser388 side chain (Fig 5.5B); and the transfer of ADP- ribose+ to the guanidyl nitrogen of actin Arg117 (proximal to NC1 of ADPribose+) which is recognized and positioned by... interaction and activation of actin by signal proteins For instance, MAPK -actin interaction was observed after microfilament disruption in human intestinal epithelial cells (Khurana and Dey, 2003; Nemeth et al., 20 04) Activated mekk1 was also demonstrated to co-localize with elements of the cytoskeleton 135 mediating c-JNK activation near or within the locale of the cytoskeletal network (English et al., . Nakajima et al., 20 04; Obara et al., 1991). Having demonstrated the actin- specific ADPRT activity of CDT, the abundance and equimolar concentration of G- and F -actin must be protected against. NC1 of ADP- ribose by negative carboxylate group of Glu380 and Ser388 side chain (Fig. 5.5B); and the transfer of ADP- ribose + to the guanidyl nitrogen of actin Arg117 (proximal to NC1 of ADP- ribose + ). positive value for streptomycin. While vancomycin and cloxacillin worked against ARTase and not NADse activity indicating interference on CDT -ADP- ribose + - actin interaction, cephradine and cefotaxime