Understanding the indirect DNA read-out specificity of I-CreI Meganuclease

The high DNA specificity of homing endonucleases makes them a powerful protein scaffold to engineer enzymes for genome manipulation. Understanding their molecular recognition of DNA is an important prerequisite to generate engineered enzymes able to cleave DNA in specific desired genome sites. Protein-DNA recognition studies have been mostly focused on specific direct contacts between amino acid side chains and bases to redesign the binding interface. However, the important role of indirect readout in the central region of the target DNA of the homing endonuclease I-CreI suggested that indirect readout may play a key role in the redesign of protein-DNA interactions. The sequences of the I-CreI central substrate region, 2NN, along with the adjacent 5NNN, are key for substrate cleavage. Here, we analyse the mechanism of target discrimination at the 5NNN region by the I-CreI protein, revealing its critical role in the location and occupancy of the catalytic metal ions, which is crucial for cleavage. Our data highlight the importance of indirect readout for target DNA cleavage, thus aiding I-CreI engineering when targeting new DNA sequences.e thank the Swiss Light Source (SLS) and ALBA beamline staff for their support. This work was supported by the Spanish MINECO (JCI-2011-09308 to R.M and CTQ2017-83810-R to F.J.B.), the Severo Ochoa Excellence Accreditation (SEV-2016-0644) and the Novo Nordisk Foundation (Grant NNF14CC0001 to G.M.),S

Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif

Heterotrimeric G proteins are quintessential signalling switches activated by nucleotide exchange on Gα. Although activation is predominantly carried out by G-protein-coupled receptors (GPCRs), non-receptor guanine-nucleotide exchange factors (GEFs) have emerged as critical signalling molecules and therapeutic targets. Here we characterize the molecular mechanism of G-protein activation by a family of non-receptor GEFs containing a Gα-binding and -activating (GBA) motif. We combine NMR spectroscopy, computational modelling and biochemistry to map changes in Gα caused by binding of GBA proteins with residue-level resolution. We find that the GBA motif binds to the SwitchII/α3 cleft of Gα and induces changes in the G-1/P-loop and G-2 boxes (involved in phosphate binding), but not in the G-4/G-5 boxes (guanine binding). Our findings reveal that G-protein-binding and activation mechanisms are fundamentally different between GBA proteins and GPCRs, and that GEF-mediated perturbation of nucleotide phosphate binding is sufficient for Gα activation

Double Monoubiquitination Modifies the Molecular Recognition Properties of p15PAF Promoting Binding to the Reader Module of Dnmt1

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its Nterminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.Spanish Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional (MINECO/FEDER) [CTQ2017-83810-R to F.J.B.]; Labex EpiGenMed, an “Investissements d”avenir’ program [ANR-10-LABX-12-01 to PB]. MOSTMicro [LISBOA-01-0145-FEDER-007660 to T.N.C. and H.M.]. A.G.M. acknowledges Spanish MINECO for predoctoral contract BE-2015-075847, and the CIC bioGUNE acknowledges MINECO for the Severo Ochoa accreditation Sev-2016-0644. The CBS-Montpellier is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INSB-04-01 and ANR-10-INSB- 05, respectively)

Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair

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Equality, Equity, and Diversity: Educational Solutions in the Basque Country

Public education is one of the greatest achievements of European countries during the twentieth century. While schooling systems neither exclusively form citizens, nor are they sufficient to alleviate all inequalities, education plays an increasingly important strategic role in relieving social problems and promoting the civic and ethical upbringing of our children. Researchers and professors at the UPV/EHU have had the privilege to design and implement important educational projects in conjunction with government of Autonomous Community of the Basque Country, which has the authority over education in its territory. This book presents the timely (in most cases since 2000) observations, research, and programs that have resulted from this cooperation. Our stress—in both our theoretical and analytical dimensions—has been on the importance of diversity, the promotion of social and human values, and respect for basic human rights. In addition, we describe the cooperation that must be fostered—and the various needs met—between all educational "agents": academic researchers, administrators, teachers, parents, and the community at large to promote equality and fairness in our society.This book was published with generous financial support from the Basque Government.Introduction: Alfonso Unceta and Concepción Medrano ? Part 1 Education in the Basque Country ? 1. Education Provision in the Basque Country by Alfonso Unceta and Andrés Davila ? 2. Addressing Basque Diversity in the Classroom: Measures to Avoid Excluding At-Risk Youth by Begoña Martínez Domínguez ? 3. Improving Social Interaction: Experimentally Validated Proposals for Psycho-educational Intervention by Maite Garaigodobil and Jone Aliri ? 4. Socialization to Prevent Gender Violence in the Basque Country by Maria José Alonso Olea, Aitor Gómez González, and Nekane Beloki Arizti ? 5. Resolution and Transformation of At-School Conflicts by Ramón Alzate Sáez de Heredia, Lucía Gorbeña, and Cristina Merino ? Part 2 Socioeducational Context in the Basque Country ? 6. Learning Communities: A Basque Egalitarian Educational Project by Maite Arandia Loroño, Isabel Martínez Domínguez, and Iñaki Santa Cruz Ayo ? 7. Migrants en Route: Community Socioeducational Action by Miguel Arriaga Landeta and Begoña Abad Miguélez ? 8. Educating from the Family: A Proposal to Connect Homes and Institutions by Enrique Arranz Freijo, Fernando Olabarrieta Artetxe, Juan Luis Martín Ayala ? 9. The Development of Values and the Media by Concepción Medrano, Ana Aierbe, and Juan Ignacio Martínez de Morentin ? Index ? List of Contributor

Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease

Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches

Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus

Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein–DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo

ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy

The redirection of T cell activity using bispecific antibodies is one of the most promising cancer immunotherapy approaches currently in development, but it is limited by cytokine storm-related toxicities, as well as the pharmacokinetics and tumor-penetrating capabilities of current bispecific antibody formats. Here, we have engineered the ATTACK (Asymmetric Tandem Trimerbody for T cell Activation and Cancer Killing), a novel T cell-recruiting bispecific antibody which combines three EGFR-binding single-domain antibodies (VHH; clone EgA1) with a single CD3-binding single-chain variable fragment (scFv; clone OKT3) in an intermediate molecular weight package. The two specificities are oriented in opposite directions in order to simultaneously engage cancer cells and T cell effectors, and thereby promote immunological synapse formation. EgA1 ATTACK was expressed as a homogenous, non-aggregating, soluble protein by mammalian cells and demonstrated an enhanced binding to EGFR, but not CD3, when compared to the previously characterized tandem bispecific antibody which has one EgA1 VHH and one OKT3 scFv per molecule. EgA1 ATTACK induced synapse formation and early signaling pathways downstream of TCR engagement at lower concentrations than the tandem VHH-scFv bispecific antibody. Furthermore, it demonstrated extremely potent, dose-dependent cytotoxicity when retargeting human T cells towards EGFR-expressing cells, with an efficacy over 15-fold higher than that of the tandem VHH-scFv bispecific antibody. These results suggest that the ATTACK is an ideal format for the development of the next-generation of T cell-redirecting bispecific antibodies

Running title: Non-toxic broad anti-tumor activity of an EGFR×4-1BB bispecific trimerbod

32 p.-4 fig.Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).Peer reviewe

A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with Fc gamma R interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8(N)/(C)EGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8(N)/(C)EGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8(N)/(C)EGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate Fc gamma R interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy