RESUMEN
Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Transducción de Señal/genética , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Eritrocitos/parasitología , Regulación del Desarrollo de la Expresión Génica , Humanos , Hidrólisis , Merozoítos/enzimología , Merozoítos/genética , Merozoítos/crecimiento & desarrollo , Fosfoproteínas/clasificación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Esquizontes/enzimología , Esquizontes/genética , Esquizontes/crecimiento & desarrollo , Factores de TiempoRESUMEN
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.
Asunto(s)
AMP Cíclico/metabolismo , Interacciones Huésped-Parásitos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Parásitos/metabolismo , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Parásitos/enzimología , Parásitos/crecimiento & desarrollo , Parásitos/ultraestructura , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
The entry of viruses into host cells is one of the key processes of infection. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show, utilizing an in vitro liposome penetration assay and cell biology, that bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosome-specific lipid lysobisphosphatidic acid for productive membrane penetration and viral entry. Further, we provide preliminary evidence that lipid lysobisphosphatidic acid facilitates pore expansion during membrane penetration, suggesting a mechanism for lipid factor requirement of BTV. This finding indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.
Asunto(s)
Virus de la Lengua Azul/metabolismo , Cápside/metabolismo , Endosomas/metabolismo , Lisofosfolípidos/metabolismo , Monoglicéridos/metabolismo , Internalización del Virus , Animales , Western Blotting , Virus de la Lengua Azul/genética , Virus de la Lengua Azul/fisiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Endocitosis , Endosomas/virología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Liposomas/metabolismo , Microscopía Confocal , Mutación , Células Sf9 , SpodopteraRESUMEN
In model organisms, type IV ATPases (P4-ATPases) require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite Plasmodium falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (termed A, B, and C) encoded by P. falciparum are required for GCα function. Using gene tagging and conditional gene disruption, we demonstrate that CDC50B and CDC50C but not CDC50A are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GCα whereas CDC50C is the binding partner of another putative P4-ATPase, phospholipid-transporting ATPase 2 (ATP2). Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GCα for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell hemoglobin and consequently hemozoin formation. IMPORTANCE Malaria morbidity arises due to successive rounds of replication of Plasmodium parasites within red blood cells. Mature daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that a unique bifunctional guanylyl cyclase, GCα, initiates egress by synthesis of cGMP. GCα has an N-terminal P4-ATPase domain of unknown function. In model organisms, P4-ATPases function through interaction with a CDC50 partner protein. Here, we investigate the role of CDC50 orthologues in P. falciparum and show that GCα binds CDC50B, an interaction that regulates egress efficiency. We also find that CDC50C is essential and binds a putative P4-ATPase, ATP2, in a complex that influences endocytosis of host hemaglobin. Our results highlight the heterogenous and critical role of CDC50 proteins in P. falciparum.
Asunto(s)
Malaria Falciparum , Malaria , Adenosina Trifosfatasas/genética , Animales , Eritrocitos/parasitología , Guanilato Ciclasa , Humanos , Malaria Falciparum/parasitología , Merozoítos/fisiología , Fosfolípidos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trofozoítos/metabolismoRESUMEN
1. Insulin-resistant states are commonly associated with a significantly higher risk of atherosclerosis. Insulin resistance has also been correlated with enhanced very low-density lipoprotein (VLDL) production, which is exacerbated by increased intestinal lipid synthesis and insulin-stimulated de novo lipogenesis. Microsomal triglyceride transfer protein (MTP) catalyses the critical step in the synthesis and secretion of VLDL and chylomicrons. The purpose of the present study was to test the hypothesis that chronic inhibition of MTP with a small molecule inhibitor would improve insulin sensitivity and reduce atherogenic risk in a genetic model of diabetic dyslipidaemia. 2. The in vivo activity of BMS-201038, a potent inhibitor of MTP, was evaluated in a model of hypertriglyceridemia induced by Triton WR1339 and corn oil in Zucker fatty rats. Triglyceride secretion rate was significantly reduced by a single dose of BMS-201038 by 35% at 0.3 mg/kg and 47% at 1 mg/kg, respectively. 3. Another group of Zucker fatty rats was dosed orally with BMS-201038 (0.3 and 1 mg/kg) for 14 days. Serum levels of triglycerides were reduced by 71% and 87%, non-esterified free fatty acids were reduced by 33% and 40%, and low-density lipoproteins by 26% and 29%, by 0.3 mg/kg and 1 mg/kg dose of BMS-201038, respectively. These serum lipid changes were accompanied by significant improvements in glucose tolerance and insulin sensitivity. In addition, lipid peroxidation in liver was reduced by 59% and 61%, and superoxide dismutase activity was increased by 11% and 45% by 0.3 mg/kg and 1 mg/kg dose of BMS-201038, respectively. Similar beneficial changes were found in aorta as well. 4. The present study provides evidence that inhibition of MTP with a small molecule inhibitor significantly improves dyslipidaemia associated with insulin resistance and reduces the atherosclerotic risk.
Asunto(s)
Aterosclerosis/etiología , Aterosclerosis/prevención & control , Bencimidazoles/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Resistencia a la Insulina , Obesidad/complicaciones , Animales , Anticolesterolemiantes/administración & dosificación , Anticolesterolemiantes/farmacología , Aterosclerosis/complicaciones , Bencimidazoles/administración & dosificación , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Metabolismo/efectos de los fármacos , Obesidad/tratamiento farmacológico , Ratas , Ratas Zucker , Factores de RiesgoRESUMEN
The composition of topical and transdermal formulations is known to determine the rate and the extent of drug delivery to and through the skin. However, to date, the role of excipients in these formulations on skin delivery of actives has received little attention from scientists in the field. Monitoring skin absorption of both drug and vehicle may provide insights into the mechanism by which excipients promote permeation and may facilitate the design of effective and safer products. Previously, we have investigated the use of quantitative Confocal Raman Spectroscopy (CRS) to investigate the delivery of an active to the skin, and we also reported the first fully quantitative study that compared this method with the well-established in vitro permeation test (IVPT) model. To further explore the potential of quantitative CRS in assessing topical delivery, the present work investigated the effects of commonly used excipients on the percutaneous absorption of a model drug, ibuprofen (IBU). Permeation of IBU and selected solvents following finite dose applications to human skin was determined in vitro and in vivo by Franz diffusion studies and quantitative CRS, respectively. The solvents used were propylene glycol (PG), dipropylene glycol (DPG), tripropylene glycol (TPG), and polyethylene glycol 300 (PEG 300). Overall, the cumulative amounts of IBU that permeated at 24 h in vitro were similar for PG, DPG, and TPG (p > 0.05). These three vehicles outperformed PEG 300 (p < 0.05) in terms of drug delivery. Concerning the vehicles, the rank order for in vitro skin permeation was DPG ≥ PG > TPG, while PEG 300 did not permeate the skin. A linear relationship between maximum vehicle and IBU flux in vitro was found, with a correlation coefficient (R2) of 0.95. When comparing in vitro with in vivo data, a positive in vitro-in vivo (IVIV) correlation between the cumulative permeation of IBU in vitro and the total amount of IBU that penetrated the stratum corneum (SC) in vivo was observed, with a Pearson correlation coefficient (R2) of 0.90. A strong IVIV correlation, R2 = 0.82, was found following the linear regression of the cumulative number of solvents permeated in vitro and the corresponding skin uptake in vivo measured with CRS. This is the first study to correlate in vivo permeation of solvents measured by CRS with data obtained by in vitro diffusion studies. The IVIV correlations suggest that CRS is a powerful tool for profiling drug and vehicle delivery from dermal formulations. Future studies will examine additional excipients with varying physicochemical properties. Ultimately, these findings are expected to lead to new approaches for the design, evaluation, and optimization of formulations that target actives to and through the skin.
RESUMEN
Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (Plasmodium spp), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.IMPORTANCE The clinical manifestations of malaria arise due to successive rounds of replication of Plasmodium parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of Plasmodium falciparum and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , GMP Cíclico/biosíntesis , Eritrocitos/parasitología , Guanilato Ciclasa/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Transducción de Señal , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/genética , GMP Cíclico/genética , Guanilato Ciclasa/genética , Humanos , Malaria/parasitología , Merozoítos/fisiología , Plasmodium falciparum/genética , Dominios Proteicos , Proteínas Protozoarias/genéticaRESUMEN
Human adaptive natural killer (NK) cells have diminished reliance on accessory cytokines for their activation whilst being efficiently activated by infected host cells in conjunction with pathogen specific antibodies. Here, we show that potent antibody-dependent NK cell responses are induced by Plasmodium falciparum infected erythrocytes (iRBC) in peripheral blood mononuclear cells (PBMC) from malaria-exposed Gambian individuals in the presence of autologous sera, which are absent in those from malaria-naïve UK individuals. However, malaria hyper-immune serum promotes rapid NK cell responses to iRBC in cells from both Gambian and UK individuals. Among Gambians, highly differentiated, adaptive (CD56dimFcεR1γ-CD57+) NK cells dominate both antibody-dependent NK cell IFN-γ responses and degranulation responses, whereas among UK individuals these responses are predominantly found within canonical, highly differentiated CD56dimFcεR1γ+CD57+ NK cells. Indeed, overall frequencies of adaptive, FcεR1γ-CD57+ NK cells are significantly higher among Gambian donors compared to HCMV-infected and HCMV-uninfected UK adults. Among UK individuals, antibody-dependent NK cell IFN-γ responses to iRBC were dependent on IL-18 whereas among Gambians, the predominant adaptive FcεR1γ- NK cell response was IL-18 (and accessory cell) independent (although the lower frequency response of canonical FcεR1γ NK cells did rely on this cytokine).
Asunto(s)
Interleucina-18/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Inmunidad Adaptativa/inmunología , Adulto , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Femenino , Humanos , Inmunidad Innata/inmunología , MasculinoRESUMEN
Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in long term in vitro culture with human red blood cells (RBCs). Its closer ancestry to other non-falciparum human malaria parasites, more balanced AT-content, larger merozoites and higher transfection efficiencies, gives P. knowlesi some key advantages over P. falciparum for the study of malaria parasite cell/molecular biology. Here, we describe the generation of marker-free CRISPR gene-edited P. knowlesi parasites, the fast and scalable production of transfection constructs and analysis of transfection efficiencies. Our protocol allows rapid, reliable and unlimited rounds of genome editing in P. knowlesi requiring only a single recyclable selection marker.
RESUMEN
Cyclic adenosine monophosphate (cAMP) is an important signalling molecule across evolution, but until recently there was little information on its role in malaria parasites. Advances in gene editing - in particular conditional genetic approaches and mass spectrometry have paved the way for characterisation of the key components of the cAMP signalling pathway in malaria parasites. This has revealed that cAMP signalling plays a critical role in invasion of host red blood cells by Plasmodium falciparum merozoites through regulating the phosphorylation of key parasite proteins by the cAMP-dependent protein kinase (PKA). These insights will help us to investigate parasite cAMP signalling as a target for novel antimalarial drugs.
Asunto(s)
AMP Cíclico/metabolismo , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Humanos , Malaria Falciparum/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de SeñalRESUMEN
The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the µ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2µ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2µ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2µ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2µ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the µ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
Asunto(s)
Antimaláricos/farmacología , Artemisininas/metabolismo , Clatrina/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Esquizontes/efectos de los fármacos , Esquizontes/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Resistencia a Medicamentos , Endocitosis/fisiología , Técnicas de Inactivación de Genes , Proteínas de la Membrana/metabolismo , Organismos Modificados Genéticamente , Plasmodium falciparum/genética , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esquizontes/genéticaRESUMEN
In vitro permeation studies using nail clippings or nail plates are commonly used in the development of transungual formulations. However, there are ethical, safety and cost issues associated with sourcing such tissues. Herein, we describe a preliminary approach is described for the design and manufacture of a human nail model surrogate based on 3D printing. To evaluate these 3D printed constructs, nails were mounted in conventional glass Franz cells and a commercial antifungal lacquer formulation containing ciclopirox olamine was applied daily to the surrogate printed surfaces for a period of 14 days. On days 8 and 14, the surfaces of the 3D printed nails were washed with ethanol to remove excess formulation. Confocal Raman spectroscopy (CRS) was used to profile the drug in the 3D printed nail. At the end of the Franz cell studies, no drug was observed in the receptor phase. CRS studies confirmed penetration of the active into the model nails with reproducible depth profiles. Our ongoing work is focused on synthesising commercial and non-commercial printable resins that can replicate the physical and chemical characteristics of the human nail. This will allow further evaluation of actives for ungual therapy and advance the development of the surrogate nail tissue model.
RESUMEN
Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted P. knowlesi strain and define parameters for optimal homology-driven repair. We establish a scalable protocol for the production of repair templates by PCR and demonstrate the flexibility of the system by tagging proteins with distinct cellular localisations. Using iterative rounds of genome-editing we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.
Asunto(s)
Edición Génica/métodos , Malaria Vivax/genética , Parásitos/genética , Plasmodium knowlesi/genética , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/metabolismo , Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Humanos , Malaria/inmunología , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/inmunología , Malaria Vivax/inmunología , Parásitos/inmunología , Parásitos/fisiología , Plasmodium knowlesi/inmunología , Plasmodium knowlesi/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismoRESUMEN
The cyclic nucleotides 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) are intracellular messengers found in most animal cell types. They usually mediate an extracellular stimulus to drive a change in cell function through activation of their respective cyclic nucleotide-dependent protein kinases, PKA and PKG. The enzymatic components of the malaria parasite cyclic nucleotide signalling pathways have been identified, and the genetic and biochemical studies of these enzymes carried out to date are reviewed herein. What has become very clear is that cyclic nucleotides play vital roles in controlling every stage of the complex malaria parasite life cycle. Our understanding of the involvement of cyclic nucleotide signalling in orchestrating the complex biology of malaria parasites is still in its infancy, but the recent advances in our genetic tools and the increasing interest in signalling will deliver more rapid progress in the coming years.
Asunto(s)
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Plasmodium/metabolismo , Transducción de Señal , Proteínas Quinasas Reguladas por Nucleótidos Cíclicos/genética , Proteínas Quinasas Reguladas por Nucleótidos Cíclicos/metabolismo , Estadios del Ciclo de Vida , Plasmodium/crecimiento & desarrollo , Plasmodium/patogenicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Human skin remains the membrane of choice when conducting in vitro studies to determine dermal penetration of active pharmaceutical ingredients or xenobiotics. However there are ethical and safety issues associated with obtaining human tissue. For these reasons synthetic membranes, cell culture models or in silico predictive algorithms have been researched intensively as alternative approaches to predict dermal exposure in man. Porcine skin has also been recommended as an acceptable surrogate for topical or transdermal delivery research. Here we examine the in vitro permeation of a model active, ibuprofen, using human or porcine skin, as well as the Parallel Artificial Membrane Permeation Assay (PAMPA) model and silicone membrane. Finite dose studies were conducted in all models using commercial ibuprofen formulations and simple volatile ibuprofen solutions. The dose applied in the PAMPA model was also varied in order to determine the amount of applied formulation which best simulates typical amounts of topical products applied by patients or consumers. Permeation studies were conducted up to 6h for PAMPA and silicone and up to 48h for human and porcine skin. Cumulative amounts permeated at 6h were comparable for PAMPA and silicone, ranging from 91 to 136µg/cm(2) across the range of formulations studied. At 48h, maximum ibuprofen permeation in human skin ranged from 11 to 38µg/cm(2) and corresponding values in porcine skin were 59-81µg/cm(2). A dose of 1µL was confirmed as appropriate for finite dose studies in the PAMPA model. The formulation which delivered the greatest amount of ibuprofen in human skin was also significantly more efficient than other formulations when evaluated in the PAMPA model. The PAMPA model also discriminated between different formulation types (i.e. gel versus solution) compared with other models. Overall, the results confirm the more permeable nature of the PAMPA, silicone membrane and porcine tissue models to ibuprofen compared with human skin. Further finite dose studies to elucidate the effects of individual excipients on the barrier properties of the PAMPA model are needed to expand the applications of this model. The range of actives that are suitable for study using the model also needs to be delineated.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Ibuprofeno/farmacocinética , Membranas Artificiales , Absorción Cutánea , Administración Cutánea , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Química Farmacéutica/métodos , Relación Dosis-Respuesta a Droga , Excipientes/química , Humanos , Ibuprofeno/administración & dosificación , Modelos Biológicos , Permeabilidad , Siliconas/química , Piel/metabolismo , Porcinos , Factores de TiempoRESUMEN
Viruses sense environmental cues such as pH to engage in membrane interactions for cell entry during infection, but how nonenveloped viruses sense pH is largely undefined. Here, we report both high- and low-pH structures of bluetongue virus (BTV), which enters cells via a two-stage endosomal process. The receptor-binding protein VP2 possesses a zinc finger that may function to maintain VP2 in a metastable state and a conserved His866, which senses early-endosomal pH. The membrane-penetration protein VP5 has three domains: dagger, unfurling and anchoring. Notably, the ß-meander motif of the anchoring domain contains a histidine cluster that can sense late-endosomal pH and also possesses four putative membrane-interaction elements. Exposing BTV to low pH detaches VP2 and dramatically refolds the dagger and unfurling domains of VP5. Our biochemical and structure-guided-mutagenesis studies support these coordinated pH-sensing mechanisms.
Asunto(s)
Virus de la Lengua Azul/efectos de los fármacos , Virus de la Lengua Azul/fisiología , Internalización del Virus/efectos de los fármacos , Secuencias de Aminoácidos , Virus de la Lengua Azul/química , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Análisis Mutacional de ADN , Endocitosis , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica/efectos de los fármacos , Dedos de ZincRESUMEN
The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention.
Asunto(s)
Virus de la Lengua Azul/fisiología , Biología Molecular , Proteínas Virales/metabolismo , Replicación Viral , Animales , Virus de la Lengua Azul/genética , Proteínas Virales/genéticaRESUMEN
Murine prion protein deleted for residues 105-125 is intrinsically neurotoxic and mediates a TSE-like phenotype in transgenic mice. Equivalent and overlapping deletions were expressed in E.coli, purified and analyzed. Among mutants spanning the region 95-135, a construct lacking solely residues 105-125 had distinct properties when compared with the full-length prion protein 23-231 or other deletions. This distinction was also apparent followed expression in eukaryotic cells. Unlike the full-length protein, all deletion mutants failed to bind to synthetic membranes in vitro. These data suggest a novel structure for the 105-125 deleted variant that may relate to its biological properties.
Asunto(s)
Priones/química , Priones/genética , Animales , Línea Celular , Membrana Celular/metabolismo , Expresión Génica , Liposomas/metabolismo , Ratones , Fosfatidilgliceroles/metabolismo , Proteínas Priónicas , Priones/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de SecuenciaRESUMEN
Peptide nanofibres (PNFs) have gained increasing interest as engineered biomaterials for drug delivery and tissue repair because of the versatility in design they offer through the self-assembly of amphiphilic peptide molecules. Their self-assembly is governed by hydrophobic interactions and hydrogen bonds between peptide sequences able to form beta-sheets. In this report, we describe the self-assembly of PNFs by using palmitoyl-peptide molecules containing two different cationic amino acid sequences and offer a description of the nanofiber physicochemical characteristics. The structural degradation of these PNFs in physiologically-relevant media was evaluated experimentally and two mechanisms are proposed. We also piloted the tracking of PNFs intracellularly in vitro, upon interaction with primary neuronal cultures, and intracranially in vivo, after stereotactic administration deep within the brain using two types of fluorescent labelled PNFs. Overall, the self-assembled PNFs were seen to internalise within neurons and be removed or degrade in the brain. Further work is needed to determine the utility of such PNFs as molecular transporters within neuronal tissue.
Asunto(s)
Nanofibras , Péptidos/química , Animales , Células Cultivadas , Ratones , Ratones Desnudos , RatasRESUMEN
Ibuprofen (IBU) has been available as a topical skin preparation for more than two decades. Its primary indication is for the relief of pain and inflammation in rheumatic disease and other musculoskeletal conditions. This article reviews the various formulation strategies which have been investigated for percutaneous IBU delivery to date. The focus is on studies which have been conducted with IBU in the free base form on human or porcine skin as data from other species are known to overestimate likely permeation in man. Emerging technologies for enhanced dermal delivery of IBU are considered including methods which require physical disruption of the membrane. The role of biophysical techniques such as Confocal Raman Spectroscopy in the rational development of IBU formulations is also discussed.