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In der vorliegenden Dissertation stand die Aufklärung der Funktion und Regulation von p21 in der Mitose im Mittelpunkt. p21 ist als Cdk-Inhibitor und Schlüsselregulator bekannt, der in viele fundamentale zelluläre Prozesse involviert ist: Zellzyklusregulation, Apoptose, Seneszenz, Zellmigration und Dynamik des Zytoskeletts, Transkription, Differenzierung sowie DNA-Reparatur, aber auch in die Umprogrammierung induzierter pluripotenter Stammzellen (Besson et al. 2008; Abbas und Dutta 2009; Jung et al. 2010).
Die unkontrollierte Proliferation von Zellen ist mit der Tumorgenese assoziiert und wird unter anderem durch die Fehlregulation von p21, aber auch durch die wichtigen mitotischen Kinasen Cdk1, im Komplex mit ihrer regulatorischen Untereinheit Cyclin B1, sowie Plk1 bedingt. Zudem ist das Fehlen von p21 oder die Fehllokalisation in das Zytoplasma mit einer schlechteren Prognose für den Patienten und Chemotherapie-Resistenz von Tumoren verbunden (Abukhdeir und Park 2008). Aufgrund der zunehmenden Inzidenz und Mortalität von Krebserkrankungen ist es daher von besonderem klinischem Interesse, die molekularen Ursachen für die Entstehung maligner Tumorerkrankungen aufzuklären. Bislang existieren kaum Studien über welche molekularen Mechanismen die Funktionen von p21, dem wichtigsten Cdk-Inhibitor, der zum Beispiel durch die Anwendung niedermolekularer Inhibitoren wie BI 2536, das sich bereits in klinischen Phase II Studien befindet (Strebhardt 2010), beeinflusst wird, während der Mitose reguliert werden.
In der vorliegenden Dissertation wurde daher die physiologische Rolle des Cdk-Inhibitors bzw. Regulators p21 während der Mitose untersucht und mit der Kinaseaktivität von Cdk1/Cyclin B1, wie auch Plk1 korreliert. Es konnte gezeigt werden, dass p21 während der Mitose stark exprimiert wird und dass mitotisches p21 in verschiedenen Krebszelllinien unabhängig von dem p53-Status in einer phosphorylierten Form vorkommt, welche mit der Aktivität von Cdk1 und weniger mit der von Cdk2 assoziiert ist. Durch Untersuchungen der isogenen HCT116-Zelllinien mit und ohne p21 wurde aufgezeigt, wie wichtig p21 für den ordnungsgemäßen Ablauf der Mitose ist. Ohne p21 sind sowohl die Anaphase wie auch die Zytokinese verlängert, die Zellen ordnen die Chromosomen fehlerhaft in der Metaphaseplatte an (congression Fehler), besitzen weitaus mehr lagging Chromosomen und fast 20 % der Zellen weisen im Versuchsverlauf Polyploidie auf. Durch den Verlust des Cdk-Regulators p21 kommt es zur Fehlregulation von Cdk1 und seiner Substrate (wie MCAK) und es treten die oben beschriebenen Probleme auf.
Weiterhin phosphoryliert Cdk1/Cyclin B1 p21 an Ser-130 in vitro und ex vivo in der frühen Phase der Mitose, der Prophase bzw. Prometaphase. Die nicht phosphorylierbare p21 Form S130A befindet sich hauptsächlich im Zellkern und führt zu vermehrtem Auftreten von congression Fehlern, während die S130D-Mutante, die die Phosphorylierung durch Cdk1 vortäuscht, schneller degradiert wird und zudem den Phänotyp der HCT116 p21-/- Zellen verstärkt. Zellen, die S130D exprimieren, benötigen mehr Zeit für das Durchlaufen der Mitose. Hier ist vor allem die Metaphase stark verlängert, aber auch Anaphase und Zytokinese. Dies führt zu congression Fehlern und zu Polyploidie. Diese Ergebnisse bestätigen, wie wichtig die zeitlich korrekte Phosphorylierung von p21 und die dadurch vermittelte Aktivierung von Cdk1/Cyclin B1 ist.
Darüber hinaus stabilisiert die Suppression von Plk1 das p21 Protein, was darauf hinweist, dass die Degradation von p21 während der Prometaphase von Plk1 kontrolliert wird. Dies wird von der Tatsache unterstützt, dass Ser-114, wie auch Ser116 von Plk1 in vitro phosphoryliert wird. Die Deregulation von p21 durch Plk1, SS114/116AA bzw. SS114/116DD induziert Chromosomenfehler, wodurch die molekularen Mechanismen, warum fehlreguliertes Plk1 die Tumorgenese fördert, hervorgehoben werden.
Nach Abschluss der bisherigen Untersuchungen steht fest, dass man sich von der starren Rolle von p21 als Tumorsuppressor und Akteur während der G1/S-Phase lösen muss. Der Cdk-Inhibitor p21 trägt entscheidend zur mitotischen Progression bei, vor allem bedingt durch die zeitlich ordnungsgemäße Inaktivierung bzw. Aktivierung von Cdk1/Cyclin B1, der Kinase, die wiederum zahlreiche für die Mitose essentielle Proteine reguliert. In Zukunft muss zum besseren Verständnis der Rolle von p21 in der Mitose die genaue Abfolge der Ereignisse unter Einbeziehung der Degradationsmechanismen eingehender untersucht werden.
Acute lymphoblastic leukemia (ALL), a neoplastic disorder of blood cells of the lymphoid lineage, is the most frequent childhood cancer. In spite of increasing survival rates, the outcome for adults, infants or relapsed patients is still less favorable, highlighting the need for novel treatment options. Reactive oxygen species (ROS) are important signaling molecules that are involved in a variety of cellular pathways. As high ROS levels lead to oxidative stress and irreversible oxidation of cellular macromolecules, the production and elimination of ROS is tightly controlled. Therefore, cells express several antioxidant molecules and enzymes, including glutathione, catalase and the thioredoxin (Trx) system, to balance ROS levels. As cancer cells were found to have increased ROS levels that could contribute to tumor progression and metastasis, they rely strongly on these antioxidant systems to prevent oxidative damage, making cancer cells especially vulnerable to ROS-inducing treatments. ROS and oxidative stress have been shown to induce programmed cell death via different pathways, however the exact mechanisms that couples oxidative signaling and cell death is not completely understood.
As a disturbance of the cellular redox homeostasis was reported during leukemia development and progression, we wanted to determine the potential of Trx inhibitors for ALL therapy. Additionally, we aimed to further understand the role of ROS and subsequent protein oxidation in the induction and execution of programmed cell death.
First, we demonstrated that the Trx1 inhibitor PX-12 induced cell death in three ALL cell lines. Further analysis of the events leading to PX-12-induced cell death in FADD-deficient (FD) Jurkat cells revealed an increase in ROS levels and oxidation-mediated dimer formation of peroxiredoxin 3 (PRDX3). Interestingly cell death was inhibited by the thiol-containing antioxidant N-acetylcysteine (NAC), but not by non-thiol-containing ROS scavengers. PX-12 treatment further induced cleavage of caspase-9 and -3 and activation of the pro-apoptotic BCL-2 protein BAK, leading us to the conclusion that mitochondria-dependent apoptosis was induced. Interestingly, we could demonstrate an important role for the BH3-only protein NOXA in the mediation of PX-12-induced apoptosis as knock-down of NOXA prevented cell death induction and BAK activation. Our findings give novel insights into the mechanism of PX-12-induced cell death in ALL cell lines and underscores the potential of PX-12 for the treatment of ALL.
To further understand the processes leading to cell death upon inhibition of the Trx system, we analyzed global protein oxidation in Jurkat FD cells upon treatment with the Trx reductase inhibitor Auranofin. In line with previous results, Auranofin induced intrinsic apoptosis that was dependent on BAK and accompanied by increased ROS levels. Using a BIAM Switch Assay followed by mass spectrometry, we demonstrated that Auranofin treatment induced oxidation of over 200 proteins. We identified several proteins whose oxidation upon Auranofin treatment was expected, like Trx1, Trx2 and several peroxiredoxins. Additionally, we verified oxidation of APAF1-interacting protein (APIP) and protein arginine N-methyltransferase (PRMT1) that are both implicated in the regulation of apoptosis. With this analysis we were able to demonstrate that Auranofin treatment leads to changes in global protein oxidation. Whether oxidation of the determined proteins changes their functionality and contributes to apoptosis induction remains to be elucidated.
As we identified BAK as an important player in PX-12- and Auranofin-induced cell death in the previous parts of this study, we wanted to further understand its involvement in ROS-mediated cell death. First analyses in wild-type (WT) and BAK-/- murine embryonic fibroblasts (MEFs) revealed that BAK was essential for Auranofin-induced cell death and that this cell death was caspase-independent in MEFs. Interestingly, BAK oxidation was induced upon treatment with Auranofin, but not upon stimulation with the apoptosis-inducing compound Etoposide. Expression of mutated BAK, with either one or both oxidation-sensitive cysteines mutated to oxidation-insensitive serines, revealed that mutating already one cysteine protected cells from Auranofin , but not Etoposide-induced cell death. Of note, mutation of the BAK BH3 domain rescued MEFs from both, Auranofin- and Etoposide-mediated cell death. The presence of cysteine residues also altered BAK interactions as observed by a mass spectrometric analysis of Auranofin-treated MEFs expressing either WT or cysteine-less BAK. We identified interactions of WT BAK with proteins involved in mitochondrial fission and vesicle transport upon Auranofin treatment. Of note, interaction with proteins involved in apoptosis, like BAX or BCL-XL, was not changed between WT and cysteine-less BAK. Our results demonstrate a critical role for BAK oxidation in Auranofin-induced cell death. Furthermore, we identified novel oxidation-dependent BAK interaction partners.
To conclude, this study highlights the potential of ROS-inducing treatments for ALL therapy and provides novel insights into the redox regulation of programmed cell death.
Epidermal growth factor (EGF) receptor belongs to the broad family of enzymatic receptors called receptor tyrosine kinases (RTKs). Generally, the binding of a ligand to these receptors leads to activation of their intracellular kinase activity that sets in motion a cascade of signaling events. In order to ensure appropriate responses to physiological stimuli, the cell is endowed with the ability to regulate signal transduction via numerous mechanisms such as dephosphorylation of the RTK and its substrates as well as downregulation of the RTK. Activation of EGFR is a potent mitogenic (proliferative) and motogenic (cell motility) signal that plays crucial roles during embryonic development and maintenance of adult tissue. EGFR signaling is primarily regulated by ligand-induced receptor internalization with subsequent degradation in lysosomes. While the complex of proteins that are recruited to EGFR after its activation is well understood, proteins that interact with the receptor in the absence of ligand binding are still not systematically studied. With the goal of identifying novel binding partners of non-activated EGFR, a membrane based yeast-two hybrid screen (MYTH) was conducted. MYTH is based on the principle of in vivo reconstitution of the N-terminus (Nub) and C-terminus (Cub) halves of ubiquitin once brought into close proximity. A chimeric protein consisting of EGFR fused to Cub and a transcription factor was used as a bait to screen Nub-tagged cDNA library. Analysis of resultant yeast transformants revealed a total of 87 proteins to interact with EGFR. Of these only 11 were previously shown to bind to EGFR. A majority of the other proteins were shown to interact with the receptor by yeast retransformation. Fifteen were confirmed to bind to EGFR by coimmunoprecipitation assays in mammalian cells. One of the novel EGFR interactors identified in the screen was histone deacetylase 6 (HDAC6). This deacetylase is localized in the cytoplasm and known to deacetylate alpha-tubulin, HSP90 and cortactin. The juxtamembrane region of EGFR binds to the Cterminus of HDAC6. Functionally, overexpression of wild type HDAC6 stabilized ligand-induced degradation of the receptor. On the other hand, deacetylase deficient or EGFR binding compromised mutants of HDAC6 were able to stabilize EGFR only partially. Downmodulation of HDAC6 expression by RNAi markedly accelerated degradation of the receptor. Taken together, HDAC6 is a negative regulator of EGFR downregulation that is dependent on its deacetylase activity and ability to bind to the receptor. Imaging studies revealed that HDAC6 does not affect internalization of EGFR from the plasma membrane but rather influences the post-endocytic trafficking of the receptor-ligand complex to lysosomes. Pulse-chase experiments using fluorophoretagged EGF showed that EGFR is transported faster towards the peri-nuclear region and delivered to late endosomes rapidly in HDAC6 depleted cells. HDAC6 is demonstrated to act, at least partly, by regulating the acetylation of alpha-tubulin. Upon EGFR activation, acetylation of alpha-tubulin on lysine 40 is progressively increased as shown by mass spectrometry and immunoblotting. Forced expression of a dominant negative mutant of alpha-tubulin, but not wild type alpha-tubulin, led to reduced speed and processive movement of early endosomes in GFP-Rab5 expressing cells. In a surprising twist, EGFR is able to phosphorylate HDAC6 on Tyr570. Phosphorylation of Tyr570 and Ser568 leads to inactivation of the deacetylase function of HDAC6 as shown by in vivo and in vitro assays. In summary, HDAC6 diminishes EGFR downregulation by slowing the transport of intracellular vesicles. The inhibitory effect is removed once HDAC6 is phosphorylated on key residues. In line with these findings, two recent reports have shown that hyper-acetylation of alpha-tubulin induced by inhibition of HDAC6 increases the transport of brain derived neurotrophic factor and JNK interacting protein-1 in different cell systems. Acetylated microtubules are more efficient in recruiting motor proteins like kinesin-1 and dynein. These findings indicate that HDAC6 plays an important regulatory role in intracellular trafficking pathways. However, several outstanding issues still remain unresolved. How does acetylation of microtubules influence vesicular trafficking? In this regard, the temporal and spatial dynamics of alpha-tubulin acetylation following EGFR activation should be studied. Furthermore, whether HDAC6 affects the trafficking of other endocytic cargos and additional organelles is an interesting question to address.
Since Inhibitor of Apoptosis (IAP) proteins are frequently dysregulated in different cancer entities and contribute to apoptosis resistance, pharmacological IAP antagonists are considered to be promising agents for the future development of cancer treatment strategies. IAP antagonists are small-molecule drugs that have been designed to mimic the interaction site of IAP proteins with their endogenous inhibitor Second mitochondrial activator of caspases (SMAC). Thus, they are frequently referred to as SMAC mimetics. Treatment with SMAC mimetics engages an apoptotic program in cancers by affecting different components of the apoptotic machinery. Besides disinhibition of caspases, SMAC mimetics trigger non-canonical nuclear factor-κB (NF-κB) signaling, which induces upregulation of tumor necrosis factor (TNF) α and other NF-κB target genes. In particular, TNFα production has been closely linked to the induction of SMAC mimetic-mediated cell death. The TNFα-dependent para/autocrine loop facilitates the formation of a cytosolic complex consisting of caspase-8, Fas-associated death domain (FADD) and Receptor-interacting protein (RIP) 1, which serves as caspase-8 activation platform and ultimately triggers induction of apoptosis. In the present study, we use the small-molecule bivalent SMAC mimetic BV6 to analyze SMAC-stimulated NF-κB signaling in cancer cell lines of different entities. Interestingly, we identify two novel NF-κB-regulated factors that are both required for SMAC mimetic-induced apoptosis in a context-dependent manner. First, we show that NF-κB-dependent upregulation of death receptor 5 (DR5) can serve as an alternative mechanism of BV6-mediated cell death. We demonstrate that BV6 treatment induces NF-κB-dependent but largely TNFα -independent apoptosis in A172 glioblastoma cells. By using an unbiased whole genome expression analysis approach, we identify DR5 as a critical NF-κB target gene, which substitutes TNFα and is indispensable for BV6-initated cell death in A172 cells. Second, we demonstrate that Interferon regulatory factor (IRF) 1 is required for BV6-induced TNFα production and apoptosis. Our study provides evidence that IRF1 closely cooperates with the NF-κB network in BV6-mediated cell death and additionally alters expression of selective SMAC mimetic-induced target genes. Furthermore, we show that BV6 treatment triggers secretion of a set of proinflammatory cytokines and increases attraction of monocytes to BV6-treated tumor cells in an IRF1-dependent manner. In summary, our work supports the notion that NF-κB-regulated factors are critically required for SMAC mimetic-initiated apoptosis. We show that IRF1 is indispensable for TNFα production and cell death in BV6-sensitive cell lines and that also DR5 can serve as a proapoptotic NF-κB-controlled factor in BV6-induced apoptosis besides TNFα. Furthermore, this study contributes to an improved understanding on non-apoptotic functions of SMAC mimetics, as IRF1 additionally influences expression levels of proinflammatory cytokines and attraction of immune cells. Thus, our work provides novel insights into the regulation of SMAC mimetic-induced signaling events, which is crucial for the translation of SMAC mimetics for use in clinical application.
Die Gentherapie bietet eine interessante alternative Behandlungsoption bei der Therapie der HIV-Infektion und könnte langfristig die Standardmedikation mit antiretroviralen Substanzen ergänzen oder ersetzen. Antivirale Genprodukte, die frühe Schritte im HIV-Replikationszyklus hemmen, bevor sich das Virus in das Genom der Zielzelle integriert hat, sind dabei besonders vielversprechend. Hierzu zählen insbesondere die von der C-terminalen heptad repeat Region des HIV-Hüllglykoproteins gp41 abgeleiteten C-Peptide, die hochwirksame Inhibitoren des Viruseintritts sind. Während des HIV-Eintrittsprozesses interagieren sie mit den viralen gp41 N-Helices und verhindern somit die Ausbildung des zur Fusion von viraler und zellulärer Membran erforderlichen Sechs-Helix-Bündels. Die Sekretion antiretroviraler C-Peptide durch genmodifizierte T-Lymphozyten in vivo birgt großes therapeutisches Potential: Nach Freisetzung in den extrazellulären Raum können die Peptide nicht nur genmodifizierte sondern auch unbehandelte Nachbarzellen vor HIV-Infektion schützen (Bystander-Effekt). Somit könnte selbst mit den heute zur Verfügung stehenden Methoden, mit denen lediglich ein Teil aller potentiellen HIV-Zielzellen modifiziert werden kann, die Virusreplikation effektiv unterdrückt werden. Im Rahmen der vorliegenden Arbeit wurden daher C-Peptid-basierte in vivo sezernierte antivirale Eintrittsinhibitoren (iSAVE) für die HIV-Gentherapie entwickelt. Kurze Peptide, wie die antiviralen C-Peptide, werden von eukaryotischen Zellen aufgrund von Größenbeschränkungen beim Eintritt in den Sekretionsweg jedoch nur schlecht sezerniert. Um die effiziente Sekretion von iSAVE-Peptiden durch genmodifizierte humane Zellen zu erreichen, wurde das C-Peptid daher verlängert. Hierbei wurde das therapeutische Peptid einerseits um nicht antiviral aktive Gerüstelemente ergänzt. Andererseits wurden Concatemer-Konstrukte generiert, in denen zwei C-Peptide jeweils über einen flexiblen oder proteolytisch spaltbaren Linker verbunden sind. Die unterschiedlichen iSAVE-Peptid-Varianten wurden in vitro in transfizierten und transduzierten Zelllinien und in primären humanen T-Lymphozyten charakterisiert. Hierbei wurden Sekretionseffizienz und Prozessierung sowie antivirale Aktivität und Bystander-Inhibition der sezernierten Peptide untersucht. Dabei zeigte sich, dass die Effizienz der C-Peptidsekretion stark mit der Peptidlänge korreliert, so dass durch Sequenzverlängerungen die Sekretion deutlich gesteigert werden konnte. Darüber hinaus waren N-Glykane für die effiziente Sekretion der C-Peptide unerlässlich. Die antiretrovirale Aktivität hingegen reduzierte sich mit zunehmender Peptidlänge dramatisch und wurde auch durch N-Glykane leicht beeinträchtigt, so dass weder die durch Gerüstelemente verlängerten C-Peptide, noch die ungespaltenen C-Peptid-Concatemere antiretrovirale Wirkung zeigten. Durch die Generierung proteolytisch spaltbarer C-Peptid-Concatemere konnten die strukturellen Erfordernisse für effiziente Sekretion mit hoher inhibitorischer Aktivität vereinbart werden. Die Prozessierung der Concatemere durch die Proprotein-Convertase Furin war allerdings nicht einfach zu erreichen. Nur das Einfügen eines flexiblen Linkers mit optimierter Furinerkennungssequenz zwischen den beiden C-Peptiden erlaubte die effiziente Spaltung in monomere Peptide mit hoher antiretroviraler Aktivität. Therapeutisch wirksame Peptidkonzentrationen dieser optimierten iSAVE-Peptide wurden sowohl von transfizierten und transduzierten Zelllinien als auch von primären humanen T-Zellen sezerniert. Nach Freisetzung in den extrazellulären Raum konnten die Peptide nicht nur genmodifizierte sondern auch unbehandelte Nachbarzellen in vitro vor HIV-1 Eintritt und Infektion schützen. Die generierten iSAVE-Peptide bilden damit eine hervorragende Grundlage für die weitere präklinische und klinische Entwicklung eines neuen Gentherapieansatzes zur Behandlung der HIV-Infektion.
Resistance in glucocorticoid-induced apoptosis is associated with poor prognosis for long term survival in childhood acute lymphoblastic leukemia (ALL). As Smac mimetics have been shown to reactivate apoptosis by antagonizing Inhibitor of Apoptosis (IAP) proteins, we investigate the potential of the Smac mimetic BV6 to overcome glucocorticoid-resistance in ALL. This study shows that BV6 synergistically cooperates with glucocorticoids to trigger apoptosis and to suppress clonogenic growth of pediatric ALL cells. Of note, the BV6/glucocorticoid combination treatment also induces cell death in cells having defects in the apoptotic signaling cascade by inducing a switch from apoptotic to necroptotic cell death. The clinical relevance of our novel combination treatment is underscored by parallel experiments in primary pediatric ALL samples, in which glucocorticoids and BV6 act together to induce cell death in a synergistic manner. Importantly, the addition of BV6 enhances the anti-leukemic effects of glucocorticoids in an in vivo mouse model of pediatric ALL without causing substantial side effects, highlighting the potency of a BV6/glucocorticoid combination treatment. In contrast, BV6 does not increase cytotoxicity of glucocorticoids against several non-malignant cell types of the lympho-hematopoietic system. Furthermore, we have identified the novel underlying mechanism of BV6/glucocorticoid-induced apoptosis by showing that BV6 and glucocorticoids synergistically act together to promote assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing cell death complex. Ripoptosome assembly is critically required for BV6/Dexamethasone-induced cell death, since genetic silencing of its members, i.e. RIP1, reduces ROS production, caspase activation and most importantly cell death induction. BV6/glucocorticoid combination treatment promotes ripoptosome assembly by inhibition of both of its negative regulators, IAP proteins and cFLIP. Thus, we identify that BV6 and glucocorticoids cooperate together to reduce cIAP1, cIAP2 and XIAP protein levels and cFLIP expression. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, since blocking antibodies for TNFα, TRAIL or CD95L or genetic silencing of their corresponding receptors fail to rescue BV6/glucocorticoid-induced cell death. In summary, this study shows that the Smac mimetic BV6 sensitizes for glucocorticoid-induced apoptosis by promoting ripoptosome assembly with important implications for the treatment of childhood ALL.
Small molecule inhibitors sensitize neuroblastoma cells for chemotherapeutic drug-induced apoptosis
(2015)
Neuroblastoma (NB) is one of the most common solid extracranial pediatric tumors, deriving from undifferentiated cells of the peripheral nervous system. It accounts for approximately 10% of all childhood cancers. High stage tumors usually show poor prognosis despite aggressive treatment such as radiotherapy or chemotherapy. Therefore, it is of utmost importance to find novel treatment strategies in order to improve existing chemotherapy protocols. Combination treatment offers advantages, as chemotherapeutic drugs can be applied in low and subtoxic doses, reducing possible side-effects. Here, we report in a two-part study that small molecule inhibitors (SMI), namely BI 2536, a PLK1 inhibitor and BV6, a SMAC mimetic (SM), sensitize neuroblastoma cells for chemotherapeutic drug-induced cell death. By using i) BI 2536 in combination with vinca alkaloids and ii) BV6 in combination with either doxorubicin or vinca alkaloids, we show that cell death is synergistically enhanced compared to monotherapy. Furthermore, combination treatment significantly reduces survival of NB cells in long-term assays, compared to single treatment. We identify that vinca alkaloid/SMI combinations induce mitotic arrest, as shown by phosphorylation of histone H3, which results in the induction of intrinsic apoptosis and inhibition of CDK1 by RO-3306 could abolish these findings. Mechanistically, upon vinca alkaloid/SMI-induced mitotic arrest, anti-apoptotic BCL-2 proteins such as MCL-1, BCL-2 or BCL-XL are degraded or inactivated by phosphorylation, which induces the activation of the proapoptotic BCL-2 family proteins BAX and BAK. The importance of the mitochondrial apoptosis pathway in vinca alkaloid/SMI-induced cell death was further highlighted by the fact that ectopic expression of BCL-2 inhibits vinca alkaloid/SMI-induced DNA fragmentation and BAK- and caspase-activation. In contrast to the vinca alkaloid/SMI cotreatment, DOX/SMI (DOX/BV6)-induced apoptosis only partially involves the mitochondrial pathway. Instead, we clarify that RIP1 is required for DOX/BV6-induced apoptosis, as pharmacological and genetic inhibition of RIP1 rescues from apoptosis induction. Although it has been shown in previous studies that SM-treatment (e.g. BV6) can induce the NF-κB pathway and auto-/paracrine TNFα production through cIAP1/2 depletion, DOX/BV6-induced apoptosis is completely independent of NF-κB activation in our setting, despite fast cIAP1 depletion. This conclusion is based on the fact that inhibition of the NF-κB pathway by exogenously expressed dominant-negative IκBα as well as application of a TNFα blocking antibody does not reduce DOX/BV6-induced cell death. In summary, we unravel two new promising treatment strategies for neuroblastoma patients by using a combination treatment of two different small molecule inhibitors, combined with well-characterized chemotherapeutic agents. Furthermore we give detailed insights into cell death pathways induced by these combination treatments, in which mitochondria and RIP1 have a differential role in chemotherapeutic drug-induced apoptosis.
Die 5-Lipoxygenase (5-LOX) stellt den Startpunkt des Leukotrienstoffwechsels dar, da sie Arachidonsäure (AA) über die 5(S)-Hydroperoxy-6-trans-8,11,14-cis-eicosatetraensäure (5-HpETE) in Leukotrien A4 (LTA4) umwandelt. 5-HpETE kann zum korrespondierenden Alkohol 5(S)-Hydroxy-6-trans-8,11,14-cis-eicosatetraensäure (5-HETE) reduziert werden. LTA4 dient als Zwischenprodukt für die Synthese von LTB4 und den Cysteinyl-gebundenden LTs LTC4, LTD4 und LTE4. LTs nehmen eine wichtige Funktion in der Immunabwehr ein, sind jedoch auch an einer Vielzahl von Krankheitsgeschehen wie z. B. Asthma bronchiale, Atherosklerose und einiger Tumorarten beteiligt. Die 5-LOX teilt sich in zwei Domänen auf: der reglatorischen, N-terminalen Domäne und der katalytischen, C-terminalen Domäne. Ihre Aktivität unterliegt einer komplexen allosterischen Regulation und kinetischen Besonderheiten wie einer Substratinhibition. In vielen Fällen ist die regulatorische PLAT-(Polycystin-1, Lipoxygenase, alpha-Toxin)-Domäne involviert. Sie ist essentiell an der Bindung von Calcium, Membranen und weiterer Faktoren wie dem Coactosin-like protein (CLP) und Dicer beteiligt. Auch eine zweite Bindungsstelle für das Substrat oder einen seiner Metaboliten wird dort vermutet. Letztlich bleibt jedoch die Regulation der 5-LOX-Aktivität durch die PLAT-Domäne unzureichend geklärt. Diese Tatsache und die fortwährende Suche nach neuen Ansatzpunkten für die 5-LOX-Inhibition bilden den Hintergrund, vor dem diese Arbeit angefertigt wurde.
Das Ziel lag in der Entwicklung einer stabilen, isolierten PLAT-Domäne und deren Charakterisierung. Es stellte sich jedoch heraus, dass sich die isolierte Domäne durch eine hohe thermische Instabilität und starke Aggregationsneigung auszeichnet. Mittels Mutationsstudien auf Basis der 5-LOX AS 1-115, verbunden mit Gelfiltrationsläufen zur Analyse der Proteinaggregation, wurde schließlich ein Konstrukt entwickelt, das in Konzentrationen < 0,5 mg/ml als Monomer vorlag: die sogenannte PLAT1-115 W75G. Ein Austausch des W75 in Glycin erhöhte ebenfalls die thermische Stabilität, so dass Versuche bei 20°C durchgeführt werden konnten. Zunächst wurden jedoch die grundlegenden Eigenschaften der Mutante untersucht. Dies umfasste die Beantwortung der Frage, ob auch die PLAT1-115 W75G Calcium bindet, sowie die Aufnahme eines Circulardichroismus-(CD)-Spektrums. Der erste Aspekt konnte mit mehreren Methoden bestätigt werden. Eine Calciumzugabe zum Laufpuffer 20 mM MOPS, 50 mM KCl pH 7,4 erhöhte konzentrationsabhängig das Elutionsvolumen der PLAT1-115 W75G auf der analytischen Gelfiltrationssäule – vermutlich durch den bekannten Einfluss von Calcium auf die Hydrophobizität der PLAT-Domäne. Zusätzlich wurde die Interaktion durch differential scanning fluorimetry (DSF) und Oberflächen-Plasmonen-Resonanz-Spektroskopie (SPR) nachgewiesen. Allerdings gelang aus verschiedenen Gründen keine Quantifizierung der Bindungsaffinität. Das CD-Spektrum bestätigte die Struktur der PLAT-Domäne als sogenanntes all-beta_protein und ermöglichte die Einordnung der PLAT1-115 W75G in die Gruppe der betaII-Proteine.
Ein weiterer Fokus dieser Arbeit lag auf der vermuteten allosterischen Fettsäurebindungsstelle in der PLAT-Domäne. Es wurde versucht, die Interaktion mittels SPR nachzuweisen. Zur Vorbereitung wurde im 5-LOX-Aktivitätstest und im DSF an der isolierten Domäne ein Detergens bestimmt, das einen möglichst geringen Einfluss auf das Protein ausübt. Dabei zeigte Octyl-beta-D-glucopyranosid (beta-OG) das vorteilhafteste Profil. Auf dieser Basis wurde die kritische Mizellbildungs-Konzentration (CMC) der AA und einiger HETEs in beta-OG-haltigen Puffern bestimmt. Die SPR-Studien ergaben jedoch keine reproduzierbaren Ergebnisse. In einem weiteren Schritt wurden die Substrathemmung des Gesamtproteins 5-LOX und der Einfluss von Calcium charakterisiert. Sowohl in Gegenwart von ~ 1 mM freiem Calcium als auch von 1 mM EDTA lag mit 20 µM AA die höchste Produktbildung nach 10-minütiger Reaktion vor. Das Detergens Tween20 (T20) hob in einer Konzentration unter seiner CMC (0,001 % m/V) in Anwesenheit von Calcium die Inhibition auf. Ohne Calcium zeigte sich auch in Gegenwart von T20 die bekannte Substratinhibition der 5-LOX einschließlich ihrer Maximalaktivität bei 20 µM AA. Diese Ergebnisse deuten darauf hin, dass Calcium eine Bindung der 5-LOX an eventuell vorhandene, negativ geladene Vesikel aus AA und Detergens vermitteln und dadurch die Substratinhibition aufheben kann. In Fällen, in denen die Substratinhibition vor dem Erreichen der AA-CMC auftritt, hat Calcium folglich keinen Einfluss.
Zuletzt wurde die Interaktion der PLAT1–115 W75G mit CLP und einem C-terminalen Fragment von Dicer untersucht. Im Crosslinking ließ sich nicht auf eine Interaktion der isolierten PLAT-Domäne mit CLP schließen. Dagegen ergaben Diamid-Crosslinking-Studien, dass die isolierte PLAT-Domäne in der Lage ist, das Dicer-Fragment zu binden. Dieses Ergebnis wurde im SPR bestätigt.
Membrane proteins (MPs) constitute about 30% of the genome and are essential in many cellular processes. In particular structural characterisation of MPs is challenged by their hydrophobic nature resulting in expression difficulties and structural instability upon extraction from the membrane. Despite these challenges, progress in sample preparation and the techniques to solve MP structures has led to 281 unique MP structures as of January 2011. Through the combination of a cell-free expression system and selective labelling strategies, this thesis aimed to advance the structure determination of α-helical MPs by NMR spectroscopy and resulted in the structure determination of a seven-ransmembrane-helix protein. Results were obtained for the 5-lipoxygenase-activating protein (FLAP) and proteorhodopsin (PR). The detergent-based cell-free expression mode proved most efficient for production of both targets, but optimisation of FLAP and PR followed different routes. The presence of a retinal cofactor in PR greatly facilitated the search for an appropriate hydrophobic environment. For structural studies, NMR spectra of FLAP indicated favourable properties of the lysolipid LPPG. In contrast, PR was stable and homogenous in the short-chain lipid diC7PC. As NMR spectra of α-helical MPs are generally characterised by broad lines and signal overlap, selective labelling strategies were essential in the assignment process of both targets. For the backbone assignment of FLAP the transmembrane segment-enhanced (TMS) labelling was developed, employing the six amino acids AFGILV. These residues cluster predominantly in transmembrane helices and form long stretches allowing a large extent of backbone assignment. Besides that, the combinatorial labelling enables identification of unique pairs in the sequence based on a mixture of 15N and 1-13C-labelled amino acids. To find the optimal labelling pattern for a given primary structure, the UPLABEL algorithm has been made available and successfully applied in the backbone assignment of PR. Both selective labelling approaches greatly benefitted from the use of a cell-free expression system to reduce isotope scrambling. Additionally, the de novo structure of PR was determined with an average backbone rmsd of 1.2 Å based on TALOS-derived backbone torsion angles, intrahelical hydrogen bond restraints and distance restraints from the NOE and paramagnetic relaxation enhancement (PRE). A major bottleneck in the NMR structure determination of MPs concerns the number of long-range distances which are often limited. In PR, side chain assignment was enabled by stereo-array isotope labelling as well as selective labelling which provided 33 long-range NOEs. These NOEs stabilised the symmetry of the seven helix bundle. With a total number of 1031, the majority of long-range distances were derived from PREs. The structure of PR reveals differences to its homologues such as the absence of an anti-parallel β-sheet between helices B and C and allows conclusions towards the mechanism of colour tuning.
According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin.
PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases.
To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites.
The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent.
In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface.
In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process.
Membrane proteins are a diverse group of proteins that serve a multitude of purposes with one of the most important ones being transport. All kinds of substrates are shuffled over biological membranes with the help of dedicated proteins enabling the transport along and against a concentration gradient. Within the group of actively transporting proteins a diverse set of proteins that rely on an electrochemical gradient to facilitate transport of a substrate against its concentration gradient can be found. Those so-called secondary active
transporters are a group on integral membrane proteins ubiquitous to all cells. They allow the transport of all kinds of substrates like nutrients, ions, other metabolites and drugs over the hydrophobic barrier created by the cellular and organellar membrane. The gradients that provide the main driving force for most of the transporters are either sodium ions or protons, although transporters utilizing other ions or organic compounds are found as well. In case of exchangers two very similar substrates are transported in opposing direction over the membrane, one against its electrochemical gradient driven by the other.
Along with a structural diversity of the transporters concerning overall shape, oligomerization and number of transmembrane elements comes a mechanistic variety though still following the principle of alternating access. In humans the malfunction of secondary active transporters can lead to a physiological disorders such as epilepsy, depression or obesity.
The focus of this thesis was the structural and functional characterization of the secondary active transporter SeCitS from Salmonella enterica, a symporter of the 2-hydroxycarboxylate family. The transport of citrate as a bivalent ion is facilitated by the flux of sodium ions that have an inward-facing gradient over the inner membrane of Salmonella enterica. Transport experiments showed that the transport ratio is two sodium ions per citrate molecule, netting in an electroneutral transport. Compared to other members of the family the specificity of the transporter towards its main substrate is very high.
Structural information on the protein was initially obtained through 2D electron crystallography, which allowed the identification of the oval shaped dimer and a first hint towards a significant conformational change that the protein undergoes during its transport cycle. Using 3D crystallography, the X-ray structure of the transporter was solved. The protein crystalizes as a stable, but conformationally asymmetric dimer. As bound citrate can be readily identified in both protomers they can be assigned into an outward- and an inward-facing conformation, with the main citrate binding site in the outward-facing conformation.
One interesting feature of the crystal structure was the large surface available for multimerization, providing a platform for tight dimerization of the two protomers. On the other hand, SeCitS did not show a true cooperativity of transport. With those two aspects taken into account the question arose if any potential crosstalk between the monomers within the dimer takes place and influences transport (negative cooperativity) or the conformational distribution within the dimer (stabilization of the protein within the membrane).
The functional approach in answering this question was the use of mutated variants of the protein for cross-linking within one monomer. Two residues were chosen respectively to lock one of either conformation to be able to test for transport activity in the remaining protomer. The suitability of the residues was derived from the crystal structure (D112 – R205 to lock the inward-facing conformation and L337 – S412 for the outward-facing conformation). After initial promising results the final variants were not stable enough to be analyzed in transport assays.
To analyze the distribution of relative conformations within the dimer the protein was reconstituted into native-like lipid environment such as nanodiscs or saposin nanoparticles to be analyzed by cryo-electron microscopy. The first images were recorded and did yield promising 2D classes where the general features of the transporter were identified. Yet, an improved preparation is required to obtain a high resolution structure.
The key functional aspects of a transporter are its ability to bind and transport its substrates. In a set of experiments those features were investigated by a radioligand transport assay and by isothermal titration calorimetry (ITC). The transport properties of the protein were assessed in a filter assay using a radioactively labeled citrate as a read-out. The protein was reconstituted into proteoliposomes and subjected to different substrate conditions. Different ions were tested in its ability to drive or inhibit transport, but only sodium ions were able to drive transport and also not hindered by the presence of other ions...
Nichtribosomale Peptid Synthetasen sind Quelle für eine Vielzahl an Sekundärmetaboliten mit antibiotischer Wirkung. Jede Synthetase besteht aus einer Abfolge von Modulen, wobei jedes Modul die nötigen Domänen für den Einbau eines Bausteins in das gebildeten Peptids enthält. Ein Ansatz zur Gewinnung neuer Peptidantibiotika, die angesichts der steigenden Zahl multiresistenter Keime dringend benötigt werden, ist der Austausch von Domänen oder Modulen. Aufgrund bisher noch nicht verstandener Selektivitäten, entweder zwischen den Domänen oder zwischen einzelnen Domänen und Zwischenstufen des gebildeten Peptids, führt dieser Ansatz jedoch in der Praxis oft zu keiner oder nur geringer Ausbeute.
Ziel der vorgelegten Arbeit war es, einige dieser Selektivitäten zu untersuchen, wobei der Fokus auf Peptidyl Carrier Proteinen Domänen (PCPs) lag. An diese Domänen sind alle Intermediate während der Reifung des Peptids kovalent über einen Phosphopantethein-Kofaktor (Ppan-Arm) gebunden.
Im ersten Teil der Arbeit sollte die Struktur einer mit einem Heptapeptid beladenen PCP mittels Lösungs-Kernspinresonanzspektroskopie (NMR) bestimmt werden. Hierbei konnte die natürliche Verknüpfung zwischen Ppan-Arm und Peptid über einen Thioester nicht verwendet werden, da diese Bindung zu Hydrolyse-anfällig war. Es konnte jedoch gezeigt werden, dass die Substitution des Thioesters durch eine nicht hydrolysierbare Amidbindung keinen Einfluss auf die Struktur hat, wodurch die Strukturbestimmung möglich war. Hierbei zeigte sich, dass die Peptid-beladene PCP in der sogenannten A/H state Konformation vorliegt, wobei das an sie gebundene Peptid frei beweglich ist. Somit scheint es wahrscheinlich, dass die PCP keine Selektivität für das an sie gebundene Peptid aufweist. Dies ist ein Unterschied zu den strukturell ähnlichen Acyl Carrier Proteinen (ACPs) aus der bakteriellen Fettsäurebiosynthese, da diese eine Bindungstasche für die an sie gebundenen Fettsäuren ausbilden.
Untersuchungen der Selektivität der Kondensationsdomäne (C Domäne) für das PCP gebundene Peptid mittels NMR-Titrationen und biochemischer Analysen konnten nicht durchgeführt werden, da sich im Laufe des Projekts zeigte, dass die aus der Synthetase herausgetrennte C Domäne katalytisch nicht aktiv war. Stattdessen sollte die Kristallstruktur einer Peptid-beladenen PCP-C Bidomäne, für welche eine katalytische Aktivität bereits gezeigt worden war, gelöst werden. Da aber bereits ein signifikanter Anteil der Bidomäne während der Expression mit dem Ppan-Arm beladen wurde, war die nötige quantitative Beladung mit dem Peptid gekoppelten Ppan-Arm in vitro nicht möglich. Eine quantitative Modifizierung mit dem Ppan-Arm in vitro war hingegen erfolgreich, und die Struktur der Ppan-beladenen Bidomäne konnte gelöst werden. Aufgrund des großen Abstands zwischen den aktiven Zentren der beiden Domänen kann es sich bei der beobachteten Orientierung nicht um jene handeln, die die beiden Domänen zueinander annehmen, wenn die C Domäne das PCP-gebundene Peptid bindet.
Im zweiten Teil der Arbeit wurde die Modifizierung einer PCP durch eine Gruppe II Phosphopantetheintransferase (PPT) untersucht. PPTs katalysieren die Übertragung des Ppan Arms auf die Seitenkette eines in PCPs konservierten Serins. In dieser Magnesium-abhängigen Reaktion dient Coenzym A (CoA) als Quelle für den Ppan-Arm. Durch Mutation des konservierten Serins in der PCP zu Alanin konnte ein stabiler Komplex aus PCP und PPT in Anwesenheit von CoA und Magnesium kristallisiert und seine Struktur bestimmt werden.
In einem Strukturmodell für den PCP/PPT Komplex war eine andere Konformation für die PCP postuliert worden, als sie in der Kristallstruktur des Komplexes zu beobachten ist. Durch Strukturbestimmung der PCP mittels Lösungs-NMR und anschließender Titrationsexperimente konnte jedoch gezeigt werden, dass sowohl die freie als auch die komplexierte PCP in Lösung ebenfalls die in der Kristallstruktur beobachtete Konformation einnehmen.
Aufgrund der gelösten Kristallstruktur konnten zwei Bereiche identifiziert werden, in denen die beiden Proteine im Komplex in direktem Kontakt zueinander stehen. Der eine Bereich ist durch eine intermolekulare Wasserstoffbrücke, der andere durch hydrophobe Wechselwirkungen zwischen den Proteinen gekennzeichnet. Durch ortsspezifische Mutagenese konnten beide Wechselwirkungen gestört werden, was sich in einer Abnahme der Komplexstabilität und einer veränderten Geschwindigkeit der Übertragung des Ppan-Arms äußerte.
Die große strukturelle Ähnlichkeit zwischen dem in dieser Arbeit untersuchten Komplex aus zwei in Bacillus vorkommenden Proteinen und einem humanen ACP/PPT Komplex legt die Vermutung nahe, dass die beobachteten Wechselwirkungen in vielen Organismen konserviert sind.
Proteostasis stressors that destabilize the cellular proteome, like heat shock, trigger transcription and translational reactions leading to the accumulation of heat shock proteins, also called molecular chaperones. During stress, induction of stress response genes is prioritized so that molecular chaperones and other stress response proteins are synthesized to cope with proteome misfolding and aggregation. In order to promote the selective translation of stress-specific genes, translation of others genes that are nonessential for cell survival has to stop. Nonessential protein-coding mRNAs accumulate in the cytosol with the associated proteins to form granular structures called stress granules (SG). These membrane-less organelles are thought to be involved in cell survival, mRNA stabilization and mRNA triage. They were proposed to form via the liquid-liquid phase separation which can be triggered by the high local concentration of RNA-binding proteins. mRNAs were long thought to simply play a scaffolding role by bringing RNA-binding proteins together and allowing their concentration and local aggregation. Recently, the active role of mRNAs in the SG assembly became apparent, too. For example, the spontaneous assembly of total yeast RNA into granules was observed, and these RNA granules showed a large overlap with SG transcriptome. Furthermore, cytosolic mRNAs can be released from polyribosomes under stress and be exposed to the cytosolic contents as free mRNAs. It has been suggested that this massive increase of free mRNA in the cytosol might overload the capacities of RNA-stabilizing proteins. The remaining free mRNA molecules would then become exposed to misfolded and aggregation-prone proteins and trigger granulation.
We investigated the role of free mRNAs in different stress conditions during the early and chronic phases of stress response and explored their involvement in SGs assembly and amlyoidogenesis. We identified and studied the interactome of a free mRNA probe incubated with heat shocked cell lysate by means of quantitative mass spectrometry. Proteomics analysis allowed us to identify 79 interactors of free mRNA. Among these interactors, we focused on the translation initiation factor eIF2α and on the RNA methyltransferase TRMT6/61A. Both interactions were verified biochemically, which confirmed that the association is enhanced in heat shocked lysate. In vitro reconstitution showed that free mRNA and TRMT6 interact directly. Ex vivo pulldowns revealed that eIF2α and TRMT6/61A interact under stress conditions and that this interaction is RNA-dependent.
TRMT6/61A is a tRNA methytransferase responsible for the methylation of the adenosine 58 at the position 1 producing m1A. However, also mRNAs have been recently found to be methylated by TRMT6/61A. Our bioinformatics analyses revealed that significantly more mRNAs enriched in SG contain the motif for methylation than SG-depleted mRNAs. We hypothesized that m1A methylation of mRNAs could constitute a tag for the mRNAs targeting to SGs. TRMT61A knock-down (KD) cell lines were generated using the CRISPR-Cas9 technique. In TRMT61A KD cells, m1A was significantly reduced on mRNAs, which correlated with an increased sensitivity of the cells to proteostasis stress. KD cells also showed defects in SG assembly. In heat shocked cells, an m1A motif-containing mRNA recovered better after returning to normal temperature than a control mRNA with mutated motif. In addition, we could isolate SGs and analyze their m1A and m6A content by mass spectrometry. While m6A content in SG mRNAs was very similar to cytosolic mRNAs, m1A was almost 8 times enriched in SGs. Thus, we could confirm experimentally the results of the bioinformatics analysis and directly support the hypothesis that m1A is a tag to direct mRNAs for sequestration. Finally, we compared amyloidogenesis in wild-type and TRMT61A KD cell lines. Cells with reduced levels of TRMT61A demonstrated an increased accumulation of transfected Aβ and an impaired aggregate clearance. Various assays led us to conclude that the lack of m1A deposition on mRNAs enhanced RNA co-aggregation with amyloids.
Based on our results, we propose a model explaining the fate of free mRNA during proteostasis stress. Upon polysome disassembly, free mRNA is released and becomes free to interact with other proteins, including the methyltransferase TRMT6/61A. TRMT6/61A methylates the freed mRNAs containing the cognate motif. The m1A tag then targets mRNAs to SGs promoting sequestration. Upon stress release, SGs disassemble, thus releasing rescued mRNAs which could now reenter translation and support cell recovery. On the other hand, non-sequestered mRNAs increasingly co-aggregate with aggregating proteins. Thus, deficiency of the N1-adenine methylation of mRNAs due to the lack of TRMT6/61A increases the amount of unpacked mRNAs. The deposition of m1A on mRNAs could then be a way to protect them during exposure to stress, to limit their co-aggregation with misfolded proteins and to allow a faster recovery upon stress release.
Employing NMR spectroscopy, it is not only possible to calculate the three dimensional structures of single proteins, but also to study dynamics and conformational changes of protein-complexes. In fact that is an important aspect, since the protein function depends on dynamics and interactions with other molecules. Therefore the study of protein-protein interactions is of highest importance for a better understanding of biological processes. Based on NMR methods, in this thesis we were able to determine protein-protein interactions within the enterobacterial Rcs signalling complex which is regulated via a phosphorelay. Originally identified as regulator of capsule synthesis, the Rcs phosphorelay is now considered to be implicated in stress response caused by disturbances in the peptidoglycan layer. Beyond that the Rcs system is involved in multiplex transcriptional networks including cell division, motility, biofilm formation and virulence. Because of such global nature and its extraordinary structural organisation involving membrane integrated sensor proteins (RcsC, RcsD), coactivators (RcsF, RcsA) and a transcription factor (RcsB), the Rcs system is one of the most remarkable phosphorelays in the family of enterobacteriacaea. During the complex phosphotransfer the histidine phosphotransferase (HPt) domain of the intermediary RcsD protein mediates the phosphotransfer between RcsC and RcsB, and probably modulates the phosphorylation state of the response regulator RcsB. Therefore the present work has been focused on the interface between RcsD and RcsB in more detail. In the first part of the thesis a new domain within the RcsD protein has been identified and structurally analysed by liquid NMR spectroscopy. RcsD is an inner membrane bound hybrid sensor like-kinase composed of a periplasmic sensor domain and a cytoplasmic portion. The cytoplasmic part contains the histidine like-kinase (HK) domain and the histidine phosphotransferase (HPt) domain. By analysis of the secondary structure in more detail, it was shown here that the two domains are intermitted by an additional 13.3 kDa domain. Corresponding to the position of the ABL (α−β−loop) domain of RcsC, located C-terminal to the RcsC-HK domain, the new identified domain was named RcsD-ABL. The central structural element of RcsD-ABL is a β-sheet composed of six strands with a β1−β2−β3−β4−β6−β5 topology and surrounded by two α-helices α1 and α2. In the second part of the thesis, RcsD-ABL is identified as a binding domain for the response regulator RcsB by NMR titration experiments. Such a binding domain for a response regulator has so far only been described for the histidine kinase CheA. In reportergene assays with β-galactosidase and ONPG as substrate it was shown that overexpression of RcsD-ABL in high amounts inhibited binding of RcsB to its target promoter. The β-galactosidase activity was reduced by 80 % with respect to cells carrying no plasmid encoding RcsD-ABL. The mapping of the binding interface was successfully achieved by chemical shift perturbations, a fast mapping protocol and selective labelling. It was shown that the interaction between RcsD-ABL and RcsB takes place via a binding interface comprising mainly the two α-helices of RcsD-ABL and the α-helices α7, α8 and α10 in the effector domain of RcsB. In the third part of the thesis, the interaction of RcsB with RcsD-ABL was related to that with RcsD-HPt. Using NMR titration experiments and ITC measurements, a comparison of the binding constants (Kd) of RcsB interacting either with the isolated RcsD-ABL (2 PM) or the isolated RcsDHPt domain (40 PM) revealed a higher affinity of RcsD-ABL to RcsB. A conjugate of RcsD-ABL-HPt interacting with RcsB decreased the Kd in the one-site fitting mode to 10 PM. However, the two-site fitting mode applied for RcsD-ABL-HPt/RcsB interaction resulted in a Kd (RcsD-ABL) of 2 PM and a Kd (RcsD-HPt) of 8 PM, indicating that RcsD-ABL enhances the binding of RcsD-HPt to RcsB. In the last part of the thesis, it was partly possible together with the data obtained from NMR titration experiments, PRE measurements and a HADDOCK protocol to develop a geometrical model for the interaction of RcsD with RcsB. In this model the receiver domain of RcsB interacts with the RcsD-HPt domain and the RcsB effector domain interacts with the RcsD-ABL domain. These results lead to surprising insights on the regulation of phosphorelays, since normally the effector domain binds to DNA. Here the effector domain is recognized by the newly identified RcsD-ABL domain. Prospectively, further investigations of phosphorylation affects and mutational studies will be of great interest.
The focus of this thesis is the integral membrane protein Escherichia coli diacylglycerol kinase (DGK). It is located within the inner membrane, where it catalyzes the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatic acid (PA). DGK is a unique enzyme, which does not share any sequence homology with typical kinases. In spite of its small size, it exhibits a notable complexity in structure and function. The aim of this thesis is the investigation of DGK’s structure and function at an atomic level directly within the native-like lipid bilayer using MAS NMR. This way, a deeper understanding of DGK’s catalytic mechanism should be obtained.
First, the preparation of DGK was optimized, leading to a sample, which provides well-resolved MAS NMR spectra. The high quality MAS NMR spectra formed the foundation for the second step, the resonance assignment of DGK’s backbone and side chains. The assignment was performed at high magnetic field (1H frequency 850 MHz). The sequential assignment of immobile domains was carried out using dipolar coupling based 3D experiments, NCACX, NCOCX and CONCA. The measurement time could be reduced by paramagnetic doping with Gd3+-DOTA in combination with an E-free probehead. The sequential assignment was mainly performed using a uniformly labelled sample (U-13C,15N-DGK). Residual ambiguities could be resolved by reverse labelling (U-13C,15N-DGK-I,L,V). Resonances could be assigned for 82% of the residues, from which 74% were completely assigned. For validation, ssFLYA was applied, which is a generally applicable algorithm for the automatic assignment of protein solid state NMR spectra. Its principal applicability for demanding systems as membrane proteins could be proven for the first time. Overall, ~90% of the manually obtained assignments could be confirmed by ssFLYA. For the completion of DGK’s assignment, J-coupling based 2D experiments, 1H-13C/15N HETCOR and 13C-13C TOBSY, were carried out to detect highly mobile residues. This way, residues of the two termini and the cytosolic loop, which were not detectable by dipolar coupling based experiments, could be assigned tentatively. Whereupon, peaks for arginine and lysine were assigned unambiguously to Arg9 and Lys12. Overall, ~84% of the residues could be assigned by the applied NMR strategy. Furthermore, a secondary structure analysis was carried out. It showed substantial similarities between wild-type DGK, its thermostable mutant determined both by MAS NMR and the crystal structure of wtDGK. However, there are few differences around the flexible regions most likely caused by the high mobility of these regions. During the assignment procedure, no systematic peak doublets or triplets were detected, indicating that the DGK trimer adopts a symmetric conformation. This is in contrast to the X-ray structure, which shows asymmetries between the three subunits. Especially, crystal packing may be a potential source for these structural asymmetries.
On the basis of the nearly complete assignment of DGK, the apo state was compared with the substrate bound states. Perturbations in peak position and intensity of the substrate bound states were analysed for all assigned residues in 3D and 2D spectra. The nucleotide-bound state was emulated by adenylylmethylenediphosphonate (AMP-PCP), a non-hydrolysable ATP analogue, whereas the DAG-bound state was mimicked by 1,2-dioctanoyl-sn-glycerol (DOG, chain length n = 8). Upon nucleotide binding, extensive chemical shift perturbations could be observed. These data provide evidence for a symmetric DGK trimer with all of its three active sites concurrently occupied. Additionally, it could be demonstrated that the nucleotide substrate induces a substantial conformational change. This most likely supports the enzyme in binding of the lipid substrate, indicating positive heteroallostery. In contrast, the overall alterations caused by DOG are very minor. They involve mainly changes in peak intensities. For DGK bound with either AMP-PCP+DOG or only AMP-PCP, a similar spectral fingerprint was observed. This implies that binding of the nucleotide seems to set the enzyme into a catalytic active state, triggering the actual phosphoryl transfer reaction.
The investigation of DGK’s remarkable stability and the cross-talk between its subunits forms the last part of this thesis. This demands for the identification of key intra- and interprotomer contacts, which are of structural or functional importance. For this purpose, 13C-13C DARR and 2D NCOCX spectra with long mixing times were recorded using high field MAS NMR. Additionally, DNP-enhanced 13C−15N TEDOR experiments were conducted on mixed labelled DGK trimers to enable the visualization of interprotomer contacts. With the applied NMR strategy, intra- (Arg32 - Trp25/ Glu28/ Ala29 and Trp112 - Ser61) and interprotomer (ArgNn,e - AspCg/ GluCd/ AsnCg) long-range interactions could be identified.
Ubiquitin is a highly conserved protein involved in several cellular processes like protein degradation, endocytosis, signal transduction and DNA repair. The discovery of ubiquitin-like proteins (UBL) and ubiquitin-like domains (ULD) increases the number of regulation pathways where the property of the ubiquitin-fold is profitable.
Autophagy is the catabolic pathway used in cells to deliver cytosolic components and dysfunctional organelles to the lysosome for degradation. MAP1LC3 proteins are ubiquitin-like proteins involved in one hand for the expansion of the autophagosome, which sequesters cytosolic substrates. In the other hand, these proteins (LC3- and GABARAP- subfamilies) bind to autophagic receptors linked to polyubiquitinated proteins aggregates. For this project, the 3D structure of the GABARAPL-1/NBR1-LIR complex was determined and confirmed that GABARAPL-1 belongs to the MAP1LC3 proteins family, structurally characterized by an ubiquitin-fold, consisting of a central beta-sheet formed by four beta-strands and two alpha-helices on one side of the beta-sheet, preceded N terminally by two alpha-helices, resulting in the formation of two hydrophobic pockets, hp1 and hp2. The autophagic receptor NBR1 interacts with GABARAPL-1 through the hp1 and hp2 with its LIR motif taking an extended beta conformation upon binding, forming an intermolecular beta-sheet with the second beta-strand of GABARAPL 1. This LC3- interacting region (LIR) consists of an Theta XX Gamma sequence preceded by acidic amino acids, with Theta and Gamma represented by any aromatic and hydrophobic residues, respectively. Interaction studies of the LIR domains of p62, Nix and NBR1 with different members of the MAP1LC3 proteins family indicate that the presence of a tryptophan in the LIR motif increases the binding affinity. Substitution to other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation, suggesting that effector proteins can interact with a wide variety of different sequences with similar and moderate binding affinities.
Additionally to be present in proteins dealing with protein folding and degradation, ubiquitin-like domain were found protein involved in the regulation of signal transduction like TBK1, a serine/threonine kinase responsible for induction of immune response. In this second project, based on the NMR chemical shifts of the TBK1 domain contained between amino acids 302 and 383, secondary structure prediction programs (TALOS and CSI) confirmed the presence of an Ubiquitin-like domain in TBK1 by identifying one alpha-helix and four beta-strands sequentially aligned like following beta-beta-alpha-beta-beta. This alignment corresponds perfectly with the secondary structure elements of Ubiquitin and proved that TBK1_ULD belongs to the UBL protein superfamily. The similarity to ubiquitin was even bigger by the presence in addition of a small beta-strand and a short helix, which are observed as the beta 5-strand and a 310-helix in Ubiquitin, respectively. The first attempts on the 3D structure determination confirmed the Ub-fold but due to the lack of assignment in TBK1_ULD, only a structure based on ubiquitin as a model was determined. Interaction studies of TBK1_ULD with the IAD-SRR domain of IRF3 showed that both side of the molecule seems involved and that the TBK1/IRF3 interaction is more complex than a one to one binding process. Unfortunately, the instability of TBK1_ULD associated to the difficulty in the purification of IAD-SRR did not allow to further study this interaction more precisely.
Finally, to overcome the difficulty encountered in NMR experiments because of low expression and/or poor solubility, an expression vector using the intrinsic property of ubiquitin was designed. Fused to proteins or peptides targets, this construct produced proteins and peptides in a larger amount than with traditional expression vectors and also with a less cost than chemical synthesis for pure labeled peptides for NMR structural studies. The presence of a hexa histidine tag was useful for the isolation and the purification of the constructs. The existence of a TEV cleavage site was created to keep the possibility of releasing the ubiquitin moiety from the expressed protein or peptide. Moreover, the ubiquitin-tag could also still be attached to the protein/peptide of interest when biophysical methods like NMR, ITC or CD spectroscopy are applied, providing the same results than for the protein/peptide moiety alone.
Electron microscopy (EM) demarcates itself from other structural biology techniques by its applicability to a large range of biological objects that spans from whole cells to individual macromolecules. In single-particle cryo-EM, frozen-hydrated samples, prepared by vitrification with liquid ethane, retain macromolecules in a medium that approximates their natural aqueous environment and that, in this way, preserves high-resolution structural information. Nonetheless, the sensitivity of biological specimens to the high-energy electron beam introduces restrictions on the total dose that can be used during imaging while avoiding significant radiation damage. Consequently, the signal-to-noise ratio attained in each individual image is very low, and structures with high-resolution detail must be recovered by averaging thousands of projections in random orientations. This is achieved through the use of image processing algorithms capable of aligning and classifying particle images through the evaluation of cross-correlation functions between each particle and a reference.
In recent years, several innovations took place in the field of single-particle cryo-EM, among which the development of direct electron detectors must be highlighted. Direct electron detectors have a better detective quantum efficiency (DQE) than both photographic film and CCD cameras, and offer a fast readout, compatible with the acquisition of movie stacks. Additionally, new image processing software has become available, with more sophisticated algorithms and designed to take advantage of the specific characteristics of the movies produced with direct electron detectors. These technological advances in both hardware and software catalyzed a revolution in single-particle cryo-EM, which is now routinely used for the determination of near-atomic structures. As a result, the range of macromolecules accessible to cryo-EM has increased drastically, as targets that were unsuitable before for imaging due to their small dimensions can now be adequately visualized and refined to high-resolution.
During my doctoral work, I have used single-particle cryo-EM to structurally characterize challenging membrane proteins, with a strong emphasis on protein complexes from aerobic respiratory chains. In chapter I of this thesis, I present my results on the bovine respirasome, a mitochondrial supercomplex composed of complexes I, III and IV. Chapter II is dedicated to the analysis of the structure of alternative complex III (ACIII) from Rhodothermus marinus, a bacterial quinol:cytochrome c/HiPIP oxidoreductase unrelated to the canonical cytochrome bc1 complex (complex III). In addition, in chapter III I describe the structure of KimA, a high-affinity potassium transporter that drives the transport of its substrate by using the energy stored in the form of a proton gradient. These three membrane proteins, with molecular weights ranging from 140 kDa to 1.7 MDa, illustrate the possibilities and limitations faced in single-particle cryo-EM.
The aerobic respiratory chain is responsible for the generation of a transmembrane difference of electrochemical potential that is then used by ATP synthase for the production of ATP or for driving solute transport over the membrane. They catalyze the transfer of electrons from a substrate, such as NADH or succinate, to molecular oxygen and use the chemical energy released in these redox reactions to drive the translocation of protons, or in some cases sodium ions, to the intermembrane space in mitochondria or the periplasm in bacteria.
In mitochondria, the respiratory chain is composed of four complexes: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). While it was for a long time believed that these complexes existed as single entities in the membrane, the use of milder procedures for protein purification and analysis revealed that respiratory complexes associate into well-ordered structures, known as supercomplexes. These have been proposed to offer different structural and functional advantages that are still controversial, including substrate channeling, stabilization of individual complexes and reduction of reactive oxygen species (ROS) production. The most thoroughly studied respiratory supercomplex has been the respirasome, conserved in higher eukaryotes and composed of one copy of complex I, a complex III dimer and one complex IV. By single-particle cryo-EM analysis, I retrieved a 9 Å map of the respirasome from Bos taurus, which allowed the accurate docking of atomic models of the three component complexes. The structure shows that complex III associates to the concave side of the membrane arm of complex I, while complex IV is located between the end of the complex I hydrophobic arm and complex III. Several defined protein-protein contacts are observed between the component complexes, which are mediated predominantly by supernumerary subunits and close to the membrane surfaces. The interactions established between complex I and complex III are extensive and may support the argument that the association of complex I into supercomplexes is required for the stabilization or even the biogenesis of this complex.
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Structural characterization of stressosome complexes by single-particle cryo-electron microscopy
(2015)
The stressosome is a Mega Dalton macromolecular complex involved in stress adaptation in bacteria. Stressosomes are considered as stress signaling hubs. They are able to perceive a variety of different stress stimuli and transduce them into one single cellular answer, which is the initialization of a transcriptional up-regulation of hundreds of different genes encoding for universal but also very specific stress response proteins.
The stressosome of Bacillus subtilis became a prime example for this intriguing stress-triggered transcriptional regulation when its architecture was determined by Single-particle cryo-electron microscopy (cryo-EM) in 2008. In Gram-positive Bacillus species, the stressosome complex senses changes in salt concentration, ethanol content, blue-light, heat or acid stress contributing to the general stress response by activation of the alternative σB factor. σB is a transcriptional promoter that initiates the transcription of over 150 general stress genes, e.g., genes that encode osmolyte transporters to counteract osmotic and chill stress. The B. subtilis stressosome (stressosome_Bc) is composed of multiple copies of the 3 proteins: RsbR, RsbS and RsbT. These three Rsb proteins (Regulator of Sigma B) are found clustered in one operon forming the conserved RST module. RsbS and RsbR are scaffold proteins comprising a STAS domain, respectively. Because these domains are dominantly associated to sulfate transporters and anti-sigma antagonist they were named STAS domains, however, they were also identified in other sensor proteins. In the stressosome they form the internal ball-shaped core, while the N-terminal globin-fold sensor domain of RsbR, protruding to the outside, facilitates stress sensing. It is assumed that the stress signal is transduced to the stressosome core via the STAS domain resulting in conformational changes of the core. These changes affect the binding of the third protein, RsbT, a serin-threonine kinase. As a direct consequence of stress sensing the RsbT kinase is released from the complex to start an activation cascade involving the stepwise activation of RsbU, V, W, and X, which are all part of the same operon, and finally of σB. In Bacillus species, several RsbR orthologs were identified varying mainly in the sequence of the N-terminal sensor domains. It is assumed that the stressosome_Bc assembles with a still unknown combination of RsbR orthologs allowing for the broad spectrum of stress stimuli that can be processed in vivo. The pathogenic bacteria Listeria monocytogenes is a close relative of Bacillus. Its potent stress response allows Listeria to survive the harsh environmental conditions during host infection and therefore the stress regulation machinery is contributing heavily to the virulence of this pathogen. In Listeria the Rsb operon is conserved and highly homologous to the Bacillus one. In the frame of this thesis, the in vitro assembly of Listeria innocua stressosomes was shown for the first time by Single-particle (SP) negative stain EM. Moreover, binding of Listeria RsbT to the assembled RsbR-RsbS complex was demonstrated biochemically.
Despite the conservation of the RST-module the entire Rsb operon is not conserved in the bacterial kingdom suggesting that signal transduction and regulation of gene expression might occur by very different mechanisms in stressosomes of different species. We have focused here on a stressosome type from the Gram-negative pathogen Vibrio vulnificus that is quite distinct from the Bacillus ones with respect to (1) the missing conservation of the Rsb operon, (2) the role of RsbT, (3) the activation of a different transcriptional promoter, and (4) the absence of additional RsbR orthologs. Interestingly, there is only one RsbR protein encoded in the genome. This one contains a Haem-group in its N-terminal domain being oxygen sensitive. It is assumed that the Vibrio stressosome perceive only oxidative stress and that regulation occurs via a diguanylate cyclase with a GAF domain that synthesizes the second messenger c-di-GMP from GTP.
We have started a structure determination of the Vibrio vulnificus stressosome by SP cryo-EM to elucidate the differences in the molecular mechanism of stress sensing in divers stressosome types. A 3D map of the oxidized (activated) Vibrio vulnificus stressosome was determined to 7.6 Å resolution revealing an increased flexibility of both the core and the N-terminal sensor domains in comparison to the Bacillus stressosome suggesting that our structure has trapped for the first time an active state of a stressosome complex. A 3D map of the stressosome core to 7 Å resolution allowed fitting of a homology model of the Vibrio stressosome based on the Bacillus stressosome as template. The conformational changes could be attributed to the entire core, which was confirmed by MD simulations.
The endoplasmic-reticulum-associated protein degradation pathway ensures quality control of newly synthesized soluble and membrane proteins of the secretory pathway. Proteins failing to fold into their native structure are processed in a multistep process and finally ubiquitinated and degraded by the proteasome in order to protect the cell from proteotoxic stress. My thesis covers structural as well as functional studies of various protein components that constitute the protein complexes that are responsible for this process.
One sub-project addressed the mechanism of glycan recognition by Yos9 as part of the ERAD substrate selection. NMR solution structures of the mannose-6-phosphate homology (MRH) domain of Yos9 both in a free and glycan bound conformation reveal a gripping movement of loop regions upon binding of correctly processed glycan structures.
The main projects focused on revealing the mechanism of efficient ubiquitin chain assembly by the ERAD ubiquitination machinery. This included the investigation of the role of the ERAD components Cue1 and Ubc7 in processive ubiquitin chain formation, how ubiquitin chain conformations change during elongation, how the conformation of a chain is impacted by interacting proteins and finally understand the activity regulation of the ERAD E2 enzyme Ubc7 by its cognate RING E3 ligases. Nuclear magnetic resonance (NMR) analysis and fluorescence-based ubiquitination assays show that the CUE domain of Cue1 contributes with its proximal binding preference as well as with its position dependent accelerating effect to efficient ubiquitin chain formation. This is required to efficiently drive degradation of substrates. Specific ubiquitin binding events dictate and coordinate the spatial arrangement of the E2 enzyme relative to the distal tip of a chain. This process can be further accelerated by RING E3 ligases that promote Ubc7 activity by more than ~20 fold via inducing allosteric changes around the catalytic cysteine. My results additionally suggest a model where Ubc7 dimerization results in proximity induced activation of the E2. This data ensures rapid diubiquitin formation that is followed by a CUE domain assisted chain elongation mechanism where Cue1 acts in an E4 like fashion.
How ubiquitin binding events can modulate the conformations of a ubiquitin chain were investigated by pulsed electron-electron double resonance (PELDOR) spectroscopy combined with molecular modeling. This shows that K48-linked diubiquitin samples a broad conformational space which can be modulated in distinct ways. The CUE domain of Cue1 uses conformational selection of pre-populated open conformations to support ubiquitin chain elongation. In contrast, deubiquitinating enzymes shift the conformational distribution to weakly or even non-populated conformations to allow cleavage of the isopeptide bond that connects adjacent ubiquitins. Ubiquitin chain elongation increases the sampled conformational space and suggests that this high conformational flexibility might contribute to efficient proteasomal recognition.
In Reaktion auf zellulären Stress wie etwa Schädigungen der DNA oder die vermehrte Aktivität von Onkogenen aktivieren vorgeschaltete Signalkaskaden den Transkriptionsfaktor (TF) p53. Dieser kann über die Aktivierung der Expression von Zielgenen wiederum die Zellteilung stoppen, die Reparatur von DNA Schäden initiieren oder in schweren Fällen die Eliminierung der Zelle durch Apoptose einleiten. Ist p53 durch Mutationen deaktiviert, können sich entartete somatische Zellen vermehren und in der Folge Krebs entstehen.
In Wirbeltieren finden sich neben p53 mit p63 und p73 zwei weitere TFs, welche während der Evolution aus dem gleichen gemeinsamen Vorläufer durch Genduplikationen hervorgegangen sind. Die drei TFs sind modular aufgebaut und alle Isoformen verfügen jeweils minimal über eine DNA Bindungsdomäne (DBD) und eine Tetramerisierungsdomäne (TD). Werden die p53 ähnlichen TFs aktiviert, lagern sie sich über die TD vermittelt zu Tetrameren zusammen, wodurch ihre DBDs kooperativ an DNA Sequenzmotive binden können. Die DBD ist auch über große phylogenetische Abstände hinweg hoch konserviert, wodurch bereits gezeigt werden konnte, dass auch primitive vielzellige Tiere bereits Homologe dieser TF Familie besitzen. Im Vergleich zur DBD variiert die Proteinsequenz der TD deutlich stärker, was andeutet, dass deren Struktur im Laufe der Evolution erhebliche Veränderungen durchlaufen hat. Diese Veränderungen aufzuklären ist das übergeordnete Forschungsvorhaben zu dem diese Dissertationsschrift beiträgt.
Ciona intestinalis (C.int.) ist eine Spezies aus dem Unterstamm der Manteltiere. Diese sind die engsten lebenden Verwandten der Wirbeltiere und C.int. ist ein populärer Modelorganismus für die Erforschung der Embryonalentwicklung. Sein Genom kodiert für zwei p53 ähnliche TFs, welche mit p53/p73-a und p53/p73-b bezeichnet werden. Die Struktur ihrer TDs wurde im Rahmen der vorliegenden Arbeit mittels Kernspinresonanz (NMR) Spektroskopie untersucht.
Die TD von menschlichem p53 (hp53) ist ein Dimer aus Dimeren. Jedes Monomer formt einen beta-Strang und eine alpha-Helix. Im primären Dimer lagern diese sich so zusammen, dass ein beta-Faltblatt entsteht und die alpha-Helices mit entgegen gesetzter Orientierung der Länge nach aneinander packen. Zwei dieser Dimer lagern sich dann so zum Tetramer zusammen, dass zwischen pol-ständigen beta-Faltblättern ein Bündel aus vier Helices entsteht. Dieses Motiv ist auch in den TDs der Ciona Proteine hochkonserviert und wird im Folgenden als Kern?TD bezeichnet. In den TDs von menschlichem p63 und p73 (hp63 und hp73) verfügt jedes Monomer an seinem C-terminus noch über eine zweite Helix. Die zweiten Helices eines jeden Dimers greifen wie Klammern um das jeweils andere primäre Dimer und stabilisieren so das Tetramer. Entscheidend für die stabile Anbindung an die Kern?TD ist dabei ein charakteristisches Tyrosin-Arginin (YR) Motiv in der zweiten Helix, welches sich auch in der Sequenz der TD von C.int. p53/p73-a wiederfindet. Analysen der Sekundärstruktur auf Basis von NMR Experimenten ergaben jedoch, dass die TD von C.int. p53/p73-a bei 25°C keine zweite Helix ausbildet. Mit Hilfe von chimären TD Peptiden, in denen Teile der Ciona Sequenz gegen die entsprechenden Abschnitte von hp73 ausgetauscht wurden, konnte gezeigt werden, dass die Kern TD von C.int. p53/p73-a fähig ist eine zweite Helix zu stabilisieren und hierfür neben dem YR Motiv auch der Sequenzabschnitt zwischen erster und zweiter Helix entscheidend ist. Stabilisierende Substitutionen in diesem Bereich bewirkten ebenso wie ein Absenken der Temperatur die Ausbildung einer zweiten Helix, welche jedoch im Gegensatz zu jener in hp73 nur transient faltet und auch nicht essentiell für die Bildung des Tetramers ist, wohl aber dessen Stabilität erhöht.
Spezifisch in der Entwicklungslinie von Ciona kam es dazu, dass eine, für eine entsprechende Vorläuferversion von C.int. p53/p73-a kodierende, mRNA spontan zurück in DNA übersetzt und ins Genom eingefügt wurde. Die durch diese Retrotransposition erzeugte neue Genkopie C.int. p53/p73-b muss demnach ursprünglich einmal für die gleiche Proteinsequenz kodiert haben, innerhalb der TD finden sich konservierte Reste jedoch nur im Bereich der Kern TD.
Von der TD von C.int. p53/p73-b wurde die molekulare Struktur in freier Lösung mittels NMR ermittelt. Diese zeigte, dass interessanterweise in der TD von C.int. p53/p73-b jedes Monomer am C-terminus eine stabil gefaltete, zweite Helix besitzt. Obwohl diese zweite Helix sich aus einer Sequenz faltet, die keinerlei Sequenzhomologie zu homologen Proteinen aus Wirbeltieren aufweist, lagert sie sich in einer Position auf die Kern TD, welche der in hp73 sehr nahe kommt. Da die primären Dimere der Kern TD aber anders als in hp63 und hp73 durch Salzbrücken miteinander verbunden sind, ist die zweite Helix jedoch nicht essentiell, um das Tetramer zu stabilisieren. Vermutlich kommt der zweiten Helix von C.int. p53/p73-b vielmehr u.a. die Aufgabe zu die Bildung von Heterotetrameren aus C.int. p53/p73-a und –b zu unterbinden.
Zusammengenommen zeigen die Ergebnisse, dass die Architektur der TD mit zweiter Helix bereits der Prototyp für die TDs aller p53 ähnlichen Proteine der Wirbel- und Manteltiere war und die als eine Art Klammer das Tetramer stabilisierende zweite Helix sich nicht erst während der Evolution der Wirbeltiere entwickelt hat.