Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T09:08:00Z (GMT). No. of bitstreams: 1 Smetana_JulianaHelenaCosta_D.pdf: 8660811 bytes, checksum: cb33e97d4c49fdce1e29094a2f6089cc (MD5) Previous issue date: 2009 / Resumo: As células respondem constantemente a uma enorme variedade de estímulos, que são interpretados e integrados por meio de redes de sinalização, dando origem a uma resposta biológica. Defeitos nesses circuitos são a causa de diversas doenças, incluindo muitos, se não todos os tipos de câncer. As fosfatases, enzimas que removem grupamentos fosfato dos substratos de quinases, dependem principalmente de subunidades regulatórias para definir sua especificidade. As fosfatases do tipo 2A constituem a subfamília PPP, que é formada por PP2A, PP4 e PP6. PP2A é a principal fosfatase solúvel de fosfosserina e fosfotreonina em células animais e é encontrada predominantemente como uma holoenzima formada por uma subunidade catalítica (C), uma subunidade regulatória (B, B', B'' ou B''') e uma de ancoragem (PR65/A). Em levedura, as fosfatases 2A desempenham um importante papel na via da quinase TOR, o que ocorre por meio da proteína essencial Tap42. A proteína Tip41 foi identificada como um parceiro de interação de Tap42 e regulador da via da quinase TOR em levedura. A homóloga de Tap42 em mamíferos, chamada de a4, está envolvida na regulação de diversos processos celulares, como diferenciação, desenvolvimento, migração celular e apoptose, por meio de seu papel conservado de regulador de fosfatases 2A. A homóloga em mamíferos de Tip41, chamada TIPRL, é uma proteína ainda pouco caracterizada. Este trabalho teve como objetivo analisar a função das proteínas a4 e TIPRL humanas e esclarecer seu papel na regulação de fosfatases 2A. A caracterização estrutural de a4 e Tap42, usando dados de SAXS, dicroísmo circular e proteólise limitada, mostrou que essas proteínas apresentam um domínio N-terminal compacto formado por a-hélices e um domínio C-terminal desestruturado. Em uma triagem de interações com a proteína TIPRL humana, identificamos as fosfatases PP2Ac, PP4c e PP6c como seus parceiros de interação, assim como os fatores de transcrição MafB e TAF10. Ao contrário do esperado a partir do modelo de levedura, a4 e TIPRL não interagem diretamente, mas formam um complexo ternário com PP2Ac. Uma triagem de substratos de fosfatases 2A regulador por TIPRL identificou os fatores de splicing SF2/ASF e SF2p32. Nossos resultados sugerem um modelo estrutural para a regulação das fosfatases 2A por a4 e mostram que TIPRL é um novo regulador comum dessas fosfatases com funções na regulação da expressão gênica. / Abstract: Cells respond constantly to a variety of stimuli, which are interpreted and integrated through signaling networks, giving rise to biological responses. Defects in this circuitry are a cause of many diseases, including cancer. Protein phosphatases are enzymes which remove phosphate groups from kinase substrates, relying mainly on regulatory subunits for their substrate specificity. Type 2A phosphatases belong to the PPP subfamily, which is formed by PP2A, PP4 and PP6. PP2A is the major soluble serine/threonine phosphatase in animal cells and is found predominantly as a heterotrimer composed of a catalytic (C), a regulatory (B, B', B'' or B''') and a scaffold (PR65/A) subunit. Type 2A phosphatases play a major role in the yeast TOR signaling pathway through their interaction with the essential protein Tap42. Tip41 was identified as a Tap42 interacting protein and regulator of the TOR pathway. a4, the mammalian orthologue of Tap42, regulates many cellular processes such as differentiation, development, cell migration and apoptosis as a conserved type 2A phosphatase regulator. TIPRL, the mammalian orthologue of Tip41, is still poorly characterized. The objective of the present work was to analyse the function of a4 and TIPRL and improve the understanding of their role as type 2A phosphatase regulators. The structural characterization of a4 using SAXS analyses, circular dichroism and limited proteolysis, showed that these proteins are formed by an a-helical N-terminal domain and an unfolded C-terminal domain. A screen for TIPRL interacting proteins identified PP2Ac, PP4c and PP6c and also the transcription factors MafB and TAF10. Unlike their yeast conterparts, a4 and TIPRL do not interact directly, but rather form a ternary complex with PP2A. A search for type 2A phosphatase substrates regulated by TIPRL identified the splicing factor SF2/ASF and its regulatory protein SF2p32. Our results suggest a structural model for the regulation of type 2A phosphatases by a4 and show that TIPRL is a novel common regulator of these phosphatases which functions in regulation of gene expression. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

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[ES] La ubiquitinación inducida por hormonas desempeña un papel crucial en la vida media de los reguladores negativos clave de la propia señalización hormonal. En la señalización por ABA, los reguladores negativos clave son las PP2Cs del clado A, como PP2CA o ABI1, y su degradación es un mecanismo complementario a la inhibición de su actividad mediada por PYR/PYL/RCAR. El ABA promueve la degradación de ABI1 a través de las E3 ligasas PUB12/13, y PP2CA a través de las E3 ligasas RGLG1/5. Sin embargo, se predice que otras E3 ligasas no identificadas también regularán la vida media de las PP2Cs del clado A. En pasos posteriores de la señalización por ABA, el ABA también induce la regulación positiva de los niveles de transcrito y proteína de las PP2Cs como un mecanismo de retroalimentación negativa. Por lo tanto, restablecer la señalización de ABA también requiere la degradación de las PP2Cs para evitar su acumulación excesiva inducida por el propio ABA. En este trabajo identificamos las proteínas BTB/POZ AND MATH DOMAIN (BPM), adaptadores del sustrato de las E3 ligasas multiméricas CULLIN3-RING E3 (CRL3), como proteínas que interactúan con las PP2Cs. BPM3 y BPM5 interactúan en el núcleo con PP2CA, así como con ABI1, ABI2 y HAB1. Además, BPM3 y BPM5 aceleran la degradación de las PP2Cs de una manera dependiente del ABA y su sobreexpresión conduce a una mayor sensibilidad al ABA. Además, las plantas mutantes bpm3 bpm5 mostraron una mayor acumulación de PP2CA, ABI1 y HAB1, lo que conduce a una sensibilidad global disminuida al ABA. Finalmente, utilizando ensayos bioquímicos y genéticos, demostramos que las BPM aumentaban la ubiquitinación de PP2CA. Dado que la formación de los complejos ternarios receptor-ABA-fosfatasa se ve notablemente afectada por la abundancia de sus componentes proteicos y la concentración de ABA, revelamos que las BPM y las E3 ligasas multiméricas CRL3 son moduladores importantes de los niveles del correceptor PP2C para regular la señalización temprana de ABA, así como durante los consiguientes pasos de restablecimiento. Al contrario que con PUB12/13, no se sabe cómo el ABA aumenta la degradación de PP2CA a través de RGLG1/5. En el caso de RGLG1, esta proteína se encuentra predominantemente en la membrana plasmática, mientras que PP2CA se encuentra predominantemente en el núcleo. Nosotros demostramos que el ABA modifica la localización subcelular de RGLG1, promoviendo la interacción nuclear con PP2CA. En primer lugar, encontramos que RGLG1 está miristoilado in vivo, lo que facilita su unión a la membrana plasmática, sin embargo, el ABA inhibe esta miristoilación. El ABA también regula negativamente a N-myristoyltransferase 1, la enzima activa y central en la miristoilación de proteínas, esto puede ayudar a promover la translocación de RGLG1 al núcleo. Allí, RGLG1 puede interactuar con ciertos receptores monoméricos del ABA, como PYL8. El reclutamiento nuclear de la E3 ligasa también fue promovido por el aumento de los niveles de proteína PP2CA y por la formación de complejos RGLG1-PYL8-PP2CA en presencia de ABA. Además, nosotros encontramos que RGLG1Gly2Ala, mutada en el sitio de miristoilación N-terminal, muestra localización nuclear constitutiva y provoca una respuesta más sensible al ABA y al estrés salino y osmótico. En resumen, proporcionamos evidencia de que una ligasa E3 puede reubicarse dinámicamente en respuesta al ABA, el estrés salino y osmótico, y el aumento de los niveles de su sustrato, lo que revela un mecanismo para explicar cómo el ABA mejora la interacción RGLG1-PP2CA y, por lo tanto, la degradación de PP2CA. / [CAT] L' ubiquitinació induïda per hormones té un paper crucial en la vida mitjana dels reguladors negatius clau de la pròpia senyalització hormonal. En la senyalització per ABA, els reguladors negatius clau són les PP2Cs del clado A, com PP2CA o ABI1, i la seva degradació és un mecanisme complementari a la inhibició de la seva activitat mediada per PYR/PYL/RCAR. El ABA promou la degradació de ABI1 a través de les E3 lligases PUB12/13, i PP2CA per mitja de les E3 lligases RGLG1/5. No obstant això, es prediu que altres E3 lligases no identificades també regularan la vida mitjana de les PP2Cs del clado A. En passos posteriors de la senyalització per ABA, l'ABA també indueix la regulació positiva de nivells de transcrit i proteïna de les PP2Cs com un mecanisme de retroalimentació negativa. Per tant, restablir la senyalització d'ABA també requereix la degradació de les PP2Cs per evitar la seva acumulació excessiva induïda pel propi ABA. En aquest treball identifiquem les proteïnes BTB/POZ AND MATH DOMAIN (BPM), adaptadors del substrat de les E3 lligases multimèriques CULLIN3-RING E3 (CRL3), com proteïnes que interactuen amb les PP2Cs. BPM3 i BPM5 interactuen en el nucli amb PP2CA, així com amb ABI1, ABI2 i HAB1. A més, BPM3 i BPM5 acceleren la degradació de les PP2Cs d'una manera dependent del ABA i la seva sobreexpressió porta a una major sensibilitat al ABA. A més, les plantes mutants bpm3 bpm5 van mostrar una major acumulació de PP2CA, ABI1 i HAB1, el que porta a una sensibilitat global disminuïda a ABA. Finalment, utilitzant assajos bioquímics i genètics, aconseguint que les BPM augmentaven l' ubiquitinació de PP2CA. Atès que la formació dels complexos ternaris receptor-ABA-fosfatasa es veu notablement afectada per l'abundància dels seus components proteics i la concentració d'ABA, revelem que les BPM i les E3 lligases multimèriques CRL3 són moduladors importants dels nivells del coreceptor PP2C per regular la senyalització primerenca de ABA, així com durant els consegüents passos de restabliment. Al contrari que amb PUB12/13, no se sap com el ABA augmenta la degradació de PP2CA a través d'RGLG1/5. En el cas de RGLG1, aquesta proteïna es troba predominantment en la membrana plasmàtica, mentre que PP2CA es troba predominantment en el nucli. Nosaltres vam demostrar que l'ABA modifica la localització subcelular de RGLG1, promovent la interacció nuclear amb PP2CA. En primer lloc, trobem que RGLG1 està miristoilado in vivo, el que facilita la seva unió a la membrana plasmàtica, però, el ABA inhibeix aquesta miristoilación. El ABA també regula negativament N-myristoyltransferase 1, l' enzim actiu i central en la miristoilación de proteïnes, això pot ajudar a promoure la translocació de RGLG1 al nucli. Allà, RGLG1 pot interactuar amb certs receptors monomèrics de l'ABA, com PYL8. El reclutament nuclear de l'E3 lligasa també va ser promogut per l'augment dels nivells de proteïna PP2CA i per la formació de complexos RGLG1-PYL8-PP2CA en presència d'ABA. A més, nosaltres trobem que RGLG1Gly2Ala, mutada en el lloc de miristoilació N-terminal, mostra localització nuclear constitutiva i provoca una resposta més sensible al ABA i l'estrès salí i osmòtic. En resum, proporcionem evidència que una ligasa E3 pot reubicar dinàmicament en resposta al ABA, l'estrès salí i osmòtic, i l'augment dels nivells de la seva substrat, el que revela un mecanisme per explicar com el ABA millora la interacció RGLG1-PP2CA i, per tant, la degradació de PP2CA. / [EN] Hormone-induced ubiquitination plays a crucial role to determine the half-life of key negative regulators of hormone signaling. In case of ABA signaling, the key negative regulators are the clade-A PP2Cs, such as PP2CA or ABI1, and their degradation is a complementary mechanism to PYR/PYL/RCAR-mediated inhibition of their activity. ABA promotes the degradation of ABI1 through the PUB12/13 E3 ligases, and PP2CA through the RGLG1/5 E3 ligases. However, other unidentified E3 ligases are predicted to regulate clade A PP2Cs half-life as well. At later steps of ABA signaling, ABA also induces upregulation of PP2C transcripts and protein levels as a negative feedback mechanism. Therefore, resetting of ABA signaling also requires PP2C degradation to avoid excessive ABA-induced accumulation of PP2Cs. In this work we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multimeric CULLIN3-RING E3 ligases (CRL3s), as PP2C-interacting proteins. BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2 and HAB1. Additionally, BPM3 and BPM5 accelerate the turnover of PP2Cs in an ABA-dependent manner and their overexpression leads to enhanced ABA sensitivity. Moreover, bpm3 bpm5 mutant plants showed increased accumulation of PP2CA, ABI1 and HAB1, which leads to global diminished ABA sensitivity. Finally, using biochemical and genetic assays we demonstrated that BPMs enhance the ubiquitination of PP2CA. Given the formation of receptor-ABA-phosphatase ternary complexes is markedly affected by the abundance of protein components and ABA concentration, we reveal that BPMs and multimeric CRL3 E3 ligases are important modulators of PP2C co-receptor levels to regulate early ABA signaling as well as the subsequent resetting steps. In contrast to PUB12/13, it was not known how ABA enhances the degradation of PP2CA by RGLG1/5. RGLG1 is predominantly found in the plasma membrane whereas PP2CA is predominant in the nucleus. We demonstrate that ABA modifies the subcellular localization of RGLG1, promoting nuclear interaction with PP2CA. Firstly, we found that RGLG1 is myristoylated in vivo, which facilitates its attachment to the plasma membrane, nevertheless, ABA inhibits its myristoylation. ABA also downregulates N-myristoyltransferase 1, the central active enzyme of protein myristoylation, which may help to promote RGLG1 translocation to the nucleus. There, RGLG1 can interact with certain monomeric ABA receptors, as PYL8. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP2CA protein levels and the formation of RGLG1-PYL8-PP2CA complexes in the presence of ABA. Additionally, we found that RGLG1Gly2Ala protein, mutated at the N-terminal myristoylation site, shows constitutive nuclear localization and causes an enhanced response to ABA and salt and osmotic stresses. In summary, we provided evidence that an E3 ligase can dynamically relocalize in response to ABA, salt and osmotic stress, and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG1-PP2CA interaction and hence PP2CA degradation. / Julián Valenzuela, J. (2020). Regulation of ABA signaling through degradation of clade A PP2Cs by the RGLG1 and CRL3 BPM E3 ligases [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137777 / TESIS

Phosphateses (EC 3.1.3.x) are a group of enzymes that hydrolyze phosphoesters. That way they affect the energetic metabolism of a cell and its regulation. Phosphatases that dephosphorylate proteins are an integral part of signaling pathways, stress responses and they modulate enzymatic activity. This thesis deals with the study of phosphatases obtained from tobacco leaves (Nicotiana tabacum, L.). Solution of enzymes was prepared by extraction in both acidic and alkaline buffers. Through the use of the chromogenic substrate pNP-phosphate it was determined that there is a higher phosphatase activity in the glycosylated fraction than in the fraction that did not bind to Con A Sepharose. The research of the ions effect on the phosphatase activity has determined that Mg2+ and Ca2+ show positive effect on the phosphatase activity while the effect of Co2+ and Mn2+ is inhibitory. The Zn2+ ions have shown no effect whatsoever. The glycosylated phosphatases also dephosphorylated low-weight-molecular substrates phosphoserine, ATP and glucose-6-phosphate. The research of protein phosphatase activity discovered the affinity to the substrate phosvitin, although neither to casein nor its tryptic cleaves. Detailed experiments have shown that the pH optima for all the substrates lie from pH 5 to 6. Glycosylated phosphatases...

Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...

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Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3 x l0-min episodes of ischemic pelleting or pre-incubation with 100 μM adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 μM), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

Indiana University-Purdue University Indianapolis (IUPUI) / Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.

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