RESUMO
Plasmodium falciparum is the main causative agent of malaria, a deadly disease that mainly affects children under five years old. Artemisinin-based combination therapies have been pivotal in controlling the disease, but resistance has arisen in various regions, increasing the risk of treatment failure. The non-mevalonate pathway is essential for the isoprenoid synthesis in Plasmodium and provides several under-explored targets to be used in the discovery of new antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) is the first and rate-limiting enzyme of the pathway. Despite its importance, there are no structures available for any Plasmodium spp., due to the complex sequence which contains large regions of high disorder, making crystallisation a difficult task. In this manuscript, we use cryo-electron microscopy to solve the P. falciparum DXPS structure at a final resolution of 2.42 Å. Overall, the structure resembles other DXPS enzymes but includes a distinct N-terminal domain exclusive to the Plasmodium genus. Mutational studies show that destabilization of the cap domain interface negatively impacts protein stability and activity. Additionally, a density for the co-factor thiamine diphosphate is found in the active site. Our work highlights the potential of cryo-EM to obtain structures of P. falciparum proteins that are unfeasible by means of crystallography.
Assuntos
Microscopia Crioeletrônica , Plasmodium falciparum , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Pentosiltransferases/metabolismo , Pentosiltransferases/química , Pentosiltransferases/genética , Pentosiltransferases/ultraestrutura , Domínios Proteicos , Modelos Moleculares , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , TransferasesRESUMO
The retromer is a cellular structure that recruits and recycles proteins inside the cell. In mammalian and yeast, the retromer components have been widely studied, but very little in parasites. In yeast, it is formed by a SNX-BAR membrane remodeling heterodimer and the cargo selecting complex (CSC), composed by three proteins. One of them, the Vps26 protein, possesses a flexible and intrinsically disordered region (IDR), that facilitates interactions with other proteins and contributes to the retromer binding to the endosomal membrane. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, the retromer actively participates during the high mobility and phagocytosis of trophozoites, but the molecular details in these events, are almost unknown. Here, we studied the EhVps26 role in phagocytosis. Bioinformatic analyses of EhVps26 revealed a typical arrestin folding structure of the protein, and a long and charged IDR, as described in other systems. EhVps26 molecular dynamics simulations (MDS) allowed us to predict binding pockets for EhVps35, EhSNX3, and a PX domain-containing protein; these pockets were disorganized in a EhVps26 truncated version lacking the IDR. The AlphaFold2 software predicted the interaction of EhVps26 with EhVps35, EhVps29 and EhSNX3, in a model similar to the reported mammalian crystals. By confocal and transmission electron microscopy, EhVps26 was found in the trophozoites plasma membrane, cytosol, endosomes, and Golgi-like apparatus. During phagocytosis, it followed the erythrocytes pathway, probably participating in cargoes selection and recycling. Ehvps26 gene knocking down evidenced that the EhVps26 protein is necessary for efficient phagocytosis.
Assuntos
Biologia Computacional , Entamoeba histolytica , Fagocitose , Proteínas de Protozoários , Entamoeba histolytica/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Biologia Computacional/métodos , Humanos , Simulação de Dinâmica Molecular , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/química , Ligação Proteica , Sequência de Aminoácidos , Eritrócitos/parasitologia , Eritrócitos/metabolismoRESUMO
OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.
Assuntos
Entamoeba histolytica , Complexo de Endopeptidases do Proteassoma , Entamoeba histolytica/enzimologia , Entamoeba histolytica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Sítios de Ligação , Leupeptinas/farmacologia , Especificidade por Substrato , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Inibidores de Proteassoma/farmacologia , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Domínio Catalítico , Ligação Proteica , Modelos MolecularesRESUMO
Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.
Assuntos
Artemisininas , Plasmodium falciparum , Ligação Proteica , Proteínas de Protozoários , Proteínas Recombinantes , Artemisininas/farmacologia , Plasmodium falciparum/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Mutação , Polimorfismo de Nucleotídeo Único , Antimaláricos/farmacologia , Resistência a Medicamentos/genéticaRESUMO
Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.
Assuntos
Antagonistas do Ácido Fólico , Tetra-Hidrofolato Desidrogenase , Triazinas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Humanos , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/química , Triazinas/farmacologia , Triazinas/química , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Proguanil/farmacologia , Proguanil/química , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/enzimologia , Benzimidazóis/farmacologia , Benzimidazóis/química , Relação Estrutura-Atividade , Antiprotozoários/farmacologia , Antiprotozoários/química , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , OxirredutasesRESUMO
Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1-400 aa), Linker (246-446 aa) and Adh (444-687 aa) to evaluate their role in virulence. The TrophozBro11-400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker226-446 also increased the virulence properties, but with lower effect than the TrophozBro11-400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh444-687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes.
Assuntos
Entamoeba histolytica , Proteínas de Protozoários , Entamoeba histolytica/patogenicidade , Entamoeba histolytica/metabolismo , Animais , Camundongos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Humanos , Virulência , Fagocitose , Domínios Proteicos , Entamebíase/parasitologia , Entamebíase/metabolismo , Cricetinae , Trofozoítos/metabolismoRESUMO
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
New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.
Assuntos
Antimaláricos , Plasmodium falciparum , Plasmodium vivax , Proteômica , Proteínas de Protozoários , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/enzimologia , Plasmodium vivax/efeitos dos fármacos , Humanos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteômica/métodos , Aminopeptidases/metabolismo , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/químicaRESUMO
The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development.
Assuntos
Anticorpos Antiprotozoários , Antígenos de Protozoários , Epitopos , Vacinas Antimaláricas , Plasmodium vivax , Proteínas de Protozoários , Receptores de Superfície Celular , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Plasmodium vivax/imunologia , Animais , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/química , Epitopos/imunologia , Epitopos/química , Camundongos , Anticorpos Antiprotozoários/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Modelos Moleculares , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Camundongos Endogâmicos BALB CRESUMO
Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It bears a significant global health burden with limited treatment options, thus calling for the development of new and effective drugs. Certain trypanosomal metabolic enzymes have been suggested to be druggable and valid for subsequent inhibition. In this study, the crystal structure of glycerol kinase from T. cruzi, a key enzyme in glycerol metabolism in this parasite, is presented. Structural analysis allowed a detailed description of the glycerol binding pocket, while comparative assessment pinpointed a potential regulatory site which may serve as a target for selective inhibition. These findings advance the understanding of glycerol metabolism in eukaryotes and provide a solid basis for the future treatment of Chagas disease.
Assuntos
Doença de Chagas , Glicerol Quinase , Trypanosoma cruzi , Trypanosoma cruzi/enzimologia , Glicerol Quinase/química , Glicerol Quinase/metabolismo , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Cristalografia por Raios X , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Modelos Moleculares , Humanos , Sítios de Ligação , Glicerol/química , Conformação ProteicaRESUMO
Repetitive elements in DNA sequences are a hallmark of Apicomplexan protozoa. A genome-wide screening for Tandem Repeats was conducted in Toxoplasma gondii and related Coccidian parasites with a novel strategy to assess compositional bias. A conserved pattern of GC skew and purine-pyrimidine bias was observed. Compositional bias was also present at the protein level. Glutamic acid was the most abundant amino acid in the purine (GA) rich cluster, while Serine prevailed in pyrimidine (CT) rich cluster. Purine rich repeats, and consequently glutamic acid abundance, correlated with high scores for intrinsically disordered protein regions/domains. Finally, variability was established for repetitive regions within a well-known rhoptry antigen (ROP1) and an uncharacterized hypothetical protein with similar features. The approach we present could be useful to identify potential antigens bearing repetitive elements.
Assuntos
Proteínas de Protozoários , Sequências de Repetição em Tandem , Toxoplasma , Toxoplasma/genética , Sequências de Repetição em Tandem/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Genoma de Protozoário , Composição de BasesRESUMO
PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.
Assuntos
Insulisina , Plasmodium falciparum , Proteínas de Protozoários , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Insulisina/genética , Insulisina/química , Insulisina/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Insulina/química , Insulina/metabolismo , Insulina/genética , Zinco/química , Zinco/metabolismo , Genoma de Protozoário , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/isolamento & purificação , Expressão Gênica , Clonagem Molecular , Antimaláricos/química , Antimaláricos/farmacologia , Ciclosporina/química , Ciclosporina/farmacologiaRESUMO
Succinate dehydrogenase (SDH) is a protein complex that functions in the tricarboxylic acid cycle and the electron transport chain of mitochondria. In most eukaryotes, SDH is highly conserved and comprises the following four subunits: SdhA and SdhB form the catalytic core of the complex, while SdhC and SdhD anchor the complex in the membrane. Toxoplasma gondii is an apicomplexan parasite that infects one-third of humans worldwide. The genome of T. gondii encodes homologues of the catalytic subunits SdhA and SdhB, although the physiological role of the SDH complex in the parasite and the identity of the membrane-anchoring subunits are poorly understood. Here, we show that the SDH complex contributes to optimal proliferation and O2 consumption in the disease-causing tachyzoite stage of the T. gondii life cycle. We characterize a small membrane-bound subunit of the SDH complex called mitochondrial protein ookinete developmental defect (MPODD), which is conserved among myzozoans, a phylogenetic grouping that incorporates apicomplexan parasites and their closest free-living relatives. We demonstrate that TgMPODD is essential for SDH activity and plays a key role in attaching the TgSdhA and TgSdhB proteins to the membrane anchor of the complex. Our findings highlight a unique and important feature of mitochondrial energy metabolism in apicomplexan parasites and their relatives.
Assuntos
Proteínas de Protozoários , Succinato Desidrogenase , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Toxoplasma/enzimologia , Succinato Desidrogenase/metabolismo , Succinato Desidrogenase/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Humanos , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitocôndrias/metabolismo , Filogenia , AnimaisRESUMO
Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. It remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.
Assuntos
Cinetocoros , Proteínas de Protozoários , Cinetocoros/metabolismo , Cinetocoros/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Modelos Moleculares , Sequência de Aminoácidos , Filogenia , Ligação Proteica , Cristalografia por Raios X , Segregação de Cromossomos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genéticaRESUMO
Plasmodium species encode a unique set of six modular proteins named LCCL lectin domain adhesive-like proteins (LAPs) that operate as a complex and that are essential for malaria parasite transmission from mosquito to vertebrate. LAPs possess complex architectures obtained through unique assemblies of conserved domains associated with lipid, protein and carbohydrate interactions, including the name-defining LCCL domain. Here, we assessed the prevalence of Plasmodium LAP orthologues across eukaryotic life. Our findings show orthologous conservation in all apicomplexans, with lineage-specific repertoires acquired through differential lap gene loss and duplication. Besides Apicomplexa, LAPs are found in their closest relatives: the photosynthetic chromerids, which encode the broadest repertoire including a novel membrane-bound LCCL protein. LAPs are notably absent from other alveolate lineages (dinoflagellates, perkinsids and ciliates), but are encoded by predatory colponemids, a sister group to the alveolates. These results reveal that the LAPs are much older than previously thought and pre-date not only the Apicomplexa but the Alveolata altogether.
Assuntos
Evolução Molecular , Filogenia , Plasmodium , Proteínas de Protozoários , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Plasmodium/genética , Plasmodium/metabolismo , Alveolados/genética , Alveolados/metabolismo , Domínios Proteicos , Apicomplexa/genética , Apicomplexa/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lectinas/químicaRESUMO
Trypanosoma evansi, the causative agent of surra, is the most prevalent pathogenic salivarian trypanosome and affects the majority of domesticated and wild animals in endemic regions. This work aimed to analyze detergent-solubilized T. evansi proteins and identify potential diagnostic biomarkers for surra. Triton X-114-extracted membrane-enriched proteins (MEP) of T. evansi bloodstream forms were analyzed using a gel-free technique (LC-ESI-MS/MS). 247 proteins were identified following the MS analysis of three biological and technical replicates. Two of these proteins were predicted to have a GPI-anchor, 100 (40%) were predicted to have transmembrane domains, and 166 (67%) were predicted to be membrane-bound based on at least one of six features: location (WolfPSORT, DeepLoc-2.0, Protcomp-9.0), transmembrane, GPI, and gene ontology. It was predicted that 76 (30%) of proteins had membrane evidence. Typical membrane proteins for each organelle were identified, among them ISG families (64, 65, and 75 kDa), flagellar calcium-binding protein, 24 kDa calflagin, syntaxins and oligosaccharyltransferase some of which had previously been studied in other trypanosomatids. T. evansi lacks singletons and exclusive orthologous groups, whereas three distinct epitopes have been identified. Data are available via ProteomeXchange with identifier PXD040594. SIGNIFICANCE: Trypanosoma evansi is a highly prevalent parasite that induces a pathological condition known as "surra" in various species of ungulates across five continents. The infection gives rise to symptoms that are not pathognomonic, thereby posing challenges in its diagnosis and leading to substantial economic losses in the livestock industry. A significant challenge arises from the absence of a diagnostic test capable of distinguishing between Trypanosoma equiperdum and T. evansi, both of which are implicated in equine diseases. Therefore, there is a pressing need to conduct research on the biochemistry of the parasite in order to identify proteins that could potentially serve as targets for differential diagnosis or therapeutic interventions.
Assuntos
Proteômica , Proteínas de Protozoários , Trypanosoma , Tripanossomíase , Trypanosoma/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/análise , Proteômica/métodos , Animais , Tripanossomíase/diagnóstico , Tripanossomíase/parasitologia , Detergentes/química , Proteínas de Membrana/química , CavalosRESUMO
The apicomplexan intracellular parasite Toxoplasma gondii is a major food borne pathogen that is highly prevalent in the global population. The majority of the T. gondii proteome remains uncharacterized and the organization of proteins into complexes is unclear. To overcome this knowledge gap, we used a biochemical fractionation strategy to predict interactions by correlation profiling. To overcome the deficit of high-quality training data in non-model organisms, we complemented a supervised machine learning strategy, with an unsupervised approach, based on similarity network fusion. The resulting combined high confidence network, ToxoNet, comprises 2,063 interactions connecting 652 proteins. Clustering identifies 93 protein complexes. We identified clusters enriched in mitochondrial machinery that include previously uncharacterized proteins that likely represent novel adaptations to oxidative phosphorylation. Furthermore, complexes enriched in proteins localized to secretory organelles and the inner membrane complex, predict additional novel components representing novel targets for detailed functional characterization. We present ToxoNet as a publicly available resource with the expectation that it will help drive future hypotheses within the research community.
Assuntos
Mapas de Interação de Proteínas , Proteínas de Protozoários , Toxoplasma , Toxoplasma/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Mapas de Interação de Proteínas/fisiologia , Biologia Computacional , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Bases de Dados de Proteínas , Aprendizado de Máquina , Análise por ConglomeradosRESUMO
The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.
Assuntos
Apicoplastos , Plasmodium falciparum , Apicoplastos/metabolismo , Apicoplastos/genética , Plasmodium falciparum/genética , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/química , Exonucleases/metabolismo , Exonucleases/genética , Replicação do DNA , Animais , Mutagênese Insercional , Especificidade da Espécie , Humanos , DNA/metabolismo , DNA/químicaRESUMO
Aminoacyl-tRNA synthetases (aaRSs) play a central role in the translation of genetic code, serving as attractive drug targets. Within this family, the lysyl-tRNA synthetase (LysRS) constitutes a promising antimalarial target. ASP3026, an anaplastic lymphoma kinase (ALK) inhibitor was recently identified as a novel Plasmodium falciparum LysRS (PfLysRS) inhibitor. Here, based on cocrystal structures and biochemical experiments, we developed a series of ASP3026 analogues to improve the selectivity and potency of LysRS inhibition. The leading compound 36 showed a dissociation constant of 15.9 nM with PfLysRS. The inhibitory efficacy on PfLysRS and parasites has been enhanced. Covalent attachment of L-lysine to compound 36 resulted in compound 36K3, which exhibited further increased inhibitory activity against PfLysRS but significantly decreased activity against ALK. However, its inhibitory activity against parasites did not improve, suggesting potential future optimization directions. This study presents a new example of derivatization of kinase inhibitors repurposed to inhibit aaRS.
Assuntos
Quinase do Linfoma Anaplásico , Antimaláricos , Lisina-tRNA Ligase , Plasmodium falciparum , Inibidores de Proteínas Quinases , Plasmodium falciparum/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Lisina-tRNA Ligase/antagonistas & inibidores , Lisina-tRNA Ligase/metabolismo , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Quinase do Linfoma Anaplásico/antagonistas & inibidores , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Antimaláricos/farmacologia , Antimaláricos/química , Relação Estrutura-Atividade , Humanos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Parasitic diseases, predominantly prevalent in developing countries, are increasingly spreading to high-income nations due to shifting migration patterns. The World Health Organization (WHO) estimates approximately 300 million annual cases of giardiasis. The emergence of drug resistance and associated side effects necessitates urgent research to address this growing health concern. In this study, we evaluated over eleven thousand pharmacological compounds sourced from the FDA database to assess their impact on the TATA-binding protein (TBP) of the early diverging protist Giardia lamblia, which holds medical significance. We identified a selection of potential pharmacological compounds for combating this parasitic disease through in silico analysis, employing molecular modeling techniques such as homology modeling, molecular docking, and molecular dynamics simulations. Notably, our findings highlight compounds DB07352 and DB08399 as promising candidates for inhibiting the TBP of Giardia lamblia. Also, these compounds and DB15584 demonstrated high efficacy against trophozoites in vitro. In summary, this study identifies compounds with the potential to combat giardiasis, offering the prospect of specific therapies and providing a robust foundation for future research.