Molecular insights into the regulation of the oncoprotein SET/TAF-Iß by extramitochondrial cytochrome c

La regulación adecuada de las respuestas celulares al estrés es fundamental para mantener la homeostasis y evitar la proliferación celular descontrolada. Entre los agentes de estrés, los estímulos genotóxicos pueden comprometer procesos celulares esenciales, tales como la replicación o la transcripción del ADN. Por lo tanto, una reparación adecuada del ADN es fundamental para evitar mutaciones o aberraciones en el genoma que puedan amenazar la viabilidad celular, provocar neoplasias o enfermedades genéticas. Los mecanismos de respuesta al daño del ADN implican una compleja red de proteínas que señalizan, ejecutan y/o regulan la reparación del ADN o la entrada de la célula en apoptosis. El citocromo c respiratorio es un ejemplo de proteína multifuncional que actúa en el mantenimiento de la homeostasis energética en la mitocondria, pero que también participa en respuestas al estrés en los principales compartimentos celulares, como son el citoplasma y el núcleo. Se ha demostrado que la translocación del citocromo c al núcleo bajo estrés genotóxico conlleva su interacción con numerosas proteínas nucleares implicadas en la reparación del ADN y la supervivencia celular. La presente Tesis Doctoral se centra en la interacción entre el citocromo c y SET/TAF-I?, una oncoproteína predominantemente nuclear que desempeña un papel clave tanto en la remodelación de la cromatina como en la respuesta al daño en el ADN, entre otros procesos celulares relacionados con la supervivencia celular. El trabajo desarrollado profundiza en los mecanismos moleculares que subyacen al reconocimiento entre ambas proteínas y cómo modulan la función de SET/TAF-I?

Cytochrome c prompts the recruitment of its nuclear partners SET/TAF-Iβ and NPM1 into biomolecular condensates

International audienceCompartmentalization of proteins by liquid-liquid phase separation (LLPS) is used bycells to control biochemical reactions spatially and temporally. Among them, therecruitment of proteins to DNA foci and nucleolar trafficking occurs by biomolecularcondensation. Within this frame, the oncoprotein SET/TAF-I plays a key role in bothchromatin remodeling and DNA damage response, as does nucleophosmin (NPM1)which indeed participates in nucleolar ribosome synthesis. Whereas phase separationby NPM1 is widely characterized, little is known about that undergone by SET/TAF-I.Here, we show that SET/TAF-I experiences phase transition together with respiratorycytochrome c (Cc), which translocates to the nucleus upon DNA damage. Here wereport the molecular mechanisms governing Cc-induced phase separation of SET/TAF-I and NPM1, where two lysine-rich clusters of Cc are essential to recognize molecularsurfaces on both partners in a specific and coordinated manner. Cc thus emerges as asmall, globular protein with sequence-encoded heterotypic phase-separationproperties

Mitochondrial cytochrome c shot towards histone chaperone condensates in the nucleus

Despite mitochondria being key for the control of cell homeostasis and fate, their role in DNA damage response is usually just regarded as an apoptotic trigger. However, growing evidence points to mitochondrial factors modulating nuclear functions. Remarkably, after DNA damage, cytochrome c (Cc) interacts in the cell nucleus with a variety of well-known histone chaperones, whose activity is competitively inhibited by the haem protein. As nuclear Cc inhibits the nucleosome assembly/disassembly activity of histone chaperones, it might indeed affect chromatin dynamics and histone deposition on DNA. Several histone chaperones actually interact with Cc Lys residues through their acidic regions, which are also involved in heterotypic interactions leading to liquid–liquid phase transitions responsible for the assembly of nuclear condensates, including heterochromatin. This relies on dynamic histone–DNA interactions that can be modulated by acetylation of specific histone Lys residues. Thus, Cc may have a major regulatory role in DNA repair by fine-tuning nucleosome assembly activity and likely nuclear condensate formation.Ministerio de Ciencia, Innovación y Universidades PGC2018-096049-B-I00Junta de Andalucía BIO198, US-1254317, US-1257019, P18-FR-3487, P18-HO-409

Association of ABCB1 and VEGFA gene polymorphisms with breast cancer susceptibility and prognosis

Breast cancer (BC) is the most common cause of cancer-related death in women worldwide. Several ABCB1 and VEGFA gene polymorphisms, such as ABCB1-G1199 T/A (rs2229109), VEGFA -634 G > C (rs2010963), VEGFA 2578 C > A (rs699947) and VEGFA 7 C > T (rs25648) have been associated with risk of BC and clinical outcomes. The purpose of this study was to evaluate the association between these gene polymorphisms and BC risk and prognosis. A retrospective case-control study was conducted, including 84 BC cases and 119 controls of Spanish (European, Caucasian) origin. ABCB1-G1199 T/A (rs2229109), VEGFA -634 G > C (rs2010963), VEGFA 2578 C > A (rs699947) and VEGFA 7 C > T (rs25648) gene polymorphisms were analysed by TaqMan®. The genotypic logistic regression model adjusted by aged revealed no association with any of the polymorphisms and BC risk, although the C-allele of VEGFA 2578 C > A showed a trend to higher BC risk in the allelic and recessive models (p = 0.055 and 0.054, respectively). There was no influence of these gene polymorphisms on overall survival (OS). The univariate Cox model showed that carriers of the A-allele for VEGFA 2578 C > A tended to have longer OS compared to CC patients (CC vs A-allele Hazard ratio (HR): 2.08; CI95 % = 0.96-4.49; p = 0.0587). There was no association between the gene polymorphisms analysed and disease-free survival (DFS). The univariate Cox model showed a trend toward a longer DFS in patients carrying ABCB1-G1199 T/A GG genotype compared to those with A-allele (GG vs A-allele HR: 0.43; CI95 % = 0.18-1.03; p = 0.0612). No influence of ABCB1-G1199 T/A (rs2229109), VEGFA -634 G > C (rs2010963), VEGFA 2578 C > A (rs699947) and VEGFA 7 C > T (rs25648) gene polymorphisms on risk of developing BC was found in our study. There was no association between the polymorphisms studied and DFS and OS

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iβ

International audienceIntrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iβ

International audienceIntrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iβ (SET/TAF-Iβ), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iβ is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iβ is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iβ:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iβ (a.k.a. SET/TAF-Iβ ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations

Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation.

International audienceThe regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins