RESUMO
Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.
Assuntos
Cadeias Pesadas de Miosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Dimerização , Microscopia Eletrônica , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Dobramento de Proteína , Ressonância de Plasmônio de Superfície , Suínos , Proteínas Supressoras de TumorRESUMO
A recent study described a recessive ATPase activating germ-line mutation in smooth-muscle myosin (smmhc/myh11) underlying the zebrafish meltdown (mlt) phenotype. The mlt zebrafish develops intestinal abnormalities reminiscent of human Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JP). To examine the role of MYH11 in human intestinal neoplasia, we searched for MYH11 mutations in patients with colorectal cancer (CRC), PJS and JP. We found somatic protein-elongating frameshift mutations in 55% of CRCs displaying microsatellite instability and in the germ-line of one individual with PJS. Additionally, two somatic missense mutations were found in one microsatellite stable CRC. These two missense mutations, R501L and K1044N, and the frameshift mutations were functionally evaluated. All mutations resulted in unregulated molecules displaying constitutive motor activity, similar to the mutant myosin underlying mlt. Thus, MYH11 mutations appear to contribute also to human intestinal neoplasia. Unregulated MYH11 may affect the cellular energy balance or disturb cell lineage decisions in tumor progenitor cells. These data challenge our view on MYH11 as a passive differentiation marker functioning in muscle contraction and add to our understanding of intestinal neoplasia.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Intestinais/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo II/genética , Miosinas de Músculo Liso/genética , Polipose Adenomatosa do Colo/genética , Sequência de Aminoácidos , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Síndrome de Peutz-Jeghers/genética , Miosinas de Músculo Liso/fisiologiaRESUMO
The single chain variable fragment (scFv) of an anti-idiotypic catalytic monoclonal antibody, 9G4H9, displaying a beta-lactamase-like activity was cloned. The recombinant protein was expressed through the periplasm in Escherichia coli in the presence or in the absence of FkpA, a chaperone-like enzyme and tested for its hydrolytic activity. The results show that the catalytic parameters for hydrolysis of ampicillin by scFv9G4H9 are clearly influenced by the presence of FkpA, indicating that the correct folding of the fragment represents a crucial step for catalysis.
Assuntos
Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Catalíticos/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Anticorpos Anti-Idiotípicos/genética , Anticorpos Anti-Idiotípicos/isolamento & purificação , Anticorpos Catalíticos/genética , Anticorpos Catalíticos/isolamento & purificação , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Clonagem Molecular , Hidrólise , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Modelos Moleculares , Mimetismo MolecularRESUMO
We previously characterized a defective-folding variant of the periplasmic maltose-binding protein, MalE31. To examine the alternative folding pathways open to the MalE31 precursor, we have analyzed the cellular fates of this aggregation-prone protein carrying altered signal sequences. Our results are most easily interpreted by a kinetic competition between exportation, folding, and degradation.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mutação , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/metabolismo , Dobramento de Proteína , Transporte Biológico , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Variação Genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Mutagênese Sítio-Dirigida , Proteínas Periplásmicas de Ligação/genética , Conformação Proteica , Sinais Direcionadores de Proteínas/genética , Frações Subcelulares/metabolismoRESUMO
9G4H9, a catalytic antibody displaying ß-lactamase-like activity, has been developed by the anti-idiotypic approach using ß-lactamase as the first antigen. Thus 9G4H9 represents the 'internal image' of ß-lactamase. We selected a cyclic peptide anchored to a bacteriophage M13 library using 9G4H9 as the target. Pep90 is a cyclic heptapeptide enclosed between two cysteine residues. We showed that Pep90 could inhibit both TEM-1 ß-lactamase (K(i) = 333 µm) and several penicillin-binding proteins (IC50 values ranging from 6-62 µm). We determined that the tryptophan residue of Pep90 is of crucial importance for its inhibitory activity. Using Pep90 as a scaffold, we generated a new class of peptidomimetics that retained inhibitory activity towards TEM-1 ß-lactamase.
Assuntos
Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Inibidores de beta-Lactamases , Anticorpos Catalíticos/química , Anticorpos Catalíticos/genética , Bacteriófago M13/genética , Domínio Catalítico , Cinética , Modelos Moleculares , Mutagênese , Biblioteca de Peptídeos , Peptídeos Cíclicos/genética , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Engenharia de Proteínas , Triptofano/química , beta-LactamasesRESUMO
Data on catalytic antibodies (abzymes) having critical roles in pathologies, for example in some auto-immune diseases accumulate at overwhelming pace. Nevertheless, the misunderstanding of how antibodies can mimic a catalytic activity may hamper the development of therapeutic tools. We thus investigated the structure function roles of residues of a catalytic antibody endowed with a beta-lactamase activity. The catalytic antibody 9G4H9 was generated using the internal image properties of anti-idiotypic antibodies. The single-chain fragment was cloned and produced in Escherichia coli. Based on the structure function knowledge of beta-lactamases pattern and on the tridimensional model of the scFv, five residues were selected for mutagenic analysis to learn about the contribution of putative residues in catalysis. Light chain mutants R24A, S26A, S28A, and E98A lost catalytic activity significantly. Mutant K27A retained catalytic activity but the estimated K(M) was affected. Kinetic outcomes support the argument that S26 and S28 function as nucleophile and E98 as general acid/base catalyst. We have selected a peptide Pep90 able to inhibit 9G4H9 catalytic activity. We also demonstrate the tryptophan and proline residues of Pep90 (YHFLWGP) are responsible for binding and inhibitory properties of Pep90 on 9G4H9. A three-dimensional model docked with Pep90 is therefore built in which critical residues of Pep90 are buried at the interface of CDR-L1 and CDR-L3 loops whereas other are exposed to the solvent.