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EhADH112 is a Bro1 domain-containing protein involved in the Entamoeba histolytica multivesicular bodies pathway.

Bañuelos C, García-Rivera G, López-Reyes I, Mendoza L, González-Robles A, Herranz S, Vincent O, Orozco E - J. Biomed. Biotechnol. (2012)

Bottom Line: EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis.Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [(35)S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain.Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, Mexico.

ABSTRACT
EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis. Here, we investigated an alternative role for EhADH112 in the MVB protein trafficking pathway by overexpressing 166 amino acids of its N-terminal Bro1 domain in trophozoites. Trophozoites displayed diminished phagocytosis rates and accumulated exogenous Bro1 at cytoplasmic vesicles which aggregated into aberrant complexes at late stages of phagocytosis, probably preventing EhADH112 function. Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [(35)S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain. Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

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Cellular location of EhADH112 and the Bro1 recombinant polypeptide in transfected trophozoites. Confocal microscopy images of permeabilized ANeo, ANeoBro1, or ANeoADH112 trophozoites incubated with (a) mαEhADH112 or (b) pαFLAG antibodies. Negative controls correspond to trophozoite preparations only incubated with secondary antibodies. Top: phase contrast images. Bottom: corresponding confocal sections. Arrowheads: small cytoplasmic vesicles.
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fig2: Cellular location of EhADH112 and the Bro1 recombinant polypeptide in transfected trophozoites. Confocal microscopy images of permeabilized ANeo, ANeoBro1, or ANeoADH112 trophozoites incubated with (a) mαEhADH112 or (b) pαFLAG antibodies. Negative controls correspond to trophozoite preparations only incubated with secondary antibodies. Top: phase contrast images. Bottom: corresponding confocal sections. Arrowheads: small cytoplasmic vesicles.

Mentions: By confocal microscopy, mαEhADH112 antibodies revealed the presence of EhADH112 at the plasma membrane of permeabilized ANeo, ANeoBro1, and ANeoADH112 trophozoites (Figure 2(a)), although fluorescence was higher in ANeoADH112 population, since these trophozoites express both, endogenous EhADH112 and exogenous EhADH112-FLAG.


EhADH112 is a Bro1 domain-containing protein involved in the Entamoeba histolytica multivesicular bodies pathway.

Bañuelos C, García-Rivera G, López-Reyes I, Mendoza L, González-Robles A, Herranz S, Vincent O, Orozco E - J. Biomed. Biotechnol. (2012)

Cellular location of EhADH112 and the Bro1 recombinant polypeptide in transfected trophozoites. Confocal microscopy images of permeabilized ANeo, ANeoBro1, or ANeoADH112 trophozoites incubated with (a) mαEhADH112 or (b) pαFLAG antibodies. Negative controls correspond to trophozoite preparations only incubated with secondary antibodies. Top: phase contrast images. Bottom: corresponding confocal sections. Arrowheads: small cytoplasmic vesicles.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303925&req=5

fig2: Cellular location of EhADH112 and the Bro1 recombinant polypeptide in transfected trophozoites. Confocal microscopy images of permeabilized ANeo, ANeoBro1, or ANeoADH112 trophozoites incubated with (a) mαEhADH112 or (b) pαFLAG antibodies. Negative controls correspond to trophozoite preparations only incubated with secondary antibodies. Top: phase contrast images. Bottom: corresponding confocal sections. Arrowheads: small cytoplasmic vesicles.
Mentions: By confocal microscopy, mαEhADH112 antibodies revealed the presence of EhADH112 at the plasma membrane of permeabilized ANeo, ANeoBro1, and ANeoADH112 trophozoites (Figure 2(a)), although fluorescence was higher in ANeoADH112 population, since these trophozoites express both, endogenous EhADH112 and exogenous EhADH112-FLAG.

Bottom Line: EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis.Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [(35)S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain.Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, DF, Mexico.

ABSTRACT
EhADH112 is an Entamoeba histolytica Bro1 domain-containing protein, structurally related to mammalian ALIX and yeast BRO1, both involved in the Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated multivesicular bodies (MVB) biogenesis. Here, we investigated an alternative role for EhADH112 in the MVB protein trafficking pathway by overexpressing 166 amino acids of its N-terminal Bro1 domain in trophozoites. Trophozoites displayed diminished phagocytosis rates and accumulated exogenous Bro1 at cytoplasmic vesicles which aggregated into aberrant complexes at late stages of phagocytosis, probably preventing EhADH112 function. Additionally, the existence of a putative E. histolytica ESCRT-III subunit (EhVps32) presumably interacting with EhADH112, led us to perform pull-down experiments with GST-EhVps32 and [(35)S]-labeled EhADH112 or EhADH112 derivatives, confirming EhVps32 binding to EhADH112 through its Bro1 domain. Our overall results define EhADH112 as a novel member of ESCRT-accessory proteins transiently present at cellular surface and endosomal compartments, probably contributing to MVB formation during phagocytosis.

Show MeSH
Related in: MedlinePlus