SRC3-mediated Calmodulin Acetylation Is Essential For Long-term Potentiation And Contextual Fear Memory

Synaptic plasticity is the important mechanism of learning and memory.It mainly includes long-term potentiation(LTP)and long-term depression(LTD).LTP is divided into early LTP and late LTP in time.Early LTP mainly involves post-translational modifications of proteins,while late LTP requires the resynthesis of proteins.A large number of studies have shown that phosphorylation is involved in the development of early LTP,and the role of other types of post-translational modifications in the formation of LTP is poorly understood.This research studies the role of protein acetylation in LTP and learning and memory.Protein acetylation mainly includes N-terminal alpha amino acetylation and lysine epsilon amino acetylation.The N-terminal alpha amino acetylation is an irreversible modification that regulates protein synthesis,stability,and localization.The lysine epsilon amino acetylation is reversible and dynamic,so it can be regarded as a switch to regulate protein function and is more important in regulating the physiological activities.Lysine acetylation levels are commonly regulated by Lysine acetyltransferases(KATs)and Lysine deacetylases(KDACs).Numerous studies have shown that neural activity can activate gene transcription by increasing acetylation of histones to promote late LTP formation and learning and memory.However,it is unclear whether synaptic acetylation is involved in early LTP and learning and memory.Calmodulin(Ca M)is a Ca²⁺sensor that is widely expressed in eukaryotic cells.Ca M binds to effector proteins such as protein kinases,G protein-coupled receptors,and ion channels,and plays a pivotal role in Ca²⁺signal transduction.Ca M plays an important role in regulating synaptic plasticity.Activation of NMDA receptors on the postsynaptic membrane causes an increase in intracellular Ca²⁺influx,Ca M is activated by Ca²⁺binding,and further binds to and activates calmodulin-dependent kinase II?(Ca MKII?).The enzymatic cascade mediated by Ca²⁺/Ca M/Ca MKII?activation plays an important role in the insertion into membrane of the AMPA receptors and the formation of early LTP.Recent studies have shown that Ca M can shuttle into the nucleus to regulate gene transcription and late LTP production through activation of Ca MKK.We used affinity mass spectrometry to find 1245 acetylated proteins and 2001acetylated lysine sites in the mouse forebrain.Among them,the frequency of Ca M acetylation was as high as 88 times,ranking 5th among all acetylated proteins.This study was to investigate the regulatory mechanism of Ca M acetylation in response to neural activity and its role in synaptic plasticity and contextual fear learning and memory.A new method for the enrichment and detection of acetylated proteins was used,combined with mass spectrometry analysis,structural biology analysis,biochemistry and molecular biology,and gene editing(CRISPR/Cas9)to study the regulatory mechanisms of dynamic modification of Ca M acetylation,and its function in synaptic plasticity and learning and memory.The main findings are as follows:1.Identify mouse brain acetylome:1245 acetylated proteins and 2001 Ac-K sites;2.SRC3 acetylates Ca M,KDAC9 and SIRT1 deacetylates Ca M;3.The acetylation of Ca M enhances the binding with Ca²⁺and Ca MKII?and promotes the activation of Ca MKII?.4.NMDAR-dependent LTP enhances Ca M acetylation by increasing the translocation of SRC3 to the membrane and interaction with Ca M;5.SRC3-mediated Ca M acetylation is necessary for long-term potentiation LTP and contextual fear memory.Through this series of experiments,we conclude that SRC3-mediated Ca M acetylation is essential for LTP and contextual fear memory.This study reveals a new molecular mechanism for the regulation of LTP and contextual fear memory,as well as a new understanding of the regulatory mechanisms and functions of protein acetylation in neural activity.