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Research - Keller Group
Scientific Background Problem A major unsolved problem connected with human metabolic diseases is that many clinically relevant metabolic  parameters are used for differential diagnosis; however the underlying pathobiochemical mechanisms for their  appearances are frequently elusive. Thus, there is a lack of comprehensive knowledge with respect to the structure and  regulation of respective metabolic pathways and/or the function of the genes involved. Background: This is especially the case for the 3-methylglutaconic acidurias (3MGAuria), a group of human inherited  diseases characterized by the accumulation of 3-methylglutaconic acid (3MGA) in patient plasma and urine. Only in  primary 3MGAuria this accumulation can be easily explained via defects in the AUH gene, resulting in the blockage of  mitochondrial leucine degradation and the cells inability to detoxify the 3MGA precursor 3-Methylglutaconyl-CoA (see  Fig 1). Fig. 1: Mitochondrial leucine catabolism is affected by 3-methylglutaconic aciduria related genetic mutations: Leucine catabolic enzymes are shown in blue colour. All depicted proteins are localised to mitochondria, except for the cytosolic BCAT1. Evidence of membrane association (M) or soluble forms (S) is shown in red letters. However, for a series of other 3MGAuria causing genes there is no pathological rationale explaining this phenotype (e.g. TAZ, OPA3, TMEM70, CLOB, DNAJC19, SERAC1). Still, it is noticeable that those genes appear to be functionally  connected to mitochondrial membranes and phospholipid homeostasis therein, especially regarding a mitochondria  exclusive class of phospholipids, the cardiolipins (CL) (see Fig. 1). Research questions and strategy Key aim of our current work is to investigate the interdependency of mitochondrial phospholipid homeostasis in  membranes with mitochondrial functioning and its impact of leucine catabolism, in order to elucidate the principal  pathological principles of respective 3MGAurias. Important steps towards successfully answer this research question are  a) the assembly of a comprehensive patient fibroblast cell line collection for 3MGAurias, b) the generation of stable  CRISPR/Cas9 knockout lines for the two 3MGAurias: MEGDEL Syndrome (SERAC1) and Barth Syndrome (TAZ), and c)  establishing the methodological LC-MS/MS platform for mitochondrial lipidomics as well as further assays for assessing  mitochondrial functions. Lipidomics platform Our recently developed LC-MS/MS platform allows in combination with mathematical modeling techniques to elucidate the composition of the mitochondrial cardiolipin composition in very great detail. Recorded MS1 data is utilized for  quantification of up to ~120 different CL species as well as dozens of monolyso-CL and oxidized CL species. At the same  time MS/MS fragment data allows us to compute detailed structural information, acyl chain distributions and lipid  molecule symmetry information for each of the quantified CL species; data which is therefore ideally suited to study  genetic perturbations of mitochondrial phospholipid homeostasis. Additionally this method has already been extended to also cover other important phospholipid lipid classes (Fig. 2). Fig. 2: LC-MS/MS analytical platform for the quantification of cardiolipin profiles. A) 2D representation of the LC-MS  elution pattern of more than 120 different cardiolipin species in the mass range of 1300-1500 m/z. B) Full MS1 spectrum at  RT=9 min shows different CL classes eluting at the same time, with different m/z. C) Details for example peak m/z 1428, 8.8  min (CL70:4): i) Natural isotope pattern ii) Double peak caused by subspecies. D) Identified phospholipid species: PA:  Phosphatidic acid, PC: Phosphatidylcholine, PE: Phosphatidylethanolamine, and PS: Phosphatidylserine (measured with a  modified HPLC gradient)   
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