No description available.

Numerous essential cellular processes, such as gene regulation and tRNA processing, are carried out by structured RNAs. While in vitro most RNAs become kinetically trapped in non-functional misfolded states that render them inactive on a biologically-relevant time scale, RNAs folding in vivo do not share this same outcome. RNAs do indeed misfold in the cell; however, chaperone proteins promote escape from these non-native states and foster folding to functional conformations. DEAD-box proteins are ATP-dependent RNA chaperone proteins that function by disrupting structure, which can facilitate structural conversions. Here, studies with both local and global focuses are used to uncover mechanistic features of DEAD-box proteins CYT-19 and Mss116p. Both of these proteins are general RNA chaperones as they each have the ability to facilitate proper folding of multiple structured RNAs. The first study probes how DEAD-box proteins interact with a simple duplex substrate. Separating the strands of a duplex is an ATP-dependent process and is central to structural disruption by DEAD-box proteins. Here, how ATP is utilized during duplex separation is monitored by comparing ATP hydrolysis rates with strand separation rates. Results indicate that one ATP molecule is sufficient for complete separation of a 6-11 base pair RNA duplex. Under some conditions, ATP binding in the absence of hydrolysis is sufficient for duplex separation. Next, focus is shifted to a more global perspective as the function of Mss116p is probed in the folding of a cognate group II intron substrate, aI5[gamma], under near-physiological conditions. Three catalytically-active constructs of aI5[gamma] are used and catalysis serves as a proxy for folding. Folding of all constructs is promoted by the presence of Mss116p and ATP. In vitro and in vivo results indicate that a local unfolding event is promoted by Mss116p, stimulating formation of the native state. Lastly, orthogonal methods that probe physical features of RNA are used to provide insight into the structural intermediates with which Mss116p acts. / text

No description available.

No description available.

Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

Neurons, astrocytes and oligodendrocytes of the central nervous system (CNS) arise from a common pool of multipotent neuroepithelial progenitor cells lining the walls of the neural tube. Initially, neuroepithelial cells undergo symmetric proliferative divisions, thereby expanding the progenitor pool and determining the size of brain compartments. At the onset of neurogenesis, a subset of progenitors switch to asymmetric or terminal symmetric neurogenic divisions. Maintenance of progenitor cell population throughout the period of neurogenesis is essential to generate the full diversity of neuronal cell types and proper histological pattern. However, the mechanisms responsible for the maintenance of progenitor cells proliferation are far from being fully understood. The family of Y-box proteins is involved in control of proliferation and transformation in various normal and pathological cellular systems, and therefore was considered as a candidate to exert such a function. Y-box proteins have a capacity to bind DNA and RNA, thereby controlling gene expression from transcription to translation. This study aimed to examine the expression of mouse Y-box protein 3 (MSY3) in the developing nervous system and elucidate its putative role in regulation of proliferation of progenitor cells. As presented in this work, the MSY3 protein in the embryonic CNS is expressed solely in progenitor cells and not neurons. Moreover, as shown by two independent approaches: morphologically, i.e. using immunofluorescence and confocal microscopy, and biochemically, MSY3 expression is downregulated concomitantly with the spatiotemporal progression of neurogenesis. Interestingly, in preliminary results it was shown that MSY3 is expressed in Dcx-positive transient amplifying precursors in germinal zones of the adult brain, and in EGF-dependent neurospheres. To evaluate whether MSY3 could regulate the neurogenesis, the levels of the MSY3 protein in the progenitors were acutely downregulated or elevated by electroporation of RNAi or MSY3 expression plasmids, respectively. Neither premature reduction of MSY3 in the neuroepithelium (E9.5-E11.5) nor prolonged expression at the developmental stage when this protein is endogenously downregulated (E10.5-14.5) did affect proliferation versus the cell cycle exit of progenitors. Moreover, in Notch1-deficient progenitors in the cerebellar anlage, which exhibit precocious differentiation, MSY3 was not prematurely downregulated, suggesting that MSY3 also is not an early marker of differentiation. Differential centrifugation, immunoprecipitation and polysomal analysis performed in this study revealed that the MSY3 protein in the developing embryo, as well as in Neuro-2A cells, is associated with RNA. On a sucrose density gradient MSY3 co-fractionates with ribosomes and actively translating polysomes, suggesting that it might have a role in regulation of translation. However, downregulation or overexpression of MSY3 in the Neuro-2A cell line did not affect global translation rates. Other researchers suggested that the MSY3 protein has the redundant function with Y-box protein 1 (YB-1). Accordingly, in our system the MSY3 protein could be co-immunoprecipitated with YB-1. Importantly, developmentally regulated expression of MSY3 is not a hallmark of general translation apparatus, as several other proteins involved in translation did not show similar downregulation. To summarise, this work showed that the MSY3 protein is a marker of proliferation of progenitor cells in the embryonic and adult brain, being absent from neurons. Discovery of the molecular mechanism by which MSY3 exerts its role in the cell could provide the link between the translational machinery and proliferation.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

No description available.

Das Ubiquitin-Proteasom-System eukaryotischer Zellen spielt eine zentrale Rolle beim Abbau von fehlgefalteten und nicht mehr benötigten Proteinen. Damit erfüllt es regulatorische Funktionen bei zellulären Prozessen wie z.B. dem Zellzyklus und der Transkription. Das Protein Proteasominhibitor 31 (PI31) wurde als Inhibitor des Proteasoms in vitro charakterisiert. Des weiteren wurde gezeigt, daß überexprimiertes PI31 im murinen System ein Modulator der Assemblierung des Immunoproteasoms (i20S) ist. Über die Funktion und Regulation von PI31 im humanen System war bisher nichts bekannt und wurde deshalb in dieser Arbeit untersucht. Es konnte gezeigt werden, daß neben dem PI31-Transkript mindestens neun weitere alternative Spleißvarianten des humanen PI31 Gens PSMF1 existieren. Die PI31-Isoformen V2 bis V10 unterscheiden sich von PI31 (V1) teils durch eine fehlende N-terminale Domäne oder einen veränderten C-Terminus. Die Isoform V5 wird als einzige gewebespezifisch in Testikeln exprimiert und ist im Zellkern lokalisiert. Ausschließlich die Überexpression der Isoform V3 führt zur Inhibition der proteasomalen Aktivität in vivo. Ein modulatorischer Einfluß von PI31 oder einer der Isoformen auf die Assemblierung des humanen i20S bestätigte sich dagegen nicht. Die Überexpression von PI31 und V3 in humanen Zellen führte indes zu einer Akkumulation und verzögerten Degradation von proteasomalen Substraten. Es wurde außerdem gezeigt, daß die Expression von humanem PI31 durch virusassoziierte Stimuli wie dsRNA und Typ I-Interferone induziert werden kann. Für die 3kb lange 3’UTR der PI31-mRNA konnte zusätzlich nachgewiesen werden, daß sie inhibitorisch auf die Expression wirkt und somit eine regulatorische Funktion besitzt. In Zusammenhang mit der von Kirk et al. (2008) gezeigten Heterodimerisierung von PI31 mit dem F-Box Protein Fbxo7, weisen die hier vorgestellten Ergebnisse auf eine Funktion von PI31 und dessen Isoformen bei der Ubiquitinierung von proteasomalen Substraten hin. / The ubiquitin–proteasome pathway is the major intracellular system for protein degradation. It plays an important role in the regulation of cellular processes like cell cycle control, signal transduction and gene transcription. The protein proteasome inhibitor 31 (PI31) was initially characterized as a potent inhibitor of proteasomal activity in vitro. Furthermore it was shown that PI31 modulates the assembly of the murine immunoproteasome (i20S). The function and regulation of PI31 in the human system is so far unexplored and therefore the topic of this study. It was shown that at least nine alternatively spliced variants of the PI31 gene PSMF1 exist additionally to the PI31 transcript. The PI31 isoforms V2 to V10 differ from PI31 (V1) in parts of the N-terminus and in a modified C-terminus. Only the isoform V5 is tissue specific expressed in testis and localized in the nucleus. After overexpression only the isoform V3 has the ability to inhibit the proteasomal activity in vivo. In contrast to the murine system neither PI31 nor the isoforms showed a modulatory effect on the assembly of the i20S. The overexpression of PI31 and V3 in human cells results instead in the accumulation and delayed degradation of proteasomal substrates. Furthermore the expression of human PI31 can be induced by virus associated stimuli like dsRNA and type I interferones. In addition, for the 3kb long 3’UTR of the PI31-mRNA an inhibitory effect on the expression and therefore a regulatory role was shown. Together with data from Kirk et al. (2008), who show the heterodimerization of PI31 with the F-box protein Fbxo7, the presented results suggest a function of PI31 and its isoforms in the process of ubiquitination of proteasomal substrates.

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy