@phdthesis{Plank2019, author = {Plank, Christina}, title = {Untersuchung von Dihydroisochinolinonderivaten als m{\"o}gliche Inhibitoren von Hsc70}, doi = {10.25972/OPUS-16265}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162655}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Einhergehend mit einer steigenden Lebenserwartung nimmt auch die Zahl der am Multiplen Myelom Erkrankten zu. Bis dato gibt es nur wenige Therapieans{\"a}tze dieser selten vorkommenden Blutkrebserkrankung. Im Zusammenhang mit der Entstehung des Multiplen Myeloms stehen vor allem zwei bedeutende Hitzeschockproteine: Hsp90 und Hsp70. Beide haben die Aufgabe, Zellen vor Apoptose zu sch{\"u}tzen. In proliferierenden Plasmazellen ist eine {\"U}berexpression an Hsp90 zu beobachten. Entwickelte Inhibitoren f{\"u}hrten zwar zu einer verminderten Hsp90-Aktivit{\"a}t, allerdings wurde diese durch eine vermehrte Expression von Hsp70 kompensiert, weshalb Myelomzellen weiterhin proliferierten. Aus diesem Grund bietet sich Hsp70 als weiterer Angriffspunkt in der Therapierung des Multiplen Myeloms an. Die bislang entwickelten Inhibitoren binden entweder an die Nukleotid- oder Substratbindedom{\"a}ne. Da beide Stellen unspezifisch sind, wurden durch virtuelles Screening potenzielle Inhibitoren f{\"u}r Hsp70 identifiziert, welche in vitro und in vivo tats{\"a}chlich Effekte hinsichtlich der Herunterregulierung von Hsp70 zeigten. Ob die entwickelten Substanzen jedoch direkt an Hsp70 binden, war die Fragestellung der vorliegenden Arbeit. In dieser Arbeit wurde untersucht, inwiefern die entwickelten Inhibitoren an Hsp70 binden und dieses inhibieren. Die humane Hsp70-Familie besitzt sechzehn Mitglieder, die alle {\"a}hnliche Aufgaben und Strukturmerkmale aufweisen. F{\"u}r die durchgef{\"u}hrten Versuche wurde die Hsp70-Isoform Hsc70 verwendet. In einem Protein-Ligand-Assay konnte gezeigt werden, dass die meisten Verbindungen durch Aggregatbildung zu einer Inhibition von Hsc70 f{\"u}hrten. Durch Zugabe von Detergenz konnten die gebildeten Aggregate aufgebrochen und so der Inhibitionseffekt aufgehoben bzw. deutlich reduziert werden. Damit konnte gezeigt werden, dass die in Zell- und Mausversuchen beobachteten Effekte vermutlich nicht auf eine direkte Inhibition von Hsc70 zur{\"u}ckzuf{\"u}hren sind. Ob diese Effekte nun ebenfalls auf Aggregatbildung beruhen oder aber ein anderes Protein als das vermutete Hsc70 inhibiert wird, was {\"u}ber eine Signalkaskade zur Inhibition von Hsc70 f{\"u}hrt, w{\"a}re eine interessante Fragestellung f{\"u}r weitere Untersuchungen. Da sowohl in NMR-Versuchen als auch dem durchgef{\"u}hrten Protein-Ligand-Assay gezeigt werden konnte, dass die vormals als potenzielle Inhibitoren entwickelten Verbindungen nur schwach aktiv sind, wurde durch Fragment-basierte Ans{\"a}tze eine andere Bindestelle f{\"u}r m{\"o}gliche Inhibitoren identifiziert. Hierbei konnte N-Acetyl-D-Glucosamin in der Nukleotidbindedom{\"a}ne von Hsc70 detektiert werden. Hieraus k{\"o}nnten sich neue Ans{\"a}tze zur Entwicklung neuartiger in silico entwickelter Hsc70-Inhibitoren ergeben. Ausgangspunkt f{\"u}r die Docking-Studien zur Entwicklung neuer Hsp70-Inhibitoren war die Kristallstruktur von bHsc70 ED 1-554, einer trunkierten Doppelmutante des nativen Hsc70. Bis dato ist diese 554 Aminos{\"a}uren umfassende Mutante die einzige Hsc70-Variante von der die Zweidom{\"a}nenstruktur kristallisiert werden konnte. F{\"u}r dieses Konstrukt wurde zun{\"a}chst ein optimiertes Aufreinigungsprotokoll entwickelt, um dann Kristallisationsversuche mit ausgew{\"a}hlten AH-Verbindungen, die in den Docking-Studien entwickelt wurden, durchzuf{\"u}hren. Hierbei konnte jedoch keine Bindung festgestellt werden. Die Kristallisation mit Ver-155008, einem bekannten Hsc70-Inhibitor, f{\"u}hrte jedoch zur ersten Zweidom{\"a}nenstruktur von Hsc70 mit gebundenem Ver-155008. Neben der obigen Fragestellung wurde außerdem untersucht, wie funktional aktiv das trunkierte Hsc70-Konstrukts ist. Hier zeigte sich, dass aufgrund des fehlenden C-Terminus zwar eine geringe Aktivit{\"a}t von 30 \% im Vergleich zur Volll{\"a}nge zu beobachten war. F{\"u}r eine nahezu vollst{\"a}ndige R{\"u}ckfaltungsaktivit{\"a}t ist aber der C-Terminus essentiell. Weiterhin konnte in ITC-Versuchen der Kd-Wert von Ver-155008 an die verwendete Mutante ermittelt werden, der dem bereits bekannten Kd von Ver-155008 an das native Hsc70 {\"a}hnlich ist.}, subject = {Hitzeschockproteine}, language = {de} } @phdthesis{Goetz2018, author = {G{\"o}tz, Silvia}, title = {Zuo1 - ein neues G-Quadruplex-bindendes Protein in \(Saccharomyces\) \(cerevisiae\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152158}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {G-Quadruplex (G4)-Strukturen sind sehr stabile und polymorphe DNA und RNA Sekund{\"a}rstrukturen mit einem konservierten Guanin-reichen Sequenzmotiv (G4-Motiv). Sie bestehen aus {\"u}bereinander gestapelten planaren G-Quartetts, in denen je vier Guanine durch Wasserstoffbr{\"u}ckenbindungen zusammengehalten werden. Da G4-Motive in Eukaryoten an bestimmten Stellen im Genom angereichert vorkommen, wird angenommen, dass die Funktion von G4-Strukturen darin besteht, biologische Prozesse positiv oder negativ zu regulieren. Aufgrund der hohen thermodynamischen Stabilit{\"a}t von G4 Strukturen ist davon auszugehen, dass Proteine in die Faltung, Stabilisierung und Entfaltung dieser Nukleins{\"a}ure-Strukturen regulatorisch involviert sind. Bis heute wurden viele Proteine in der Literatur beschrieben, die G4-Strukturen entwinden k{\"o}nnen. Jedoch konnten bisher nur wenige Proteine identifiziert werden, die in vivo die Faltung f{\"o}rdern oder G4-Strukturen stabilisieren. Durch Yeast One-Hybrid (Y1H)-Screenings habe ich Zuo1 als neues G4 bindendes Protein identifiziert. In vitro Analysen best{\"a}tigten diese Interaktion und es stellte sich heraus, dass Zuo1 G4-Strukturen stabilisiert. {\"U}bereinstimmend mit den in vitro Daten konnte gezeigt werden, dass Zuo1 signifikant an G4-Motive im Genom von Saccharomyces ceresivisiae bindet. Genomweit {\"u}berlappen G4-Motive, an die Zuo1 bindet, mit Stellen, an denen die DNA Replikation zum Stillstand kommt und vermehrt DNA Sch{\"a}den vorkommen. Diese Ergebnisse legen nahe, dass Zuo1 eine Funktion w{\"a}hrend der DNA Reparatur oder in Zusammenhang mit dem Vorankommen der DNA Replikationsgabel hat, indem G4-Strukturen stabilisiert werden. Diese Hypothese wird außerdem durch genetische Experimente gest{\"u}tzt, wonach in Abwesenheit von Zuo1 die Genominstabilit{\"a}t zunimmt. Aufgrund dieser Daten war es m{\"o}glich ein Model zu entwickeln, bei dem Zuo1 w{\"a}hrend der S-Phase G4-Strukturen bindet und stabilisiert wodurch die DNA Replikation blockiert wird. Diese Interaktion findet neben Stellen schadhafter DNA statt und unterst{\"u}tzt somit DNA Reparatur-Prozesse wie beispielsweise die Nukleotidexzisionsreparatur. Als weiteres potentielles G4-bindendes Protein wurde Slx9 in Y1H-Screenings identifiziert. In vitro Experimente zeigten zwar, dass Slx9 mit h{\"o}herer Affinit{\"a}t an G4-Strukturen bindet im Vergleich zu anderen getesteten DNA Konformationen, jedoch wurde in S. cerevisiae genomweit keine signifikante Bindung an G4-Motive festgestellt.}, subject = {Saccharomyces cerevisiae}, language = {de} } @phdthesis{Wanzek2016, author = {Wanzek, Katharina}, title = {The investigation of the function of repair proteins at G-quadruplex structures in \(Saccharomyces\) \(cerevisiae\) revealed that Mms1 promotes genome stability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142547}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu \& Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity.}, subject = {Quadruplex-DNS}, language = {en} } @article{WaldholmWangBrodinetal.2011, author = {Waldholm, Johan and Wang, Zhi and Brodin, David and Tyagi, Anu and Yu, Simei and Theopold, Ulrich and {\"O}stlund Farrants, Ann Kristin and Visa, Neus}, title = {SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in \(Drosophila\) \(melanogaster\)}, series = {BMC Molecular Biology}, volume = {12}, journal = {BMC Molecular Biology}, number = {46}, doi = {10.1186/1471-2199-12-46}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142613}, pages = {1-12}, year = {2011}, abstract = {Background: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. Results: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. Conclusions: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.}, language = {en} } @article{YinBrocherFischeretal.2011, author = {Yin, Jun and Brocher, Jan and Fischer, Utz and Winkler, Christoph}, title = {Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa}, series = {Molecular neurodegeneration}, volume = {6}, journal = {Molecular neurodegeneration}, number = {56}, doi = {10.1186/1750-1326-6-56}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141090}, pages = {1-17}, year = {2011}, abstract = {Background: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results: We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion: Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.}, language = {en} } @article{HaddadChenZhangetal.2011, author = {Haddad, Dana and Chen, Nanhai G. and Zhang, Qian and Chen, Chun-Hao and Yu, Yong A. and Gonzalez, Lorena and Carpenter, Susanne G. and Carson, Joshua and Au, Joyce and Mittra, Arjun and Gonen, Mithat and Zanzonico, Pat B. and Fong, Yuman and Szalay, Aladar A.}, title = {Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus}, series = {Journal of Translational Medicine}, volume = {9}, journal = {Journal of Translational Medicine}, number = {36}, doi = {10.1186/1479-5876-9-36}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140847}, pages = {1-14}, year = {2011}, abstract = {Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153. Methods: GLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free (124)I radiotracer. Results: GLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via (124)I-PET. Conclusion: Insertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.}, language = {en} } @article{KilincEhrigPessianetal.2016, author = {Kilinc, Mehmet Okyay and Ehrig, Klaas and Pessian, Maysam and Minev, Boris R. and Szalay, Aladar A.}, title = {Colonization of xenograft tumors by oncolytic vaccinia virus (VACV) results in enhanced tumor killing due to the involvement of myeloid cells}, series = {Journal of Translational Medicine}, volume = {14}, journal = {Journal of Translational Medicine}, number = {340}, doi = {10.1186/s12967-016-1096-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168914}, year = {2016}, abstract = {Background The mechanisms by which vaccinia virus (VACV) interacts with the innate immune components are complex and involve different mechanisms. iNOS-mediated NO production by myeloid cells is one of the central antiviral mechanisms and this study aims to investigate specifically whether iNOS-mediated NO production by myeloid cells, is involved in tumor eradication following the virus treatment. Methods Human colon adenocarcinoma (HCT-116) xenograft tumors were infected by VACV. Infiltration of iNOS\(^{+}\) myeloid cell population into the tumor, and virus titer was monitored following the treatment. Single-cell suspensions were stained for qualitative and quantitative flow analysis. The effect of different myeloid cell subsets on tumor growth and colonization were investigated by depletion studies. Finally, in vitro culture experiments were carried out to study NO production and tumor cell killing. Student's t test was used for comparison between groups in all of the experiments. Results Infection of human colon adenocarcinoma (HCT-116) xenograft tumors by VACV has led to recruitment of many CD11b\(^{+}\) ly6G\(^{+}\) myeloid-derived suppressor cells (MDSCs), with enhanced iNOS expression in the tumors, and to an increased intratumoral virus titer between days 7 and 10 post-VACV therapy. In parallel, both single and multiple rounds of iNOS-producing cell depletions caused very rapid tumor growth within the same period after virus injection, indicating that VACV-induced iNOS\(^{+}\) MDSCs could be an important antitumor effector component. A continuous blockade of iNOS by its specific inhibitor, L-NIL, showed similar tumor growth enhancement 7-10 days post-infection. Finally, spleen-derived iNOS+ MDSCs isolated from virus-injected tumor bearing mice produced higher amounts of NO and effectively killed HCT-116 cells in in vitro transwell experiments. Conclusions We initially hypothesized that NO could be one of the factors that limits active spreading of the virus in the cancerous tissue. In contrast to our initial hypothesis, we observed that PMN-MDSCs were the main producer of NO through iNOS and NO provided a beneficial antitumor effect, The results strongly support an important novel role for VACV infection in the tumor microenvironment. VACV convert tumor-promoting MDSCs into tumor-killing cells by inducing higher NO production.}, language = {en} } @phdthesis{Brosi2021, author = {Brosi, Cornelia}, title = {Functional characterization of the TTF complex and its role in neurodevelopmental disorders}, doi = {10.25972/OPUS-15778}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157783}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The eukaryotic gene expression requires extensive regulations to enable the homeostasis of the cell and to allow dynamic responses due to external stimuli. Although many regulatory mechanisms involve the transcription as the first step of the gene expression, intensive regulation occurs also in the post-transcriptional mRNA metabolism. Thereby, the particular composition of the mRNPs plays a central role as the components associated with the mRNA form a specific "mRNP code" which determines the fate of the mRNA. Many proteins which are involved in this regulation and the mRNA metabolism are affected in diseases and especially neurological disorders often result from an aberrant mRNP code which leads to changes in the regulation and expression of mRNPs. The focus of this work was on a trimeric protein complex which is termed TTF complex based on its subunits TDRD3, TOP3β and FMRP. Biochemical investigations revealed that the three components of the TTF complex are nucleo-cytosolic shuttle proteins which localize in the cytoplasm at the steady-state, associate with mRNPs and are presumably connected to the translation. Upon cellular stress conditions, the TTF components concentrate in stress granules. Thus, the TTF complex is part of the mRNP code, however its target RNAs and function are still completely unknown. Since the loss of functional FMRP results in the fragile X syndrome and TOP3β is associated with schizophrenia and intellectual disability, the TTF complex connects these phenotypically related neuro-psychiatric disorders with each other on a molecular level. Therefore, the aim of this work was to biochemically characterize the TTF complex and to define its function in the mRNA metabolism. In this work, evidence was provided that TDRD3 acts as the central unit of the TTF complex and directly binds to FMRP as well as to TOP3β. Thereby, the interaction of TDRD3 and TOP3β is very stable, whereas FMRP is a dynamic component. Interestingly, the TTF complex is not bound directly to mRNA, but is recruited via the exon junction complex (EJC) to mRNPs. This interaction is mediated by a specific binding motif of TDRD3, the EBM. Upon biochemical and biological investigations, it was possible to identify the interactome of the TTF complex and to define the role in the mRNA metabolism. The data revealed that the TTF complex is mainly associated with "early" mRNPs and is probably involved in the pioneer round of translation. Furthermore, TOP3β was found to bind directly to the ribosome and thus, establishes a connection between the EJC and the translation machinery. A reduction of the TTF components resulted in selective changes in the proteome in cultured cells, whereby individual protein subsets seem to be regulated rather than the global protein expression. Moreover, the enzymatic analysis of TOP3β indicated that TOP3β is a type IA topoisomerase which can catalytically attack not only DNA but also RNA. This aspect is particularly interesting with regard to the connection between early mRNPs and the translation which has been revealed in this work. The data obtained in this work suggest that the TTF complex plays a role in regulating the metabolism of an early mRNP subset possibly in the course of the pioneer round of translation. Until now, the link between an RNA topoisomerase and the mRNA metabolism is thereby unique and thus provides a completely new perspective on the steps in the post-transcriptional gene expression and its regulation.}, subject = {Messenger-RNP}, language = {en} } @phdthesis{Bedenk2018, author = {Bedenk, Kristina}, title = {Biochemische und strukturelle Charakterisierung der Genexpressionsmaschinerie des Vaccinia Virus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135538}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Die Familie der Pockenviren zeichnet sich durch ein komplexes DNA Genom aus und hat großes medizinisches Potential. Am eindrucksvollsten ist dies f{\"u}r das Vaccinia-Virus (VACV) belegt, welches nicht nur als Pocken-Impfstoff eingesetzt wird, sondern auch als onkolytisches Virus in der Tumorbiologie. VACV hat einen außergew{\"o}hnlichen Replikationszyklus, welcher ausschließlich im Zytoplasma der Wirtszelle stattfindet. Somit ist die gesamte virale Genexpressionsmaschinerie v{\"o}llig unabh{\"a}ngig von kernvermittelten Reaktionen des Wirts und somit auch aus Sicht der Grundlagenforschung von gr{\"o}ßtem Interesse. Die Schl{\"u}sselkomponente der viralen Genexpression ist die makromolekulare DNA-abh{\"a}ngige RNA Polymerase (vvRPO), deren Untereinheiten allesamt Virus-kodiert sind. Zwar wurden in den letzten Jahren Protokolle zur biochemischen und funktionellen Charakterisierung der vvRPO etabliert, ein detailliertes Wissen {\"u}ber deren Zusammenlagerung in vivo und die r{\"a}umlichen und zeitlichen Interaktionen mit den Transkriptions- bzw. Prozessierungsfaktoren sind aber weitgehend unbekannt. Diese Arbeit umfasst Untersuchungen zur strukturellen und funktionellen Charakterisierung der vvRPO und seiner assoziierten Faktoren. Grundlage hierf{\"u}r war die Etablierung eines Reinigungsprotokolls mithilfe eines neu konstruierten rekombinanten VACV (GLV-1h439). Diese Strategie erlaubte es hoch-molekulare native vvRPO Komplexe zu isolieren. Ein transkriptions-inaktiver Komplex (Komplex I) mit einer kalkulierten Masse von 575 kDa bestand aus den acht Untereinheiten des vvRPO Holoenzyms und den Polymerase-assoziierten Faktoren RAP94 und D6. Ein zweiter, transkriptionell aktiver Komplex (Komplex II) mit einer Masse von 803 kDa enthielt, neben dem Holoenzym der vvRPO, noch weitere Faktoren, die prim{\"a}r die Erkennung der DNA-Matrize und die Prozessierung der naszierenden RNA vermitteln. Hierbei handelt es sich um RAP94, das virale Capping Enzym bestehend aus den zwei Untereinheiten D1 und D12, A7 und dem Terminationsfaktor NPH I. Interessanterweise enthielt dieser Komplex zus{\"a}tzlich mit E11 eine bislang unbekannte weitere Protein-Komponente, sowie tRNAGln und tRNAArg. Der isolierte Kompelx II ist daher ein Ribonukleoprotein (RNP). Die Verf{\"u}gbarkeit von hoch-reinen vvRPO Komplexen erlaubte es erstmals deren strukturelle Architektur zu untersuchen. Hierf{\"u}r wurden drei experimentelle Ans{\"a}tze, die klassische R{\"o}ntgenstrukturanalyse, die Kryo-Elektronenmikroskopie (Kryo-EM) und Quervernetzungssstudien miteinander kombiniert. Die Strukturen der Komplexe I und II haben eine Aufl{\"o}sung von 11-12 {\AA}, wobei auff{\"a}llig war, dass beide eine markante strukturelle {\"A}hnlichkeit zur eukaryotischen RNA Polymerase II aufwiesen. Dar{\"u}ber hinaus gelang es zus{\"a}tzliche Bereiche im Komplex II zu definieren, welche die Polymerase-assoziierten Prozessierungsfaktoren beherbergen. Zudem konnte die atomare Struktur von E11, mittels R{\"o}ntgenstrukturanalyse bei einer Aufl{\"o}sung von 1,9 {\AA}, gel{\"o}st werden. Das E11 Protein besitzt ein neuartiges Faltungsmuster und weist einen intensiven Dimerisierungskontakt auf, welcher sich {\"u}ber vier ß-Faltbl{\"a}tter ausbildet. Die im Rahmen dieser Arbeit erhaltenen Daten legen die Grundlage f{\"u}r ein detailliertes Verst{\"a}ndnis der r{\"a}umlichen Organisation der viralen Transkriptonsmaschinerie. Dar{\"u}ber hinaus werden sie funktionelle Studien erm{\"o}glichen, welche die Rolle der einzelnen Proteine, sowie der tRNAs bei der mRNA Synthese kl{\"a}ren helfen.}, subject = {Vaccinia-Virus}, language = {de} } @article{StepniakKaestnerPoggietal.2015, author = {Stepniak, Beata and K{\"a}stner, Anne and Poggi, Giulia and Mitjans, Marina and Begemann, Martin and Hartmann, Annette and Van der Auwera, Sandra and Sananbenesi, Farahnaz and Kr{\"u}ger-Burg, Dilja and Matuszko, Gabriela and Brosi, Cornelia and Homuth, Georg and V{\"o}lzke, Henry and Benseler, Fritz and Bagni, Claudia and Fischer, Utz and Dityatev, Alexander and Grabe, Hans-J{\"o}rgen and Rujescu, Dan and Fischer, Andre and Ehrenreich, Hannelore}, title = {Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes}, series = {EMBO Molecular Medicine}, volume = {7}, journal = {EMBO Molecular Medicine}, number = {12}, doi = {10.15252/emmm.201505696}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136893}, pages = {1565-1579}, year = {2015}, abstract = {Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60\% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high-versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.}, language = {en} } @article{SpiveyDeGiorgiZhaoetal.2012, author = {Spivey, Tara L. and De Giorgi, Valeria and Zhao, Yingdong and Bedognetti, Davide and Pos, Zoltan and Liu, Qiuzhen and Tomei, Sara and Ascierto, Maria Libera and Uccellini, Lorenzo and Reinboth, Jennifer and Chouchane, Lotfi and Stroncek, David F. and Wang, Ena and Marincola, Francesco M.}, title = {The stable traits of melanoma genetics: an alternate approach to target discovery}, series = {BMC Genomics}, volume = {13}, journal = {BMC Genomics}, number = {156}, doi = {10.1186/1471-2164-13-156}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-131992}, year = {2012}, abstract = {Background: The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level. We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number. Results: Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis. Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05). We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers. We then tested whether the genes could categorize 112 melanoma metastases. Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A). This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer. The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy. An intermediate third class (C) was further identified. The three phenotypes were confirmed by unsupervised principal component analysis. Conclusions: This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy.}, language = {en} } @article{GrossHennardMasourisetal.2012, author = {Gross, Henrik and Hennard, Christine and Masouris, Ilias and Cassel, Christian and Barth, Stephanie and Stober-Gr{\"a}sser, Ute and Mamiani, Alfredo and Moritz, Bodo and Ostareck, Dirk and Ostareck-Lederer, Antje and Neuenkirchen, Nils and Fischer, Utz and Deng, Wen and Leonhardt, Heinrich and Noessner, Elfriede and Kremmer, Elisabeth and Gr{\"a}sser, Friedrich A.}, title = {Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {8}, doi = {10.1371/journal.pone.0042106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133707}, year = {2012}, abstract = {The Epstein-Barr Virus (EBV) -encoded EBNA2 protein, which is essential for the in vitro transformation of B-lymphocytes, interferes with cellular processes by binding to proteins via conserved sequence motifs. Its Arginine-Glycine (RG) repeat element contains either symmetrically or asymmetrically di-methylated arginine residues (SDMA and ADMA, respectively). EBNA2 binds via its SDMA-modified RG-repeat to the survival motor neurons protein (SMN) and via the ADMA-RG-repeat to the NP9 protein of the human endogenous retrovirus K (HERV-K (HML-2) Type 1). The hypothesis of this work was that the methylated RG-repeat mimics an epitope shared with cellular proteins that is used for interaction with target structures. With monoclonal antibodies against the modified RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA-antibody to hnRNP K was demonstrated using E. coli expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV-infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that the methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties.}, language = {en} } @article{GowdaGodderKmieciaketal.2011, author = {Gowda, Madhu and Godder, Kamar and Kmieciak, Maciej and Worschech, Andrea and Ascierto, Maria-Libera and Wang, Ena and Francesco M., Marincola and Manjili, Masoud H.}, title = {Distinct signatures of the immune responses in low risk versus high risk neuroblastoma}, series = {Journal of Translational Medicine}, volume = {9}, journal = {Journal of Translational Medicine}, number = {170}, doi = {10.1186/1479-5876-9-170}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135147}, pages = {1-8}, year = {2011}, abstract = {Background: Over 90\% of low risk (LR) neuroblastoma patients survive whereas less than 30\% of high risk (HR) patients are long term survivors. Age (children younger than 18 months old) is associated with LR disease. Considering that adaptive immune system is well developed in older children, and that T cells were shown to be involved in tumor escape and progression of cancers, we sought to determine whether HR patients may tend to show a signature of adaptive immune responses compared to LR patients who tend to have diminished T-cell responses but an intact innate immune response. Methods: We performed microarray analysis of RNA extracted from the tumor specimens of HR and LR patients. Flow cytometry was performed to determine the cellular constituents in the blood while multiplex cytokine array was used to detect the cytokine profile in patients' sera. A HR tumor cell line, SK-N-SH, was also used for detecting the response to IL-1 beta, a cytokines which is involved in the innate immune responses. Results: Distinct patterns of gene expression were detected in HR and LR patients indicating an active T-cell response and a diminished adaptive immune response, respectively. A diminished adaptive immune response in LR patients was evident by higher levels of IL-10 in the sera. In addition, HR patients had lower levels of circulating myeloid derived suppressor cells (MDSC) compared with a control LR patient. LR patients showed slightly higher levels of cytokines of the innate immune responses. Treatment of the HR tumor line with IL-1b induced expression of cytokines of the innate immune responses. Conclusions: This data suggests that adaptive immune responses may play an important role in the progression of HR disease whereas innate immune responses may be active in LR patients.}, language = {en} } @article{WeibelBasseLuesebrinkHessetal.2013, author = {Weibel, Stephanie and Basse-Luesebrink, Thomas Christian and Hess, Michael and Hofmann, Elisabeth and Seubert, Carolin and Langbein-Laugwitz, Johanna and Gentschev, Ivaylo and Sturm, Volker J{\"o}rg Friedrich and Ye, Yuxiang and Kampf, Thomas and Jakob, Peter Michael and Szalay, Aladar A.}, title = {Imaging of Intratumoral Inflammation during Oncolytic Virotherapy of Tumors by \(^{19}\)F-Magnetic Resonance Imaging (MRI)}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0056317}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130311}, pages = {e56317}, year = {2013}, abstract = {Background Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy. Methodology/Principal Findings The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors. Conclusions/Significance These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.}, language = {en} } @article{KrehanHeubeckMenzeletal.2012, author = {Krehan, Mario and Heubeck, Christian and Menzel, Nicolas and Seibel, Peter and Sch{\"o}n, Astrid}, title = {RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {16}, doi = {10.1093/nar/gks476}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130648}, pages = {7956- 7966}, year = {2012}, abstract = {RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.}, language = {en} } @article{WangChenMinevetal.2013, author = {Wang, Huiqiang and Chen, Nanhai G. and Minev, Boris R. and Zimmermann, Martina and Aguilar, Richard J. and Zhang, Qian and Sturm, Julia B. and Fend, Falko and Yu, Yong A. and Cappello, Joseph and Lauer, Ulrich M. and Szalay, Aladar A.}, title = {Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains}, series = {PLoS ONE}, volume = {8}, journal = {PLoS ONE}, number = {9}, doi = {10.1371/journal.pone.0071105}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130059}, pages = {e71105}, year = {2013}, abstract = {Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.}, language = {en} } @phdthesis{Pelz2015, author = {Pelz, Jann-Patrick}, title = {Strukturbiologische Untersuchungen zur Chaperone-vermittelten Zusammenlagerung spleißosomaler U-snRNPs}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116973}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Durch die Spleißreaktion werden nicht-kodierende Sequenzelemente (Introns) aus eukaryotischen Vorl{\"a}ufer-mRNAs entfernt und die kodierenden Sequenzelemente (Exons) miteinander zu einem offenen Leserahmen verbunden. Dieser zentrale Prozessierungsschritt w{\"a}hrend der eukaryotischen Genexpression wird durch das Spleißosom katalysiert, das aus den vier kleinen nukle{\"a}ren Ribonucleoproteinpartikeln (snRNPs) U1, U2, U4/U6 und U5, sowie einer Vielzahl weiterer Proteinfaktoren gebildet wird. Alle snRNPs besitzen eine gemeinsame ringf{\"o}rmige Kernstruktur, die aus sieben gemeinsamen Sm-Proteinen (SmB/B'-D1-D2-D3-E-F-G) besteht, die ein einzelstr{\"a}ngiges Sequenzmotiv auf der snRNAs binden. W{\"a}hrend sich diese, als Sm-Core-Dom{\"a}ne bezeichnete Struktur in vitro spontan ausbilden kann, erfolgt die Zusammenlagerung in vivo in einem assistierten und hochregulierten Prozess. Dieser ist abh{\"a}ngig von insgesamt mindestens 12 trans-agierenden Faktoren, die in den PRMT5- und SMN-Komplexen organisiert sind. Der PRMT5-Komplex agiert in der fr{\"u}hen Phase der Zusammenlagerung, indem er die Sm-Proteine durch die Untereinheit pICln rekrutiert und die symmetrische Methylierung von Argininresten in den C terminalen Schw{\"a}nzen von SmB/B', SmD1 und SmD3 katalysiert. Als Resultat dieser fr{\"u}hen Phase befinden sich die Sm-Proteine SmD1-D2-E-F-G und SmB/B'-D3 in zwei getrennten und durch pICln organisierten Komplexen. W{\"a}hrend SmB/B'-D3-pICln am PRMT5-Komplex gebunden bleibt, existiert der zweite Komplex als freies Intermediat mit einem Sedimentationskoeffizienten von 6S. Diese Intermediate k{\"o}nnen nicht mit RNA assoziieren, sodass f{\"u}r die Fortsetzung des Zusammenlagerungsprozesses die Interaktion der Sm-Proteine mit pICln aufgel{\"o}st werden muss. Dies geschieht in der sp{\"a}ten Phase der Sm-Core-Zusammenlagerung, in der die Sm-Proteine vom SMN-Komplex (bestehend aus SMN, Gemin2-8 und unrip) {\"u}bernommen werden und pICln dissoziiert wird. Dadurch werden die Sm-Proteine f{\"u}r ihre Interaktion mit der snRNA aktiviert und k{\"o}nnen auf die Sm-Bindestelle transferiert werden, wodurch die Formierung des Sm-Core abgeschlossen wird. Im Rahmen dieser Arbeit konnten mit Hilfe einer Kombination r{\"o}ntgenkristallographischer und elektronenmikroskopischer Methoden zwei wichtige Intermediate dieses Zusammenlagerungs-prozesses strukturbiologisch charakterisiert werden. Bei diesen Intermediaten handelt es sich um den 6S-Komplex, sowie um ein Sm-Protein-Transferintermediat mit einem Sedimentations-koeffizienten von 8S. In diesem ist der 6S-Komplex an zwei zentrale Untereinheiten des SMN-Komplexes (SMN und Gemin2) gebunden, w{\"a}hrend pICln den Komplex noch nicht verlassen hat. Der 8S-Komplex stellt daher ein „gefangenes" Intermediat zwischen der fr{\"u}hen und sp{\"a}ten Phase der Zusammenlagerung dar. Zun{\"a}chst gelang es eine erste Kristallform des rekombinant hergestellten 8S-Komplexes zu erhalten, die jedoch keine Strukturl{\"o}sung erlaubte. Durch eine kombinierte Optimierung der Kristallisationsbedingung und der verwendeten Proteine wurde eine weitere {\"a}hnliche Kristallform erhalten, mit der die Kristallstruktur des 8S-Komplexes gel{\"o}st werden konnte. Die Kristallisation des 6S-Komplexes gelang im Anschluss auf Basis der Hypothese, dass Kristalle beider Komplexe aufgrund der kompositionellen Verwandtschaft zwischen 6S und 8S auch {\"A}hnlichkeiten in der Architektur ihrer Kristallgitter aufweisen k{\"o}nnten. Daher wurden innerhalb von pICln gezielt Aminos{\"a}uren substituiert, die sich innerhalb von Kristallkontakten der 8S-Kristalle befanden und konformationell eingeschr{\"a}nkt waren. Mit entsprechend rekonstituierten 6S-Pr{\"a}parationen konnten dann zwei Kristallformen erzeugt werden, die eine Strukturl{\"o}sung des 6S-Komplexes erm{\"o}glichten. Durch die Kristallstruktur des 6S-Komplexes konnte f{\"u}r pICln eine strukturelle Mimikry der Sm-Proteine identifiziert werden. Diese erm{\"o}glicht eine Bindung der Sm-Proteine und eine fr{\"u}hzeitige topologische Organisation des Sm-Pentamers D1-D2-F-E-G in einer geschlossenen hexameren Ringstruktur. Die Kristallstruktur des 8S-Komplexes zeigt, wie der SMN-Komplex {\"u}ber Gemin2 an das Sm-Pentamer bindet. In Kombination mit einer EM-Struktur des 8S-Komplexes gelang es weiterhin, einen plausiblen Mechanismus f{\"u}r die Elimination von pICln und die Aktivierung der Sm-Proteine f{\"u}r die snRNA-Bindung zu formulieren. Somit konnten diese Arbeiten zu einem besseren Verst{\"a}ndnis der Funktionen von trans-agierenden Faktoren bei Zusammenlagerung von RNA-Protein-Komplexen in vivo beitragen.}, subject = {Spleißosom}, language = {de} } @article{HofmannWeibelSzalay2014, author = {Hofmann, Elisabeth and Weibel, Stephanie and Szalay, Aladar A.}, title = {Combination treatment with oncolytic Vaccinia virus and cyclophosphamide results in synergistic antitumor effects in human lung adenocarcinoma bearing mice}, doi = {10.1186/1479-5876-12-197}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110168}, year = {2014}, abstract = {Background The capacity of the recombinant Vaccinia virus GLV-1h68 as a single agent to efficiently treat different human or canine cancers has been shown in several preclinical studies. Currently, its human safety and efficacy are investigated in phase I/II clinical trials. In this study we set out to evaluate the oncolytic activity of GLV-1h68 in the human lung adenocarcinoma cell line PC14PE6-RFP in cell cultures and analyzed the antitumor potency of a combined treatment strategy consisting of GLV-1h68 and cyclophosphamide (CPA) in a mouse model of PC14PE6-RFP lung adenocarcinoma. Methods PC14PE6-RFP cells were treated in cell culture with GLV-1h68. Viral replication and cell survival were determined by plaque assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Subcutaneously implanted PC14PE6-RFP xenografts were treated by systemic injection of GLV-1h68, CPA or a combination of both. Tumor growth and viral biodistribution were monitored and immune-related antigen profiling of tumor lysates was performed. Results GLV-1h68 efficiently infected, replicated in and lysed human PC14PE6-RFP cells in cell cultures. PC14PE6-RFP tumors were efficiently colonized by GLV-1h68 leading to much delayed tumor growth in PC14PE6-RFP tumor-bearing nude mice. Combination treatment with GLV-1h68 and CPA significantly improved the antitumor efficacy of GLV-1h68 and led to an increased viral distribution within the tumors. Pro-inflammatory cytokines and chemokines were distinctly elevated in tumors of GLV-1h68-treated mice. Factors expressed by endothelial cells or present in the blood were decreased after combination treatment. A complete loss in the hemorrhagic phenotype of the PC14PE6-RFP tumors and a decrease in the number of blood vessels after combination treatment could be observed. Conclusions CPA and GLV-1h68 have synergistic antitumor effects on PC14PE6-RFP xenografts. We strongly suppose that in the PC14PE6-RFP model the enhanced tumor growth inhibition achieved by combining GLV-1h68 with CPA is due to an effect on the vasculature rather than an immunosuppressive action of CPA. These results provide evidence to support further preclinical studies of combining GLV-1h68 and CPA in other highly angiogenic tumor models. Moreover, data presented here demonstrate that CPA can be combined successfully with GLV-1h68 based oncolytic virus therapy and therefore might be promising as combination therapy in human clinical trials.}, language = {en} } @phdthesis{Nube2013, author = {Nube, Jacqueline Sui Lin}, title = {Comparative Analysis of Vaccinia Virus-Encoded Markers Reflecting Actual Viral Titres in Oncolytic Virotherapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85689}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Using viruses to treat cancer is a novel approach to an age-old disease. Oncolytic viruses are native or recombinant viruses that have the innate or enhanced capability to infect tumour cells, replicate within the tumour microenvironment and subsequently lyse those cells. One representative, the vaccinia virus (VACV), belongs to the orthopoxvirus genus of the Poxviridae family. GLV-1h68, a recombinant and attenuated vaccinia virus devel- oped by the Genelux Corporation, is a member of this family currently being tested in various phase I/II clinical trials under the name GL-ONC1. It has been shown to specif- ically replicate in tumour cells while sparing healthy tissue and to metabolise prodrug at or transport immunological payloads to the site of affliction. Since imaging modalities offer little insight into viral replication deep within the body, and because oncolytic virotherapy is dependent on replication within the target tissue, the need for a monitoring system is evident. Pharmacokinetic analysis of this oncolytic agent was to give insight into the dynamics present in tumours during treatment. This, in turn, would give clinicians the opportunity to monitor the efficacy as early as possible after the onset of treatment, to observe treatment progression and possibly to gauge prognosis, without resorting to invasive procedures, e.g. biopsies. A criteria for viable biomarkers was that it had to be directly dependent on viral replica- tion. Ideally, a marker for treatment efficacy would be specific to the treatment modality, not necessarily the treatment type. Such a marker would be highly detectable (high sen- sitivity), specific for the treatment (high specificity), and present in an easily obtained specimen (blood). Taking this into consideration, the biomarkers were chosen for their potential to be indicators of viral replication. Thus, the biomarkers analysed in this thesis are: the native proteins expressed by the viral genes A27L and B5R, the virally encoded recombinant proteins β-galactosidase, β-glucuronidase, green fluorescent protein (GFP), carboxypeptidase G2 (CPG2) and carcinoembryonic antigen (CEA). Each marker is under the control of one of five different promoters present. All recombinant viruses used in this thesis express A27L, B5R, GFP and β-glucuronidase and all are derived from the parental virus GLV-1h68. In addition to these markers, GLV-1h68 expresses β-galactosidase; GLV-1h181 expresses CPG2. [...]}, subject = {Onkolyse}, language = {en} } @phdthesis{Flegler2022, author = {Flegler, Vanessa Judith}, title = {Application of electron cryomicroscopy for structural and functional studies on the mechanosensitive channels of small conductance}, doi = {10.25972/OPUS-26897}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268979}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues. In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration. The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.}, language = {en} } @article{TsonevaMinevFrentzenetal.2017, author = {Tsoneva, Desislava and Minev, Boris and Frentzen, Alexa and Zhang, Qian and Wege, Anja K. and Szalay, Aladar A.}, title = {Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis}, series = {Molecular Therapy Oncolytics}, volume = {5}, journal = {Molecular Therapy Oncolytics}, doi = {10.1016/j.omto.2017.03.001}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170786}, pages = {41-61}, year = {2017}, abstract = {Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56\(^{bright}\) NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.}, language = {en} } @phdthesis{Amelingmeier2022, author = {Amelingmeier, Florian}, title = {Identifizierung und Untersuchung TOP-mRNA - bindender Faktoren}, doi = {10.25972/OPUS-28923}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289231}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie ben{\"o}tigt und daher mittels vielf{\"a}ltiger Mechanismen streng reguliert wird. Ein Beispiel hierf{\"u}r stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche haupts{\"a}chlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5'-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv erm{\"o}glicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. N{\"a}hrstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird. Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation erm{\"o}glicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erw{\"a}gung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP - Sequenzen identifiziert werden, welche sich f{\"u}r Strukturanalysen eignen w{\"u}rden. Als weiterer TOP-interagierender Faktor wurde {\"u}ber verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert. Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. W{\"a}hrend gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7-Konstrukt sowie 7SK-Konstrukte f{\"u}r Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK - Interaktion aus{\"u}ben, welcher in weiteren Studien ber{\"u}cksichtigt werden sollte.}, subject = {Proteinbiosynthese}, language = {de} } @article{TolayBuchberger2022, author = {Tolay, Nazife and Buchberger, Alexander}, title = {Role of the ubiquitin system in stress granule metabolism}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {7}, issn = {1422-0067}, doi = {10.3390/ijms23073624}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284061}, year = {2022}, abstract = {Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin ('ubiquitylation'), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.}, language = {en} } @article{TolayBuchberger2021, author = {Tolay, Nazife and Buchberger, Alexander}, title = {Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system}, series = {Life Science Alliance}, volume = {4}, journal = {Life Science Alliance}, number = {5}, doi = {10.26508/lsa.202000927}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259810}, pages = {e202000927}, year = {2021}, abstract = {Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.}, language = {en} } @phdthesis{Huber2023, author = {Huber, Hannes}, title = {Biochemical and functional characterization of DHX30, an RNA helicase linked to neurodevelopmental disorder}, doi = {10.25972/OPUS-28050}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280505}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease "Neurodevelopmental Disorder with severe Motor Impairment and Absent Language" (NEDMIAL;OMIM\#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3' → 5' RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome's mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5'TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.}, language = {en} } @article{BenhalevyGuptaDananetal.2017, author = {Benhalevy, Daniel and Gupta, Sanjay K. and Danan, Charles H. and Ghosal, Suman and Sun, Hong-Wei and Kazemeier, Hinke G. and Paeschke, Katrin and Hafner, Markus and Juranek, Stefan A.}, title = {The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation}, series = {Cell Reports}, volume = {18}, journal = {Cell Reports}, number = {12}, doi = {10.1016/j.celrep.2017.02.080}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171122}, pages = {2979-2990}, year = {2017}, abstract = {The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.}, language = {en} } @article{PakniaChariStarketal.2016, author = {Paknia, Elham and Chari, Ashwin and Stark, Holger and Fischer, Utz}, title = {The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs}, series = {Cell Reports}, volume = {16}, journal = {Cell Reports}, number = {12}, doi = {10.1016/j.celrep.2016.08.047}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162420}, pages = {p3103-3112}, year = {2016}, abstract = {The formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated.}, language = {en} } @incollection{DasZografakisOeljeklausetal.2023, author = {Das, Hirakjyoti and Zografakis, Alexandros and Oeljeklaus, Silke and Warscheid, Bettina}, title = {Analysis of Yeast Peroxisomes via Spatial Proteomics}, series = {Peroxisomes}, booktitle = {Peroxisomes}, edition = {accepted version}, publisher = {Springer}, doi = {10.1007/978-1-0716-3048-8_2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-327532}, publisher = {Universit{\"a}t W{\"u}rzburg}, pages = {13-31}, year = {2023}, abstract = {Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.}, language = {en} } @article{SendellPriceTulenkoPetterssonetal.2023, author = {Sendell-Price, Ashley T. and Tulenko, Frank J. and Pettersson, Mats and Kang, Du and Montandon, Margo and Winkler, Sylke and Kulb, Kathleen and Naylor, Gavin P. and Phillippy, Adam and Fedrigo, Olivier and Mountcastle, Jacquelyn and Balacco, Jennifer R. and Dutra, Amalia and Dale, Rebecca E. and Haase, Bettina and Jarvis, Erich D. and Myers, Gene and Burgess, Shawn M. and Currie, Peter D. and Andersson, Leif and Schartl, Manfred}, title = {Low mutation rate in epaulette sharks is consistent with a slow rate of evolution in sharks}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-42238-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357827}, year = {2023}, abstract = {Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10\(^{-10}\) per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.}, language = {en} } @article{HaddadSocciChenetal.2016, author = {Haddad, Dana and Socci, Nicholas and Chen, Chun-Hao and Chen, Nanhai G and Zhang, Qian and Carpenter, Susanne G and Mittra, Arjun and Szalay, Aladar A and Fong, Yuman}, title = {Molecular network, pathway, and functional analysis of-time dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy}, series = {Molecular Therapy — Oncolytics}, volume = {3}, journal = {Molecular Therapy — Oncolytics}, doi = {10.1038/mto.2016.8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165855}, pages = {16008}, year = {2016}, abstract = {Background: Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy. Methods: Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software. Results: Differential gene analysis revealed a total of 12,412 up- and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways. Conclusions: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.}, language = {en} } @phdthesis{PrietoGarcia2022, author = {Prieto Garc{\´i}a, Cristian}, title = {USP28 regulates Squamous cell oncogenesis and DNA repair via ΔNp63 deubiquitination}, doi = {10.25972/OPUS-27033}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270332}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer resistance toward therapies inducing DNA damage. SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited, and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities, the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a potential alternative to improve the therapeutic response and clinical outcomes of SCC patients. However, ∆Np63 is considered "undruggable." As is commonly observed in transcription factors, ∆Np63 does not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of different pathways and cellular processes, making it difficult to counteract its function by targeting downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin-proteasome system (UPS), the development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target ∆Np63 in SCC treatment. This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability, and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time, ∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in preclinical mouse models. Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance, thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28 inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such as Cisplatin.}, language = {en} } @article{VeepaschitViswanathanBordonneetal.2021, author = {Veepaschit, Jyotishman and Viswanathan, Aravindan and Bordonne, Remy and Grimm, Clemens and Fischer, Utz}, title = {Identification and structural analysis of the Schizosaccharomyces pombe SMN complex}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {13}, doi = {10.1093/nar/gkab158}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259880}, pages = {7207-7223}, year = {2021}, abstract = {The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.}, language = {en} } @phdthesis{Kawan2024, author = {Kawan, Mona}, title = {The membrane trafficking protein myoferlin is a novel interactor of p97}, doi = {10.25972/OPUS-28121}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281218}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2024}, abstract = {p97 uses the energy of ATP hydrolysis to unfold and thereby segregate proteins. It is involved in various cellular processes such as proteasomal degradation, DNA damage repair, autophagy, and endo-lysosomal trafficking. The specificity for these processes is controlled by more than 30 regulatory cofactors. Interactions of p97 with cofactors and target proteins are known to be highly dynamic and transient. To identify new interaction partners and to uncover novel cellular functions of p97, the interactome of endogenous p97 was determined by using in cellulo crosslinking followed by immunoprecipitation and mass spectrometry. Myoferlin (MYOF) was identified as a novel interactor of p97 and the interaction was validated in reciprocal immunoprecipitation experiments for different cell lines. The ferlin family member MYOF is a tail-anchored membrane protein containing multiple C2 domains. MYOF is involved in various membrane repair and trafficking processes such as the endocytic recycling of cell surface receptors. The MYOF interactome was determined by mass spectrometry. Among others, the p97 cofactor PLAA, CD71 and Rab14 were identified as common interactors of p97 and MYOF. Immunoprecipitation experiments with PLAA KO cells revealed that the interaction between MYOF and p97 depends on PLAA. Immunofluorescence microscopy showed a co-localization of MYOF with Rab14 and Rab11, which are both involved in endocytic recycling pathways. Furthermore, immunofluoroscence experiments revealed that MYOF and the p97 cofactor PLAA are localized to Rab14- and Rab5-positive endosomal compartments. Using p97 inhibitors and p97 trapping mutants, the presence of p97 at MYOF-positive and Rab14-positive structures could be demonstrated. Consistent with this finding, the endocytic recycling of transferrin was delayed upon inhibition of p97. Taken together, this work identified MYOF as a novel interactor of p97 and suggests a role for p97 in the recycling of endocytic cargo.}, subject = {Endosom}, language = {en} }