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Biochemical characterization of presynaptic membrane protein complexes

dc.contributor.advisorJahn, Reinhard Prof. Dr.
dc.contributor.authorNinov, Momchil
dc.titleBiochemical characterization of presynaptic membrane protein complexesde
dc.contributor.refereeJahn, Reinhard Prof. Dr.
dc.description.abstractengAt chemical synapses, synaptic vesicles, membrane-enclosed entities filled with neurotransmitters, undergo exocytosis at restricted areas of the presynaptic membrane called active zones (AZ). The core AZ proteins form a heterogeneous dynamic network in functional collaboration with many soluble and integral proteins. This network, called cytomatrix at the active zone (CAZ), represents a biochemical challenge due to its limited detergent solubilization. In addition, there is a big gap in the knowledge about the extractability of proteins associated with the presynaptic cytomatrix. Often, analyses focused on the extraction of a particular protein of interest and the identification of co-purified proteins without any further validation of the findings. However, till date, no report has systematically investigated the solubilization of synaptic membranes by detergents. Our main goal was to characterize biochemically presynaptic membrane protein complexes. For this reason, I established a systematic protocol for non-denaturing solubilization of synaptic membranes. Moreover, the extractability of broad spectrum of presynaptic and postsynaptic proteins was assessed by immunoblots. The solubilization protocol considered experimental parameters like rotor type, centrifugation time as well as nature of the starting material. Interestingly, I found that the most proteins (integral, soluble and scaffolding) associated with the presynaptic CAZ were better extracted than the postsynaptic density-associated proteins. Importantly, for first time, a complete or partial extraction of very large cytomatrix proteins such as Piccolo and Bassoon using cholate, taurodeoxycholate and dodecyl-β-D-maltoside detergents was achieved. Moreover, the use of these detergents under optimized solubilization conditions allowed the extraction of all core AZ constituents as well as of many regulatory and scaffolding proteins, ion channels and receptors. Furthermore, I used density gradient centrifugation and size exclusion chromatography for separation of membrane-derived extracts. These results suggested possible preservation of protein-protein interactions during the solubilization process regardless of the used detergent. Additionally, in order to validate the established extraction protocol, I studied the presynaptic membrane protein syntaxin 1 in more detail. The target was immunoprecipitated from cholate extracts under two different conditions. The proteins, co-immunopurified with syntaxin 1, were analyzed by quantitative mass spectrometry. The use of stringent parameters in the statistical evaluation of the proteomics data allowed identification of 158 and 275 proteins above the threshold margin under high and low salt conditions, respectively. Both groups of proteins overlapped almost completely and emphasized the reproducibility of the data. In line with our goals, more than 95% of the identified proteins, that passed the filtering criteria, were membrane proteins. Many of these proteins were shown in earlier studies to interact directly with syntaxin 1 or form multimeric complexes with its participation. These results supported the idea of successful membrane protein extraction under preservation of protein-protein interactions. They also underline the usefulness of our protocol for the study of other presynaptic proteins and their
dc.contributor.coRefereeUrlaub, Henning Prof. Dr.
dc.contributor.thirdRefereeBrose, Nils Prof. Dr.
dc.contributor.thirdRefereeWienands, Jürgen Prof. Dr.
dc.contributor.thirdRefereeMoser, Tobias Prof. Dr.
dc.contributor.thirdRefereeRizzoli, Silvio Prof. Dr.
dc.subject.engmass spectrometryde
dc.subject.engsyntaxin 1de
dc.subject.engtransmembrane proteinde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de
dc.identifier.ppn1004916353 1000142922

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