RESUMEN
Arginine metabolism mediated by arginases plays a critical role in cell and tissue function. The arginine hydrolysis is deeply involved in the urea cycle, which helps the kidney excrete ammonia from blood. Upregulation of arginases affects microenvironment stability due to the presence of excess urea in blood. To regulate the arginase activities properly, a synthetic peptide based on the structure of human arginase I was designed and assessed. Preliminary data shows it inhibits human arginase I and II with an IC50 of 2.4 ± 0.3 and 1.8 ± 0.1 mmol, respectively. Our kinetic analysis indicates the inhibition is not competitive with substrate - suggesting an allosteric mechanism. This result provides a step towards specific inhibitors design.
Asunto(s)
Arginasa/antagonistas & inhibidores , Péptidos/química , Regulación Alostérica , Arginasa/química , Arginasa/metabolismo , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Aspartato Carbamoiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Aspartato Carbamoiltransferasa/química , Aspartato Carbamoiltransferasa/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Simulación del Acoplamiento Molecular , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacosRESUMEN
Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.
Asunto(s)
Proteínas Fúngicas/metabolismo , Peroxinas/metabolismo , Pichia/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Proteínas Fúngicas/química , Modelos Moleculares , Peroxinas/química , Pichia/química , Unión Proteica , Conformación Proteica , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (PfMDH) are reported. In order to gain a deeper understanding of the function and role of PfMDH, the protein was purified to homogeneity. The purified protein crystallized in space group P1, with unit-cell parameters a = 72, b = 157, c = 159 Å, α = 105, ß = 101, γ = 95°. The resulting crystals diffracted to a maximal resolution of 2.24 Å and the structure has been solved by molecular replacement, with 16 monomers in the asymmetric unit. The 16 monomers are arranged into four independent tetramers, in agreement with previous reports demonstrating the tetrameric solution state of PfMDH. The X-ray structure of PfMDH is expected to clarify the differences in catalysis by PfMDH compared with other MDH family members and to provide a basis for the structure-based design of specific PfMDH inhibitors as well as general MDH inhibitors.
Asunto(s)
Malato Deshidrogenasa/química , Plasmodium falciparum/enzimología , Cristalización , Cristalografía por Rayos XRESUMEN
Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, was initially discovered as a cyanide detoxification enzyme. However, it was recently also found to be a genetic predictor of resistance to obesity-related type 2 diabetes. Diabetes type 2 is characterized by progressive loss of adequate ß-cell insulin secretion and onset of insulin resistance with increased insulin demand, which contributes to the development of hyperglycemia. Diabetic complications have been replicated in adult hyperglycemic zebrafish, including retinopathy, nephropathy, impaired wound healing, metabolic memory, and sensory axonal degeneration. Pancreatic and duodenal homeobox 1 (Pdx1) is a key component in pancreas development and mature beta cell function and survival. Pdx1 knockdown or knockout in zebrafish induces hyperglycemia and is accompanied by organ alterations similar to clinical diabetic retinopathy and diabetic nephropathy. Here we show that pdx1-knockdown zebrafish embryos and larvae survived after incubation with thiosulfate and no obvious morphological alterations were observed. Importantly, incubation with hTST and thiosulfate rescued the hyperglycemic phenotype in pdx1-knockdown zebrafish pronephros. Activation of the mitochondrial TST pathway might be a promising option for therapeutic intervention in diabetes and its organ complications.
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Diabetes Mellitus Tipo 2 , Hiperglucemia , Pronefro , Animales , Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/complicaciones , Modelos Teóricos , Pronefro/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Tiosulfatos , Pez Cebra/metabolismoRESUMEN
Introduction: Malaria is one of the most prevalent human infections worldwide with over 40% of the world's population living in malaria-endemic areas. In the absence of an effective vaccine, emergence of drug-resistant strains requires urgent drug development. Current methods applied to drug target validation, a crucial step in drug discovery, possess limitations in malaria. These constraints require the development of techniques capable of simplifying the validation of Plasmodial targets.Areas covered: The authors review the current state of the art in techniques used to validate drug targets in malaria, including our contribution - the protein interference assay (PIA) - as an additional tool in rapid in vivo target validation.Expert opinion: Each technique in this review has advantages and disadvantages, implying that future validation efforts should not focus on a single approach, but integrate multiple approaches. PIA is a significant addition to the current toolset of antimalarial validation. Validation of aspartate metabolism as a druggable pathway provided proof of concept of how oligomeric interfaces can be exploited to control specific activity in vivo. PIA has the potential to be applied not only to other enzymes/pathways of the malaria parasite but could, in principle, be extrapolated to other infectious diseases.
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Antimaláricos/farmacología , Descubrimiento de Drogas , Malaria/tratamiento farmacológico , Animales , Desarrollo de Medicamentos , Humanos , Malaria/parasitología , Plasmodium/aislamiento & purificación , Plasmodium/parasitología , Estudios de Validación como AsuntoRESUMEN
Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.
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Antimaláricos , Aspartato Carbamoiltransferasa/genética , Naftiridinas/farmacología , Plasmodium falciparum , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Ácido Aspártico , Plasmodium falciparum/enzimología , Plasmodium falciparum/genéticaRESUMEN
Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), also known as Rhodanese, is a mitochondrial enzyme which catalyzes the transfer of sulfur in several molecular pathways. After its initial identification as a cyanide detoxification enzyme, it was found that its functions also include sulfur metabolism, modification of ironsulfur clusters and the reduction of antioxidants glutathione and thioredoxin. TST deficiency was shown to be strongly related to the pathophysiology of metabolic diseases including diabetes and obesity. This review summarizes research related to the enzymatic properties and functions of TST, to then explore the association between the effects of TST on mitochondria and development of diseases such as diabetes and obesity.
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Antioxidantes/metabolismo , Enfermedades Metabólicas/genética , Azufre/metabolismo , Tiosulfato Azufretransferasa/genética , Glutatión/metabolismo , Humanos , Proteínas Hierro-Azufre/genética , Enfermedades Metabólicas/enzimología , Enfermedades Metabólicas/patología , Selenio/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tiosulfato Azufretransferasa/metabolismoRESUMEN
The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.
RESUMEN
Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.
Asunto(s)
Antimaláricos/química , Descubrimiento de Drogas , Malato Deshidrogenasa/química , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Antimaláricos/farmacología , Sitios de Unión , Secuencia Conservada , Expresión Génica , Humanos , Malato Deshidrogenasa/antagonistas & inhibidores , Malato Deshidrogenasa/genética , Modelos Moleculares , Conformación Molecular , Mutación , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Unión Proteica , Proteínas Recombinantes , Especificidad por SustratoRESUMEN
Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85â Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.
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Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Peroxinas/química , Peroxinas/metabolismo , Pichia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Cristalización/métodos , Proteínas Fúngicas/genética , Proteínas de Transporte de Membrana/genética , Peroxinas/genética , Pichia/genética , Unión Proteica/fisiología , Proteínas de Saccharomyces cerevisiae/genética , Difracción de Rayos X/métodosRESUMEN
BACKGROUND: The validation of drug targets in malaria and other human diseases remains a highly difficult and laborious process. In the vast majority of cases, highly specific small molecule tools to inhibit a proteins function in vivo are simply not available. Additionally, the use of genetic tools in the analysis of malarial pathways is challenging. These issues result in difficulties in specifically modulating a hypothetical drug target's function in vivo. OBJECTIVE: The current "toolbox" of various methods and techniques to identify a protein's function in vivo remains very limited and there is a pressing need for expansion. New approaches are urgently required to support target validation in the drug discovery process. METHOD: Oligomerisation is the natural assembly of multiple copies of a single protein into one object and this self-assembly is present in more than half of all protein structures. Thus, oligomerisation plays a central role in the generation of functional biomolecules. A key feature of oligomerisation is that the oligomeric interfaces between the individual parts of the final assembly are highly specific. However, these interfaces have not yet been systematically explored or exploited to dissect biochemical pathways in vivo. RESULTS AND CONCLUSION: This mini review will describe the current state of the antimalarial toolset as well as the potentially druggable malarial pathways. A specific focus is drawn to the initial efforts to exploit oligomerisation surfaces in drug target validation. As alternative to the conventional methods, Protein Interference Assay (PIA) can be used for specific distortion of the target protein function and pathway assessment in vivo.
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Antimaláricos/farmacología , Sistemas de Liberación de Medicamentos , Malaria Falciparum/tratamiento farmacológico , Proteínas/metabolismo , Antimaláricos/uso terapéutico , Intercambio Genético , Descubrimiento de Drogas , Técnicas de Silenciamiento del Gen , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genéticaRESUMEN
The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.
Asunto(s)
Proteína Coatómero/química , Cristalografía por Rayos X , Humanos , Conformación ProteicaRESUMEN
The de novo pyrimidine-biosynthesis pathway of Plasmodium falciparum is a promising target for antimalarial drug discovery. The parasite requires a supply of purines and pyrimidines for growth and proliferation and is unable to take up pyrimidines from the host. Direct (or indirect) inhibition of de novo pyrimidine biosynthesis via dihydroorotate dehydrogenase (PfDHODH), the fourth enzyme of the pathway, has already been shown to be lethal to the parasite. In the second step of the plasmodial pyrimidine-synthesis pathway, aspartate and carbamoyl phosphate are condensed to N-carbamoyl-L-aspartate and inorganic phosphate by aspartate transcarbamoylase (PfATC). In this paper, the 2.5â Å resolution crystal structure of PfATC is reported. The space group of the PfATC crystals was determined to be monoclinic P21, with unit-cell parameters a = 87.0, b = 103.8, c = 87.1â Å, α = 90.0, ß = 117.7, γ = 90.0°. The presented PfATC model shares a high degree of homology with the catalytic domain of Escherichia coli ATC. There is as yet no evidence of the existence of a regulatory domain in PfATC. Similarly to E. coli ATC, PfATC was modelled as a homotrimer in which each of the three active sites is formed at the oligomeric interface. Each active site comprises residues from two adjacent subunits in the trimer with a high degree of evolutional conservation. Here, the activity loss owing to mutagenesis of the key active-site residues is also described.
Asunto(s)
Aspartato Carbamoiltransferasa/química , Ácido Aspártico/química , Carbamoil Fosfato/química , Plasmodium falciparum/química , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Aspartato Carbamoiltransferasa/genética , Aspartato Carbamoiltransferasa/metabolismo , Ácido Aspártico/metabolismo , Sitios de Unión , Carbamoil Fosfato/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Modelos Moleculares , Mutación , Plásmidos/química , Plásmidos/metabolismo , Plasmodium falciparum/enzimología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Pyridoxal kinases (PdxK) catalyze the phosphorylation of vitamin B6 precursors. Thus, these enzymes are an essential part of many metabolic processes in all organisms. The protozoan parasite Plasmodium falciparum (the main causative agent of Malaria tropica) possesses a unique de novo B6-biosynthesis pathway in addition to a interconversion pathway based on the activity of plasmodial PdxK (PfPdxK). The role of PdxK in B6 salvage has prompted previous authors to suggest PdxK as a promising target for structure-based antimalarial drug design. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of PfPdxK are reported. PfPdxK crystals have been grown in space group P21, with unit-cell parameters a=52.7, b=62.0, c=93.7â Å, ß=95°. A data set has been collected to 2â Å resolution and an initial molecular-replacement solution is described.