Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development.

BACKGROUND: With the increasing resistance of malaria parasites to available drugs, there is an urgent demand to develop new anti-malarial drugs. Calpain inhibitor, ALLN, is proposed to inhibit parasite proliferation by suppressing haemoglobin degradation. This provides Plasmodium calpain as a potential target for drug development. Pf-calpain, a cysteine protease of Plasmodium falciparum, belongs to calpain-7 family, which is an atypical calpain not harboring Ca2+-binding regulatory motifs. In this present study, in order to establish the screening system for Pf-calpain specific inhibitors, the active form of Pf-calpain was first identified. METHODS: Recombinant Pf-calpain including catalytic subdomain IIa (rPfcal-IIa) was heterologously expressed and purified. Enzymatic activity was determined by both fluorogenic substrate assay and gelatin zymography. Molecular homology modeling was carried out to address the activation mode of Pf-calpain in the aspect of structural moiety. RESULTS: Based on the measurement of enzymatic activity and protease inhibitor assay, it was found that the active form of Pf-calpain only contains the catalytic subdomain IIa, suggesting that Pf-calpain may function as a monomeric form. The sequence prediction indicates that the catalytic subdomain IIa contains all amino acid residues necessary for catalytic triad (Cys-His-Asn) formation. Molecular modeling suggests that the Pf-calpain subdomain IIa makes an active site, holding the catalytic triad residues in their appropriate orientation for catalysis. The mutation analysis further supports that those amino acid residues are functional and have enzymatic activity. CONCLUSION: The identified active form of Pf-calpain could be utilized to establish high-throughput screening system for Pf-calpain inhibitors. Due to its unique monomeric structural property, Pf-calpain could be served as a novel anti-malarial drug target, which has a high specificity for malaria parasite. In addition, the monomeric form of enzyme may contribute to relatively simple synthesis of selective inhibitors.

Protein kinase CK2/PTEN pathway plays a key role in platelet-activating factor-mediated murine anaphylactic shock.

Platelet-activating factor (PAF) is a major mediator in the induction of fatal hypovolemic shock in murine anaphylaxis. This PAF-mediated effect has been reported to be associated with PI3K/Akt-dependent eNOS-derived NO. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is phosphatidylinositol phosphate phosphatase, which negatively controls PI3K by dephosphorylating the signaling lipid, phosphatidylinositol 3,4,5-triphosphate. In this study, we examined the possible involvement of PTEN in PAF-mediated anaphylactic shock. Induction of anaphylaxis or PAF injection resulted in a rapid decrease in PTEN activity, followed by increases in PI3K activity and phosphorylation of Akt and eNOS. Systemic administration of adenoviruses carrying PTEN cDNA (adenoviral PTEN), but not the control AdLacZ, not only attenuated anaphylactic symptoms, but also reversed anaphylaxis- or PAF-induced changes in PTEN and PI3K activities, as well as phosphorylation of Akt and eNOS. We found that the decreased PTEN activity was associated with PTEN phosphorylation, the latter effect being prevented by the protein kinase CK2 inhibitor, DMAT. DMAT also inhibited anaphylactic symptoms as well as the anaphylaxis- or PAF-mediated PTEN/PI3K/Akt/eNOS signaling cascade. CK2 activity was increased by PAF. The present data provide, as the key mechanism underlying anaphylactic shock, PAF triggers the upstream pathway CK2/PTEN, which ultimately leads to the activation of PI3K/Akt/eNOS. Therefore, CK2/PTEN may be a potent target in the control of anaphylaxis and other many PAF-mediated pathologic conditions.

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum.

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.

Antimalarial effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal by the inhibition of Plasmodium falciparum Calpain.

The biological understanding of malaria parasites has increased considerably over the past two decades with the discovery of many potential targets for the development of new antimalarial drugs. Calpain, a cysteine protease of Plasmodium falciparum, is believed to be a central mediator essential for parasitic activity. However, the utility of calpain as a potential anti-malarial target in P. falciparum has not been fully determined. In the present study, we determined the effect of N-acetyl-L-Leucyl-L-leucyl-L-norleucinal (ALLN)-treatment on the expression of calpain in erythrocytic stages of P. falciparum and its usefulness as an antimalarial chemotherapeutic agent. ALLN was shown to have low toxicity to HeLa cells but high toxicity to malaria. ALLN inhibited the expression of calpain in ring, trophozoite and schizont stages when treated for 48 h. Also, after 48 h, samples were characterized by 6.15% and 0% parasitemia without ALLN treatment and with ALLN treatment, respectively. Brightfield and confocal microscopy revealed that ALLN treatment affects merozoite maturation. As ALLN concentration increased from 1 muM to 100 microM, ring stage parasites did not mature into the schizont stage. When ALLN treatment was continued for 48 h, it also significantly inhibited the maturation of ring-stage parasites into trophozoite or schizont stages and survival of malarial parasites. Taken together, these findings suggest that ALLN inhibit the maturation and survival of P. falciparum and calpain expression, and thus has potential utility as an antimalarial chemotherapeutic agent.

Mycobacterium abscessus D-alanyl-D-alanine dipeptidase induces the maturation of dendritic cells and promotes Th1-biased immunity.

Mycobacterium abscessus, a member of the group of non-tuberculous mycobacteria, has been identified as an emerging pulmonary pathogen in humans. However, little is known about the protective immune response of antigenpresenting cells, such as dendritic cells (DCs), which guard against M. abscessus infection. The M. abscessus gene MAB1843 encodes D-alanyl-D-alanine dipeptidase, which catalyzes the hydrolysis of D-alanyl-D-alanine dipeptide. We investigated whether MAB1843 is able to interact with DCs to enhance the effectiveness of the host's immune response. MAB1843 was found to induce DC maturation via toll-like receptor 4 and its downstream signaling pathways, such as the mitogen-activated protein kinase and nuclear factor kappa B pathways. In addition, MAB1843-treated DCs stimulated the proliferation of T cells and promoted Th1 polarization. Our results indicate that MAB1843 could potentially regulate the immune response to M. abscessus, making it important in the development of an effective vaccine against this mycobacterium. [BMB Reports 2016; 49(10): 554-559].

Rapid degradation of Pseudomonas fluorescens 1-aminocyclopropane-1-carboxylic acid deaminase proteins expressed in transgenic Arabidopsis.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is commonly produced by plant growth-promoting rhizobacteria (PGPR) and has been suggested to facilitate the growth and stress tolerance of hosts via a reduction in levels of ethylene. However, the regulatory mechanism of ACC deaminase (AcdS) protein within host plant cells is largely unknown. Here, we demonstrated beneficial effects and post-translational modification of PGPR-originated AcdS proteins in plants. Compared with the wild-type, transgenic Arabidopsis expressing the Pseudomonas fluorescens acdS (PfacdS) gene displayed increased root elongation and reduced sensitivity to 10 µM exogenous ACC, an ethylene precursor. Arabidopsis expressing PfacdS also showed increased tolerance to high salinity (150 mM NaCl). PfAcdS proteins accumulated in transgenic Arabidopsis were rapidly degraded, which was potentially mediated by the 26S proteasome pathway. The degradation of PfAcdS was alleviated in the presence of exogenous ACC. In conclusion, our data suggest that the plant growth-promoting effects of bacterial AcdS proteins are potentially modulated via protein turnover inside the host plant cells. Such post-translational modification plays a physiological role in the mutualistic interactions between microorganisms and plants in the rhizospheric and/or endospheric niche.

1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas fluorescens promoting the growth of Chinese cabbage and its polyclonal antibody.

Bacterial 1-aminocyclopropane-1-carboxlyate (ACC) deaminase (AcdS) is an enzyme that cleaves ACC, a precursor of the plant hormone ethylene, into α-ketobutyrate and ammonia. The acdS gene was cloned from Pseudomonas fluorescens, which was capable of improving the seedling of Chinese cabbage under salinity condition. The recombinant AcdS (rAcdS) exhibited optimal activity at pH 8.5 and 30°C. Strong activity was sustained at up to 100 mM NaCl. The polyclonal anti-P. fluorescens AcdS antibody was produced in a rabbit that had been immunized with the purified rAcdS. This antibody successfully recognized the homologous antigens derived from the total proteins of isolated plant growth-promoting microorganisms. A statistically significant correlation was observed between the intensity of hybridization signal and AcdS activity measured by a biochemical method, suggesting its application as a useful indicator for active deaminases.

The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4.

In this study, we show that Mycobacterium avium subsp. paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-α, and IL-1ß) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naïve T cells to polarized CD4(+) and CD8(+) T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of CD4(+) and CD8(+) T cells.

A potential protein adjuvant derived from Mycobacterium tuberculosis Rv0652 enhances dendritic cells-based tumor immunotherapy.

A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from Mycobacterium tuberculosis Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4+ and CD8+ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8+ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.

Brevicompanine E reduces lipopolysaccharide-induced production of proinflammatory cytokines and enzymes in microglia by inhibiting activation of activator protein-1 and nuclear factor-kappaB.

Excessive release of proinflammatory cytokines by activated microglia can cause neurotoxicity in neurodegenerative diseases. We found that Brevicompanine E (BE), isolated from a deep ocean sediment derived fungus Penicillium sp., inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) production in microglia. Moreover, electrophoretic mobility shift assay (EMSA) demonstrated that BE attenuated nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) DNA binding activity in LPS-induced microglia. Consistent with this finding, BE inhibited LPS-induced IkappaBalpha degradation, NF-kappaB nuclear translocation, and also Akt, c-Jun NH2-terminal kinase (JNK) phosphorylation. Thus, BE may be potentially useful for modulating neuroinflammation.

ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease.

Chromatin remodeling and transcription regulation are tightly controlled under physiological conditions. It has been suggested that altered chromatin modulation and transcription dysfunction may play a role in the pathogenesis of Huntington's disease (HD). Increased histone methylation, a well established mechanism of gene silencing, results in transcriptional repression. ERG-associated protein with SET domain (ESET), a histone H3 (K9) methyltransferase, mediates histone methylation. We show that ESET expression is markedly increased in HD patients and in transgenic R6/2 HD mice. Similarly, the protein level of trimethylated histone H3 (K9) was also elevated in HD patients and in R6/2 mice. We further demonstrate that both specificity protein 1 (Sp1) and specificity protein 3 (Sp3) act as transcriptional activators of the ESET promoter in neurons and that mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic, interferes with the DNA binding of these Sp family transcription factors, suppressing basal ESET promoter activity in a dose dependent manner. The combined pharmacological treatment with mithramycin and cystamine down-regulates ESET gene expression and reduces hypertrimethylation of histone H3 (K9). This polytherapy significantly ameliorated the behavioral and neuropathological phenotype in the R6/2 mice and extended survival over 40%, well beyond any existing reported treatment in HD mice. Our data suggest that modulation of gene silencing mechanisms, through regulation of the ESET gene is important to neuronal survival and, as such, may be a promising treatment in HD patients.