The general paradigm centers on the aggregation of overproduced αSyn into progressively larger amyloids that cause cell toxicity, with other aspects of αSyn cellular distribution and function remaining unclear. Nevertheless, understanding the role of αSyn in PD remains challenging. The intrinsically disordered neuronal protein α-synuclein (αSyn) is found accumulated in a fibrillar form in Lewy bodies, a hallmark of Parkinson’s disease (PD). Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies. Experiments with live HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. In electrophysiology experiments, addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on the αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Recently we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the Voltage-Dependent Anion Channel (VDAC) of the outer mitochondrial membrane. Involvement of alpha-synuclein (αSyn) in Parkinson’s disease (PD) is complicated and difficult to trace on cellular and molecular levels.
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