Semisynthesis Of Phosphorylated α-Synuclein And Effects Of Phosphorylation On Metal Ion Binding

Parkinson’s disease（PD）is the second most common neurodegenerative disease,affecting at least 0.3%of the global population and more than 3%of people over the age of80.In its early stage,PD is mainly characterized by movement disorders.Patients show a series of movement retardation,stiffness and fatigue,postural instability and tremor,as well as non-movement symptoms that may precede movement symptoms,including rapid eye movement sleep disorder,constipation and low olfaction.PD is characterized by the formation of neural inclusions called Lewy bodies（LB）,which are mainly composed of insoluble fibers and aggregated formsα-synuclein（α-syn）.α-Syn is a presynaptic neuron protein,which is genetically and neuropathologically related to PD.α-Syn may be involved in the pathogenesis of PD in many ways,but its abnormal soluble oligomer conformation is generally considered to be a toxic substance,which mediates the destruction of intracellular homeostasis and neuronal death by affecting various intracellular targets,including synaptic function.In addition,secretedα-syn may have harmful effects on adjacent cells.α-Syn is a small protein with 140 amino acids,which is divided into three different regions:positively charged N-terminal region,central hydrophobic region with high aggregation tendency and highly acidic C-terminal domain.It has been proposed that the C-terminal plays a role in theα-syn transportation to the nucleus and metal ions can interact withα-syn.In addition,the majority ofα-syn post translational modifications（PTMs）,including phosphorylation and nitration,occur in the C-terminal region,such as Y125 and S129.Moreover,these modification sites are close to binding sites of metal ions.As the molecular mechanisms of metal ions and phosphorylation ofα-syn remain elusive,this study aims to investigate the interaction of metal ions and phosphorylation sites,as well as their potential roles in PD pathogenesis.1.To assess the structure and function ofα-syn with site-specific PTM,this study employed a general and efficient semisynthesis strategy,which can introduce single or multiple PTMs at specific site.In addition,it allows preparation of C-terminal modifiedα-syn at mini gram scale,which is sufficient for detailed biophysical and biochemical analyses.2.Using fluorescent spectrometry,UV-Vis spectrometry,isothermal calory titration and mass spectrometry,this study investigated the interaction between metal ions,i.e.,Fe（II）,Cu（II）and Fe（III）,and phosphorylatedα-syn segments（p Y125,p S129 and p Y125+p S129）.Results showed that phosphorylation increases the binding between metal ions andα-syn segments,compared to wild-type（WT）.In addition,Fe（III）showed higher binding affinity than those of Fe（II）and Cu（II）.3.α-Syn segments with different phosphorylation sites can enhance their binding to metal ions.However,it is still unknown which phosphorylation site is the major contributor.Here,using fluorescent spectrometry,UV-Vis spectrometry,isothermal calory titration and mass spectrometry,this study showed thatα-syn segment binds the lowest amount of metal ions when S129 is phosphorylated while it binds the highest amount of metal ions when both Y125 and S129 are phosphorylated.4.As the binding between different phosphorylatedα-syn and Fe（II）can alter the physiological function in organisms,this study employed the UV-Vis spectrometry to investigation the impact of their binding on H₂O₂generation.Results showed that phosphorylatedα-syn inhibits the production of H₂O₂,and the inhibitory effect increases with the binding affinity between metal ions andα-syn segments.