Embargoed until 1 January 2011 / The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.

Embargoed until 1 January 2011 / The filamin family act as scaffolding proteins associating with actin filmanents, acting through a highly conserved actin binding domain (ABD). The ABD of the filamins is homologous to that found in other F-actin binding proteins such as dystrophin. Mutations in the filamin A gene cause a wide range of disease symptoms in humans reflecting the diversity of the roles that filamin A has in cell structure and signalling pathways. The diseases fall into two separate phenotypic groups. Periventricular nodular heterotopia (PVNH) generally results from the complete loss of filamin A protein, and affects the central nervous system. The clinically separate otopalatodigital disorders (OPD) spectrum disorders are skeletal disorders and were hypothesised to be gain of function phenotype diseases. At the beginning of this work, there was very little structural data available for the human filamins, and none for the crucial highly conserved actin binding domain. This lack of structural data limited the interpretation of the biochemical and genetic data and constrained our understanding of the disease associated mutations that cluster in this domain. These studies aimed to provide insights into the structure and mechanism of actin binding domains, and thus provide a better understanding of the diseases caused when this domain is mutated. A secondary structural analysis and crystal structures of the wildtype and OPD2 associated mutant ABDs were obtained. The overall fold of the three proteins was equivalent as determined by circular dichroism spectroscopy and x-ray crystallography. The ABD from filamin A E254K showed 3.7 fold increased F-actin affinity, accompanied by a reduced thermostability (of 5.6 °C). Western blotting of OPD2, frontometaphyseal dysplasia (FMD) and PVNH patient fibroblast lysates showed similar levels of filamin A compared to the control cells. In addition the OPD and PVNH patient fibroblasts were able to adhere to fibronectin and migrate with an equivalent rate to control cells. Together these results have allowed correlations to be developed between structure, protein stability, actin affinity, cellular phenotype and the overall clinical phenotype. Showing that, at least in one example, OPD2 may be due to an increased actin affinity providing further evidence for a gain of function mechanism of OPD2.

Certain hydrogel forming de novo proteins that utilize different crosslinking methods are studied experimentally on a rheometer. The stress reaxation modulus of CRC, a telechelic, triblock protein, is shown to be that of a stretched exponential function with a value of β ≅ 0.5. The insertion of an integrin binding domain and changes in pH within the range 6.5–8.5 are shown not to significantly affect the resulting rheological behavior. A selective chemical crosslinker is used on CRC hydrogel systems and is shown to change the rheological behavior of the system to that of a combination of a chemically and physically crosslinked system. Chemically crosslinked hydrogels composed of W6, a wheat gluten-based protein, demonstrate a storage modulus weakly dependent on the angular frequency that is much greater than the loss modulus, with a modulus concentration dependence of c^9/4.

Nuclear hormone receptors (NHR) are important transcription factors that regulate development and metabolism in the large group of animals. Caenorhabditis elegans contains 284 nuclear receptors, which is unusually large amount compared to receptors of Drosophila melanogaster (18) and humans (48). 15 receptors of the C. elegans have homologous receptor structure with receptors of D. melanogaster and mammals. The remaining 269 NHR are specific to nematodes and belong to the group of supplementary nuclear receptors (SupNRs), the evolutionary precursor of the HNF4 - an important transcription factor in humans. In this work we describe the nuclear hormone receptor nhr-97 C. elegans, whose expression and function have not yet been studied. The gene is encoded in the genome of C. elegans and is among SupNRs. Nhr-97 consists of two isoforms A and B, whose expression in C. elegans tissues is different. Localization of gene expression in vivo was determined using lines expressing nhr-97:: GFP. For the A isoform expression of nhr-97::GFP was localized in neurons in the pharynx and the tail, in the intestine and hypodermis, in isoform B in the pharynx, in neurons around the corpus of pharynx, the head mesodermal cell and in anal sphincter. Nhr-97 expression during development of C. elegans was determined by...

Spumaretroviren, oder Foamyviren (FV), unterscheiden sich von Orthoretroviren durch mehrere Besonderheiten in ihrer Replikationsstrategie. Das Partikel-assoziierte Hüllglykoprotein (Env-Protein) des „Prototype Foamy Virus“ (PFV) ist im Vergleich zu anderen retroviralen Hüllglykoproteinen einzigartig. Die Koexpression des PFV Env-Proteins für die PFV-Partikelfreisetzung ist essenziell und die spezifische Funktion kann nicht von heterologen viralen Env-Proteinen übernommen werden. Das Env-Protein des PFV durchläuft eine für ein Membranglykoprotein ungewöhnliche Biosynthese. Das Env-Vorläuferprotein besitzt zu Beginn eine Typ-III-Membrantopologie, bei der der N- und der C-Terminus im Zytoplasma lokalisiert sind. Während des Transports zur Zelloberfläche wird es posttranslational durch bisher unbekannte zelluläre Proteasen in mindestens drei Untereinheiten gespalten. Das N-terminale Signalpeptid bzw. Leader-Peptid (LP) hat eine Typ-II-Membrantopologie, mit dem N-Terminus im Zytoplasma und dem C-Terminus im Lumen, wohingegen die Transmembran (TM)-Untereinheit eine Typ-IMembrantopologie besitzt, bei der der N-Terminus im Lumen und der C-Terminus im Zytoplasma lokalisiert sind. Die interne Oberflächen (SU)-Untereinheit assoziiert vermutlich im Lumen mit der extrazellulären Domäne der TM-Untereinheit. Im Rahmen dieser Arbeit wurde der Beweis erbracht, dass Furin oder Furin-ähnliche Proteasen und nicht der Signalpeptidase-Komplex für beide proteolytischen Spaltungen verantwortlich sind. Durch die N-terminale Sequenzierung der SU- und der TM-Untereinheit eines aufgereinigten PFV Env-Immunoadhäsionsproteins wurden N-terminal von beiden Spaltstellen Furin- Konsensussequenzen identifiziert. Mutationsanalysen von zwei sich in diesem Bereich überlappenden minimalen Furin-Konsensussequenzen an der PFV LP/SU-Spaltstelle im wildtypischen PFV Env-Protein bestätigten die Ergebnisse der N-terminalen Sequenzierung und bewiesen, dass nur die erste Spaltstelle genutzt wird. Obwohl diese Mutanten aufgrund geringerer Partikelfreisetzung einen signifikanten Verlust der Infektiosität zeigten, wurde keine Korrelation zur Inhibierung der Spaltung beobachtet, da andere Mutanten mit normaler LP/SU-Spaltung einen ähnlichen Defekt besaßen. Virale Env-Proteine initiieren den Eintritt membranumhüllter Viren in die Wirtszelle durch die Bindung an zelluläre Rezeptoren. Dabei führen Konformationsänderungen in den Env- Proteinen zum Verschmelzen der Virusmembran mit der Zellmembran und weiterhin zur Aufnahme des Kapsids in das Zytoplasma der Wirtszelle. Die foamyviralen Env-Proteine sind in dieser Hinsicht keine Ausnahme und vermitteln die Anheftung an die Wirtszelle durch die Bindung an den bisher unbekannten zellulären Rezeptor. Der zelluläre foamyvirale Rezeptor ist vermutlich ein ubiquitäres Molekül, denn bisher konnte keine Zelllinie identifiziert werden, die gegen FV-Infektionen resistent ist. Bislang existieren nur sehr wenig strukturelle und funktionelle Informationen der extrazellulären Domänen des PFV Env-Proteins. Deshalb wurde im Hauptteil dieser Arbeit die PFV Env-Rezeptorbindungsdomäne (RBD) charakterisiert. Hierfür wurden rekombinante PFV Env-Immunoadhäsionsproteine verwendet und deren Bindungskapazitäten an Zielzellen in der durchflusszytometrischen Analyse bestimmt. Untersuchungen zeigten, dass sowohl die extrazelluläre Domäne der C-terminalen TM-Untereinheit als auch der Transport der Immunoadhäsionsproteine durch das spezifische PFV Env LP zum sekretorischen Weg für die Bindung an Zielzellen entbehrlich sind und ließen vermuten, dass die PFV Env-RBD innerhalb der SU-Untereinheit lokalisiert ist. N- und C-terminale Deletionsanalysen der PFV Env SU-Untereinheit enthüllten eine minimale kontinuierliche RBD von AS 225 bis 555. Interne Deletionen im PFV Env-Protein von AS 397 bis 483 wurden im Gegensatz zu deletierten Regionen von AS 262 bis 300 und AS 342 bis 396 ohne signifikanten Einfluss auf die Wirtszellbindung in Immunoadhäsionsproteinen toleriert. Die Analyse der Immunoadhäsionsproteine mit einzelnen substituierten Cysteinen in der PFV Env SU-Untereinheit zeigten, dass nur die Immunoadhäsionsproteine, die in der nicht essenziellen Region von AS 397 bis 483 lokalisierte Cysteine ersetzt hatten, eine Restbindungskapazität behielten. Interessanterweise zeigte die Analyse von verschiedenen N-Glykosylierungsmutanten eine bedeutende Rolle der Kohlenhydratkette an Position N391 im PFV Env-Protein entweder hinsichtlich der direkten Interaktion mit dem zellulären Rezeptor oder für die korrekte Faltung der PFV Env-RBD. Diese Ergebnisse weisen darauf hin, dass ein diskontinuierliches Sequenzmotiv von AS 225 bis 396 und AS 484 bis 555 für die Bildung der PFV Env-RBD essenziell ist und die darin lokalisierte potenzielle achte N-Glykosylierungsstelle eine entscheidende Rolle bei der Wirtszellbindung spielt. / Spumaretroviruses or foamy viruses (FVs) use a replication pathway with features distinctive from orthoretroviruses. The particle-associated envelope (Env) glycoprotein of prototype foamy virus (PFV) is unique compared to other retroviral envelope proteins since its coexpression is strictly required for the FV particle release process and its function cannot be replaced by heterologous viral glycoproteins. The PFV Env glycoprotein shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N-and C-terminus located in the cytoplasm. During its transport to the cell surface, it is posttranslationally processed by yet-unidentified cellular proteases into at least three subunits. The N-terminal signal or leader peptide (LP) has a type II membrane topology, whereas the C-terminal transmembrane (TM) subunit has a type I membrane topology. The internal surface (SU) subunit presumably associates with extracellular domains of TM on the luminal side. Here we provide strong evidence that furin itself or furin-like proteases and not the signal peptidase complex are responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping minimal furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect. Viral Env proteins initiate entry of membrane enveloped viruses into cells by binding to cell surface receptors followed by conformational changes leading to membrane fusion and delivery of the genome containing viral capsid to the cytoplasm. The Env glycoproteins of FVs are no exception and mediate attachment to host cells through binding to an yet unknown ubiquitous cellular receptor molecule because no cell type is currently known that is resistant to FV entry. Little structural and functional information on the extracellular domains of PFV Env is available. In this study we characterized the PFV Env receptor-binding-domain (RBD) by flow-cytometric analysis of recombinant PFV Env immunoadhesin binding to target cells. Analysis showed that the extracellular domains of the C-terminal TM subunit as well as targeting of the recombinant immunoadhesins by the cognate LP to the secretory pathway were dispensable for target cell binding suggesting that the PFV Env RBD is contained within the SU subunit. N- and C- terminal deletion analysis of the SU domain revealed an minimal continuous RBD spanning aa 225-555, however internal deletions covering the region from aa 397-483, but not aa 262-300 or aa 342-396, were tolerated without significant influence on host cell binding. Analysis of individual cysteine point mutants in PFV Env SU revealed that only most of those located in the non-essential region from aa 397-483 retained residual binding activity. Interestingly, analysis of various N-glycosylation site mutants suggests an important role of the carbohydrate chain attached to N391 either for direct interaction with the cellular receptor or for correct folding of the PFV Env RBD. Taken together these results suggest that a bipartite sequence motif spanning aa 225-396 and aa 484-555 is essential for formation of the PFV Env RBD, with N-glycosylation site 8 playing a crucial role for host cell binding.

Indiana University-Purdue University Indianapolis (IUPUI) / SETMAR is a chimeric protein that originates from the fusion of a SET domain to the mariner Hsmar1 transposase. This fusion event occurred approximately 50 million years ago, after the split of an anthropoid primate ancestor from the prosimians. Thus, SETMAR is only expressed in anthropoid primates, such as humans, apes, and New World monkeys. Evolutionary sequence analyses have revealed that the DNA-binding domain, one of the two functional domains in the Hsmar1 transposase, has been subjected to a strong purifying selection. Consistent with these analyses, SETMAR retains robust binding specificity to its ancestral terminal inverted repeat (TIR) DNA. In the human genome, this TIR sequence is dispersed in over 1500 perfect or nearly perfect sites. Given that many DNA-binding domains of transcriptional regulators are derived from transposases, we hypothesized that SETMAR may play a role in gene regulation. In this thesis, we determined the crystal structures of the DNA-binding domain bound to both its ancestral TIR DNA and a variant TIR DNA sequence at 2.37 and 3.07 Å, respectively. Overall, the DNA-binding domain contains two helix-turn-helix (HTH) motifs linked by two AT-hook motifs and dimerizes through its HTH1 motif. In both complexes, minor groove interactions with the AT-hook motifs are similar, and major groove interactions with HTH1 involve a single residue. However, four residues from HTH2 participate in nucleobase-specific interactions with the TIR and only two with the variant DNA sequence. Despite these differences in nucleobase-specific interactions, the DNA-binding affinities of SETMAR to TIR or variant TIR differ by less than two-fold. From cell-based studies, we found that SETMAR represses firefly luciferase gene expression while the DNA-binding deficient mutant does not. A chromatin immunoprecipitation assay further confirms that SETMAR binds the TIR sequence in cells. Collectively, our studies suggest that SETMAR functions in gene regulation.

No description available.

The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%–90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.

Esta tesis se interesa por distinguir entre los efectos directos de los receptores nucleares y aquellos mediados por las rutas de transducción de señales en la transcripción de genes en respuesta a hormona y proliferación celular. Para esto, nosotros hemos expresado establemente en una línea celular T47Dy desprovista de PR, formas variantes marcadas de la isoforma B del PR en regiones involucradas bien en la unión al DNA(PRB-DBD), en su habilidad para interaccionar con ER y activar la cascada c-Src/Erk (PRB-ERID), o la incapacidad de reclutar coactivadores. La expresión génica en respuesta a progesterona en líneas celulares expresando los PRB salvaje y mutantes ha sido estudiada un microarray con 750 genes de cáncer de mama. Los resultados definen conjuntos de genes regulados en respuesta a hormona por los diferentes modos de acción del PRB, también genes dónde las rutas nucleares y no genómicas cooperan. Por último, se ha centrado la atención en la participación del gen Ciclina D1 (CCND1) en proliferación celular por hormona, el modo de acción del PR en su activación y el análisis de las regiones promotoras dónde PR se une. / This these is interested on distinguishing between direct effects of nuclear receptors and those mediated by signal transduction pathways on transcription of hormone-responsive genes and cell proliferation. For this, it stablies expressed in the PR-negative T47Dy breast cancer cell line, tagged forms of the PRB mutated at regions involved either in DNA binding, in its ability to interact with ER and activate the c-Src/Erk cascade, or the recruitment of coactivators. Gene expression in response to progestins in cell lines expressing wild type or mutant PRB has been studied by a 750 genes-containing breast cancer customized cDNA microarray. Our results define the subsets of hormoneresponsive genes regulated by the different modes of action of PRB, as well as genes where the nuclear and nongenomic pathways of PRB cooperate. Finally, it has focused the attention on the involvement of Cyclin D1 gene (CCND1) activation by hormone on cell proliferation, the mode of action of PR on its activation and the analysis of promoter regions where PR binds.

Le transport et la traduction localisée des ARN messagers sont observés chez plusieurs organismes et sont requis pour de multiples phénomènes tels la mémoire, la division cellulaire asymétrique et l’établissement des axes durant le développement. Staufen, une protéine liant l’ARN double-brin, a été identifié dans un premier temps chez la mouche à fruits Drosophila melanogaster. Il a été montré, chez cet organisme, que Staufen est requis pour la localisation des messagers bicoid et oskar aux pôles antérieur et postérieur de l’ovocyte, respectivement. Également, Staufen est requis afin que la répression traductionnelle du messager oskar soit levée une fois qu’il est bien localisé. Chez les mammifères, Stau1 est une protéine ubiquiste qui est présente dans des complexes prenant la forme de granules dans les dendrites des neurones. Également, Stau1 peut interagir de façon indépendante de l’ARN avec le ribosome et cofractionner tant avec la sous-unité 40S qu’avec la sous-unité 60S du ribosome dans un gradient de saccharose. L’implication de Stau1 dans un mécanisme permettant la dérépression traductionnelle de certains ARNm chez les mammifères était donc une voie d’investigation intéressante. Nous avons donc décidé de vérifier si Stau1 mammifère avait la capacité de stimuler la traduction d’un ARNm cellulaire via un mécanisme régulé. Au moment où cette thèse a été entreprise, aucun ARNm cellulaire lié par Stau1 n’avait été identifié chez les mammifères. Des structures d’ARN double-brin ont donc été employées afin de réprimer la traduction d’un ARNm rapporteur. C’est ainsi que nous avons montré que Stau1 peut stimuler la traduction d’un ARNm lorsqu’il lie celui-ci dans sa région 5’ non-traduite. Par la suite, en employant des micropuces d’ADN, nous avons identifié des messagers cellulaires dont la distribution dans les polysomes lourds est modifiée par Stau1. En effet, un groupe de messagers est enrichi dans les polysomes lourds suite à une surexpression de Stau1, ce qui suggère que Stau1 stimule la traduction de cette population d’ARNm. Afin d’identifier un mécanisme potentiel de régulation de l’activité traductionnelle de Stau1, nous nous sommes intéressés à la capacité d’auto-association de cette protéine. Nous avons montré que Stau1, tout comme plusieurs protéines liant l’ARN double-brin, est en mesure de s’associer à lui-même, et ce, d’une façon indépendante de l’ARN. Nous avons identifié les déterminants impliqués mettant ainsi au jour un nouveau mécanisme pouvant influencer les activités cellulaires de Stau1. Les résultats présentés dans cette thèse suggèrent donc que Stau1 est en mesure de stimuler la traduction d’une sous-population précise d’ARN messagers au sein de la cellule permettant ainsi de jeter un regard nouveau sur l’implication de cette protéine dans divers phénomènes au sein de l’organisme. / Transport and local translation of RNA are found in several organisms and are required for multiple phenomena such as memory, asymmetric cell division and establishment of the axis during development. Staufen, a double-stranded RNA binding protein, was first identified in Drosophila melanogaster. In the fruitfly, it was shown that Staufen is required for the proper localization of the bicoid and oskar transcripts to the anterior and posterior ends of the oocyte, respectively. It was also found that Staufen is important for the translational derepression of oskar once it is adequately localized. In mammals, Stau1 is a ubiquitous protein found in granules in the dendrites of neurons. Also, Stau1 can bind the ribosome in a RNA-independent manner and cofractionates with both ribosomal subunits in a sucrose gradient. The implication of Stau1 in a mechanism allowing translational derepression of certain RNAs in mammals was therefore an interesting path to explore. Accordingly, we decided to verify if mammalian Stau1 had the capacity to stimulate the translation of cellular RNAs through a regulated mechanism. When this thesis was initiated, no cellular RNA target of Stau1 had been identified in mammals. Therefore, double-stranded RNA structures were used to repress the translation of a reporter mRNA. With this model, we showed that Stau1 can stimulate the translation of a transcript when it is bound to its 5’ UTR. With the use of DNA microarrays, we identified cellular mRNAs which distribution in heavy polysomes was altered by Stau1. When Stau1 is overexpressed, this group of mRNAs is enriched heavy polysomes, suggesting a translational stimulation of this population by Stau1. To identify a regulatory mechanism that could influence Stau1’s translational activity, we studied the self-association capacity of this protein. We showed that Stau1, like several double-stranded RNA binding proteins, can self-associate in a RNA-independent manner. We have identified the determinants required for this interaction that as the potential to be important for the regulation of the cellular activities of Stau1. The results presented in this thesis suggest that Stau1 can stimulate the translation of a specific subset of mRNAs in the cell, letting us look at Stau1’s implication in different processes from a new point of view.