131 research outputs found

    From cilia to cancer: the two splicing variants of the human TBCCD1 gene

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    Funds are from Instituto Politécnico de Lisboa, IPL/2017/CILIOPAT/ESTeSL.Almost all human genes that contain multiple exons undergo alternative splicing. Therefore, a single gene can originate multiple mRNA isoforms which causes a dramatic increase in the variability of the expected proteome. Noteworthy, phenotypic variability and disease susceptibility in human populations are related to alternative splicing. Published work from our group identified a new human centrosomal protein, TBCC domain-containing 1 (TBCCD1). Our studies revealed that this gene undergoes alternative splicing producing at least two transcripts encoding proteins. Here we analyze the differential functions of the two splicing variants (TBCCD1v1 and TBCCD1v2). Both variants present distinct cellular localization being TBCCD1v1 essentially centrosomal, whereas TBCCD1v2 is cytoplasmatic. The screening for TBCCD1v2 proximity interactome using BioID identified 19 proteins that functionally group in kinetochore, MT/cilia, and DNA-binding proteins. Striking, the overexpression of TBCCD1v2 decreases the levels of the kinetochore protein CENP-M, a protein upregulated in tumors. On the other hand, the TBCCD1v1 is involved in MT organization and is required to maintain the distal structure of the mother centriole. Our BioID screening for TBCCD1v1 interactors revealed 82 distinct proteins including several well-known proteins encoded by ciliopathy genes. A wider analysis of how TBCCD1v1 levels impact cellular physiological proteome showed that the group of proteins presenting fold changes in their levels vs control cells is enriched in proteins involved in focal adhesions, namely HSPA5/GRP-78/BiP, PDIA3, RPS10, MSN, TGM2, and PPP1R12A. Together our results show that we are still far from having a complete picture of the functional importance of TBCCD1 and how its deregulation may be associated not only with the development of ciliopathies but also with more common diseases like cancer.info:eu-repo/semantics/publishedVersio

    The maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1

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    Centrosomes are organelles consisting of two structurally and functionally distinct centrioles, with the mother centriole having complex distal (DA) and subdistal appendages (SDA). Despite their importance, how appendages are assembled and maintained remains unclear. This study investigated human TBCCD1, a centrosomal protein essential for centrosome positioning, to uncover its localization and role at centrioles. We found that TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from SDA to DA and extending inside and outside the centriole lumen. TBCCD1 depletion caused centrosome mispositioning, which was partially rescued by taxol, and the loss of microtubules (MTs) anchored to centrosomes. TBCCD1 depletion also reduced levels of SDA proteins involved in MT anchoring such as Centriolin/CEP110, Ninein, and CEP170. Additionally, TBCCD1 was essential for the correct positioning of motile cilia basal bodies and associated structures in Paramecium. This study reveals that TBCCD1 is an evolutionarily conserved protein essential for centriole and basal body localization and appendage assembly and maintenance. A BioID screening also linked TBCCD1 to ciliopathy-associated protein networks.info:eu-repo/semantics/publishedVersio

    TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning

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    In animal cells the centrosome is actively positioned at the cell center in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here we report the first characterization of human TBCCD1, a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE-1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1 depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound-healing assays. However, the microtubule nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization

    DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus

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    International audienceBrown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups

    Estudo da proteína humana centrossomal TBCCD1: determinação de domínios funcionais

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    Tese de mestrado em Bioqu√≠mica, apresentada √† Universidade de Lisboa, atrav√©s da Faculdade de Ci√™ncias, 2011O centrossoma √© o principal centro organizador de microt√ļbulos das c√©lulas animais, desempenhando fun√ß√Ķes celulares essenciais no processo de divis√£o celular, uma vez que regula a nuclea√ß√£o e organiza√ß√£o espacial dos microt√ļbulos, estando tamb√©m implicado no posicionamento de organelos na c√©lula, como o complexo de Golgi, no estabelecimento da polaridade celular, na migra√ß√£o e ades√£o celulares e na ciliog√©nese. Nas c√©lulas animais o centrossoma encontra-se posicionado no centro da c√©lula em estreita associa√ß√£o com o n√ļcleo. Os cofactores da tubulina (TBCA-E) s√£o prote√≠nas que participam na via de folding da tubulina e possuem fun√ß√Ķes relacionadas com o citoesqueleto, desempenhando pap√©is essenciais nas c√©lulas eucariotas. A prote√≠na TBCCD1 (TBCC-domain containing protein 1) √© uma prote√≠na centrossomal relacionada com o TBCC e com a prote√≠na RP2, uma vez que possui os dom√≠nios funcionais TBCC e CARP, por√©m n√£o parece possuir actividade de GAP para a tubulina. O TBCCD1 √© um potencial regulador do posicionamento do centrossoma em estreita interac√ß√£o com o n√ļcleo e da organiza√ß√£o citoplasm√°tica. Neste trabalho descrevemos a identifica√ß√£o do dom√≠nio respons√°vel pela localiza√ß√£o centrossomal da prote√≠na TBCCD1 humana, constitu√≠do pelos primeiros 20 res√≠duos de amino√°cidos da sua regi√£o N-terminal. Em c√©lulas humanas observ√°mos que a express√£o da prote√≠na TBCCD1 com uma muta√ß√£o pontual no res√≠duo de prolina na posi√ß√£o 24 leva √† deslocaliza√ß√£o da ő≥-tubulina do centrossoma. Verific√°mos tamb√©m que tr√™s muta√ß√Ķes pontuais distintas nos motivos VxPX e KRAK da prote√≠na causam uma menor efici√™ncia na forma√ß√£o de c√≠lios prim√°rios. Concluindo, a prote√≠na centrossomal TBCCD1 humana parece ter uma liga√ß√£o √† ő≥-tubulina, contudo ainda n√£o est√° esclarecido se esta interac√ß√£o √© directa ou indirecta, podendo a ő≥-tubulina ser um parceiro molecular do TBCCD1. O TBCCD1 dever√° tamb√©m ter um papel essencial in vivo resultante do seu envolvimento na manuten√ß√£o da liga√ß√£o do centrossoma ao n√ļcleo e no processo de ciliog√©nese.The centrosome is the major microtubule organizing center in animal cells, playing an essential role in cellular processes such cell division, since it regulates the nucleation and spatial organization of microtubules, and is also implicated in organelle positioning in the cell, such as the Golgi apparatus, cell polarity establishment, cell migration and adhesion and ciliogenesis. In animal cells, the centrosome is positioned in the center of the cell in close association with the nucleus. The tubulin cofactors (TBCA-E) are proteins involved in tubulin folding pathway that have emerged as proteins with crucial roles in eukaryotic cells related to the cytoskeleton. The TBCCD1 protein (TBCC domain-containing protein 1) is a centrossomal protein related to TBCC and RP2 protein, since it contains the TBCC and CARP domains. However, TBCCD1 probably doesn‚Äôt have a GAP activity towards tubulin. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization. In this work we described the identification of the centrosome targeting motif of the human TBCCD1, corresponding to the first 20 amino acid residues of its N-terminus region. Our studies performed in mammalian cell lines revealed that the expression of TBCCD1 with a point mutation in the proline residue at position 24 leads to the mis-localization of ő≥-tubulin from the centrosome. Furthermore, we also found that three distinct point mutations in the motifs VxPX and KRAK lead to a lower efficiency of transfected cells to assemble primary cilia. Also, the obtained results clearly show that the human centrossomal TBCCD1 protein seems to have an interaction with ő≥-tubulin, but whether this is direct or indirect is still not clear. However, our results support the idea that ő≥-tubulin will probably be a molecular partner of TBCCD1. The TBCCD1 should also have an essential role in vivo resulting from their involvement in maintaining the nucleus-centrosome association and its involvement in ciliogenesis

    " Caracterização da proteína centrossomal TBCCD1 durante o desenvolvimento embrionário do peixe zebra".

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    Mestrado em Ciências do Mar - Recursos MarinhosMaster's degree in Marine Sciences - Marine Resource

    Fun√ß√Ķes celulares da prote√≠na centrossomal humana TBCCD1: prote√≠nas interatuantes e papel na din√Ęmica dos microt√ļbulos

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    Tese de mestrado em Bioqu√≠mica, apresentada √† Universidade de Lisboa, atrav√©s da Faculdade de Ci√™ncias, 2015A prote√≠na humana centrossomal TBCCD1 est√° envolvida na liga√ß√£o do centrossoma ao n√ļcleo. A diminui√ß√£o dos n√≠veis do TBCCD1 causa a quebra do eixo de polaridade intr√≠nseca ‚ÄúN√ļcleo-Centrossoma-Complexo de Golgi‚ÄĚ em consequ√™ncia da anormal localiza√ß√£o do centrossoma na periferia da c√©lula a qual √© acompanhada por uma desorganiza√ß√£o do complexo de Golgi. Recentemente, foram identificadas variantes de splicing alternativo desta prote√≠na que se localizam exclusivamente no citoplasma. Neste trabalho pretendeu-se esclarecer as fun√ß√Ķes da prote√≠na TBCCD1 e da TBCCD1 variante 2, atrav√©s i) do estudo do efeito da deple√ß√£o da TBCCD1 na din√Ęmica dos microt√ļbulos e no citoesqueleto de actina e ii) da identifica√ß√£o das suas prote√≠nas interatuantes, recorrendo ao m√©todo de BioID. Apesar de a TBCCD1 ter um dom√≠nio que a associa com a actina e interagir com v√°rias prote√≠nas que t√™m como fun√ß√Ķes polimerizar os filamentos de actina, em particular nas jun√ß√Ķes c√©lula-c√©lula e de ades√£o, n√£o foi poss√≠vel identificar nenhuma rela√ß√£o clara entre a TBCCD1 e os filamentos de actina nas c√©lulas depletadas de TBCCD1. Este trabalho permitiu concluir que a TBCCD1 tem um papel importante na estabiliza√ß√£o dos microt√ļbulos em c√©lulas hTERT-RPE-1, em particular de uma subpopula√ß√£o de microt√ļbulos que rodeia o n√ļcleo e que se localiza preferencialmente do lado do centrossoma. Esta subpopula√ß√£o de microt√ļbulos regula parcialmente o posicionamento do centrossoma em rela√ß√£o ao n√ļcleo nestas c√©lulas. Observou-se ainda que a deple√ß√£o da TBCCD1 causa a dispers√£o da prote√≠na centrossomal CEP170, que se associa a esta subpopula√ß√£o de microt√ļbulos, e fica dispersa no citoplasma das c√©lulas depletadas de TBCCD1. Neste trabalho foi tamb√©m poss√≠vel estabelecer o interatoma da TBCCD1 e da TBCCD1 variante 2. Assim, observou-se que a TBCCD1 interatua com prote√≠nas com distintas fun√ß√Ķes celulares, estando principalmente envolvida na din√Ęmica dos microt√ļbulos, com a integridade do centrossoma e na polaridade celular, particularmente no estabelecimento e manuten√ß√£o das jun√ß√Ķes celulares. A an√°lise do interatoma, conjuntamente com os resultados obtidos no estudo da din√Ęmica dos microt√ļbulos, sugere que a TBCCD1 seja uma prote√≠na adaptadora que promove a intera√ß√£o entre diferentes prote√≠nas. A TBCCD1 variante 2, por outro lado, interage sobretudo com chaperones moleculares, prote√≠nas mitocondriais e centrom√©ricas. Embora √† primeira vista estas intera√ß√Ķes possam parecer n√£o estar relacionadas, uma an√°lise mais detalhada mostra que a TBCCD1 variante 2 poder√° estar envolvida na montagem e organiza√ß√£o de estruturas complexas como os cinetocoros e a organiza√ß√£o do genoma mitocondrial atrav√©s da intera√ß√£o direta com os diferentes componentes ou atrav√©s da intera√ß√£o com chaperones moleculares.The human centrosomal protein TBCCD is involved in the connection between the centrosome and the nucleus. Lower TBCCD1 levels lead to a disrupture of the intrinsic cell polarity axis ‚ÄúNucleus-Centrosome-Golgi Apparatus‚ÄĚ due to an abnormal localization of the centrosome in the cell periphery, which is accompanied by the fragmentation of the Golgi apparatus. Recently, TBCCD1 splicing variants that localize exclusively in the cytoplasm have been identified. In this work we aimed at clarifying what are the cellular functions of TBCCD1 and TBCCD1 variant 2 by i) studying the effect of TBCCD1 depletion in the microtubule network and actin cytoskeletons dynamics and ii) identifying their interacting proteins using the BioID method. Although TBCCD1 has a domain able to associate this protein with actin and TBCCD1 interacts with several proteins that have a role in actin polymerization, particularly in cell-cell and adhesion junctions, it was not possible to identify a clear relationship between TBCCD1 and the actin filaments in TBCCD1-depleted cells. This work allowed us to conclude that TBCCD1 has an important role in microtubule stabilization in hTERT-RPE-1 cells, in particularly in a microtubule subpopulation surrounding the nucleus which is localized preferentially near the centrosome. This microtubule subpopulation partially regulates the centrosome-nucleus positioning. We also observed that TBCCD1 depletion causes cytoplasmic dispersion of the centrosomal protein CEP170, which is associated with this microtubule subpopulation. In this work we also established the TBCCD1 and TBCCD1 variant 2 interactomes. TBCCD1 interacts with several proteins with distinct functions, mainly involved in microtubule dynamics, centrosome integrity and cell polarity, and also in the establishment and maintenance of cell junctions. Both the interactome analysis and the results obtained with the microtubule dynamic studies suggest that TBCCD1 may function as a promoter of the interaction between distinct proteins. TBCCD1 variant 2, in other hand, interacts mainly with molecular chaperones and mitochondrial and centromeric proteins. Although it may seem that these interactions are not related with which other, a more detailed analysis shows that TBCCD1 variant 2 protein can be involved in the assembly and organization of complex structures, such as the kinetochores and the organization of the mitochondrial genome either through the direct interaction with the different components or through the interaction with molecular chaperones

    TBCCD1

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    Estudo da fun√ß√£o das prote√≠nas variantes da TBCCD1 humana, uma prote√≠na chave na liga√ß√£o do centrossoma ao n√ļcleo

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    Tese de mestrado em Bioqu√≠mica, apresentada √† Universidade de Lisboa, atrav√©s da Faculdade de Ci√™ncias, 2015Com a identifica√ß√£o e o estudo do gene humano tbccd1, foi identificada uma prote√≠na, a TBCCD1, por ele codificada. Esta prote√≠na foi ent√£o descrita como sendo uma prote√≠na centrossomal, que se localiza tamb√©m nos corpos basais de c√≠lios prim√°rios e no corpo m√©dio. Foram identificados dois novos transcritos do tbccd1 resultantes de splicing alternativo, nomeadamente, variante 2 e variante 3, sendo que o transcrito variante 1 codifica a prote√≠na TBCCD1 variante 1 inicialmente descrita. O knockdown do tbccd1 utilizando siRNAs provoca v√°rios fen√≥tipos em c√©lulas hTERT RPE-1, como o aumento da dist√Ęncia centrossoma-n√ļcleo, a fragmenta√ß√£o do complexo de Golgi e a diminui√ß√£o na velocidade de migra√ß√£o em ensaios de fecho da ferida. Assim, este trabalho teve como principal objetivo o estudo da fun√ß√£o biol√≥gica de cada uma das variantes identificadas. Estudou-se a localiza√ß√£o celular de cada uma das variantes identificadas em diferentes linhas celulares humanas. Observou-se que as prote√≠nas TBCCD1 variante 2 e TBCCD1 variante 3 t√™m uma localiza√ß√£o citoplasm√°tica em todas as situa√ß√Ķes analisadas. Pelo contr√°rio, como tinha j√° sido observado, a prote√≠na TBCCD1 variante 1 localiza-se no centrossoma, no corpo m√©dio e nos corpos basais de c√≠lios prim√°rios. Curiosamente, observou-se que a localiza√ß√£o desta prote√≠na no corpo m√©dio √© dependente dos microt√ļbulos, ao contr√°rio do que acontece com a sua localiza√ß√£o centrossomal. Esta observa√ß√£o sugere ent√£o uma intera√ß√£o com microt√ļbulos do corpo m√©dio onde existe uma acumula√ß√£o de microt√ļbulos acetilados. Observou-se tamb√©m que a sobre express√£o das prote√≠nas TBCCD1 variante 1 ou TBCCD1 variante 2 provoca uma diminui√ß√£o nos n√≠veis de őĪ-tubulina acetilada, assim como de ő≥-tubulina e de ő≤-actina. J√° a sobre express√£o da prote√≠na TBCCD1 variante 3 n√£o parece afetar os n√≠veis dos componentes do citoesqueleto analisados. Estudou-se tamb√©m se o fen√≥tipo do aumento da dist√Ęncia centrossoma-n√ļcleo pode ser provocado por apenas uma das variantes ou resulta da a√ß√£o combinada de mais do que uma variante. Para isso, realizaram-se ensaios de recupera√ß√£o do fen√≥tipo, tendo-se observado que as prote√≠nas TBCCD1 variante 1 e TBCCD1 variante 2 revertem parcialmente o fen√≥tipo em estudo. Para al√©m disso, realizou-se um estudo recorrendo a RT-qPCR no qual se observou que as variantes t√™m uma fun√ß√£o de regula√ß√£o entre si. A sobre express√£o das prote√≠nas TBCCD1 variante 1 ou TBCCD1 variante 2 leva √Ä altera√ß√£o dos n√≠veis dos transcritos das variantes 1, 2 e 3. J√° a sobre express√£o da prote√≠na TBCCD1 variante 3 afeta apenas os n√≠veis do transcrito variante 3 end√≥geno. Em conjunto, os resultados obtidos neste estudo indicam que as prote√≠nas codificadas pelos tr√™s transcritos identificados dever√£o ter diferentes fun√ß√Ķes celulares. Para al√©m disso, as prote√≠nas TBCCD1 variante 1 e TBCCD1 variante 2 afetam os n√≠veis de őĪ-tubulina acetilada, ő≥-tubulina e ő≤-actina, o que pode afetar, por exemplo, a organiza√ß√£o do complexo de Golgi e a migra√ß√£o celular, atrav√©s da regula√ß√£o da din√Ęmica dos microt√ļbulos. Assim, estes resultados contribuem de forma decisiva para a compreens√£o dos fen√≥tipos observados nas experi√™ncias do knockdown do tbccd1.When the human gene tbccd1 was identified and studied, a protein, TBCCD1, that it encoded was also identified. This protein was then described as a centrossomal protein that also localizes at the basal bodies of primary cilia and to the midbody. Two new transcripts of tbccd1 originated by alternative splicing were identified, namely, variant 2 and variant 3, and it is now known that variant 1 encodes the TBCCD1 protein initially described. The knockdown of tbccd1 using siRNAs causes several phenotypes in hTERT RPE-1 cells, such as the increase in centrosome-nucleus distance, fragmentation of the Golgi apparatus and a decrease of the cell migration in wound healing assays. The main goal of this work was to study the biological function of each of the identified tbccd1 variants. In this study, the cellular localization of each of the variants in different human cell lines was accessed. TBCCD1 variant 2 and TBCCD1 variant 3 proteins have a cytoplasmic localization in all the situations analyzed. As it has already been described, TBCCD1 variant 1 protein localizes at the centrosome, the basal bodies of primary cilia and to the midbody. Interestingly, it was shown that the midbody localization of this protein is dependent on microtubules, while its centrossomal localization is not. This observation suggested an interaction of this protein with a population of microtubules in the midbody where they are highly acetylated. Overexpression of TBCCD1 variant 1 or TBCCD1 variant 2 causes a decrease on acetylated őĪ-tubulin levels, and also on ő≥-tubulin and ő≤-actin levels. The overexpression of TBCCD1 variant 3 does not seem to affect the levels of any of the cytoskeleton components analyzed. It was also studied if the increased centrosome. Nucleus distance phenotype is caused by only one or a combination of the identified variants. In order to do this, we performed phenotype rescue assays, and it was shown that overexpression of TBCCD1 variant 1 or TBCCD1 variant 2 can partially rescue this phenotype. Moreover, we used RT-qPCR to study whether the identified variants have a regulatory function between them, and showed that TBCCD1 variant 1 or TBCCD1 variant 2 overexpression affects the levels of the three transcripts. On the other hand, the overexpression of TBCCD1 variant 3 only affects variant 3 endogenous transcript levels. Taken together, the results obtained in this study indicate that the proteins encoded by the three transcripts might have different cellular functions. Furthermore, TBCCD1 variant 1 and TBCCD1 variant 2 affect acetylated őĪ-tubulin, ő≥-tubulin and ő≤-actin levels which may be implicated on, for example, Golgi apparatus organization and cell migration by the regulation of microtubules dynamics. Therefore, this results contributed to the comprehension of the causes of the tbccd1 knockdown phenotypes

    Estudo da prote√≠na humana centrossomal TBCCD1 em condi√ß√Ķes de stress oxidativo causadas por H202 em estado estacion√°rio

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    Tese de mestrado, Bioqu√≠mica (Bioqu√≠mica M√©dica), Universidade de Lisboa, Faculdade de Ci√™ncias, 2010O per√≥xido de hidrog√©nio (H2O2) embora seja um oxidante relativamente fraco, √© capaz de alterar o n√≠vel de oxida√ß√£o dos grupos ti√≥is ao reagir com res√≠duos de ciste√≠nas. Essas propriedades tornam o H2O2 uma das esp√©cies reactivas de oxig√©nio (ERO) com potencialidade para participar em vias de sinaliza√ß√£o. Deste modo, o desencadeamento de uma determinada via de sinaliza√ß√£o, ou a sua modula√ß√£o, dever√° ocorrer pelo aumento tempor√°rio das concentra√ß√Ķes de H2O2, como por exemplo, durante a resposta inflamat√≥ria. Por outro lado, a exposi√ß√£o prolongada a algumas ERO t√™m vindo a ser relacionada com o in√≠cio, progress√£o e desenvolvimento tumoral. Muitos dos tumores s√£o caracterizados por aneuploidia e anormalidades centrossomais. Por exemplo, c√©lulas de mam√≠fero expostas a H2O2, por bolus addition, apresentam centrossomas supra-numer√°rios. Neste contexto iniciou-se o estudo da influ√™ncia de concentra√ß√Ķes sinalizadoras de H2O2 nos n√≠veis de duas prote√≠nas centrossomais, a prote√≠na TBCCD1 (TBCC-domain containing protein 1) e a y‚ąítubulina nas linhas celulares humanas HeLa e hTERT-RPE-1. O TBCCD1 √© um potencial regulador do posicionamento do centrossoma e da organiza√ß√£o citoplasm√°tica, a y‚ąítubulina √© uma prote√≠na chave na nuclea√ß√£o dos microt√ļbulos nos centros organizadores de microt√ļbulos, como o centrossoma. Por western blot mostrou-se, em ambas as linhas celulares, o aumento dos n√≠veis de TBCCD1 ao longo do tempo de exposi√ß√£o de H2O2 em estado estacion√°rio. A an√°lise da velocidade de consumo de H2O2 em c√©lulas hTERT-RPE-1 que sobre-expressam a prote√≠na de fus√£o TBCCD1-GFP mostrou um rela√ß√£o directa desta com o aumento dos n√≠veis de TBCCD1-GFP. Esta prote√≠na, na presen√ßa de H2O2, varia de modo an√°logo √† prote√≠na end√≥gena. Estudos similares mostraram a diminui√ß√£o dos n√≠veis de y‚ąítubulina ao longo do tempo de exposi√ß√£o de H2O2. Por microscopia de imunofluoresc√™ncia indirecta observou-se tamb√©m a diminui√ß√£o dos n√≠veis desta prote√≠na no centrossoma em c√©lulas HeLa. Observou-se ainda uma altera√ß√£o da capacidade de nuclea√ß√£o dos microt√ļbulos pelo centrossoma e uma modifica√ß√£o na organiza√ß√£o destes ap√≥s repolimeriza√ß√£o em c√©lulas previamente tratadas com nocodazole e ap√≥s remo√ß√£o desta. No conjunto, estes resultados mostram que ambos os n√≠veis destas prote√≠nas variam na presen√ßa de baixos concentra√ß√Ķes de H2O2, constituindo este trabalho uma abordagem inicial do efeito do H2O2 em concentra√ß√Ķes reguladoras no centrossoma.The hydrogen peroxide (H2O2) although a relatively weak oxidant, is highly reactive with sulfhydryl groups. These properties make the H2O2 a reactive oxygen species (ROS) with the potential to participate as a regulator in signaling pathways. Thus, the temporary increase in the concentrations of H2O2 should trigger or modulate a signal pathway, such as during the inflammatory response. Moreover, prolonged exposure to some ROS has been related with the initiation, progression and tumor development. Many tumors are characterized by aneuploidy and centrosomal abnormalities. For example, mammalian cells exposed to H2O2 by bolus addition showed supra-numerary centrosomes. In this context we began the study of the influence of signaling concentrations of H2O2 in the levels of two centrosomal proteins, protein TBCCD1 (TBCC domain-containing protein 1) and y ‚ąí tubulin in HeLa and hTERT-RPE-1 human cell lines. The TBCCD1 is a potential regulator of the positioning of the centrosome and cytoplasmic organization and the y ‚ąí tubulin is a key protein in the nucleation of microtubules on microtubule organizing centers, such as the centrosome. By western blot, we detected in both cell lines, increased levels of TBCCD1 over time of exposure to a steady state of H2O2. The analysis of H2O2 consumption by intact hTERT-RPE-1 cells that over-expresses the fusion protein TBCCD1-GFP showed a direct relationship of this with increased levels of TBCCD1-GFP. Noteworthy, in the presence of H2O2, the TBCCD1-GFP levels, showed the same variation that those of the endogenous protein. Similar studies showed that ‚ąítubulin levels decreased of upon an exposition to H2O2. By immunolocalization microscopy we observed in HeLa cells a decrease of y ‚ąí tubulin‚Äôs levels at the centrosome. To investigate if this observation has an impact in the ability of centrosomes to nucleate microtubules we have treated HeLa cells, previously exposed to steady-state levels of H2O2, with the antimitotic drug nocodozole. After washing out this microtubule depolymerizing agent we follow microtubule repolymerization by immunofluorescence microscopy. We observed that cells treated with H2O2 present a different ability to nucleate microtubules in comparison to control cells. At end these cells presented a distinct organization of the microtubule cytoskeleton. Taken together the results presented in this work show that a low concentration of H2O2 promotes a variation of the TBCCD1 and y ‚ąí tubulin‚Äôs levels. Therefore, this work constitutes an initial approach to the study of the impact at the centrosome of H2O2 at levels expected for a signaling role
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