Analysis Of Differential Expression And Interactions Of Proteins In Brain Synaptosomes

The mammal brain is the center for integrating information and controlling various physiological activities and behaviors. Synapses are the sites for functional connection of the neurons in brain. Many physiological and biochemical processes related to the functions of synapses, such as transmitter release and activation, transmitter receptor modulation, signal transduction cascades and so on, are all performed by the intricate network of synaptic proteins cooperatively. Therefore, a deep investigation into the synaptic proteins and their interactions as well as the differential expression of the synaptic proteins under various physiological and pathological conditions would be helpful for further understanding the structure and function of the synapses. Our present research, using proteomics and the related technologies, focused on the protein interactions involved in the synaptic transmission in mammal brain synaptosomes and the differentially expressed synaptic proteins during the accumulation of advanced glycation end-products, with the aims to discover and characterize new proteins that interact with special receptors and ion channels and deepen our understanding of the molecular mechanism of synaptic transmission.There have been evidences showing that administration of high-level D-galactose into mice could lead to the accumulation of advanced glycation end-products (AGEs) that would in turn induce the development of diabetes and diabetes-associated complications. For example, diabetic neuropathy is one of the diabetes complications, which deteriorates the patient's learning and memory abilities due to the altered expression of synaptic proteins and protein complexes. To evaluate the AGE-induced differential expression of synaptic proteins in synapsomes, proteomic methods blue native/SDS-PAGE and iTRAQ were used to screen for differentially expressed synaptic proteins in cerebral cortex of C57 BL/6 mice administrated with high-level D-galactose. In total, the expression level of 84 proteins was changed during AGE accumulation. The significantly differentially expressed proteins are mainly involved in neurotransmission, energy metabolism and signal transduction pathway, suggesting that during AGEs accumulation the energy metabolism and neurotransmission in synapses were attenuated. Elucidation of the differentially expressed synaptic proteins induced by the AGEs accumulation opens a new window to the mechanism of learning and memory impairments in diabetic neuropathy and provides references for treatment of the diseases.Synaptotagminâ… , an important synaptic vesicle membrane protein involved in Ca2+-regulated exocytosis, senses calcium influx and triggers synaptic vesicle fusion and neurotransmitter release. Despite extensive studies, the accurate function and mechanism of synaptotagminâ… in the regulation of synaptic transmission remain incompletely understood. Presence of distinct protein-interacting domains in synaptotagmin I has led to the suggestion that they associate with proteins that are involved in synaptic transmission, thereby exerting regulation action. This study was directed towards identifying possible new binding partners of synaptotagminâ… . As a result, a small GTP-binding protein Rab3A was found to interact with synaptotagminâ… by blue native PAGE. This result was further confirmed by co-immunoprecipitation from rat brain synaptic plasma membrane protein extracts, which showed that Rab3A interacts strongly with synaptotagmin I in a Ca2+-independent manner. Then we utilized site-directed mutation and GST pull down to identify the key site of Rab3A binding. It was found that KKKK motif in the C2B domain of synaptotagmin I was essential for Rab3A binding, and inositol hexakisphosphate modulated Rab3 A binding to synaptotagmin I. In view of that Rab3A can inhibit neurotransmitter release and that Rab3A and syntaxin in t-SNAREs complex (syntaxin/SNAP-25) bind to the same motif in synaptotagmin I, it was speculated that Rab3A might inhibit the membrane fusion and neurotransmitter release through competing with syntaxin in binding to the same site in the C2B domain of synaptotagmin I. An in vitro competitive experiment was designed to demonstrate this speculation. The present study shows that Rab3A is a new interacting partner of synaptotagmin I and the interaction between these two proteins plays an important role in the regulation of membrane fusion in synaptic transmission, thus sheding light on the fundamental problem of regulated synaptic transmission.The voltage-gated potassium channel Kvl.4 is an important A-type potassium channel and modulates the excitability of neurons in center nerve system.The expression and location in membranes, channel kenetics and post-translational modification of Kv1.4 are all regulated by its interacting proteins. Although there have been some research reports on the interacting proteins of Kv1.4, there are still many problems to be resolved. In the present research, synaptotagmin I was found to form a native complex in a Ca2+-dependent fashion with Kv1.4 in presynaptic membrane in rat hippocampus by immunoprecipitation combined with mass spectrometric analysis.This indicated that synaptotagminâ… was an interacting protein of Kv1.4, which was further demonstrated by the experimental result that co-immunoprecipitation of Kv1.4 was achieved by antibody against synaptotagminâ… . Immonuhistochemical experiment demonstrated that synaptotagminâ… and Kv1.4 protein had co-location in the CA3 region of hippocampus. In addition, using patch clamp technique, synaptotagminâ… was found to delay the inactivation of Kv1.4 in HEK 293 cells, without effect on its activation kinetics; The interaction of synaptotagminâ… with Kv1.4 was Ca2+-dependent. Synaptotagminâ… was shown to be an intrinsic auxiliary subunit of native Kv1.4 channel, and the observed physical and functional interactions between them might play a certain role in synaptic efficacy and neuronal excitability. The above experimental results provide some new clues for probing into the regulation of neurotransmission.