<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Investigation of the lipid environment of the mammalian transamidase complex"^^ . "The glycosylphosphatidylinositol (GPI) anchor is a lipid moiety attached to over 150 human proteins\r\nand plays a crucial role in cell surface display at the plasma membrane. The transamidase\r\ncomplex, consisting of the subunits PIGK/S/T/U and GPAA1, is localised to the endoplasmic reticulum\r\n(ER) and is responsible for attachment of the GPI-anchor to receiving proteins. Previously,\r\ninteractions between the transamidase complex subunit GPAA1 and a sphingolipid have been\r\nshown. The aim of this thesis was to establish an experimental setup to analyse protein lipid interactions\r\nof the transamidase complex.\r\nThese interactions were assessed via in-gel fluorescence and western blot quantification through\r\nclick chemistry with bifunctional sphingolipids combined with transient expression of transamidase\r\nsubunits. Thin layer chromatography was used to verify the metabolic status of exogenously\r\nadded photoactivatable and clickable sphingosine (pacSph) at given time points. Results\r\nconfirmed the previous reported interaction of GPAA1. The validity of this outcome was limited\r\nto GPAA1 only due to uncertainty with regards to the transient expression, such as affecting\r\nthe stoichiometry of the transamidase complex and mislocalisation of the transiently overexpressed\r\nGPAA1. To circumvent these limitations, CRISPR/Cas9-based gene editing was performed\r\nto insert a C-terminal tag in the genomic region of GPAA1. This allowed to establish a cellular\r\nmodel to study sphingolipid and cholesterol interactions with the endogenous transamidase\r\ncomplex. Gene edited tagging of GPAA1 did not interfere with synthesis and trafficking of GPIanchor\r\nproteins to the cell surface. The analysis of the composition of the transamidase complex\r\nin this cell line by co-immunoprecipitation showed only PIGK/T/S to interact with GPAA1, but\r\nnot PIGU. When styrene maleic anhydride (SMA) co-polymer was used to extract the transamidase\r\ncomplex and its native lipid environment, even PIGU was detected, suggesting that PIGU\r\nmight be only loosely attached to the transamidase complex. Furthermore, first studies on mass\r\nspectrometric analysis of lipids extracted from an SMA-immunoprecipitation suggested that this\r\napproach allows to determine lipids in direct proximity of the transamaidase complex. Finally,\r\nGPAA1-pacSph metabolite interaction could be inhibited through the ceramide synthase inhibitor\r\nFB1, while the glucosylceramide synthase inhibitor PPMP did not interfere with the interaction.\r\nThis suggested that ceramide or a metabolite upstream of this lipid is the interaction partner of\r\nGPAA1.\r\nIn summary, the established cellular model verified previously published results in an endogenous\r\nsetting. For the future, this model lays the foundation for quantitative determination of the\r\nlipid environment of the transamidase complex. Furthermore, this system might also provide\r\nstructural information via single particle cryo-electron microscopy of affinity purified GPAA1\r\ncomplexes."^^ . "2020" . . . . . . . "Thomas Johannes"^^ . "Roxlau"^^ . "Thomas Johannes Roxlau"^^ . . . . . . "Investigation of the lipid environment of the mammalian transamidase complex (PDF)"^^ . . . "2019.02.26_Dissertation_RoxlauThomas(PDF_A).pdf"^^ . . . "Investigation of the lipid environment of the mammalian transamidase complex (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #27099 \n\nInvestigation of the lipid environment of the mammalian transamidase complex\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .