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Aproximacion funcional a CERKL, un gen causante de la retinosis pigmentaria, mediante el estudio de la localización intracelular de la proteína y la identificación de sus partners proteicos y no proteicos

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Aproximacion funcional a CERKL, un gen causante de la retinosis pigmentaria, mediante el estudio de la localización intracelular de la proteína y la identificación de sus partners proteicos y no proteicos

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dc.contributor.advisor	Knecht Roberto, Erwin Carlos
dc.contributor.advisor	Perez Jimenez, Eva
dc.contributor.author	Fathinajafabadi Nasresfahani, Alihamze
dc.contributor.other	Departament de Bioquímica i Biologia Molecular	es_ES
dc.date.accessioned	2015-05-19T08:11:47Z
dc.date.available	2015-05-20T03:45:05Z
dc.date.issued	2015	es_ES
dc.date.submitted	21-05-2015	es_ES
dc.identifier.uri	http://hdl.handle.net/10550/43888
dc.description.abstract	Within the last two and a half decades, technological advances have led to a progress in gene identification of retinal diseases. However, the function of a high number of the over hundred retinal diseases and fifty RP genes known remains to be characterized. Functional studies have been hampered mainly by the lack of suitable animal and cellular models, the intracellular complexity and specific metabolic demands of the retinal cells and the extensive genetic, allelic and clinical heterogeneity associated with the majority of retinal degenerative disorders. Within this context, CERKL, an autosomal recessive RP and CRD -causing gene, stands among the genes whose function awaits characterization. In fact, repeated attempts by several groups to assign a ceramide kinase function to CERKL have proved fruitless. Therefore and to gain some insight into CERKL function we investigated here in detail the subcellular localization and the interaction partners of the CERKL full length protein isoform (CERKLa). The protein was localized both in the nucleus and in the cytoplasm of various cell types, in agreement with other studies. Although the CERKL distribution appeared to be mainly homogeneous, some aggregates became apparent in the cytoplasm of transiently transfected cells. A systematic analysis in cultured cells using different markers of various cell compartments revealed that CERKL aggregates colocalized with SGs, which are cytoplasmic complexes of mRNAs and mRNA-binding proteins such as those that regulate mRNA translation and stability. These granules are induced by stress conditions and provide the cells with a mechanism to stop protein synthesis and promote a quick recovery of proteostasis when the stress disappears. Targeting to SGs under stress conditions would agree with CERKL protective role from apoptosis induced by oxidative stress. In addition to SGs, CERKL was also found associated with P-bodies, which are involved in mRNA degradation and are in dynamic equilibrium with SGs. Also in support to a CERKL contribution to stress response and protection of photoreceptors is the punctuated CERKL labeling noticed after a light stress in the outer nuclear layer of rat photoreceptors. The CERKL-C125W mutant, which does not enter the nucleus, is not found in SGs. In fact, the localization of wild type CERKL to SGs seems to depend on nuclear import/export of the protein and on mRNA transcription. Therefore, we reasoned that CERKL has a role in the nucleus before it associates with SGs, probably related to RNA transport. In fact, CERKL subcellular localization studies have shown strong accumulation in the nucleoli. Whether this is related to particular protein isoforms or depends on the cellular state deserves further study. Actually, there are other genes mutated in RP that are involved in RNA metabolism at the nucleus, such as the spliceosome components PRPF31, PRPF8 and PRPF3, or the RNA splicing factor RP9. Recently, in age-related macular degeneration, SFRS10 has been reported to regulate alternative splicing of stress response genes under hypoxic conditions. In the cytosol, apart from the localization of CERKL in aggregates, the protein was mostly found diffusely distributed. This was particularly evident in HeLa cells stably expressing this protein. Since SGs are in dynamic equilibrium with polysomes, we analyzed whether this diffuse localization of CERKL could correspond to its binding to polysomes. Using sucrose gradients, CERKL localized to the polysomal pellet and to a greater extent in the soluble fractions that contained postpolysomal mRNPs. Contrary to polysomes, the association of CERKL to these fractions was not sensitive to EDTA or puromycin and was only sensitive to RNase A at high salt concentrations. Therefore, it appears that CERKL in these fractions is associated with quite compact mRNPs. The association of CERKL to mRNPs can occur by protein-RNA and protein-protein interactions. Our data show that CERKL directly interacts with mRNA through its N-terminal region and that this link involves the 5′cap structure of mRNA. Since in the proteomic analysis we did not observe any interaction between CERKL and the two mammalian proteins known to bind directly to the 5′cap structure, elF4E and nCBC [25], and since eIF4E was not detected in immunoprecipitation assays (data not shown), the CERKL-mRNA association is probably direct. However, we cannot exclude an interaction with a yet unidentified component of the 5′cap structure. In addition, CERKL was found to bind to proteins of the translation machinery, such as PABP, eIF3B, HSP70 and the ribosomal protein RPS3, all of which are found in SGs and in other mRNPs, such as neuronal and transport granules. Except for eIF3B, the interaction of CERKL with these proteins was mRNA-dependent, mostly forming compact RNPs. Interestingly, the CERKL-C125W mutant did not interact with eIF3B and its binding to the other proteins was not sensitive to RNase A even at high salt concentrations, indicating the absence of RNA in these complexes. This together with the restricted nuclear/cytoplasmic shutling ability shown for this mutant could explain why it is not found in Sgs. CERKL-containing compact mRNPs were found associated with microtubules and in the cytoskeletal fraction. In fact, CERKL contains a PH domain that is also present in proteins that bind to microtubules. This association could be found here in distal compartments of differentiated neural cells. These results suggest a role of this protein in the transport of the mRNAs present in these structures, although this needs to be further investigated. In this regard, it is known that a compact packaging of mRNAs into mRNPs is essential to protect the mRNAs and to guarantee their survival until translation occurs. The assembly of these mRNPs starts while the mRNA is still being transcribed. This explains the requirements of an active transcription, the nuclear import/export cycle of CERKL and why the CERKL C125W mutant is unable to form these complexes. Although we have highlighted a new cellular role of CERKL and shown that a pathological mutant lacks these features, further studies are needed to substantiate the in vivo role of CERKL variants in the retina, focusing on the relationship between the protein isoforms, their intracellular localization and the disease phenotype.	en_US
dc.description.abstract	La proteína CERKL se encuentra localizada tanto en el núcleo como en el citoplasma. En el citoplasma, CERKL se distribuye de manera difusa y también formando unos agregados que en su mayor parte se distribuyen en la periferia nuclear. Esos agregados colocalizan con marcadores de gránulos de estrés y su cantidad aumenta sometiendo las células a estrés (arsenito de sodio o choque térmico) y disminuye tras un tratamiento con cicloheximida (un inhibidor de la síntesis de proteínas que desensambla los gránulos de estrés). La entrada y salida de CERKL del núcleo es necesaria para su localización en los gránulos de estrés y, por eso, el mutante patológico C125W que está ausente de los núcleos no colocaliza con los marcadores de gránulos de estrés. CERKL se acumula en el núcleo y su presencia en los gránulos de estrés disminuye considerablemente cuando se tratan las células con leptomicina B (un inhibidor de la salida de proteínas del núcleo) o con actinomicina D y α-amanitina (inhibidores de la transcripción). CERKL colocaliza también con otros componentes celulares conteniendo ribonucleoproteínas, como son los cuerpos P, los polisomas y unas partículas ribonucleoproteicas compactas. La asociación de CERKL en los polisomas se pierde cuando se tratan con EDTA y RNasa A que los desensamblan, mientras que en las partículas ribonucleoproteicas compactas eso solo ocurre cuando los tratamientos anteriores se realizan a concentraciones salinas elevadas que reducen su compactación. CERKL interacciona con: i) proteínas de la maquinaria de traducción de los RNAs mensajeros, como son eIF3B, PABP o RPS3, ii) chaperonas que intervienen en el plegamiento de las proteínas recién sintetizadas, como son HSP70 o HSP90, iii) las tubulinas α y β de los microtúbulos, y iv) proteínas de la membrana plasmática, como son la filagrina, la desmoplaquina y la desmogleína. Además, CERKL interacciona con proteínas que intervienen en el metabolismo del DNA o del RNA, como la nucleofosmina, en el transporte al núcleo, como la importina 4, y en la unión a lípidos, como la apolipoproteína D. La interacción de CERKL con PABP, RPS3 y HSP70, pero no con eIF3B, se pierde tras un tratamiento con RNasa A en presencia de concentraciones salinas elevadas. Estos resultados son consistentes con que la mayoría de esas interacciones ocurren en partículas ribonucleoproteicas compactas y que la interacción de CERKL con PABP, RPS3 y HSP70 depende de la integridad de los RNAs mensajeros, mientras que la interacción con eIF3B podría ocurrir directamente. El mutante patológico C125W de CERKL es incapaz de interaccionar con la proteína eIF3B y sus interacciones con PABP, HSP70 y RPS3 no se pierden tras un tratamiento con RNasa A. Esto sugiere que este mutante presenta anormalidades en la formación de las partículas ribonucleoproteicas compactas, probablemente debido a su incapacidad de unirse eficazmente a los RNAs mensajeros. CERKL interacciona con componentes del complejo 48S de preinicio de la traducción, tales como las proteínas eIF3B, eIF3G y eIF3I, con proteínas ribosomales de la subunidad 40S, como las RPS3, RPS5 y RPSA y con factores no proteicos, como el residuo 7-metil-guanosina del extremo 5´ del RNA mensajero.	es_ES
dc.format.extent	138 p.	es_ES
dc.language.iso	es	es_ES
dc.subject	CERKL	es_ES
dc.subject	retinosis pigmentaria	es_ES
dc.subject	RNA mensajero	es_ES
dc.title	Aproximacion funcional a CERKL, un gen causante de la retinosis pigmentaria, mediante el estudio de la localización intracelular de la proteína y la identificación de sus partners proteicos y no proteicos	es_ES
dc.type	doctoral thesis	es_ES
dc.subject.unesco	UNESCO::CIENCIAS MÉDICAS	es_ES
dc.subject.unesco	UNESCO::QUÍMICA	es_ES
dc.embargo.terms	0 days	es_ES