Inhibition of cysteine cathepsin B and L activation in astrocytes contributes to neuroprotection against cerebral ischemia via blocking the tBid-mitochondrial apoptotic signaling pathway.

The roles of cathepsins in the ischemic astrocytic injury remain unclear. Here, we test the hypothesis that activation of cathepsin B and L contributes to the ischemic astrocyte injury via the tBid-mitochondrial apoptotic signaling pathways. In the rat models of pMCAO, CA-074Me or Clik148, a selective inhibitor of cathepsin B or cathepsin L, reduced the infarct volume, improved the neurological deficits and increased the MAP2 and GFAP levels. In OGD-induced astrocyte injury, CA-074Me or Clik148 decreased the LDH leakage and increased the GFAP levels. In the ischemic cortex or OGD-induced astrocytes injury, Clik148 or CA-074Me reversed pMCAO or OGD-induced increase in active cathepsin L or cathepsin B at 3 h or 6 h, increase in tBid, reduction in mitochondrial cytochrome-c (Cyt-c) and increase in cytoplastic Cyt-c and active caspase-3 at 12-24 h of the late stage of pMCAO or OGD. CA-074Me or Clik148 also reduced cytosolic and mitochondrial tBid, increased mitochondrial Cyt-c and decreased cytoplastic Cyt-c and active caspase-3 at 6 h of the early stage of Bid activation. CA-074Me or Clik148 blocked the pMCAO-induced release of cathepsin B or L from the lysosomes into the cytoplasm and activation of caspase-3 in ischemic astrocytes at 12 h after ischemia. Concurrent inhibition of cathepsin B and cathepsin L provided better protection on the OGD-induced astrocytic apoptosis than obtained with separate use of each inhibitor. These results suggest that inhibition of the cysteine cathepsin B and cathepsin L activation in ischemic astrocytes contributes to neuroprotection via blocking the tBid-mitochondrial apoptotic signaling pathway.

Cathepsin L activity controls adipogenesis and glucose tolerance.

Cysteine proteases play an important part in human pathobiology. This report shows the participation of cathepsin L (CatL) in adipogenesis and glucose intolerance. In vitro studies demonstrate the role of CatL in the degradation of the matrix protein fibronectin, insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R), essential molecules for adipogenesis and glucose metabolism. CatL inhibition leads to the reduction of human and murine pre-adipocyte adipogenesis or lipid accumulation, protection of fibronectin from degradation, accumulation of IR and IGF-1R beta-subunits, and an increase in glucose uptake. CatL-deficient mice are lean and have reduced levels of serum glucose and insulin but increased levels of muscle IR beta-subunits, fibronectin and glucose transporter (Glut)-4, although food/water intake and energy expenditure of these mice are no less than their wild-type littermates. Importantly, the pharmacological inhibition of CatL also demonstrates reduced body weight gain and serum insulin levels, and increased glucose tolerance, probably due to increased levels of muscle IR beta-subunits, fibronectin and Glut-4 in both diet-induced obese mice and ob/ob mice. Increased levels of CatL in obese and diabetic patients suggest that this protease is a novel target for these metabolic disorders.

Promiscuous processing of human alphabeta-protryptases by cathepsins L, B, and C.

Human α- and ß-protryptase zymogens are abundantly and selectively produced by mast cells, but the mechanism(s) by which they are processed is uncertain. ß-Protryptase is sequentially processed in vitro by autocatalysis at R(-3) followed by cathepsin (CTS) C proteolysis to the mature enzyme. However, mast cells from CTSC-deficient mice successfully convert protryptase (pro-murine mast cell protease-6) to mature murine mast cell protease-6. α-Protryptase processing cannot occur by trypsin-like enzymes due to an R(-3)Q substitution. Thus, biological mechanisms for processing these zymogens are uncertain. ß-Tryptase processing activity(ies) distinct from CTSC were partially purified from human HMC-1 cells and identified by mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL also directly process α-protryptase (Q(-3)) and mutated ß-protryptase (R(-3)Q) as well as wild-type ß-protryptase to maturity, indicating no need for autocatalysis, unlike the CTSC pathway. Heparin promoted tryptase tetramer formation and protected tryptase from degradation by CTSB and CTSL. Thus, CTSL and CTSB are capable of directly processing both α- and ß-protryptases from human mast cells to their mature enzymatically active products.

Processing of human protryptase in mast cells involves cathepsins L, B, and C.

Human ß-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized tetramer. ß-Protryptase in solution can be directly processed to the mature enzyme by cathepsin (CTS) L and CTSB, and sequentially processed by autocatalysis at R(-3), followed by CTSC proteolysis. However, it is uncertain which CTS is involved in protryptase processing inside human mast cells, because murine bone marrow-derived mast cells from CTSC-deficient mice convert protryptase (pro-mouse mast cell protease-6) to mature mouse mast cell protease-6. This finding suggests that other proteases are important for processing human ß-protryptase. In the current study, reduction of either CTSB or CTSL activity inside HMC-1 cells by short hairpin RNA silencing or CTS-specific pharmacologic inhibitors substantially reduced mature ß-tryptase formation. Similar reductions of tryptase levels in primary skin-derived mast cells were observed with these pharmacologic inhibitors. In contrast, protryptase processing was minimally reduced by short hairpin RNA silencing of CTSC. A putative pharmacologic inhibitor of CTSC markedly reduced tryptase levels, suggesting an off-target effect. Skin mast cells contain substantially greater amounts of CTSL and CTSB than do HMC-1 cells, the opposite being found for CTSC. Both CTSL and CTSB colocalize to the secretory granule compartment of skin mast cells. Thus, CTSL and CTSB are central to the processing of protryptase(s) in human mast cells and are potential targets for attenuating production of mature tryptase in vivo.

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD(+) synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3.

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.

Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin.

The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.

Structure-based development of specific inhibitors for individual cathepsins and their medical applications.

Specific inhibitors for individual cathepsins have been developed based on their tertiary structures of X-ray crystallography. Cathepsin B-specific inhibitors, CA-074 and CA-030, and cathepsin L specific inhibitors, CLIK-148 and CLIK-195, were designed as the epoxysuccinate derivatives. Cathepsin S inhibitor, CLIK-060, and cathepsin K inhibitor, CLIK-166, were synthesized. These inhibitors can use in vitro and also in vivo, and show no toxicity for experimental animals by the amounts used as the cathepsin inhibitor. Various cathepsins are used in the processing of antigenic proteins. The CLIK-060 treatment to the autoimmune disease, Sjögren model mice, led to strongly suppress the expression of the pathological symptoms. Cathepsins L or K participates to the degradation of bone collagen. The CLIK-148 protects osteoporosis in animals and also protects the bone metastasis of cancer cells. Cathepsin L also enhances insulin-induced glucose uptake into 3T3-L1 adipocytes, suggesting cathepsin L plays the roles in adipogenesis and glucose tolerance in type 2 diabetes.

Structural basis for the kexin-like serine protease from Aeromonas sobria as sepsis-causing factor.

The anaerobic bacterium Aeromonas sobria is known to cause potentially lethal septic shock. We recently proposed that A. sobria serine protease (ASP) is a sepsis-related factor that induces vascular leakage, reductions in blood pressure via kinin release, and clotting via activation of prothrombin. ASP preferentially cleaves peptide bonds that follow dibasic amino acid residues, as do Kex2 (Saccharomyces cerevisiae serine protease) and furin, which are representative kexin family proteases. Here, we revealed the crystal structure of ASP at 1.65 A resolution using the multiple isomorphous replacement method with anomalous scattering. Although the overall structure of ASP resembles that of Kex2, it has a unique extra occluding region close to its active site. Moreover, we found that a nicked ASP variant is cleaved within the occluding region. Nicked ASP shows a greater ability to cleave small peptide substrates than the native enzyme. On the other hand, the cleavage pattern for prekallikrein differs from that of ASP, suggesting the occluding region is important for substrate recognition. The extra occluding region of ASP is unique and could serve as a useful target to facilitate development of novel antisepsis drugs.

Genetic and pharmacologic alteration of cathepsin expression influences reovirus pathogenesis.

The cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt), Ctsb(-/-), Ctsl(-/-), and Ctss(-/-) mice with the virulent reovirus strain T3SA+. The survival rate of Ctsb(-/-) mice was enhanced in comparison to that of wt mice, whereas the survival rates of Ctsl(-/-) and Ctss(-/-) mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed in Ctsl(-/-) and Ctss(-/-) mice in comparison to the levels for wt and Ctsb(-/-) mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.

Retinal pigment epithelium-derived CTLA-2alpha induces TGFbeta-producing T regulatory cells.

T cells that encounter ocular pigment epithelium in vitro are inhibited from undergoing TCR-triggered activation, and instead acquire the capacity to suppress the activation of bystander T cells. Because retinal pigment epithelial (RPE) cells suppress T cell activation by releasing soluble inhibitory factors, we studied whether soluble factors also promote the generation of T regulatory (Treg) cells. We found that RPE converted CD4(+) T cells into Treg cells by producing and secreting CTLA-2alpha, a cathepsin L (CathL) inhibitor. Mouse rCTLA-2alpha converted CD4(+) T cells into Treg cells in vitro, and CTLA-2alpha small interfering RNA-transfected RPE cells failed to induce the Treg generation. RPE CTLA-2alpha induced CD4(+)CD25(+)Foxp3(+) Treg cells that produced TGFbeta in vitro. Moreover, CTLA-2alpha produced by RPE cells inhibited CathL activity in the T cells, and losing CathL activity led to differentiation to Treg cells in some populations of CD4(+) T cells. In addition, T cells in the presence of CathL inhibitor increased the expression of Foxp3. The CTLA-2alpha effect on Treg cell induction occurred through TGFbeta signaling, because CTLA-2alpha promoted activation of TGFbeta in the eye. These results show that immunosuppressive factors derived from RPE cells participate in T cell suppression. The results are compatible with the hypothesis that the eye-derived Treg cells acquire functions that participate in the establishment of immune tolerance in the posterior segment of the eye.

Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii.

The crystal structure of the highly thermostable l-aspartate oxidase (LAO) from the hyperthermophilic archaeon Sulfolobus tokodaii was determined at a 2.09 A resolution. The factors contributing to the thermostability of the enzyme were analyzed by comparing its structure to that of Escherichia coli LAO. Like E. coli LAO, the S. tokodaii enzyme consists of three domains: an FAD-binding domain, an alpha+beta capping domain, and a C-terminal three-helix bundle. However, the situation of the linker between the FAD-binding domain and C-terminal three-helix bundle in S. tokodaii LAO is completely different from that in E. coli LAO, where the linker is situated near the FAD-binding domain and has virtually no interaction with the rest of the protein. In S. tokodaii LAO, this linker is situated near the C-terminal three-helix bundle and contains a beta-strand that runs parallel to the C-terminal strand. This results in the formation of an additional beta-sheet, which appears to reduce the flexibility of the C-terminal region. Furthermore, the displacement of the linker enables formation of a 5-residue ion-pair network between the FAD-binding and C-terminal domains, which strengthens the interdomain interactions. These features might be the main factors contributing to the high thermostability of S. tokodaii LAO.

Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity.

The cysteine endoprotease cathepsin S mediates degradation of the MHC class II invariant chain Ii in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for Sjögren syndrome. Specific inhibitor of cathepsin S (Clik60) in vitro markedly impaired presentation of an organ-specific autoantigen, 120-kDa alpha-fodrin, by interfering with MHC class II-peptide binding. Autoantigen-specific T cell responses were significantly and dose-dependently inhibited by incubation with Clik60, but not with inhibitor s of cathepsin B or L. Clik60 treatment of mouse salivary gland cells selectively inhibited autopeptide-bound class II molecules. Moreover, the treatment with Clik60 in vivo profoundly blocked lymphocytic infiltration into the salivary and lacrimal glands, abrogated a rise in serum autoantibody production, and led to recovery from autoimmune manifestations. Thus, inhibition of cathepsin S in vivo alters autoantigen presentation and development of organ-specific autoimmunity. These data identify selective inhibition of cysteine protease cathepsin S as a potential therapeutic strategy for autoimmune disease processes.

Crystal structure of archaeal highly thermostable L-aspartate dehydrogenase/NAD/citrate ternary complex.

The crystal structure of the highly thermostable L-aspartate dehydrogenase (L-aspDH; EC 1.4.1.21) from the hyperthermophilic archaeon Archaeoglobus fulgidus was determined in the presence of NAD and a substrate analog, citrate. The dimeric structure of A. fulgidus L-aspDH was refined at a resolution of 1.9 A with a crystallographic R-factor of 21.7% (R(free) = 22.6%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. Structural comparison of the A. fulgidus L-aspDH/NAD/citrate ternary complex and the Thermotoga maritima L-aspDH/NAD binary complex showed that A. fulgidus L-aspDH assumes a closed conformation and that a large movement of the two loops takes place during substrate binding. Like T. maritima L-aspDH, the A. fulgidus enzyme is highly thermostable. But whereas a large number of inter- and intrasubunit ion pairs are responsible for the stability of A. fulgidus L-aspDH, a large number of inter- and intrasubunit aromatic pairs stabilize the T. maritima enzyme. Thus stabilization of these two L-aspDHs appears to be achieved in different ways. This is the first detailed description of substrate and coenzyme binding to L-aspDH and of the molecular basis of the high thermostability of a hyperthermophilic L-aspDH.

Catechin derivatives: specific inhibitor for caspases-3, 7 and 2, and the prevention of apoptosis at the cell and animal levels.

Tea-catechin derivatives are shown to inhibit activities of caspases-3, 2 and 7 in vitro, and prevented experimental apoptosis at the cell and animal levels. Epigallo-catechin-gallate showed the strongest inhibition at 1 x 10(-7)M to these caspases, but cysteine cathepsins and caspase-8 were not inhibited. Caspase-3 inhibition showed a 2nd-order allosteric-type, but the inhibition of caspases-2 and 7 showed a non-competitive-type. The apoptosis-test using cultured HeLa cells was inhibited by these catechins. In rat hepatocytes, apoptosis was induced by d-galactosamine in vivo. In this case, caspase-3 activity in the cytoplasm, the serum aminotransferases and dUTP nick formation detected by TUNNEL-staining were effects, and these elevations were suppressed by administration of catechin.

A novel apoptosis cascade mediated by lysosomal lactoferrin and its participation in hepatocyte apoptosis induced by D-galactosamine.

A new apoptosis cascade mediated by lysosomal lactoferrin was found in apoptotic liver induced by d-galactosamine. Caspase-3 and lactoferrin were increased in the apoptotic liver cytoplasm and serum transaminases were elevated. Recombinant lactoferrin stimulated procaspase-3 processing at 10(-6)-10(-7)M to an extent similar to that by granzyme B in vitro. Lactoferrin changed procaspase-3 structure susceptible to the processing. Synthetic peptide Y(679)-K(695) in lactoferrin molecule inhibited the processing of procaspase-3 by lactoferrin. Lactoferrin in lysosomes was decreased and lactoferrin released into cytoplasm was increased quantitatively in d-galactosamine induced apoptotic liver, and procaspase-3 in cytoplasm was processed to caspase-3.

The first crystal structure of hyperthermostable NAD-dependent glutamate dehydrogenase from Pyrobaculum islandicum.

The extremely thermostable NAD-dependent glutamate dehydrogenase (NAD-GluDH) from Pyrobaculum islandicum, a member of the Crenarchaeota, was crystallized, and its 3D structure has been determined by X-ray diffraction methods. The homohexameric structure of Pb. islandicum glutamate dehydrogenase (Pis-GluDH) was solved and refined at a resolution of 2.9A with a crystallographic R-factor of 19.9% (Rfree 26.0%). The structure indicates that each subunit consists of two domains separated by a deep cleft containing an active site. The secondary structural elements and catalytically important residues of the enzyme were highly conserved among the NAD(P)-dependent GluDHs from other sources. A structural comparison of Pis-GluDH with other NAD(P)-dependent GluDHs suggests that a significant difference in the alpha8-loop-alpha9 region of this enzyme is associated with its coenzyme specificity. From the analysis of the 3D structure, hydrophobic interactions between intersubunits were found to be important features for the enzyme oligomerization. It has been reported that Pis-GluDH is highly thermostable, like the GluDH of the hyperthermophilic archaeum Pyrococcus furiosus, and the increase in the intersubunit ion pair networks is responsible for the extreme thermostability of the Pc. furiosus enzyme. However, the number of intersubunit ion pairs in the Pis-GluDH molecules is much smaller than those of the Pc. furiosus GluDH. The number of hydrophobic interactions at the intersubunit interfaces were increased and responsible for the extremely high thermostability. This indicates that the major molecular strategy for high thermostability of the GluDHs may be different for each hyperthermophile.

Processing of human cathepsin D is independent of its catalytic function and auto-activation: involvement of cathepsins L and B.

The current mechanism proposed for the processing and activation of the 52 kDa lysosomal aspartic protease cathepsin D (cath-D) is a combination of partial auto-activation generating a 51 kDa pseudo-cath-D, followed by enzyme-assisted maturation involving cysteine and/or aspartic proteases and yielding successively a 48 kDa intermediate and then 34 + 14 kDa cath-D mature species. Here we have investigated the in vivo processing of human cath-D in a cath-D-deficient fibroblast cell line in order to determine whether its maturation occurs through already active cath-D and/or other proteases. We demonstrate that cellular cath-D is processed in a manner independent of its catalytic function and that auto-activation is not a required step. Moreover, the cysteine protease inhibitor E-64 partially blocks processing, leading to accumulation of 52-48 kDa cath-D intermediates. Furthermore, two inhibitors, CLICK148 and CA-074Met, specific for the lysosomal cath-L and cath-B cysteine proteases induce accumulation of 48 kDa intermediate cath-D. Finally, maturation of endocytosed pro-cath-D is also independent of its catalytic function and requires cysteine proteases. We therefore conclude that the mechanism of cath-D maturation involves a fully-assisted processing similar to that of pro-renin.

Significance of 32-kDa cathepsin L secreted from cancer cells.

It is recognized that many cancer cells secrete cathepsin L to degrade the components of extracellular matrices and basement membranes, thus promoting tumor invasion and metastasis. However, very little information is available concerning the secreted forms of cathepsin L and their possible role in human cancer. We initially demonstrated that approximately 10-fold higher mature cathepsin L activity was secreted in a medium of human fibrosarcoma (HT 1080) cells, compared with their intracellular activity. A 32-kDa major-activity band, together with a 41-kDa faint-activity band, was detected in the medium by our newly developed gelatin zymography. The two forms were further confirmed to be cathepsin L by immunoblot analysis. Both were apparently secreted directly from the cells, as neither was affected when the cells were cultured in the presence of various kinds of proteinase inhibitors. Human tumor necrosis factor-alpha (TNF-alpha) stimulated not only the production of the 32-kDa cathepsin L, but also its secretion. Moreover, the 32-kDa cathepsin L activities in 3 colon and 2 lung cancer tissues were significantly higher than in normal tissues. Based on the foregoing, there are good reasons to speculate that the 32-kDa cathepsin L found in HT 1080 cell medium is involved in cancer invasion and metastasis.

A second novel dye-linked L-proline dehydrogenase complex is present in the hyperthermophilic archaeon Pyrococcus horikoshii OT-3.

Two distinguishable activity bands for dye-linked l-proline dehydrogenase (PDH1 and PDH2) were detected when crude extract of the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was run on a polyacrylamide gel. After purification, PDH1 was found to be composed of two different subunits with molecular masses of 56 and 43 kDa, whereas PDH2 was composed of four different subunits with molecular masses of 52, 46, 20 and 8 kDa. The native molecular masses of PDH1 and PDH2 were 440 and 101 kDa, respectively, indicating that PDH1 has an alpha4beta4 structure, while PDH2 has an alphabetagammadelta structure. PDH2 was found to be similar to the dye-linked l-proline dehydrogenase complex from Thermococcus profundus, but PDH1 is a different type of enzyme. After production of the enzyme in Escherichia coli, high-performance liquid chromatography showed the PDH1 complex to contain the flavins FMN and FAD as well as ATP. Gene expression and biochemical analyses of each subunit revealed that the beta subunit bound FAD and exhibited proline dehydrogenase activity, while the alpha subunit bound ATP, but unlike the corresponding subunit in the T. profundus enzyme, it exhibited neither proline dehydrogenase nor NADH dehydrogenase activity. FMN was not bound to either subunit, suggesting it is situated at the interface between the alpha and beta subunits. A comparison of the amino-acid sequences showed that the ADP-binding motif in the alpha subunit of PDH1 clearly differs from that in the alpha subunit of PDH2. It thus appears that a second novel dye-linked l-proline dehydrogenase complex is produced in P. horikoshii.

Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin.

Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals. It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton. Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues. The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region. However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT. The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380. Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity. At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase. Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction.