Membrane proteins are molecules that are associated with the membrane of the cell or organelle. They possess fundamental functions in the cell. They are flexible enough to change conformationally to perform their functions. Among the membrane proteins G-protein coupled receptors are most diverse and large group. G protein-coupled receptors (GPCRs) are integral membrane proteins used by cells to convert extracellular signals into intracellular responses, including responses to hormones, neurotransmitters, and responses to visual, olfactory, and taste cues. In the retina of the mammals, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is essential for dim vision, and interruption of this process leads to congenital stationary night blindness (CSNB) in human. In metabotropic signaling cascade of retinal cells, initiated by the mGluR6 receptor, the generation of depolarizing responses to light-induced changes are due to neurotransmitter glutamate release from the photoreceptor axonal terminals. There are several proteins involved in transducing pathway, among which is GPR179 receptor. GPR179 is an orphan receptor, a member of the G protein-coupled receptor (GPCR) superfamily, which has been shown to be important for the synaptic responses of ON-bipolar cells of the retina and the most important component of metabotropic signaling cascade. Evidence showed that mutations or loss of GPR179 leads to CSNB. GPR179 protein interacts with components of the signaling cascade of ON-bipolar cells. Determination of three-dimensional structure of GPR179 taking advantage of the recent advances in single particle cryo-electron microscopy, could provide with a molecular understanding of its function in this signaling process. This thesis work was focused on identifying the best conditions for the expression of GPR179 in a suitable host system and a purification protocol to obtain a protein with the stability and purity requirements for structural studies. The different approaches carried put in this thesis work involved detergent based purification or the use of a more native-like environment such as reconstitution into apolipoprotein-based lipid nanodisc. The expression of GPR179 was carried out in HEK293F expression system first in small and subsequently in large scale. We generated different constructs of GPR179 containing GFP and affinity tags and Pikachurin, which is a dystroglycan ligand that complexes with GPR179 for photoreceptor synaptic transmission. The GFP fusion to the constructs were employed to facilitate the expression analysis of these constructs through Fluorescence-detection size exclusion chromatography (FSEC) before purification. The expression analysis was done in HEK293F and HEK293-EBNA to compare the expression of GPR179 in both the expression system. We set up a purification pipeline for GPR179 that consists of Immobilized metal ion affinity chromatography (IMAC) with cobalt and nickel resin followed by Size exclusion chromatography. The affinity tags we used for purification were Histidine tag and Strep tag. Yield and protein quality were used to identify the best purification system. In parallel, we performed reconstitution of detergent purified GPR179 in soybean nanodiscs assembled with scaffold protein MSP 2N2. Despite technical difficulties related to reconstituted protein recovery we managed to obtain a sample suitable for negative staining electron microscopy. In conclusion, this work identified a suitable expression system for GPR179 in HEK239F mammalian cells and provided a layout for purification protocol for the GPR179 protein. The best results were obtained using a GFP-fusion construct for screening conditions and a strep tag for higher purification quality. Moreover, protein reconstituted in nanodiscs will provide a valid alternative for GPR179 structural studies in a lipidic environment.
Le proteine di membrana sono molecole associate alla membrana della cellula o dell'organello. Nella retina dei mammiferi, la trasmissione sinaptica tra i fotorecettori a bastoncello eccitati dalla luce e i neuroni bipolari ON a valle è essenziale per la visione fioca e l'interruzione di questo processo porta alla cecità notturna stazionaria congenita (CSNB) nell'uomo. Nella cascata di segnalazione metabotropica delle cellule retiniche, avviata dal recettore mGluR6, la generazione di risposte depolarizzanti ai cambiamenti indotti dalla luce è dovuta al rilascio del glutammato del neurotrasmettitore dai terminali assonali dei fotorecettori. Ci sono diverse proteine coinvolte nella via di trasduzione, tra cui il recettore GPR179. GPR179 è un recettore orfano, un membro della superfamiglia del recettore accoppiato a proteine G (GPCR), che ha dimostrato di essere importante per le risposte sinaptiche delle cellule bipolari ON della retina e il componente più importante della cascata di segnalazione metabotropica. Le prove hanno mostrato che le mutazioni o la perdita di GPR179 portano a CSNB. La proteina GPR179 interagisce con i componenti della cascata di segnalazione delle cellule ON-bipolari. La determinazione della struttura tridimensionale di GPR179 sfruttando i recenti progressi nella microscopia crioelettronica a particella singola, potrebbe fornire una comprensione molecolare della sua funzione in questo processo di segnalazione.Questo lavoro di tesi si è concentrato sull'identificazione delle migliori condizioni per l'espressione di GPR179 in un sistema ospite adatto e unprotocollo di purificazione per ottenere una proteina con i requisiti di stabilità e purezza per studi strutturali. I diversi approcci svolti in questo lavoro di tesi hanno coinvolto la purificazione a base di detergenti o l'uso di un ambiente più simile a quello nativo come la ricostituzione in nanodisc lipidici a base di apolipoproteine. L'espressione di GPR179 è stata effettuata nel sistema di espressione HEK293F prima in piccola scala e successivamente in larga scala. Abbiamo generato diversi costrutti di GPR179 contenenti GFP e tag di affinità e Pikachurin, che è un ligando distroglicano che si complessa con GPR179 per la trasmissione sinaptica dei fotorecettori. La fusione GFP con i costrutti è stata impiegata per facilitare l'analisi dell'espressione di questi costrutti attraverso la cromatografia a esclusione dimensionale con rilevamento della fluorescenza (FSEC) prima della purificazione. L'analisi dell'espressione è stata eseguita in HEK293F e HEK293-EBNA per confrontare l'espressione di GPR179 in entrambi i sistemi di espressione.Abbiamo istituito una pipeline di purificazione per GPR179 che consiste in cromatografia di affinità con ioni metallici immobilizzati (IMAC) con resina di cobalto e nichel seguita da cromatografia di esclusione dimensionale. I tag di affinità che abbiamo utilizzato per la purificazione erano il tag di istidina e il tag di strept. La resa e la qualità delle proteine sono state utilizzate per identificare il miglior sistema di purificazione. In parallelo, abbiamo eseguito la ricostituzione del detergente GPR179 purificato in nanodischi di soia assemblati con proteina scaffold MSP 2N2. Nonostante le difficoltà tecniche legate al recupero delle proteine ricostituite siamo riusciti ad ottenere un campione adatto alla microscopia elettronica a colorazione negativa.In conclusione, questo lavoro ha identificato un sistema di espressione adatto per GPR179 nelle cellule di mammifero HEK239F e ha fornito un layout per il protocollo di purificazione per la proteina GPR179. I migliori risultati sono stati ottenuti utilizzando un costrutto di fusione GFP per condizioni di screening e un tag streptococco per una maggiore qualità di purificazione. Inoltre, la proteina ricostituita in nanodiscs fornirà una valida alternativa per gli studi strutturali GPR179 in ambiente lipidico.
Expression and purification trials of retinal receptor GPR179 for structural studies.
MURUGAN, ABIRLA
2021/2022
Abstract
Membrane proteins are molecules that are associated with the membrane of the cell or organelle. They possess fundamental functions in the cell. They are flexible enough to change conformationally to perform their functions. Among the membrane proteins G-protein coupled receptors are most diverse and large group. G protein-coupled receptors (GPCRs) are integral membrane proteins used by cells to convert extracellular signals into intracellular responses, including responses to hormones, neurotransmitters, and responses to visual, olfactory, and taste cues. In the retina of the mammals, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is essential for dim vision, and interruption of this process leads to congenital stationary night blindness (CSNB) in human. In metabotropic signaling cascade of retinal cells, initiated by the mGluR6 receptor, the generation of depolarizing responses to light-induced changes are due to neurotransmitter glutamate release from the photoreceptor axonal terminals. There are several proteins involved in transducing pathway, among which is GPR179 receptor. GPR179 is an orphan receptor, a member of the G protein-coupled receptor (GPCR) superfamily, which has been shown to be important for the synaptic responses of ON-bipolar cells of the retina and the most important component of metabotropic signaling cascade. Evidence showed that mutations or loss of GPR179 leads to CSNB. GPR179 protein interacts with components of the signaling cascade of ON-bipolar cells. Determination of three-dimensional structure of GPR179 taking advantage of the recent advances in single particle cryo-electron microscopy, could provide with a molecular understanding of its function in this signaling process. This thesis work was focused on identifying the best conditions for the expression of GPR179 in a suitable host system and a purification protocol to obtain a protein with the stability and purity requirements for structural studies. The different approaches carried put in this thesis work involved detergent based purification or the use of a more native-like environment such as reconstitution into apolipoprotein-based lipid nanodisc. The expression of GPR179 was carried out in HEK293F expression system first in small and subsequently in large scale. We generated different constructs of GPR179 containing GFP and affinity tags and Pikachurin, which is a dystroglycan ligand that complexes with GPR179 for photoreceptor synaptic transmission. The GFP fusion to the constructs were employed to facilitate the expression analysis of these constructs through Fluorescence-detection size exclusion chromatography (FSEC) before purification. The expression analysis was done in HEK293F and HEK293-EBNA to compare the expression of GPR179 in both the expression system. We set up a purification pipeline for GPR179 that consists of Immobilized metal ion affinity chromatography (IMAC) with cobalt and nickel resin followed by Size exclusion chromatography. The affinity tags we used for purification were Histidine tag and Strep tag. Yield and protein quality were used to identify the best purification system. In parallel, we performed reconstitution of detergent purified GPR179 in soybean nanodiscs assembled with scaffold protein MSP 2N2. Despite technical difficulties related to reconstituted protein recovery we managed to obtain a sample suitable for negative staining electron microscopy. In conclusion, this work identified a suitable expression system for GPR179 in HEK239F mammalian cells and provided a layout for purification protocol for the GPR179 protein. The best results were obtained using a GFP-fusion construct for screening conditions and a strep tag for higher purification quality. Moreover, protein reconstituted in nanodiscs will provide a valid alternative for GPR179 structural studies in a lipidic environment.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15183