Identifizierung von In-vivo Interaktionspartnern von PMP22 im peripheren Nerven der Ratte
Identification of in-vivo interaction partners of PMP22 in the peripheral nerves of rats
Doctoral thesis
Date of Examination:2023-03-21
Date of issue:2023-03-20
Advisor:Prof. Dr. Michael Werner Sereda
Referee:Prof. Dr. Michael Werner Sereda
Referee:Prof. Dr. Henning Urlaub
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Abstract
English
The most common hereditary neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A), is caused by a duplication of the gene coding for the peripheral myelin protein 22 (PMP22). Despite long lasting scientific investigations, the function of this transmembrane protein and thereby pathophysiological mechanisms of the disease remain obscure and treatment options are limited. In order to elucidate the physiological and pathophysiological function of PMP22 and thereby suggest new treatment approaches, the investigation of its protein-protein interaction partners is meaningful. In the given work, we established an immunoprecipitation mass-spectrometry approach to identify in-vivo protein-protein interaction partners of Pmp22 in the sciatic nerves of rats. In order to allow the application of denaturing detergents and conserve also transient and low-affinity interactions we employed protein crosslinking covalently linking Pmp22 to the proteins in close proximity. We optimized the solubilization of Pmp22 in the sciatic nerve and the immunoprecipitation of Pmp22 from the nerve homogenate with and without crosslinker. The detergents applied in this work were differently effective in solubilizing Pmp22. In crosslinked homogenate, Pmp22 could be solubilized only using sodium dodecyl sulfate-polyacrylamide while non-crosslinked samples were most effectively solubilized with dodecyl-beta-D-maltoside. The immunoprecipitation protocol was further successfully optimized in terms antibody and dynabead amounts. As a pilot experiment, we analyzed the immunoprecipitation eluate using liquid chromatography tandem mass-spectrometry investigating the potential interactom of Pmp22 in rats’ sciatic nerves. The analysis of this pilot experiment considering (1) specific enrichment and (2) Gene Ontology terms suggested a potential function of Pmp22 and its protein interaction partners in cell-cell adhesions, focal adhesions, the actin cytoskeleton and intracellular signaling pathways. Namely, myelin protein zero, periaxin, 14-3-3 protein and the actin-binding protein cofilin-1 were among others specifically enriched in the Pmp22 immunoprecipitation eluate. Interactions of Pmp22 with myelin protein zero and periaxin could explain the function of Pmp22 as a stabilizer of the myelin architecture. The interaction with 14-3-3 and/or cofilin-1 could furthermore represent the molecular correlate of the disrupted shaping, structure and polarity of Schwann cells in the development of peripheral nerves in CMT1A. Within follow-up investigations using the given works’ Pmp22 immunoprecipitation protocol and crosslinking approach, these potential protein interaction partners could be validated thereby paving the way for novel treatment strategies in CMT1A.
Keywords: Immunoprecipitation Mass-spectrometry; Myelin; Charcot-Marie-Tooth 1A