The overall objective of the work described in this thesis was to characterise the three genes Hv14.3.3c, HvMAPKK1 and HvFKBP41, in terms of a role in defence and stress response signalling. These genes had previously been found to be differentially expressed in compatible versus incompatible interactions of barley with the fungus Rhynchosporium secalis, suggesting a possible role in the plant defence response, while current literature suggests these genes may also play a role in signal transduction, possibly under a broad range of stresses, including abiotic as well as biotic. Two main approaches were undertaken to characterise gene function: expression analysis and the identification of protein-protein interactions. To facilitate expression analysis, full length cDNA fragments of each gene were first obtained using bioinformatics, RACE and genomic walking techniques. Expression was then investigated using quantitative real-time RT-PCR. The results of the expression analysis confirmed that the candidate genes were in fact differentially expressed during infection, suggesting a role in the defence response of barley against R. secalis. Analysing their expression in the context of other stresses and treatments, namely frost, drought and ABA, indicated their role may not be limited only to biotic stress, but include abiotic stress as well. To investigate the possibility that these genes are involved in signalling during the defence response, protein-protein interaction techniques such as yeast two-hybrid and affinity pulldowns were used to identify interacting proteins in an attempt to place the genes within a known signalling network and build and extend on these networks. Y2H screening was used successfully to identify two putative interactors of Hv14.3.3c; an EPSP (5-enolpyruvylshikimate-3-phosphate) synthase and a putative wound-induced protein, and two interactors of HvFKBP41; a Rab-type GTPase and the same wound-induced protein. From what is known about the function of these genes in the literature, they fit well with a role in stress response signalling and the potential to be involved in signalling networks with the candidate gene products and also with each other. Through the trial of many different affinity pulldown techniques, a method for identifying interacting proteins from plant extracts was successfully established, however, issues with protein identification meant that interacting proteins were not identified using this technique. Steps were then made towards confirming the interactions identified using the Y2H system. Full length cDNA sequences of the identified interactors were obtained and expression analysis performed, in the aim of investigating co-expression patterns between the genes encoding the interacting proteins and the three candidate genes, to support a potential interaction. To confirm the Hv14.3.3c-HvEPSP interaction, co-immunoprecipitation and BRET were then used, however confirmation was unsuccessful due to issues with non-specific binding in co-immunoprecipitation and technical issues trying to establish the BRET analysis system in barley. In summary, the results of this study place the candidate genes Hv14.3.3c, HvMAPKK1 and HvFKBP41 as players in signal transduction during the plant defence/stress response. With the identification of previously uncharacterised protein interactions, some progress has also been made towards placing these genes within known signalling networks and identifying potential downstream genes that could possibly play a more specific role in defence response signalling and therefore be potential targets for the generation of resistant or stress tolerant plants. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2009

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

Os estudos em reprodução e desenvolvimento das plantas, especialmente voltados ao pistilo, são de grande interesse agronômico, econômico e científico. Em nosso laboratório, recentemente, foi identificado e caracterizado SCI1 (Stigma/style Cell-cycle Inhibitor 1), um inibidor do ciclo celular que atua de forma tecido específica no pistilo de Nicotiana tabacum L. e Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). Foi identificada a proteína NtCDKG;2 (N. tabacum Cyclin-dependent Kinase 2) como parceira de interação de NtSCI1 (N . tabacum SCI1), em um ensaio de pull-down (STRINI, 2014). A literatura aponta que os inibidores de ciclo celular regulam o ciclo através da inibição de CDK, o que sugere que NtSCI1 possa regular o ciclo celular através da inibição de NtCDKG;2. O presente estudo mostra análises detalhadas da localização de GFP-NtCDKG;2 em células epiteliais de N. benthamiana. Verificou-se que a proteína NtCDKG;2 está presente no nucleoplasma e também co-localiza em speckles nucleares. Em cultura de células BY2 expressando GFP-NtCDKG;2 de forma estável, foi observado que, durante a metáfase e anáfase, a proteína NtCDKG;2 está junto ao fuso acromático. Adicionalmente, ensaios de BiFC (Bi-molecular Fluorescence Complementation) realizados neste trabalho mostram que a interação entre as proteínas NtCDKG;2 e NtSCI1 ocorre em uma região localizada na periferia nucleolar, durante a interfase. Também foram identificadas possíveis isoformas de NtCDKG;2. A possibilidade da ocorrência de isoformas sugere que, de maneira análoga à sua homóloga em humanos, as isoformas resultantes de NtCDKG;2 possam atuar em diferentes processos. Em busca de parceiros de interação de NtCDKG;2, para identificar em que vias esta proteína atua, foi realizado um screening de uma biblioteca de cDNAs de estigmas e estiletes de N. tabacum, no sistema de duplo-híbrido em leveduras (Y2H). Através desse ensaio, foram identificados diversos parceiros envolvidos com transcrição e processamento de RNA. Dentre as proteínas identificadas, cuja interação foi confirmada neste trabalho, destaca-se a proteína NtCDKF;1, uma proteína que fosforila o CTD da RNA Polimerase II e, dessa forma, auxilia a transcrição e o splicing cotranscricional (HAJHEIDARI et al., 2012). O presente trabalho mostra também a interação entre NtCDKG;2 e a proteína NtCBP1, uma proteína que possui um papel importante na regulação inicial da transcrição de proteínas mediadoras do crescimento do tubo polínico (LI et al., 2015). xx Adicionalmente, o screening de Y2H possibilitou a identificação da interação entre NtCDKG;2 e NtRanBP1, uma proteína chave na formação do fuso acromático que, em humanos, interage com uma isoforma homóloga a NtCDKG;2, a CDK11p46 (MIKOLAJCZYK et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011). Análises in silico realizadas com a sequência de aminoácidos de NtCDKG;2 apontaram motivos de interação com proteína do tipo F-Box, ciclina, CDK, fosfatase, 14-3-3, BRCA1 e indicaram o local provável de interação do complexo CDK-Ciclina com o respectivo inibidor. Foi testada e comprovada a interação entre NtCDKG;2 e a 14-3-3D, por Y2H, uma parceira de NtSCI1. Outra lacuna que precisava ser preenchida é referente à regulação da expressão de NtSCI1. Com este intuito, foram realizadas análises in silico para identificar elementos cis-regulatórios na sequência genômica de NtSCI1. Essas análises indicaram a presença de importantes elementos cis-regulatórios relacionados à identidade meristemática (como WUSCHEL e AINTEGUMENTA), identidade do carpelo (AGAMOUS, BELL) e progressão do ciclo celular (E2F e CDC5). Algumas considerações podem ser feitas associando os resultados obtidos a estudos feitos paralelamente em nosso laboratório: 1) Compilando a localização de NtCDKG;2 em splicing speckles e sua interação com os diferentes parceiros de interação relacionados à transcrição e splicing, sugere-se que NtCDKG;2 também atue nos processos transcricionais e de splicing. 2) Considerando a localização subcelular de NtCDKG;2 durante as diferentes fases do ciclo celular, às análises in silico dessa proteína que identificaram sua possível interação com BRCA1, além da interação confirmada com a proteína NtRanBP1, é possível sugerir que NtCDKG;2 atue, direta ou indiretamente, na organização do fuso acromático de plantas. 3) Propõem-se que NtSCI1 regule a proliferação celular no pistilo através da interação com NtCDKG;2 que se dá no nucléolo das células. Dessa forma, NtSCI1 prenderia NtCDKG;2 no nucléolo e inibiria sua atuação, como na organização do fuso acromático, o que acarretaria inibição da divisão celular. 4) Devido aos motivos cis-regulatórios encontrados na sequência genômica de NtSCI1 e o efeito que a proteína possui desde as fases iniciais do desenvolvimento do pistilo, sugere-se que a expressão desse gene seja regulada por elementos diretamente envolvidos no controle do término do meristema floral e nas vias de desenvolvimento de órgãos florais. / Studies on plant reproduction and development, specifically those related to the pistil, are of great agronomic, economic and scientific interest. In our laboratory, we recently identified and characterized SCI1 (Stigma/style Cell-cycle Inhibitor 1), an inhibitor of the cell cycle which acts tissuespecifically in the pistil of Nicotiana tabacum L. and Arabidopsis thaliana (L.) Heynh. (DEPAOLI et al., 2011; DEPAOLI; DORNELAS; GOLDMAN, 2014). The NtCDKG;2 (N. tabacum Cyclin-dependent Kinase G; 2) protein was identified as an interaction partner of NtSCI1 (N. tabacum SCI1) in a pulldown assay (STRINI, 2014). The literature suggests that cell cycle inhibitors control the cycle through the inhibition of CDKs, indicating that NtSCI1 might control cell cycle by inhibiting NtCDKG;2. This study shows detailed analysis of GFP-NtCDKG;2 localization in leaf cells of N. benthamiana. The analysis shows that NtCDKG;2 is present in the nucleoplasm and also co-localizes with nuclear speckles. In BY2 cell culture stably expressing GFP-NtCDKG;2, it was observed that NtCDKG;2 is at the achromatic spindle during metaphase and anaphase. Additionally, BiFC (Bimolecular Fluorescence Complementation) assays performed in this study have shown that the interaction of NtCDKG;2 and NtSCI1 occurs in the nucleolar periphery during interphase. Putative isoforms of NtCDKG;2 were also identified. The possible occurrence of these isoforms suggests that, in a similar way to its human homologue, NtCDKG;2 putative isoforms could act in different processes. To identify in which processes this protein could act, a search for NtCDKG;2 interaction partners was performed through the screening of a N. tabacum stigma and style cDNA library in the yeast two-hybrid (Y2H) system. Several partners identified through this assay have roles in RNA transcription and processing. Among the identified partners with interaction confirmed during this work, stands out the NtCDKF;1 protein, a CDK that phosphorylates the RNA polymerase II CTD, and thus, supports transcription and co-transcriptional splicing (HAJHEIDARI et al., 2012). This study also shows the interaction of NtCDKG;2 with NtCBP1, a protein which has an important role in the transcriptional regulation of genes encoding proteins mediating pollen tube growth (LI et al., 2015). Furthermore, the Y2H screening allowed the identification of the interaction of NtCDKG;2 with NtRanBP1, a key protein in the formation of the achromatic spindle which, in humans, interacts with the CDK11p46 isoform (MIKOLAJCZYK xxii et al., 2003; YOKOYAMA et al., 2008; ZHANG; DAWE, 2011), a homologue of NtCDKG;2. In silico analysis of the amino acid sequence of NtCDKG;2 revealed motifs of predicted interaction with F-box proteins, cyclins, CDKs, phosphatases, 14-3-3s, BRCA1, and also pointed the region where the CDK-cyclin complex might interact with its respective inhibitor. The interaction of NtCDKG;2 with 14-3-3D, a known partner of NtSCI1, was tested and confirmed by Y2H. Another gap that needed to be filled is related to the regulation of NtSCI1 expression. To address this issue, in silico analysis to identify cis-regulatory elements was performed in NtSCI1 genomic region. These analyses revealed the presence of important cis-regulatory elements related to meristem identity (such as WUSCHEL and AINTEGUMENTA), carpel identity (AGAMOUS, BELL), and cell cycle progression (E2F and CDC5). Taken together results from this study and parallel studies performed in our laboratory, a few remarks can be made: 1) Taken the localization of NtCDKG;2 in splicing speckles, and its interaction with different proteins involved in transcription and splicing, it is suggested that NtCDKG;2 also has roles on these processes; 2) Considering the subcellular localization of NtCDKG;2 during the different cell cycle phases, the in silico analysis of this protein that predicts its interaction with BRCA1, and the confirmed interaction with NtRanBP1 protein, it is possible to suggest that NtCDKG;2 has a direct or indirect role in the organization of the achromatic spindle in plants; 3) It is proposed that NtSCI1 regulates cell proliferation in the pistil through its interaction with NtCDKG;2, which occurs in the nucleolus. Thus, NtSCI1 could hold NtCDKG;2 in the nucleolus, inhibiting its actions, such as in the organization of the achromatic spindle, resulting in cell division arrest. 4) Due to the cis-regulatory elements found in the genomic sequence of NtSCI1, and the effect of this protein since the initial stages of pistil development, it is suggested that its expression is regulated by elements directly involved in the control of the floral meristem termination and pathways of floral organ development.

Seasonal and pandemic Influenza virus infections cause about three to five million cases of severe illness and about 250,000 to 500,000 deaths world-wide annually according to the WHO. Although investigated intensively, Influenza virus pathogenesis is still not very well understood and hard to predict. Influenza A viruses contain a segmented, single-(-) stranded RNA genome encoding at least 10 different proteins and are highly diverse due to hypermutation and reassortment. In previous work, 56 viral genes from six different influenza A virus isolates had been cloned and genome-wide screened for virus-host protein interactions using yeast-two hybrid technology and several human and chicken cDNA libraries, leading to the identification of 127 high-confidence cellular interactors of which 40 have also been identified by RNA interference in other studies. In this thesis, two of the cellular interactors identified which both bound to the viral multifunctional protein NS1, TRBP and PACT, were further investigated with regard to their role in virus life cycle. These two proteins are known to be involved in miRNA silencing and PKR regulation. Both interactions between NS1 and TRBP and NS1 and PACT were confirmed by co-immunoprecipitation, and both TRBP and PACT co-localized with NS1 in a cytosolic compartment. NS1 was also found to be present in the RISC complex in pull-down assays with the RISC core component Ago2. In functional assays, NS1 dose-dependently inhibited RNA silencing. Although no differences in TRBP-binding between NS1 proteins of various different influenza strains could be detected in direct mating Y2H assays, they varied with regard to their inhibitory activity on RNA silencing. TRBP and PACT alone were unable to restore NS1-induced inhibition of RNA silencing activity, however both together restored RNA silencing. Moreover, the siRNA knockdown of PACT abolished the association of NS1 with Ago2, and NS1 competitively inhibited the binding of TRBP and PACT to Ago2. The depletion of either TRBP or PACT led to an inhibition of influenza virus replication. The depletion of TRBP also lifted cellular IFNβ level without infection. However, the knockdown of TRBP but not PACT blocked IFNβ production and increased cell viability post infection. These results indicate that NS1 inhibits the binding of PACT and TRBP to the RISC complex and thereby inhibits miRNA-induced gene silencing. The hypothesis that TRBP supports influenza replication potentially by regulating PKR regulation and IFNβ induction requires further investigation. In conclusion, this study provides evidence for the complexity of virus-host interactions and the dual role of viral proteins in activating both positive and negative regulatory cellular mechanisms.

Veränderungen in phosphorylierungsabhängigen Signalwegen, Akkumulation von Proteinaggregaten im Gehirn und neuronaler Zelltod sind Neurodegenerationskennzeichen und Indikatoren für überlappende molekulare Mechanismen. Um Einblicke in die involvierten Signalwege zu erhalten, wurde mit Hilfe eines modifizierten Hefe-Zwei-Hybrid (Y2H)-Systems für 71 Proteine, die mit neurologischen Erkrankungen assoziiert sind, proteomweit nach Protein-Protein Interaktionen (PPIs) gesucht. Für 21 dieser Proteine wurden PPIs identifiziert. Das Gesamtnetzwerk besteht aus 79 Proteinen und 90 PPIs von denen 5 phosphorylierungsabhängig sind. Ein Teil dieser PPIs wurde in unabhängigen Interaktionsassays mit einer Validierungsrate von 66 % getestet. Der netzwerkbasierte Versuch verbindet erfolgreich neurologische Erkrankungen untereinander aber auch mit zellulären Prozessen. Ser/Thr-Kinase abhängige PPIs verknüpfen zum Beispiel das Parkinson Protein 7 (PARK7, DJ1) mit den E3 Ligase Komponenten ASB3 und RNF31 (HOIP). Die Funktion dieser Proteine bekräftigt den Zusammenhang zwischen dem Ubiquitin-Proteasom-System und der Parkinson Krankheit (PD). Neurofibromin 2 (NF2, merlin) Isoformen und PARK7 interagieren mit der regulatorischen PI3K Untereinheit p55-gamma (PIK3R3). Diese PPIs basieren auf Tyr-Kinase Aktivität im modifizierten Y2H System und funktionellen PIK3R3 pTyr-Erkennungsmodulen (SH2 Domänen) in co-IP und Venus PCA Versuchen. Dies verknüpft den PI3K/AKT Überlebenssignalweg mit zwei unterschiedlichen neurologischen Erkrankungsphenotypen: dem PD assoziierten neuronalen Zelltod und der Neurofibromatose Typ 2-assoziierten Tumorentstehung. Die vergleichende Beobachtung von PIK3R3, AOF2 (KDM1A, LSD1) Interaktionen auf NF2 Isoformlevel offenbart eine Bevorzugung von Isoform 7 bei zytoplasmatischer Lokalisation, wohingegen Isoform 1 PPIs an der Membran lokalisiert sind. Das modifizierungsabhängige und isoformspezifische PPI Netzwerk ermöglichte neue Hypothesen zu molekularen Pathomechanismen. / Alterations in phosphorylation-dependent signalling pathways, accumulation of aggregated proteins in the brain and neuronal apoptosis are common to neurodegeneration and implicate overlapping molecular mechanism. To gain insight into involved pathways, a modified yeast-two hybrid (Y2H) system was applied to screen 71 proteins associated with neurological disorders in a proteome-wide manner. For 21 of these proteins interactions were identified including 5 phosphorylation-dependent ones. In total, the network connected 79 proteins through 90 protein-protein interactions (PPIs). A fraction of these Y2H PPIs was tested in secondary interaction assays with a validation rate of 66 %. The described network-based approach successfully identified proteins associated with more than one disorder and cellular functions connected to specific disorders. In particular, the network revealed Ser/Thr kinase-dependent PPIs between the Parkinson protein 7 (PARK7, DJ1) and the E3 ligase components ASB3 and RNF31 (HOIP). The function of these proteins further substantiates the established connection between Parkinson’s disease (PD) and ubiquitination-mediated proteasome (dis)functions. Neurofibromin 2 (NF2, merlin) isoforms and PARK7 were identified as PI3K regulatory subunit p55-gamma (PIK3R3) interactors. These PPIs required Tyr kinase coexpression in the modified Y2H system and functional PIK3R3 pTyr-recognition modules (SH2 domains) in co-IP and Venus PCA experiments. This finding implicates the PI3K/AKT survival pathway in PD-associated neuronal apoptosis and Neurofibromatosis type 2-associated tumour formation. Investigation of PIK3R3, AOF2 (KDM1A, LSD1) and EMILIN1 PPIs on NF2 isoform level revealed preferential isoform 7 binding and cytoplasmic or membrane localisation of these PPIs for isoform 7 or 1, respectively. The generated modification-dependent and isoform-specific PPI network triggered many hypotheses on the molecular mechanisms implicated in neurological disorders.

Die Muscle-RING-finger (MuRF) Proteine sind E3-Ubiquitin-Ligasen, die im Muskelgewebe den Ubiquitin-Proteasom-System abhängigen Abbau von Proteinen vermitteln. MuRF1 wird in der Muskelatrophie verstärkt synthetisiert, was zu einem gesteigerten Proteinabbau und damit zum Verlust von Muskelmasse führt. Zudem sind Mäuse, denen MuRF1 fehlt vor Muskelatrophie geschützt. E3-Ubiquitin-Ligasen fungieren oftmals in Multiproteinkomplexen. Dies wurde für MuRF-Proteine bisher nicht gezeigt. Aufgrund dessen sollten neue MuRF-Multiproteinkomplex assoziierte Faktoren mittels Hefe-Zwei-Hybrid-System und SILAC AP-MS identifiziert und deren Einfluss auf die MuRF-Funktion charakterisiert werden. Es wurden sowohl neue als auch publizierte MuRF-Interaktionspartner (Iap) gefunden. Von den neu entdeckten MuRF-Iap wurde der Fokus auf WDR42A gelegt, da das Protein mit beiden Methoden identifiziert wurde und zudem funktionell hoch interessant ist. WDR42A homologe Proteine bilden zirkuläre β-Propeller Strukturen die Multiproteinkomplexe koordinieren. Die Interaktion zwischen MuRF-Proteinen und WDR42A wurde mittels Ko-IP Experimenten und Kolokalisationsstudien bestätigt. Cycloheximid-Abbau-Experimente deuten darauf hin, dass WDR42A kein MuRF1 Substrat-Protein ist. Da die MuRF-Proteine spezifisch im Muskel hergestellt werden, sollte überprüft werden ob WDR42A ebenfalls im Muskelgewebe synthetisiert wird. Es wurde gezeigt, dass WDR42A ubiquitär sowie im Muskelgewebe und in immortalisierten Muskelzellen hergestellt wird. Analog zu MuRF1 wird WDR42A in der Denervations-induzierten Skelettmuskelatrophie und der Muskelentwicklung verstärkt synthetisiert. Die Herunterregulation von WDR42A mittels siRNA in C2C12 Myotuben schützte diese Zellen vor dem Auftreten von Atrophie. Diese Ergebnisse zeigen, dass WDR42A wie MuRF1 an der Entstehung von Muskelatrophie beteiligt ist. Aufgrund der WDR42A Domänenstruktur wird vermutet, dass WDR42A als Scaffolding-Protein MuRF1-Multiproteinkomplexe reguliert. / The muscle-RING-finger (MuRF) proteins are E3 ubiquitin ligases which coordinate the ubiquitin-proteasome system dependent protein degradation in muscle tissue. MuRF1 is up-regulated under muscle atrophy conditions. This leads to enhanced proteolysis and thereby to loss of muscle mass and strength. Furthermore are MuRF1 knockout mice resistant to muscle atrophy. E3 ubiquitin ligases often operate in multi-protein complexes. This has not been shown for MuRF proteins. Therefore we used yeast-two-hybrid and SILAC-AP-MS to identify and subsequently characterize new MuRF multi-protein complex associated proteins. We found new and also published MuRF interaction partners (Iap) with both methods. Amongst the new Iap, we focused on WDR42A, because it was found with both techniques and his interesting functional potential. WDR42A exhibits seven consecutive arranged WD40-repeat domains. This domain arrangement leads in homologues proteins to the formation of seven-bladed β-propeller structures, which act as protein interaction platforms that coordinate multi-protein complexes. The protein interaction between the MuRFs and WDR42A was confirmed with Co-IP and co-localization experiments. Cycloheximide decay experiments indicated that WDR42A is not a MuRF1 substrate protein. The MuRF proteins are muscle specific, therefore we tested if WDR42A is also synthetized in muscle tissue. We could show that WDR42A is ubiquitously, but also in muscle tissue as well as in immortalized muscle cells produced. WDR42A is similar to MuRF1 up-regulated under denervation-induced skeletal muscle atrophy as well as in muscle development. Furthermore are C2C12 myotubes resistant to muscle atrophy after siRNA down-regulation of WDR42A. These results demonstrate that WDR42A is like MuRF1 important for the development of muscle atrophy. Due to the domain structure of WDR42A, we hypothesize that WDR42A regulates MuRF1 multi protein complexes as scaffolding protein.

No description available.

Molecular biology has entered an era of systematic and automated experimentation. High-throughput techniques have moved biology from small-scale experiments focused on specific genes and proteins to genome and proteome-wide screens. One result of this endeavor is the compilation of complex networks of interacting proteins. Molecular biologists hope to understand life's complex molecular machines by studying these networks. This thesis addresses tree open problems centered upon their analysis and quality assessment. First, we introduce power graph analysis as a novel approach to the representation and visualization of biological networks. Power graphs are a graph theoretic approach to lossless and compact representation of complex networks. It groups edges into cliques and bicliques, and nodes into a neighborhood hierarchy. We demonstrate power graph analysis on five examples, and show its advantages over traditional network representations. Moreover, we evaluate the algorithm performance on a benchmark, test the robustness of the algorithm to noise, and measure its empirical time complexity at O (e1.71)- sub-quadratic in the number of edges e. Second, we tackle the difficult and controversial problem of data quality in protein interaction networks. We propose a novel measure for accuracy and completeness of genome-wide protein interaction networks based on network compressibility. We validate this new measure by i) verifying the detrimental effect of false positives and false negatives, ii) showing that gold standard networks are highly compressible, iii) showing that authors' choice of confidence thresholds is consistent with high network compressibility, iv) presenting evidence that compressibility is correlated with co-expression, co-localization and shared function, v) showing that complete and accurate networks of complex systems in other domains exhibit similar levels of compressibility than current high quality interactomes. Third, we apply power graph analysis to networks derived from text-mining as well to gene expression microarray data. In particular, we present i) the network-based analysis of genome-wide expression profiles of the neuroectodermal conversion of mesenchymal stem cells. ii) the analysis of regulatory modules in a rare mitochondrial cytopathy: emph{Mitochondrial Encephalomyopathy, Lactic acidosis, and Stroke-like episodes} (MELAS), and iii) we investigate the biochemical causes behind the enhanced biocompatibility of tantalum compared with titanium.

Die vorliegende Arbeit beschäftigt sich mit verschiedenen Aspekten der Assoziation von Saccharose-Synthase (SUS) mit subzellulären Strukturen. Durch cDNA-Durchmusterungen konnten proteinogene Bindepartner von SUS sowie Aktin identifiziert und zum Teil verifiziert werden. Die dritte Isoform SuS3 aus Mais wurde auf molekularer Ebene identifiziert und das rekombinante Protein biochemisch charakterisiert. Trotz signifikanter Sequenzunterschiede zwischen den SUS-Isoformen, wurden ähnliche katalytische Eigenschaften und mögliche posttranslationale Modifikationen der Enzyme nachgewiesen, darunter die Redox-Modifikation der Enzymaktivität und das Potential zur reversiblen Phosphorylierung. Der Einfluss der Phoshorylierung von SUS auf dessen enzymatische und assoziative Aktivität wurde mittels mutagenisiertem Protein untersucht und zeigte kein stark verändertes Verhalten infolge der Mutationen. Eine metabolische Regulation der SUS-Aktin-Wechselwirkung durch Zucker konnte bestätigt und die katalytische Aktivität von SUS in Gegenwart von Aktin gezeigt werden. Assoziationsstudien von Aktin mit synthetischen Peptiden sowie immunologische Untersuchungen lieferten Hinweise für die Aktinbindedomäne in SUS. Co-Pelletierungsexperimente zeigten die Assoziation von SUS mit Mikrotubuli aus Rinderhirn. In vitro konkurriert SUS mit Aldolase um die Bindung an Miktotubuli. Als proteinogene Bindepartner von SUS wurden einige im Kohlenhydratstoffwechsel sowie im 26S-Proteasom-Komplex involvierte Proteine identifiziert. Ebenso wurde eine Glutathion-Peroxidase identifiziert, die ubiquitäre Transkriptakkumulation dokumentiert und die katalytische Aktivität des rekombinanten Proteins gezeigt. Eine weitere cDNA-Durchmusterung führte zur Identifikation verschiedener glykolytischer Enzyme als potentielle Interaktionspartner von Mais-Aktin sowie zu Bindepartnern, die nach Sequenzanalyse Domänen mit Homologien zu bekannten ABPs aus tierischen Organismen zeigten.

Proteinmethylierung spielt eine immer größere Rolle in der Regulierung zellulärer Prozesse. Die Entwicklung effizienter proteomweiter Methoden zur Detektion von Methylierung auf Proteinen ist limitiert und technisch schwierig. In dieser Arbeit haben wir einen neuen Hefe-Zwei-Hybrid-Ansatz (Y2H) entwickelt, der Proteine, die miteinander wechselwirken, mit Hilfe von Sequenzierungen der zweiten Generation identifiziert (Y2H-Seq). Der neue Y2H-Seq-Ansatz wurde systematisch mit dem Y2H-Seq-Ansatz verglichen. Dafür wurde ein Bait-Set von 8 Protein-Arginin-Methyltransferasen, 17 Protein-Lysin-Methyltransferasen und 10 Demethylasen gegen 14,268 Prey-Proteine getestet. Der Y2H-Seq-Ansatz ist weniger arbeitsintensiv, hat eine höhere Sensitivität als der Standard Y2H-Matrix-Ansatz und ist deshalb besonders geeignet, um schwache Interaktionen zwischen Substraten und Protein-Methyltransferasen zu detektieren. Insgesamt wurden 523 Wechselwirkungen zwischen 22 Bait-Proteinen und 324 Prey-Pr oteinen etabliert, darunter 11 bekannte Methyltransferasen-Substrate. Netzwerkanalysen zeigen, dass Methyltransferasen bevorzugt mit Transkriptionsregulatoren, DNA- und RNA-Bindeproteinen wechselwirken. Diese Daten repräsentieren das erste proteomweite Wechselwirkungsnetzwerk über Protein-Methyltransferasen und dienen als Ressource für neue potentielle Methylierungssubstrate. In einem in vitro Methylierungsassay wurden exemplarisch mit Hilfe massenspektrometrischer Analysen die methylierten Aminosäurereste einiger Kandidatenproteine bestimmt. Von neun getesteten Proteinen waren sieben methyliert, zu denen gehören SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP und WDR42A. Wahrscheinlich sind viele Methylierungssubstrate im Netzwerk vorhanden. Das vorgestellte Protein-Protein-Wechselwirkungsnetzwerk zeigt, dass Proteinmethylierung sehr unterschiedliche zelluläre Prozesse beeinflusst und ermöglicht die Aufstellung neuer Hypothesen über die Regulierung Molekularer Mechanismen durch Methylierung. / Protein methylation on arginine and lysine residues is a largely unexplored posttranslational modification which regulates diverse cellular processes. The development of efficient proteome-wide approaches for detecting protein methylation is limited and technically challenging. We developed a novel workload reduced yeast-two hybrid (Y2H) approach to detect protein-protein interactions utilizing second generation sequencing. The novel Y2H-seq approach was systematically evaluated against our state of the art Y2H-matrix screening approach and used to screen 8 protein arginine methyltransferases, 17 protein lysine methyltransferases and 10 demethylases against a set of 14,268 proteins. Comparison of the two approaches revealed a higher sensitivity of the new Y2H-seq approach. The increased sampling rate of the Y2H-seq approach is advantageous when assaying transient interactions between substrates and methyltransferases. Overall 523 interactions between 22 bait proteins and 324 prey proteins were identified including 11 proteins known to be methylated. Network analysis revealed enrichment of transcription regulator activity, DNA- and RNA-binding function of proteins interacting with protein methyltransferases. The dataset represents the first proteome-wide interaction network of enzymes involved in methylation and provides a comprehensively annotated resource of potential new methylation substrates. An in vitro methylation assay coupled to mass spectrometry revealed amino acid methylation of candidate proteins. Seven of nine proteins tested were methylated including SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP and WDR42A indicating that the interaction network is likely to contain many putative methyltransferase substrate pairs. The presented protein-protein interaction network demonstrates that protein methylation is involved in diverse cellular processes and can inform hypothesis driven investigation into molecular mechanisms regulated through methylation.