Fatty acid-2 hydroxylase (FA2H) is a 43 kDa transmembrane protein residing in the endoplasmic reticulum. As a monooxygenase it is responsible for the alpha–hydroxylation of fatty acids, which are later incorporated into sphingolipids, thereby generating 2-OH sphingolipids. FA2H and 2-OH sphingolipids show a quite ubiquitous tissue distribution, with high amounts detectable in brain, spinal cord, skin, testis, ovary, kidney, stomach and intestine. Especially important is their role in myelin, where up to 60% of the total amount of galactoylceramide and sulfatide are alpha- hydroxylated. The importance of this modification is emphasized by the observation that humans with a FA2H deficiency develop a spastic paraplegia (autosomal recessive spastic paraplegia 35 (SPG35)). Moreover, aged FA2H-KO mice present with a similar phenotype, showing an axonal and myelin sheath degeneration in spinal cord and later also in sciatic nerves. While the symptoms of FA2H deficiency have been well described, so far almost nothing is known about the exact disease mechanisms. Furthermore, there is no knowledge about the proteins biological regulation and its protein microenvironment. Thus, in the first part of the thesis FA2H-KO mice of different ages (6, 13 & 17 months) were examined for changes in their CNS- and PNS-myelin protein composition, which might help to better understand the observed pathology. This was achieved using a TMT 6-plex gel-free quantitative mass spectrometry approach. In the CNS this led to the identification of various protein alterations, of which some were already present in 6-month-old mice. The most prominent ones, which were also verified by Western blot, were upregulations of C1qb, C4b, ApoE, GFAP, tau protein, hinting at previously unknown roles of inflammation, astrogliosis and tau aggregation in the pathology of FA2H deficiency. In addition, a strong upregulation of one structural myelin membrane protein, Opalin/TMEM10, was observed as well. Because not much is known about this protein’s function so far, its possible role in the pathology of FA2H deficiency should be elucidated by further experiments. The PNS-myelin analysis also allowed for the identification of various protein changes. Those were mainly restricted to 13- and 17-month-old animals, which is in line with the late onset of PNS- demyelination. Unfortunately, because many of the measured changes were not consistent and thus less confident, further experiments are still needed for verification. The second part of this thesis concentrated on the discovery of protein interaction partners of FA2H. This was done, because the pathology may not be caused by the absence of 2-OH sphingolipids, but rather the loss of certain protein interactions of FA2H. For the identification, two complementary affinity purification-based screening strategies were applied in combination with quantitative mass spectrometry (SILAC-lP). In addition, a selection of the interaction partners was afterwards successfully verified by Western blotting and bimolecular fluorescence complementation (BiFC). Finally, this for the first time allowed a description of FA2H interaction partners in mammals. Interestingly, many are involved in metabolism, transport and regulation of synthesis of sphingolipids, indicating a tight coupling of enzymes participating in these processes. Furthermore, with PGRMC1 and 2, two promising regulators of FA2H’s activity were identified
