RESUMEN
Neoadjuvant chemotherapy (NACT) for breast cancer (BC) often results in pathologic complete response (pCR), i.e., the complete elimination of visible cancer cells. It is unclear whether the use of ultrasensitive genetic methods may still detect residual BC cells in complete responders. Breast carcinomas arising in BRCA1 mutation carriers almost always carry alterations of the TP53 gene thus providing an opportunity to address this question. The analysis of consecutive BC patients treated by NACT revealed a higher pCR rate in BRCA1-driven vs. BRCA1-wildtype BCs (13/24 (54%) vs. 29/192 (15%), p < 0.0001). Twelve pre-/post-NACT tissue pairs obtained from BRCA1 mutation carriers were available for the study. While TP53 mutation was identified in all chemonaive tumors, droplet digital PCR (ddPCR) analysis of the post-NACT tumor bed revealed the persistence of this alteration in all seven pCR-non-responders but in none of five pCR responders. Eleven patients provided to the study post-NACT tissue samples only; next-generation sequencing (NGS) analysis revealed mutated TP53 copies in all six cases without pCR but in none of five instances of pCR. In total, TP53 mutation was present in post-NACT tissues in all 13 cases without pCR, but in none of 10 patients with pCR (p < 0.000001). Therefore, the lack of visible tumor cells in the post-NACT tumor bed is indeed a reliable indicator of the complete elimination of transformed clones. Failure of ultrasensitive methods to identify patients with minimal residual disease among pCR responders suggests that the result of NACT is a categorical rather than continuous variable, where some patients are destined to be cured while others ultimately fail to experience tumor eradication.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Terapia Neoadyuvante/métodos , Mutación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteína BRCA1/genéticaRESUMEN
We have used the technique of polarized microfluorimetry to obtain new insight into the pathogenesis of skeletal muscle disease caused by the Gln147Pro substitution in ß-tropomyosin (Tpm2.2). The spatial rearrangements of actin, myosin and tropomyosin in the single muscle fiber containing reconstituted thin filaments were studied during simulation of several stages of ATP hydrolysis cycle. The angular orientation of the fluorescence probes bound to tropomyosin was found to be changed by the substitution and was characteristic for a shift of tropomyosin strands closer to the inner actin domains. It was observed both in the absence and in the presence of troponin, Ca2+ and myosin heads at all simulated stages of the ATPase cycle. The mutant showed higher flexibility. Moreover, the Gln147Pro substitution disrupted the myosin-induced displacement of tropomyosin over actin. The irregular positioning of the mutant tropomyosin caused premature activation of actin monomers and a tendency to increase the number of myosin cross-bridges in a state of strong binding with actin at low Ca2+.
Asunto(s)
Sustitución de Aminoácidos , Contracción Muscular , Miotonía Congénita/genética , Tropomiosina/química , Actinas/química , Adenosina Trifosfato/metabolismo , Animales , Calcio/química , Calcio/metabolismo , Células Cultivadas , Humanos , Simulación de Dinámica Molecular , Miosinas/química , Miosinas/metabolismo , Dominios Proteicos , Conejos , Tropomiosina/genética , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismoRESUMEN
The E41K mutation in TPM2 gene encoding muscle regulatory protein beta-tropomyosin is associated with nemaline myopathy and cap disease. The mutation results in a reduced Ca2+-sensitivity of the thin filaments and in muscle weakness. To elucidate the structural basis of the reduced Ca2+-sensitivity of the thin filaments, we studied multistep changes in spatial arrangement of tropomyosin (Tpm), actin and myosin heads during the ATPase cycle in reconstituted fibers, using the polarized fluorescence microscopy. The E41K mutation inhibits troponin's ability to shift Tpm to the closed position at high Ca2+, thus restraining the transition of the thin filaments from the "off" to the "on" state. The mutation also inhibits the ability of S1 to shift Tpm to the open position, decreases the amount of the myosin heads bound strongly to actin at high Ca2+, but increases the number of such heads at low Ca2+. These changes may contribute to the low Ca2+-sensitivity and muscle weakness. As the mutation has no effect on troponin's ability to switch actin monomers on at high Ca2+ and inhibits their switching off at low Ca2+, the use of reagents that increase the Ca2+-sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Actinas/química , Sustitución de Aminoácidos , Animales , Humanos , Técnicas In Vitro , Contracción Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Mutantes/química , Miopatías Nemalínicas/genética , Miopatías Nemalínicas/metabolismo , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Conejos , Tropomiosina/químicaRESUMEN
Substitution of Arg for Gly residue in 91th position in ß-tropomyosin caused by a point mutation in TPM2 gene is associated with distal arthrogryposis, characterized by a high Ca2+-sensitivity of myofilament and contracture syndrome. To understand the mechanisms of this defect, we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres, using the polarized fluorescence microscopy. The mutation was shown to markedly decrease the bending stiffness of ß-tropomyosin in the thin filaments. In the absence of the myosin heads the mutation did not alter the ability of troponin to shift tropomyosin to the blocked position and to switch actin monomers off at low Ca2+. During the ATPase cycle the movement of the mutant tropomyosin is restrained, it is located near the open position, which allows strong binding of the myosin heads to actin even at low Ca2+. This may be the reason for both high Ca2+-sensitivity and contractures associated with the Arg91Gly mutation. The use of reagents that decrease the Ca2+sensitivity of the troponin complex may not be appropriate to restore muscle function in patients with this mutation.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Fibras Musculares de Contracción Rápida/fisiología , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sustitución de Aminoácidos , Animales , Arginina/genética , Arginina/metabolismo , Células Cultivadas , Glicina/genética , Glicina/metabolismo , Mutagénesis Sitio-Dirigida , Miosinas/metabolismo , Conejos , Tropomiosina/genéticaRESUMEN
Effects of the Ala155Thr substitution in hydrophobic core of tropomyosin Tpm1.1 on conformational rearrangements of the components of the contractile system (Tpm1.1, actin and myosin heads) were studied by polarized fluorimetry technique at different stages of the actomyosin ATPase cycle. The proteins were labelled by fluorescent probes and incorporated into ghost muscle fibres. The substitution violated the blocked and closed states of thin filaments stimulating abnormal displacement of tropomyosin to the inner domains of actin, switching actin on and increasing the relative number of the myosin heads in strong-binding state. Furthermore, the mutant tropomyosin disrupted the major function of troponin to alter the distribution of the different functional states of thin filaments. At low Ca2+ troponin did not effectively switch thin filament off and the myosin head lost the ability to drive the spatial arrangement of the mutant tropomyosin. The information about tropomyosin flexibility obtained from the fluorescent probes at Cys190 indicates that this tropomyosin is generally more rigid, that obviously prevents tropomyosin to bend and adopt the appropriate conformation required for proper regulation.
Asunto(s)
Miosinas/química , Tropomiosina/química , Animales , Polarización de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Point mutations R167H and K168E in tropomyosin Tpm1.1 (TM) disturb Ca2+-dependent regulation of the actomyosin ATPase. To understand mechanisms of this defect we studied multistep changes in mobility and spatial arrangement of tropomyosin, actin and myosin heads during the ATPase cycle in reconstituted ghost fibres using the polarized fluorescence microscopy. It was found that both mutations disturbed the mode of troponin operation in the fibres. At high Ca2+, troponin increased the fraction of actin monomers that were in the "switched on" state, but both mutant tropomyosins were shifted toward the outer actin domains, which decreased the fraction of strongly bound myosin heads throughout the ATPase cycle. At low Ca2+, the R167H-TM was located close to the outer actin domains, which reduced the number of strongly-bound myosin heads. However, under these conditions troponin increased the number of actin monomers that were switched on. The K168E-TM was displaced far to the outer actin domains and troponin binding decreased the fraction of switched on actin monomers, but the proportion of the strongly bound myosin heads was abnormally high. Thus, the mutations differently disturbed transmission of conformational changes between troponin, tropomyosin and actin, which is essential for the Са2+-dependent regulation of the thin filament.
Asunto(s)
Tropomiosina/química , Tropomiosina/genética , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Adenosina Trifosfatasas/química , Animales , Calcio/química , Masculino , Microscopía Fluorescente , Mutación , Miosinas/química , Miosinas/genética , Mutación Puntual , Unión Proteica , Conformación Proteica , Conejos , Proteínas Recombinantes/químicaRESUMEN
Amino acid substitutions: Arg167His, Arg167Gly and Lys168Glu, located in a consensus actin-binding site of the striated muscle tropomyosin Tpm1.1 (TM), were used to investigate mechanisms of the thin filament regulation. The azimuthal movement of TM strands on the actin filament and the responses of the myosin heads and actin subunits during the ATPase cycle were studied using fluorescence polarization of muscle fibres. The recombinant wild-type and mutant TMs labelled with 5-IAF, 1,5-IAEDANS-labelled S1and FITC-phalloidin F-actin were incorporated into the ghost muscle fibres to acquire information on the orientation of the probes relative to the fibre axis. The substitutions Arg167Gly and Lys168Glu shifted TM strands into the actin filament centre, whereas Arg167His moved TM towards the periphery of the filament. In the presence of Arg167Gly-TM and Lys168Glu-TM the fraction of actin monomers that were switched on and the number of the myosin heads strongly bound to F-actin were abnormally high even under conditions close to relaxation. In contrast, Arg167His-TM decreased the fraction of switched on actin and reduced the formation of strongly bound myosin heads throughout the ATPase cycle. We concluded that the altered TM-actin contacts destabilized the thin filament and affected the actin-myosin interactions.
Asunto(s)
Adenosina Trifosfatasas/química , Miosinas/química , Tropomiosina/química , Tropomiosina/genética , Actinas/química , Animales , Arginina/química , Glutamina/química , Glicina/química , Histidina/química , Lisina/química , Masculino , Microscopía Fluorescente , Mutación , Nucleótidos , Faloidina/química , Conejos , Proteínas Recombinantes/química , TemperaturaRESUMEN
We have investigated the effect of the E41K, R91G, and E139del ß-tropomyosin (TM) mutations that cause congenital myopathy on the position of TM and orientation of actin monomers and myosin heads at different mimicked stages of the ATPase cycle in troponin-free ghost muscle fibers by polarized fluorimetry. A multi-step shifting of wild-type TM to the filament center accompanied by an increase in the amount of switched on actin monomers and the strongly bound myosin heads was observed during the ATPase cycle. The R91G mutation shifts TM further towards the inner and outer domains of actin at the strong- and weak-binding stages, respectively. The E139del mutation retains TM near the inner domains, while the E41K mutation captures it near the outer domains. The E41K and R91G mutations can induce the strong binding of myosin heads to actin, when TM is located near the outer domains. The E139del mutation inhibits the amount of strongly bound myosin heads throughout the ATPase cycle.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Enfermedades Musculares/metabolismo , Miosinas/metabolismo , Tropomiosina/metabolismo , Actinas/química , Animales , Humanos , Músculo Esquelético/metabolismo , Enfermedades Musculares/genética , Mutación , Miosinas/química , Conformación Proteica , Conejos , Tropomiosina/química , Tropomiosina/genéticaRESUMEN
To investigate how TM stabilization induced by the Gly126Arg mutation in skeletal α-TM or in smooth muscle ß-TM affects the flexibility of TMs and their position on troponin-free thin filaments, we labelled the recombinant wild type and mutant TMs with 5-IAF and F-actin with FITC-phalloidin, incorporated them into ghost muscle fibres and studied polarized fluorescence at different stages of the ATPase cycle. It has been shown that in the myosin- and troponin-free filaments the Gly126Arg mutation causes a shift of TM strands towards the outer domain of actin, reduces the number of switched on actin monomers and decreases the rigidity of the C-terminus of α-TM and increases the rigidity of the N-terminus of ß-TMs. The binding of myosin subfragment-1 to the filaments shifted the wild type TMs towards the inner domain of actin, decreased the flexibility of both terminal parts of TMs, and increased the number of switched on actin monomers. Multistep alterations in the position of α- and ß-TMs and actin monomers in the filaments and in the flexibility of TMs and F-actin during the ATPase cycle were observed. The Gly126Arg mutation uncouples a correlation between the position of TM and the number of the switched on actin monomers in the filaments.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Mutación , Tropomiosina/genética , Tropomiosina/metabolismo , Actinas/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceínas/metabolismo , Humanos , Faloidina/metabolismo , Estructura Secundaria de Proteína , Tropomiosina/químicaRESUMEN
Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type α-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.
Asunto(s)
Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Subfragmentos de Miosina/metabolismo , Fragmentos de Péptidos/metabolismo , Tropomiosina/química , Actomiosina/química , Actomiosina/genética , Actomiosina/metabolismo , Sustitución de Aminoácidos , Animales , Asparagina/genética , Ácido Aspártico/genética , Colorantes Fluorescentes/química , Ácido Glutámico/genética , Glicina/genética , Humanos , Contracción Muscular , Mutación , Naftalenosulfonatos , Estructura Secundaria de Proteína , Conejos , Espectrometría de Fluorescencia , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin.
Asunto(s)
Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Actomiosina/química , Nucleótidos de Adenina/farmacología , Adenosina Trifosfatasas/química , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Polarización de Fluorescencia , Técnicas In Vitro , Modelos Biológicos , Moluscos/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Proteínas Musculares/química , Miosinas/química , Miosinas/metabolismo , Fosforilación , Conformación Proteica , ConejosRESUMEN
To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift tropomyosin strands further towards the open position and to change the affinity of tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca(2+)-sensitivity caused by these mutations which initiates the disease remodeling.
Asunto(s)
Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Tropomiosina/química , Actinas/química , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Sustitución de Aminoácidos , Ácido Aspártico/genética , Ácido Glutámico/genética , Humanos , Mutación , Conformación Proteica , Tropomiosina/genética , Tropomiosina/metabolismoRESUMEN
Deletion of Glu139 in ß-tropomyosin caused by a point mutation in TPM2 gene is associated with cap myopathy characterized by high myofilament Ca2+-sensitivity and muscle weakness. To reveal the mechanism of these disorders at molecular level, mobility and spatial rearrangements of actin, tropomyosin and the myosin heads at different stages of actomyosin cycle in reconstituted single ghost fibres were investigated by polarized fluorescence microscopy. The mutation did not alter tropomyosin's affinity for actin but increased strongly the flexibility of tropomyosin and kept its strands near the inner domain of actin. The ability of troponin to switch actin monomers "on" and "off" at high and low Ca2+, respectively, was increased, and the movement of tropomyosin towards the blocked position at low Ca2+ was inhibited, presumably causing higher Ca2+-sensitivity. The mutation decreased also the amount of the myosin heads which bound strongly to actin at high Ca2+ and increased the number of these heads at relaxation; this may contribute to contractures and muscle weakness.