The Study On The Structures Of The Scaffolding And Intra Membrane Domain Of Caveolin-1and Their Interactions With Cholesterol

Caveolae are flask-shaped invaginations of the plasma membranes. Caveolinshave been identified as integral membrane proteins and an important structuralcomponent of caveolae. The caveolin protein family has three members, caveolin-1,-2, and-3. Caveolin-1is involved in cellular endocytosis, cholesterol homeostasis andsignal transduction, and plays a role in controlling cell growth, development andproliferation. Caveolin-1has also been associated with some pathophysiologicalprocesses, such as inflammation, atherosclerosis, oncogenic transformation,myocardial hypertrophy and senescence. Cholesterol is crucial to the formation andmaintenance of caveolae. Early studies showed that the caveolae are rich of sterolsand caveolin binds cholesterol tightly in an equal ratio. The decrease in the amount ofcholesterol in caveolae will flatting the structure of caveolae and affecting the signaltransduction of some receptors.Caveolin-1is anchored to the membrane with a hydrophobic membrane-insertingsegment (102–122) and three palmitoyl chains attached to Cys residues (residues133,143and156). Caveolin-1forms an unusual hairpin-shaped structure in phospholipidmembrane rather than a membrane-spanning α-helix. The formation of thehairpin-shaped structure brings both the N-and C-terminal domains of the protein intothe cytoplasm. This asymmetric insertion of caveolin-1into the lipid bilayer perturbsthe inner leaflet more than outer leaflet, leading to an unbalanced stretching to themembrane and curving the membrane to create caveolae. The residue Pro110in theIMD has been shown to be important for the formation of a hairpin structure of theprotein in the membrane. The helical bend will disappear with the mutation of Pro110.The most prominent domain of caveolin-1is the CSD, as residues within it not onlyare responsible for membrane binding but also are critical for oligomerization, proteininteractions, and cholesterol recognition. The CSD contains a sequenceV94-T-K-Y-W-F-Y-R101that is considered a cholesterol recognition/interaction sequence (CRAC). Another structural feature of caveolin-1is its formation of highmolecular mass oligomers containing15monomers. The oligomerization ofcaveolin-1is associated closely with the scaffolding domain.In order to gain further insight into the structure of caveolin-1and the role of theCRAC motif in the interactions of the protein with cholesterol, we studied in thisthesis the structures of the intramembrane domain and the scaffolding domain ofcaveolin-1and the interactions of these domains with cholesterol using differentialscanning calorimetry (DSC), fluorescence, circular dichroism (CD) and attenuatedtotal reflection fourier transform infrared (ATR-FTIR) spectra. We characterized thesecondary structures of these domains, explored the effects of the domains on thedistributions of cholesterol in the membrane, and investigated the roles of CRACmotif in inducing the packing of cholesterol in membrane. We also determined threedimensional structures of the intramembrane peptides and the oligomerization of thepeptides using NMR, CD and SDS-PAGE techniques. Three CRAC-containingpeptides, corresponding to caveolin-194–101,82–101and93–126, oneCRAC-excluding peptide corresponding to caveolin-1101–126and the Y97Isubstituents of three CRAC-containing peptides, were used in the study. Thedetergents sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) as wellas the organic solvent1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were chosen asmembrane-mimetic environments in the studies of the structures and assemblies of theintramembrane peptides, and the phopholipid1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was chosen as liposome-mimic in the studies ofpeptide-cholesterol interactions.The DSC studies demonstrated that all the caveolin-1peptides studied induced aredistribution of cholesterol in DPPC membrane. However, the redistributions ofcholesterol in the DPPC membrane were different in the presence of different peptides.Of the three CRAC-containing peptides, the peptide93–126was particularly effectivein promoting cholesterol segregation, while the peptide82–101was less potent inpromoting the formation of domains than the peptide93–126, but was more potentthan the peptide94–101. Whereas the formation of domains was facilitated by the peptide Cav-1(93–126) in the DPPC membrane with cholesterol, a dispersivedistribution of cholesterol instead of clustering of cholesterol in the lipid membranewas mediated by Cav-1(101–126). The Y97I mutation reduced the potency of thepeptides in sequestering cholesterol into domains. The fluorescence results showedthat the presence of Chol induced a movement of the peptides in the DPPC lipidmembrane from a deeper position in the absence of Chol to a more solvent exposedposition. For the CRAC-containing peptides, the Trp residues of the wild-typepeptides undergo a more significant change in environments from the Chol-freemembrane to the Chol-containing membrane than those of the mutants, possiblythrough more preferential interaction of the wild-type peptides with Chol than themutants. However, the peptide Cav-1(101–126) that has no CRAC motif showed alower magnitude of change either in fluorescence intensity or in Ksv from theChol-containing membrane to the Chol-free membrane than the peptideCav-1(93–126) that contains the CRAC motif. The CD and ATR-FTIR resultsdemonstrated that the peptides94–101,82–101and93–126formed predominantlyrandom,-sheet and-helix conformations, respectively. Different conformations ofthe three peptides in the membrane may be a factor contributing to their differenteffects on the distribution of Chol in the lipid membrane. The interaction of theCRAC motif with Chol may have a stabilizing effect on the-helix of theintramembrane peptide and the-sheet of the scaffolding domain peptide. The Y97Imutation resulted in partly unfolding of helix in the CRAC motif. All these resultssuggested that the CRAC segment is an crucial caveolin-cholesterol interactiondomain and the residue Y97may play a role in the interaction of caveolin-1withcholesterol.The three dimensional structures of two peptides from the membrane-insertingdomain (caveolin-193–126and101–126) were obtained by NMR and the dynamiccalculation. Both structures were characterized by a ‘helix-break-helix’ motif, inwhich two helices span over Leu103–Phe107and Ile117–His126and a loop rangingGly108–Gly116is sandwiched in between. The glycine residues at position108and116were found to be important for the break of the helical structure. All the CD, SDS-PAGE and NMR results showed an evidence that the CRAC-containing peptideCav-1(93–126) was more aggregated than the CRAC-excluding peptideCav-1(101–126), suggesting that the CRAC-motif may play a promoting role in theaggregation of the peptide.