Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane. We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated. Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.

The secretory pathway of G. max performs a prominent role in its defense to H. glycines parasitism, a finding reinforced by the identification of the SNARE-associated gene ALPHA-SNAP-5 being rhg1. Other proteins performing important roles in secretion are Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA which bind to form a trimeric complex that imports proteins into the ER for their eventual secretion. Comparative analyses to the Saccharomyces cerevisiae Sec61ALPHA, Sec61BETA and Sec61GAMMA proteins, respectively, shows G. max has 4 Sec61-ALPHA, 9 Sec61-BETA and4 Sec61-GAMMA proteins. At least one paralog from each gene family is expressed in H. glycines-parasitized root cells in G. max, but while undergoing a defense process. The overexpression of one selected Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA in the H. glycines-susceptible genotype G. max[Williams 82/PI 518671] leads to an engineered defense response. In contrast, RNAi of the same selected Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA genes in the H. glycines-resistant genotype G. max[Peking/PI 548402] leads to an engineered susceptible response. The combined opposite outcomes of the Sec61-ALPHA, Sec61-BETA and Sec61-GAMMA gene overexpression and RNAi provides evidence that they function in the defense process, consistent with the hypothesis that the G. max secretion system plays a role in its defense to H. glycines parasitism.

During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain.

Im Rahmen der vorliegenden Arbeit wurde eine mögliche Beteiligung der Protein-Translokase des Endoplasmatischen Retikulums aus Canis familiaris an der Vermittlung eines passiven Calcium-Ausstromes aus dem ER-Lumen untersucht. Der Sec61-Komplex konstituiert eine ionenpermeable Pore im Translokon des Endoplasmatischen Retikulums. Der Kanal zeigt eine hohe Dynamik im Schaltverhalten mit einer Vielzahl von Unterleitwerten, deren Mittelwerte gut mit publizierten Daten übereinstimmen. Zudem besitzt die Pore eine geringe Anionenselektivität in Experimenten mit KCl-Lösungen. Unter Verwendung von CaCl2- und MgCl2-Elektrolyten steigt diese deutlich an, was in vivo den Sec61-vermittelten, passiven Calcium-Ausstrom aus dem ER limitieren kann. Calmodulin (CaM) konnte im Rahmen der vorliegenden Arbeit als potenter Effektor des Sec61-Kanales identifiziert werden. Das Protein vermittelt ein Calcium-abhängiges, nahezu vollständiges Schließen des Kanals, während Calcium-freies ApoCalmodulin keinen Effekt auf den Offenzustand hat. Mittels Fluoreszenz-Korrelations-Spektroskopie konnte gezeigt werden, dass ein IQ-Motiv als putative Calmodulin-Bindestelle im cytosolischen N-Terminus der Sec61alpha-Untereinheit Ca2 -CaM mit nanomolarer Affinität bindet, eine Interaktion mit ApoCaM hingegen erst bei wesentlich höheren Konzentrationen stattfindet. Die CaM-vermittelte, negative Feedback-Regulation des Sec61-Komplexes durch Calcium legt einen CDI- (calcium-dependent inactivation) Mechanismus nahe, der die Membranbarriere des Endoplasmatischen Retikulums auch in Anwesenheit des weiten Translokonkanals aufrecht erhält.Vergleichende Experimente haben zudem ergeben, dass der Sec61-Kanal aus Saccharomyces cerevisiae im Hinblick auf die grundsätzlichen elektrophysiologischen Eigenschaften übereinstimmende Charakteristika mit dem Komplex aus Canis familiaris zeigt.

Im Gegensatz zur den monomeren Leaderpeptidasen der bakteriellen Plasmamembran bestehen die eukaryotischen Signalpeptidasen der ER-Membran aus einem heteromeren Protein-Komplex. In der Hefe S. cerevisiae setzt sich die Signalpeptidase aus den vier Membranproteinen Sec11p, Spc1p, Spc2p und Spc3p zusammen. Neben der zur prokaryontischen Leaderpeptidase homologen Untereinheit Sec11p wird auch Spc3p benötigt um die Spaltungsfunktion in der Zelle auszuüben. Die Deletion von SPC3 führt zu einer lethalen Akkumulation von sekretorischen Vorstufenproteinen in vivo, sowie zum Verlust der Spaltungsaktivität in vitro. Spc1p und Spc2p sind nicht essentiell für die Hefe. Für Spc2p konnte jedoch gezeigt werden, daß die Signalpeptidase über die Spc2p Untereinheit mit den b-Untereinheiten des Sec61-Komplexes und des Ssh1-Komplexes interagiert. Vermutlich wird es so dem Komplex ermöglicht, während des Translokationsprozesses engen Kontakt zu der im Translokationskanal befindlichen Signalsequenz aufzunehmen. Im zweiten Teil der Arbeit wurden neue Komponenten aus der ER Membran von Säugern aufgereinigt. Dabei wurde ein ribosomenfreier Sec61-Komplex entdeckt, der mit zwei weiteren Membranproteinen assoziiert ist. Die beiden neuen Membranproteine weisen Homologien zu essentiellen Untereinheiten des postranslational aktiven Sec-Komplexes der Hefe S. cerevisiae auf. Die Rolle des neu entdeckten Säugerkomplexes während der Proteintranslokation ist noch unbekannt, in der Arbeit werden mögliche Funktionen des Komplexes diskutiert. / In contrast to the monomer leaderpeptidase of the prokaryotic plasmamembrane, the eukaryotic signalpeptidase of the ER-membrane is a heteromer protein complex. In yeast the signalpeptidase consist of the four subunits Sec11p, Spc1p, Spc2p and Spc3p. Additional to Sec11p also Spc3p is essential for cell growth and cell life. The depletion of Spc3p cause lethal accumulation of precursor proteins in vivo and lost of cleavage activity in vitro. Spc1p and Spc2p are not essential for the cell. We show here, that the Spc2p subunit interacts with the ß-subunits of the Sec61- and the Ssh1-complex. These data implicate that Spc2p facilitates the interactions between different components of the translocation site. In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occurco-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

La sécrétion des protéines est un processus essentiel à la vie. Chez les eucaryotes, les protéines sécrétées transitent dans le réticulum endoplasmique par le pore de translocation. Le translocon est composé de trois sous-unités fondamentales nommées Sec61α, β et γ chez les mammifères, ou Sec61p, Sbh1p et Sss1p chez les levures. Tandis que le rôle des sous-unités α et γ est bien connu, celui de la sous-unité β demeure énigmatique. Plusieurs phénotypes distincts sont associés à cette protéine dans différents organismes, mais le haut niveau de conservation de séquence suggère plutôt une fonction universelle conservée. Récemment, Feng et al. (2007) ont montré que le domaine transmembranaire (TMD) de Sbh1p était suffisant pour complémenter plusieurs phénotypes associés à la délétion du gène chez Saccharomyces cerevisiae, suggérant un rôle important de cette région. L’objectif de mon projet de recherche consiste à étudier la fonction biologique de la sous-unité β du translocon et de son TMD chez Schizosaccharomyces pombe. Dans cette levure, j’ai découvert que le gène sbh1+ n’était pas essentiel à la viabilité à 30oC, mais qu’il était requis pour la croissance à basse température. La délétion de sbh1+ entraîne une sensibilité aux stress de la paroi cellulaire et une diminution de la sécrétion des protéines à 23oC. La surexpression de Sbh1p diminue elle aussi la sécrétion des protéines et altère la morphologie cellulaire. Ces phénotypes sont distincts de ceux observés chez S. cerevisiae, où la délétion des deux paralogues de Sec61β entraîne une sensibilité à haute température plutôt qu’à basse température. Malgré cela, les homologues de Sec61β de S. pombe et de S. cerevisiae sont tout deux capables de complémenter la thermosensibilité respective de chaque levure. La complémentation est possible même avec l’homologue humain de Sec61β, indiquant la conservation d’une fonction de Sec61β de la levure à l’homme. Remarquablement, le TMD de Sec61β de S. pombe, de S. cerevisiae et de l’humain sont suffisants pour complémenter la délétion génomique autant chez la levure à fission que chez la levure à bourgeons. Globalement, ces observations indiquent que le TMD de Sec61β exerce une fonction cellulaire conservée à travers les espèces. / Protein secretion is an essential biological process. In eukaryotes, secreted proteins transit into the endoplasmic reticulum through the translocon pore. The core of the translocation channel is composed of three subunits called Sec61α, β and γ in mammals, or Sec61p, Sbh1p and Sss1p in yeasts. While the role of the α and γ subunit is well understood, the function of the β subunit remains ill-defined. Although numerous species-specific phenotypes have been reported for this protein, the striking sequence conservation among species argue in favour of a universal role. Recently, Feng et al. (2007) reported the surprising finding that the transmembrane domain (TMD) of Sbh1p was sufficient to complement different functions of the entire protein in Saccharomyces cerevisiae, suggesting an important role for this region. The aim of my project was to explore the biological function of the translocon β subunit and its TMD in Schizosaccharomyces pombe. In this yeast, we found that the sbh1+ gene is unessential for viability at 30oC, but is required for growth at low temperature. Knockout of sbh1+ results in sensitivity to cell-wall stress and reduced protein secretion at 23oC. Overexpression of Sbh1p also diminishes protein secretion and results in an elongated cell shape. These phenotypes contrast with those observed S. cerevisiae, as deletion of both Sec61β paralogs in this yeast results in heat sensitivity instead of cold sensitivity. Nevertheless, Sec61β homologs from both S. pombe and S. cerevisiae complement the respective temperature sensitivity of either yeast. This functional complementation can also be accomplished by the human homolog of the translocon β subunit, indicating that a fundamental function of Sec61β is conserved from yeast to human. Remarkably, the TMD of Sec61β homologs from S. pombe, S. cerevisiae and human are sufficient to complement the gene knockout in both fission and budding yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species.

Der Eintritt löslicher oder membranständiger Proteine in den Sekretionsweg beginnt mit dem Transport der Proteine durch die Membran des Endoplasmatischen Retikulums (ER). Die Translokationspore wird durch den heterotrimeren Sec61-Komplex gebildet. Homologe Membranproteine der alpha- und gamma-Untereinheit dieses Komplexes sind bislang in allen daraufhin untersuchten prokaryotischen und eukaryotischen Organismen gefunden worden. Es handelt sich somit um einen entwicklungsgeschichtlich hoch konservierten Mechanismus des Proteintransports durch Membranen. Zahlreiche Untersuchungen in den letzten Jahren haben uns mittlerweile ein komplexes Bild des Translokationsgeschehens vermittelt. Die vorliegende Arbeit beleuchtet einen völlig neuen Aspekt der Proteintranslokation: Es zeigt sich, daß Gene, die für Komponenten des Sec61-Komplexes kodieren, in sehr unterschiedlichen eukaryotischen Organismen unabhängig voneinander dupliziert wurden und sich im folgenden nebeneinander weiterentwickelten. Die Untersuchung dieser sog. paralogen Gene und ihrer Genprodukte zum einen in Säugerzellen und zum anderen in der Hefe Saccharomyces cerevisiae ist Gegenstand der Arbeit. In Säugerzellen finden sich zwei sehr ähnliche SEC61a-Gene (knapp 80% Identität auf Nukleinsäureebene, etwa 94% auf Aminosäureebene), deren Expression in verschiedenen Organen und Embryonalstadien der Maus analysiert wurde. In allen bislang getesteten Geweben wurden beide Gene exprimiert. Deutliche Unterschiede zeigten sich allerdings in der Stärke der Expression: Während die Expressionshöhe des bereits bekannten SEC61a-I-Gens relativ konstant war, variierte die des paralogen SEC61a-II-Gens zwischen den einzelnen Organen. Der selektive Vorteil zweier paraloger SEC61a-Gene für den Säugerorganismus liegt somit möglicherweise in der unterschiedlichen Regulierbarkeit ihrer Expression. In Saccharomyces cerevisiae finden sich paraloge Proteine und dafür kodierende Gene sowohl für die alpha-Untereinheit Sec61p als auch für die beta-Untereinheit Sbh1p des heterotrimeren Sec61p-Komplexes. Der Grad der Identität liegt mit 32% zwischen Sec61p und Ssh1p (Sec sixty-one homolog 1) allerdings erheblich niedriger als in Säugerzellen. Sbh1p und Sbh2p (Sec sixty-one beta homolog 2) sind zu etwa 50% identisch. Für die gamma-Untereinheit Sss1p wurde kein paraloges Protein gefunden. Es konnte gezeigt werden, daß die beiden, hier neu beschriebenen Proteine Ssh1p und Sbh2p zusammen mit Sss1p einen eigenständigen, heterotrimeren Komplex, den Ssh1p-Komplex, bilden, der ebenfalls in der ER-Membran lokalisiert ist und eine Funktion beim Proteintransport durch diese Membran hat. Im Gegensatz zu SEC61 ist SSH1 nicht essentiell, wenn auch das Ssh1p für normale Wachstumsraten erforderlich ist. Die Deletion des SBH2-Gens zeigt keinen Phänotyp, was allerdings für die des SBH1-Gens ebenfalls gilt. Erst das Fehlen beider Proteine führt zu einem temperatur-abhängigen Wachstumsphänotyp und zu Defekten in der Translokation. Der entscheidende Unterschied zwischen dem Sec61p- und dem Ssh1p-Komplex scheint darin zu bestehen, daß der Sec61p-Komplex modulartig einsetzbar ist: als trimerer Komplex bei der kotranslationalen Translokation und im heptameren Sec-Komplex beim posttranslationalen Proteintransport. Demgegenüber deuten die Untersuchungen des Ssh1p-Komplexes darauf hin, daß dieser nicht mit dem tetrameren Sec62p-Sec63p-Komplex zum heptameren Sec-Komplex assoziieren kann. Vielmehr läßt er sich in Assoziation mit membrangebundenen Ribosomen nachweisen, was auf eine ausschließliche Funktion des Ssh1p-Komplexes bei der kotranslationalen Translokation schließen läßt. / Soluble or membrane proteins entering the secretory pathway are first translocated across the endoplasmic reticulum (ER) membrane. The proteins move through a channel formed by the heterotrimeric Sec61-complex. Homologues of the alpha- and gamma-subunit of this complex have been found in a wide variety of procaryotic and eucaryotic organisms indicating an evolutionary highly conserved mechanism for translocating proteins across membranes. Several recent investigations contributed to a complex understanding of this process. The work presented here sheds light on a completely new aspect of protein translocation across the ER-membrane: Interestingly genes coding for components of the Sec61-complex are found to have been duplicated independently in diverse eucaryotic organisms giving rise to so called paralogous genes (= intraspecies homologues) co-evolving after duplication. These paralogous genes and their gene products in mammalian cells as well as in the yeast Saccharomyces cerevisiae have been analyzed in detail. Mammalian cells possess two very similar SEC61a-genes (about 80% identity on nucleic acid level, about 94% on protein level). Their expression has been analyzed for several murine organs and embryonic stages. All tissues tested so far showed expression of both genes though the level of expression differed significantly: whereas expression of the already known SEC61a-I-gene seemed to be more or less constant across different tested organs, expression levels of the paralogous SEC61a-II-gene were not. Consequently the ability to differentially regulate the expression of the two paralogous SEC61a-genes may be of selective advantage for the mammalian organism. In the yeast Saccharomyces cerevisiae paralogous proteins of the alpha-subunit Sec61p as well as for the beta-subunit Sbh1p of the heterotrimeric Sec61p-complex can be found. The paralogous alpha-subunits Sec61p and Ssh1p (Sec sixty-one homolog 1) are less well conserved (32% identity) than the mammalian paralogues. Sbh1p and Sbh2p (Sec sixty-one beta homolog 2) are 50% identical. No paralogous protein was found for the gamma-subunit Sss1p. Instead, it could be shown that the new proteins Ssh1p and Sbh2p together with Sss1p are found in a heterotrimeric complex, the Ssh1p-complex, which like the Sec61p-complex localizes to the ER-membrane and has a function in translocating proteins across this membrane. In contrast to Sec61p Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and show translocation defekts in vivo as well as in vitro. The most intriguing difference between the Sec61p-and the Ssh1p-complex might be that the Sec61p-complex has a role in both co- and post-translational translocation pathways, as a separate entity and as part of the heptameris Sec-complex, respectively. However, there was no evidence for the Ssh1p-complex being part of an heptameric complex together with the tetrameric Sec62p-Sec63p-complex, but association of the trimeric Ssh1p-complex with membrane-bound ribosomes could be shown, making it likely that the Ssh1p-complex functions exclusively in the co-translational pathway of protein transport.

La sécrétion des protéines est un processus essentiel à la vie. Chez les eucaryotes, les protéines sécrétées transitent dans le réticulum endoplasmique par le pore de translocation. Le translocon est composé de trois sous-unités fondamentales nommées Sec61α, β et γ chez les mammifères, ou Sec61p, Sbh1p et Sss1p chez les levures. Tandis que le rôle des sous-unités α et γ est bien connu, celui de la sous-unité β demeure énigmatique. Plusieurs phénotypes distincts sont associés à cette protéine dans différents organismes, mais le haut niveau de conservation de séquence suggère plutôt une fonction universelle conservée. Récemment, Feng et al. (2007) ont montré que le domaine transmembranaire (TMD) de Sbh1p était suffisant pour complémenter plusieurs phénotypes associés à la délétion du gène chez Saccharomyces cerevisiae, suggérant un rôle important de cette région. L’objectif de mon projet de recherche consiste à étudier la fonction biologique de la sous-unité β du translocon et de son TMD chez Schizosaccharomyces pombe. Dans cette levure, j’ai découvert que le gène sbh1+ n’était pas essentiel à la viabilité à 30oC, mais qu’il était requis pour la croissance à basse température. La délétion de sbh1+ entraîne une sensibilité aux stress de la paroi cellulaire et une diminution de la sécrétion des protéines à 23oC. La surexpression de Sbh1p diminue elle aussi la sécrétion des protéines et altère la morphologie cellulaire. Ces phénotypes sont distincts de ceux observés chez S. cerevisiae, où la délétion des deux paralogues de Sec61β entraîne une sensibilité à haute température plutôt qu’à basse température. Malgré cela, les homologues de Sec61β de S. pombe et de S. cerevisiae sont tout deux capables de complémenter la thermosensibilité respective de chaque levure. La complémentation est possible même avec l’homologue humain de Sec61β, indiquant la conservation d’une fonction de Sec61β de la levure à l’homme. Remarquablement, le TMD de Sec61β de S. pombe, de S. cerevisiae et de l’humain sont suffisants pour complémenter la délétion génomique autant chez la levure à fission que chez la levure à bourgeons. Globalement, ces observations indiquent que le TMD de Sec61β exerce une fonction cellulaire conservée à travers les espèces. / Protein secretion is an essential biological process. In eukaryotes, secreted proteins transit into the endoplasmic reticulum through the translocon pore. The core of the translocation channel is composed of three subunits called Sec61α, β and γ in mammals, or Sec61p, Sbh1p and Sss1p in yeasts. While the role of the α and γ subunit is well understood, the function of the β subunit remains ill-defined. Although numerous species-specific phenotypes have been reported for this protein, the striking sequence conservation among species argue in favour of a universal role. Recently, Feng et al. (2007) reported the surprising finding that the transmembrane domain (TMD) of Sbh1p was sufficient to complement different functions of the entire protein in Saccharomyces cerevisiae, suggesting an important role for this region. The aim of my project was to explore the biological function of the translocon β subunit and its TMD in Schizosaccharomyces pombe. In this yeast, we found that the sbh1+ gene is unessential for viability at 30oC, but is required for growth at low temperature. Knockout of sbh1+ results in sensitivity to cell-wall stress and reduced protein secretion at 23oC. Overexpression of Sbh1p also diminishes protein secretion and results in an elongated cell shape. These phenotypes contrast with those observed S. cerevisiae, as deletion of both Sec61β paralogs in this yeast results in heat sensitivity instead of cold sensitivity. Nevertheless, Sec61β homologs from both S. pombe and S. cerevisiae complement the respective temperature sensitivity of either yeast. This functional complementation can also be accomplished by the human homolog of the translocon β subunit, indicating that a fundamental function of Sec61β is conserved from yeast to human. Remarkably, the TMD of Sec61β homologs from S. pombe, S. cerevisiae and human are sufficient to complement the gene knockout in both fission and budding yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species.

Orientador: Luciano Resende Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-07-27T08:14:21Z (GMT). No. of bitstreams: 1 MartelliJunior_Hercilio_M.pdf: 3416342 bytes, checksum: 1e4781599e6c56d26cf0a8c2c2bb6f6e (MD5) Previous issue date: 2000 / Resumo: Fibromatose Gengival Hereditária (FGH) representa uma condição oral incomum (1:750.000), caracterizada por um aumento gengival fibrótico generalizado. Manifesta-se como entidade clínica isolada ou como parte de síndromes, produzindo excessiva quantidade de colágeno e outras moléculas da matriz extra-celular. Hsp47 é uma chaperone residente no retículo endoplasmático (RE) que liga especificamente a moléculas de colágeno, enquanto Sec61 a representa uma proteína transmembrânica com ativa participação na condução de cadeias polipeptídicas nascentes para o lúmen do RE. Este trabalho descreve a participação das proteínas Hsp47 e Sec61? em culturas de fibroblastos provenientes de uma única família portadora de FGH e de pacientes com gengiva normal (GN) nos eventos de translação/translocação de colágeno tipo I. Ensaios de Western blot mostraram uma produção aumentada de Hsp47 em fibroblastos de FGH, comparado a fibroblastos de GN em condições de homeostasia e em situações de estresse térmico. Além disso, foi demonstrado produção de Sec61? nas linhagens celulares, FGH e GN, porém sem diferenças nos padrões de produção. A maior produção de Hsp47 pode estar envolvida na proteção da degradação intra reticular de colágeno, podendo ser um dos fatores responsáveis pela fibrose característica desta doença. Embora os mecanismos biológicos responsáveis pela FGH sejam ainda desconhecidos, o conhecimento da participação destas proteínas na regulação da biosíntese de colágeno pode ser importante para o entendimento de condições genéticas, como a FGH / Abstract: Study of the sinthesis of Hsp47 and Sec61??during the events of translacions/translocations of collagen type I in fibroblasts from hereditary gingival fibromatosis Hereditary Gingival Fibromatosis (HGF) represents an uncommon oral condition (l :750,000) characterized fibrous gingival enlargement. HGF can manifest as an isolated clinical entity or as part of a syndrome. The gingiva of patients with HGF produce excessive amount of collagen and other extracellular matrix. Hsp47 is an endoplasmic reticulun (ER) resident chaperone which binds specifically to collagen molecules, and Sec61? represents a transmembranous protein with active role in conducting of nascent polypeptide chain into the ER. This study describes the role of Hsp47 and Sec61? during the events of translationltranslocation of collagen type I in fibroblasts from patients with HGF and patients presenting normal gingiva (NG). Western blot assays demonstrated an increased production of Hsp47 in fibroblasts HGF as compared to NG cells under stress and unstressed conditions. In addition, Sec61? was evenly found in both cell types showing no marked variations in quantity in both stressed or unstressed situations. The more increased production of Hsp47 may related to a collagen degradation protective mechanism inside the ER. This can be one of the factors responsible for the fibrous features of HGF. Although the exact biological mechanisms involved in HGF are still unknown, the study of these ER proteins role in regulating collagen biosynthesis may be important for understanding hereditary conditions such as HGF / Mestrado / Mestre em Biologia e Patologia Buco-Dental

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) respectively act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the non-selective interaction between the ribosome and the Sec61 complex. The Sec61 complex, serving as both the high affinity ribosome receptor and the translocation channel, is performing two very different functions which presumably requires different activity domains within the Sec61 complex. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the N and C-terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between a loss of ribosome binding activity and the digestion of the C-terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether ribosome binding to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind ribosomes with high affinity at physiological ionic strength remain active in SRP-independent translocation indicating that ribosome binding and translocation promotion are separable activities of the Sec61 complex. Translocation promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.