Membrane proteins are found in all kingdoms of life and are essential for cellular interactions with the environment. Although a large research effort have been put into this group many membrane proteins remains uncharacterized, both in terms of function and evolutionary history. We have estimated the component of α-helical membrane proteins within the human proteome; the membrane proteome. We found that the human membrane proteome make up 27% of all protein, which we could classify the majority of into 234 families and further into three major functional groups: receptors, transporters or enzymes. We extended this analysis by determining the membrane proteome of 24 organisms that covers all major groups of eukaryotes. This comprehensive membrane protein catalog of over 100,000 proteins was utilized to determine the evolutionary history of all membrane protein families throughout eukaryotes.  We also investigated the evolutionary history across eukaryotes of the antiviral Interferon induced transmembrane proteins (IFITM) and the G protein-coupled receptor (GPCR) superfamily in detail.  We identified ten novel human homologs to the IFITM proteins, which together with the known IFITMs forms a family that we call the Dispanins. Using phylogenetic analysis we show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and animals, and that the family later expanded in vertebrates into four subfamilies. The GPCR superfamily was mined across eukaryotic species and we present evidence for a common origin for four of the five main human GPCR families; Rhodopsin, Frizzled, Adhesion and Secretin in the cAMP receptor family that was found in non-metazoans and invertebrates, but has been lost in vertebrates. Here we present the first accurate estimation of the human proteome together with comprehensive functional and evolutionary classification and extend it to organisms that represents all major eukaryotic groups. Moreover, we identify a novel protein family, the Dispanins, which has an evolutionary history that has been formed by horizontal gene transfer from bacteria followed by expansions in the animal lineage. We also study the evolution of the GPCR superfamily throughout eukaryotic evolution and provide a comprehensive model of the evolution and relationship of these receptors.

abstract: G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set. The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

No description available.

O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos. / The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.

<p>Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.</p>

O sistema olfatório de mamífero pode discriminar milhares de odores presentes no meio ambiente. Aproximadamente 1000 diferentes receptores olfatórios (ORs) são expressos no epitélio olfatório (OE) do nariz, Os ORs detectam os odores e transmitem os sinais resultantes para o bulbo olfatório (OB) no cérebro. Os ORs pertencem a super família dos receptores acoplados a proteína G (GPCR) e apresentam sete domínios transmembrânicos putativos. Por razões desconhecidas, os ORs são retidos no retículo endoplasmático quando expressos em linhagens de células de mamíferos heterólogas. Provavelmente, proteínas acessórias sejam requeridas para o endereçamento dos Ors para a superficie celular. No presente estudo, utilizamos o OR M93 para estudar os mecanismos de expressão de um ORo A dissertação teve como objetivos específicos: (l) construção de um vetor para expressão do OR M93 em fusão com GFP em levedura e análise de sua localização celular; (2) identificar proteínas expressas no epitélio olfatório de camundongo que interajam com os ORs. A análise por microscopia de fluorescência revelou que a expressão do OR M93 fusionado a GFP demonstrou um padrão de fluorescência que sugere a retenção do OR M93 no retículo endoplasmático. Nós utilizamos o sistema de duplo híbrido em levedura para varrer uma biblioteca de cDNA de epitélio olfatório de camundongo com uma isca correspondente à região N-terminal do OR M93. Quatro proteínas candidatas foram identificadas: HLA-B associado ao transcrito 3 (BAT-3/ Scythe), superfamília transmembrana 4 (membro CD82), superfamília transmembrana 4 (membro OAP-I) e sindecan (membro SDC2) (\"GenBank accession numbers\": BC026647, D14883, BC0430n e BC047144). A análise da hibridação in situ destas proteínas, revelou que a proteína OAP-1 é a melhor candidata a interação com OR M93. Dessa maneira, nós indicamos a proteína OAP-1 como possível proteína candidata a auxiliar o OR a ser expresso de maneira funcional em sistemas heterólogos. / The mammalian olfactory system can discrim inate thousands of odorants present in the environrnent. Approximately 1000 different olfactory receptors (ORs) are expressed in the olfactory epithelium (OE) of the nose. The ORs detect odorants and transmit the resulting signals to the olfactory bulb (OB) of the brain. ORs belong to the G-protein-coupled receptor (GPCR) super family and have seven putative transmembrane domains. For unknown reasons, the ORs are retained in the endoplasmatic reticulum when expressed in heterologous mammalian cell lines. Probably accessory proteins are required for the sorting of the ORs to the cell surface. In the present work, we used the OR M93 to study the mechanisms of OR expression. Our goals were to (1) construct an expression vector for OR M93 in fusion with GFP in yeast and (2) to identify proteins expressed in the mouse OE that interact with ORs. The analysis by fluorescence microscopy suggested that OR M93 in fusion with GFP was retained in the endoplasmic reticulum (ER) of yeast. We used the yeast two-hybrid system to screen a mouse OE cDNA library with a bait corresponding to the N-terminal region ofthe üR M93. Four potential candidates were identified: HLA-B associated transcript 3 (BAT-3/Scythe), transmembrane 4 superfamily (CD82 member), transmembrane 4 superfamily (TSPN-3 member) and syndecan (SDC2). In situ hybridization analysis suggests that OAP-l protein represents the best candidate for interaction with OR M93. We suggest the OAP-l protein could be an accessory protein required for the sorting of the ORs to the cell surface in heterologous cell lines.

Les β-arrestines jouent un rôle important dans la transduction du signal par les récepteurs couplés aux protéines G (RCPG). Nous montrons dans cette thèse que les β-arrestines exercent des régulations plus complexes et subtiles qu'on ne le pensait jusque-là sur la voie AMPc/PKA/CREB qui est activée par les RCPGs couplés à Gs. Nous montrons que les β-arrestines interagissent directement la PKAcat et contribuent à sa translocation nucléaire. De plus, nous mettons en évidence une interaction β-arrestine/CREB qui conduit à la formation d'un complexe transcriptionnellement actif sous l’action de l’agoniste. D’autre part, nous avons constaté que les β-arrestines interagissent directement avec PKAcat, p70S6K et Src via un même site et lesquelles sont donc potentiellement mutuellement exclusives. Nous avons ensuite mesuré l’impact d’une mutation et d’un polymorphisme du R-FSH sur la signalisation dépendante des β-arrestines, notamment grâce à l’utilisation de senseurs FRET et BRET. / Β-arrestins play an important role in G protein-coupled receptor (GPCR)-induced signal transduction. In this thesis, we show here that β-arrestins exert more complex and subtle regulation than previously thought on the cAMP/PKA/CREB pathway which is activated by Gs-coupled GPCRs. We demonstrate that β-arrestins directly interact with PKAcat and promote its translocation to the nucleus. Moreover, we provide evidence that β-arrestins directly interact with CREB thereby forming a transcriptionally active complex upon agonist stimulation. We also found that PKAcat, p70S6K and Src all directly interact with β-arrestins through the same interaction site and are therefore potential mutually exclusive interactions. We then measured the impact of a point mutation and of a polymorphism in the FSH-R on β-arrestin-dependent signaling, in part using FRET and BRET sensors.

Afin d’améliorer la stabilité plasmatique de peptides, nous avons développé une nouvelle stratégie basée sur l’introduction d’une chaîne fluorocarbonée dans la séquence d’un peptide natif. En appliquant le concept à l’apeline-17, un peptide présentant un intérêt potentiel pour le traitement de maladies cardiovasculaires, nous avons amélioré sa stabilité plasmatique de 4 min à plus de 24 h ainsi que son efficacité in vivo. L’étude du mécanisme de stabilisation a permis de mettre en évidence la liaison de la fluoroapeline à l’albumine, conduisant à la protection du peptide vis-à-vis de la protéolyse. Le concept a été appliqué à d’autres peptides tels que l’apeline-13, l’angiotensine II, l’ocytocine et la spexine, démontrant ainsi l’étendue et les limitations de la méthode. Enfin, nous avons également conçu des sondes fluorescentes « turn-on » originales capables de révéler leur fluorescence uniquement après liaison au récepteur ciblé. Ces sondes pourront nous servir, par la suite, pour l’étude in vivo de la biodistribution des fluoropeptides. / In order to improve the plasma stability of peptides, we have developed a new strategy based on the introduction of a fluorocarbon chain in the sequence of a native peptide. By applying this concept to apelin-17, a peptide showing a potential interest for the treatment of cardiovascular diseases, we have improved its plasma stability from 4.6 min to more than 24 h as well as its in vivo efficacy. The mechanism leading to the increase of plasma stability has been carefully investigated demonstrating the binding of the fluoroapeline to the albumin, leading to protection towards roteolysis. The concept has been applied to other peptides such as apelin-13, angiotensin II, oxytocin and spexine, showing the extension and the limitations of this method. Finally, we have designed original fluorescent fluorogenic probes which turn on their fluorescence only after binding to the targeted receptor. These probes could be used for in vivo biodistribution studies of fluoropeptides.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins in animals and represent the largest family of druggable targets in the human genome. Therefore, it is of no surprise that the molecular mechanisms of GPCR activation and signal transduction have attracted close attention for the past few decades. Several stabilizing interactions within the GPCR transmembrane (TM) domain helices regulate receptor activation. An example is a salt bridge between 2 highly conserved amino acids at the bottom of TM3 and TM6 that has been characterized for a large number of GPCRs. Through structural modeling and molecular dynamics (MD) simulations, we predicted several electrostatic interactions to be involved in metabotropic glutamate receptor 2 (mGlu2R) activation. To experimentally test these predictions, we employed a charge reversal mutagenesis approach to disrupt predicted receptor electrostatic intramolecular interactions as well as intermolecular interactions between the receptor and G proteins. Using two electrode voltage clamp in Xenopus laevis oocytes expressing mutant receptors and G-proteins, we revealed novel electrostatic interactions, mostly located around intracellular loops 2 and 3 of mGlu2R, that are critical for both receptor and G-protein activation. These studies contribute to elucidating the molecular determinants of mGluRs activation and conformational coupling to G-proteins, and can likely be extended to include other classes of GPCRs.