Molecular recognition between PCNA and proteins involved in DNA replication and repair: p15 and p12

162 p.El antígeno nuclear de células en proliferación (PCNA) es un factor esencial en replicación y reparación de ADN en eucariotas. Forma un anillo homotrimérico que rodea y se desliza a lo largo del ADN, actuando como una plataforma de anclaje de enzimas que modifican el ADN y de proteínas reguladoras a la horquilla de replicación. Estas interacciones ocurren frecuentemente a través de una secuencia consenso de aminoácidos denominada motivo PIP que se une a un bolsillo hidrofóbico de PCNA. En esta tesis doctoral se estudian proteínas de unión a PCNA implicadas en la replicación y reparación del ADN, con el fin último de entender sus interacciones y función utilizando una aproximación estructural. Por un lado, se ha realizado un exhaustivo análisis conformacional de la proteína p15PAF doblemente monoubiquitinada y el impacto de esta modificación postraduccional en su unión a PCNA y a ADN.También ha caracterizado la interacción de la subunidad p12 de la polimerasa delta (pol ¿) humana con PCNA mediante RMN, calorimetría y cristalografía. Los resultados presentados en esta tesis contribuyen a entender mejor la organización y función de componentes esenciales de las maquinarias de replicación y reparación del ADN en la célula

The P. aeruginosa effector Tse5 forms membrane pores disrupting the membrane potential of intoxicated bacteria

The type VI secretion system (T6SS) of Pseudomonas aeruginosa injects effector proteins into neighbouring competitors and host cells, providing a fitness advantage that allows this opportunistic nosocomial pathogen to persist and prevail during the onset of infections. However, despite the high clinical relevance of P. aeruginosa, the identity and mode of action of most P. aeruginosa T6SS-dependent effectors remain to be discovered. Here, we report the molecular mechanism of Tse5-CT, the toxic auto-proteolytic product of the P. aeruginosa T6SS exported effector Tse5. Our results demonstrate that Tse5-CT is a pore-forming toxin that can transport ions across the membrane, causing membrane depolarisation and bacterial death. The membrane potential regulates a wide range of essential cellular functions; therefore, membrane depolarisation is an efficient strategy to compete with other microorganisms in polymicrobial environments.We gratefully acknowledge the Laboratories of Dr. Daniel Ladant (Institut Pasteur, Paris) and Dr. Victor de Lorenzo (Centro Nacional de Biotecnologia, Madrid) for the plasmids received (pKTop and pSEVA plasmids, respectively). Also, we would like to acknowledge the Laboratory of Dr. Joseph Mougous for the P. aeruginosa strains received. The technical assistance from Cristina Civantos and Adrian Ruiz is also very much appreciated. We acknowledge the FGCZ for the mass spectrometry analyses and the technical support (Functional Genomics Center Zurich (FGCZ), University/ETH Zurich). D.A.-J. acknowledges support by the MINECO Contracts CTQ2016-76941-R and PID2021-127816NB-I00, Fundacion Biofisica Bizkaia, the Basque Excellence Research Centre (BERC) programme, and IT709-13 and IT1745-22 of the Basque Government, and Fundacion BBVA. A.G.-M. acknowledges the financial support received from the Spanish Ministry of Universities and the Grants for the requalification of the Spanish university system for 2021-2023, financed by the European Union-Next Generation EU-Margarita Salas Modality. A.A. acknowledges support from the Spanish Ministry of Science and Innovation (Project 2019-108434GB-I00 funded by MCIN/AEI/10.13039/501100011033), Generalitat Valenciana (project AICO/2020/066) and Universitat Jaume I (project UJI-B2018-53). M.Q.-M. acknowledges support from the Spanish Ministry of Science and Innovation (Project IJC2018-035283-I funded by MCIN/AEI/10.13039/501100011033) and Universitat Jaume I (project UJI-A2020-21). P.B acknowledges the financial support received from the Spanish Ministry of Science and Innovation through the Ramon y Cajal Programme (contract RYC2019-026551-I)

Molecular recognition between PCNA and proteins involved in DNA replication and repair: p15 and p12

162 p.El antígeno nuclear de células en proliferación (PCNA) es un factor esencial en replicación y reparación de ADN en eucariotas. Forma un anillo homotrimérico que rodea y se desliza a lo largo del ADN, actuando como una plataforma de anclaje de enzimas que modifican el ADN y de proteínas reguladoras a la horquilla de replicación. Estas interacciones ocurren frecuentemente a través de una secuencia consenso de aminoácidos denominada motivo PIP que se une a un bolsillo hidrofóbico de PCNA. En esta tesis doctoral se estudian proteínas de unión a PCNA implicadas en la replicación y reparación del ADN, con el fin último de entender sus interacciones y función utilizando una aproximación estructural. Por un lado, se ha realizado un exhaustivo análisis conformacional de la proteína p15PAF doblemente monoubiquitinada y el impacto de esta modificación postraduccional en su unión a PCNA y a ADN.También ha caracterizado la interacción de la subunidad p12 de la polimerasa delta (pol ¿) humana con PCNA mediante RMN, calorimetría y cristalografía. Los resultados presentados en esta tesis contribuyen a entender mejor la organización y función de componentes esenciales de las maquinarias de replicación y reparación del ADN en la célula

Human PCNA Structure, Function and Interactions

© 2020 by the authors.Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and repair. It forms a homotrimeric ring that embraces the DNA and slides along it, anchoring DNA polymerases and other DNA editing enzymes. It also interacts with regulatory proteins through a sequence motif known as PCNA Interacting Protein box (PIP-box). We here review the latest contributions to knowledge regarding the structure-function relationships in human PCNA, particularly the mechanism of sliding, and of the molecular recognition of canonical and non-canonical PIP motifs. The unique binding mode of the oncogene p15 is described in detail, and the implications of the recently discovered structure of PCNA bound to polymerase δ are discussed. The study of the post-translational modifications of PCNA and its partners may yield therapeutic opportunities in cancer treatment, in addition to illuminating the way PCNA coordinates the dynamic exchange of its many partners in DNA replication and repair.This research was funded by grant CTQ2017-83810-R from MCIU/AEI/FEDER, UEPeer reviewe

The P. aeruginosa type VI secretion system effector Tse5 forms ion-selective membrane pores that disrupt the membrane potential of intoxicated cells

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.Pseudomonas aeruginosa is an opportunistic pathogen of great clinical impact, being the main cause of acute and chronic infections in immunosuppressed and cystic fibrosis patients. This virulence is largely due to the secretion systems possessed by the bacteria, among which the Type 6 Secretion System (T6SS) in P. aeruginosa stands out 1. The T6SS is a nanomachine that assembles inside the bacteria and injects severa effectors/toxins into target cells 2. The large number of effectors and their functional diversity play a crucial role in the virulence of the infection caused by this gram-negative bacterium. One of these effectors is the Type VI secretion system exported effector 5 (Tse5) which was described to have bacteriolytic activity although its molecular activity is still unknown 3,4. In this work we studied the molecular function of Tse5 effector. To do so, we performed growth inhibition curves to determine if it has a bacteriolytic or bacteriostatic effect. We hypothesised that the toxic domain of Tse5 (Tse5-CT) exerts its toxic activity on the membrane since its immunity protein (Tsi5) inserts into the inner bacterial membrane to neutralise the toxicity 3. To define if this toxin can increase cell permeability or disrupt membrane potential of the target bacteria, we carried out flow cytometry experiments in Pseudomonas putida. Furthermore, we designed a Tse5-CT deletion mutants with a dual reporter localize at the C-terminus that allowed to identify transmembrane regions. Our data indicate that Tse5-CT expression has a bacteriolytic effect on Pseudomonas putida cells that can be reversed by co-expression of the cognate immunity protein Tsi5. Moreover, Tse5 toxin inserts into the membrane through transmembrane and amphipathic domains and disrupts membrane potential of target cells. The results obtained in this study help to gain insight regarding the function and the mechanisms of action Tse5 effector and eventually could help to develop drugs that mimic its behaviour

The effector Tse5 of P. aeruginosa forms ion-selective membrane pores that disrupt the membrane potential of intoxicated bacteria

Póster presentado en Gordon Research Conference on Microbial Toxins and Pathogenicity, celebrado en Southbridge (USA) entre el 10 y el 15 de julio de 2022

Structural analysis of ING3 protein and histone H3 binding

25 p.-6 fig.-2 tab.Proteins belonging to the ING family regulate the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at Lysine 4 (H3K4me3). This modification is recognized by the Plant HomeoDomain (PHD) present at the C-terminal region of the five ING proteins. ING3 facilitates acetylation of histones H2A and H4 by the NuA4-Tip60 MYST histone acetyl transferase complex, and it has been proposed to be an oncoprotein. The crystal structure of the N-terminal domain of ING3 shows that it forms homodimers with an antiparallel coiled-coil fold. The crystal structure of the PHD is similar to those of its four homologs. These structures explain the possible deleterious effects of ING3 mutations detected in tumors. The PHD binds histone H3K4me3 with low-micromolar, and binds the non-methylated histone with a 54-fold reduced affinity. Our structure explains the impact of site directed mutagenesis experiments on histone recognition. These structural features could not be confirmed for the full-length protein as solubility was insufficient for structural studies, but the structure of its folded domains suggest a conserved structural organization for the ING proteins as homodimers and bivalent readers of the histone H3K4me3 mark.FJB and MF-G were supported by grants PID2020-113225GB-I00 and PRE2018-085788 funded by MCIN/AEI/10.13039/501100011033. AG-M. acknowledges the financial support received from the Spanish Ministry of Universities and the Grants for the requalification of the Spanish university system for 2021-2023, funded by the European Union-Next Generation EU-Margarita Salas Modality.Peer reviewe

An in vitro system of autologous lymphocytes culture that allows the study of homeostatic proliferation mechanisms in human naive CD4 T-cells.

The size of peripheral T-cell pool is kept constant throughout life. However, a decline in lymphocyte numbers is a feature of several human disorders, in which fast and slow homeostatic proliferation play a crucial role. Several in vitro and in vivo models have been developed to study such processes. Nevertheless, self- and commensal- antigens, well-known triggers of homeostatic proliferation, have not been examined in these models. We have designed an in vitro culture of human T-cells exposed to rIL7 and autologous antigen-presenting cells (aAPC) that allows the simultaneous characterization of the different types of homeostatic proliferation. Using our model, we first confirmed that both rIL7 and aAPC are survival signals ultimately leading to homeostatic proliferation. In addition, we explored the modulation of different anti-apoptotic, proliferative, activation and homing markers during fast and slow homeostatic proliferation. Finally, different subsets of Treg were generated during homeostatic proliferation in our model. In summary, our in vitro system is able to simultaneously reproduce both types of homeostatic proliferation of human naive CD4 T-cells, and allows the characterization of these processes. Our in vitro system is a useful tool to explore specific features of human homeostatic proliferation in different human lymphopenia-related disorders and could be used as a cell therapy approach

Structural and functional insights into the delivery of a bacterial Rhs pore-forming toxin to the membrane

Abstract Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial Rearrangement hot spot (Rhs) proteins represent a widespread example of toxin polymorphism. Here, we present the 2.45 Å cryo-electron microscopy structure of Tse5, an Rhs protein central to Pseudomonas aeruginosa type VI secretion system-mediated bacterial competition. This structural insight, coupled with an extensive array of biophysical and genetic investigations, unravels the multifaceted functional mechanisms of Tse5. The data suggest that interfacial Tse5-membrane binding delivers its encapsulated pore-forming toxin fragment to the target bacterial membrane, where it assembles pores that cause cell depolarisation and, ultimately, bacterial death