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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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EhADH112 major presence in membrane subcellular fractions and EhADH112 interaction with EhVps32. (a) Location of EhADH112 in wild-type trophozoites subcellular fractions. Proteins (50 μg) from different fractions of trophozoite extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot assays were performed using pαEhADH243 and mαactin antibodies and corresponding peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibodies. Membranes were revealed by chemiluminescence. Left: SN1 and P1 fractions resulting after total proteins (TP) centrifugation at 250 ×g for 30 min in a mannitol/sucrose gradient. Middle: SN2 and P2 fractions resulting after SN1 ultracentrifugation at 40 000 ×g for 60 min. Right: P3 and P4 fractions obtained after ultracentrifugation of solubilized P1 at 40 000 ×g for 60 min. (b) Binding of EhADH112 to EhVps32 through the Bro1 domain. GST or GST-EhVps32 proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro synthesized [35S]-EhADH112 or [35S]-EhADH112 derivatives. Left: purified GST and GST-EhVps32 fusion proteins or [35S]-EhADH112 and [35S]-truncated derivatives (autoradiography, lanes 3, 4 and 5) used for binding experiments (8% of the total reaction mixture). Right: pulled-down proteins electrophoresed on 10% SDS-polyacrylamide and detected by autoradiography.
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fig7: EhADH112 major presence in membrane subcellular fractions and EhADH112 interaction with EhVps32. (a) Location of EhADH112 in wild-type trophozoites subcellular fractions. Proteins (50 μg) from different fractions of trophozoite extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot assays were performed using pαEhADH243 and mαactin antibodies and corresponding peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibodies. Membranes were revealed by chemiluminescence. Left: SN1 and P1 fractions resulting after total proteins (TP) centrifugation at 250 ×g for 30 min in a mannitol/sucrose gradient. Middle: SN2 and P2 fractions resulting after SN1 ultracentrifugation at 40 000 ×g for 60 min. Right: P3 and P4 fractions obtained after ultracentrifugation of solubilized P1 at 40 000 ×g for 60 min. (b) Binding of EhADH112 to EhVps32 through the Bro1 domain. GST or GST-EhVps32 proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro synthesized [35S]-EhADH112 or [35S]-EhADH112 derivatives. Left: purified GST and GST-EhVps32 fusion proteins or [35S]-EhADH112 and [35S]-truncated derivatives (autoradiography, lanes 3, 4 and 5) used for binding experiments (8% of the total reaction mixture). Right: pulled-down proteins electrophoresed on 10% SDS-polyacrylamide and detected by autoradiography.

Mentions: To investigate whether native EhADH112 remains as a soluble or insoluble membrane-associated protein in wild-type trophozoites, we carried out the procedure described by Aley et al. [34] followed by Western blot assays, using rabbit polyclonal antibodies against the last 243 amino acids of EhADH112 (pαEhADH243), which detect the carboxy-terminus of the adhesin. pαEhDH243 antibodies recognized the expected 78 kDa band corresponding to the EhADH112 molecular weight in crude extracts (TP) obtained after disruption of trophozoites incubated with concanavalin A in the presence of protease inhibitors (Figure 7(a), left panel, lane 1). After centrifugation at 250 ×g for 30 min on a mannitol/sucrose gradient, EhADH112 appeared in the supernatant (SN1), which contains vesicles, small membrane fragments, and soluble proteins (Figure 7(a), left panel, lane 2). We also detected a weak band in the pellet (P1), which contains large fragments of plasma membranes and cell debris (Figure 7(a), left panel, lane 3). As a control, we used mouse monoclonal antibodies against actin (mαactin), which reacted with the corresponding 43 kDa protein in all fractions (Figure 7(a), left panel, lanes 1 to 3).


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)

EhADH112 major presence in membrane subcellular fractions and EhADH112 interaction with EhVps32. (a) Location of EhADH112 in wild-type trophozoites subcellular fractions. Proteins (50 μg) from different fractions of trophozoite extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot assays were performed using pαEhADH243 and mαactin antibodies and corresponding peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibodies. Membranes were revealed by chemiluminescence. Left: SN1 and P1 fractions resulting after total proteins (TP) centrifugation at 250 ×g for 30 min in a mannitol/sucrose gradient. Middle: SN2 and P2 fractions resulting after SN1 ultracentrifugation at 40 000 ×g for 60 min. Right: P3 and P4 fractions obtained after ultracentrifugation of solubilized P1 at 40 000 ×g for 60 min. (b) Binding of EhADH112 to EhVps32 through the Bro1 domain. GST or GST-EhVps32 proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro synthesized [35S]-EhADH112 or [35S]-EhADH112 derivatives. Left: purified GST and GST-EhVps32 fusion proteins or [35S]-EhADH112 and [35S]-truncated derivatives (autoradiography, lanes 3, 4 and 5) used for binding experiments (8% of the total reaction mixture). Right: pulled-down proteins electrophoresed on 10% SDS-polyacrylamide and detected by autoradiography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303925&req=5

fig7: EhADH112 major presence in membrane subcellular fractions and EhADH112 interaction with EhVps32. (a) Location of EhADH112 in wild-type trophozoites subcellular fractions. Proteins (50 μg) from different fractions of trophozoite extracts were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot assays were performed using pαEhADH243 and mαactin antibodies and corresponding peroxidase-labeled anti-rabbit or anti-mouse IgG secondary antibodies. Membranes were revealed by chemiluminescence. Left: SN1 and P1 fractions resulting after total proteins (TP) centrifugation at 250 ×g for 30 min in a mannitol/sucrose gradient. Middle: SN2 and P2 fractions resulting after SN1 ultracentrifugation at 40 000 ×g for 60 min. Right: P3 and P4 fractions obtained after ultracentrifugation of solubilized P1 at 40 000 ×g for 60 min. (b) Binding of EhADH112 to EhVps32 through the Bro1 domain. GST or GST-EhVps32 proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro synthesized [35S]-EhADH112 or [35S]-EhADH112 derivatives. Left: purified GST and GST-EhVps32 fusion proteins or [35S]-EhADH112 and [35S]-truncated derivatives (autoradiography, lanes 3, 4 and 5) used for binding experiments (8% of the total reaction mixture). Right: pulled-down proteins electrophoresed on 10% SDS-polyacrylamide and detected by autoradiography.
Mentions: To investigate whether native EhADH112 remains as a soluble or insoluble membrane-associated protein in wild-type trophozoites, we carried out the procedure described by Aley et al. [34] followed by Western blot assays, using rabbit polyclonal antibodies against the last 243 amino acids of EhADH112 (pαEhADH243), which detect the carboxy-terminus of the adhesin. pαEhDH243 antibodies recognized the expected 78 kDa band corresponding to the EhADH112 molecular weight in crude extracts (TP) obtained after disruption of trophozoites incubated with concanavalin A in the presence of protease inhibitors (Figure 7(a), left panel, lane 1). After centrifugation at 250 ×g for 30 min on a mannitol/sucrose gradient, EhADH112 appeared in the supernatant (SN1), which contains vesicles, small membrane fragments, and soluble proteins (Figure 7(a), left panel, lane 2). We also detected a weak band in the pellet (P1), which contains large fragments of plasma membranes and cell debris (Figure 7(a), left panel, lane 3). As a control, we used mouse monoclonal antibodies against actin (mαactin), which reacted with the corresponding 43 kDa protein in all fractions (Figure 7(a), left panel, lanes 1 to 3).

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