RESUMO
The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico , GMP Cíclico , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Fosforilação , GMP Cíclico/metabolismo , Malária/parasitologia , Malária/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Animais , Plasmodium falciparum/metabolismo , Plasmodium falciparum/genética , Humanos , Transdução de Sinais , Eritrócitos/parasitologia , Eritrócitos/metabolismoRESUMO
Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein ß-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in ß-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent ß-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.
Assuntos
Antimaláricos/farmacologia , Cisteína Proteases/metabolismo , Plasmodium falciparum/metabolismo , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/metabolismo , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Humanos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Proteólise , Proteínas de Protozoários/antagonistas & inibidores , Serina Proteases/metabolismo , Espectrina/metabolismoRESUMO
Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.
Assuntos
Antimaláricos/farmacologia , Ácidos Borônicos/farmacologia , Peptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/química , Subtilisinas/química , Antimaláricos/síntese química , Sítios de Ligação , Ácidos Borônicos/síntese química , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Expressão Gênica , Humanos , Cinética , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/fisiologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/síntese química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Subtilisinas/antagonistas & inibidores , Subtilisinas/genética , Subtilisinas/metabolismo , TermodinâmicaRESUMO
Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACß) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.
Assuntos
AMP Cíclico/metabolismo , Interações Hospedeiro-Parasita , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Parasitos/metabolismo , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Parasitos/enzimologia , Parasitos/crescimento & desenvolvimento , Parasitos/ultraestrutura , Fosfoproteínas/metabolismo , Fosforilação , Fosfosserina/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/patogenicidade , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.
Assuntos
Malária Falciparum/genética , Plasmodium falciparum/genética , Serina Proteases/química , Subtilisina/genética , Sequência de Aminoácidos/genética , Animais , Eritrócitos/parasitologia , Genoma/genética , Humanos , Estágios do Ciclo de Vida/genética , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Plasmodium falciparum/patogenicidade , Proteólise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Serina Proteases/genética , Subtilisina/química , Vacúolos/parasitologiaRESUMO
The malaria parasite Plasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole (PV). The most abundant P. falciparumâ PV protein, called SERA5, is essential in blood stages and possesses a papain-like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA5 possesses a Ser residue (Ser596) at the position of the canonical catalytic Cys of papain-like proteases, and the function of SERA5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the Ser596-containing parasite-derived or recombinant protein. However, substitution of Ser596 with a Cys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA5 can bind peptides. Using targeted homologous recombination in P. falciparum, we substituted Ser596 with Ala with no phenotypic consequences, proving that SERA5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA5 with an affinity-purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C-terminally tag the SERA5 gene, or replace Ser596 with a bulky Arg residue. Our findings show that SERA5 plays an indispensable but non-enzymatic role in the P. falciparum blood-stage life cycle.
Assuntos
Antígenos de Protozoários/metabolismo , Malária Falciparum/parasitologia , Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Motivos de Aminoácidos , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Humanos , Estágios do Ciclo de Vida , Malária Falciparum/sangue , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Reprodução AssexuadaRESUMO
The malaria merozoite invades erythrocytes in the vertebrate host. Iterative rounds of asexual intraerythrocytic replication result in disease. Proteases play pivotal roles in erythrocyte invasion, but little is understood about their mode of action. The Plasmodium falciparum malaria merozoite surface sheddase, PfSUB2, is one such poorly characterized example. We have examined the molecular determinants that underlie the mechanisms by which PfSUB2 is trafficked initially to invasion-associated apical organelles (micronemes) and then across the surface of the free merozoite. We show that authentic promoter activity is important for correct localization of PfSUB2, likely requiring canonical features within the intergenic region 5' of the pfsub2 locus. We further demonstrate that trafficking of PfSUB2 beyond an early compartment in the secretory pathway requires autocatalytic protease activity. Finally, we show that the PfSUB2 transmembrane domain is required for microneme targeting, while the cytoplasmic domain is essential for surface translocation of the protease to the parasite posterior following discharge from micronemes. The interplay of pre- and post-translational regulatory elements that coordinate subcellular trafficking of PfSUB2 provides the parasite with exquisite control over enzyme-substrate interactions.
Assuntos
Epitopos/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Subtilisinas/metabolismo , Epitopos/genética , Epitopos/imunologia , Eritrócitos/imunologia , Eritrócitos/metabolismo , Expressão Gênica/genética , Expressão Gênica/imunologia , Malária Falciparum/genética , Malária Falciparum/imunologia , Merozoítos/imunologia , Merozoítos/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Transporte Proteico/genética , Transporte Proteico/imunologia , Proteólise , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Subtilisinas/genética , Subtilisinas/imunologiaRESUMO
The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Merozoítos/fisiologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/metabolismo , Transdução de Sinais/fisiologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Organelas/metabolismoRESUMO
Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.
Assuntos
Membrana Celular/metabolismo , Eritrócitos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico Ativo , Membrana Celular/genética , Eritrócitos/parasitologia , Células HL-60 , Humanos , Lipídeos de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Vacúolos/metabolismo , Vacúolos/parasitologiaRESUMO
BACKGROUND: The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS: Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS: We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS: Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE: Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.
Assuntos
Ácido Aspártico Endopeptidases , Plasmodium falciparum , Proteínas de Protozoários , Plasmodium falciparum/enzimologia , Plasmodium falciparum/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Ácido Aspártico Endopeptidases/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Proteólise , Humanos , Subtilisinas/metabolismo , Domínio Catalítico , Domínios Proteicos , Malária Falciparum/parasitologia , Malária Falciparum/metabolismo , Eritrócitos/parasitologia , Eritrócitos/metabolismoRESUMO
Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.
Assuntos
Antimaláricos , Ácidos Borônicos , Plasmodium falciparum , Complexo de Endopeptidases do Proteassoma , Proteínas de Protozoários , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Humanos , Relação Estrutura-Atividade , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Ácidos Borônicos/síntese química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Antimaláricos/farmacologia , Antimaláricos/química , Antimaláricos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/síntese química , SubtilisinasRESUMO
The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte.
Assuntos
Cisteína Proteases/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Cisteína Proteases/genética , Ativação Enzimática , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/fisiologia , Proteólise , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Subtilisinas/genética , Vacúolos/enzimologia , Vacúolos/ultraestruturaRESUMO
The malaria parasite Plasmodium falciparum replicates within an intraerythrocytic parasitophorous vacuole (PV). Rupture of the host cell allows release (egress) of daughter merozoites, which invade fresh erythrocytes. We previously showed that a subtilisin-like protease called PfSUB1 regulates egress by being discharged into the PV in the final stages of merozoite development to proteolytically modify the SERA family of papain-like proteins. Here, we report that PfSUB1 has a further role in 'priming' the merozoite prior to invasion. The major protein complex on the merozoite surface comprises three proteins called merozoite surface protein 1 (MSP1), MSP6 and MSP7. We show that just before egress, all undergo proteolytic maturation by PfSUB1. Inhibition of PfSUB1 activity results in the accumulation of unprocessed MSPs on the merozoite surface, and erythrocyte invasion is significantly reduced. We propose that PfSUB1 is a multifunctional processing protease with an essential role in both egress of the malaria merozoite and remodelling of its surface in preparation for erythrocyte invasion.
Assuntos
Eritrócitos/parasitologia , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Parasitos/enzimologia , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Consenso , Eritrócitos/efeitos dos fármacos , Humanos , Merozoítos/enzimologia , Dados de Sequência Molecular , Parasitos/efeitos dos fármacos , Peptídeos/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Subtilisinas/antagonistas & inibidores , Subtilisinas/químicaRESUMO
The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.
Assuntos
Anticorpos Antiprotozoários/farmacologia , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Recombinação Homóloga , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
PfSUB1, a subtilisin-like protease of the human malaria parasite Plasmodium falciparum, is known to play important roles during the life cycle of the parasite and has emerged as a promising antimalarial drug target. In order to provide a detailed understanding of the origin of binding determinants of PfSUB1 substrates, we performed molecular dynamics simulations in combination with MM-GBSA free energy calculations using a homology model of PfSUB1 in complex with different substrate peptides. Key interactions, as well as residues that potentially make a major contribution to the binding free energy, are identified at the prime and nonprime side of the scissile bond and comprise peptide residues P4 to P2'. This finding stresses the requirement for peptide substrates to interact with both prime and nonprime side residues of the PfSUB1 binding site. Analyzing the energetic contributions of individual amino acids within the peptide-PfSUB1 complexes indicated that van der Waals interactions and the nonpolar part of solvation energy dictate the binding strength of the peptides and that the most favorable interactions are formed by peptide residues P4 and P1. Hot spot residues identified in PfSUB1 are dispersed over the entire binding site, but clustered areas of hot spots also exist and suggest that either the S4-S2 or the S1-S2' binding site should be exploited in efforts to design small molecule inhibitors. The results are discussed with respect to which binding determinants are specific to PfSUB1 and, therefore, might allow binding selectivity to be obtained.
Assuntos
Plasmodium falciparum/química , Proteínas de Protozoários/química , Subtilisinas/química , Sítios de Ligação , Eletroquímica , Ligação de Hidrogênio , Modelos Moleculares , Peptídeos/química , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.
Assuntos
Antimaláricos , Peptidomiméticos , Camundongos , Animais , Antimaláricos/farmacologia , Antimaláricos/metabolismo , Peptidomiméticos/farmacologia , Peptidomiméticos/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/metabolismo , Ácido Aspártico Endopeptidases , Plasmodium falciparum/metabolismo , Proteínas de ProtozoáriosRESUMO
Widespread resistance to many antimalarial therapies currently in use stresses the need for the discovery of new classes of drugs with new modes of action. The subtilisin-like serine protease SUB1 controls egress of malaria parasites (merozoites) from the parasite-infected red blood cell. As such, SUB1 is considered a prospective target for drugs designed to interrupt the asexual blood stage life cycle of the malaria parasite. Inhibitors of SUB1 have potential as wide-spectrum antimalarial drugs, as a single orthologue of SUB1 is found in the genomes of all known Plasmodium species. This mini-perspective provides a short overview of the function and structure of SUB1 and summarizes all of the published SUB1 inhibitors. The inhibitors are classified by the methods of their discovery, including both rational design and screening.
Assuntos
Antimaláricos , Malária , Plasmodium , Antimaláricos/química , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Eritrócitos/metabolismo , Humanos , Malária/tratamento farmacológico , Malária/parasitologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Serina , Inibidores de Serina Proteinase , Subtilisinas/química , Subtilisinas/metabolismoRESUMO
The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here, we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.
Malaria kills over half a million people every year worldwide. A single-celled parasite called Plasmodium falciparum is responsible for the most lethal form of the disease. This malaria-causing agent is carried by mosquitos which transmit the parasite to humans through their bite. Once in the bloodstream, the parasite enters red blood cells and starts to replicate so it can go on to infect other cells. Like our cells, P. falciparum is surrounded by a membrane, and further membranes surround a number of its internal compartments. To make these protective coats, the parasite has to gather a nutrient called choline to form an important building block in the membrane. The parasite gets most of its choline by absorbing and digesting a molecule known as lysoPC found in the bloodstream of its host. However, it was unclear precisely how the parasite achieves this. To address this question, Ramaprasad, Burda et al. used genetic and metabolomic approaches to study how P. falciparum breaks down lysoPC. The experiments found that mutant parasites that are unable to make an enzyme called GDPD were able to infect red blood cells, but failed to grow properly once inside the cells. The mutant parasites took up less choline and, as a result, also made fewer membrane building blocks. The team were able to rescue the mutant parasites by supplying them with large quantities of choline, which allowed them to resume growing. Taken together, the findings of Ramaprasad, Burda et al. suggest that P. falciparum uses GDPD to extract choline from lysoPC when it is living in red blood cells. More and more P. falciparum parasites are becoming resistant to many of the drugs currently being used to treat malaria. One solution is to develop new therapies that target different molecules in the parasite. Since it performs such a vital role, GDPD may have the potential to be a future drug target.
Assuntos
Malária Falciparum , Malária , Parasitos , Animais , Parasitos/metabolismo , Colina/metabolismo , Plasmodium falciparum/genética , Glicerilfosforilcolina/metabolismo , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target.
Assuntos
Peptídeo Hidrolases/metabolismo , Plasmodium falciparum/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Estágios do Ciclo de Vida/fisiologia , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Plasmodium falciparum/crescimento & desenvolvimento , Estrutura Terciária de ProteínaRESUMO
Host cell invasion by apicomplexan pathogens such as the malaria parasite Plasmodium spp. and Toxoplasma gondii involves discharge of proteins from secretory organelles called micronemes and rhoptries. In Toxoplasma a protein complex comprising the microneme apical membrane antigen 1 (AMA1), two rhoptry neck proteins, and a protein called Ts4705, localises to the moving junction, a region of close apposition between parasite and host cell during invasion. Antibodies against AMA1 prevent invasion and are protective in vivo, and so AMA1 is of widespread interest as a malaria vaccine candidate. Here we report that the AMA1 complex identified in Toxoplasma is conserved in Plasmodium falciparum. We demonstrate that the invasion-inhibitory monoclonal antibody (mAb) 4G2, which recognises P. falciparum AMA1 (PfAMA1), cannot bind when PfAMA1 is in a complex with its partner proteins. We further show that a single completely conserved PfAMA1 residue, Tyr251, lying within a conserved hydrophobic groove adjacent to the mAb 4G2 epitope, is required for complex formation. We propose that mAb 4G2 inhibits invasion by preventing PfAMA1 from interacting with other components of the invasion complex. Our findings should aid the rational design of subunit malaria vaccines based on PfAMA1.