h1

TY  - THES
AU  - Willnow, Sophie
TI  - Enzymatische Alkinfunktionalisierung zur Markierung und Identifizierung von Protein-Methyltransferase-Substraten
CY  - Aachen
PB  - Publikationsserver der RWTH Aachen University
M1  - RWTH-CONV-143845
SP  - VIII, 168 S. : Ill., graph. Darst.
PY  - 2013
N1  - Aachen, Techn. Hochsch., Diss., 2012
AB  - After translation, proteins are modified in many ways by being coupled covalently to chemical groups or other proteins. These posttranslational modifications modulate enzyme functions and can transmit signals, e.g. in the histone code. Protein methylation is one example of a posttranslational modification. The transfer of one to three methyl groups from the cofactor S-adenosyl-L-methionine (AdoMet) to different amino acid residues, e.g. lysine, arginine or glutamine, is catalyzed by methyltransferases (MTases). The methylation of amino acids does not result in a major change of conformation, but rather creates new interaction sites for so-called methyl effectors. These can recognize the methylation state of a protein and translate this signal into a cellular response. The methylation is reversed by demethylases. To date, the small size and the poor detectability of radioactively labeled methyl groups prevent a comprehensive analysis of MTases and their substrates. Thus we took the task to develop a novel two-step labeling method for MTase substrates and with this approach, labeled the substrates of selected MTases in complex samples. Protein MTases not only accept the natural cofactor AdoMet, but can also transfer elongated side chains from synthetic analogs to their substrates. A small side chain and an activating double or triple bond located in proximity to the substitution center provide a sufficient high transfer activity. Also, the exchange of the central sulfur atom with selenium leads to a considerable increase in activity. In this work, a collection of cofactor analogs was tested with the protein MTase Dim-5. Only with the cofactor analog SeAdoPropyne, containing a propargyl group, a complete modification of a test peptide was achieved. This is due to the small side chain and the acceptable stability of the cofactor analog, as well as the activation by the selenonium center and the neighboring triple bond. Other cofactor analogs tested showed lower reaction rates and did not lead to full conversion. Terminal alkynes can be coupled to azides in the Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC), resulting in the formation of triazoles. Reacting under mild conditions with very high rates, the CuAAC can be used to couple alkyne-functionalized proteins to azide-derivatized labeling reagents. The CuAAC is a bioorthogonal reaction and its use for protein labeling was investigated in this work. It became clear that especially the ligand tris-(hydroxypropyltriazolylmethyl)amine (THPTA) accelerated the reaction and protected proteins against Cu(I)-mediated degradation. In our two-step labeling approach protein substrates were first alkyne-functionalized using SeAdoPropyne. Subsequently, proteins were labeled chemically using CuAAC with azide-derivatized fluorophores or biotin. This two-step method was successfully applied to different MTases and their substrates: to the lysine MTases Dim-5, Clr4, G9a and Set7/9, the arginine MTase PRMT1, and the glutamine MTase PrmC. Thus, SeAdoPropyne can be utilized by many protein MTases with different substrate specificities. Lastly, the two-step labeling approach was embedded into standard proteomic methods to develop a procedure for the isolation and identification of substrates of protein MTases. Labeling in protein lysate was analyzed using bacterial MTase PrmC by identifying its two known substrates RF1 and RF2. Subsequent to fluorescence labeling samples were separated via 2D gel electrophoresis followed by mass spectrometry-based identification of spots of interest. This way, RF2 could be identified as a substrate. In addition, biotin-labeled proteins were isolated from lysate using magnetic particles and were analyzed by mass spectrometry. Here, RF1 could be identified as a substrate. Finally, substrates of human lysine MTase Set7/9 were labeled on a protein array leading to almost 300 still to be confirmed substrate candidates. In principle, the two-step labeling method can be linked to proteomic methods. However, all techniques lack sensitivity, which needs to be enhanced in further studies. All in all, the two-step labeling method can be effectively used to label protein MTase substrates. In the future it should be applied to the identification of novel substrates.
KW  - Click-Chemie (SWD)
KW  - Epigenetik (SWD)
KW  - Methyltransferase <S> (SWD)
KW  - Posttranslationale Änderung (SWD)
KW  - Selen (SWD)
KW  - Adenosylmethioninanaloga (SWD)
LB  - PUB:(DE-HGF)11
UR  - https://publications.rwth-aachen.de/record/228595
ER  -