Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Expert Opin Drug Deliv ; 19(8): 927-944, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35899474

RESUMO

INTRODUCTION: The development of long-acting injectables (LAIs) for protein and peptide therapeutics has been a key challenge over the last 20 years. If these molecules offer advantages due to their high specificity and selectivity, their controlled release may confer several additional benefits in terms of extended half-life, local delivery, and patient compliance. AREA COVERED: This manuscript aims to give an overview of peptide and protein-based LAIs from an industrial perspective, describing both approved and promising technologies (with exceptions of protein engineering strategies and devices), their advantages and potential improvements to aid their access to the market. EXPERT OPINION: Many LAIs have been developed for peptides, with formulations on the market for several decades. On the contrary, LAIs for proteins are still far from the market and issues related to manufacturing and sterilization of these products still need to be overcome. In situ forming depots (ISFDs), whose simple manufacturing conditions and easy administration procedures (without reconstitution) are strong advantages, appear as one of the most promising technologies for the delivery of these molecules. In this regard, the approval of ELIGARD® in the early 2000's (which still requires a complex reconstitution process), paved the way for the development of second-generation, ready-to-use ISFD technologies like BEPO® and FluidCrystal®.


Assuntos
Peptídeos , Proteínas , Preparações de Ação Retardada , Humanos , Injeções
2.
J Control Release ; 341: 578-590, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34915070

RESUMO

Monoclonal antibodies (mAbs) are large size molecules that have demonstrated high therapeutic potential for the treatment of cancer or autoimmune diseases. Despite some excellent results, their intravenous administration results in high plasma concentration. This triggers off-target effects and sometimes poor targeted tissue distribution. To circumvent this issue, we investigated a local controlled-delivery approach using an in situ forming depot technology. Two clinically relevant mAbs, rituximab (RTX) and daratumumab (DARA), were formulated using an injectable technology based on biodegradable PEG-PLA copolymers. The stability and controlled release features of the formulations were investigated. HPLC and mass spectrometry revealed the preservation of the protein structure. In vitro binding of formulated antibodies to their target antigens and to their cellular FcγRIIIa natural killer cell receptor was fully maintained. Furthermore, encapsulated RTX was as efficient as classical intravenous RTX treatment to inhibit the in vivo tumor growth of malignant human B cells in immunodeficient NSG mice. Finally, the intra-articular administration of the formulated mAbs yielded a sustained local release associated with a lower plasma concentration compared to the intra-articular delivery of non-encapsulated mAbs. Our results demonstrate that the utilization of this polymeric technology is a reliable alternative for the local delivery of fully functional clinically relevant mAbs.


Assuntos
Polímeros , Animais , Preparações de Ação Retardada/química , Camundongos , Polímeros/química
3.
J Control Release ; 336: 457-468, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34214596

RESUMO

The generation of acylated impurities has represented an important hurdle in the development of long acting injectables for therapeutic peptides using biocompatible polymers with a polyester moiety. We investigated here an in situ forming depot (ISFD) technology that uses polyethylene glycol - polyester copolymers and a solvent exchange mechanism to promote depot formation. This technology has shown promise in formulating small molecules as well as therapeutic proteins. In the present work, using the well-known somatostatin analog octreotide acetate (OctAc) as a model molecule, we evaluated this delivery platform to release therapeutic peptides. Peptide acylation was found to be pronounced in the formulation, while it was very limited once the depot was formed and during the release process. The octreotide acylation pattern was fully characterized by LC-MS/MS. Moreover, it was demonstrated that exchanging the acetate anion with more hydrophobic counterions like pamoate or lauryl sulfate allowed to greatly improve the peptide stability profile, as well as the formulation release performance. Finally, the in vivo evaluation through pharmacokinetics studies in rat of these new octreotide salts in ISFD formulations showed that octreotide was quantifiable up to four weeks post-administration with a high bioavailability and an acceptable initial burst.


Assuntos
Octreotida , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida , Cinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Tecnologia
4.
Nucleic Acid Ther ; 31(3): 208-219, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-32678992

RESUMO

Antisense-mediated exon skipping constitutes a promising new modality for treatment of Duchenne Muscular Dystrophy (DMD), which is caused by gene mutations that typically introduce a translation stop codon in the dystrophin gene, thereby abolishing production of functional dystrophin protein. The exon removal can restore translation to produce a shortened, but still partially functional dystrophin protein. Peptide nucleic acid (PNA) as a potential antisense drug has previously been shown to restore the expression of functional dystrophin by splice modulation in the mdx mouse model of DMD. In this study, we compare systemic administration of a 20-mer splice switching antisense PNA oligomer through intravenous (i.v.) and subcutaneous (s.c.) routes in the mdx mice. Furthermore, the effect of in situ forming depot technology (BEPO®) and PNA-oligonucleotide formulation was studied. In vivo fluorescence imaging analysis showed fast renal/bladder excretion of the PNA (t½ ∼ 20 min) for i.v. administration, while s.c. administration showed a two to three times slower excretion. The release from the BEPO depot exhibited biphasic kinetics with a slow release (t½ ∼ 10 days) of 50% of the dose. In all cases, some accumulation in kidneys and liver could be detected. Formulation of PNA as a duplex hybridization complex with a complementary phosphorothioate oligonucleotide increased the solubility of the PNA. However, none of these alternative administration methods resulted in significantly improved antisense activity. Therefore, either more sophisticated formulations such as designed nanoparticles or conjugation to delivery ligands must be utilized to improve both pharmacokinetics as well as tissue targeting and availability. On the other hand, the results show that s.c. and BEPO depot administration of PNA are feasible and allow easier, higher, and less frequent dosing, as well as more controlled release, which can be exploited both for animal model studies as well as eventually in the clinic in terms of dosing optimization.


Assuntos
Distrofia Muscular de Duchenne , Ácidos Nucleicos Peptídicos , Animais , Distrofina/genética , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Ácidos Nucleicos Peptídicos/genética , Oligonucleotídeos Fosforotioatos
5.
Mol Cancer Ther ; 17(9): 1927-1940, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29891487

RESUMO

Small therapeutic proteins represent a promising novel approach to treat cancer. Nevertheless, their clinical application is often adversely impacted by their short plasma half-life. Controlled long-term delivery of small biologicals has become a challenge because of their hydrophilic properties and in some cases their limited stability. Here, an in situ forming depot-injectable polymeric system was used to deliver BiJ591, a bispecific T-cell engager (BiTE) targeting both prostate-specific membrane antigen (PSMA) and the CD3 T-cell receptor in prostate cancer. BiJ591 induced T-cell activation, prostate cancer-directed cell lysis, and tumor growth inhibition. The use of diblock (DB) and triblock (TB) biodegradable polyethylene glycol-poly(lactic acid; PEG-PLA) copolymers solubilized in tripropionin, a small-chain triglyceride, allowed maintenance of BiJ591 stability and functionality in the formed depot and controlled its release. In mice, after a single subcutaneous injection, one of the polymeric candidates, TB1/DB4, provided the most sustained release of BiJ591 for up to 21 days. Moreover, the use of BiJ591-TB1/DB4 formulation in prostate cancer xenograft models showed significant therapeutic activity in both low and high PSMA-expressing tumors, whereas daily intravenous administration of BiJ591 was less efficient. Collectively, these data provide new insights into the development of controlled delivery of small therapeutic proteins in cancer. Mol Cancer Ther; 17(9); 1927-40. ©2018 AACR.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Polímeros/química , Neoplasias da Próstata/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Antígenos de Superfície/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Complexo CD3/imunologia , Linhagem Celular Tumoral , Liberação Controlada de Fármacos , Glutamato Carboxipeptidase II/imunologia , Humanos , Masculino , Camundongos SCID , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos
6.
Nucleic Acids Res ; 40(13): 6367-79, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22467209

RESUMO

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.


Assuntos
Efeitos da Posição Cromossômica , Enzimas de Restrição do DNA/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/química , Marcação de Genes , Engenharia Genética , Genoma Humano , Humanos , Mutagênese
7.
Nucleic Acids Res ; 39(14): 6124-36, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21482539

RESUMO

Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders. Previous studies have shown strong crosstalk between different residues and regions of the DNA binding interface. To investigate this phenomenon, we systematically combined mutations from three groups of amino acids in the DNA binding regions of the I-CreI HE. Our results confirm that important crosstalk occurs throughout this interface in I-CreI. Detailed analysis of success rates identified a nearest-neighbour effect, with a more pronounced level of dependence between adjacent regions. Taken together, these data suggest that combinatorial engineering does not necessarily require the identification of separable functional or structural regions, and that groups of amino acids provide acceptable building blocks that can be assembled, overcoming the context dependency of the DNA binding interface. Furthermore, the present work describes a sequential method to engineer tailored HEs, wherein three contiguous regions are individually mutated and assembled to create HEs with engineered specificity.


Assuntos
Enzimas de Restrição do DNA/química , Proteínas de Ligação a DNA/química , Sítios de Ligação , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Mutação , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Especificidade por Substrato
8.
Nucleic Acids Res ; 39(2): 729-43, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20846960

RESUMO

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.


Assuntos
Reparo do DNA , Enzimas de Restrição do DNA/química , Genes RAG-1 , Linhagem Celular , DNA/química , Clivagem do DNA , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Marcação de Genes , Loci Gênicos , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Engenharia de Proteínas , Recombinação Genética
9.
Nucleic Acids Res ; 38(6): 2006-18, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20026587

RESUMO

Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities. We demonstrate the modularity of the chimeric DmoCre meganuclease previously described, by successfully assembling mutants with locally altered specificities affecting both I-DmoI and I-CreI subdomains in order to create active meganucleases with altered specificities. Moreover these new engineered DmoCre variants appear highly specific and present a low toxicity level, similar to I-SceI, and can induce efficient homologous recombination events in mammalian cells. The DmoCre based meganucleases can therefore offer new possibilities for various genome engineering applications.


Assuntos
Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Animais , Células CHO , Sobrevivência Celular , Técnicas de Química Combinatória , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Mutagênese , Mutação , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato/genética
10.
Nucleic Acids Res ; 37(16): 5405-19, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19584299

RESUMO

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.


Assuntos
Enzimas de Restrição do DNA/genética , Marcação de Genes , Genes RAG-1 , Animais , Células CHO , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/metabolismo , Dimerização , Engenharia Genética , Humanos , Recombinação Genética , Imunodeficiência Combinada Severa/genética
11.
Proc Natl Acad Sci U S A ; 105(44): 16888-93, 2008 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-18974222

RESUMO

Homing endonucleases, also known as meganucleases, are sequence-specific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 A resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.


Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo I/química , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/metabolismo , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Engenharia de Proteínas/métodos , Especificidade por Substrato
12.
Chemistry ; 14(31): 9530-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18833547

RESUMO

Heptosides are found in important bacterial glycolipids such as lipopolysaccharide (LPS), the biosynthesis of which is targeted for the development of novel antibacterial agents. This work describes the synthesis of a fluorinated analogue of ADP-L-glycero-beta-D-manno-heptopyranose, the donor substrate of the heptosyl transferase WaaC, which catalyzes the incorporation of this carbohydrate into LPS. Synthetically, the key step for the preparation of ADP-2F-heptose is the simultaneous and stereoselective installation of both the fluorine atom at C-2 and the phosphoryl group at C-1 through a selectfluor-mediated (selectfluor=1-chloromethyl-4-fluorodiazoniabicyclo[2.2.2]octane bis(triflate)) electrophilic addition/nucleophilic substitution involving a heptosylglycal. Therefore, we detail in this article 1) the stereoselective preparation of the key intermediates heptosylglycals, 2) the development of a new fluorophosphorylation procedure allowing an excellent beta-gluco stereoselectivity with "all-equatorial" glycals, 3) the synthesis of the target ADP-2F-heptose, and 4) some comments on the contacts observed between the fluorine atom of the final molecule and the protein in the crystallographic structure of heptosyltransferase WaaC.


Assuntos
Difosfato de Adenosina/química , Compostos de Flúor/síntese química , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Polissacarídeos/química , Compostos de Flúor/química , Modelos Moleculares , Estrutura Molecular , Fosforilação , Proteínas/química , Estereoisomerismo
13.
J Mol Biol ; 371(1): 49-65, 2007 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-17561112

RESUMO

Meganucleases are sequence-specific endonucleases which recognize large (>12 bp) target sites in living cells and can stimulate homologous gene targeting by a 1000-fold factor at the cleaved locus. We have recently described a combinatorial approach to redesign the I-CreI meganuclease DNA-binding interface, in order to target chosen sequences. However, engineering was limited to the protein regions shown to directly interact with DNA in a base-specific manner. Here, we take advantage of I-CreI natural degeneracy, and of additional refinement steps to extend the number of sequences that can be efficiently cleaved. We searched the sequence of the human XPC gene, involved in the disease Xeroderma Pigmentosum (XP), for potential targets, and chose three sequences that differed from the I-CreI cleavage site over their entire length, including the central four base-pairs, whose role in the DNA/protein recognition and cleavage steps remains very elusive. Two out of these targets could be cleaved by engineered I-CreI derivatives, and we could improve the activity of weak novel meganucleases, to eventually match the activity of the parental I-CreI scaffold. The novel proteins maintain a narrow cleavage pattern for cognate targets, showing that the extensive redesign of the I-CreI protein was not made at the expense of its specificity. Finally, we used a chromosomal reporter system in CHO-K1 cells to compare the gene targeting frequencies induced by natural and engineered meganucleases. Tailored I-CreI derivatives cleaving sequences from the XPC gene were found to induce high levels of gene targeting, similar to the I-CreI scaffold or the I-SceI "gold standard". This is the first time an engineered homing endonuclease has been used to modify a chromosomal locus.


Assuntos
Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Marcação de Genes , Engenharia de Proteínas , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cricetinae , Cricetulus , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Genes Reporter , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
14.
EMBO J ; 26(10): 2594-604, 2007 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-17464289

RESUMO

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.


Assuntos
Colicinas/química , Colicinas/metabolismo , Colicinas/genética , Cristalografia por Raios X , Peso Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Análise Espectral Raman
15.
Nucleic Acids Res ; 34(22): e149, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17130168

RESUMO

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.


Assuntos
Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Engenharia de Proteínas/métodos , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Interpretação Estatística de Dados , Dimerização , Genes RAG-1 , Humanos , Mutação , Nucleotídeos/metabolismo , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Especificidade por Substrato
16.
J Mol Biol ; 363(2): 383-94, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16963083

RESUMO

Lipopolysaccharides constitute the outer leaflet of the outer membrane of Gram-negative bacteria and are therefore essential for cell growth and viability. The heptosyltransferase WaaC is a glycosyltransferase (GT) involved in the synthesis of the inner core region of LPS. It catalyzes the addition of the first L-glycero-D-manno-heptose (heptose) molecule to one 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue of the Kdo2-lipid A molecule. Heptose is an essential component of the LPS core domain; its absence results in a truncated lipopolysaccharide associated with the deep-rough phenotype causing a greater susceptibility to antibiotic and an attenuated virulence for pathogenic Gram-negative bacteria. Thus, WaaC represents a promising target in antibacterial drug design. Here, we report the structure of WaaC from the Escherichia coli pathogenic strain RS218 alone at 1.9 A resolution, and in complex with either ADP or the non-cleavable analog ADP-2-deoxy-2-fluoro-heptose of the sugar donor at 2.4 A resolution. WaaC adopts the GT-B fold in two domains, characteristic of one glycosyltransferase structural superfamily. The comparison of the three different structures shows that WaaC does not undergo a domain rotation, characteristic of the GT-B family, upon substrate binding, but allows the substrate analog and the reaction product to adopt remarkably distinct conformations inside the active site. In addition, both binary complexes offer a close view of the donor subsite and, together with results from site-directed mutagenesis studies, provide evidence for a model of the catalytic mechanism.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Glicosiltransferases/química , Heptoses/química , Estrutura Terciária de Proteína , Difosfato de Adenosina/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compostos de Flúor/química , Compostos de Flúor/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Heptoses/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Dobramento de Proteína , Alinhamento de Sequência
17.
Mol Microbiol ; 51(4): 1027-37, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14763978

RESUMO

RmpM is a putative peptidoglycan binding protein from Neisseria meningitidis that has been shown to interact with integral outer membrane proteins such as porins and TonB-dependent transporters. Here we report the 1.9 A crystal structure of the C-terminal domain of RmpM. The 150-residue domain adopts a betaalphabetaalphabetabeta fold, as first identified in Bacillus subtilis chorismate mutase. The C-terminal RmpM domain is homologous to the periplasmic, C-terminal domain of Escherichia coli OmpA; these domains are thought to be responsible for non-covalent interactions with peptidoglycan. From the structure of the OmpA-like domain of RmpM, we suggest a putative peptidoglycan binding site and identify residues that may be essential for binding. Both the crystal structure and solution experiments indicate that RmpM may exist as a dimer. This would promote more efficient peptidoglycan binding, by allowing RmpM to interact simultaneously with two glycan chains through its C-terminal, OmpA-like binding domain, while its (structurally uncharacterized) N-terminal domain could stabilize oligomers of porins and TonB-dependent transporters in the outer membrane.


Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli , Neisseria meningitidis/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Corismato Mutase/química , Cristalografia por Raios X , Dimerização , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Dados de Sequência Molecular , Peptidoglicano/metabolismo , Porinas/química , Ligação Proteica , Subunidades Proteicas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
18.
J Mol Biol ; 332(2): 353-68, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12948487

RESUMO

Escherichia coli possesses a TonB-dependent transport system, which exploits the iron-binding capacity of citrate and its natural abundance. Here, we describe three structures of the outer membrane ferric citrate transporter FecA: unliganded and complexed with iron-free or diferric dicitrate. We show the structural mechanism for discrimination between the iron-free and ferric siderophore: the binding of diferric dicitrate, but not iron-free dicitrate alone, causes major conformational rearrangements in the transporter. The structure of FecA bound with iron-free dicitrate represents the first structure of a TonB-dependent transporter bound with an iron-free siderophore. Binding of diferric dicitrate to FecA results in changes in the orientation of the two citrate ions relative to each other and in their interactions with FecA, compared to the binding of iron-free dicitrate. The changes in ligand binding are accompanied by conformational changes in three areas of FecA: two extracellular loops, one plug domain loop and the periplasmic TonB-box motif. The positional and conformational changes in the siderophore and transporter initiate two independent events: ferric citrate transport into the periplasm and transcription induction of the fecABCDE transport genes. From these data, we propose a two-step ligand recognition event: FecA binds iron-free dicitrate in the non-productive state or first step, followed by siderophore displacement to form the transport-competent, diferric dicitrate-bound state in the second step.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Ácido Cítrico/metabolismo , Proteínas de Escherichia coli , Compostos Férricos/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/metabolismo , Ácido Cítrico/química , Cristalografia por Raios X , Escherichia coli/metabolismo , Ligantes , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência
19.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 5): 784-91, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11976489

RESUMO

The estimation of the twinning ratio in crystals of two proteins, bacteriorhodopsin and a truncated form of p67(phox), is described. Various approaches are combined to detect and determine accurately the twinning ratio. For each protein, three data sets from crystals exhibiting twinning ratios ranging from 0 to almost 50% are analysed. Self-rotation functions and R(sym) values considering space groups of higher symmetry are indicative of twinning. Precise values of the twinning ratios are derived from Yeates' statistical approaches and Britton plots. The twinning ratios are also obtained by analysing the second-, third- and fourth-order moments of the intensity distribution. The twinning ratios obtained from the various approaches are compared. Second-, third- and fourth-order moments of the intensity distribution are useful indicators for evaluating the twinning precisely in order to correct the intensities or to screen rapidly for non-twinned crystals.


Assuntos
Bacteriorodopsinas/química , Fosfoproteínas/química , Cristalografia por Raios X , Halobacterium salinarum/química , Neutrófilos/química , Conformação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...