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Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog.

ABSTRACT: Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite Entamoeba histolytica. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding of EhRab8A to EhCdc50 was also confirmed by reciprocal immunoprecipitation and blue-native polyacrylamide gel electrophoresis, the latter of which revealed an 87 kDa complex. Indirect immunofluorescence imaging with and without Triton X100 showed that endogenous EhCdc50 localized on the surface in the absence of permeabilizing agent but was observed on the intracellular structures and overlapped with the ER marker Bip when Triton X100 was used. Overexpression of N-terminal HA-tagged EhCdc50 impaired its translocation to the plasma membrane and caused its accumulation in the ER. As reported previously in other organisms, overexpression and accumulation of Cdc50 in the ER likely inhibited surface transport and function of the plasma membrane lipid flippase P4-ATPase. Interestingly, HA-EhCdc50-expressing trophozoites gained resistance to miltefosine, which is consistent with the prediction that HA-EhCdc50 overexpression caused its accumulation in the ER and mislocalization of the unidentified lipid flippase. Similarly, EhRab8A gene silenced trophozoites showed increased resistance to miltefosine, supporting EhRab8A-dependent transport of EhCdc50. This study demonstrated for the first time that EhRab8A mediates the transport of EhCdc50 and lipid flippase P4-ATPase from the ER to the plasma membrane.

SUBMITTER: Hanadate Y

PROVIDER: S-EPMC6321534 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog.

Hanadate Yuki Y Saito-Nakano Yumiko Y Nakada-Tsukui Kumiko K Nozaki Tomoyoshi T

International journal of molecular sciences 20181130 12

Membrane traffic plays a pivotal role in virulence in the enteric protozoan parasite <i>Entamoeba histolytica</i>. EhRab8A small GTPase is a key regulator of membrane traffic at the endoplasmic reticulum (ER) of this protist and is involved in the transport of plasma membrane proteins. Here we identified the binding proteins of EhRab8A. The Cdc50 homolog, a non-catalytic subunit of lipid flippase, was identified as an EhRab8A binding protein candidate by affinity coimmunoprecipitation. Binding o ...[more]

PMID: 30513690

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Project description:BACKGROUND: The polyamines putrescine, spermidine, and spermine are organic cations that are required for cell growth and differentiation. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is a highly regulated enzyme. METHODOLOGY AND RESULTS: To use this enzyme as a potential drug target, the gene encoding putative ornithine decarboxylase (ODC)-like sequence was cloned from Entamoeba histolytica, a protozoan parasite causing amoebiasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 1,242 bp encoding a putative protein of 413 amino acids with a calculated molecular mass of 46 kDa and a predicted isoelectric point of 5.61. The E. histolytica putative ODC-like sequence has 33% sequence identity with human ODC and 36% identity with the Datura stramonium ODC. The ORF is a single-copy gene located on a 1.9-Mb chromosome. The recombinant putative ODC protein (48 kDa) from E. histolytica was heterologously expressed in Escherichia coli. Antiserum against recombinant putative ODC protein detected a band of anticipated size approximately 46 kDa in E. histolytica whole-cell lysate. Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ODC, had no effect on the recombinant putative ODC from E. histolytica. Comparative modeling of the three-dimensional structure of E. histolytica putative ODC shows that the putative binding site for DFMO is disrupted by the substitution of three amino acids-aspartate-332, aspartate-361, and tyrosine-323-by histidine-296, phenylalanine-305, and asparagine-334, through which this inhibitor interacts with the protein. Amino acid changes in the pocket of the E. histolytica enzyme resulted in low substrate specificity for ornithine. It is possible that the enzyme has evolved a novel substrate specificity. CONCLUSION: To our knowledge this is the first report on the molecular characterization of putative ODC-like sequence from E. histolytica. Computer modeling revealed that three of the critical residues required for binding of DFMO to the ODC enzyme are substituted in E. histolytica, resulting in the likely loss of interactions between the enzyme and DFMO.

Dataset Information

Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog.

Publications

Identification and Characterization of the Entamoeba Histolytica Rab8a Binding Protein: A Cdc50 Homolog.

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