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1.
Biochim Biophys Acta ; 1848(7): 1619-1628, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25883090

RESUMEN

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.


Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/metabolismo , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Interacciones Huésped-Parásitos , Humanos , Immunoblotting , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Confocal , Microscopía Electrónica de Rastreo , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Espectrina/metabolismo , Resonancia por Plasmón de Superficie
2.
FASEB J ; 28(7): 3103-13, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24706359

RESUMEN

The genomes of malaria parasites (Plasmodium spp.) contain a family of genes encoding proteins with a Plasmodium helical interspersed subtelomeric (PHIST) domain, most of which are predicted to be exported into the parasite-infected human red blood cell (iRBC). Here, using transgenic parasites and a combination of cellular, biochemical, and biophysical assays, we have characterized and determined the function of a novel member of the PHIST protein family in Plasmodium falciparum, termed lysine-rich membrane-associated PHISTb (LyMP). LyMP was shown to associate directly with the cytoskeleton of iRBCs where it plays a role in their abnormal ability to adhere to a protein expressed on vascular endothelial cells, resulting in sequestration. Deletion of LyMP dramatically reduced adhesion of iRBCs to CD36 by 55%, which was completely restored to wild-type levels on complementation. Intriguingly, in the absence of LyMP, formation of RBC membrane knobs and the level of surface exposure of the parasites' major cytoadhesive ligand, PfEMP1, were identical to those for the parental parasite line, demonstrating for the first time an additional mechanism that enhances cytoadherence of iRBCs beyond those already recognized. Our findings identify LyMP as a previously unknown RBC cytoskeletal-binding protein that is likely to be of major significance in the complex pathophysiology of falciparum malaria.-Proellocks, N. I., Herrmann, S., Buckingham, D. W., Hanssen, E., Hodges, E. K., Elsworth, B., Morahan, B. J., Coppel, R. L., Cooke, B. M. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum infected red blood cells.


Asunto(s)
Adhesión Celular/fisiología , Citoesqueleto/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Lisina/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Membrana Celular/metabolismo , Membrana Celular/parasitología , Citoesqueleto/parasitología , Endotelio Vascular/metabolismo , Endotelio Vascular/parasitología , Humanos , Malaria Falciparum/metabolismo , Malaria Falciparum/parasitología , Proteínas de la Membrana/metabolismo , Unión Proteica/fisiología
3.
Biochim Biophys Acta ; 1798(5): 861-71, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20132790

RESUMEN

Intra-erythrocytic Plasmodium falciparum malaria parasites synthesize and export numerous proteins into the red blood cell (RBC) cytosol, where some bind to the RBC membrane skeleton. These interactions are responsible for the altered antigenic, morphological and functional properties of parasite-infected red blood cells (IRBCs). Plasmodium falciparum protein 332 (Pf332) is a large parasite protein that associates with the membrane skeleton and who's function has recently been elucidated. Using recombinant fragments of Pf332 in in vitro interaction assays, we have localised the specific domain within Pf332 that binds to the RBC membrane skeleton to an 86 residue sequence proximal to the C-terminus of Pf332. We have shown that this region partakes in a specific and saturable interaction with actin (K(d)=0.60 microM) but has no detectable affinity for spectrin. The only exported malaria protein previously known to bind to actin is PfEMP3 but here we demonstrate that there is no competition for actin-binding between PfEMP3 and Pf332, suggesting that they bind to different target sequences in actin.


Asunto(s)
Citoesqueleto/metabolismo , Membrana Eritrocítica , Eritrocitos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Actinas/metabolismo , Animales , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestructura , Eritrocitos/citología , Eritrocitos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas Protozoarias/genética , Espectrina/metabolismo
4.
J Cell Biol ; 172(6): 899-908, 2006 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-16520384

RESUMEN

The high mortality of Plasmodium falciparum malaria is the result of a parasite ligand, PfEMP1 (P. falciparum) erythrocyte membrane protein 1), on the surface of infected red blood cells (IRBCs), which adheres to the vascular endothelium and causes the sequestration of IRBCs in the microvasculature. PfEMP1 transport to the IRBC surface involves Maurer's clefts, which are parasite-derived membranous structures in the IRBC cytoplasm. Targeted gene disruption of a Maurer's cleft protein, SBP1 (skeleton-binding protein 1), prevented IRBC adhesion because of the loss of PfEMP1 expression on the IRBC surface. PfEMP1 was still present in Maurer's clefts, and the transport and localization of several other Maurer's cleft proteins were unchanged. Maurer's clefts were altered in appearance and were no longer found as close to the periphery of the IRBC. Complementation of mutant parasites with sbp1 led to the reappearance of PfEMP1 on the IRBC surface and the restoration of adhesion. Our results demonstrate that SBP1 is essential for the translocation of PfEMP1 onto the surface of IRBCs and is likely to play a pivotal role in the pathogenesis of P. falciparum malaria.


Asunto(s)
Antígenos de Superficie/metabolismo , Proteínas Portadoras/genética , Eritrocitos/parasitología , Proteínas de la Membrana/genética , Orgánulos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Adhesión Celular/genética , Regulación hacia Abajo/genética , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestructura , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Exocitosis/genética , Regulación de la Expresión Génica/fisiología , Humanos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Orgánulos/ultraestructura , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Transporte de Proteínas/genética
5.
Analyst ; 135(3): 525-30, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20174705

RESUMEN

The effects of fixation and dehydration on the distribution of heme-based molecules inside red blood cells and the structural integrity of the cells have been investigated using Raman mapping and AFM topographic imaging. A strong correlation was observed between the thickness of the cells as determined from AFM images and the intensity of the characteristic heme bands in the Raman maps, demonstrating that heme compounds are relatively evenly distributed inside dried and fixed cells in the majority of cases. The exception occurred when cells were dried in phosphate buffered saline, where more hemichrome appears close to the periphery of the cell despite the AFM image showing a plateau like topography. Using neat formaldehyde solution as a fixative is inadequate for a complete structural preservation and results in diffusion of hemoglobin into the surrounding area. However, a mixture of formaldehyde (3%) and glutaraldehyde (0.1%) in buffer was found to be sufficient to retain the structural integrity of cells with minimal autofluorescence. This protocol was also suitable for red blood cells infected with Plasmodium falciparum parasites, and preserved the characteristic knob-like structures on the infected red blood cell surface.


Asunto(s)
Eritrocitos/química , Microscopía de Fuerza Atómica/métodos , Espectrometría Raman/métodos , Eritrocitos/parasitología , Formaldehído/química , Glutaral/química , Hemo/química , Humanos , Malaria Falciparum/diagnóstico , Plasmodium falciparum/aislamiento & purificación
6.
Int J Parasitol ; 44(5): 319-28, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24530877

RESUMEN

Alteration of the adhesive and mechanical properties of red blood cells caused by infection with the malaria parasite Plasmodium falciparum underpin both its survival and extreme pathogenicity. A unique family of parasite putative exported kinases, collectively called FIKK (Phenylalanine (F) - Isoleucine (I) - Lysine (K) - Lysine (K)), has recently been implicated in these pathophysiological processes, however, their precise function in P. falciparum-infected red blood cells or their likely role in malaria pathogenesis remain unknown. Here, for the first time, we demonstrate that one member of the FIKK family, FIKK4.2, can function as an active kinase and is localised in a novel and distinct compartment of the parasite-infected red blood cell which we have called K-dots. Notably, targeted disruption of the gene encoding FIKK4.2 (fikk4.2) dramatically alters the parasite's ability to modify and remodel the red blood cells in which it multiplies. Specifically, red blood cells infected with fikk4.2 knockout parasites were significantly less rigid and less adhesive when compared with red blood cells infected with normal parasites from which the transgenic clones had been derived, despite expressing similar levels of the major cytoadhesion ligand, PfEMP1, on the red blood cell surface. Notably, these changes were accompanied by dramatically altered knob-structures on infected red blood cells that play a key role in cytoadhesion which is responsible for much of the pathogenesis associated with falciparum malaria. Taken together, our data identifies FIKK4.2 as an important kinase in the pathogenesis of P. falciparum malaria and strengthens the attractiveness of FIKK kinases as targets for the development of novel next-generation anti-malaria drugs.


Asunto(s)
Eritrocitos/parasitología , Fosfotransferasas/metabolismo , Plasmodium falciparum/enzimología , Factores de Virulencia/metabolismo , Técnicas de Inactivación de Genes , Humanos , Fosfotransferasas/genética , Plasmodium falciparum/genética , Transporte de Proteínas , Factores de Virulencia/genética
7.
Mol Microbiol ; 56(4): 990-1003, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15853885

RESUMEN

During erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3-disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1-KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3-KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 degrees C resulted in 33% culture inhibition of resa1-KO parasites while marginally impacting parental and resa3-KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1-KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.


Asunto(s)
Calor , Mutación , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Antígenos CD36/metabolismo , Forma de la Célula , Citoesqueleto/metabolismo , Eritrocitos/citología , Eritrocitos/parasitología , Humanos , Malaria Falciparum , Plasmodium falciparum/genética , Saimiri/parasitología
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