| Myasthenia gravis（MG）is a rare autoimmune disease characterized by the destruction of the postsynaptic membrane（endplate membrane）in the neuromuscular junction position and is generally considered to be an abnormality of the receptor.The main clinical manifestations of skeletal muscle repeated contraction fatigue,rest after the relief.MG incidence of 8-20/10 million,the prevalence rate of 50/10 million.MG patients suffering from quality of life and quality of life decline,the progress of the disease is the loss of labor and self-care ability,serious symptoms can even endangerlife.The neuromuscular junctions mainly include anterior membrane structure,a posterior membrane structure,and a gap structure between the two.The postsynaptic membrane surface aggregates a large number of acetylcholine receptors（AChR）,and other proteins associated with acetylcholine receptor（AChR）function.In the physiological case,the nerve impulses reach the anterior axon of the anterior axon,the synaptic vesicle moved to the presynaptic membrane site with its fusion,the synaptic vesicle neurotransmitter-Ann released into the gap,and then the role In the posterior membrane of the AchR,calcium channel transient opening,Ca2+influx,presynaptic membrane release acetylcholine,acetylcholine and acetylcholine receptor（AChR）binding to the presynaptic membrane depolarization,generating action potential,action potential re-stimulation Proposed submucosal membrane to excite the proliferation of skeletal muscle cells,causing the depolarization of skeletal muscle membrane.When the sarcolemma of the sarcolemma to reach the threshold potential level of the onset of action potential,causing muscle contraction.The distribution of acetylcholine receptors in the neuromuscular junctions is a key factor in the neuromuscular conduction of the postsynaptic membrane.LRP4,MuSK and nAChR together form the end plate membrane specific LRP4-MuSK-nAChR functional unit,plays an important role in the process of neuromuscular impulse chemical transfer:Argin is a neuronal derived heparin-sulfate protein Polyglycans,which are synthetic and released extracellular matrix proteins for motor nerve endings,are also regulators of the interaction between LRP4 and MuSK,both of which produce binding.Agrin binds to the firstβ-helix domain of LRP4 to directly induce the conformational changes of LRP4 and enhances the interaction between LRP4and MuSK,resulting in phosphorylation of MuSK,induction of extensive aggregation of nAChR in the postsynaptic membrane,Membrane of the ACh neurotransmitter combined to complete the nerve impulse conduction.LRP4 is a member of the low density lipoprotein receptor（LDLR）protein family.Studies have shown that sorting nexin 17（SNX17）,located in the primary endosome,binds to the intracellular domain of LDLR during LDL and LDLR binding to induce lysosomal degradation,Re-use of the plasma membrane into the recycling of the way to accelerate the uptake of cells on the LDL.LRP4 and MuSK to raise nAChR to the postsynaptic membrane region,is the key to complete the neuromuscular excitatory transmission,the autoantibody can lead to the occurrence of MG.Our previous study found that MGX patients with abnormal expression of SNX17,which is intracellular adapter protein,can mediate the majority of LDLR molecular endocytosis process,to avoid degradation of lysosomes,accelerated LDLR is recycled to the membrane.The number of SNX17 expression in thymus tissue of MG patients was increased in the preoperative laboratory.The spatial distribution of nAChR in the intercostal muscle of MG patients was found to be irrelevant to the red linearity profile by laser confocal microscopy.The internal network configuration showed vacuolar changes.If you can repair the end plate membrane receptor,muscle normal systolic function is restored,will MG patients with disease rehabilitation play a positive role.SNX17 is one of the most characteristic members of the sorting nexins（SNXs）and is highly compatible with PtdIns（3）P.SNX17 is distributed across all tissues and a variety of different types of cells and is an adapter protein present in the primary endosomes.In addition to having a PX domain,SNX17 is the only family member in SNXs that has the FERM domain and the particularly carboxyl terminal.PX domain specificity interacts with phosphatidylinositol phosphate to localize SNXs within cell vesicles.The FERM domain is responsible for binding proteins that play a role in protein sorting and cell membrane transport.The FERM domain of SNX17 recognizes the NPxY motifs of LDLR,low density lipoprotein receptor-related protein and LRP1 LDLR family members,so that the receptor protein on the plasma membrane surface Can be reused efficiently.Previous studies have shown that the NPxY motif of the cytoplasmic tail of the LDLR family is necessary for SNX17 recognition,whereas LRP4 also contains the NPxY motif,suggesting that it has the same ability to bind to SNX17 as LRP1.Previously,the interaction between SNX17 and LRP4 was confirmed by immunoprecipitation.In this paper,GST pull down experiment will be used to verify the direct interaction between SNX17 and LRP4.So that SNX17-mediated LRP4 receptor recirculation,re-use to the sarcolemma,the recovery of LRP4-MuSK-nAChR functional unit is possible for the treatment of MG to provide a new target.Objective:To explore the mechanism of SNX17 mediating the recycling of LRP4receptor,promoting the aggregation of nAChR and restoring LRP4-MuSK-nAChR functional unit.Methods:1PCR technique was used to amplify the intracellular domain of LRP4.The gene sequence of LRP4 was obtained from Gene Bank.Primers were designed using Pre mier5.0.The primers were 5’-TCGCGGATCCTACAGACACAAAAAATCCAAGTTCA CTGA-3’,and the downstream primer was 5’-ACCGCTCGAGTTAGACCTGGCTCT CTGAGGAGAGC-3’;2Construction of prokaryotic expression plasmid of pGEX-4T-2/LRP4 intracellular domain by molecular cloning technique;3Recombinant pGEX-4T-2/LRP4 expression plasmid was transformed into E.coli BL21strain.The expressed product was induced by IPTG and analyzed by SDS-PAGE;4Purified pGEX-4T-2/LRP4 fusion protein;5The fusion protein of pGEX-4T-2/LRP4 was detected by Western Blotting;6Construction ofMyc-SNX17 Eukaryotic Expression Vector and Transfection of Myc-SNX17 to HEK293;7 Myc-SNX17 protein was detected by Western blotting;8GST pull down.Results:1Construction of recombinant plasmid pGEX-4T-2/LRP4;2 pGEX-4T-2/LRP4 fusion protein induced expression by IPTG;3 The fusion protein pGEX-4T-2/LRP4 was identify by Western Blotting;4 Construction of eukaryotic expression vector of Myc-SNX17 and its expression,Myc-SNX17 was transfected into HEK293 and the protein was extracted;5 GST pull down assay of fusion protein pGEX-4T-2/LRP4and Myc-SNX17.Conclusion:SNX17 interacts with LRP4 in vitro to enable SNX17 to mediate LRP4 receptor recirculation,re-use to the sarcolemma,and make it possible to restore LRP4-MuSK-nAChR functional units... |
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