RESUMO
Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.
Assuntos
Bacteroides fragilis/genética , Inflamação/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Bacteroides fragilis/patogenicidade , Ilhas Genômicas/genética , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloendopeptidases/genética , Microbiota , Neoplasias/genética , Neoplasias/patologia , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Two Drosophila myosin II point mutations (D45 and Mhc(5)) generate Drosophila cardiac phenotypes that are similar to dilated or restrictive human cardiomyopathies. Our homology models suggest that the mutations (A261T in D45, G200D in Mhc(5)) could stabilize (D45) or destabilize (Mhc(5)) loop 1 of myosin, a region known to influence ADP release. To gain insight into the molecular mechanism that causes the cardiomyopathic phenotypes to develop, we determined whether the kinetic properties of the mutant molecules have been altered. We used myosin subfragment 1 (S1) carrying either of the two mutations (S1(A261T) and S1(G200D)) from the indirect flight muscles of Drosophila. The kinetic data show that the two point mutations have an opposite effect on the enzymatic activity of S1. S1(A261T) is less active (reduced ATPase, higher ADP affinity for S1 and actomyosin subfragment 1 (actin · S1), and reduced ATP-induced dissociation of actin · S1), whereas S1(G200D) shows increased enzymatic activity (enhanced ATPase, reduced ADP affinity for both S1 and actin · S1). The opposite changes in the myosin properties are consistent with the induced cardiac phenotypes for S1(A261T) (dilated) and S1(G200D) (restrictive). Our results provide novel insights into the molecular mechanisms that cause different cardiomyopathy phenotypes for these mutants. In addition, we report that S1(A261T) weakens the affinity of S1 · ADP for actin, whereas S1(G200D) increases it. This may account for the suppression (A261T) or enhancement (G200D) of the skeletal muscle hypercontraction phenotype induced by the troponin I held-up(2) mutation in Drosophila.
Assuntos
Actinas , Difosfato de Adenosina/metabolismo , Cardiomiopatia Restritiva , Proteínas de Drosophila , Modelos Cardiovasculares , Miosina Tipo II , Mutação Puntual , Actinas/genética , Actinas/metabolismo , Actomiosina/genética , Actomiosina/metabolismo , Animais , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Músculos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Fenótipo , Troponina I/genética , Troponina I/metabolismoRESUMO
The subfragment 2/light meromyosin "hinge" region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Miofibrilas/química , Subfragmentos de Miosina/genética , Subfragmentos de Miosina/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Fenômenos Biomecânicos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Voo Animal/fisiologia , Humanos , Cinética , Microscopia Eletrônica , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/fisiologia , Miofibrilas/fisiologia , Subfragmentos de Miosina/química , Miosina Tipo II/genética , Sarcômeros/química , Sarcômeros/fisiologia , Difração de Raios XRESUMO
We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K(1)k(+2)) and ADP affinity (K(AD)), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Éxons , Miosinas , Isoformas de Proteínas , Actomiosina/química , Actomiosina/genética , Actomiosina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Voo Animal , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Músculo Esquelético/metabolismo , Miofibrilas/ultraestrutura , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/ultraestrutura , Miofibrilas/metabolismo , Miosinas/química , Miosinas/genética , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , TransgenesRESUMO
Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.