Proteomics

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Coactosin phosphorylation controls Entamoeba histolytica 1 cell membrane protusions and cell motility


ABSTRACT: Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix (ECM). Transcriptional silencing of the E. histolytica matrix surface metalloprotease EhMSP-1 produces hyper-adherent, less motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1 silenced phenotype, here we compared the phosphoproteome of EhMSP-1 silenced and vector control trophozoites using quantitative mass spectrometry based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1 silenced and control cells, including EhCoactosin, a member of the ADF/Cofilin family of actin binding proteins, which was hyperphosphorylated at serine 147 (S147). Regulated over-expression of wild type, phosphomimetic (S147D), and non phosphorylatable (S147A) EhCoactosin variants was used to test if S147 phosphorylation functions in control of E. histolytica actin dynamics. Each of the over-expressed proteins co37 localized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing EhCoactosin S147D formed more and poorly coordinated small membrane protrusions compared to control or S147A expressing cells, while trophozoites overexpressing EhCoactosin S147A were significantly more motile within a model of mammalian ECM. Therefore, although EhCoactosin’s actin-binding ability appeared unaffected by S147 phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1 silenced trophozoites, and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Entamoeba Histolytica Hm-1:imss Entamoeba Histolytica Entamoeba Histolytica Hm-1:imss-a Entamoeba Histolytica Hm-1:imss-b

TISSUE(S): Cell Culture

DISEASE(S): Amebiasis

SUBMITTER: Ying Wai Lam  

LAB HEAD: Christopher Huston

PROVIDER: PXD018276 | Pride | 2020-07-15

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Differential_Phosphoproteomics.msf Msf
Differential_Phosphoproteomics.pep.xml Pepxml
Differential_Proteomics.msf Msf
Differential_Proteomics.pep.xml Pepxml
Differential_Proteomics.prot.xml Xml
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