Добірка наукової літератури з теми "UBTD1"

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.

Au cours de ma thèse, j'ai travaillé sur le modèle d'organoïde 3D en suivant deux objectifs principaux : i) développer des outils génétiques pour modifier le génome des organoïdes et ii) déchiffrer le rôle de la ubiquitin domain-containing protein 1 (UBTD1) dans le développement des organoïdes de la prostate.L'ingénierie du génome est devenue ces dernières années plus accessible grâce aux endonucléases programmables par ARN telles que le système CRISPR-Cas9. Cependant, l'utilisation de cette technologie d'édition dans des organes synthétiques appelés "organoïdes" reste très inefficace. Ceci est principalement dû aux méthodes de livraison utilisées pour la machinerie CRISPR-Cas9, qui sont principalement réalisées par électroporation de RNP contenant le complexe CAS9-gRNA, une procédure toxique pour les organoïdes. Nous décrivons ici l'utilisation de la technologie "Nanoblade" pour réaliser l'édition du génome dans les organoïdes. Les nanoblades ont dépassé de loin les niveaux de knock-out (KO) obtenus avec d'autres techniques utilisées jusqu'à présent pour la livraison de la machinerie d'édition de gènes. Nous avons atteint jusqu'à 80 % de knockout génétique dans les organoïdes après traitement avec les nanoblades. Nous avons atteint un niveau élevé de KO médié par les nanoblades pour le gène codant le récepteur des androgènes (AR) et le gène du cystic fibrosis transmembrane conductance regulator (CFTR) avec des nanoblades contenant un seul ARNg ou un double ARNg. Plus important encore, contrairement à d'autres méthodes d'édition de gènes, ce résultat a été obtenu sans toxicité pour les organoïdes. En outre, il ne faut que quatre semaines pour obtenir des lignées stables KO pour un gène dans les organoïdes et aucun INDELS indésirable évident dans un site hors cible du génome n'a été détecté. En conclusion, les nanoblades simplifient et permettent une édition rapide du génome dans les organoïdes avec peu ou pas d'effets secondaires.La morphogenèse et le remodelage des tissus sont des processus finement régulés, régis par les adhésions entre cellules. Cependant, le contrôle spatial et temporel des molécules d'adhésion reste partiellement inexploré. Nous avons étudié ici le rôle d'UBTD1 comme modulateur de la force des adhérences dans l'épithélium de la prostate. Nous avons montré que la régulation négative d'UBTD1 perturbe l'auto-organisation des cellules en trois dimensions. Inversement, nous avons démontré que la surexpression d'UBTD1 induit des monocouches épithéliales plus régulières et augmente la tension de la surface cellulaire. Les analyses transcriptomiques ont révélé un profil d'expression génique des protéines impliquées dans les jonctions cellulaires affectées par la modulation d'UBTD1. En utilisant le modèle d'organoïde de prostate, nous avons montré que l'expression d'UBTD1 dans les cellules luminales perturbait la formation de lumen dans les organoïdes de prostate de souris. Enfin, en utilisant une approche de co- immunoprécipitation couplée à la spectrométrie de masse, nous avons montré que UBTD1 interagit avec des partenaires impliqués dans les jonctions cellule-cellule et que ces interactants voient leur expression modulée par la dérégulation de UBTD1. Nos résultats montrent qu'une protéine impliquée dans les processus de dégradation des protéines régule la force des jonctions d'adhérence
During my thesis, I worked on the 3D organoid model following two main objectives: i) developing genetic tools to modify the genome of organoids and ii) deciphering the role of ubiquitin domain-containing protein 1 (UBTD1) in the development of prostate organoids . Genome engineering has become in the last few years more accessible thanks to the RNA programmable endonucleases such as the CRISPR-Cas9 system. However, using this editing technology in synthetic organs called ‘organoids’ is still very inefficient. This is mainly due to the delivery methods used for the CRISPR-Cas9 machinery, which are predominantly performed by electroporation of RNPs containing the CAS9-gRNA complex, a procedure toxic for the organoids. Here we describe the use of the ‘Nanoblade’ technology to accomplish genome editing in organoids. Nanoblades outperformed by far knockout (KO) levels achieved with other techniques used to date for delivery of the gene editing machinery. We reached up to 80% of gene knockout in organoids after treatment with nanoblades. We achieved high-level nanoblade-mediated KO for the androgen receptor (AR) encoding gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene with single gRNA or dual gRNA containing nanoblades. Most importantly, in contrast to other gene editing methods, this was obtained without toxicity for the organoids. Moreover, it requires only four weeks to obtain stable lines KO for a gene in organoids and no obvious unwanted INDELS in off-target site in the genome were detected. In conclusion, nanoblades simplify and allow rapid genome editing in organoids with little to no side-effects.Morphogenesis and tissue remodeling are finely regulated processes governed by cell-cell adhesions. However, the spatial and temporal control of adhesion molecules remains partially unexplored. Here we studied the role of UBTD1 as a modulator of the strength of adherens in the prostate epithelium. We showed that down-regulation of UBTD1 disrupted the self- organization of cells in three dimensions. Conversely, we demonstrated that overexpression of UBTD1 induced more regular epithelial monolayers and increased cell surface tension. Transcriptomic analyses revealed a gene expression profile of proteins involved in cell junctions affected by UBTD1 modulation. Using the prostate organoid model, we showed that UBTD1 expression in luminal cells disrupted cyst formation in mouse prostate organoids. Finally using a co-immunoprecipitation approach coupled to mass spectrometry, we showed that UBTD1 interacts with partners involved in cell-cell junctions and that these interactants have their expression modulated by UBTD1 deregulation. Our results show that a protein involved in protein degradation processes regulates the strength of adherens junctions

Molecular Biology and Genetics
M.S.
p97, a member of the AAA ATPase (ATPases Associated with diverse cellular Activities) family of proteins, exists as a hexamer with two centrally located ATPase domains D1 and D2. The ATPase function of p97 is the means by which mechanical force is applied to substrates, consequently changing their conformation. As a highly abundant protein within cells, p97 has been shown to function in multiple pathways whereby specificity is directed via adaptor proteins. The largest family of bona fide p97 adaptors is the `Ubiquitin regulatory X' (UBX) domain containing family of adaptors. In addition to the UBX domain containing family of adaptors, proteins containing a PUB domain have been implicated in binding to p97. The p97 adaptor protein UBXD1 contains both a UBX and PUB domain, however the UBX domain does not participate in binding p97 due to absence of the conserved motif required for binding. Recently, a highly conserved region within the first 150 amino acids of the UBXD1 N-terminus has been shown to participate in p97 binding (Kern et al., 2009). The cellular function of UBXD1 remains largely unknown. One of the focuses of the Haines laboratory is to elucidate the function of UBXD1. Unpublished results from within the laboratory suggest UBXD1 to be defective at interacting with p97 mutants found in Inclusion Body Myopathy associated with Paget's Disease of Bone and Frontotemporal Dementia (IBMPFD) and Amyotrophic Lateral Sclerosis (ALS) disease, leading to a disruption of the autophagy pathway. These results suggest a role for UBXD1 as a human disease relevant protein. Search of a Single Nucleotide Polymorphism (SNP) database for polymorphisms within conserved regions of UBXD1 was carried out. Interestingly, the SNP database revealed a polymorphic variant within a conserved region of the PUB domain. The SNP was found within the asparagine residue of the evolutionarily conserved surface patch, resulting in an asparagine(N) to serine(S) substitution, termed N184S. The two main objectives of my master's thesis project are 1) to characterize this UBXD1 polymorphic variant in terms of p97 binding capabilities and determine the prevalence of the N184S PUB domain SNP within the population and 2) identification/verification of UBXD1 interacting proteins that may provide a clue for UBXD1 function in autophagy and define domains required for association. As a result of interaction-based studies, I have been able to show a severe loss-of-binding phenotype by the UBXD1 N184S polymorphic variant. While the N184S PUB domain SNP could not be validated within my sample population, the interaction data led to the discovery of potential UBXD1 interacting proteins. Additionally, proteomics data generated by Dr. Dale Haines in the lab of Dr. Raymond Deshaies revealed ERGIC-53 to be a novel binding partner of UBXD1. Through interaction-based studies, I have been able to determine the regions required for the interaction between UBXD1 and ERGIC-53 as well as propose a possible role for p97 UBXD1 complexes in autophagy.
Temple University--Theses

Molecular Biology and Genetics
Ph.D.
p97 is a member of the AAA family of proteins (ATPase Associated with various cellular Activities). It is a highly conserved and abundant protein and functions in numerous ubiquitin-mediated processes including ERAD. Endoplasmic Reticulum Associated Degradation is the process by which misfolded/ubiquitinated proteins translocate out of the ER and migrate to the proteasome for degradation. p97 maintains substrate misfolding and mediates its exit from the ER and trafficking to the 26S proteasome. It also plays important roles in protein trafficking, the cell-cycle, apoptosis and homeotypic Golgi Apparatus and Endoplasmic Reticulum membrane fusion after mitosis. In addition, p97 plays a role in the aggresome-autophagy degradation pathway, which handles the ubiquitin-mediated destruction of aggregate-prone, misfolded, cytosolic proteins. p97 mutation is the causative alteration in the disorder, IBMPFD, which is marked by defects in autophagy. This broad diversity of function is mediated through p97's interaction with a large group of adaptor proteins. Many of these adaptors harbor both p97 interaction motifs and ubiquitin association domains. However, more than half of known p97 adaptors do not. Their function is largely unknown. UBXD1 is one known adaptor for p97 that does not have a ubiquitin association domain (UBA), and has been shown to have decreased interaction with IBMPFD mutant p97R155H and p97A232E. Recently, it has been suggested to perform a role in protein trafficking, specifically in monoubiquitinated caveolin-1 internalization and trafficking to the endosome. A novel high abundance UBXD1 interacting partner has been identified via solution-based mass spectrometric analyses. ERGIC-53, the namesake of the ER-Golgi Intermediate Compartment, has been shown to be involved in bi-directional trafficking between the ER and Golgi. The association between UBXD1 and ERGIC-53 is unique among UBX family members. Deletional analysis has shown that unlike p97, the ERGIC-53-UBXD1 interaction takes place in the extreme amino terminus of UBXD1, (within the first 10 amino acids) which is predicted by computer modeling to form a hydrophobic binding pocket. Further site-directed mutagenesis work has clearly shown four amino acids (3 highly hydrophobic) are crucial for maintaining this interaction. They have been modeled to form a conserved alpha-helix. ßCOPI, a primary member of the COPI coatomer complex which is involved in protectively coating ERGIC-53 positive vesicles, is also thought to be involved with the ERGIC-53-UBXD1-p97 pathway. ßCOPI has been identified as a UBXD1-independent interactor with p97. Modest UBXD1 over- expression using a ponasterone inducible system has shown that UBXD1 modulates ERGIC-53 localization. Additionally, a functional link between UBXD1, p97 and ERGIC-53 in autophagy has been discovered through the use of a highly efficient, miR30-based, inducible knockdown system. Upon individual knockdown of UBXD1, p97 and ERGIC-53, autophagic markers p62 and LC3-II accumulate at relatively high levels in normal culture conditions, strongly suggesting a role in mediating basal autophagy. However, when placed under starvation conditions, autophagy progresses and p62 is degraded. It is speculated from these studies that a p97/UBXD1 complex plays a role in regulating the trafficking of ERGIC-53 positive vesicles and this activity plays an important role in autophagy.
Temple University--Theses

Durante i miei 3 anni di dottorato, mi sono interessato sullo studio di Eff/UbcD1, una proteina altamente conservata nota per essere coinvolta nella protezione dei telomeri in Drosophila. Mutazioni nel gene eff/UbcD1, che codifica per un enzima E2 evolutivamente conservato, causano un'alta frequenza di fusioni telomeriche (TFs) nelle cellule mitotiche di Drosophila, indicando che Eff/UbcD1 è richiesto per la protezione dei telomeri. Tuttavia si sa molto poco sui meccanismi molecolari alla base di questa funzione. Recenti lavori hanno dimostrato che Eff/UbcD1 è una componente della cromatina che suui cromosomi politenici fortemente colocalizza con HP1, un ben noto fattore cromatino che è anche richiesto per prevenire TFs. Nella prima parte della mia tesi, descriverl i risultati della relazione genetica e molecolare tra UbcD1 e HP1. Descriverò che la perdita di UbcD1 nei mutanti HP1 causa una forte diminuzione delle TFs rispetto al singolo mutante HP1, suggerendo che le funzioni protetttive ai telomeri di entrambe le proteine sono fortemente interconnesse. La mia analisi biochimica rivela che UbcD1 interagisce e ubiquitina HP1 e HP1 è capace di legare l'ubiquitina in maniera non covalente. Inoltre dimostrerò come UbcD1 promuova l'arricchimento di una nuova forma di HP1 che migra lentamente (sHP1) precedentemente non caratterizzata. Inoltre, descriverò una inedita relazione funzionale tra HP1, il complesso MRN e l'istone H3. Attraverso mutagenesi sito specifica, ho identificato almeno 2 diverse lisine richieste per l'ubiquitinazione di HP1. Di queste, la lisina 151, appare essere un residuo chiave per l'ubiquitinazione della forma sHP1 e cosa ancora più importante, dimostrerò che l'ubiquitinazione della lisina 151 è richiesta per regolare la stabilità sia del complesso MRN e dell'istone H3. Nella seconda parte della mia tesi, mostrerò dei risultati che indicano un ruolo di UbcD1 nella riparazione del danno al DNA. Descriverl che la perdita di UbcD1, ma non dell'ortologo di Rad6 di Drosophila, UbcD6, dà radioresistenza dopo irradiamento con raggi X. La mia analisi citologica sui cromosomi mitotici rivela che nelle cellule mitotiche dei mutanti ubcd1 le frequenze di rotture cromosomiche (CBs), aberrazioni cromosomiche (CABs) e i foci indotti da irradiamento (IRIF) sono tutte significativamente ridotte rispetto al controllo. Tuttavia, il numero delle CBs e CABs indotte dai raggi X aumenta a seguito dell'overespressione di UbcD1 indicando che Ubcd1 gioca un ruolo negativo nel riparo al danno al DNA. Inoltre, la mia analisi per western blot indica anche che il complesso MRN è richiesto per la stabilità di ubcd1. In aggiunta, esperimenti di Co-IP rivelano che ubcd1 interagisce con Rad50, un membro del complesso MRN. Tuttavia, i livelli proteici di Rad50 non sono alterati nei mutanti ubcd1, indicando che se ubcd1 è coinvolto nell'ubiquitinazione di rad50, questa modifica non è richiesta per la degradazione di rad50. Presi nel loro insieme, i miei dati suggeriscono che ubcd1 gioca dei ruoli importanti nella regolazione della stabilità cromosomica interagendo sia con fattori telomeri che della risposta al danno al DNA.
During my three PhD years, I focused my interest on the study of Eff/UbcD1, a well conserved protein known to be involved in telomere protection in Drosophila. Mutations in the effete/UbcD1 gene, that encodes a highly evolutionarily conserved ubiquitin-conjugating enzyme, lead to frequent Telomeric Fusions (TFs) in Drosophila mitotic cells, indicating that Effete/UbcD1 is required for telomere protection. However, little is known about the molecular mechanism underlying this function. Recent work has shown that Effete/UbcD1 is a chromatin component that on polytene chromosomes strongly co-localizes with Heterochromatin Protein 1 (HP1), a well-known chromatin factor that is also required to prevent TFs. In the first part of my thesis, I describe results on the genetic and molecular relationship between UbcD1 and HP1. Intriguingly, I describe that loss of Eff/UbcD1 in a Hp1 mutant background yields to a frequency of TFs lower than that observed in the single Hp1 mutants, suggesting that protective functions at telomeres of both Eff/UbcD1 and HP1 are strongly interconnected. My biochemical analyses reveal that Eff/UbcD1 physically interacts with and ubiquitinates HP1 and that HP1 itself is able to bind ubiquitin in a non covalent manner. Furthermore, I also demonstrate that Eff/UbcD1 promotes the enrichment of a novel and slow migrating isoform of HP1 (sHP1) that was not described before. Moreover, I describe a previously uncharacterized functional relationship involving HP1, the MRN complex and histone H3. By site-specific mutagenesis, I identified at least 2 different lysines required for HP1 ubiquitination. Of these, Lys151 arises as a key residue for ubiquitination of sHP1 and more importantly, I demonstrate that Lys151 ubiquitination is required for regulating the stability of both MRN complex and histone H3. In the second part of my thesis, I also show results that indicate a role for Eff/UbcD1 in DNA repair. I describe that loss of Eff/UbcD1, but not the Drosophila Rad6 ortholog UbcD6, confers radioresistance after X-ray irradiation. My cytological analysis of mitotic chromosomes reveals that in Eff/UbcD1 mitotic cells the frequencies of chromosome breaks (CBs), chromosome aberrations (CABs) and irradiation induced foci (IRIF) are all significantly reduced after X-ray compared to control. However, the number of X-ray induced CBS and CABs increases when Eff/UbcD1 is overexpressed indicating that Eff/UbcD1 plays a negative role in the DNA damage response (DDR) and/or repair. Moreover, my western blot analysis also indicate that the MRN complex is required for Eff/UbcD1 stability. In addition, Co-IP experiments reveal that Eff/UbcD1 interacts with the MRN complex member Rad50. However, Rad50 protein levels are not affected in eff/UbcD1 mutants, indicating that if Eff/UbcD1 is involved in Rad50 ubiquitination, this modification is not required for Rad50 degradation. Taken together, my results suggest that Eff/UbcD1 plays important roles in the regulation of chromosome stability by interacting with both telomeric and DDR factors.

Die essenzielle, Ubiquitin-selektive ATPase p97 reguliert eine Vielzahl unterschiedlicher Prozesse in Eukaryoten. Dazu zählen Proteinqualitätskontrolle, DNA-Reparatur, Signaltransduktion, Zellzykluskontrolle, Autophagie sowie das endolysosomale System. Diese unterschiedlichen Funktionen von p97 werden durch die Bindung von Kofaktoren engmaschig gesteuert und kontrolliert. Die größte und am besten untersuchte Gruppe von p97-Kofaktoren sind die Proteine der UBX Familie. Diese zeichnen sich durch den Besitz einer UBX-Domäne aus, welche die Bindung an p97 vermittelt. Das in höheren Eukaryoten konservierte Familienmitglied UBXD1 besitzt darüber hinaus mit einer PUB-Domäne und einem VIM-Motiv noch mindestens zwei weitere p97-Bindemodule. UBXD1 kann an Vesikel des endolysosomalen Degradationssytems lokalisieren, seine genauen zellulären Funktionen sind jedoch noch weitgehend unbekannt. Ziel dieser Arbeit war die funktionelle Charakterisierung von humanem UBXD1. Dafür wurden Kandidaten eines zuvor durchgeführten Yeast-Two-Hybrid-Screens auf ihre Two Hybrid-Interaktion mit unterschiedlichen UBXD1-Varianten getestet. Darüber hinaus wurde durch Immunpräzipitationsexperimente untersucht, ob die Kandidatenproteine auch in Säugerzellen mit UBXD1 interagieren. Als vielversprechende neue Bindungspartner von UBXD1 wurden so die Ubiquitin-Ligase TRIAD3A und das Ubiquitin-editierende Protein A20 identifiziert. Desweiteren konnte gezeigt werden, dass die Interaktion zwischen UBXD1 und A20 von einer funktionellen PUB Domäne und dem siebten Zinkfinger Motiv von A20 abhängig ist. Da sowohl TRIAD3A als auch A20 negative Regulatoren des NF B Signalweges sind, wurde daraufhin untersucht, ob auch UBXD1 eine Funktion in diesem Signalweg besitzt. Tatsächlich war in UBXD1-depletierten HeLa 57A-Zellen die NF B-abhängige Expression eines Reportgens nach Aktivierung des Signalweges durch TNF, IL-1, Doxorubicin und H2O2 stark reduziert. Dabei spricht die verringerte Aktivierung nach unterschiedlichen Stimuli für eine generelle Rolle von UBXD1 im NF B Signalweg. Durch quantitative Echtzeit-PCR konnte gezeigt werden, dass in HeLa- und HEK293T-Zellen nach UBXD1-Depletion auch die Expression endogener NF B Zielgene verringert ist. Da in UBXD1-depletierten Zellen nach Stimulation mit TNF oder IL-1 bereits die Kerntranslokation des NF B-Transkriptionsfaktor p65 reduziert ist, ist davon auszugehen, dass UBXD1 an einer früheren Phase der Aktivierung des Signalweges beteiligt ist. Möglicherweise ist dies darauf zurückzuführen, dass UBXD1 bekannte Funktionen von A20 reguliert und etwa die Bindung von A20 an Vesikel des endolysosomalen Systems oder an lineare Ubiquitinketten beeinflusst. Diese Arbeit beschreibt somit eine neue Funktion des p97-Kofaktors UBXD1 im NF B-Signalweg
The essential, ubiquitin-selective ATPase p97 regulates a variety of cellular processes in eukaryotes. Among others, these include protein quality control, DNA repair, signal-transduction, cell cycle control, autophagy and the endolysosomal system. The distinct functions of p97 are tightly controlled by regulatory cofactors. UBX domain-containing proteins are the largest and best studied group of p97 cofactors . They are characterized by a UBX domain, which mediates binding to p97. The family-member UBXD1 is highly conserved in higher eukaryotes and possesses at least two additional p97 binding modules, a PUB domain and a VIM motif. While UBXD1 can localize to vesicles of the endolysosomal degradation system, its exact cellular function is still poorly understood. The aim of this study was the functional characterisation of human UBXD1. To that end, candidates of a previous yeast two-hybrid screen were tested for their two-hybrid interaction with different UBXD1 variants. Immunoprecipitation experiments were used to analyse if the candidates also interact with UBXD1 in mammalian cells. This led to the identification of the ubiquitin-ligase TRIAD3A and the ubiquitin-editing protein A20 as promising new binding partners of UBXD1. Moreover, it could be demonstrated that the interaction between UBXD1 and A20 depends on a functional PUB domain and the seventh zinc finger motif of A20. Because both TRIAD3A and A20 are negative regulators of the NF-B signaling pathway, it was subsequently tested if UBXD1 also has a function in NF-B signaling. Indeed, UBXD1-depleted HeLa 57A cells showed a strongly reduced NF B dependent expression of a reporter gene after activation of the signaling pathway by TNF, IL-1, Doxorubicin and H2O2. The reduced activity observed after various stimuli argues for a general role of UBXD1 in the NF-B signaling pathway. Quantitative real-time PCR demonstrated that the expression of endogenous NF-B target genes in HeLa and HEK293T cells was also reduced upon UBXD1-depletion. Since the nuclear translocation of the NF-B subunit p65 upon stimulation with TNF or IL-1was also reduced in UBXD1-depleted cells, UBXD1 is likely to participate in an earlier phase of NF-B activation. It is possible that UBXD1 regulates a known function of A20 and influences for example the binding of A20 to endocytic vesicles or to linear ubiquitin chains. In summary, this work describes a novel function of the p97 cofactor UBXD1 as a positive regulator of the NF-B signaling pathway