2024-03-29T09:48:41Zhttps://www.tdx.cat/oai/requestoai:www.tdx.cat:10803/3951832017-09-21T04:47:44Zcom_10803_183col_10803_235
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Stability and folding of G-protein coupled receptors associated to degenerative diseases
[Barcelona] :
Universitat Politècnica de Catalunya,
2016
Accés lliure
http://hdl.handle.net/10803/395183
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Dong, Xiaoyun,
autor
1 recurs en línia (196 pàgines)
Tesi
Doctorat
Universitat Politècnica de Catalunya. Departament d'Enginyeria Agroalimentària i Biotecnologia
2016
Universitat Politècnica de Catalunya. Departament d'Enginyeria Agroalimentària i Biotecnologia
Tesis i dissertacions electròniques
Garriga Solé, Pere,
supervisor acadèmic
Ramon Portés, Eva,
supervisor acadèmic
TDX
G-protein coupled receptors (GPCRs) are the largest membrane protein superfamily encoded by the human genome and represent the largest class of drug targets for a wide range of pathological conditions. Two GPCRs members, the visual pigment rhodopsin associated with the retinal degenerative disease retinitis pigmentosa (RP), and the muscarinic acetylcholine (ACh) 3 receptor (M3R) associated with Alzheimer’s disease (AD), are studied in this thesis.
Rhodopsin is the prototypical visual photoreceptor mediating scotopic vision and distributed throughout the retina. Upon illumination, the bound chromophore 11-cis-retinal isomerizes to all-trans-retinal and triggers the visual signaling cascade. Mutations found in rhodopsin, such as G90V and N55K, are responsible for the retinal degenerative disease RP. To counteract the low structural stability of these mutants, and to provide a deeper understanding of the molecular mechanisms leading to visual dysfunction, artificial membranes in the form of DMPC/DHPC bicelles and DDHA-PC liposomes were prepared. DMPC/DHPC bicelles and DDHA-PC liposomes provided a native-like bilayer environment, which preserved rhodopsin wild type and mutants structure and increased their thermal stability to varying degrees compared to the usual dodecyl maltoside (DM) detergent. Furthermore, chromophore regeneration of G90V and N55K mutants in DMPC/DHPC bicelles condition was enhanced compared to DM condition. Moreover, the kinetics for the active state metarhodopsin II (Meta II) decay indicated that retinal release rates of G90V and N55K mutants became faster in the presence of DMPC/DHPC bicelles and DDHA-PC liposomes compared to DM condition. The addition of hydroxylamine upon Meta II complete decay of WT and G90V in bicelles increased fluorescence intensity, suggesting that retinal can be retained inside the binding pocket. DMPC/DHPC bicelles and DDHA-PC liposomes provided stable conditions so that G90V opsin, obtained after Meta II completely decay, was able to regenerate upon the addition of exogenous retinal. On the other hand, N55K was not able to regenerate, indicating that the molecular mechanisms associated to this mutant has important differences which may be associated with their specific clinical phenotypes.
The interactions between rhodopsin and arrestin, and between M3R and tau protein are studied in their association with the degenerative diseases, RP and AD respectively. Active rhodopsin bound R175E mutant arrestin and slowed down the retinal release from the binding pocket.
Arrestin binding assays on mutants associated to RP would help uncover mechanisms related to visual cascade termination, not studied so far.
M3R plays a role in muscarinic ACh signal transmission and on ion channels function especially in the central nervous system (CNS). In the M3R-tau interaction studies, M3R WT and mutants N132G and D518N did change the location of tau from the cytoplasm to the membrane when they were coexpressed in HEK293T cells. M3R mutants D518K and K523Q were affected when coexpressed with tau and trafficked from the membrane to the cytoplasm. These shifts in location likely result from the interaction between tau and M3R WT and mutants. This finding provides new clues about the specific tau binding/recognition sites on M3R and the possible involvement of such interaction in the pathophysiology of AD.
Overall, the artificial membranes DMPC/DHPC bicelles and DDHA-PC liposomes systems provide a better bilayer environment to stabilize rhodopsin WT and mutants than DM detergent environment thus reverting their intrinsic thermal sensitivity. The different behavior of G90V and N55K in artificial membranes could be associated with their specific clinical phenotypes. On the other side, the results obtained on the interaction between GPCRs and other proteins provide a foundation for further studies associated with GPCRs mutants and degenerative diseases
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