|Summary||The biogenic monoamines serotonin (5-HT), histamine (HA), dopamine (DA) and norepinephrine (NE) act as neurotransmitters and hormones in controlling crucial functions of the mammalian organism. A recently discovered posttranslational modification (serotonylation) involves the covalent binding of 5 HT to protein-bound glutamine (Q) residues of small G proteins. This reaction, catalysed by the enzyme family of transglutaminases (TGM), results in a change in the G protein’s GTP hydrolysis activity.
In the attempt to clarify whether this phenomenon is restricted to 5 HT, the four monoamines 5 HT, HA, DA and NE were investigated both in vitro and in cell culture. Eight small and two heterotrimeric GTPases, as well as phospholipase A2 (PLA2), were analysed in combination with TGM1, 2, 3 and fXIIIA as well as four monoamines revealing that the TGM-dependent incorporation of monoamines (monoaminylation) is a specific reaction. HA incorporation (histaminylation) proved to be most prominent, followed by DA, NE and 5 HT. Histaminylation of small and heterotrimeric GTPases results in a functional change, shown using GTP hydrolysis experiments and effector binding studies. As a result of the diminished intrinsic and GAP-mediated hydrolysis, Gαo1 and Cdc42 become constitutively activated and remain in the active, GTP-bound state. This then leads to an increased affinity to the binding partners RGS4 and Pak3. PLA2 histaminylation in contrast results in a significantly elevated lypolytic activity.
The fact that the catalytic centres of most GTPases contain a conserved Q residue suggested that the residue is specifically modified by monoaminylation. Using a mass spectronomy approach, this hypothesis could be substantiated as fragment ion spectra of Gαo1, Gαq and Rab18 revealed the modified catalytic Q residue. Histaminylation can therefore be regarded as a novel TGM-dependent regulatory posttranslational modification of signalling proteins.
Furthermore, eight cell lines of different origin were characterized in respect of the expression of four monoamine transporters and seven TGM, and monoamine uptake as well as protein incorporation were measured. The assessment of TGM dependence was possible using the competitive inhibitor cysteamine. The results suggest that monoaminylation occurs in most cell lines in varying amount. Dopaminylation was most pronounced, but NE, HA and 5 HT incorporation was also detectable. According to these findings, monoaminylation is taking place in many different cell types.
In order to identify signal transduction pathways regulated by this modification, PC12 and 3T3 L1 cells were challenged with the DA derivative DA-biotin, which was developed in the course of this thesis, or the commercial 5-(biotinamido)pentylamine. In both cases, covalently modified proteins were isolated and identified by mass spectronomy, revealing that a multitude of proteins are monoaminylation substrates. Among them are proteins involved in protein biosynthesis and folding, glycolysis, fatty acid metabolism, signal transduction and mitochondrial function. Of the potentially modified proteins, Rab1b, NPM1 and AnxA2 were analysed in detail verifying the monoaminylation of NPM1 and Rab1b in PC12 cells.
With the help of the results presented here, a detailed analysis of protein monoaminylation as a novel posttranslational modification capable of fine-tuning multifarious cellular functions is now possible.