RESUMO
Endemic Burkitt lymphoma (eBL) is characterized by an oncogenic IGH/c-MYC translocation and Epstein-Barr virus (EBV) positivity, and is epidemiologically linked to Plasmodium falciparum malaria. Both EBV and malaria are thought to contribute to eBL by inducing the expression of activation-induced cytidine deaminase (AID), an enzyme involved in the IGH/c-MYC translocation. AID/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC) family enzymes have recently emerged as potent mutagenic sources in a variety of cancers, but apart from AID, their involvement in eBL and their regulation by EBV and P. falciparum is unknown. Here, we show that upon inoculation with EBV, human B cells strongly upregulate the expression of enzymatically active APOBEC3B and APOBEC3G. In addition, we found significantly increased levels of APOBEC3A in B cells of malaria patients, which correlated with parasite load. Interestingly, despite the fact that APOBEC3A, APOBEC3B, and APOBEC3G caused c-MYC mutations when overexpressed in HEK293T cells, a mutational enrichment in eBL tumors was only detected in AID motifs. This suggests that even though the EBV- and P. falciparum-directed immune response triggers the expression and activity of several AID/APOBEC members, only the upregulation of AID has oncogenic consequences, while the induction of the APOBEC3 subfamily may primarily have immunoprotective functions.
Assuntos
Desaminases APOBEC , Linfoma de Burkitt , Citidina Desaminase , Infecções por Vírus Epstein-Barr , Malária Falciparum , Desaminases APOBEC/genética , Desaminase APOBEC-3G , Linfoma de Burkitt/enzimologia , Linfoma de Burkitt/genética , Citidina Desaminase/genética , Infecções por Vírus Epstein-Barr/enzimologia , Infecções por Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4 , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/genética , Antígenos de Histocompatibilidade Menor , MutagênicosRESUMO
The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.
Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/químicaRESUMO
Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1ß, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.
Assuntos
Inflamação/enzimologia , Macrófagos/enzimologia , Malária Cerebral/enzimologia , Malária Falciparum/enzimologia , Estresse Oxidativo , Plasmodium falciparum/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Xantina Oxidase/metabolismo , Caspase 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Inflamação/sangue , Inflamação/parasitologia , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos , Macrófagos/parasitologia , Malária Cerebral/sangue , Malária Cerebral/parasitologia , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Cytochrome P450 (CYP) enzymes are essential in the metabolism of most drugs used today. Single nucleotide polymorphism(s) occurring in CYP genes can adversely affect drug pharmacokinetics, efficacy, and safety. Individuals carrying the CYP2C8*2 c.805A > T (CYP2C8*2; rs11572103) allele have impaired amodiaquine metabolism, increased risk of amodiaquine-related adverse events, and may promote the selection of drug-resistant parasite strains. This study investigated the distribution of the CYP2C8*2 allele in Brazzaville, Republic of Congo, where artesunate + amodiaquine is used as the second-line treatment for uncomplicated Plasmodium falciparum malaria. METHODS: A total of 285 febrile children visiting the Marien Ngouabi paediatric hospital were genotyped for CYP2C8*2 using PCR-restriction fragment length polymorphism (PCR-RFLP). The allele frequencies and genotype distribution were determined. RESULTS: The CYP2C8*2 allele was successfully genotyped in 75% (213/285) of the study participants. The CYP2C8*2A allele had a frequency of 63%, whereas the CYP2C8*2T allele had a frequency of 37%. Genotypes CYP2C8*2AA (rapid metabolizer), CYP2C8*2AT (intermediate metabolizer), and CYP2C8*2TT (poor metabolizer) were observed in 44%, 38%, and 18% of the investigated participants, respectively. CONCLUSIONS: This study gives the first description of CYP2C8*2 allele distribution in the Republic of Congo and highlights the potential risk of amodiaquine-related adverse events. Information from this study will be beneficial during pharmacovigilance investigations.
Assuntos
Amodiaquina/uso terapêutico , Antimaláricos/uso terapêutico , Citocromo P-450 CYP2C8/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Alelos , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Congo , Combinação de Medicamentos , Feminino , Frequência do Gene , Genótipo , Humanos , Lactente , Malária Falciparum/enzimologia , Masculino , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antimaláricos/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Proteínas de Transporte de Cátions/antagonistas & inibidores , Eritrócitos/enzimologia , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Sódio/metabolismo , Adenosina Trifosfatases/genética , Animais , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/genética , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Homeostase , Humanos , Transporte de Íons , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genéticaRESUMO
AIM: In Plasmodium falciparum malaria, the clinical manifestation of acute kidney injury (AKI) is commonly associated with acute tubular necrosis (ATN) in the kidney tissues. Renal tubular cells often exhibit various degrees of cloudy swelling, cell degeneration, and frank necrosis. To study individual cell death, this study evaluates the degree of renal tubular necrosis in association with apoptosis in malarial kidneys. METHODS: Kidney tissues from P. falciparum malaria with AKI (10 cases), and without AKI (10 cases) were evaluated for tubular pathology. Normal kidney tissues from 10 cases served as controls. Tubular necrosis was assessed quantitatively in kidney tissues infected with P. falciparum malaria, based on histopathological evaluation. In addition, the occurrence of apoptosis was investigated using cleaved caspase-3 marker. Correlation between tubular necrosis and apoptosis was analyzed. RESULTS: Tubular necrosis was found to be highest in P. falciparum malaria patients with AKI (36.44% ± 3.21), compared to non-AKI (15.88% ± 1.63) and control groups (2.58% ± 0.39) (all p < 0.001). In the AKI group, the distal tubules showed a significantly higher degree of tubular necrosis than the proximal tubules (p = 0.021) and collecting tubules (p = 0.033). Tubular necrosis was significantly correlated with the level of serum creatinine (r = 0.596, p = 0.006), and the occurrence of apoptosis (r = 0.681, p = 0.001). CONCLUSION: In malarial AKI, the process of apoptosis occurs in ATN.
Assuntos
Injúria Renal Aguda/enzimologia , Caspase 3/análise , Túbulos Renais/enzimologia , Malária Falciparum/enzimologia , Injúria Renal Aguda/parasitologia , Injúria Renal Aguda/patologia , Apoptose , Biomarcadores/sangue , Biópsia , Estudos de Casos e Controles , Creatinina/sangue , Ativação Enzimática , Humanos , Imuno-Histoquímica , Necrose do Córtex Renal/enzimologia , Necrose do Córtex Renal/parasitologia , Necrose do Córtex Renal/patologia , Túbulos Renais/parasitologia , Túbulos Renais/patologia , Malária Falciparum/parasitologia , Malária Falciparum/patologia , NecroseRESUMO
The cAMP-dependent protein kinase PKA is a well-characterized member of the serine-threonine protein AGC kinase family and is the effector kinase of cAMP signaling. As such, PKA is involved in the control of a wide variety of cellular processes including metabolism, cell growth, gene expression and apoptosis. cAMP-dependent PKA signaling pathways play important roles during infection and virulence of various pathogens. Since fluxes in cAMP are involved in multiple intracellular functions, a variety of different pathological infectious processes can be affected by PKA signaling pathways. Here, we highlight some features of cAMP-PKA signaling that are relevant to Plasmodium falciparum-infection of erythrocytes and present an update on AKAP targeting of PKA in PGE2 signaling via EP4 in Theileria annulata-infection of leukocytes and discuss cAMP-PKA signling in Toxoplasma.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Malária Falciparum/enzimologia , Plasmodium falciparum/metabolismo , Sistemas do Segundo Mensageiro , Theileria annulata/metabolismo , Theileriose/enzimologia , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , AMP Cíclico/metabolismo , Humanos , Malária Falciparum/patologia , Theileriose/patologiaRESUMO
Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein.
Assuntos
Malária Falciparum/enzimologia , Plasmodium falciparum/genética , RecQ Helicases/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , DNA de Protozoário/genética , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , RNA de Cadeia Dupla/genética , RecQ Helicases/química , RecQ Helicases/metabolismoRESUMO
Protein post-translational modifications (PTM) are commonly used to regulate biological processes. Protein S-acylation is an enzymatically regulated reversible modification that has been shown to modulate protein localization, activity and membrane binding. Proteome-scale discovery on Plasmodium falciparum schizonts has revealed a complement of more than 400 palmitoylated proteins, including those essential for host invasion and drug resistance. The wide regulatory affect on this species is endorsed by the presence of 12 proteins containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) that is associated with palmitoyl-transferase activity. Genetic interrogation of these enzymes in Apicomplexa has revealed essentiality and distinct localization at cellular compartments; these features are species specific and are not observed in yeast. It is clear that palmitoylation has an elaborate role in Plasmodium biology and opens intriguing questions on the functional consequence of this group of acylation modifications and how the protein S-acyl transferases (PATs) orchestrate molecular events.
Assuntos
Lipoilação/genética , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Serina C-Palmitoiltransferase/metabolismo , Acilação/genética , Animais , Malária Falciparum/genética , Malária Falciparum/parasitologia , Ácido Palmítico/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Estrutura Terciária de Proteína , Serina C-Palmitoiltransferase/genéticaRESUMO
BACKGROUND: Plasmodium falciparum serine repeat antigen 5 (PfSERA5) is an abundant blood stage protein that plays an essential role in merozoite egress and invasion. The native protein undergoes extensive proteolytic cleavage that appears to be tightly regulated. PfSERA5 N-terminal fragment is being developed as vaccine candidate antigen. Although PfSERA5 belongs to papain-like cysteine protease family, its catalytic domain has a serine in place of cysteine at the active site. METHODS: In the present study, we synthesized a number of peptides from the N- and C-terminal regions of PfSERA5 active domain and evaluated their inhibitory potential. RESULTS: The final proteolytic step of PfSERA5 involves removal of a C-terminal ~6kDa fragment that results in the generation of a catalytically active ~50kDa enzyme. In the present study, we demonstrate that two of the peptides derived from the C-terminal ~6kDa region inhibit the parasite growth and also cause a delay in the parasite development. These peptides reduced the enzyme activity of the recombinant protein and co-localized with the PfSERA5 protein within the parasite, thereby indicating the specific inhibition of PfSERA5 activity. Molecular docking studies revealed that the inhibitory peptides interact with the active site of the protein. Interestingly, the peptides did not have an effect on the processing of PfSERA5. CONCLUSIONS: Our observations indicate the temporal regulation of the final proteolytic cleavage step that occurs just prior to egress. GENERAL SIGNIFICANCE: These results reinforce the role of PfSERA5 for the intra-erythrocytic development of malaria parasite and show the role of carboxy terminal ~6kDa fragments in the regulation of PfSERA5 activity. The results also suggest that final cleavage step of PfSERA5 can be targeted for the development of new anti-malarials.
Assuntos
Antígenos de Protozoários/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Proteólise , Antígenos de Protozoários/genética , Eritrócitos/metabolismo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Peptídeos/química , Peptídeos/farmacologia , Plasmodium falciparum/genética , Estrutura Terciária de ProteínaRESUMO
The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca(2+)-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large-scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1-9 µm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 µm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6-specific inhibitors with good antimalarial activity.
Assuntos
Antimaláricos/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/isolamento & purificação , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Animais , Western Blotting , ATPases Transportadoras de Cálcio/metabolismo , Humanos , Técnicas In Vitro , Malária Falciparum/enzimologia , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Ca2+-ATPase expression in 15 selected isolates from malaria patients at the University College Hospital (UCH) Ibadan and two cloned strains (W2-chloroquine resistant, D6-chloroquine sensitive) of P.falciparum was assessed using spectrophotometric assay method. The kinetics of activity of Ca2+- ATPase in three isolates (NCP 14, NCP5, NCP1) and two clones (W2, D6) also assessed. 12% SDS-PAGE analysis of total proteins in one isolate (NCP14) and two clones (W2, D6) was also investigated. All the selected isolates and the two cloned strains exhibited measurable Ca2+-ATPase activity. The Ca2+-ATPase activity in cloned strain D6 (6.50 + 0.74mmolPi/min/mg protein) was higher than in cloned strain W2 (3.93 + 0.61mmolPi/min/mg protein. The Ca2+-ATPase activity in isolates from malaria patients varied widely (1.95 + 0.74 - 21.56 +1.43mmolPi/min/mg protein). The kinetic constants obtained for the two cloned strains showed that clone W2 had a higher Vmax (Vmax = 363mmolPi/min/mg protein) than clone D6 (Vmax = 74mmolPi/min/mg protein). All the isolates and the two cloned strains showed similar affinity for ATP (Km ~ 10mM). Scan of SDS-PAGE gel of total proteins in the isolate and cloned strains showed the presence of oligopeptide bands of molecular weights range of 148-176 kDa; 116-123 kDa respectively. These suggest the presence of predicted polypeptide of Ca2+-ATPase nature of molecular weight estimate of 139 kDa. The study agrees with previous findings that Ca2+-ATPase is functionally expressed in P.falciparum, The study also indicates that Ca2+-ATPase functional expression may vary with isolate or clone but the ATP binding mechanism to the enzyme is similar in all isolates and clones of P. falciparum. The study further suggests a possible association between acquisition of chloroquine resistance and Ca2+-ATPase functional expression in P. falciparum.
Assuntos
ATPases Transportadoras de Cálcio/química , Clonagem Molecular , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , ATPases Transportadoras de Cálcio/isolamento & purificação , Cloroquina/farmacologia , Resistência a Medicamentos , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Malária Falciparum/genética , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificaçãoRESUMO
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, Glu²¹°- Lys4°³) in falcipain-2, and one salt bridge (Arg²°²-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.
Assuntos
Cisteína Endopeptidases/genética , Ativação Enzimática/genética , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Hemoglobinas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Malária Falciparum/parasitologia , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.
Assuntos
Aminopeptidases/química , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Aminoácidos/química , Aminoácidos/metabolismo , Aminopeptidases/metabolismo , Animais , Domínio Catalítico , Cristalografia por Raios X/métodos , Eritrócitos/metabolismo , Humanos , Malária Falciparum/parasitologia , Peptídeos/química , Peptídeos/metabolismo , Proteólise , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
In the malarial parasite Plasmodium falciparum, a multifunctional phosphoethanolamine methyltransferase (PfPMT) catalyzes the methylation of phosphoethanolamine (pEA) to phosphocholine for membrane biogenesis. This pathway is also found in plant and nematodes, but PMT from these organisms use multiple methyltransferase domains for the S-adenosylmethionine (AdoMet) reactions. Because PfPMT is essential for normal growth and survival of Plasmodium and is not found in humans, it is an antiparasitic target. Here we describe the 1.55 Å resolution crystal structure of PfPMT in complex with AdoMet by single-wavelength anomalous dispersion phasing. In addition, 1.19-1.52 Å resolution structures of PfPMT with pEA (substrate), phosphocholine (product), sinefungin (inhibitor), and both pEA and S-adenosylhomocysteine bound were determined. These structures suggest that domain rearrangements occur upon ligand binding and provide insight on active site architecture defining the AdoMet and phosphobase binding sites. Functional characterization of 27 site-directed mutants identifies critical active site residues and suggests that Tyr-19 and His-132 form a catalytic dyad. Kinetic analysis, isothermal titration calorimetry, and protein crystallography of the Y19F and H132A mutants suggest a reaction mechanism for the PMT. Not only are Tyr-19 and His-132 required for phosphobase methylation, but they also form a "catalytic" latch that locks ligands in the active site and orders the site for catalysis. This study provides the first insight on this antiparasitic target enzyme essential for survival of the malaria parasite; however, further studies of the multidomain PMT from plants and nematodes are needed to understand the evolutionary division of metabolic function in the phosphobase pathway of these organisms.
Assuntos
Metiltransferases/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Adenosina/análogos & derivados , Adenosina/química , Substituição de Aminoácidos , Antimaláricos/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/química , Etanolaminas/química , Humanos , Cinética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/enzimologia , Malária Falciparum/genética , Metiltransferases/antagonistas & inibidores , Metiltransferases/genética , Mutação de Sentido Incorreto , Fosforilcolina/química , Plasmodium falciparum/genética , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genéticaRESUMO
Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antiprotozoários/farmacologia , Benzoquinonas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma/enzimologia , Tripanossomíase/tratamento farmacológico , Acetilação/efeitos dos fármacos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Modelos Animais de Doenças , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/genética , Camundongos , Plasmodium berghei/enzimologia , Plasmodium berghei/genética , Plasmodium falciparum/genética , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma/genética , Tripanossomíase/enzimologia , Tripanossomíase/genéticaRESUMO
Severe falciparum malaria remains a major killer in tropical countries. Central in the pathophysiology is mechanical obstruction in the microcirculation caused by cytoadherence and sequestration of parasitized erythrocytes. However, the pathogenesis of many features complicating severe malaria, including coma, renal failure and thrombocytopenia, remains incompletely understood. These disease manifestations are also key features of thrombotic thrombocytopenic purpura, a life-threatening disease strongly associated with a deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS13. We measured plasma ADAMTS13 activity, VWF antigen and VWF propeptide levels in 30 patients with severe falciparum malaria, 12 patients with uncomplicated falciparum malaria and 14 healthy Bangladeshi controls. In patients with severe malaria ADAMTS13 activity levels were markedly decreased in comparison to normal controls (mean [95%CI]: 23% [20-26] vs. 64% [55-72]) and VWF antigen and propeptide concentrations were significantly elevated (VWF antigen: 439% [396-481] vs. 64% [46-83]; VWF propeptide: 576% [481-671] vs. 69% [59-78]). In uncomplicated malaria VWF levels were also increased compared to healthy controls but ADAMTS13 activity was normal. The results suggest that decreased ADAMTS13 activity in combination with increased VWF concentrations may contribute to the complications in severe malaria.
Assuntos
Proteínas ADAM/deficiência , Coagulação Sanguínea , Malária Falciparum/sangue , Malária Falciparum/enzimologia , Precursores de Proteínas/sangue , Proteína ADAMTS13 , Adulto , Bangladesh , Biomarcadores/sangue , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Hemoglobinas/metabolismo , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Regulação para Cima , Adulto Jovem , Fator de von WillebrandRESUMO
In Plasmodium falciparum-infected red blood cells (RBCs), the flavoenzyme glutathione reductase (GR) regenerates reduced glutathione, which is essential for antioxidant defense. GR utilizes NADPH produced in the pentose phosphate shunt by glucose-6-phosphate dehydrogenase (G6PD). Thus, conditions affecting host G6PD or GR induce increased sensitivity to oxidants. Hereditary G6PD deficiency is frequent in malaria endemic areas and provides protection against severe malaria. Furthermore, GR deficiency resulting from insufficient saturation of the enzyme with its prosthetic group FAD is common. Based on these naturally occurring phenomena, GR of malaria parasites and their host cells represent attractive antimalarial drug targets. Recently we were given the opportunity to examine invasion, growth, and drug sensitivity of three P. falciparum strains (3D7, K1, and Palo Alto) in the RBCs from three homozygous individuals with total GR deficiency resulting from mutations in the apoprotein. Invasion or growth in the GR-deficient RBCs was not impaired for any of the parasite strains tested. Drug sensitivity to chloroquine, artemisinin, and methylene blue was comparable to parasites grown in GR-sufficient RBCs and sensitivity towards paraquat and sodium nitroprusside was only slightly enhanced. In contrast, membrane deposition of hemichromes as well as the opsonizing complement C3b fragments and phagocytosis were strongly increased in ring-infected RBCs of the GR-deficient individuals compared to ring-infected normal RBCs. Also, in one of the individuals, membrane-bound autologous IgGs were significantly enhanced. Thus, based on our in vitro data, GR deficiency and drug-induced GR inhibition may protect from malaria by inducing enhanced ring stage phagocytosis rather than by impairing parasite growth directly.
Assuntos
Glutationa Redutase/deficiência , Glutationa Redutase/genética , Malária Falciparum/complicações , Malária Falciparum/enzimologia , Estudos de Casos e Controles , Complemento C3/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Feminino , Predisposição Genética para Doença , Glutationa/metabolismo , Homozigoto , Humanos , Imunoglobulina G/metabolismo , Concentração Inibidora 50 , Pessoa de Meia-Idade , Fagocitose , Plasmodium falciparum/metabolismoRESUMO
Plasmodium falciparum is a purine auxotroph requiring hypoxanthine as a key metabolic precursor. Erythrocyte adenine nucleotides are the source of the purine precursors, making adenosine deaminase (ADA) a key enzyme in the pathway of hypoxanthine formation. Methylthioadenosine (MTA) is a substrate for most malarial ADAs, but not for human ADA. The catalytic site specificity of malarial ADAs permits methylthiocoformycin (MT-coformycin) to act as a Plasmodium-specific transition state analogue with low affinity for human ADA [Tyler, P. C., Taylor, E. A., Frohlich, R. G. G., and Schramm, V. L. (2007) J. Am. Chem. Soc. 129, 6872-6879]. The structural basis for MTA and MT-coformycin specificity in malarial ADAs is the subject of speculation [Larson, E. T., et al. (2008) J. Mol. Biol. 381, 975-988]. Here, the crystal structure of ADA from Plasmodium vivax (PvADA) in a complex with MT-coformycin reveals an unprecedented binding geometry for 5'-methylthioribosyl groups in the malarial ADAs. Compared to malarial ADA complexes with adenosine or deoxycoformycin, 5'-methylthioribosyl groups are rotated 130 degrees . A hydrogen bonding network between Asp172 and the 3'-hydroxyl of MT-coformycin is essential for recognition of the 5'-methylthioribosyl group. Water occupies the 5'-hydroxyl binding site when MT-coformycin is bound. Mutagenesis of Asp172 destroys the substrate specificity for MTA and MT-coformycin. Kinetic, mutagenic, and structural analyses of PvADA and kinetic analysis of five other Plasmodium ADAs establish the unique structural basis for its specificity for MTA and MT-coformycin. Plasmodium gallinaceum ADA does not use MTA as a substrate, is not inhibited by MT-coformycin, and is missing Asp172. Treatment of P. falciparum cultures with coformycin or MT-coformycin in the presence of MTA is effective in inhibiting parasite growth.
Assuntos
Adenosina Desaminase/metabolismo , Coformicina/análogos & derivados , Coformicina/química , Coformicina/metabolismo , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Adenosina Desaminase/química , Animais , Antimaláricos/química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Coformicina/farmacologia , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/prevenção & controle , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.