Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Cystic Fibrosis (CF) is an autosomal recessive disease that arises from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. The deletion of phenylalanine-508 (ΔF508-CFTR) is the most prevalent CF mutation and results in a misfolded protein that fails to exit the endoplasmic reticulum (ER). Previous studies demonstrated that mutation of a di-arginine based ER retention motif (R553AR555) in the first nucleotide binding domain (NBD1) rescues the trafficking defect of ΔF508-CFTR. We hypothesized that if the R553AR555 motif mediates retention of the ΔF508-CFTR protein, peptides that mimic this motif should antagonize mistrafficking mediated by aberrant exposure of the endogenous R553AR555 motif. We generated a peptide bearing the R553AR555 motif (CF-RXR) and conjugated it to the cell penetrating peptide Tat (CPP-CF-RXR) to facilitate intracellular delivery and investigated its efficacy in rescuing the mistrafficking and function of ΔF508-CFTR. Using a variety of biochemical and functional assays we demonstrate that the CPP-CF-RXR peptide is effective at increasing surface expression of ΔF508-CFTR in baby hamster kidney (BHK) and human embryonic kidney (HEK) cell lines. Furthermore, the increased surface expression is accompanied by an increase in its functional expression as a chloride channel. Using Ussing chamber assays, we demonstrate that the CPP-CF-RXR peptide improved ΔF508-CFTR channel function in respiratory epithelial tissues obtained from CF patients. Additionally, we investigated the effects of small molecules on mediating biosynthetic rescue of a ΔF508-CFTR construct bearing the additional mutations R553K and R555K (ΔFRK-CFTR) to inactivate the R553AR555 motif. Interestingly, mutation of the R553AR555 motif exerts an additive effect with correctors VRT-325 and Corrector 4a. Taken together, our data suggests that abnormal accessibility of the RXR motif present in NBD1 is a key determinant of the mistrafficking of the major CF causing mutant.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La chaîne invariante (Ii) agit à titre de chaperon dans l’assemblage et le trafic des molécules du complexe majeur d’histocompatibilité de classe II (CMHII). Chez l’humain, les deux isoformes prédominantes, p33 et p35, diffèrent par la présence d’un motif di-arginine (RXR). Ce dernier permet la rétention de p35 au réticulum endoplasmique (RE) jusqu’à son masquage par une molécule de CMHII. La chaîne invariante forme des trimères auxquels s’associent successivement jusqu’à trois dimères αß de CMHII résultant en la formation de pentamères, heptamères et nonamères. Toutefois, la stœchiométrie exacte des complexes Ii-CMHII qui quittent le RE et le mécanisme permettant le masquage du motif RXR demeurent un sujet de débats. Dans un premier temps, nous avons examiné par une approche fonctionnelle la stœchiométrie des complexes formés autour de p33 et de p35. Nous avons observé que p35 engendre la formation de complexes nonamériques (αßIi)3 et permet l’incorporation de différents isotypes de CMHII autour d’un même trimère de p35 alors que p33 facilite la formation de pentamères (αß)1Ii3. Lors de l’étude du masquage du motif RxR par les CMHII, nous avons montré que son inactivation requiert une interaction directe (en cis) entre les sous-unités p35 et CMHII, résultant en une rétention des trimères de p35 insaturés au RE. Aussi, nous avons observé que contrairement aux complexes p33-CMHII, les complexes p35-CMHII sont retenus au RE lorsque coexprimés avec la protéine NleA de la bactérie Escherichia coli entérohémorragique. Comme l’expression de NleA interfère avec la formation des vésicules COPII responsable de l’export du RE, nous supposons que la sortie du RE des complexes p35-CMHII dépend des vésicules COPII alors que la sortie des complexes formés autour de l’isoforme p33 est indépendante de la formation de ces vésicules. La trimérisation d’Ii représente la toute première étape dans la formation des complexes Ii-CMHII. Deux domaines d’Ii permettent la formation de trimères; le domaine de trimérisation (TRIM) et le domaine transmembranaire (TM). Nous nous sommes intéressés à la nécessité de ces domaines dans la trimérisation de la chaîne et la formation subséquente de complexes avec les CMHII. Nous avons démontré que le domaine TRIM n’est pas essentiel à la trimérisation de la chaîne, à la formation de pentamères et de nonamères ainsi qu’au trafic adéquat de ces complexes Ii-CMHII dans la cellule. En absence des domaines TM d’Ii et des CMHII, nous avons observé la formation de complexes pseudo-nonamériques. Ceci suppose que la présence de ce domaine n’est pas un prérequis à la formation de nonamères. En conséquence, la présence d’un seul domaine de trimérisation de Ii est requise pour la formation de trimères et de complexes nonamériques. L’ensemble de nos résultats démontrent que la fonction de p35 n’est pas redondante à celle de p33. p35 influence de manière distincte le trafic des CMHII puisqu’il affecte la stœchiométrie des sous-unités incorporées aux complexes Ii- CMHII. / The invariant chain (Ii) assists in the folding and trafficking of MHC class II molecules (MHCII). Four different isoforms of the human Ii have been described (p33, p35, p41 and p43). The main isoforms, p33 and p35, differ by the presence of a di-arginine (RXR) endoplasmic reticulum (ER) retention motif in p35. This motif is inactivated upon binding of MHCII. In the ER, p33 and p35 assemble into trimers before associating with MHCII. The sequential binding of up to three MHCII αß dimers to Ii trimers results in the formation of pentamers, heptamers and nonamers. However, the exact stoichiometry of the Ii-MHCII complex and the mechanism allowing shielding of the ER retention motif remain a matter of debate. To shed light on these issues, we chose a functional approach to examine the stoichiometry of complexes formed around the p33 and p35 isoforms. We showed that p35 promotes formation of nonameric complexes (αßIi)3 while formation of pentameric complexes (αß)1Ii3 was observed for p33. We then showed that formation of nonameric complexes can result in the inclusion of distinct MHCII isotypes around a single trimeric p35 scaffold. When answering the question wetter masking of the p35 RXR motif by MHCII results in the formation of nonamers, we showed that the actual inactivation of motif requires a direct cis-interaction between p35 and the MHCII, precluding ER egress of unsaturated p35 trimers. Interestingly, as opposed to p33-MHCII complexes, p35-MHCII complexes remained in the ER when co-expressed with the NleA protein of enterohaemorrhagic Escherichia coli. Expression of this bacterial protein is thought to interfere with the formation of COPII vesicles, leading to the conjecture that p35-MHCII and p33-MHCII complexes exit the ER in a COPII-dependant and COPII-independent manner, respectively. The trimerization of Ii represents the very first step in the formation of Ii-MHCII complex. Two domains of Ii, the trimerization domain (TRIM) and the transmembrane (TM) domain have been shown to trigger the trimerization of the chain. We focused our attention on the requirement of the two trimerization domains in Ii self-association and in the formation of pentameric and nonameric complexes. We showed that the TRIM domain of Ii is not essential for the chain’s trimerization, formation of pentamers and nonamers and for proper traffic with MHCII molecules. In absence of the Ii and MHCII TM domains, we observed the formation of a nonamer-like structure hereby suggesting that the presence of this domain is not a prerequisite for nomamer complex formation. Consequently, our results showed that either Ii trimerization domains are sufficient for Ii trimer formation and nonameric complex trafficking. Taken together, our results demonstrate that the function of the p35 isoform is not redundant, influencing distinctively MHCII trafficking as the subunit stoichiometry of oligomeric Ii/MHCII complexes is affected by p35.