Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Developing Inhibitors of the p47phox-p22phox Protein-Protein Interaction by Fragment-Based Drug Discovery.

Nicotinamide adenine dinucleotide phosphate oxidase isoform 2 is an enzyme complex, which generates reactive oxygen species and contributes to oxidative stress. The p47phox-p22phox interaction is critical for the activation of the catalytical NOX2 domain, and p47phox is a potential target for therapeutic intervention. By screening 2500 fragments using fluorescence polarization and a thermal shift assay and validation by surface plasmon resonance, we found eight hits toward the tandem SH3 domain of p47phox (p47phoxSH3A-B) with KD values of 400-600 µM. Structural studies revealed that fragments 1 and 2 bound two separate binding sites in the elongated conformation of p47phoxSH3A-B and these competed with p22phox for binding to p47phoxSH3A-B. Chemical optimization led to a dimeric compound with the ability to potently inhibit the p47phoxSH3A-B-p22phox interaction (Ki of 20 µM). Thereby, we reveal a new way of targeting p47phox and present the first report of drug-like molecules with the ability to bind p47phox and inhibit its interaction with p22phox.