| Background?-actin is one of the key cytoskeletal protein in almost all cell types.Various studies reported that ?-actin is distributed at the axon terminals and dendritic spines in neurons.In a growing neuron,?-actin is abundantly enriched at the tip of the neuronal axons,regulating the growth of axons.During the maturation of neuron,part of the ?-actin is concentrated in dendritic spines,which is critical for neuronal functioning.As a neurocytoskeletal protein,?-actin is not only essential for the shaping of its morphology but also important for brain activities e.g.learning and memory formation as well as behavioral changes.In the earlier phase of my study,I accidently found out that ?-actin is likely modified by protein palmitoylation.Interestingly,it has never been reported that ?-actin is palmitoylated.ObjectiveIn this study,we sought to examine first,whether ?-actin is palmitoylated and second,what is the molecular relevance of palmitoylated-?-actin.This study will extend our understanding of an unknown regulatory mechanism of ?-actin from the perspective of post-translational modification-protein palmitoyaltion.Methods1.Confirmation of ?-actin palmitoylation: Palmitoylated-?-actin is verified in vitro and in vivo e.g.293 T cells and mouse brain tissue.2.Identifying the specific residue(s)of ?-actin palmitoylation: varieties of different mutants were generated and overexpressed in 293 T cells,and identified by comparing the signal strength of ?-actin palmitoylation modification.3.Reconfirmation of ?-actin palmitoylation: The ?-actin protein was collected and determined by mass spectrometry.4.Identiying the specific palmitoylation sites within ?-actin: Mutants were constructed based on biochemical results.After overexpressing in 293 T cells,the level of ?-actin palmitoylation was compared and the data was analyzed.5.Screening the depalmitoylation enzymes of ?-actin: Overexpress all 5 known depalmitoylation enzymes,comparing the level of ?-actin palmitoylation.6.Screening the palmitoylation enzymes of ?-actin: Overexpress all known 23 palmitoylation enzyme,comparing the level of ?-actin palmitoylation.7.The function of ?-actin palmitoylation in cells: WT and mutatant-actin were expressed in different cell types and corresponding cell morphology and cytoskeleton were compared and analyzed.8.Effect of point-mutation of ?-actin in cells:Point-mutated ?-actin were expressed in different cell types and corresponding cell morphology and cytoskeleton were compared and analyzed.Results1.?-actin is a palmitoylated protein.2.?-actin is multi-palmitoylated.3.PPT1 and PPT2 might be involved in the depalmitoylation of ?-actin.4.zDHHC17,zDHHC18,zDHHC23,zDHHC25 might be involved in the palmitoylation of ?-actin.5.?-actin palmitoylation plays important roles in the remodeling of cytoskeleton.Conclusion?-actin is indeed palmitoylated,which seems essential for the maintenance of the membrane protrusions,although the underlying mechanism still awaited to be addressed. |
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