• John Bush

      Articles written in Journal of Biosciences

    • A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

      Entsar Saheb Ithay Biton Katherine Maringer John Bush

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      Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.

    • Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions?

      Entsar Saheb Wendy Trzyna John Bush

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      Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

    • Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

      Katherine Maringer Azure Yarbrough Sunder Sims-Lucas Entsar Saheb Sanaa Jawed John Bush

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      Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

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