50 results on '"Sijwali PS"'
Search Results
2. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy
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Klionsky, Dj, Abdelmohsen, K, Abe, A, Abedin, Mj, Abeliovich, H, Acevedo Arozena, A, Adachi, H, Adams, Cm, Adams, Pd, Adeli, K, Adhihetty, Pj, Adler, Sg, Agam, G, Agarwal, R, Aghi, Mk, Agnello, M, Agostinis, P, Aguilar, Pv, Aguirre-Ghiso, J, Airoldi, Em, Ait-Si-Ali, S, Akematsu, T, Akporiaye, Et, Al-Rubeai, M, Albaiceta, Gm, Albanese, C, Albani, D, Albert, Ml, Aldudo, J, Algül, H, Alirezaei, M, Alloza, I, Almasan, A, Almonte-Beceril, M, Alnemri, Es, Alonso, C, Altan-Bonnet, N, Altieri, Dc, Alvarez, S, Alvarez-Erviti, L, Alves, S, Amadoro, G, Amano, A, Amantini, C, Ambrosio, S, Amelio, I, Amer, Ao, Amessou, M, Amon, A, An, Z, Anania, Fa, Andersen, Su, Andley, Up, Andreadi, Ck, Andrieu-Abadie, N, Anel, A, Ann, Dk, Anoopkumar-Dukie, S, Antonioli, M, Aoki, H, Apostolova, N, Aquila, S, Aquilano, K, Araki, K, Arama, E, Aranda, A, Araya, J, Arcaro, A, Arias, E, Arimoto, H, Ariosa, Ar, Armstrong, Jl, Arnould, T, Arsov, I, Asanuma, K, Askanas, V, Asselin, E, Atarashi, R, Atherton, Ss, Atkin, 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D, Sinha, S, Sinicrope, Fa, Sirko, A, Sirohi, K, Sishi, Bj, Sittler, A, Siu, Pm, Sivridis, E, Skwarska, A, Slack, R, Slaninová, I, Slavov, N, Smaili, Ss, Smalley, Ks, Smith, Dr, Soenen, Sj, Soleimanpour, Sa, Solhaug, A, Somasundaram, K, Son, Jh, Sonawane, A, Song, C, Song, F, Song, Hk, Song, Jx, Song, W, Soo, Ky, Sood, Ak, Soong, Tw, Soontornniyomkij, V, Sorice, M, Sotgia, F, Soto-Pantoja, Dr, Sotthibundhu, A, Sousa, Mj, Spaink, Hp, Span, Pn, Spang, A, Sparks, Jd, Speck, Pg, Spector, Sa, Spies, Cd, Springer, W, Clair, Ds, Stacchiotti, A, Staels, B, Stang, Mt, Starczynowski, Dt, Starokadomskyy, P, Steegborn, C, Steele, Jw, Stefanis, L, Steffan, J, Stellrecht, Cm, Stenmark, H, Stepkowski, Tm, Stern, St, Stevens, C, Stockwell, Br, Stoka, V, Storchova, Z, Stork, B, Stratoulias, V, Stravopodis, Dj, Strnad, P, Strohecker, Am, Ström, Al, Stromhaug, P, Stulik, J, Su, Yx, Su, Z, Subauste, Cs, Subramaniam, S, Sue, Cm, Suh, Sw, Sui, X, Sukseree, S, Sulzer, D, Sun, Fl, Sun, J, Sun, Sy, Sun, Y, 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Winklhofer, Kf, Witkin, Ss, Wohlgemuth, Se, Wollert, T, Wolvetang, Ej, Wong, E, Wong, Gw, Wong, Rw, Wong, Vk, Woodcock, Ea, Wright, Kl, Wu, C, Wu, D, Wu, Gs, Wu, J, Wu, M, Wu, S, Wu, Wk, Wu, Y, Wu, Z, Xavier, Cp, Xavier, Rj, Xia, Gx, Xia, T, Xia, W, Xia, Y, Xiao, H, Xiao, J, Xiao, S, Xiao, W, Xie, Cm, Xie, Z, Xilouri, M, Xiong, Y, Xu, C, Xu, F, Xu, H, Xu, J, Xu, L, Xu, X, Xu, Y, Xu, Zx, Xu, Z, Xue, Y, Yamada, T, Yamamoto, A, Yamanaka, K, Yamashina, S, Yamashiro, S, Yan, B, Yan, X, Yan, Z, Yanagi, Y, Yang, Ds, Yang, Jm, Yang, L, Yang, M, Yang, Pm, Yang, P, Yang, Q, Yang, W, Yang, Wy, Yang, X, Yang, Y, Yang, Z, Yao, Mc, Yao, Pj, Yao, X, Yao, Z, Yasui, Ls, Ye, M, Yedvobnick, B, Yeganeh, B, Yeh, Es, Yeyati, Pl, Yi, F, Yi, L, Yin, Xm, Yip, Ck, Yoo, Ym, Yoo, Yh, Yoon, Sy, Yoshida, K, Yoshimori, T, Young, Kh, Yu, H, Yu, Jj, Yu, Jt, Yu, J, Yu, L, Yu, Wh, Yu, Xf, Yu, Z, Yuan, J, Yuan, Zm, Yue, By, Yue, J, Yue, Z, Zacks, Dn, Zacksenhaus, E, Zaffaroni, N, Zaglia, T, Zakeri, Z, Zecchini, V, Zeng, J, Zeng, M, Zeng, Q, Zervos, As, Zhang, Dd, Zhang, F, Zhang, G, Zhang, Gc, Zhang, H, Zhang, J, Zhang, Jp, Zhang, L, Zhang, My, Zhang, X, Zhang, Xd, Zhang, Y, Zhao, M, Zhao, Wl, Zhao, X, Zhao, Yg, Zhao, Y, Zhao, Yx, Zhao, Z, Zhao, Zj, Zheng, D, Zheng, Xl, Zheng, X, Zhivotovsky, B, Zhong, Q, Zhou, Gz, Zhou, G, Zhou, H, Zhou, Sf, Zhou, Xj, Zhu, H, Zhu, Wg, Zhu, W, Zhu, Xf, Zhu, Y, Zhuang, Sm, Zhuang, X, Ziparo, E, Zois, Ce, Zoladek, T, Zong, Wx, Zorzano, A, and Zughaier, Sm
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- 2016
3. Oleuropein activates autophagy to circumvent anti-plasmodial defense.
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Sharma P, Tandel N, Kumar R, Negi S, Sharma P, Devi S, Saxena K, Chaudhary NR, Saini S, Kumar R, Chandel BS, Sijwali PS, and Tyagi RK
- Abstract
Antimalarial drug resistance and unavailability of effective vaccine warrant for newer drugs and drug targets. Hence, anti-inflammatory activity of phyto-compound (oleuropein; OLP) was determined in antigen (LPS)-stimulated human THP-1 macrophages (macrophage model of inflammation; MMI). Reduction in the inflammation was controlled by the PI3K-Akt1 signaling to establish the "immune-homeostasis." Also, OLP treatment influenced the cell death/autophagy axis leading to the modulated inflammation for extended cell survival. The findings with MII prompted us to detect the antimalarial activity of OLP in the wild type (3D7), D10-expressing GFP-Atg18 parasite, and chloroquine-resistant (Dd2) parasite. OLP did not show the parasite inhibition in the routine in vitro culture of P. falciparum whereas OLP increased the antimalarial activity of artesunate. The molecular docking of autophagy-related proteins, investigations with MMI, and parasite inhibition assays indicated that the host activated the autophagy to survive OLP pressure. The challenge model of P. berghei infection showed to induce autophagy for circumventing anti-plasmodial defenses., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)
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- 2024
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4. Plasmodium falciparum contains functional SCF and CRL4 ubiquitin E3 ligases, and CRL4 is critical for cell division and membrane integrity.
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Rizvi Z, Reddy GS, Gorde SM, Pundir P, Das D, and Sijwali PS
- Subjects
- Humans, Cell Division, Cullin Proteins metabolism, Ubiquitin metabolism, Ubiquitination, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
Protein ubiquitination is essential for cellular homeostasis and regulation of several processes, including cell division and genome integrity. Ubiquitin E3 ligases determine substrate specificity for ubiquitination, and Cullin-RING E3 ubiquitin ligases (CRLs) make the largest group among the ubiquitin E3 ligases. Although conserved and most studied in model eukaryotes, CRLs remain underappreciated in Plasmodium and related parasites. To investigate the CRLs of human malaria parasite Plasmodium falciparum, we generated parasites expressing tagged P. falciparum cullin-1 (PfCullin-1), cullin-2 (PfCullin-2), Rbx1 (PfRbx1) and Skp1 (PfSkp1). PfCullin-1 and PfCullin-2 were predominantly expressed in erythrocytic trophozoite and schizont stages, with nucleocytoplasmic localization and chromatin association, suggesting their roles in different cellular compartments and DNA-associated processes. Immunoprecipitation, in vitro protein-protein interaction, and ubiquitination assay confirmed the presence of a functional Skp1-Cullin-1-Fbox (PfSCF) complex, comprising of PfCullin-1, PfRbx1, PfSkp1, PfFBXO1, and calcyclin binding protein. Immunoprecipitation, sequence analysis, and ubiquitination assay indicated that PfCullin-2 forms a functional human CRL4-like complex (PfCRL4), consisting of PfRbx1, cleavage and polyadenylation specificity factor subunit_A and WD40 repeat proteins. PfCullin-2 knock-down at the protein level, which would hinder PfCRL4 assembly, significantly decreased asexual and sexual erythrocytic stage development. The protein levels of several pathways, including protein translation and folding, lipid biosynthesis and transport, DNA replication, and protein degradation were significantly altered upon PfCullin-2 depletion, which likely reflects association of PfCRL4 with multiple pathways. PfCullin-2-depleted schizonts had poorly delimited merozoites and internal membraned structures, suggesting a role of PfCRL4 in maintaining membrane integrity. PfCullin-2-depleted parasites had a significantly lower number of nuclei/parasite than the normal parasites, indicating a crucial role of PfCRL4 in cell division. We demonstrate the presence of functional CRLs in P. falciparum, with crucial roles for PfCRL4 in cell division and maintaining membrane integrity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rizvi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
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- 2024
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5. Reinvestigation of diphenylmethylpiperazine analogues of pyrazine as new class of Plasmodial cysteine protease inhibitors for the treatment of malaria.
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Madhav H, Reddy GS, Rizvi Z, Jameel E, Patel TS, Rahman A, Yadav V, Fatima S, Heyat F, Pal K, Minju-Op A, Subbarao N, Bhattacharjee S, Dixit BC, Sijwali PS, and Hoda N
- Abstract
Malaria eradication is still a global challenge due to the lack of a broadly effective vaccine and the emergence of drug resistance to most of the currently available drugs as part of the mainline artemisinin-based combination therapy. A variety of experimental approaches are quite successful in identifying and synthesizing new promising pharmacophore hybrids with distinct mechanisms of action. Based on our recent findings, the current study demonstrates the reinvestigation of a series of diphenylmethylpiperazine and pyrazine-derived molecular hybrids. Pyrazine-derived molecular hybrids were screened to investigate the antiplasmodial activity on drug-susceptible Pf 3D7 and drug-resistant Pf W2 strains. The selected compounds were shown to be potent dual inhibitors of cysteine protease Pf FP2 and Pf FP3. Time-course parasitic development study demonstrated that compounds were able to arrest the growth of the parasite at the early trophozoite stage. The compounds did not show hemolysis of red blood cells and showed selectivity to the parasite compared with the mammalian Vero and A5489 cell lines. The study underlined HR5 and HR15 as a new class of Plasmodial falcipain inhibitors with an IC
50 of 6.2 μM and 5.9 μM for Pf FP2 and 6.8 μM and 6.4 μM for Pf FP3, respectively. Both compounds have antimalarial efficacy with IC50 values of 3.05 μM and 2.80 μM for the Pf 3D7 strain, and 4.35 μM and 3.39 μM for the Pf W2 strain, respectively. Further structural optimization may turn them into potential Plasmodial falcipain inhibitors for malaria therapeutics., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (This journal is © The Royal Society of Chemistry.)- Published
- 2024
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6. Development of diphenylmethylpiperazine hybrids of chloroquinoline and triazolopyrimidine using Petasis reaction as new cysteine proteases inhibitors for malaria therapeutics.
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Madhav H, Patel TS, Rizvi Z, Reddy GS, Rahman A, Rahman MA, Ahmedi S, Fatima S, Saxena K, Manzoor N, Bhattacharjee S, Dixit BC, Sijwali PS, and Hoda N
- Subjects
- Animals, Humans, Plasmodium falciparum, Erythrocytes, Mammals, Antimalarials chemistry, Malaria drug therapy, Cysteine Proteases
- Abstract
Malaria is a widespread infectious disease, causing nearly 247 million cases in 2021. The absence of a broadly effective vaccine and rapidly decreasing effectiveness of most of the currently used antimalarials are the major challenges to malaria eradication efforts. To design and develop novel antimalarials, we synthesized a series of 4,7-dichloroquinoline and methyltriazolopyrimidine analogues using a multi-component Petasis reaction. The synthesized molecules (11-31) were screened for in-vitro antimalarial activity against drug-sensitive and drug-resistant strains of Plasmodium falciparum with an IC
50 value of 0.53 μM. The selected compounds were screened to evaluate in-vitro and in-silico enzyme inhibition efficacy against two cysteine proteases, PfFP2 and PfFP3. The compounds 15 and 17 inhibited PfFP2 with an IC50 = 3.5 and 4.8 μM, respectively and PfFP3 with an IC50 = 4.9 and 4.7 μM, respectively. Compounds 15 and 17 were found equipotent against the Pf3D7 strain with an IC50 value of 0.74 μM, whereas both were displayed IC50 values of 1.05 μM and 1.24 μM for the PfW2 strain, respectively. Investigation of effect of compounds on parasite development demonstrated that compounds were able to arrest the growth of the parasites at trophozoite stage. The selected compounds were screened for in-vitro cytotoxicity against mammalian lines and human red-blood-cell (RBC), which demonstrated no significant cytotoxicity associated with the molecules. In addition, in silico ADME prediction and physiochemical properties supported the drug-likeness of the synthesized molecules. Thus, the results highlighted the diphenylmethylpiperazine group cast on 4,7-dichloroquinoline and methyltriazolopyrimidine using Petasis reaction may serve as models for the development of new antimalarial agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)- Published
- 2023
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7. Plasmodium DDI1 is a potential therapeutic target and important chromatin-associated protein.
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Tanneru N, Nivya MA, Adhikari N, Saxena K, Rizvi Z, Sudhakar R, Nagwani AK, Atul, Mohammed Abdul Al-Nihmi F, Kumar KA, and Sijwali PS
- Subjects
- Animals, Humans, Mice, Ubiquitin genetics, Ubiquitin metabolism, DNA Damage, DNA, Chromatin, Plasmodium falciparum genetics, Plasmodium falciparum metabolism, Protozoan Proteins genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Antimalarials, HIV Protease Inhibitors, Plasmodium genetics
- Abstract
DNA damage inducible 1 protein (DDI1) is involved in a variety of cellular processes including proteasomal degradation of specific proteins. All DDI1 proteins contain a ubiquitin-like (UBL) domain and a retroviral protease (RVP) domain. Some DDI1 proteins also contain a ubiquitin-associated (UBA) domain. The three domains confer distinct activities to DDI1 proteins. The presence of a RVP domain makes DDI1 a potential target of HIV protease inhibitors, which also block the development of malaria parasites. Hence, we investigated the DDI1 of malaria parasites to identify its roles during parasite development and potential as a therapeutic target. DDI1 proteins of Plasmodium and other apicomplexan parasites share the UBL-RVP domain architecture, and some also contain the UBA domain. Plasmodium DDI1 is expressed across all the major life cycle stages and is important for parasite survival, as conditional depletion of DDI1 protein in the mouse malaria parasite Plasmodium berghei and the human malaria parasite Plasmodium falciparum compromised parasite development. Infection of mice with DDI1 knock-down P. berghei was self-limiting and protected the recovered mice from subsequent infection with homologous as well as heterologous parasites, indicating the potential of DDI1 knock-down parasites as a whole organism vaccine. Plasmodium falciparum DDI1 (PfDDI1) is associated with chromatin and DNA-protein crosslinks. PfDDI1-depleted parasites accumulated DNA-protein crosslinks and showed enhanced susceptibility to DNA-damaging chemicals, indicating a role of PfDDI1 in removal of DNA-protein crosslinks. Knock-down of PfDDI1 increased susceptibility to the retroviral protease inhibitor lopinavir and antimalarial artemisinin, which suggests that simultaneous inhibition of DDI1 could potentiate antimalarial activity of these drugs. As DDI1 knock-down parasites confer protective immunity and it could be a target of HIV protease inhibitors, Plasmodium DDI1 is a potential therapeutic target for malaria control., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2023
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8. A comparative study of antibody response, virus neutralization efficiency & metabolites in SARS-CoV-2-infected adults & children.
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Sudhakar R, Dontamala S, Bingi TC, Gorde S, Panda A, Rizvi Z, Reddy GS, Kaswan S, Bhattacharjee M, Kumar D, Nivya MA, Mesipogu RR, Varadarajan KS, Patel AB, Gupta D, Harshan KH, Tallapaka KB, and Sijwali PS
- Subjects
- Male, Female, Animals, Chlorocebus aethiops, Humans, Child, Vero Cells, Spike Glycoprotein, Coronavirus, Antibody Formation, Antibodies, Viral, SARS-CoV-2, COVID-19
- Abstract
Background & Objectives: COVID-19 has been a global pandemic since early 2020. It has diverse clinical manifestations, but consistent immunological and metabolic correlates of disease severity and protection are not clear. This study was undertaken to compare seropositivity rate, antibody levels against nucleocapsid and spike proteins, virus neutralization and metabolites between adult and child COVID-19 patients., Methods: Plasma samples from naïve control (n=14) and reverse transcription (RT)-PCR positive COVID-19 participants (n=132) were tested for reactivity with nucleocapsid and spike proteins by ELISA, neutralization of SARS-CoV-2 infectivity in Vero cells and metabolites by
[1] H nuclear magnetic resonance (NMR) spectroscopy., Results: An ELISA platform was developed using nucleocapsid and spike proteins for COVID-19 serosurvey. The participants showed greater seropositivity for nucleocapsid (72%) than spike (55.3%), and males showed higher seropositivity than females for both the proteins. Antibody levels to both the proteins were higher in intensive care unit (ICU) than ward patients. Children showed lower seropositivity and antibody levels than adults. In contrast to ICU adults (81.3%), ICU children (33.3%) showed lower seropositivity for spike. Notably, the neutralization efficiency correlated with levels of anti-nucleocapsid antibodies. The levels of plasma metabolites were perturbed differentially in COVID-19 patients as compared with the naive controls., Interpretation & Conclusions: Our results reflect the complexity of human immune response and metabolome to SARS-CoV-2 infection. While innate and cellular immune responses are likely to be a major determinant of disease severity and protection, antibodies to multiple viral proteins likely affect COVID-19 pathogenesis. In children, not adults, lower seropositivity rate for spike was associated with disease severity.- Published
- 2022
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9. Bazedoxifene, a Postmenopausal Drug, Acts as an Antimalarial and Inhibits Hemozoin Formation.
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Sudhakar R, Adhikari N, Pamnani S, Panda A, Bhattacharjee M, Rizvi Z, Shehzad S, Gupta D, and Sijwali PS
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- Animals, Female, Heme metabolism, Heme pharmacology, Heme therapeutic use, Hemeproteins, Hemoglobins, Humans, Indoles, Male, Mice, Plasmodium falciparum, Postmenopause, Raloxifene Hydrochloride pharmacology, Raloxifene Hydrochloride therapeutic use, Selective Estrogen Receptor Modulators pharmacology, Selective Estrogen Receptor Modulators therapeutic use, Tamoxifen pharmacology, Tamoxifen therapeutic use, Antimalarials pharmacology, Antimalarials therapeutic use, Malaria parasitology, Malaria, Falciparum drug therapy, Neoplasms, Osteoporosis, Postmenopausal drug therapy
- Abstract
Despite a remarkable improvement in health care and continued drug discovery efforts, malaria control efforts are continuously challenged by the emergence of drug-resistant parasite strains. Given a long and risky development path of new drugs, repurposing existing drugs for the treatment of malaria is an attractive and shorter path. Tamoxifen, a selective estrogen receptor modulator (SERM) for the treatment and prevention of estrogen receptor-positive breast cancer, possesses antibacterial, antifungal, and antiparasitic activities. Hence, we assessed tamoxifen, raloxifene, and bazedoxifene, which represent the first-, second-, and third-generation SERMs, respectively, for antimalarial activity. Raloxifene and bazedoxifene inhibited the erythrocytic development of Plasmodium falciparum with submicromolar 50% inhibitory concentration (IC
50 ) values. Among the three, bazedoxifene was the most potent and also decreased P. berghei infection in female mice but not in male mice. However, bazedoxifene similarly inhibited P. falciparum growth in erythrocytes of male and female origin, which highlights the importance of sex-specific host physiology in drug efficacy. Bazedoxifene was most potent on early ring-stage parasites, and about 35% of the treated parasites did not contain hemozoin in the food vacuole. Bazedoxifene-treated parasites had almost 34% less hemozoin content than the control parasites. However, both control and bazedoxifene-treated parasites had similar hemoglobin levels, suggesting that bazedoxifene inhibits hemozoin formation and that toxicity due to accumulation of free heme could be a mechanism of its antimalarial activity. Because bazedoxifene is in clinical use and bazedoxifene-chloroquine combination shows an additive antiparasitic effect, bazedoxifene could be an adjunctive partner of currently used antimalarial regimens. IMPORTANCE The emergence and spread of drug-resistant strains of the human malaria parasite Plasmodium falciparum has necessitated new drugs. Selective estrogen receptor modulators are in clinical use for the prevention and treatment of breast cancer and postmenopausal osteoporosis. We demonstrate that bazedoxifene, a third-generation selective estrogen receptor modulator, has potent inhibitory activity against both susceptible and drug-resistant strains of Plasmodium falciparum. It also blocked the development of Plasmodium berghei in mice. The inhibitory effect was strongest on the ring stage and resulted in the inhibition of hemozoin formation, which could be the major mechanism of bazedoxifene action. Hemozoin is a nontoxic polymer of heme, which is a by-product of hemoglobin degradation by the malaria parasite during its development within the erythrocyte. Because bazedoxifene is already in clinical use for the treatment of postmenopausal osteoporosis, our findings support repurposing of bazedoxifene as an antimalarial.- Published
- 2022
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10. Dissecting Plasmodium yoelii Pathobiology: Proteomic Approaches for Decoding Novel Translational and Post-Translational Modifications.
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Rex DAB, Patil AH, Modi PK, Kandiyil MK, Kasaragod S, Pinto SM, Tanneru N, Sijwali PS, and Prasad TSK
- Abstract
Malaria is a vector-borne disease. It is caused by Plasmodium parasites. Plasmodium yoelii is a rodent model parasite, primarily used for studying parasite development in liver cells and vectors. To better understand parasite biology, we carried out a high-throughput-based proteomic analysis of P. yoelii . From the same mass spectrometry (MS)/MS data set, we also captured several post-translational modified peptides by following a bioinformatics analysis without any prior enrichment. Further, we carried out a proteogenomic analysis, which resulted in improvements to some of the existing gene models along with the identification of several novel genes. Analysis of proteome and post-translational modifications (PTMs) together resulted in the identification of 3124 proteins. The identified PTMs were found to be enriched in mitochondrial metabolic pathways. Subsequent bioinformatics analysis provided an insight into proteins associated with metabolic regulatory mechanisms. Among these, the tricarboxylic acid (TCA) cycle and the isoprenoid synthesis pathway are found to be essential for parasite survival and drug resistance. The proteogenomic analysis discovered 43 novel protein-coding genes. The availability of an in-depth proteomic landscape of a malaria pathogen model will likely facilitate further molecular-level investigations on pre-erythrocytic stages of malaria., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
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11. Plasmodium falciparum Atg18 localizes to the food vacuole via interaction with the multi-drug resistance protein 1 and phosphatidylinositol 3-phosphate.
- Author
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Sudhakar R, Das D, Thanumalayan S, Gorde S, and Sijwali PS
- Subjects
- Amodiaquine pharmacology, Animals, Antimalarials pharmacology, Autophagy genetics, Autophagy-Related Proteins metabolism, Biological Transport, Chloroquine pharmacology, Erythrocytes drug effects, Erythrocytes parasitology, Gene Expression Regulation, Humans, Malaria parasitology, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Inbred BALB C, Multidrug Resistance-Associated Proteins metabolism, Plasmodium berghei growth & development, Plasmodium berghei metabolism, Plasmodium falciparum growth & development, Plasmodium falciparum metabolism, Protein Binding, Protozoan Proteins metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Vacuoles drug effects, Autophagy-Related Proteins genetics, Multidrug Resistance-Associated Proteins genetics, Phosphatidylinositol Phosphates metabolism, Plasmodium berghei genetics, Plasmodium falciparum genetics, Protozoan Proteins genetics, Vacuoles metabolism
- Abstract
Autophagy, a lysosome-dependent degradative process, does not appear to be a major degradative process in malaria parasites and has a limited repertoire of genes. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 bind PI3P and play an essential role in autophagosome formation. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is likely a shared feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, wild type ScAtg18 interacted with PI3P, but its expression in P. falciparum showed complete cytoplasmic localization, indicating additional requirement for food vacuole localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the immunoprecipitates of PfAtg18 and P. berghei Atg18, and also interacted with PfAtg18. In contrast with PfAtg18, ScAtg18 did not interact with MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine caused cytoplasmic localization of PfAtg18, suggesting that these target PfAtg18 transport pathway. Thus, PI3P and MDR1 are critical mediators of PfAtg18 localization., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2021
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12. Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates.
- Author
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Bhattacharjee M, Adhikari N, Sudhakar R, Rizvi Z, Das D, Palanimurugan R, and Sijwali PS
- Subjects
- Cullin Proteins genetics, Databases, Genetic, NEDD8 Protein genetics, Plasmodium falciparum genetics, Protozoan Proteins genetics, Cullin Proteins metabolism, NEDD8 Protein metabolism, Plasmodium falciparum metabolism, Protozoan Proteins metabolism
- Abstract
A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles during parasite development. NEDD8 is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulates diverse cellular processes. Although neddylation is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. We characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Ala/Gly76Ala) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 (rub1Δ) or NEDD8 conjugating E2 enzyme (ubc12Δ). The PfNEDD8 immunoprecipitate also contained S. cerevisiae cullin cdc53, further substantiating cullins as physiological substrates of PfNEDD8. Our findings lay ground for investigation of specific roles and drug target potential of neddylation in malaria parasites.
- Published
- 2020
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13. Synthesis and efficacy of pyrvinium-inspired analogs against tuberculosis and malaria pathogens.
- Author
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Gaikwad VR, Karale UB, Govindarajalu G, Adhikari N, Krishna EV, Krishna VS, Misra S, Sriram D, Sijwali PS, and Rode HB
- Subjects
- Antimalarials chemical synthesis, Antimalarials chemistry, Antitubercular Agents chemical synthesis, Antitubercular Agents chemistry, Dose-Response Relationship, Drug, Malaria, Falciparum drug therapy, Malaria, Falciparum parasitology, Microbial Sensitivity Tests, Molecular Structure, Parasitic Sensitivity Tests, Pyrvinium Compounds chemical synthesis, Pyrvinium Compounds chemistry, Structure-Activity Relationship, Tuberculosis drug therapy, Tuberculosis microbiology, Antimalarials pharmacology, Antitubercular Agents pharmacology, Mycobacterium tuberculosis drug effects, Plasmodium falciparum drug effects, Pyrvinium Compounds pharmacology
- Abstract
Herein, we report the synthesis and evaluation of pyrvinium-based antimalarial and antitubercular compounds. Pyrvinium is an FDA approved drug for the treatment of pinworm infection, and it has been reported to have antiparasitic and antimicrobial activities. Pyrvinium contains quinoline core coupled with pyrrole. We replaced the pyrrole with various aryl or heteroaryl substituents to generate pyrvinium analogs. The profiling of these compounds against malaria parasite P. falciparum 3D7 revealed analogs with better antimalarial activity than pyrvinium pamoate. Compound 14 and 16 showed IC
50 of 23 nM and 60 nM against P. falciparum 3D7, respectively. These compounds were also effective against drug-resistant malaria parasite P. falciparum Dd2 with IC50 of 53 nM and 97 nM, respectively. The cytotoxicity against CHO-K1, HEK and NRK-49F cells revealed better selectivity index for these new analogs compared to pyrvinium. Additionally, this series of compounds showed activity against M. tuberculosis H37Rv; particularly compounds 10, 13, 14 and 16 showed equipotent antitubercular activity to that of pyrvinium pamoate. The compounds 14 and 16 should be taken forward as leads for further optimization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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14. Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function.
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Bhowmick K, Tehlan A, Sunita, Sudhakar R, Kaur I, Sijwali PS, Krishnamachari A, and Dhar SK
- Subjects
- Animals, Humans, Plasmodium falciparum pathogenicity, Protozoan Proteins metabolism, p300-CBP Transcription Factors metabolism
- Abstract
The pathogenesis of human malarial parasite Plasmodium falciparum is interlinked with its timely control of gene expression during its complex life cycle. In this organism, gene expression is partially controlled through epigenetic mechanisms, the regulation of which is, hence, of paramount importance to the parasite. The P. falciparum (Pf)-GCN5 histone acetyltransferase (HAT), an essential enzyme, acetylates histone 3 and regulates global gene expression in the parasite. Here, we show the existence of a novel proteolytic processing for PfGCN5 that is crucial for its activity in vivo We find that a cysteine protease-like enzyme is required for the processing of PfGCN5 protein. Immunofluorescence and immuno-electron microscopy analysis suggest that the processing event occurs in the vicinity of the digestive vacuole of the parasite following its trafficking through the classical ER-Golgi secretory pathway, before it subsequently reaches the nucleus. Furthermore, blocking of PfGCN5 processing leads to the concomitant reduction of its occupancy at the gene promoters and a reduced H3K9 acetylation level at these promoters, highlighting the important correlation between the processing event and PfGCN5 activity. Altogether, our study reveals a unique processing event for a nuclear protein PfGCN5 with unforeseen role of a food vacuolar cysteine protease. This leads to a possibility of the development of new antimalarials against these targets.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)
- Published
- 2020
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15. Identification of antimalarial leads with dual falcipain-2 and falcipain-3 inhibitory activity.
- Author
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Rana D, Kalamuddin M, Sundriyal S, Jaiswal V, Sharma G, Das Sarma K, Sijwali PS, Mohmmed A, Malhotra P, and Mahindroo N
- Subjects
- Antimalarials chemistry, Dose-Response Relationship, Drug, Enzyme Inhibitors chemistry, Molecular Docking Simulation, Molecular Structure, Parasitic Sensitivity Tests, Plasmodium falciparum enzymology, Structure-Activity Relationship, Antimalarials pharmacology, Cysteine Endopeptidases metabolism, Enzyme Inhibitors pharmacology, Plasmodium falciparum drug effects
- Abstract
Falcipains (FPs), cysteine proteases in the malarial parasite, are emerging as the promising antimalarial drug targets. In order to identify novel FP inhibitors, we generated a pharmacophore derived from the reported co-crystal structures of inhibitors of Plasmodium falciparum Falcipain-3 to screen the ZINC library. Further, the filters were applied for dock score, drug-like characters, and clustering of similar structures. Sixteen molecules were purchased and subject to in vitro enzyme (FP-2 and FP-3) inhibition assays. Two compounds showed in vitro inhibition of FP-2 and FP-3 at low µM concentration. The selectivity of the inhibitors can be explained based on the predicted interactions of the molecule in the active site. Further, the inhibitors were evaluated in a functional assay and were found to induce morphological changes in line with their mode of action arresting Plasmodium development. Compound 15 was most potent inhibitor identified in this study., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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16. Lyse-Reseal Erythrocytes for Transfection of Plasmodium falciparum.
- Author
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Govindarajalu G, Rizvi Z, Kumar D, and Sijwali PS
- Subjects
- DNA genetics, Electroporation economics, Electroporation methods, Erythrocytes parasitology, Gene Transfer Techniques, Humans, Malaria, Falciparum parasitology, Plasmids genetics, Transfection economics, Erythrocytes metabolism, Plasmodium falciparum genetics, Transfection methods
- Abstract
Simple and efficient transfection methods for genetic manipulation of Plasmodium falciparum are desirable to identify, characterize and validate the genes with therapeutic potential and better understand parasite biology. Among the available transfection techniques for P. falciparum, electroporation-based methods, particularly electroporation of ring-infected RBCs is routinely used. Nonetheless, transfection of P. falciparum remains a resource-intensive procedure. Here, we report a simple and economic transfection method for P. falciparum, which is termed as the lyse-reseal erythrocytes for transfection (LyRET). It involved lysis of erythrocytes with a hypotonic RBC lysis buffer containing the desired plasmid DNA, followed by resealing by adding a high salt buffer. These DNA-encapsulated lyse-reseal erythrocytes were mixed with P. falciparum trophozoite/schizont stages and subjected to selection for the plasmid-encoded drug resistance. In parallel, transfections were also done by the methods utilizing electroporation of DNA into uninfected RBCs and parasite-infected RBCs. The LyRET method successfully transfected 3D7 and D10 strains with different plasmids in 63 of the 65 attempts, with success rate similar to transfection by electroporation of DNA into infected RBCs. The cost effectiveness and comparable efficiency of LyRET method makes it an alternative to the existing transfection methods for P. falciparum, particularly in resource-limited settings.
- Published
- 2019
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17. Interaction with complement proteins and dendritic cells implicates LCCL domain-containing proteins (CCps) of malaria parasites in immunomodulation.
- Author
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Sijwali PS
- Subjects
- Animals, Carrier Proteins, Complement System Proteins, Dendritic Cells, Humans, Immunomodulation, Plasmodium falciparum immunology, Malaria, Parasites, Plasmodium
- Abstract
The evasion of host immune defense is critical for pathogens to invade, establish infection and perpetuate in the host. The complement system is one of the first lines of innate immune defense in humans that destroys pathogens in the blood circulation. Activation of the complement system through direct encounter with pathogens or some other agents leads to osmolysis of pathogens, clearance of soluble immune complexes and recruitment of lymphocytes at the site of activation. Although malaria parasites are not exposed to the complement system owing to their intracellular development for most part of their life cycle in the human host, the extracellular stages must face the complement system of human or mosquito or both. In a recent issue of the Biochemical Journal , Sharma et al. reported that P lasmodium falciparum LCCL domain-containing protein 1 (PfCCp1) inhibited activation of the classical complement pathway and down-regulated effector responses of dendritic cells, which implicate PfCCp1 and related proteins in immunomodulation of the host that likely benefits the parasite. PfCCp1 belongs to a multi-domain protein family that exists as multimeric protein complexes. It needs to be investigated whether PfCCp1 or its multimeric protein complexes have an immunomodulatory effect in vivo and on the mosquito complement system., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2018
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18. Biotransformation of Artemisinin to 14-Hydroxydeoxyartemisinin: C-14 Hydroxylation by Aspergillus flavus.
- Author
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Ponnapalli MG, Sura MB, Sudhakar R, Govindarajalu G, and Sijwali PS
- Subjects
- Antimalarials chemistry, Antimalarials metabolism, Biotransformation, Hydroxylation, Magnetic Resonance Spectroscopy, Molecular Structure, Artemisinins chemistry, Artemisinins metabolism, Aspergillus flavus metabolism
- Abstract
The biotransformation of the front-line antimalarial drug, artemisinin (1) by the filamentous fungus Aspergillus flavus MTCC-9167 was investigated. Incubation of compound 1 with A. flavus afforded a new hydroxy derivative (2) along with three known metabolites (3-5). The new compound was characterized as 14-hydroxydeoxyartemisinin (2) by extensive spectroscopic data analysis (IR,
1 H and13 C NMR, HSQC, HMBC, COSY, NOESY, and HR-ESIMS). The known metabolites were identified as deoxyartemisinin (3), artemisinin G (4), and 4α-hydroxydeoxyartemisinin (5). This is the first report of hydroxylation of a secondary methyl of artemisinin at C-14 by the fungus A. flavus, which is synthetically not accessible. In addition, these compounds were evaluated for their in vitro antiplasmodial activity. Artemisinin G (4) exhibited IC50 values in the submicromolar range, which was better than those of the nonperoxidic metabolites.- Published
- 2018
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19. A conserved human DJ1-subfamily motif (DJSM) is critical for anti-oxidative and deglycase activities of Plasmodium falciparum DJ1.
- Author
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Nair DN, Prasad R, Singhal N, Bhattacharjee M, Sudhakar R, Singh P, Thanumalayan S, Kiran U, Sharma Y, and Sijwali PS
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Catalysis, Conserved Sequence, Humans, Plasmodium falciparum chemistry, Plasmodium falciparum genetics, Protein Deglycase DJ-1 genetics, Protozoan Proteins genetics, Sequence Alignment, Oxidative Stress, Plasmodium falciparum enzymology, Plasmodium falciparum metabolism, Protein Deglycase DJ-1 chemistry, Protein Deglycase DJ-1 metabolism, Protozoan Proteins chemistry, Protozoan Proteins metabolism
- Abstract
Plasmodium falciparum DJ1 (PfDJ1) belongs to the DJ-1/ThiJ/PfpI superfamily whose members are present in all the kingdoms of life and exhibit diverse cellular functions and biochemical activities. The common feature of the superfamily is the class I glutamine amidotransferase domain with a conserved redox-active cysteine residue, which mediates various activities of the superfamily members, including anti-oxidative activity in PfDJ1 and human DJ1 (hDJ1). As the superfamily members represent diverse functional classes, to investigate if there is any sequence feature unique to hDJ1-like proteins, sequences of the representative proteins of different functional classes were compared and analysed. A novel motif unique to PfDJ1 and several other hDJ1-like proteins, with the consensus sequence of TSXGPX5FXLX5L, was identified that we designated as the hDJ1-subfamily motif (DJSM). Several mutations that have been associated with Parkinson's disease are also present in DJSM, suggesting its functional importance in hDJ1-like proteins. Mutations of the conserved residues of DJSM of PfDJ1 did not significantly affect overall secondary structure, but caused both a significant loss (S151A and P154A) and gain (L168A) of anti-oxidative activity. We also report that PfDJ1 has deglycase activity, which was significantly decreased in its mutants of the catalytic cysteine (C106A) and DJSM (S151A and P154A). Episomal expression of the catalytic cysteine (C106A) or DJSM (P154A) mutant decreased growth rates of parasites as compared to that of wild type parasites or parasites expressing wild type PfDJ1. S151 appears to properly position the nucleophilic elbow containing C106 and P154 forms a hydrogen bond with C106, which could be a reason for the loss of activities of PfDJ1 upon their mutations. Taken together, DJSM delineates PfDJ1 and other hDJ1-subfamily proteins from the remaining superfamily, and is critical for anti-oxidative and deglycase activities of PfDJ1., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
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20. Synthesis and evaluation of anti-plasmodial and cytotoxic activities of epoxyazadiradione derivatives.
- Author
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Ashok Yadav P, Pavan Kumar C, Siva B, Suresh Babu K, Allanki AD, Sijwali PS, Jain N, and Veerabhadra Rao A
- Subjects
- Animals, Antimalarials chemical synthesis, Antimalarials isolation & purification, Antineoplastic Agents chemical synthesis, Antineoplastic Agents isolation & purification, Cell Line, Tumor, Cell Proliferation drug effects, Humans, Limonins chemical synthesis, Limonins isolation & purification, Malaria drug therapy, Malaria, Falciparum drug therapy, Mice, Mice, Inbred BALB C, Neoplasms drug therapy, Plasmodium berghei drug effects, Plasmodium falciparum drug effects, Antimalarials chemistry, Antimalarials pharmacology, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Azadirachta chemistry, Limonins chemistry, Limonins pharmacology
- Abstract
Epoxyazadiradione (1), a major compound derived from Neem oil, showed modest anti-plasmodial activity against CQ-resistant and CQ-sensitive strains of the most virulent human malaria parasite P. falciparum. A series of analogues were synthesized by modification of the key structural moieties of this high yield natural product. Out of the library of all compounds tested, compounds 3c and 3g have showed modest anti-plasmodial activity against CQ-sensitive (IC
50 2.8 ± 0.29 μM and 1.5 ± 0.01 μM) and CQ-resistant strains (IC50 1.3 ± 1.08 μM and 1.2 ± 0.14), while compounds 3k, 3l and 3m showed modest activity against CQ-sensitive strain of P. falciparum with IC50 values of 2.3 ± 0.4 μM, 2.9 ± 0.1 μM and 1.7 ± 0.06 μM, respectively. Additionally, cytotoxic properties of these derivatives against SIHA, PANC 1, MDA-MB-231, and IMR-3 cancer cell lines were also studied and the results indicated that low cytotoxic potentials of all the derivatives which indicating the high selectivity index of the compounds., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)- Published
- 2017
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21. A Novel and Conserved Plasmodium Sporozoite Membrane Protein SPELD is Required for Maturation of Exo-erythrocytic Forms.
- Author
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Al-Nihmi FM, Kolli SK, Reddy SR, Mastan BS, Togiri J, Maruthi M, Gupta R, Sijwali PS, Mishra S, and Kumar KA
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Anopheles parasitology, Erythrocytes metabolism, Female, Gene Dosage, Gene Expression Regulation, Green Fluorescent Proteins, Hep G2 Cells, Humans, Immunity, Immunization, Life Cycle Stages, Liver parasitology, Malaria immunology, Malaria parasitology, Malaria transmission, Membrane Proteins chemistry, Membrane Proteins genetics, Mice, Inbred C57BL, Phenotype, Protein Domains, Protozoan Proteins chemistry, Protozoan Proteins genetics, Protozoan Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Salivary Glands metabolism, Species Specificity, Sporozoites growth & development, Conserved Sequence, Erythrocytes parasitology, Membrane Proteins metabolism, Plasmodium berghei growth & development, Plasmodium berghei metabolism, Sporozoites metabolism
- Abstract
Plasmodium sporozoites are the infective forms of malaria parasite to vertebrate host and undergo dramatic changes in their transcriptional repertoire during maturation in mosquito salivary glands. We report here the role of a novel and conserved Plasmodium berghei protein encoded by PBANKA_091090 in maturation of Exo-erythrocytic Forms (EEFs) and designate it as Sporozoite surface Protein Essential for Liver stage Development (PbSPELD). PBANKA_091090 was previously annotated as PB402615.00.0 and its transcript was recovered at maximal frequency in the Serial Analysis of the Gene Expression (SAGE) of Plasmodium berghei salivary gland sporozoites. An orthologue of this transcript was independently identified in Plasmodium vivax sporozoite microarrays and was designated as Sporozoite Conserved Orthologous Transcript-2 (scot-2). Functional characterization through reverse genetics revealed that PbSPELD is essential for Plasmodium liver stage maturation. mCherry transgenic of PbSPELD localized the protein to plasma membrane of sporozoites and early EEFs. Global microarray analysis of pbspeld ko revealed EEF attenuation being associated with down regulation of genes central to general transcription, cell cycle, proteosome and cadherin signaling. pbspeld mutant EEFs induced pre-erythrocytic immunity with 50% protective efficacy. Our studies have implications for attenuating the human Plasmodium liver stages by targeting SPELD locus.
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- 2017
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22. Synthesis and evaluation of naphthyl bearing 1,2,3-triazole analogs as antiplasmodial agents, cytotoxicity and docking studies.
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Balabadra S, Kotni M, Manga V, Allanki AD, Prasad R, and Sijwali PS
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- Antiprotozoal Agents chemical synthesis, Antiprotozoal Agents chemistry, Antiprotozoal Agents toxicity, Crystallography, X-Ray, Enzyme Assays, Folic Acid Antagonists chemical synthesis, Folic Acid Antagonists chemistry, Folic Acid Antagonists toxicity, HEK293 Cells, Humans, Molecular Docking Simulation, NADP metabolism, Naphthalenes chemical synthesis, Naphthalenes chemistry, Naphthalenes toxicity, Plasmodium falciparum drug effects, Pyrimethamine chemistry, Triazoles chemical synthesis, Triazoles chemistry, Triazoles toxicity, Antiprotozoal Agents pharmacology, Folic Acid Antagonists pharmacology, Naphthalenes pharmacology, Triazoles pharmacology
- Abstract
Novel series of naphthyl bearing 1,2,3-triazoles (4a-t) were synthesized and evaluated for their in vitro antiplasmodial activity against pyrimethamine (Pyr)-sensitive and resistant strains of Plasmodium falciparum. The synthesized compounds were assessed for their cytotoxicity employing human embryonic kidney cell line (HEK-293), and none of them was found to be toxic. Among them 4j, 4k, 4l, 4m, 4n, 4t exhibited significant antiplasmodial activity in both strains, of which compounds 4m, 4n and 4t (∼3.0-fold) displayed superior activity to Pyr against resistant strain. Pyr and selected compounds (4n, 4p and 4t) that repressed parasite development also inhibited PfDHFR activity of the soluble parasite extract, suggesting that anti-parasitic activity of these compounds is a result of inhibition of the parasite DHFR. In silico studies suggest that activity of these compounds might be enhanced due to π-π stacking., (Copyright © 2016 Elsevier Ltd. All rights reserved.)
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- 2017
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23. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
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Mostowy S, Motori E, Mottet D, Mottram JC, Moussa CE, Mpakou VE, Mukhtar H, Mulcahy Levy JM, Muller S, Muñoz-Moreno R, Muñoz-Pinedo C, Münz C, Murphy ME, Murray JT, Murthy A, Mysorekar IU, Nabi IR, Nabissi M, Nader GA, Nagahara Y, Nagai Y, Nagata K, Nagelkerke A, Nagy P, Naidu SR, Nair S, Nakano H, Nakatogawa H, Nanjundan M, Napolitano G, Naqvi NI, Nardacci R, Narendra DP, Narita M, Nascimbeni AC, Natarajan R, Navegantes LC, Nawrocki ST, Nazarko TY, Nazarko VY, Neill T, Neri LM, Netea MG, Netea-Maier RT, Neves BM, Ney PA, Nezis IP, Nguyen HT, Nguyen HP, Nicot AS, Nilsen H, Nilsson P, Nishimura M, Nishino I, Niso-Santano M, Niu H, Nixon RA, Njar VC, Noda T, Noegel AA, Nolte EM, Norberg E, Norga KK, Noureini SK, Notomi S, Notterpek L, Nowikovsky K, Nukina N, Nürnberger T, O'Donnell VB, O'Donovan T, O'Dwyer PJ, Oehme I, Oeste CL, Ogawa M, Ogretmen B, Ogura Y, Oh YJ, Ohmuraya M, Ohshima T, Ojha R, Okamoto K, Okazaki T, Oliver FJ, Ollinger K, Olsson S, Orban DP, Ordonez P, Orhon I, Orosz L, 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Pessin JE, Peters GJ, Petersen M, Petrache I, Petrof BJ, Petrovski G, Phang JM, Piacentini M, Pierdominici M, Pierre P, Pierrefite-Carle V, Pietrocola F, Pimentel-Muiños FX, Pinar M, Pineda B, Pinkas-Kramarski R, Pinti M, Pinton P, Piperdi B, Piret JM, Platanias LC, Platta HW, Plowey ED, Pöggeler S, Poirot M, Polčic P, Poletti A, Poon AH, Popelka H, Popova B, Poprawa I, Poulose SM, Poulton J, Powers SK, Powers T, Pozuelo-Rubio M, Prak K, Prange R, Prescott M, Priault M, Prince S, Proia RL, Proikas-Cezanne T, Prokisch H, Promponas VJ, Przyklenk K, Puertollano R, Pugazhenthi S, Puglielli L, Pujol A, Puyal J, Pyeon D, Qi X, Qian WB, Qin ZH, Qiu Y, Qu Z, Quadrilatero J, Quinn F, Raben N, Rabinowich H, Radogna F, Ragusa MJ, Rahmani M, Raina K, Ramanadham S, Ramesh R, Rami A, Randall-Demllo S, Randow F, Rao H, Rao VA, Rasmussen BB, Rasse TM, Ratovitski EA, Rautou PE, Ray SK, Razani B, Reed BH, Reggiori F, Rehm M, Reichert AS, Rein T, Reiner DJ, Reits E, Ren J, Ren X, Renna M, Reusch JE, Revuelta JL, Reyes L, Rezaie AR, Richards RI, Richardson DR, Richetta C, Riehle MA, Rihn BH, Rikihisa Y, Riley BE, Rimbach G, Rippo MR, Ritis K, Rizzi F, Rizzo E, Roach PJ, Robbins J, Roberge M, Roca G, Roccheri MC, Rocha S, Rodrigues CMP, Rodríguez CI, de Cordoba SR, Rodriguez-Muela N, Roelofs J, Rogov VV, Rohn TT, Rohrer B, Romanelli D, Romani L, Romano PS, Roncero MI, Rosa JL, Rosello A, Rosen KV, Rosenstiel P, Rost-Roszkowska M, Roth KA, Roué G, Rouis M, Rouschop KM, Ruan DT, Ruano D, Rubinsztein DC, Rucker EB 3rd, Rudich A, Rudolf E, Rudolf R, Ruegg MA, Ruiz-Roldan C, Ruparelia AA, Rusmini P, Russ DW, Russo GL, Russo G, Russo R, Rusten TE, Ryabovol V, Ryan KM, Ryter SW, Sabatini DM, Sacher M, Sachse C, Sack MN, Sadoshima J, Saftig P, Sagi-Eisenberg R, Sahni S, Saikumar P, Saito T, Saitoh T, Sakakura K, Sakoh-Nakatogawa M, Sakuraba Y, Salazar-Roa M, Salomoni P, Saluja AK, Salvaterra PM, Salvioli R, Samali A, Sanchez AM, Sánchez-Alcázar JA, Sanchez-Prieto R, Sandri M, Sanjuan MA, Santaguida S, Santambrogio L, Santoni G, Dos Santos CN, Saran S, Sardiello M, Sargent G, Sarkar P, Sarkar S, Sarrias MR, Sarwal MM, Sasakawa C, Sasaki M, Sass M, Sato K, Sato M, Satriano J, Savaraj N, Saveljeva S, Schaefer L, Schaible UE, Scharl M, Schatzl HM, Schekman R, Scheper W, Schiavi A, Schipper HM, Schmeisser H, Schmidt J, Schmitz I, Schneider BE, Schneider EM, Schneider JL, Schon EA, Schönenberger MJ, Schönthal AH, Schorderet DF, Schröder B, Schuck S, Schulze RJ, Schwarten M, Schwarz TL, Sciarretta S, Scotto K, Scovassi AI, Screaton RA, Screen M, Seca H, Sedej S, Segatori L, Segev N, Seglen PO, Seguí-Simarro JM, Segura-Aguilar J, Seki E, Sell C, Seiliez I, Semenkovich CF, Semenza GL, Sen U, Serra AL, Serrano-Puebla A, Sesaki H, Setoguchi T, Settembre C, Shacka JJ, Shajahan-Haq AN, Shapiro IM, Sharma S, She H, Shen CK, Shen CC, Shen HM, Shen S, Shen W, Sheng R, Sheng X, Sheng ZH, Shepherd TG, Shi J, Shi Q, Shi Q, Shi Y, Shibutani S, Shibuya K, Shidoji Y, Shieh JJ, Shih CM, Shimada Y, Shimizu S, Shin DW, Shinohara ML, Shintani M, Shintani T, Shioi T, Shirabe K, Shiri-Sverdlov R, Shirihai O, Shore GC, Shu CW, Shukla D, Sibirny AA, Sica V, Sigurdson CJ, Sigurdsson EM, Sijwali PS, Sikorska B, Silveira WA, Silvente-Poirot S, Silverman GA, Simak J, Simmet T, Simon AK, Simon HU, Simone C, Simons M, Simonsen A, Singh R, Singh SV, Singh SK, Sinha D, Sinha S, Sinicrope FA, Sirko A, Sirohi K, Sishi BJ, Sittler A, Siu PM, Sivridis E, Skwarska A, Slack R, Slaninová I, Slavov N, Smaili SS, Smalley KS, Smith DR, Soenen SJ, Soleimanpour SA, Solhaug A, Somasundaram K, Son JH, Sonawane A, Song C, Song F, Song HK, Song JX, Song W, Soo KY, Sood AK, Soong TW, Soontornniyomkij V, Sorice M, Sotgia F, Soto-Pantoja DR, Sotthibundhu A, Sousa MJ, Spaink HP, Span PN, Spang A, Sparks JD, Speck PG, Spector SA, Spies CD, Springer W, Clair DS, Stacchiotti A, Staels B, Stang MT, Starczynowski DT, Starokadomskyy P, Steegborn C, Steele JW, Stefanis L, Steffan J, Stellrecht CM, Stenmark H, Stepkowski TM, Stern ST, Stevens C, Stockwell BR, Stoka V, Storchova Z, Stork B, Stratoulias V, Stravopodis DJ, Strnad P, Strohecker AM, Ström AL, Stromhaug P, Stulik J, Su YX, Su Z, Subauste CS, Subramaniam S, Sue CM, Suh SW, Sui X, Sukseree S, Sulzer D, Sun FL, Sun J, Sun J, Sun SY, Sun Y, Sun Y, Sun Y, Sundaramoorthy V, Sung J, Suzuki H, Suzuki K, Suzuki N, Suzuki T, Suzuki YJ, Swanson MS, Swanton C, Swärd K, Swarup G, Sweeney ST, Sylvester PW, Szatmari Z, Szegezdi E, Szlosarek PW, Taegtmeyer H, Tafani M, Taillebourg E, Tait SW, Takacs-Vellai K, Takahashi Y, Takáts S, Takemura G, Takigawa N, Talbot NJ, Tamagno E, Tamburini J, Tan CP, Tan L, Tan ML, Tan M, Tan YJ, Tanaka K, Tanaka M, Tang D, Tang D, Tang G, Tanida I, Tanji K, Tannous BA, Tapia JA, Tasset-Cuevas I, Tatar M, Tavassoly I, Tavernarakis N, Taylor A, Taylor GS, Taylor GA, Taylor JP, Taylor MJ, Tchetina EV, Tee AR, Teixeira-Clerc F, Telang S, Tencomnao T, Teng BB, Teng RJ, Terro F, Tettamanti G, Theiss AL, Theron AE, Thomas KJ, Thomé MP, Thomes PG, Thorburn A, Thorner J, Thum T, Thumm M, Thurston TL, Tian L, Till A, Ting JP, Titorenko VI, Toker L, Toldo S, Tooze SA, Topisirovic I, Torgersen ML, Torosantucci L, Torriglia A, Torrisi MR, Tournier C, Towns R, Trajkovic V, Travassos LH, Triola G, Tripathi DN, Trisciuoglio D, Troncoso R, Trougakos IP, Truttmann AC, Tsai KJ, Tschan MP, Tseng YH, Tsukuba T, Tsung A, Tsvetkov AS, Tu S, Tuan HY, Tucci M, Tumbarello DA, Turk B, Turk V, Turner RF, Tveita AA, Tyagi SC, Ubukata M, Uchiyama Y, Udelnow A, Ueno T, Umekawa M, Umemiya-Shirafuji R, Underwood BR, Ungermann C, Ureshino RP, Ushioda R, Uversky VN, Uzcátegui NL, Vaccari T, Vaccaro MI, Váchová L, Vakifahmetoglu-Norberg H, Valdor R, Valente EM, Vallette F, Valverde AM, Van den Berghe G, Van Den Bosch L, van den Brink GR, van der Goot FG, van der Klei IJ, van der Laan LJ, van Doorn WG, van Egmond M, van Golen KL, Van Kaer L, van Lookeren Campagne M, Vandenabeele P, Vandenberghe W, Vanhorebeek I, Varela-Nieto I, Vasconcelos MH, Vasko R, Vavvas DG, Vega-Naredo I, Velasco G, Velentzas AD, Velentzas PD, Vellai T, Vellenga E, Vendelbo MH, Venkatachalam K, Ventura N, Ventura S, Veras PS, Verdier M, Vertessy BG, Viale A, Vidal M, Vieira HL, Vierstra RD, Vigneswaran N, Vij N, Vila M, Villar M, Villar VH, Villarroya J, Vindis C, Viola G, Viscomi MT, Vitale G, Vogl DT, Voitsekhovskaja OV, von Haefen C, von Schwarzenberg K, Voth DE, Vouret-Craviari V, Vuori K, Vyas JM, Waeber C, Walker CL, Walker MJ, Walter J, Wan L, Wan X, Wang B, Wang C, Wang CY, Wang C, Wang C, Wang C, Wang D, Wang F, Wang F, Wang G, Wang HJ, Wang H, Wang HG, Wang H, Wang HD, Wang J, Wang J, Wang M, Wang MQ, Wang PY, Wang P, Wang RC, Wang S, Wang TF, Wang X, Wang XJ, Wang XW, Wang X, Wang X, Wang Y, Wang Y, Wang Y, Wang YJ, Wang Y, Wang Y, Wang YT, Wang Y, Wang ZN, Wappner P, Ward C, Ward DM, Warnes G, Watada H, Watanabe Y, Watase K, Weaver TE, Weekes CD, Wei J, Weide T, Weihl CC, Weindl G, Weis SN, Wen L, Wen X, Wen Y, Westermann B, Weyand CM, White AR, White E, Whitton JL, Whitworth AJ, Wiels J, Wild F, Wildenberg ME, Wileman T, Wilkinson DS, Wilkinson S, Willbold D, Williams C, Williams K, Williamson PR, Winklhofer KF, Witkin SS, Wohlgemuth SE, Wollert T, Wolvetang EJ, Wong E, Wong GW, Wong RW, Wong VK, Woodcock EA, Wright KL, Wu C, Wu D, Wu GS, Wu J, Wu J, Wu M, Wu M, Wu S, Wu WK, Wu Y, Wu Z, Xavier CP, Xavier RJ, Xia GX, Xia T, Xia W, Xia Y, Xiao H, Xiao J, Xiao S, Xiao W, Xie CM, Xie Z, Xie Z, Xilouri M, Xiong Y, Xu C, Xu C, Xu F, Xu H, Xu H, Xu J, Xu J, Xu J, Xu L, Xu X, Xu Y, Xu Y, Xu ZX, Xu Z, Xue Y, Yamada T, Yamamoto A, Yamanaka K, Yamashina S, Yamashiro S, Yan B, Yan B, Yan X, Yan Z, Yanagi Y, Yang DS, Yang JM, Yang L, Yang M, Yang PM, Yang P, Yang Q, Yang W, Yang WY, Yang X, Yang Y, Yang Y, Yang Z, Yang Z, Yao MC, Yao PJ, Yao X, Yao Z, Yao Z, Yasui LS, Ye M, Yedvobnick B, Yeganeh B, Yeh ES, Yeyati PL, Yi F, Yi L, Yin XM, Yip CK, Yoo YM, Yoo YH, Yoon SY, Yoshida K, Yoshimori T, Young KH, Yu H, Yu JJ, Yu JT, Yu J, Yu L, Yu WH, Yu XF, Yu Z, Yuan J, Yuan ZM, Yue BY, Yue J, Yue Z, Zacks DN, Zacksenhaus E, Zaffaroni N, Zaglia T, Zakeri Z, Zecchini V, Zeng J, Zeng M, Zeng Q, Zervos AS, Zhang DD, Zhang F, Zhang G, Zhang GC, Zhang H, Zhang H, Zhang H, Zhang H, Zhang J, Zhang J, Zhang J, Zhang J, Zhang JP, Zhang L, Zhang L, Zhang L, Zhang L, Zhang MY, Zhang X, Zhang XD, Zhang Y, Zhang Y, Zhang Y, Zhang Y, Zhang Y, Zhao M, Zhao WL, Zhao X, Zhao YG, Zhao Y, Zhao Y, Zhao YX, Zhao Z, Zhao ZJ, Zheng D, Zheng XL, Zheng X, Zhivotovsky B, Zhong Q, Zhou GZ, Zhou G, Zhou H, Zhou SF, Zhou XJ, Zhu H, Zhu H, Zhu WG, Zhu W, Zhu XF, Zhu Y, Zhuang SM, Zhuang X, Ziparo E, Zois CE, Zoladek T, Zong WX, Zorzano A, and Zughaier SM
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- Animals, Biological Assay methods, Computer Simulation, Humans, Autophagy physiology, Biological Assay standards
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- 2016
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24. Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates.
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Kolla VK, Prasad R, Sayyad Z, Atul, Shah AY, Allanki AD, Navale R, Singhal N, Tanneru N, Sudhakar R, Venkatesan V, Deshmukh MV, and Sijwali PS
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- Amino Acid Sequence, Catalytic Domain, Cathepsin L chemistry, Cysteine Endopeptidases genetics, Hemoglobins metabolism, Humans, Hydrogen-Ion Concentration, Hydrolysis, Leucine chemistry, Leucine genetics, Malaria, Falciparum parasitology, Molecular Sequence Data, Mutagenesis, Site-Directed, Peptides chemistry, Protozoan Proteins genetics, Substrate Specificity, Coumarins metabolism, Cysteine Endopeptidases chemistry, Dipeptides metabolism, Plasmodium falciparum enzymology, Protozoan Proteins chemistry
- Abstract
Falcipain-3 (FP3) is an essential and drug target cysteine protease of the most lethal human malaria parasite Plasmodium falciparum. FP3 and its majority of homologs in malaria parasites prefer Leu at the P2 position in substrates and inhibitors, whereas its major host homolog cathepsin L prefers Phe. However, FP3 is much less active on peptide substrates and has negligible activity against a P2 Arg-containing substrate (Z-RR-AMC) compared to its paralog falcipain-2A (FP2A). To identify the specificity determinants, the S2/3 pocket residues of FP3 were substituted with the corresponding residues in FP2 or cathepsin L, and the wild type and mutant proteases were assessed for hydrolysis of peptide and protein substrates. Our results indicate that the S2 pocket residues I94 and P181 of FP3 are chiefly responsible for its P2 Leu preference and negligible activity for Z-RR-AMC, respectively. E243 in FP3 and the corresponding residue D234 in FP2 have a key role in Z-RR-AMC hydrolysing activity, possibly through stabilization of side chain interactions, as their substitution with Ala abolished the activity. Several FP3 mutants, which retained P2 Leu preference and showed similar or more activity than wild type FP3 on peptide substrates, degraded haemoglobin less efficiently than wild type FP3, suggesting that multiple residues contribute to haemoglobinase activity. Furthermore, P181 and E243 appear to contribute to the optimum activity of FP3 in the food vacuole milieu (≈pH 5.5). The identification of residues determining specificity of FP3 could aid in developing specific inhibitors of FP3 and its homologs in malaria parasites., (Copyright © 2015 Elsevier B.V. All rights reserved.)
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- 2015
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25. Characterization of the autophagy marker protein Atg8 reveals atypical features of autophagy in Plasmodium falciparum.
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Navale R, Atul, Allanki AD, and Sijwali PS
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- Adenine analogs & derivatives, Adenine pharmacology, Amino Acid Sequence, Antimalarials pharmacology, Apicoplasts drug effects, Autophagy drug effects, Autophagy-Related Protein 8 Family, Chloroquine pharmacology, Doxycycline pharmacology, Erythrocytes drug effects, Erythrocytes parasitology, Gene Expression Regulation, Humans, Leucine analogs & derivatives, Leucine pharmacology, Life Cycle Stages drug effects, Life Cycle Stages genetics, Microfilament Proteins genetics, Molecular Sequence Data, Pepstatins pharmacology, Plasmodium falciparum drug effects, Plasmodium falciparum genetics, Plasmodium falciparum growth & development, Protease Inhibitors pharmacology, Protozoan Proteins genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Sequence Alignment, Sequence Homology, Amino Acid, Apicoplasts physiology, Autophagy genetics, Genome, Protozoan, Microfilament Proteins metabolism, Plasmodium falciparum metabolism, Protozoan Proteins metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.
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- 2014
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26. Genetic ablation of plasmoDJ1, a multi-activity enzyme, attenuates parasite virulence and reduces oocyst production.
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Singhal N, Mastan BS, Kumar KA, and Sijwali PS
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- Amino Acid Sequence, Animals, Artemisinins pharmacology, Enzyme Activation drug effects, Enzyme Activation genetics, Female, Gene Knockout Techniques, Humans, Male, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Multigene Family, Mutation, Oocysts drug effects, Plasmodium berghei genetics, Plasmodium falciparum genetics, Protozoan Proteins physiology, Rats, Rats, Wistar, Virulence drug effects, Virulence genetics, Cysteine Endopeptidases genetics, Oocysts enzymology, Plasmodium berghei enzymology, Plasmodium berghei pathogenicity, Plasmodium falciparum enzymology, Plasmodium falciparum pathogenicity, Protozoan Proteins genetics
- Abstract
Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, which we have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.
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- 2014
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27. Emergence of pyrido quinoxalines as new family of antimalarial agents.
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Chandra Shekhar A, Shanthan Rao P, Narsaiah B, Allanki AD, and Sijwali PS
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- Antimalarials chemical synthesis, Antimalarials chemistry, Dose-Response Relationship, Drug, Erythrocytes drug effects, Erythrocytes microbiology, Humans, Molecular Structure, Pyrazines chemical synthesis, Pyrazines chemistry, Quinoxalines chemical synthesis, Quinoxalines chemistry, Structure-Activity Relationship, Antimalarials pharmacology, Plasmodium falciparum drug effects, Pyrazines pharmacology, Quinoxalines pharmacology
- Abstract
A series of novel N-alkyl dihydro pyrido quinoxaline derivatives were synthesized using Gould-Jacobs reaction and evaluated their antimalarial activity in vitro against chloroquine sensitive (3D7) and drug resistant (Dd2) strains of Plasmodium falciparum. Among the compounds tested, 10 compounds were more potent than their structural standard analog ciprofloxacin, including 2 derivatives 5e and 5h, which showed 3.3-7.4 times more potency than ciprofloxacin against both the parasite strains. The results are encouraging and a lead molecule may emerge which is useful alone or in combination therapy., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)
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- 2014
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28. Blocking Plasmodium falciparum development via dual inhibition of hemoglobin degradation and the ubiquitin proteasome system by MG132.
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Prasad R, Atul, Kolla VK, Legac J, Singhal N, Navale R, Rosenthal PJ, and Sijwali PS
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- Cysteine Endopeptidases metabolism, Drug Synergism, Erythrocytes parasitology, Humans, Plasmodium falciparum metabolism, Plasmodium falciparum physiology, Proteasome Inhibitors pharmacology, Hemoglobins metabolism, Leupeptins pharmacology, Plasmodium falciparum drug effects, Plasmodium falciparum growth & development, Proteasome Endopeptidase Complex metabolism, Proteolysis drug effects, Ubiquitin metabolism
- Abstract
Among key potential drug target proteolytic systems in the malaria parasite Plasmodium falciparum are falcipains, a family of hemoglobin-degrading cysteine proteases, and the ubiquitin proteasomal system (UPS), which has fundamental importance in cellular protein turnover. Inhibition of falcipains blocks parasite development, primarily due to inhibition of hemoglobin degradation that serves as a source of amino acids for parasite growth. Falcipains prefer P2 leucine in substrates and peptides, and their peptidyl inhibitors with leucine at the P2 position show potent antimalarial activity. The peptidyl inhibitor MG132 (Z-Leu-Leu-Leu-CHO) is a widely used proteasome inhibitor, which also has P2 leucine, and has also been shown to inhibit parasite development. However, the antimalarial targets of MG132 are unclear. We investigated whether MG132 blocks malaria parasite development by inhibiting hemoglobin degradation and/or by targeting the UPS. P. falciparum was cultured with inhibitors of the UPS (MG132, epoxomicin, and lactacystin) or falcipains (E64), and parasites were assessed for morphologies, extent of hemoglobin degradation, and accumulation of ubiquitinated proteins. MG132, like E64 and unlike epoxomicin or lactacystin, blocked parasite development, with enlargement of the food vacuole and accumulation of undegraded hemoglobin, indicating inhibition of hemoglobin degradation by MG132, most likely due to inhibition of hemoglobin-degrading falcipain cysteine proteases. Parasites cultured with epoxomicin or MG132 accumulated ubiquitinated proteins to a significantly greater extent than untreated or E64-treated parasites, indicating that MG132 inhibits the parasite UPS as well. Consistent with these findings, MG132 inhibited both cysteine protease and UPS activities present in soluble parasite extracts, and it strongly inhibited recombinant falcipains. MG132 was highly selective for inhibition of P. falciparum (IC50 0.0476 µM) compared to human peripheral blood mononuclear cells (IC50 10.8 µM). Thus, MG132 inhibits two distinct proteolytic systems in P. falciparum, and it may serve as a lead molecule for development of dual-target inhibitors of malaria parasites.
- Published
- 2013
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29. Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents.
- Author
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Tadigoppula N, Korthikunta V, Gupta S, Kancharla P, Khaliq T, Soni A, Srivastava RK, Srivastava K, Puri SK, Raju KS, Wahajuddin, Sijwali PS, Kumar V, and Mohammad IS
- Subjects
- Animals, Antimalarials pharmacokinetics, Antimalarials pharmacology, Benzopyrans pharmacokinetics, Benzopyrans pharmacology, Catalytic Domain, Chalcones pharmacokinetics, Chalcones pharmacology, Chromans chemical synthesis, Chromans pharmacokinetics, Chromans pharmacology, Cysteine Endopeptidases chemistry, Cysteine Proteinase Inhibitors chemical synthesis, Cysteine Proteinase Inhibitors pharmacokinetics, Cysteine Proteinase Inhibitors pharmacology, Malaria drug therapy, Male, Mice, Molecular Docking Simulation, Parasitic Sensitivity Tests, Plasmodium falciparum drug effects, Plasmodium yoelii, Rats, Rats, Sprague-Dawley, Small Molecule Libraries, Structure-Activity Relationship, Antimalarials chemical synthesis, Benzopyrans chemical synthesis, Chalcones chemical synthesis, Crotalaria chemistry
- Abstract
Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.
- Published
- 2013
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30. The Ionic and hydrophobic interactions are required for the auto activation of cysteine proteases of Plasmodium falciparum.
- Author
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Sundararaj S, Singh D, Saxena AK, Vashisht K, Sijwali PS, Dixit R, and Pandey KC
- Subjects
- Amino Acid Sequence, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Hemoglobins metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Malaria, Falciparum parasitology, Protein Binding, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Structure-Activity Relationship, Cysteine Endopeptidases genetics, Enzyme Activation genetics, Malaria, Falciparum enzymology, Plasmodium falciparum enzymology
- Abstract
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are major hemoglobinases and potential antimalarial drug targets. Our previous studies demonstrated that these enzymes are equipped with specific domains for specific functions. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. As with many proteases, falcipain-2 and falcipain-3 are synthesized as inactive zymogens. However, it is not known how these enzymes get activated for hemoglobin hydrolysis. In this study, we are presenting the first evidence that salt bridges and hydrophobic interactions are required for the auto activation of cysteine proteases of P.falciparum. To investigate the mechanism of activation of these enzymes, we expressed the wild type protein as well as different mutants in E.coli. Refolding was assessed by circular dichroism. Both CD and trans activation data showed that the wild type enzymes and mutants are rich in secondary structures with similar folds. Our study revealed that prodomain-mature domain of falcipain-2 and falcipain-3 interacts via salt bridges and hydrophobic interactions. We mutated specific residues of falcipain-2 and falcipain-3, and evaluated their ability to undergo auto processing. Mutagenesis result showed that two salt bridges (Arg¹⁸⁵- Glu²²¹, Glu²¹⁰- Lys⁴⁰³) in falcipain-2, and one salt bridge (Arg²⁰²-Glu²³⁸) in falcipain-3, play crucial roles in the activation of these enzymes. Further study revealed that hydrophobic interactions present both in falcipain-2 (Phe²¹⁴ Trp⁴⁴⁹ Trp⁴⁵³) and falcipain-3 (Phe²³¹ Trp⁴⁵⁷ Trp⁴⁶¹) also play important roles in the activation of these enzymes. Our results revealed the interactions involved in auto processing of two major hemoglobinases of malaria parasite.
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- 2012
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31. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.
- Author
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Prasad R, Atul, Soni A, Puri SK, and Sijwali PS
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- Amino Acid Sequence, Animals, Cysteine Proteinase Inhibitors pharmacology, Cytoskeletal Proteins metabolism, Enzyme Stability drug effects, Erythrocytes drug effects, Erythrocytes metabolism, Erythrocytes parasitology, Haplorhini parasitology, Hemoglobins metabolism, Humans, Hydrogen-Ion Concentration drug effects, Hydrolysis drug effects, Kinetics, Leucine analogs & derivatives, Leucine pharmacology, Molecular Sequence Data, Papain chemistry, Parasites drug effects, Parasites growth & development, Plasmodium knowlesi drug effects, Plasmodium knowlesi growth & development, Protein Transport drug effects, Proteolysis drug effects, Protozoan Proteins chemistry, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Sequence Alignment, Substrate Specificity drug effects, Malaria parasitology, Papain metabolism, Parasites enzymology, Plasmodium knowlesi cytology, Plasmodium knowlesi enzymology, Protozoan Proteins metabolism
- Abstract
Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.
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- 2012
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32. Functional evaluation of Plasmodium export signals in Plasmodium berghei suggests multiple modes of protein export.
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Sijwali PS and Rosenthal PJ
- Subjects
- Amino Acid Motifs, Animals, Erythrocytes metabolism, Erythrocytes parasitology, Green Fluorescent Proteins, Peptides, Protein Sorting Signals, Protein Transport, Vacuoles parasitology, Plasmodium berghei chemistry, Protozoan Proteins metabolism
- Abstract
The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.
- Published
- 2010
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33. Falcipain cysteine proteases require bipartite motifs for trafficking to the Plasmodium falciparum food vacuole.
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Subramanian S, Sijwali PS, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Cell Membrane metabolism, Cysteine Endopeptidases physiology, Erythrocytes metabolism, Erythrocytes parasitology, Green Fluorescent Proteins metabolism, Hemoglobins chemistry, Humans, Hydrolysis, Models, Biological, Molecular Sequence Data, Plasmodium falciparum, Sequence Homology, Amino Acid, Vacuoles metabolism, Cysteine Endopeptidases chemistry
- Abstract
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 hydrolyze hemoglobin in an acidic food vacuole to provide amino acids for erythrocytic malaria parasites. Trafficking to the food vacuole has not been well characterized. To study trafficking of falcipains, which include large membrane-spanning prodomains, we utilized chimeras with portions of the proteases fused to green fluorescent protein. The prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole. Using serial truncations, deletions, and point mutations, we showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the falcipain-2 prodomain were required for efficient food vacuolar trafficking. Mutants with altered trafficking were arrested at the plasma membrane, implicating trafficking via this structure. Our results indicate that falcipains utilize a previously undescribed bipartite motif-dependent mechanism for targeting to a hydrolytic organelle, suggesting inhibition of this unique mechanism as a new means of antimalarial chemotherapy.
- Published
- 2007
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34. A chimeric cysteine protease of Plasmodium berghei engineered to resemble the Plasmodium falciparum protease falcipain-2.
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Singh A, Walker KJ, Sijwali PS, Lau AL, and Rosenthal PJ
- Subjects
- Amino Acid Substitution, Animals, Mutant Chimeric Proteins, Protozoan Proteins genetics, Cysteine Endopeptidases genetics, Plasmodium berghei enzymology, Plasmodium falciparum enzymology, Protein Engineering methods
- Abstract
The cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential targets for chemotherapy directed against Plasmodium falciparum, the most important human malaria parasite. Most in vivo evaluations of candidate antimalarials are conducted in murine malaria models, and falcipain homologs from rodent malaria parasites differ importantly from falcipain-2 and falcipain-3. We expressed berghepain-2, the single homolog of falcipain-2 and falcipain-3 of the rodent parasite P. berghei, in Escherichia coli, and characterized the refolded active enzyme. Berghepain-2 was biochemically very similar to the previously characterized rodent plasmodial protease vinckepain-2, but differed from falcipain-2 and falcipain-3 in its fine substrate and inhibitor specificity. We then used homology modeling and evolutionary trace analysis to predict key amino acids that mediate functional differences between falcipain-2 and berghepain-2. Thirteen amino acids were sequentially altered to replace berghepain-2 residues with those in falcipain-2. Mutant enzymes varied in activity and sensitivity to inhibitors. A berghepain-2 mutant with eight substitutions retained good activity and demonstrated fine substrate and inhibitor sensitivity more similar to that of falcipain-2 than berghepain-2. These results suggest that, to facilitate drug discovery, we can produce mutant animal model malaria parasites with biochemical properties more like those of the key drug target, P. falciparum.
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- 2007
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35. Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum.
- Author
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Sijwali PS, Koo J, Singh N, and Rosenthal PJ
- Subjects
- Animals, Blotting, Western, Cell Culture Techniques, Cysteine Endopeptidases genetics, Cysteine Proteinase Inhibitors, Erythrocytes parasitology, Hemoglobins metabolism, Humans, Plasmodium falciparum genetics, Plasmodium falciparum physiology, Transfection, Cysteine Endopeptidases metabolism, Plasmodium falciparum enzymology
- Abstract
Erythrocytic stages of the malaria parasite Plasmodium falciparum express four related papain-family cysteine proteases, termed falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases, but determination of the specific roles of these and other falcipains has been incomplete. To better characterize biological roles, we attempted disruption of each falcipain gene in the same strain (3D7) of P. falciparum. Disruption of falcipain-1, falcipain-2, and falcipain-2' was achieved. In each case knockouts multiplied at the same rate as wild-type parasites. The morphologies of erythrocytic falcipain-1 and falcipain-2' knockout parasites were indistinguishable from those of wild-type parasites. In contrast, consistent with previous results, falcipain-2 knockout trophozoites developed swollen, hemoglobin-filled food vacuoles, indicative of a block in hemoglobin hydrolysis and were, compared to wild-type parasites, twice as sensitive to cysteine protease inhibitors and over 1000 times more sensitive to an aspartic protease inhibitor. The falcipain-3 gene could not be disrupted, but replacement with a tagged functional copy was readily achieved, strongly suggesting that falcipain-3 is essential to erythrocytic parasites. Our data suggest key roles for falcipain-2 and falcipain-3 in the development of erythrocytic malaria parasites and a complex interplay between P. falciparum cysteine and aspartic proteases.
- Published
- 2006
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36. Structural basis for unique mechanisms of folding and hemoglobin binding by a malarial protease.
- Author
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Wang SX, Pandey KC, Somoza JR, Sijwali PS, Kortemme T, Brinen LS, Fletterick RJ, Rosenthal PJ, and McKerrow JH
- Subjects
- Amino Acid Sequence, Animals, Crystallography, X-Ray, Cystatins chemistry, Cystatins metabolism, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Humans, Models, Molecular, Molecular Sequence Data, Protein Binding, Sequence Alignment, Sequence Homology, Amino Acid, Cysteine Endopeptidases chemistry, Hemoglobins metabolism, Plasmodium falciparum enzymology, Protein Folding, Protein Structure, Tertiary
- Abstract
Falcipain-2 (FP2), the major cysteine protease of the human malaria parasite Plasmodium falciparum, is a hemoglobinase and promising drug target. Here we report the crystal structure of FP2 in complex with a protease inhibitor, cystatin. The FP2 structure reveals two previously undescribed cysteine protease structural motifs, designated FP2(nose) and FP2(arm), in addition to details of the active site that will help focus inhibitor design. Unlike most cysteine proteases, FP2 does not require a prodomain but only the short FP2(nose) motif to correctly fold and gain catalytic activity. Our structure and mutagenesis data suggest a molecular basis for this unique mechanism by highlighting the functional role of two Tyr within FP2(nose) and a conserved Glu outside this motif. The FP2(arm) motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2(arm) that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2(nose) and FP2(arm) are found only in related plasmodial proteases, suggesting that they confer malaria-specific functions.
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- 2006
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37. Plasmodium falciparum: biochemical characterization of the cysteine protease falcipain-2'.
- Author
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Singh N, Sijwali PS, Pandey KC, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Cysteine Endopeptidases biosynthesis, Cysteine Endopeptidases genetics, Cysteine Proteinase Inhibitors pharmacology, Electrophoresis, Polyacrylamide Gel, Gene Expression Regulation, Enzymologic, Hemoglobins metabolism, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Plasmodium falciparum genetics, Substrate Specificity, Cysteine Endopeptidases chemistry, Plasmodium falciparum enzymology
- Abstract
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential antimalarial drug targets. The falcipain-2' gene was identified recently and is nearly identical in sequence to falcipain-2. The product of this gene has not been studied previously. The mature protease domain of falcipain-2' was expressed in Escherichia coli, purified, and refolded to active enzyme. Functional analysis revealed similar biochemical properties to those of falcipain-2, including pH optima (pH 5.5-7.0), reducing requirements, and substrate preference. Studies with cysteine protease inhibitors showed similar inhibition of falcipain-2 and falcipain-2', although specificities were not identical. Considering activity against the presumed biological substrate, both enzymes readily hydrolyzed hemoglobin. Our results confirm that falcipain-2' is an active hemoglobinase and suggest that falcipain-2 and falcipain-2' play similar roles in erythrocytic parasites but that, for promising cysteine protease inhibitors, it will be important to confirm activity against this additional target.
- Published
- 2006
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38. The Plasmodium falciparum cysteine protease falcipain-2 captures its substrate, hemoglobin, via a unique motif.
- Author
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Pandey KC, Wang SX, Sijwali PS, Lau AL, McKerrow JH, and Rosenthal PJ
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Caseins chemistry, Cysteine Proteinase Inhibitors chemistry, Dose-Response Relationship, Drug, Drug Design, Hemoglobins metabolism, Humans, Hydrolysis, Kinetics, Leucine pharmacology, Macromolecular Substances, Models, Molecular, Molecular Sequence Data, Mutation, Peptides chemistry, Protein Binding, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Time Factors, Antimalarials pharmacology, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases physiology, Hemoglobins chemistry, Leucine analogs & derivatives, Plasmodium falciparum enzymology
- Abstract
Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a beta-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif (Delta10FP2). Delta10FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, Delta10FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (FP2E-64) formed a complex with hemoglobin, but Delta10FP2E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of Delta10FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but Delta10FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.
- Published
- 2005
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39. Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites.
- Author
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Sijwali PS, Kato K, Seydel KB, Gut J, Lehman J, Klemba M, Goldberg DE, Miller LH, and Rosenthal PJ
- Subjects
- Animals, Base Sequence, Cysteine Endopeptidases genetics, Cysteine Proteinase Inhibitors pharmacology, DNA, Protozoan genetics, Erythrocytes parasitology, Gene Targeting, Genes, Protozoan, Humans, In Vitro Techniques, Malaria, Falciparum drug therapy, Malaria, Falciparum parasitology, Plasmodium falciparum genetics, Plasmodium falciparum growth & development, Cysteine Endopeptidases physiology, Plasmodium falciparum enzymology
- Abstract
Among potential new targets for antimalarial chemotherapy are Plasmodium falciparum cysteine proteases, known as falcipains. Falcipain-2 and falcipain-3 are food vacuole hemoglobinases that may have additional functions. The function of falcipain-1 remains uncertain. To better characterize the role of falcipain-1 in erythrocytic parasites, we disrupted the falcipain-1 gene and characterized recombinant parasites. Disruption of the falcipain-1 gene was confirmed with Southern blots, and loss of expression of falcipain-1 was confirmed with immunoblots and by loss of labeling with a specific protease inhibitor. Compared with wild-type parasites, falcipain-1 knockout parasites developed normally, with the same morphology, multiplication rate, and invasion efficiency, and without significant differences in sensitivity to cysteine protease inhibitors. In wild-type and knockout parasites, cysteine protease inhibitors blocked hemoglobin hydrolysis in trophozoites, with a subsequent block in rupture of erythrocytes by mature schizonts, but they did not inhibit erythrocyte invasion by merozoites. Our results indicate that although falcipain-1 is expressed by erythrocytic parasites, it is not essential for normal development during this stage or for erythrocyte invasion.
- Published
- 2004
- Full Text
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40. Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum.
- Author
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Sijwali PS and Rosenthal PJ
- Subjects
- Animals, Hydrolysis, Plasmids, Plasmodium falciparum enzymology, Plasmodium falciparum growth & development, RNA, Messenger genetics, Transcription, Genetic, Transfection, Cysteine Endopeptidases genetics, Gene Deletion, Gene Expression Regulation, Enzymologic genetics, Hemoglobins metabolism, Plasmodium falciparum genetics
- Abstract
Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to acquire free amino acids and maintain parasite homeostasis. Hemoglobin hydrolysis appears to be a cooperative process requiring cysteine proteases (falcipains) and aspartic proteases (plasmepsins), but the specific roles of different enzymes in this process are unknown. We previously showed that falcipain-2 is a major trophozoite food vacuole cysteine protease. To characterize the specific role of falcipain-2, we disrupted the falcipain-2 gene and assessed the effect of this alteration. Falcipain-2-knockout trophozoites had markedly diminished cysteine protease activity and swollen, dark staining food vacuoles, consistent with a block in hemoglobin hydrolysis, as caused by cysteine protease inhibitors. However, more mature stages of knockout parasites were indistinguishable from wild-type parasites and developed normally. The knockout parasites had decreased and delayed expression of falcipain-2, which appeared to be directed by increased transcription of a second copy of the gene (falcipain-2'). Expression of other falcipains and plasmepsins was similar in wild-type and knockout parasites. Compared with wild-type, knockout parasites were about 3 times more sensitive to the cysteine protease inhibitors E-64 and leupeptin, and over 50-fold more sensitive to the aspartic protease inhibitor pepstatin. Our results assign a specific function for falcipain-2, the hydrolysis of hemoglobin in trophozoites. In addition, they highlight the cooperative action of cysteine and aspartic proteases in hemoglobin degradation by malaria parasites.
- Published
- 2004
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41. Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax.
- Author
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Na BK, Shenai BR, Sijwali PS, Choe Y, Pandey KC, Singh A, Craik CS, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Antimalarials pharmacology, Cloning, Molecular, Cysteine Endopeptidases chemistry, Erythrocyte Membrane metabolism, Hemoglobins metabolism, Hydrolysis, Membrane Proteins metabolism, Molecular Sequence Data, Protease Inhibitors pharmacology, Protein Folding, Protein Structure, Tertiary, Sequence Alignment, Substrate Specificity, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Plasmodium vivax enzymology
- Abstract
Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
- Published
- 2004
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42. Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2.
- Author
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Pandey KC, Sijwali PS, Singh A, Na BK, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Catalytic Domain, Cloning, Molecular, Disulfides chemistry, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Escherichia coli metabolism, Immunoblotting, Kinetics, Leupeptins chemistry, Molecular Sequence Data, Plasmodium enzymology, Protein Folding, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Tyrosine chemistry, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Plasmodium falciparum enzymology
- Abstract
The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.
- Published
- 2004
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43. Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites.
- Author
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Singh A, Shenai BR, Choe Y, Gut J, Sijwali PS, Craik CS, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Animals, Base Sequence, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Malaria parasitology, Molecular Sequence Data, Mutation, Papain chemistry, Peptide Library, Plasmodium falciparum genetics, Protein Folding, Protozoan Proteins chemistry, Protozoan Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Rodent Diseases parasitology, Rodentia, Sequence Homology, Amino Acid, Substrate Specificity, Cysteine Endopeptidases genetics, Helminth Proteins, Plasmodium falciparum enzymology, Protozoan Proteins genetics
- Abstract
Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.
- Published
- 2002
- Full Text
- View/download PDF
44. Folding of the Plasmodium falciparum cysteine protease falcipain-2 is mediated by a chaperone-like peptide and not the prodomain.
- Author
-
Sijwali PS, Shenai BR, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Catalysis, Cysteine Endopeptidases chemistry, DNA Primers, Molecular Sequence Data, Sequence Homology, Amino Acid, Cysteine Endopeptidases metabolism, Molecular Chaperones metabolism, Plasmodium falciparum enzymology, Protein Folding
- Abstract
Papain-family cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are hemoglobinases and potential drug targets. Available data suggest that papain-family proteases require prodomains for correct folding into functional conformations. However, in prior studies of falcipain-2, an Escherichia coli-expressed construct containing only a small portion of the prodomain refolded efficiently, suggesting that this enzyme differs in this regard from other papain-family enzymes. To better characterize the determinants of folding for falcipain-2, we expressed multiple pro- and mature constructs of the enzyme in E. coli and assessed their abilities to refold. Mature falcipain-2 refolded into active protease with very similar properties to those of proteins resulting from the refolding of proenzyme constructs. Deletion of a 17-amino acid amino-terminal segment of the mature protease yielded a construct incapable of correct folding, but inclusion of this segment in trans allowed folding to active falcipain-2. The prodomain was a potent, competitive, and reversible inhibitor of mature falcipain-2 (K(i) 10(-10) m). Our results identify a chaperone-like function of an amino-terminal segment of mature falcipain-2 and suggest that protease inhibition, but not the mediation of folding, is a principal function of the falcipain-2 prodomain.
- Published
- 2002
- Full Text
- View/download PDF
45. Cysteine proteases of malaria parasites: targets for chemotherapy.
- Author
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Rosenthal PJ, Sijwali PS, Singh A, and Shenai BR
- Subjects
- Animals, Cysteine Endopeptidases genetics, Cysteine Proteinase Inhibitors pharmacology, Cysteine Proteinase Inhibitors therapeutic use, Humans, Malaria enzymology, Malaria genetics, Parasites enzymology, Parasites genetics, Cysteine Endopeptidases metabolism, Drug Delivery Systems methods, Malaria drug therapy, Parasites drug effects
- Abstract
New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.
- Published
- 2002
- Full Text
- View/download PDF
46. Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3.
- Author
-
Sijwali PS, Shenai BR, Gut J, Singh A, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Cloning, Molecular, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Erythrocytes enzymology, Erythrocytes parasitology, Hemoglobins metabolism, Humans, Hydrolysis, Molecular Sequence Data, Plasmodium falciparum genetics, Plasmodium falciparum growth & development, Protein Folding, Protein Processing, Post-Translational genetics, Sequence Homology, Nucleic Acid, Cysteine Endopeptidases biosynthesis, Cysteine Endopeptidases chemistry, Helminth Proteins, Plasmodium falciparum enzymology
- Abstract
In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P(2) position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.
- Published
- 2001
- Full Text
- View/download PDF
47. Recombinant falcipain-2 cleaves erythrocyte membrane ankyrin and protein 4.1.
- Author
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Dua M, Raphael P, Sijwali PS, Rosenthal PJ, and Hanspal M
- Subjects
- Animals, Cysteine Endopeptidases genetics, Plasmodium falciparum pathogenicity, Recombinant Proteins metabolism, Substrate Specificity, Ankyrins metabolism, Cysteine Endopeptidases metabolism, Cytoskeletal Proteins, Erythrocyte Membrane metabolism, Membrane Proteins metabolism, Neuropeptides, Plasmodium falciparum enzymology
- Published
- 2001
- Full Text
- View/download PDF
48. Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2.
- Author
-
Sijwali PS, Brinen LS, and Rosenthal PJ
- Subjects
- Animals, Arginine metabolism, Crystallization, Cysteine Endopeptidases genetics, Cysteine Endopeptidases isolation & purification, Electrophoresis, Polyacrylamide Gel, Enzyme Stability, Glycerol metabolism, Hydrogen-Ion Concentration, Inclusion Bodies metabolism, Plasmodium falciparum genetics, Protein Conformation, Protein Folding, Protein Renaturation, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Plasmodium falciparum enzymology
- Abstract
The Plasmodium falciparum cysteine protease falcipain-2 is a potential new target for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematic assessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. A systematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiency varied markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidized glutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
49. Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum.
- Author
-
Shenai BR, Sijwali PS, Singh A, and Rosenthal PJ
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cysteine Endopeptidases genetics, Cysteine Endopeptidases metabolism, Hemoglobins metabolism, Molecular Sequence Data, Protein Folding, Recombinant Proteins chemistry, Substrate Specificity, Cysteine Endopeptidases chemistry, Helminth Proteins, Plasmodium falciparum enzymology
- Abstract
Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.
- Published
- 2000
- Full Text
- View/download PDF
50. Cloning and sequence analysis of the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium cynomolgi bastianelli.
- Author
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Sijwali PS, Malhotra P, Puri SK, and Chauhan VS
- Subjects
- Amino Acid Sequence, Animals, Cell Adhesion Molecules chemistry, Cell Adhesion Molecules genetics, Cloning, Molecular, Conserved Sequence, Molecular Sequence Data, Plasmodium cynomolgi chemistry, Sequence Alignment, Sequence Analysis, Genes, Protozoan, Plasmodium cynomolgi genetics, Protozoan Proteins chemistry, Protozoan Proteins genetics
- Published
- 1997
- Full Text
- View/download PDF
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