CHARLES UNIVERSITY IN PRAGUE THE FACULTY OF NATURAL SCIENCE Department of Physical and Macromolecular Chemistry The summary of the doctoral thesis Study of interactions of forkhead transcription factor FOXO4 with DNA and the 14-3-3 protein RNDr. Petr Vácha Scientific supervisor: Prof. RNDr. Tomáš Obšil, Ph.D. Prague 2015 Abstract This doctoral thesis deals with the interaction of human forkhead transcription factor FOXO4 with DNA and regulating 14-3-3 protein respectively. The main aim of this work was detailed characterization of interaction between DNA binding domain of protein FOXO4 with two canonical DNA sequences and further clarifying the role of the 14-3-3 protein in the regulation of activity of protein FOXO4. FOXO transcription factors are potent activators of the transcription of genes, which affect a variety of cellular processes. FOXO4 protein belongs to the family of forkhead transcription factor, which is a group of several tens of proteins, whose common feature is a highly conserved DNA- binding domain. Summary of the DNA binding specificity of these proteins, namely what precisely determines the small differences in the binding properties of individual forkhead proteins, despite the large amount of available structural data remains still unclear. Therefore, detailed characterization of...

This dissertation is focused on 30 kDa protein phosducin (Pdc) and on the regulation of its function through the interaction with 28 kDa adaptor protein 14-3-3. These two proteins participate in G-protein signal transduction pathways, especially in the process of light signal transduction. It is assumed that Pdc binds to the Gtβγ complex of G-protein called transducin and through this interaction it inhibits the reassociation of Gtβγ with Gtα thus reducing the visual signal transfer. This process is thought to participate in a long- term light adaptation. The regulation of Pdc function is further regulated by its phosphorylation and subsequent binding to the 14-3-3 protein. It has been speculated that the 14-3-3 binding plays a key role in the inhibition of the interaction between phosphorylated Pdc (Pdc-PP) and Gtβγ. The formation of the 14-3-3/Pdc-PP complex leads to the reassociation of Gtβγ with Gtα and consequently to the amplification of visual signal transfer. Nevertheless, the mechanism by which the 14-3-3 protein binding inhibits the interaction between Pdc and Gtβγ remains elusive. The main aims of this dissertation were: (i) to investigate the structure of Pdc in its apo-state (in the absence of the binding partner) and in the complex with 14-3-3, and (ii) to suggest the mechanism of the...

EN Protein-protein interactions have an exceptional position among other mechanisms in the regulation of signal transduction. Their systematic investigation is very important and logical step in the process of understanding to the transduction and its mechanisms at a molecular level. During my Ph.D. I was particularly interested in three important processes. ASK1 kinase is well-known initiator of the apoptosis. Under physiological conditions it is maintained in an inactive state by its two interaction partners the 14-3-3 protein and TRX1. These two proteins dissociate in the presence of reactive oxygen species by unclear mechanism and the kinase is therefore activated. The next process is an interaction between the 14-3-3 protein and phosducin and investigation of their role in the G protein signalling especially important in the biochemistry of vision. The third process is an activation of protein Nth1 through the interaction with Bmh1, yeast analog of the 14-3-3 protein, and calcium cations. I employed various biophysical method, particularly analytical ultracentrifugation, in order to explain molecular mechanisms of described processes. These techniques were used to solve the low-resolution structures of complexes TRX1 and the 14-3-3 protein with corresponding binding domains of ASK1. These...

Regulation of class IIa histone deacetylases (HDACs) phosphorylation is crucial because it provides the opportunity to control important developmental processes associated with these key enzymes. Indeed, the transcriptional repressor activity of class IIa HDAC is controlled via their phosphorylation-dependent nucleo-cytoplasmic shuttling. While a lot of efforts have been directed towards the identification of the inactivating kinases that phosphorylate class IIa HDACs, the identity of the antagonist phosphatase remained an open question. During this work, we found that protein phosphatase 2A (PP2A) is responsible for dephosphorylating the class IIa HDACs member HDAC7, thereby regulating its subcellular localization and repressor activity. In order to validate our model, functional consequences of these findings was illustrated during the two main biological processes involving HDAC7, i.e. T-cells apoptosis during negative selection and endothelial cells angiogenic activities during vascular network formation. Cellular PP2A represents a large population of trimeric holoenzymes containing a variable regulatory subunit, whose identity has a crucial role in determining the specificity of PP2A catalytic activity. In an effort to characterize the regulation of HDAC7 dephopshorylation, we identified the relevant PP2A holoenzyme regulating HDAC7 function during vasculogenesis and we found that, among diverse regulatory subunit isoforms, PP2A-Bα uniquely regulates endothelial cell angiogenic properties. PP2A-Bα silencing using small interfering RNAs results in a significant inhibition of endothelial cell tube formation and migration. These results establish PP2A, and more precisely the Bα containg PP2A holoenzyme, as an essential element in the regulation of the class IIa HDAC HDAC7 and unravel a first developmental function for the PP2A regulatory subunit Bα in the genesis of blood vessels.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Fyzikální chemie Mgr. Lenka Řežábková Studium úlohy proteinů 14-3-3 v regulaci G-proteinové signalizace Role of the 14-3-3 proteins in the regulation of G-protein signaling Disertační práce Školitel: doc. RNDr. Tomáš Obšil, Ph.D. Konzultanti: doc. RNDr. Petr Heřman, CSc. doc. RNDr. Jaroslav Večeř, CSc. Praha, 2012 Abstract The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of their binding partners in various signaling pathways that control diverse physiological and pathological processes such as signal transduction, metabolic pathways, cell cycle and apoptosis. More than 300 different cellular proteins from diverse eukaryotic organisms have been described as binding partners for the 14-3-3 proteins. During my Ph.D., I was particularly interested in the role of 14-3-3 proteins in the regulation of G protein signaling pathway. The 14-3-3 proteins affect the G protein signaling via the interaction with negative regulators of G protein cascade - the RGS proteins and phosducin. I employed both biochemical and biophysical approaches to understand how the activity and function of RGS3/14-3-3 and phosducin/14-3-3 complexes are regulated. I solved the low-resolution solution structure of...

The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...

Our understanding of non-histone acetylation as a means of cellular regulation is in its infancy. Using a mass spectrometry approach we identified acetylated lysine residues and monitored acetylation changes across the proteome as a consequence of metabolic stress (hypoxia). We observed changes in acetylation status of non-histone lysines in tumor cells. Through the use of small molecule inhibitors of histone deacetylase enzymes (HDACs) and siRNA screening identified HDAC6 as a pro-survival regulator of lysine acetylation during hypoxia. The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, K49 and K120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the K49/K120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well-described interacting partners, Bad and AS160, which triggers their dephosphorylation at S112 and T642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and S112/T642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.

No description available.

A modulação da forma e tamanho em órgãos vegetais depende do controle temporal e espacial de divisão e expansão celular. Entretanto, pouco se sabe a respeito dos mecanismos moleculares que regulam este processo durante o desenvolvimento floral. O estudo da via de sinalização de SCI1 (Stigma/style Cell Cycle Inhibitor 1) pode contribuir para o entendimento do processo de crescimento das flores. Este gene produz uma proteína, localizada no nucléolo, que está relacionada à inibição da proliferação celular no estigma e no estilete de Nicotiana tabacum, modulando o tamanho destes órgãos florais. Experimentos feitos para a identificação de parceiros de interação de SCI1 identificaram as proteínas 14-3-3A e 14-3-3D de N. tabacum como candidatas à interação. A família 14-3-3 é composta de proteínas altamente conservadas, que formam dímeros em sua conformação nativa e são responsáveis pela modulação da atividade das mais variadas proteínas em resposta a sinais intracelulares. Por isso, estas proteínas estão associadas à regulação de uma série de processos, incluindo o metabolismo, transcrição, ciclo celular, entre outros. Neste contexto, o presente trabalho teve como objetivo estudar as proteínas 14-3-3A e 14-3-3D de N. tabacum e o seu papel durante o desenvolvimento floral. Os resultados aqui obtidos revelaram que a 14-3-3A possui localização citoplasmática e nuclear, enquanto a 14-3-3D se distribui apenas no citoplasma. Também foi evidenciado que estas proteínas são capazes de formar homodímeros e heterodímeros entre si. Os homodímeros de 14-3-3A estão distribuídos no citoplasma e no núcleo, enquanto os homodímeros de 14-3-3D e heterodímeros se encontram apenas no citoplasma. Adicionalmente, a interação in vivo entre a 14-3-3A e SCI1 foi confirmada por BiFC, apresentando localização nuclear, fora do nucléolo. Análises in silico da sequência de aminoácidos de SCI1 identificaram duas regiões putativas de reconhecimento por proteínas 14-3-3s. Estas regiões estão sendo analisadas funcionalmente por meio de ensaios de BiFC com sequências mutadas de SCI1. A análise deste conjunto de resultados, juntamente com outros resultados obtidos em nosso laboratório, sugere que apenas homodímeros de 14-3-3D e heterodímeros formados entre 14-3-3A e 14-3-3D sejam capazes de interagir com SCI1. Adicionalmente, a localização nuclear dessa interação difere daquelas observadas para SCI1 e para as 14-3-3s individualmente, sugerindo que as 14-3-3s migrem para o núcleo para interagir com SCI1. Nossa hipótese é de que as proteínas 14-3-3s possam modular a localização subnuclear de SCI1. Com o objetivo de levantar dados a respeito das possíveis funções desempenhadas pelas proteínas 14-3-3A e 14-3-3D de N. tabacum, foram identificados os grupos de possíveis ortólogos dessas proteínas em A. thaliana, O. sativa, S. lycopersicum, S. tuberosum e N. benthamiana. Esta análise mostrou que os ortólogos às 14-3-3A e D em Arabidopsis estão associados ao ciclo celular, o que sugere que as proteínas de tabaco possam ter conservado essa função. Além disso, também foram produzidas plantas transgênicas silenciadas para cada uma dessas 14-3-3s de maneira independente. A análise dos fenótipos das plantas transgênicas não levou à elaboração de uma hipótese definitiva sobre a função dessas 14-3-3s no desenvolvimento floral. No entanto, algumas plantas transgênicas apresentaram estruturas menores, especialmente as pétalas, sugerindo que estas proteínas podem estar envolvidas no controle do tamanho de órgãos vegetais. / The modulation of size and shape in plant organs depends on temporal and spatial control of cell division and expansion. However, the molecular mechanisms that regulate this process during floral development are poorly understood. The study of SCI1 (Stigma/style Cell Cycle Inhibitor 1) signaling pathway can contribute to the understanding of the flower growing process. This gene produces a protein which is located in the nucleolus and is related to the inhibition of cell proliferation in the Nicotiana tabacum stigma and style, modulating the size of these organs. Experiments performed to identify SCI1 interaction partners have identified the N. tabacum 14-3-3A and 14-3-3D proteins as interaction candidates. The 14-3-3 family is composed of highly conserved proteins, which form dimers in their native conformation and are responsible to modulate the activity of a large variety of proteins in response to intracellular signals. Therefore, these proteins are associated to the regulation of several processes, including metabolism, transcription, and cell cycle, among others. In this context, the present work aimed to study the N. tabacum 14-3-3A and 14-3-3D proteins and their role during flower development. The results here obtained revealed that 14-3-3A is located in the nucleus and the cytosol, while 14-3-3D protein is distributed only in the cytosol. It was also shown that these proteins can form homodimers and heterodimers with each other. Homodimers of 14-3-3A are distributed in nucleus and cytosol, while 14-3-3D homodimers and heterodimers are located only in the cytosol. Furthermore, the in vivo interaction between SCI1 and 14-3-3A was confirmed by BiFC, showing nuclear localization, outside the nucleolus. In silico analyzes of SCI1 amino acid sequence identified two putative regions of recognition by 14-3-3 proteins. These regions are being evaluated by BiFC assays with SCI1 mutated sequences. The analyses of this set of results, together with other results obtained in our laboratory, suggests that only 14-3-3D homodimers and heterodimers between 14-3-3A and 14-3-3D are capable to interact with SCI1. Moreover, the nuclear localization of this interaction differs from the ones observed for SCI1 and for the 14-3-3s individually, which suggests that the 14-3-3s migrate to the nucleus to interact with SCI1. Our hypothesis is that the 14-3-3 proteins can modulate the subnuclear localization of SCI1. To obtain data concerning the possible roles of the N. tabacum 14-3-3A and 14-3-3D proteins, groups of possible orthologous of these proteins in A. thaliana, O. sativa, S. lycopersicum, S. tuberosum and N. benthamiana were identified. This analysis has shown that the orthologs of 14-3-3A and D in Arabidopsis are associated to the cell cycle, suggesting that the tobacco proteins might have conserved this function. Furthermore, transgenic plants silenced for each of the 14-3-3s independently were also produced. Phenotype analyzes of transgenic plants did not lead to a definitive hypothesis about the function of these 14-3-3s during floral development. However, some transgenic plants exhibited smaller structures, specially petals, which suggests that these proteins may be involved in the size control of plant organs.

A modulação da forma e tamanho em órgãos vegetais depende do controle temporal e espacial de divisão e expansão celular. Entretanto, pouco se sabe a respeito dos mecanismos moleculares que regulam este processo durante o desenvolvimento floral. O estudo da via de sinalização de SCI1 (Stigma/style Cell Cycle Inhibitor 1) pode contribuir para o entendimento do processo de crescimento das flores. Este gene produz uma proteína, localizada no nucléolo, que está relacionada à inibição da proliferação celular no estigma e no estilete de Nicotiana tabacum, modulando o tamanho destes órgãos florais. Experimentos feitos para a identificação de parceiros de interação de SCI1 identificaram as proteínas 14-3-3A e 14-3-3D de N. tabacum como candidatas à interação. A família 14-3-3 é composta de proteínas altamente conservadas, que formam dímeros em sua conformação nativa e são responsáveis pela modulação da atividade das mais variadas proteínas em resposta a sinais intracelulares. Por isso, estas proteínas estão associadas à regulação de uma série de processos, incluindo o metabolismo, transcrição, ciclo celular, entre outros. Neste contexto, o presente trabalho teve como objetivo estudar as proteínas 14-3-3A e 14-3-3D de N. tabacum e o seu papel durante o desenvolvimento floral. Os resultados aqui obtidos revelaram que a 14-3-3A possui localização citoplasmática e nuclear, enquanto a 14-3-3D se distribui apenas no citoplasma. Também foi evidenciado que estas proteínas são capazes de formar homodímeros e heterodímeros entre si. Os homodímeros de 14-3-3A estão distribuídos no citoplasma e no núcleo, enquanto os homodímeros de 14-3-3D e heterodímeros se encontram apenas no citoplasma. Adicionalmente, a interação in vivo entre a 14-3-3A e SCI1 foi confirmada por BiFC, apresentando localização nuclear, fora do nucléolo. Análises in silico da sequência de aminoácidos de SCI1 identificaram duas regiões putativas de reconhecimento por proteínas 14-3-3s. Estas regiões estão sendo analisadas funcionalmente por meio de ensaios de BiFC com sequências mutadas de SCI1. A análise deste conjunto de resultados, juntamente com outros resultados obtidos em nosso laboratório, sugere que apenas homodímeros de 14-3-3D e heterodímeros formados entre 14-3-3A e 14-3-3D sejam capazes de interagir com SCI1. Adicionalmente, a localização nuclear dessa interação difere daquelas observadas para SCI1 e para as 14-3-3s individualmente, sugerindo que as 14-3-3s migrem para o núcleo para interagir com SCI1. Nossa hipótese é de que as proteínas 14-3-3s possam modular a localização subnuclear de SCI1. Com o objetivo de levantar dados a respeito das possíveis funções desempenhadas pelas proteínas 14-3-3A e 14-3-3D de N. tabacum, foram identificados os grupos de possíveis ortólogos dessas proteínas em A. thaliana, O. sativa, S. lycopersicum, S. tuberosum e N. benthamiana. Esta análise mostrou que os ortólogos às 14-3-3A e D em Arabidopsis estão associados ao ciclo celular, o que sugere que as proteínas de tabaco possam ter conservado essa função. Além disso, também foram produzidas plantas transgênicas silenciadas para cada uma dessas 14-3-3s de maneira independente. A análise dos fenótipos das plantas transgênicas não levou à elaboração de uma hipótese definitiva sobre a função dessas 14-3-3s no desenvolvimento floral. No entanto, algumas plantas transgênicas apresentaram estruturas menores, especialmente as pétalas, sugerindo que estas proteínas podem estar envolvidas no controle do tamanho de órgãos vegetais. / The modulation of size and shape in plant organs depends on temporal and spatial control of cell division and expansion. However, the molecular mechanisms that regulate this process during floral development are poorly understood. The study of SCI1 (Stigma/style Cell Cycle Inhibitor 1) signaling pathway can contribute to the understanding of the flower growing process. This gene produces a protein which is located in the nucleolus and is related to the inhibition of cell proliferation in the Nicotiana tabacum stigma and style, modulating the size of these organs. Experiments performed to identify SCI1 interaction partners have identified the N. tabacum 14-3-3A and 14-3-3D proteins as interaction candidates. The 14-3-3 family is composed of highly conserved proteins, which form dimers in their native conformation and are responsible to modulate the activity of a large variety of proteins in response to intracellular signals. Therefore, these proteins are associated to the regulation of several processes, including metabolism, transcription, and cell cycle, among others. In this context, the present work aimed to study the N. tabacum 14-3-3A and 14-3-3D proteins and their role during flower development. The results here obtained revealed that 14-3-3A is located in the nucleus and the cytosol, while 14-3-3D protein is distributed only in the cytosol. It was also shown that these proteins can form homodimers and heterodimers with each other. Homodimers of 14-3-3A are distributed in nucleus and cytosol, while 14-3-3D homodimers and heterodimers are located only in the cytosol. Furthermore, the in vivo interaction between SCI1 and 14-3-3A was confirmed by BiFC, showing nuclear localization, outside the nucleolus. In silico analyzes of SCI1 amino acid sequence identified two putative regions of recognition by 14-3-3 proteins. These regions are being evaluated by BiFC assays with SCI1 mutated sequences. The analyses of this set of results, together with other results obtained in our laboratory, suggests that only 14-3-3D homodimers and heterodimers between 14-3-3A and 14-3-3D are capable to interact with SCI1. Moreover, the nuclear localization of this interaction differs from the ones observed for SCI1 and for the 14-3-3s individually, which suggests that the 14-3-3s migrate to the nucleus to interact with SCI1. Our hypothesis is that the 14-3-3 proteins can modulate the subnuclear localization of SCI1. To obtain data concerning the possible roles of the N. tabacum 14-3-3A and 14-3-3D proteins, groups of possible orthologous of these proteins in A. thaliana, O. sativa, S. lycopersicum, S. tuberosum and N. benthamiana were identified. This analysis has shown that the orthologs of 14-3-3A and D in Arabidopsis are associated to the cell cycle, suggesting that the tobacco proteins might have conserved this function. Furthermore, transgenic plants silenced for each of the 14-3-3s independently were also produced. Phenotype analyzes of transgenic plants did not lead to a definitive hypothesis about the function of these 14-3-3s during floral development. However, some transgenic plants exhibited smaller structures, specially petals, which suggests that these proteins may be involved in the size control of plant organs.