Enzymatic regulation of CCL2 bioactivity in neuroinflammation

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The present proposal focuses on the regulation of neuroinflammatory effects mediated by the C-C motif chemokine CCL2. We hypothesize that the biological activity of CCL2 is tuned by enzyme-catalyzed post-translational modification. On the one hand, N-terminal CCL2 truncation by dipeptidyl peptidase IV/CD26 (DP4) initiates further proteolytical degradation and abolishes its biological activity. On the other hand, N-terminal pyroglutamate modification catalyzed by the glutaminyl cyclases QC and/or isoQC protects CCL2 from proteolytical degradation and increases its biological activity. Thus, we consider the tandem of the QC/isoQC and DP4 enzymes as pro- and anti-inflammatory molecular check-points in neuroinflammation, as they may specifically modulate CCL2 activity and the functional outcome in neuroinflammatory conditions in vivo.We propose to exemplify these regulated enzymatic actions in brain disease by specifically investigating (i) the regulation of expression and enzyme activity of QC/isoQC and DP4 and generation of stabilized/degraded CCL2 variants in brain as well as the activation of local microglia and infiltrating peripheral monocytes in an inflammatory stroke mouse model, (ii) the consequences of QC/isoQC, DP4 and CCL2 ablation in knock-out mice on the above markers and on the functional outcome such as infarct size and neurological motor deficits in the ischemia model, (iii) the mechanisms and kinetics of microglia and monocyte recruitment by modified CCL2 variants. This includes the analysis of CCL2 binding to its primary receptor CCR2, which is located on monocytes and macrophages, the activation of intracellular ERK1/2 signaling cascade and chemotaxis. Thus, the proposal targets an important aspect related to the endogenous resolution of inflammation by enzyme processing pathways, which might be associated with a number of brain diseases. Furthermore, the results provide implications for the use of clinically relevant enzyme inhibitors in neuroinflammatory conditions.