Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold.

Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous recombination, an error-free mechanism, or by non-homologous end joining, a process susceptible to introducing errors in the repaired sequence. The type of DNA cleavage might alter the balance between these two alternatives. The use of "nickases" producing a specific single strand break instead of a double strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional.

Blocking ephrinB2 with highly specific antibodies inhibits angiogenesis, lymphangiogenesis, and tumor growth.

Membrane-anchored ephrinB2 and its receptor EphB4 are involved in the formation of blood and lymphatic vessels in normal and pathologic conditions. Eph/ephrin activation requires cell-cell interactions and leads to bidirectional signaling pathways in both ligand- and receptor-expressing cells. To investigate the functional consequences of blocking ephrinB2 activity, 2 highly specific human single-chain Fv (scFv) Ab fragments against ephrinB2 were generated and characterized. Both Ab fragments suppressed endothelial cell migration and tube formation in vitro in response to VEGF and provoked abnormal cell motility and actin cytoskeleton alterations in isolated endothelial cells. As only one of them (B11) competed for binding of ephrinB2 to EphB4, these data suggest an EphB-receptor-independent blocking mechanism. Anti-ephrinB2 therapy reduced VEGF-induced neovascularization in a mouse Matrigel plug assay. Moreover, systemic administration of ephrinB2-blocking Abs caused a drastic reduction in the number of blood and lymphatic vessels in xenografted mice and a concomitant reduction in tumor growth. Our results show for the first time that specific Ab-based ephrinB2 targeting may represent an effective therapeutic strategy to be used as an alternative or in combination with existing antiangiogenic drugs for treating patients with cancer and other angiogenesis-related diseases.

Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics.

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO(-) present in TBCB, which is similar to the EEY/F-COO(-) element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.

Non-specific protein-DNA interactions control I-CreI target binding and cleavage.

Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.

Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B.

E2 ubiquitin conjugases are ~20kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-C(ca) was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-C(ca) towards non-canonical E3 ligases. Binding of E2-C(ca) to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ~400nM, as shown by fluorescence and ITC. Furthermore, binding of E2-C(ca) to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-C(ca) comprises the first ß-strand and nearby residues.

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.

Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev.

Non-specifically bound nucleic acid contaminants are an unwanted feature of recombinant RNA-binding proteins purified from Escherichia coli (E. coli). Removal of these contaminants represents an important step for the proteins' application in several biological assays and structural studies. The method described in this paper is a one-step protocol which is effective at removing tightly bound nucleic acids from overexpressed tagged HIV-1 Rev in E. coli. We combined affinity chromatography under denaturing conditions with subsequent on-column refolding, to prevent self-association of Rev while removing the nucleic acid contaminants from the end product. We compare this purification method with an established, multi-step protocol involving precipitation with polyethyleneimine (PEI). As our tailored protocol requires only one-step to simultaneously purify tagged proteins and eliminate bound cellular RNA and DNA, it represents a substantial advantage in time, effort, and expense.

Dynamics and Cleavability at the alpha-cleavage site of APP(684-726) in different lipid environments.

The occurrence of late-onset Alzheimer's disease has been related to the lipid homeostasis. We tested whether the membrane lipid environment affects the dynamics and cleavability of a model peptide corresponding to the amino acid sequence 684-726 of the amyloid precursor protein APP reconstituted in liposomes. Solid-state NMR with (2)H-Ala(713), which is located within the putative transmembrane domain, suggested that the peptide observes less rotational motion in egg phosphatidylcholine (PhC) membranes than in dimyristoyl-phosphatidylcholine (DMPC) bilayers above the main phase transition temperature T(c). The residue (15)N-Ala(692), which is in the vicinity of the alpha-cleavage site, i.e., Lys(687), showed less motion after reconstitution in distearoyl-phosphatidylcholine liposomes

Conformational Selection and Induced Fit Mechanisms in the Binding of an Anticancer Drug to the c-Src Kinase.

Understanding the conformational changes associated with the binding of small ligands to their biological targets is a fascinating and meaningful question in chemistry, biology and drug discovery. One of the most studied and important is the so-called "DFG-flip" of tyrosine kinases. The conserved three amino-acid DFG motif undergoes an "in to out" movement resulting in a particular inactive conformation to which "type II" kinase inhibitors, such as the anti-cancer drug Imatinib, bind. Despite many studies, the details of this prototypical conformational change are still debated. Here we combine various NMR experiments and surface plasmon resonance with enhanced sampling molecular dynamics simulations to shed light into the conformational dynamics associated with the binding of Imatinib to the proto-oncogene c-Src. We find that both conformational selection and induced fit play a role in the binding mechanism, reconciling opposing views held in the literature. Moreover, an external binding pose and local unfolding (cracking) of the aG helix are observed.

Alpha-tocopherol influences the lipid membrane affinity of desipramine in a pH-dependent manner.

Phopholipidosis is a lipid storage disorder caused by cationic amphiphilic drugs (CADs) characterized by the lysosomal accumulation of phospholipids and drug. alpha-Tocopherol (alpha-Toc) has a reversible effect on phospholipidosis in rats and cell culture. We studied the influence of alpha-Toc on the partitioning of the CAD desipramine in a liposome/buffer system using equilibrium dialysis with the following lipid compositions: egg phosphatidylcholine (PhC) or wheat germ phosphatidylinositol (PhI) or a combination of PhC, PhI and cholesterol, containing between 1.5 and 20% (mol per mol total lipids) of alpha-Toc, alpha-tocopherol acetate (alpha-TocAc), 2,2,5,7,8-pentamethyl-6-chromanol (PMC) or cholesterol. alpha-Toc (1.5%) enhanced the partition coefficient of neutral desipramine by up to 1.1 log units while it had no influence on the partitioning of the ionized compound. In the PhC liposome system, at pH 7.4 logD increased with increasing alpha-Toc concentrations but was unchanged at pH 4.5. Similar effects were found with PMC while alpha-TocAc or cholesterol, between 1.5 and 20%, had no influence on the partitioning of desipramine. From these results we postulate that in vivo, alpha-Toc could mediate a redistribution of CADs from lysosomal membranes (pH approximately 4.5) to membranes and lipoproteins at physiological pH.

Backbone assignment of the tyrosine kinase Src catalytic domain in complex with imatinib.

The Src tyrosine kinase is the paradigm of an oncogenic kinase, and of regulation by intramolecular inhibitory interactions, as well as an important anticancer target due to its roles in cell proliferation and metastasis. The assignment of backbone (1)H(N), (13)C(α), (13)CO, and (15)N, and sidechain (13)C(ß) resonances of the catalytic domain of Src (283 residues) in complex with the anticancer drug Imatinib is reported here. Consistent with previous X-ray studies of the same complex, most signals from the activation loop are not detected, indicating that, even in the presence of the drug, it probably adopts highly heterogeneous conformations in intermediate exchange. For the rest of the polypeptide backbone, assignments have been completed for ~88% of residues, with only a few solvent-exposed amides remaining unassigned.

The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved.

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so.

Helical and flat structures from chiral dendronized rectangular oligo(phenylene ethynylene)s.

The synthesis of two chiral amphiphiles and their self-assembling features in solution and onto surfaces is reported. The different degree of interdigitation of the paraffinic substituents has an enormous influence in the chirality of the aggregates. Thus, while oligo(phenylene ethynylene) 1 shows a bisignated Cotton effect in solution and P-type helices onto surfaces, compound 2 lacks in any dichroic response at room temperature and self-assembles into flat ribbons.