Functional analysis of Rab7D small GTPase of Entamoeba histolytica

概要

［課程－２］
審査の結果の要旨
氏名 ラトナ ワユニ

EhRab7D, a small GTPase previously identified from isolated phagosomes, was
hypothesized to have function in trogocytosis (internalization of the prey by nibbling) and
phagocytosis, and thus in pathogenic mechanisms. To characterize the biological role of
EhRab7D, overexpression and gene silencing E. histolytica strains were established, and
assays for trogocytosis, phagocytosis, pinocytosis, motility, cellular adhesion, as well as
RNA seq analysis, were performed using these strains. EhRab7D interacting proteins
were identified using GST-EhRab7D in affinity purification then in silico analysis of
EhRab7D protein and prediction of its secondary structure were also performed to
discover its unique features among EhRab7 isotypes. The main results of the study as
follows:
1. EhRab7D was found to localize to small vesicles in the cytosol, but not trogo- and
phagosomes.
2. Repression of EhRab7D gene expression by gene silencing caused enhancement
of trogocytosis and phagocytosis of CHO cells, while overexpression of EhRab7D
caused reduction of the ingestion.
3. Repression of EhRab7D gene expression by gene silencing caused reduction of
motility and cellular adhesion.
4. Repression of EhRab7D gene expression by gene silencing caused enhancement
of pinocytosis.
5. Twenty-seven proteins were identified as effector candidates of EhRab7D through
affinity purification. Among 27 candidates, 11 of them were related to actin
including Arp2/3 protein. These effector candidates interact with EhRab7D in
GDP-dependent manner.
6. Affinity purification of GST-EhRab7D did not only show proteins related to
vesicular traffic and cytoskeletal rearrangement, many metabolism-related
proteins were identified to interact with EhRab7D-GDP. These proteins include
phosphoglycerate, acetyl-CoA synthetase, cysteine synthase, glyceraldehyde-3phosphate dehydrogenase, malic enzyme, and oxidative stress related protein
(thioredoxin reductase). Some metabolic proteins were also found to bind to

EhRab7D-GTP including alcohol dehydrogenase 3, elongation factor 1-alpha, and
oxidative stress related proteins (peroxiredoxin).
7. In silico analysis of EhRab7D identified a unique acidic amino acid stretch
composing the natural disordered region in the carboxyl terminus. In addition,
amino acid substitutions within the previously identified effector binding surface
of mammalian Rab7 were identified.
In this study, role of EhRab7D in actin-related processes (trogocytosis,
phagocytosis, pinocytosis, motility and cellular adhesion) has been investigated in E.
histolytica. Rab7 small GTPases are involved in endosome/phagosome maturation and
widely conserved among eukaryotes and known to regulate membrane traffic for
lysosome biogenesis. Thus, my findings add novel insights into the role of Rab7 as a
regulator of actin cytoskeleton.
よって本論文は博士（保健学）の学位請求論文として合格と認められる。