Browsing by Author "Moorhead, Greg B. G."

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    Arabidopsis At5g39790 encodes a chloroplast-localized, carbohydrate-binding, coiled-coil domain-containing putative scaffold protein
    (BioMed Central, 2008) Lohmeier-Vogel, Elke M.; Kerk, David; Nimick, Mhairi; Wrobel, Susan; Vickerman, Lori; Muench, Douglas G.; Moorhead, Greg B. G.
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    Biochemical characterization of the first step in ether lipid biosynthesis: acylation of dihydroxyacetone phosphate
    (2020-01) Chilije, Maxwell F. J.; Zaremberg, Vanina; Edwards, Robert A.; Moorhead, Greg B. G.
    Glycerolipid biosynthesis is essential for cellular growth and proliferation. It is an indispensable pathway in many living organisms. In mammals four GPAT isoforms are known to catalyze the first step in glycerolipid biosynthesis which involves acylation of glycerol-3-phosphate to lysophosphatidic acid. GPATs 1 and 2 localize to the mitochondria while GPATs 3 and 4 localize to the ER. This redundancy is poorly understood. A fifth acyltransferase, DHAPAT, known to metabolize dihydroxyacetone phosphate, resides in peroxisomes. The principal function of DHAPAT in mammals is the biosynthesis of ether-based lipids which are widely distributed in the body and noticeably abundant in membranes of the nervous system. Compared to the four GPATs, DHAPAT is understudied. Current knowledge associating DHAPAT deficiency with many human disorders has re-kindled the interest in the research community. Symptoms of DHAPAT deficiency have been observed in many peroxisomal disorders including Zellweger syndrome and Rhizomelic Chondrodysplasia Punctata type 2. Understanding of this enzyme together with the roles played by ether lipids, could help find ways of alleviating some of the problems associated with these disorders. In the current study, we have performed a biochemical characterization of animal DHAPATs using heterologous expression in the yeast Saccharomyces cerevisiae. We confirmed that DHAPAT is a peripheral membrane protein. Interestingly, DHAP acylation alone was sufficient to support life of a yeast strain devoid of endogenous GPATs (sct1Δ gpt2Δ). This phenotype was therefore used as part of a functional in vivo assay to identify regions of the enzyme required for membrane binding. These investigations unveiled a critical role of the amino end of the protein in mediating the association of DHAPAT to membranes. Surprisingly, in vivo imaging of yeast expressing animal DHAPATs revealed association of the enzyme with LDs. This feature was also conserved in the human DHAPAT overexpressed in HeLa cells. Our findings support a dual localization of DHAPAT in peroxisomes and LDs. Therefore, in addition to the well accepted peroxisomal targeting of DHAPAT, a conserved LD targeting mechanism also exists, mediated by the amino terminus of the protein.
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    Characterization of interactions between DmsD, DmsA, and TatB for the docking step for the bacterial twin-arginine translocase
    (2019-09-12) Levchenko, Elina; Turner, Raymond Joseph; Noskov, Sergei Yu; Moorhead, Greg B. G.
    Tat-pathway is the primary translocation system which deals with fully-folded proteins that all bear a “twin arginine” motif with a consensus sequence S/TRRXFLK. Three major Escherichia coli components are TatA, TatB and TatC, where the last two comprise a functional unit responsible for cargo docking. My project utilized a heterotrimer Dimethyl Sulfoxide (DMSO) reductase as a model system with two constituents (DmsA and DmsB) requiring assistance from a DmsD chaperone to reach the translocon. My goal was to study the order of events during the docking of DmsA onto the TatB and potential involvement of DmsD. The work supports that DmsD mediates the PMF-dependent transfer of the substrate to the translocase system. It was also shown in vitro (differential scanning fluorimetry; circular dichroism; chromatography) and in silico that DmsD has binding sites on its surface for DmsA and TatB, and they are distinct and potentially regulated by Mg2+ and GNP.
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    Displacement affinity chromatography of protein phosphatase one (PP1) complexes
    (2008-11-10) Moorhead, Greg B. G.; Trinkle-Mulcahy, Laura; Nimick, Mhairi; De Wever, Veerle; Campbell, David G.; Gourlay, Robert; Lam, Yun Wah; Lamond, Angus I.
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    Protein phosphatase 2A-B56 regulates mitosis by interacting with LS/TPI/V motif containing proteins
    (2019-10-18) Chaudhuri, Sibapriya; Moorhead, Greg B. G.; Zaremberg, Vanina; Ng, Kenneth Kai Sing; Holmes, Charles F. B.
    Reversible protein phosphorylation is an important post translational modification that controls diverse signaling pathways including the eukaryotic cell cycle signaling. Protein phosphatase 2A (PP2A) is a highly conserved protein phosphatase that removes phosphate groups from serine/threonine residues on proteins. PP2A is a trimeric enzyme consisting of a catalytic or C subunit, a scaffolding or A subunit and a regulatory or B subunit. In humans, B56 is one of the four families of B subunits. PP2A-B56 is an important mitotic protein phosphatase that is essential for proper execution of several mitotic events such as alignment and segregation of sister chromatids. The primary objective of this study was to understand the mechanisms that controls interaction between PP2A-B56 and its mitotic interactors. This study shows that PP2A-B56 interacts with important mitotic regulators through a LS/TPI/V motif. The LS/TPI/V proteins are widespread in the human proteome and are conserved across eukaryotes. The LS/TPI/V proteins take part in multiple signaling pathways including the eukaryotic cell cycle. The interaction between PP2A-B56 and the LS/TPI/V proteins occur in an isoform dependent and phosphorylation dependent manner. Among the five isoforms of B56, B56 gamma 3 and B56 delta have a preference for binding to dephosphorylated LS/TPI/V peptides. The LS/TPI/V motif gets phosphorylated as the cell enters prophase and gets dephosphorylated at mitotic exit. This phosphorylation event is controlled by Aurora Kinase B. B56 delta has a preference for binding to LS/TPI/V proteins when the motif is dephosphorylated. This preference is contributed by the C terminal tail of B56 delta.
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    Regulation of Epithelial Cell Plasticity by a SUMO-TGFβ Signaling Axis
    (2019-12) Chanda, Ayan; Bonni, Shirin; Moorhead, Greg B. G.; Morris, Don G.; Arcellana-Panlilio, Mayi Y.; Godbout, Roseline
    Background Protein post-translational modification by the small ubiquitin-like modifier (SUMO), or SUMOylation, can regulate the stability, subcellular localization or interactome of a protein substrate with key consequences for cellular processes including the Epithelial-Mesenchymal Transition (EMT). The secreted factor Transforming Growth Factor beta (TGFβ) is a potent inducer of EMT in development and homeostasis. Importantly, the ability of TGFβ to induce EMT has been implicated in promoting cancer invasion and metastasis, resistance to chemo/radio therapy and maintenance of cancer stem cells. Interestingly, TGFβ-induced EMT and the SUMO system intersect with important implications for cancer formation and progression, and novel therapeutics identification. The transcriptional coregulator Ski-related novel protein N (SnoN), a negative regulator of TGFβ signaling axis, is a SUMO substrate. Interestingly, Protein Inhibitor of Activated STAT 1 (PIAS1) and Transcriptional Intermediary Factor 1 gamma (TIF1γ) are two distinct SUMO E3 ligases that bind and promote the SUMOylation of SnoN to suppress TGFβ-induced EMT. PIAS1 has been shown to act in a SUMO E3 ligase-dependent manner to suppress the invasion and metastatic growth of breast cancer cells. These results raised the key questions of the significance of the role of the two distinct SUMO E3 ligases PIAS1 and TIF1γ in regulating SnoN SUMOylation and suppressing TGFβ-induced EMT in mammary non-transformed epithelial cells and breast carcinomas. Hypothesis I hypothesize that PIAS1 and TIF1γ cooperate to promote SnoN SUMOylation to suppress EMT by the TGFβ-Smad pathway with potential relevance for breast cancer metastasis and prognosis. Results In this thesis, evidence is provided suggesting that the protein abundance and nuclear localization of PIAS1 act as survival biomarkers in breast cancer patients in a tissue microarray analysis. Accordingly, further results indicate that PIAS1 acts via SUMOylation of SnoN to suppress the invasive growth of triple negative breast cancer cells in 3D-organoid culture. In other studies, findings are provided that support the idea that SnoN promotes the formation of PIAS1-SnoN-TIF1γ multiprotein complex, which promotes SnoN SUMOylation, and its ability to suppress TGFβ-induced EMT in breast epithelial and cancer cells in 3D. Mechanistic studies suggest that SUMOylation promotes SnoN binding to the epigenetic regulators histone deacetylase 1 (HDAC1) and histone acetylase p300 in such a manner that leads to the suppression of EMT induced by the TGFβ-Smad-pathway in non-transformed and cancerous mammary cell-derived 3D organoids. Conclusions The novel findings in this thesis reveal the importance of a SUMO E3 ligase complex comprising PIAS1 and TIF1γ that enhances the SUMOylation of SnoN with impact on specific epigenetic regulators that control EMT in normal and cancer cells. These findings can lead to the discovery of novel biomarkers and therapeutics in breast and potentially other epithelial cell-derived cancers.
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    Role of Prolactin in a Preclinical Model of Breast Cancer Mediated Osteolysis.
    (2019-08-29) Gopinathan, Sesha Gopal; Shemanko, Carrie S.; Moorhead, Greg B. G.; Hollenberg, Morley Donald
    At the advanced stage, breast cancer metastasizes to the bone and initiates the vicious cycle of cancer by actively inducing osteoclast differentiation which causes excessive bone degradation. Prolactin (PRL) is a hormone involved in key functions such as mammary gland development and bone homeostasis. We demonstrated that PRL also stimulates breast cancer mediated osteoclastogenesis. The overall goal of my project is to study the role of PRL in breast cancer mediated bone degradation using mouse models. The breast cancer cells chosen for this study were engineered to emit bioluminescence and secrete PRL (MCF7-BGL hPRL) or not (MCF7-BGL-EV). During cell line characterization, MCF7-BGL-hPRL cells demonstrated better osteoclast differentiation via TRAP+ staining, indicating osteolysis potential. Using western blots, the level of PRL secreted by MCF7-BGL-hPRL and the presence of long isoform of PRLR on both the cell lines were quantified. Further, a slight fold difference in the bioluminescence signal intensity between these two cell line was identified and taken into consideration for in vivo experiments. Simultaneously, the role of PRL in osteoblast differentiation and its facilitation of osteoclast differentiation was studied and it showed no influence. Mouse tibia injected with MCF7-BGL-hPRL and MCF7-BGL-EV cells respectively achieved bioluminescence endpoint at 3 weeks and the microCT analysis at 6 weeks revealed higher bone damage in MCF7-BGL-hPRL injected tibia compared to the MCF7-BGL-EV injected tibia. However, a second experiment where the BLI endpoint reached only at 6 weeks showed relatively less bone loss for the same time point. The cumulative results from both the experiments show significant loss in bone mineral density and difference in trabecular thickness in MCF7-BGL-hPRL injected and its uninjected control tibiae, but no difference was observed between the two cell lines. This in vivo study of PRL induced breast cancer mediated osteolysis was never performed before and the valuable information learned from my study outcome sets a platform for future studies.