| Caveolae is a special type of invagination in plasma membrane with important functions in membrane integrity,cellular transportation and signal transduction.The biogenesis and functions of caveolae are dependent on caveolin,a type of integral membrane protein.Caveolin-3 is a important member of the caveolin protein family and specifically expressed in skeletal muscle,cardiac muscle,smooth muscle and diaphragm muscle.The N-terminal cytoplasmic domain(NCD)of the caveolin contains potential oligomeric region.It is not unclear that how the oligomeric region of caveolin polymerize.There are many muscle diseases-related mutations in caveolin-3,which are enriched in the NCD.Currently,less is known about how these mutations would cause structure changes and malfunctions of caveolin-3.In addition,caveolin-1 can interact with cholesterol,take part in tansportation of cholesterol and maintain balance of cholesterol in plasma membrane.It is not revealed that whether caveolin-3 can bind cholesterol and which domain is the binding area.Therefore,experiments expected to elucidate about scientific issues were devised and carried out in this research,concrete contents as follows:(1)Expression,purification and polymeric characteristic for caveoin-3 NCD were studied.Since caveolin-3 has hydrophobic property of membrane protein,it is difficult to express,purify and dissolve in aqueous phase for full-length caveolin-3.However,caveolin-3 NCD is in cytoplasm area with proper hydrophily.So caveolin-3 NCD is a breakthrogh for researching properties of caveolin-3 protein.We first tried to express caveolin-3 NCD in E.coli,purified functional caveolin-3 NCD and studied its polymeric character.The monomeric caveolin-3 NCD fomed stable dimers by hydrogen-bond interaction firstly;then the dimers aggregated to form granular low-molecular-weight sub-oligomers by hydrophobic effect;the low-molecular-weight sub-oligomers formed to rod shaped high-molecular-weight oligomers by hydrophobic effect and disulfide bond;the state of high-molecular-weight oligomers was unstable and could form larger oligomers,even product precipitation.(2)Structural change and polymerization ability were analyzed for caveoin-3 NCD fused to charged tags on either N-or C-terminal of caveolin-3 NCD respectively.Caveolin-3 NCD with different tags were expressed in E.coli and purified.Oligomerization of the caveolin-3 NCD were investigated through transmission electron microscope(TEM).Solubility of caveolin-3 NCD with negtive tag was higher than with positive tag,and the solubility went down with increasing positive charges of tag.Most of caveolin-3 NCDs with different tags exist in dimers prior to forming polymers.When fused with positive tags on N-termianl of the protein,caveolin-3 NCD presented as granular state,otherwise they presented as threadiness.(3)Structural change and state of aggregation were analyzed for 12 mutations of caveolin-3 NCD.The mutations were expressed in E.coli,checked by electrophoresis and purified by SEC.State of dimer aggregation of four matations,R27Q?P29L?N33K and V44 E,were different from wild-type caveolin-3 NCD.There were not any structure changes of dimmer of the other mutations observed.The wild-type caveolin-3 formed fibrous structure with NCD uniform diameter,but the oligomeric state of mutations changed by Different degrees.(4)Structures and polymerizing abilities of caveolin-3 peptides was analyzed.The caveolin-3(57-74)peptides,potential binding domain,and its mutations were synthesized.The structures and the polymerizing abilities were tested by gel electrophoresis,circular dichroism and electron microscopy.The peptide caveolin-3(55-74)and its mutations formed dimers in higher concentration,while kept free state in lower concentration.Compared to wild-type caveolin-3(55-74),secondary structures of the mutations changed in different degrees.Each peptide could form high-molecular-weight oligomers in higher concentration and granular oligomers in lower conectration.(5)Interaction experiment of caveolin-3 NCD with cholesterol.Throgh conprecipitation experiment of purifed caveolin-3 NCD and liposome with cholesterol at different ratios,it was discovered that there was ineraction between caveolin-3 NCD and cholesterol.This result seggested that caveolin-3 binding cholesterol by its NCD,similar to caveolin-1.Overall,caveolin-3 assemble into dimer,then the dimers aggregate to sub-oligomers in partical state,and lastly the sub-polymers become oligomers in rod state;caveolin-3 NCD with different tags had different oligomerization features,suggesting dose and position of charge can affect assembling of caveolin-3;defferent mutation of caveolin-3 NCD had different aggregation state,especially dimer state,and these changes may cause different muscular diseases due to different pathopoiesis;the aggregation domain can be narrowed the scope of pepetide 67-74 amino acid sequence;through interaction experiment between purified caveolin-3 NCD and cholesterol,binding domain of caveoin-3 with cholestol was identified. |
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