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1.
Cell ; 183(2): 335-346.e13, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33035452

RESUMO

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.


Assuntos
Músculo Esquelético/metabolismo , Miosinas de Músculo Esquelético/efeitos dos fármacos , Miosinas de Músculo Esquelético/genética , Adulto , Animais , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Espasticidade Muscular/genética , Espasticidade Muscular/fisiopatologia , Músculo Esquelético/fisiologia , Miosinas/efeitos dos fármacos , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Ratos , Ratos Wistar , Miosinas de Músculo Esquelético/metabolismo
2.
Trends Biochem Sci ; 43(9): 700-713, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30057142

RESUMO

Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Compostos Heterocíclicos de 4 ou mais Anéis , Doenças Musculares , Miosinas/antagonistas & inibidores , Neoplasias , Doenças Neurodegenerativas , Cicatrização/efeitos dos fármacos , Animais , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Doenças Musculares/tratamento farmacológico , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Miosinas/metabolismo , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia
3.
J Pharmacol Exp Ther ; 376(3): 358-373, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33468641

RESUMO

Blebbistatin, para-nitroblebbistatin (NBleb), and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, whereas AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. SIGNIFICANCE STATEMENT: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences, highlighting their potential in drug development targeting myosin-2 family proteins.


Assuntos
Absorção Fisico-Química , Descoberta de Drogas , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Miosinas/antagonistas & inibidores , Animais , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/toxicidade , Humanos , Simulação de Dinâmica Molecular , Miosinas/química , Conformação Proteica , Ratos , Distribuição Tecidual
4.
Nucleic Acids Res ; 46(8): 3967-3980, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29481689

RESUMO

Formation of RAD51 filaments on single-stranded DNA is an essential event during homologous recombination, which is required for homology search, strand exchange and protection of replication forks. Formation of nucleoprotein filaments (NF) is required for development and genomic stability, and its failure is associated with developmental abnormalities and tumorigenesis. Here we describe the structure of the human RAD51 NFs and of its Walker box mutants using electron microscopy. Wild-type RAD51 filaments adopt an 'open' conformation when compared to a 'closed' structure formed by mutants, reflecting alterations in helical pitch. The kinetics of formation/disassembly of RAD51 filaments show rapid and high ssDNA coverage via low cooperativity binding of RAD51 units along the DNA. Subsequently, a series of isomerization or dissociation events mediated by nucleotide binding state creates intrinsically dynamic RAD51 NFs. Our findings highlight important a mechanistic divergence among recombinases from different organisms, in line with the diversity of biological mechanisms of HR initiation and quality control. These data reveal unexpected intrinsic dynamic properties of the RAD51 filament during assembly/disassembly, which may be important for the proper control of homologous recombination.


Assuntos
DNA de Cadeia Simples/metabolismo , Rad51 Recombinase/metabolismo , Rad51 Recombinase/ultraestrutura , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Evolução Biológica , Microscopia Crioeletrônica , Humanos , Cinética , Modelos Moleculares , Mutação , Rad51 Recombinase/genética
5.
Proc Natl Acad Sci U S A ; 114(4): E466-E475, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28069956

RESUMO

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.


Assuntos
DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinação Homóloga , RecQ Helicases/metabolismo , Reparo do DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Sequências Repetidas Invertidas , RecQ Helicases/química
6.
Nucleic Acids Res ; 45(20): 11878-11890, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29059328

RESUMO

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.


Assuntos
DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , RecQ Helicases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Magnetismo , Modelos Moleculares , Conformação de Ácido Nucleico , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , RecQ Helicases/química
7.
Trends Biochem Sci ; 38(7): 364-71, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23768997

RESUMO

The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Modelos Moleculares , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Fatores de Transcrição Winged-Helix/metabolismo , Animais , DNA/química , Proteínas de Ligação a DNA/química , Humanos , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , RNA/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição Winged-Helix/química
8.
Methods ; 108: 24-39, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27133766

RESUMO

Helicases and translocases are nucleic acid (NA)-based molecular motors that use the free energy liberated during the nucleoside triphosphate (NTP, usually ATP) hydrolysis cycle for unidirectional translocation along their NA (DNA, RNA or heteroduplex) substrates. Determination of the kinetic and thermodynamic parameters of their mechanoenzymatic cycle serves as a basis for the exploration of their physiological behavior and various cellular functions. Here we describe how recent adaptations of fluorescence-based solution kinetic methods can be used to determine practically all important mechanistic parameters of NA-based motor proteins. We outline practically useful analysis procedures for equilibrium, steady-state and transient kinetic data. This analysis can be used to quantitatively characterize the enzymatic steps of the NTP hydrolytic cycle, the binding site size, stoichiometry and energetics of protein-NA interactions, the rate and processivity of translocation along and unwinding of NA strands, and the mechanochemical coupling between these processes. The described methods yield insights into the functional role of the enzymes, and also greatly aid the design and interpretation of single-molecule experiments as well as the engineering of enzymatic properties for biotechnological applications.


Assuntos
Trifosfato de Adenosina/genética , DNA Helicases/genética , Ácidos Nucleicos/genética , Trifosfato de Adenosina/química , Sítios de Ligação , DNA/genética , DNA Helicases/química , Fluorescência , Hidrólise , Cinética , Ácidos Nucleicos/química , RNA/genética , Termodinâmica
9.
Nucleic Acids Res ; 43(2): 1090-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25539922

RESUMO

The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom's syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.


Assuntos
Difosfato de Adenosina/metabolismo , DNA/metabolismo , RecQ Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , Cinética
10.
Proc Natl Acad Sci U S A ; 109(25): 9804-9, 2012 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-22665805

RESUMO

Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre-steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.


Assuntos
DNA de Cadeia Simples/metabolismo , RecQ Helicases/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Reparo do DNA , Ativação Enzimática , Hidrólise , Transporte Proteico , Espectrometria de Fluorescência , Triptofano/metabolismo
11.
FASEB J ; 27(12): 4954-64, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24005907

RESUMO

Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.


Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , RecQ Helicases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , DNA de Cadeia Simples/química , Humanos , Hidrólise , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , RecQ Helicases/metabolismo
12.
Nucleic Acids Res ; 40(9): 3952-63, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22253018

RESUMO

Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.


Assuntos
RecQ Helicases/química , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Terciária de Proteína , Rad51 Recombinase/metabolismo , Recombinases Rec A/química , RecQ Helicases/metabolismo , Zinco/química
13.
Proc Natl Acad Sci U S A ; 107(15): 6799-804, 2010 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-20351242

RESUMO

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.


Assuntos
Difosfato de Adenosina/química , Dictyostelium/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/química , Miosinas/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Corantes Fluorescentes/química , Cinética , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Conformação Molecular , Conformação Proteica , Coelhos
14.
Nucleic Acids Res ; 38(7): e102, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20123728

RESUMO

Quantitative determination of enzymatic rates, processivity and mechanochemical coupling is a key aspect in characterizing nucleotide triphosphate (NTP)-driven nucleic acid motor enzymes, for both basic research and technological applications. Here, we present a streamlined analytical method suitable for the determination of all key functional parameters based on measurement of NTP hydrolysis during interaction of motor enzymes with the nucleic acid track. The proposed method utilizes features of kinetic time courses of NTP hydrolysis that have not been addressed in previous analyses, and also accounts for the effect of protein traps used in kinetic experiments on processivity. This analysis is suitable for rapid and precise assessment of the effects of mutations, physical conditions, binding partners and other effectors on the functioning of translocases, helicases, polymerases and other NTP-consuming processive nucleic acid motors.


Assuntos
Enzimas/metabolismo , Modelos Biológicos , Nucleotídeos/metabolismo , Heparina/metabolismo , Hidrólise , Cinética , Polifosfatos/metabolismo
15.
Nucleic Acids Res ; 38(13): 4404-14, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20211839

RESUMO

BLM, one of the human RecQ helicases, plays a fundamental role in homologous recombination-based error-free DNA repair pathways, which require its translocation and DNA unwinding activities. Although translocation is essential in vivo during DNA repair processes and it provides a framework for more complex activities of helicases, including strand separation and nucleoprotein displacement, its mechanism has not been resolved for any human DNA helicase. Here, we present a quantitative model for the translocation of a monomeric form of BLM along ssDNA. We show that BLM performs translocation at a low adenosine triphosphate (ATP) coupling ratio (1 ATP consumed/1 nucleotide traveled) and moderate processivity (with a mean number of 50 nucleotides traveled in a single run). We also show that the rate-limiting step of the translocation cycle is a transition between two ADP-bound enzyme states. Via opening of the helicase core, this structural change may drive the stepping of BLM along the DNA track by a directed inchworm mechanism. The data also support the conclusion that BLM performs double-stranded DNA unwinding by fully active duplex destabilization.


Assuntos
DNA de Cadeia Simples/metabolismo , RecQ Helicases/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinética , Transporte Proteico
16.
Nat Commun ; 13(1): 654, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115525

RESUMO

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom's syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM's multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM's interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Cruciforme/química , DNA Cruciforme/genética , Proteínas de Ligação a DNA/genética , Humanos , Cinética , Modelos Genéticos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , RecQ Helicases/genética , Reparo de DNA por Recombinação/genética
17.
FASEB J ; 24(11): 4480-90, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20631329

RESUMO

Active site loops that are conserved across superfamilies of myosins, kinesins, and G proteins play key roles in allosteric coupling of NTP hydrolysis to interaction with track filaments or effector proteins. In this study, we investigated how the class-specific natural variation in the switch-2 active site loop contributes to the motor function of the intracellular transporter myosin-5. We used single-molecule, rapid kinetic and spectroscopic experiments and semiempirical quantum chemical simulations to show that the class-specific switch-2 structure including a tyrosine (Y439) in myosin-5 enables rapid processive translocation along actin filaments by facilitating Mg(2+)-dependent ADP release. Using wild-type control and Y439 point mutant myosin-5 proteins, we demonstrate that the translocation speed precisely correlates with the kinetics of nucleotide exchange. Switch-2 variants can thus be used to fine-tune translocation speed while maintaining high processivity. The class-specific variation of switch-2 in various NTPase superfamilies indicates its general role in the kinetic tuning of Mg(2+)-dependent nucleotide exchange.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Transporte Proteico , Actinas/metabolismo , Animais , Simulação por Computador , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Mutação/genética , Nucleotídeos/metabolismo , Fosfatos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/genética
18.
J Pharm Biomed Anal ; 204: 114246, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34271288

RESUMO

Simple and consistent chiral HPLC methods for the efficient separation of enantiomeric blebbistatin derivatives, namely parent compound blebbistatin and derivatives 4-nitroblebbistatin, 4-aminoblebbistatin, 4-dimethylaminoblebbistatin, and 4-t-butylblebbistatin were developed using cellulose tris(3,5-dimethylphenylcarbamate) as a stationary phase (Lux cellulose-1 column). Blebbistatin, 4-aminoblebbistatin, and 4-dimethylaminoblebbistatin racemates were well-separated in normal-phase HPLC conditions while 4-nitroblebbistatin and 4-t-butylblebbistatin were effectively separated in both normal- and reversed-phase HPLC conditions. Furthermore, the order of elution of enantiopure compounds was found to be independent of mobile phase compositions and conditions used, and solely depends on the interaction between the enantiomer and the chiral stationary phase. We found that despite the chiral center being present far from the D-ring in the blebbistatin structure, the D-ring substitutions prominently affect the chiral separation. Ex vivo racemization studies of the most popular blebbistatin derivative (S)-(-)-4-aminoblebbistatin in rat blood and brain tissues revealed that the compound does not convert into the inactive enantiomer. This confirms that (S)-(-)-4-aminoblebbistatin is a useful tool compound in cellular and molecular biology studies without the risks of racemization and degradation effects.


Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis , Vertebrados , Animais , Cromatografia Líquida de Alta Pressão , Ratos , Estereoisomerismo
19.
Sci Rep ; 10(1): 13341, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769996

RESUMO

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2's allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2's upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins.


Assuntos
Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Linhagem Celular Tumoral , Difusão , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Humanos , Ratos
20.
Theranostics ; 10(12): 5341-5356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32373216

RESUMO

Acute ischemic stroke treatment faces an unresolved obstacle as capillary reperfusion remains insufficient after thrombolysis and thrombectomy causing neuronal damage and poor prognosis. Hypoxia-induced capillary constriction is mediated by actomyosin contraction in precapillary smooth muscle cells (SMCs) therefore smooth muscle myosin-2 could be an ideal target with potentially high impact on reperfusion of capillaries. Methods: The myosin-2 inhibitor para-aminoblebbistatin (AmBleb) was tested on isolated human and rat arterioles to assess the effect of AmBleb on vasodilatation. Transient middle cerebral artery occlusion (MCAO) was performed on 38 male Wistar rats followed by local administration of AmBleb into the ischemic brain area. Development of brain edema and changes in cerebrovascular blood flow were assessed using MRI and SPECT. We also tested the neurological deficit scores and locomotor asymmetry of the animals for 3 weeks after the MCAO operation. Results: Our results demonstrate that AmBleb could achieve full relaxation of isolated cerebral arterioles. In living animals AmBleb recovered cerebral blood flow in 32 out of the 65 affected functional brain areas in MCAO operated rats, whereas only 8 out of the 67 affected areas were recovered in the control animals. Animals treated with AmBleb also showed significantly improved general and focal deficit scores in neurological functional tests and showed significantly ameliorated locomotor asymmetry. Conclusion: Direct inhibition of smooth muscle myosin by AmBleb in pre-capillary SMCs significantly contribute to the improvement of cerebral blood reperfusion and brain functions suggesting that smooth muscle myosin inhibition may have promising potential in stroke therapies as a follow-up treatment of physical or chemical removal of the occluding thrombus.


Assuntos
Isquemia Encefálica/fisiopatologia , Infarto da Artéria Cerebral Média/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Isquemia Encefálica/diagnóstico por imagem , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Coelhos , Ratos , Ratos Wistar , Acidente Vascular Cerebral/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único
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