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1.
Genes Dev ; 29(10): 1000-5, 2015 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-25956905

RESUMO

Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Elucidating the mechanisms that regulate these motor proteins is central to understanding genome maintenance processes. Here, we show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair. Mechanistically, scMHF relieves the inhibition imposed by the structural maintenance of chromosome protein Smc5 on Mph1 activities relevant to replication-associated repair through binding to Mph1 but not DNA. Thus, scMHF is a function-specific enhancer of Mph1 that enables flexible response to different genome repair situations.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , RNA Helicases DEAD-box/metabolismo , DNA/genética , Reparo do DNA , Genoma Fúngico/genética , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
Mol Cell ; 37(6): 879-86, 2010 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-20347429

RESUMO

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/metabolismo , Histonas/metabolismo , Dobramento de Proteína , Multimerização Proteica , Linhagem Celular Tumoral , DNA/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Humanos , Ligação Proteica
3.
Nucleic Acids Res ; 42(2): 906-17, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24150939

RESUMO

The Hop2-Mnd1 complex functions with the DMC1 recombinase in meiotic recombination. Hop2-Mnd1 stabilizes the DMC1-single-stranded DNA (ssDNA) filament and promotes the capture of the double-stranded DNA partner by the recombinase filament to assemble the synaptic complex. Herein, we define the action mechanism of Hop2-Mnd1 in DMC1-mediated recombination. Small angle X-ray scattering analysis and electron microscopy reveal that the heterodimeric Hop2-Mnd1 is a V-shaped molecule. We show that the protein complex harbors three distinct DNA binding sites, and determine their functional relevance. Specifically, the N-terminal double-stranded DNA binding functions of Hop2 and Mnd1 co-operate to mediate synaptic complex assembly, whereas ssDNA binding by the Hop2 C-terminus helps stabilize the DMC1-ssDNA filament. A model of the Hop2-Mnd1-DMC1-ssDNA ensemble is proposed to explain how it mediates homologous DNA pairing in meiotic recombination.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Recombinação Homóloga , Meiose/genética , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Camundongos , Mutação Puntual , Multimerização Proteica , Estrutura Terciária de Proteína , Recombinases/metabolismo
4.
Bioconjug Chem ; 26(3): 520-8, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25714504

RESUMO

Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 µg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery.


Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala/métodos , Agregados Proteicos/fisiologia , Animais , Cabras , Humanos , Camundongos
5.
J Biol Chem ; 287(2): 1566-75, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22115747

RESUMO

During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control.


Assuntos
Cromátides/metabolismo , Cromossomos Fúngicos/metabolismo , Meiose/fisiologia , Rad51 Recombinase/metabolismo , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromátides/genética , Cromossomos Fúngicos/genética , DNA Helicases , Enzimas Reparadoras do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Mutação , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Antibodies (Basel) ; 6(3)2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31548527

RESUMO

Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".

7.
Org Lett ; 7(7): 1203-6, 2005 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-15787467

RESUMO

[reaction: see text] Conformationally constrained side chain-bridged cyclic peptides were prepared using bis-carboxylic acid ring spacers. These macrocyles were designed to inhibit protein-protein interactions mediated by the third PDZ domain (PDZ3) of a mammalian neuronal protein, PSD-95. Isothermal titration calorimetry (ITC) experiments measured dissociation constants in the low micromolar range. For each compound, the change in entropy (TdeltaS) of binding either is comparable in magnitude to the enthalpy change (deltaH) or is the predominant driving force for association.


Assuntos
Peptídeos Cíclicos/química , Conformação Proteica , Termodinâmica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína 4 Homóloga a Disks-Large , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Ratos
8.
Org Lett ; 6(20): 3429-32, 2004 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-15387515

RESUMO

[structure: see text] Isothermal titration calorimetry (ITC) is used to study the thermodynamic consequences of systematically modifying the hydrophobic character of a single residue in a series of protein-binding ligands. By substituting standard and nonproteinogenic aliphatic amino acids for the C-terminal valine of the hexapeptide KKETEV, binding to the third PDZ domain (PDZ3) of the PSD-95 protein is characterized by distinct changes in the Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) parameters. One notable observation is that peptide binding affinity can be improved with a nonstandard residue.


Assuntos
Aminoácidos/química , Proteínas do Tecido Nervoso/química , Peptídeos/química , Conformação Proteica , Termodinâmica , Sítios de Ligação , Calorimetria/métodos , Técnicas de Química Combinatória , Ligantes , Estrutura Molecular , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo
9.
Nat Commun ; 5: 2987, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24390579

RESUMO

The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anaemia pathway of the DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA-binding mode of MHF. The structure suggests that MHF prefers branched DNA over double-stranded DNA because it engages two duplex arms. Biochemical analyses verify that MHF preferentially engages DNA forks or various four-way junctions independent of the junction-site structure. Furthermore, genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results offer insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Dano ao DNA/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Cristalografia por Raios X , DNA Helicases/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína
11.
DNA Repair (Amst) ; 10(10): 1034-43, 2011 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-21880555

RESUMO

The budding yeast Mph1 protein, the putative ortholog of human FANCM, possesses a 3' to 5' DNA helicase activity and is capable of disrupting the D-loop structure to suppress chromosome arm crossovers in mitotic homologous recombination. Similar to FANCM, genetic studies have implicated Mph1 in DNA replication fork repair. Consistent with this genetic finding, we show here that Mph1 is able to mediate replication fork reversal, and to process the Holliday junction via DNA branch migration. Moreover, Mph1 unwinds 3' and 5' DNA Flap structures that bear key features of the D-loop. These biochemical results not only provide validation for a role of Mph1 in the repair of damaged replication forks, but they also offer mechanistic insights as to its ability to efficiently disrupt the D-loop intermediate.


Assuntos
RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Replicação do DNA/genética , DNA/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , RNA Helicases DEAD-box/genética , DNA/genética , DNA/metabolismo , DNA Helicases/genética , Reparo do DNA/genética , DNA Cruciforme/química , DNA Cruciforme/genética , Recombinação Homóloga , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética
12.
Nat Struct Mol Biol ; 17(10): 1255-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20871616

RESUMO

Homologous recombination mediated by RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-strand breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2: to enhance RAD51's ability to form the D loop. We show that PALB2 binds DNA and physically interacts with RAD51. Notably, although PALB2 alone stimulates D-loop formation, it has a cooperative effect with RAD51AP1, an enhancer of RAD51. This stimulation stems from the ability of PALB2 to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results demonstrate the multifaceted role of PALB2 in chromosome damage repair. Because PALB2 mutations can cause cancer or Fanconi anemia, our findings shed light on the mechanism of tumor suppression in humans.


Assuntos
Proteína BRCA2/fisiologia , Neoplasias da Mama/metabolismo , Reparo do DNA/fisiologia , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Rad51 Recombinase/fisiologia , Recombinação Genética/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas Reguladoras de Apoptose , Proteína BRCA2/química , Proteínas de Ligação a DNA/química , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Humanos , Complexos Multiproteicos , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Rad51 Recombinase/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
13.
Bioorg Med Chem Lett ; 17(22): 6147-50, 2007 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-17890086

RESUMO

A series of multivalent peptides, with the ability to simultaneously bind two separate PDZ domain proteins, has been designed, synthesized, and tested by isothermal titration calorimetry (ITC). The monomer sequences, linked with succinate, varied in length from five to nine residues. The thermodynamic binding parameters, in conjunction with results from mass spectrometry, indicate that a ternary complex is formed in which each peptide arm binds two equivalents of the third PDZ domain (PDZ3) of the neuronal protein PSD-95.


Assuntos
Domínios PDZ , Peptídeos/síntese química , Peptídeos/metabolismo , Sequência de Aminoácidos , Ligantes , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Termodinâmica
14.
Biochemistry ; 46(21): 6340-52, 2007 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-17474715

RESUMO

The thermodynamic parameters associated with the binding of several series of linear peptides to the third PDZ domain (PDZ3) of the postsynaptic density 95 protein (PSD-95) have been measured using isothermal titration calorimetry (ITC). Two strategies were pursued in developing these binding ligands: (1) systematic N-terminal truncation of sequences derived from the C-terminal regions of identified PDZ3-binding proteins (CRIPT, neuroligin-1, and citron) and (2) selective mutation of specific positions within a consensus hexapeptide (KKETEV) known to bind PDZ3. Each synthetically prepared peptide was used to titrate PDZ3, which yielded the changes in Gibbs free energy (DeltaG), enthalpy (DeltaH), and entropy (TDeltaS) for the binding event. Selected peptides were subjected to additional analysis, which entailed (1) measuring the change in heat capacity (DeltaCp) upon association, to assess the character of the binding interface, and (2) constructing thermodynamic double mutant cycles, to determine the presence of cooperative effects. From the first series, the CRIPT protein proved to be the better source for higher affinity sequences. From the second series, enhanced binding was associated with peptides that closely adhered to the established motif for class I PDZ domain C-termini, X-(T/S)-X-(V/I/L), and more specifically to a narrower motif of X-T-X-V. Further, in both series a length of six residues was necessary and sufficient to capture maximal affinity. In addition, there were significant influences upon binding by modifying the abutting "X" positions. The cumulative results provide greater detail into the specific nature of ligand binding to PDZ3 and will assist in the development of selective molecular probes for the study of this and structurally homologous PDZ domains.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Termodinâmica , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria , Sequência Consenso , Proteína 4 Homóloga a Disks-Large , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Titulometria
15.
Bioorg Med Chem Lett ; 14(6): 1385-8, 2004 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-15006367

RESUMO

Inspired by structure-based design and tailored for combinatorial preparation, a series of novel cyclic peptides has been developed to yield binding ligands for the third PDZ domain (PDZ3) of PSD-95. These side chain-side chain bridged peptides permit the systematic expansion or contraction of ring size, which is intended to maximize the conformational diversity of the ensemble. Isothermal titration calorimetry (ITC) was used to measure the dissociation constants (K(d)) and associated thermodynamic binding parameters.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Termodinâmica , Sítios de Ligação/fisiologia , Moléculas de Adesão Celular , Ligantes , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Conformação Proteica
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