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Indiana University-Purdue University Indianapolis (IUPUI) / Anbu is a bacterial protein with significant homology to the sub-units of the 20S proteasome and is predicted to be a novel bacterial proteasome. The goal of this project was to determine if the recombinant Anbu protein from Pseudomonas aeruginosa is a proteasome. Anbu from P. aeruginosa was successfully cloned, expressed and purified. In order to determine the catalytic activity of Anbu, the purified protein was tested with a variety of substrates and conditions. The targets analyzed included fluorescently-labeled substrates, denatured proteins, diubiquitin, and a peptide library in the hopes of obtaining a useful model substrate. Experiments were also conducted to determine what role Anbu has in the cell. Western analysis was performed on the cell lysate of wild type P. aeruginosa and insertional mutants to detect Anbu expression. The level of biofilm formation was compared between the wild type and mutants. Cultures were grown under stress conditions including the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione. Growth rates were monitored in an attempt to detect a phenotypic difference between the wild type and the mutants lacking Anbu, HslV, and the other proteins of interest. While a substrate for Anbu has yet to be found, this protein was found to assemble into a larger structure and P. aeruginosa lacking Anbu was sensitive to the oxidative stress of diamide and the nitrosative stress of S-nitrosoglutathione.

Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...

Recently, linear ubiquitin assembly complex (LUBAC)-mediated linear ubiquitination has come into focus due to its emerging role in activation of NF-κB in different biological contexts. However, the role of LUBAC in LMP1 signaling leading to NF-κB and interferon regulatory factor 7 (IRF7) activation has not been investigated. We show here that RNF31, the key component of LUBAC, interacts with LMP1 and IRF7 in Epstein-Barr virus (EBV)-transformed cells and that LUBAC stimulates linear ubiquitination of NEMO and IRF7. Consequently, LUBAC is required for LMP1 signaling to full activation of NF-κB but inhibits LMP1-stimulated IRF7 transcriptional activity. The protein levels of RNF31 and LMP1 are correlated in EBV-transformed cells. Knockdown of RNF31 in EBV-transformed IB4 cells by RNA interference negatively regulates the expression of the genes downstream of LMP1 signaling and results in a decrease of cell proliferation. These lines of evidence indicate that LUBAC-mediated linear ubiquitination plays crucial roles in regulating LMP1 signaling and functions. IMPORTANCE We show here that LUBAC-mediated linear ubiquitination is required for LMP1 activation of NF-κB but inhibits LMP1-mediated IRF7 activation. Our findings provide novel mechanisms underlying EBV-mediated oncogenesis and may have a broad impact on IRF7-mediated immune responses.

In Eukaryoten werden sekretorische Proteine an zytoplasmatischen Ribosomen synthetisiert und in das endoplasmatische Retikulum (ER) transportiert, wo sie ihre biologisch aktive Struktur erhalten. Defekte Proteine, die durch Fehler in diesem Reifungsprozess entstehen, werden über den Prozess der „ER-assoziierten Protein Degradation“ (ERAD) abgebaut. Eine zentrale Komponente dieses Abbauweges ist die HRD‑Ligase, ein membranständiger Proteinkomplex, der das E3‑Enzym Hrd1 enthält. Einige publizierte Arbeiten lassen eine Beteiligung der Transmembrandomäne von Hrd1 an einem neuartigen Transportsystem für den Export von fehlgefalteten Proteinen aus dem ER vermuten. Im Rahmen dieser Arbeit konnten neue Erkenntnisse über die Bedeutung der Transmembranregion von Hrd1 für den Abbau von ERAD‑Substraten in dem Modellorganismus Saccharomyces cerevisiae gewonnen werden. Die Substitution von zwei Aminosäuren in der dritten Transmembranhelix von Hrd1 (Hrd1E84L, H101L) hemmt spezifisch den Abbau von fehlgefalteten ER-luminalen Proteinen, während die Prozessierung membrangebundener Hrd1-Substrate weitestgehend unbeeinflusst blieb. Daher zeigt die Hrd1‑Variante wahrscheinlich eine Störung im Transport von luminalen ERAD‑Substraten durch die ER Membran. Biochemische Analysen zeigen keine starken Veränderungen bei der Zusammensetzung der HRD-Ligase durch die Hrd1‑Variante. Allerdings ließen sich bei Anwendung von zielgerichtetem in vivo photocrosslinking deutliche Veränderungen in der räumlichen Orientierung der Ligaseuntereinheit Der1 zu Hrd1E84L, H101L beobachten. Da Der1 ausschließlich für den Abbau ER-luminaler ERAD-Substrate benötigt wird ist anzunehmen, dass die korrekte Ausrichtung von Der1 zu Hrd1 innerhalb der ER-Membran eine Voraussetzung für den Transport von ERAD‑Substraten in das Zytoplasma ist. Die Ergebnisse dieser Arbeit zeigen erstmals einen direkten Einfluss der Transmembranregion von Hrd1 auf den Abbau luminaler ERAD‑Zielproteine. / In eukaryotes, secretory proteins are synthesized on cytoplasmic ribosomes and transported into the endoplasmic reticulum (ER), where they obtain their biologically active structure. Defect proteins that arise from errors in this maturation process are degraded by the “ER-associated protein degradation” (ERAD) pathway. A central component of ERAD is the HRD-ligase, a membrane-bound protein complex that contains the ubiquitin ligase Hrd1. Some published work raised speculations on an involvement of this trans-membrane domain in a novel transport system for the export of misfolded proteins from the ER. In the course of this work new insights for the importance of the transmembrane domain of Hrd1 for the degradation of ERAD substrates in the model organism Saccharomyces cerevisiae were obtained. The substitution of two amino acids in the third trans-membrane helix of Hrd1 (Hrd1E84L, H101L) specifically impairs the turnover of misfolded ER-luminal proteins whereas the processing of membrane-bound Hrd1 substrates remained largely unaffected. The Hrd1 variant therefore displays most likely a defect in the transport of luminal ERAD substrates through the ER membrane. No major changes in the assembly of the HRD-ligase by the Hrd1 variant were detected. Still, in site specific in vivo photo-crosslinking assays substantial changes in the spatial orientation of the ligase subunit Der1 towards Hrd1E84L, H101L were detected. Der1 is exclusively required for the degradation of ER-luminal ERAD substrates. This implies that the proper alignment of Hrd1 and Der1 within the ER membrane is a prerequisite for the transport of ERAD substrates into the cytoplasm. This work shows for the first time a direct involvement of the trans-membrane region of Hrd1 in the degradation of luminal ERAD client proteins.

Fehlgefaltete Proteine des sekretorischen Weges werden aus dem Endoplasmatischen Retikulum (ER) in das Zytosol transportiert und dort durch das Ubiquitin-Proteasom-System abgebaut. Dieser Qualitätskontrollmechanismus wird als Endoplasmatisches Retikulum-assoziierte Proteindegradation bezeichnet (ERAD). In der Bäckerhefe Saccharomyces cerevisiae stellt die HRD-Ubiquitinligase eine zentrale Komponente dieses Abbausystems dar. Eine Untereinheit dieses Multienzymkomplexes ist das ER-ständige Membranprotein Der1, das über den Faktor Usa1 an die Ubiquitinligase Hrd1 rekrutiert wird und ausschließlich für den Abbau löslicher luminaler ERAD-Substrate notwendig ist. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der C-Terminus von Der1 die Interaktion zu Usa1 und damit die Rekrutierung des Proteins zur HRD-Ligase vermittelt. Usa1 wirkt nicht nur als Rekrutierungsfaktor, sondern induziert auch die Der1-Oligomerisierung. Punktmutationen in den Transmembrandomänen von Der1 beeinträchtigen die Dislokation luminaler Substratproteine aus dem ER. Um weitere Hinweise für eine Beteiligung von Der1 beim Substrattransport zu erhalten, wurde die Methode des zielgerichtetem in vivo photocrosslinking für Der1 angewendet. Hierbei wurden bestimmte Positionen von Der1 mit dem photoreaktiven Aminosäureanalogon p-Benzoylphenylalanin markiert, was die Ausbildung von Quervernetzungen von Der1 zu Interaktionspartnern nach einer UV-Bestrahlung ermöglichte. Schließlich konnte auf diese Weise eine räumliche Nähe der luminal exponierten Bereiche von Der1 zum Substratrezeptor Hrd3 gezeigt werden, während die Transmembransegmente Quervernetzungen zu Hrd1 ausbildeten. Beide Bereiche von Der1 konnten zudem mit einem luminalen ERAD-Substrat quervernetzt werden. Anhand dieser Ergebnisse wurde somit erstmals eine direkte Beteiligung von Der1 insbesondere in den ersten Schritten der Substratdislokation gezeigt, was eine Funktion von Der1 als zentrale Komponente des Exportkomplexes nahelegt. / Newly synthesized proteins of the secretory pathway are subjected to an efficient quality control system in the endoplasmic reticulum. In order to prevent a harmful aggregation misfolded proteins are exported via a largely unknown mechanism into the cytosol and degraded by the ubiquitin-proteasome system in a process termed ER associated degradation (ERAD). In the yeast Saccharomyces cerevisiae the HRD-ligase constitutes a central component of ERAD. A subunit of this multi-enzyme complex is the small multispanning membrane protein Der1, which is exclusively required for the degradation of misfolded ER luminal proteins but dispensable for the turnover of membrane-bound substrates. In this study a short conserved motif in the cytosolic carboxyterminus of Der1 was identified that mediates the binding to the HRD-ligase. Moreover, co-immunoprecipitation experiments show that Der1 forms oligomers, which relies on its assembly into the degradation complex. Mutations in the transmembrane domains of Der1 block the export of soluble proteins across the ER-membrane. To further investigate the function of Der1 in substrate dislocation an in vivo site-specific photocrosslinking approach was applied. Various positions of Der1 were labelled with the photoreactive amino acid analogue p-benzoyl-phenylalanine followed by UV irradiation of living cells expressing these Der1 constructs. The crosslinking experiments reveal a spatial proximity of ER luminal exposed parts of Der1 to the substrate receptor Hrd3. By contrast, the membrane-embedded domains of Der1 reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to a client protein. In summary the data of this work imply that multimeric Der1 initiates the export of aberrant polypeptides from the ER-lumen by threading such molecules into the ER-membrane and routing them to Hrd1 for ubiquitylation.

Les domaines de transactivation (TAD) acides sont présents dans plusieurs protéines oncogéniques, virales et dans des facteurs de différenciation de cellules souches. Ces domaines acides contrôlent la transcription à travers une myriade d’interactions avec divers partenaires ce qui provoque l’activation de la transcription ou leur propre élimination. Cependant, dans la dernière décennie, de plus en plus de recherches ont démontré que les TAD possédaient un sous-domaine activation/dégradation (DAD) responsable pour une fonction d'activation de la transcription dépendante de la dégradation de la protéine. Un tel phénomène peut être accompli par plusieurs moyens tels que des modifications post-traductionnelles, l’association à des cofacteurs ou la formation d’un réseau d’interaction complexe en chaînes. Or, aucune preuve concrète n’a pu clairement démontrer le fonctionnement de la dépendance paradoxale entre ces deux fonctions sur un activateur de transcription. Le DAD, a été observé dans plusieurs facteurs de transcription incluant la protéine suppresseur de tumeur p53 et le facteur de différenciation érythrocyte EKLF. Un aspect particulier des DAD est que la composition de leur séquence d’acide aminé est fortement similaire à celle des domaines de liaison à l’ubiquitine (UBD) qui jouent un rôle clé dans le contrôle de la transcription à travers leur interaction non-covalente avec l’ubiquitine. Ainsi, dans ce mémoire, nous avons étudié la possibilité que les TAD acides soient capables d’agir comme UBD pour réguler leur fonction paradoxale à travers des interactions non-covalentes avec l’ubiquitine. L’analyse est faite en utilisant la résonnance magnétique nucléaire (RMN) ainsi qu’avec des essais fonctionnels de dégradation. En somme, cette étude amène une plus grande compréhension des protéines impliquées dans le contrôle des TAD et caractérise le tout premier exemple de TAD capable d’interagir avec l’ubiquitine. / Acidic transactivating domains have been shown to be potential targets for a number of different therapies but their dynamic nature and their ability to bind many interacting partners has made it difficult to fully understand their functioning mechanisms. What we do know about these domains is that they readily control transcription through a myriad of interactions capable of either activating specific aspects of their function or simply, signal for their own demise. Within the acidic TADs lies an unusual degradation/activation domain (DAD) capable of activating transcription at the cost of its degradation. In other words, DAD transcriptional activation is dependent on the degradation of the protein. Such a phenomenon could be explained by a wide variety of hypotheses like the play of post-translational modifications, co-factors, or maybe just a really sophisticated time scaled network of interactions. However, no concrete explanation of how this dual dependent functioning domain works has yet to surface. The DAD has been observed within acidic TADs of several transcription factors including the tumor suppressor p53 and the red blood cell differentiation factor EKLF. Interestingly though, the amino acid sequence composition of DADs share a strong similarity with several types of sequences from domains that bind ubiquitin (UBDs). These domains have been shown in the past to, in addition to their role in degradation, play a key role in regulating transcription through non-covalent interaction with ubiquitin. Hence, in this project, we investigated weather acidic TADs had the ability to function as UBDs and form non-covalent interactions with ubiquitin and also to determine the functional significance of this interaction in regards to the dual function of acidic TADs.

Au cours de cette thèse, nous avons étudié deux protéines par RMN : la créatine kinase musculaire (CK-MM) et le domaine UIM-SH3 de la protéine STAM2, seuls ou en interaction avec leurs partenaires. La CK-MM est une enzyme active sous forme dimérique. Elle appartient à la famille des guanidino-kinases et intervient dans le processus énergétique de la cellule. Le but de l’étude était d’élucider le mode de fonctionnement de la CK-MM. Pour cela, nous avons enregistré des expériences de relaxation R1, R2 et des expériences de perturbation de déplacement chimique sur la CK-MM libre et complexée avec MgADP et sous forme TSAC. Ces expériences montrent que la boucle 320s, spécifique à la reconnaissance des substrats, possède une dynamique rapide en absence de substrats et une dynamique ralentie en présence de substrats. La fixation des substrats dans les sites actifs de la CK-MM induit des modifications conformationelles importantes. La protéine STAM2 est composée de deux UBDs : VHS, et UIM et d’un domaine SH3 connu pour interagir avec des déubiquitinases UBPY et AMSH. Cette protéine est impliquée dans la voie de dégradation lysosomale. L’objectif de cette étude est la caractérisation du complexe SH3/ubiquitine. Pour cela, nous avons enregistré des expériences de perturbation de déplacement chimique et de relaxation R1, R2 et nOes sur le complexe UIM-SH3/ubiquitine. Ces expériences mettent en évidence que les domaines UIM et SH3 sont capables d’interagir chacun avec une ubiquitine, avec une affinité de l’ordre de la centaine de micromolaire. L’interface entre les UBDs et l’ubiquitine implique majoritairement des résidus hydrophobes et conservés / In this thesis, we study two proteins by NMR: the muscular creatine kinase (CK-MM) and the SH3 domain of STAM2 protein, in the free and complexed forms. CK-MM is an active homodimeric enzyme which belongs to the guanidino-phosphagen-kinase family. This enzyme is involved in energetic process in the cell. The aim of this study is to elucidate the functional mode of the CK-MM. For this purpose, we measured R1 and R2 relaxation rates and chemical shit perturbation experiments on the substrate-free CK-MM, the CK-MM/MgADP complex, and the inhibitory ternary complex CK-MM/MgADP-creatine-nitrate. The experiments show that the loop 320s, specific recognition of the substrates, possesses a fast dynamic in absence of substrates (in the order of nano-picosecond) and a slower dynamic in presence of creatine-MgADP-nitrate ion. The binding of the substrate in the two active sites induces of significant conformational modification of the CK-MM. STAM2 protein consists in two ubiquitin binding domains (VHS and UIM) and a SH3 domain which interacts with deubiquinating enzymes AMSH and UBPY. This protein is involved in the lysosomal degradation pathway. The aim of this study is the characterization of the interaction between SH3 domain of STAM2 and ubiquitin. For this, we recorded the R1, R2, nOes relaxation experiments and chemical shift perturbation experiments on the UIM-SH3/ubiquitin complex. These experiments show that SH3 and UIM domains interact each with a single ubiquitin, with affinity of the order of hundred micromolars. The interface between these UBDs and ubiquitin, involves mainly hydrophobic and conserved amino-acids

Germ-line mutations in the breast cancer susceptibility gene BRCA1 strongly increase the risk of developing breast and ovarian cancer in women. Different hypothesis have been proposed to explain this tissue specificity. One of the most argued hypothesis is the one that proposes a link between BRCA1 and ovarian hormones' action. Much data have been published in the last years pointing to an important role of progesterone receptor (PR) in inducing normal mammary development and also breast cancer formation. This study aimed to search for a functional relationship between BRCA1 and PR in breast cancer cells. We have found that BRCA1 inhibits the transcriptional activity of PR. We have investigated in more detail the mechanism of this effect. BRCA1 and PR interact in vivo in a ligand-independent fashion. Most importantly, BRCA1 alters the ligand-independent and dependent degradation of PR protein through its ubiquitination and this might have a direct effect on the level of PR recruitment on regulated promoters. BRCA1 is recruited to the hormone-responsive regions of PR-target genes and affects the presence of histone deacetylase activity and the level of monoubiquitinated histone H2A, linking BRCA1 action with chromatin status. These findings support a connection between BRCA1, the principal tumour suppressor responsible for familial breast cancer, and the progesterone receptor transcriptional activity. This relationship can be hypothesized to be reflected in the BRCA1-related breast tumourigenesis. / Mutaciones germinales en el gen breast cancer susceptibility gene BRCA1 aumentan altamente el riesgo de padecer cáncer de mama y ovario en mujeres. Se han propuesto diferentes hipótesis para explicar esta especificidad de tejido. Una de las hipótesis más argumentadas es la que propone una relación entre BRCA1 y la acción de las hormonas ováricas. En los últimos años se han publicado numerosos datos señalando al papel esencial del receptor de progesterona (PR) en la inducción del desarrollo normal de la mama y en la formación del cáncer de mama. Este estudio pretendía buscar una relación funcional entre BRCA1 y PR en células de cáncer de mama. Hemos demostrado que BRCA1 inhibe la actividad transcripcional de PR. Hemos investigado en más detalle el mecanismo de este efecto. BRCA1 y PR interaccionan in vivo de una manera independiente de ligando. Y lo que es más, BRCA1 altera la degradación independiente y dependiente de ligando de PR a través de su ubiquitinización y esto podría tener un efecto directo en el nivel de reclutamiento de PR en promotores regulados. BRCA1 es reclutado a las regiones de respuesta a hormona de genes diana de PR y afecta la presencia de actividad histona desacetilasa y el nivel de histona H2A monoubiquitinada, estableciendo un enlace entre la acción de BRCA1 y el estado de la cromatina. Estos hallazgos apoyan una conexión entre BRCA1, el principal supresor de tumor responsable del cáncer de mama hereditario, y la actividad transcripcional del receptor de progesterona. Se puede hipotetizar que esta relación se ve reflejada en el proceso de tumorigénesis BRCA1-dependiente.