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1.
J Mol Cell Cardiol ; 186: 125-137, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38008210

RESUMO

N-terminal cardiac myosin-binding protein C (cMyBP-C) domains (C0-C2) bind to thick (myosin) and thin (actin) filaments to coordinate contraction and relaxation of the heart. These interactions are regulated by phosphorylation of the M-domain situated between domains C1 and C2. In cardiomyopathies and heart failure, phosphorylation of cMyBP-C is significantly altered. We aimed to investigate how cMyBP-C interacts with myosin and actin. We developed complementary, high-throughput, C0-C2 FRET-based binding assays for myosin and actin to characterize the effects due to 5 HCM-linked variants or functional mutations in unphosphorylated and phosphorylated C0-C2. The assays indicated that phosphorylation decreases binding to both myosin and actin, whereas the HCM mutations in M-domain generally increase binding. The effects of mutations were greatest in phosphorylated C0-C2, and some mutations had a larger effect on actin than myosin binding. Phosphorylation also altered the spatial relationship of the probes on C0-C2 and actin. The magnitude of these structural changes was dependent on C0-C2 probe location (C0, C1, or M-domain). We conclude that binding can differ between myosin and actin due to phosphorylation or mutations. Additionally, these variables can change the mode of binding, affecting which of the interactions in cMyBP-C N-terminal domains with myosin or actin take place. The opposite effects of phosphorylation and M-domain mutations is consistent with the idea that cMyBP-C phosphorylation is critical for normal cardiac function. The precision of these assays is indicative of their usefulness in high-throughput screening of drug libraries for targeting cMyBP-C as therapy.


Assuntos
Citoesqueleto de Actina , Actinas , Proteínas de Transporte , Actinas/metabolismo , Fosforilação , Citoesqueleto de Actina/metabolismo , Miosinas/genética , Miosinas/metabolismo , Mutação
2.
J Biol Chem ; 299(12): 105369, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37865311

RESUMO

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.


Assuntos
Proteínas de Transporte , Descoberta de Drogas , Insuficiência Cardíaca , Miofibrilas , Bibliotecas de Moléculas Pequenas , Humanos , Actinas/metabolismo , Descoberta de Drogas/métodos , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Miosinas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Miofibrilas/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Técnicas Biossensoriais , Adenosina Trifosfatases/metabolismo , Músculo Esquelético/metabolismo , Proteínas Recombinantes/metabolismo , Ativação Enzimática/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência
3.
J Mol Cell Cardiol ; 166: 116-126, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35227736

RESUMO

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating contractile dysfunction in heart failure. Mimicking the structural dynamics of phosphorylated cMyBP-C by small-molecule drug binding could lead to therapies that modulate cMyBP-C conformational states, and thereby function, to improve contractility. We have developed a human cMyBP-C biosensor capable of detecting intramolecular structural changes due to phosphorylation and mutation. Using site-directed mutagenesis and time-resolved fluorescence resonance energy transfer (TR-FRET), we substituted cysteines in cMyBP-C N-terminal domains C0 through C2 (C0-C2) for thiol-reactive fluorescent probe labeling to examine C0-C2 structure. We identified a cysteine pair that upon donor-acceptor labeling reports phosphorylation-sensitive structural changes between the C1 domain and the tri-helix bundle of the M-domain that links C1 to C2. Phosphorylation reduced FRET efficiency by ~18%, corresponding to a ~11% increase in the distance between probes and a ~30% increase in disorder between them. The magnitude and precision of phosphorylation-mediated TR-FRET changes, as quantified by the Z'-factor, demonstrate the assay's potential for structure-based high-throughput screening of compounds for cMyBP-C-targeted therapies to improve cardiac performance in heart failure. Additionally, by probing C1's spatial positioning relative to the tri-helix bundle, these findings provide new molecular insight into the structural dynamics of phosphoregulation as well as mutations in cMyBP-C. Biosensor sensitivity to disease-relevant mutations in C0-C2 was demonstrated by examination of the hypertrophic cardiomyopathy mutation R282W. The results presented here support a screening platform to identify small molecules that regulate N-terminal cMyBP-C conformational states.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Insuficiência Cardíaca , Proteínas de Transporte , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Mutação , Fosforilação
4.
J Biol Chem ; 297(1): 100840, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34052227

RESUMO

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Ensaios de Triagem em Larga Escala , Miocárdio/metabolismo , Actinas/química , Animais , Técnicas Biossensoriais , Calorimetria , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligação Proteica , Coelhos , Sarcômeros/metabolismo , Fatores de Tempo
5.
J Biol Chem ; 294(44): 16228-16240, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31519753

RESUMO

Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated protein that influences actin-myosin interactions. cMyBP-C alters myofilament structure and contractile properties in a protein kinase A (PKA) phosphorylation-dependent manner. To determine the effects of cMyBP-C and its phosphorylation on the microsecond rotational dynamics of actin filaments, we attached a phosphorescent probe to F-actin at Cys-374 and performed transient phosphorescence anisotropy (TPA) experiments. Binding of cMyBP-C N-terminal domains (C0-C2) to labeled F-actin reduced rotational flexibility by 20-25°, indicated by increased final anisotropy of the TPA decay. The effects of C0-C2 on actin TPA were highly cooperative (n = ∼8), suggesting that the cMyBP-C N terminus impacts the rotational dynamics of actin spanning seven monomers (i.e. the length of tropomyosin). PKA-mediated phosphorylation of C0-C2 eliminated the cooperative effects on actin flexibility and modestly increased actin rotational rates. Effects of Ser to Asp phosphomimetic substitutions in the M-domain of C0-C2 on actin dynamics only partially recapitulated the phosphorylation effects. C0-C1 (lacking M-domain/C2) similarly exhibited reduced cooperativity, but not as reduced as by phosphorylated C0-C2. These results suggest an important regulatory role of the M-domain in cMyBP-C effects on actin structural dynamics. In contrast, phosphomimetic substitution of the glycogen synthase kinase (GSK3ß) site in the Pro/Ala-rich linker of C0-C2 did not significantly affect the TPA results. We conclude that cMyBP-C binding and PKA-mediated phosphorylation can modulate actin dynamics. We propose that these N-terminal cMyBP-C-induced changes in actin dynamics help explain the functional effects of cMyBP-C phosphorylation on actin-myosin interactions.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Miosinas Cardíacas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Polarização de Fluorescência/métodos , Humanos , Medições Luminescentes/métodos , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Fosforilação , Ligação Proteica/fisiologia , Coelhos , Rotação , Sarcômeros/metabolismo
6.
J Mol Cell Cardiol ; 125: 140-148, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30359561

RESUMO

RATIONALE: Mutations in the gene encoding the sarcomeric protein cardiac myosin-binding protein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy (HCM). Mouse models targeting cMyBP-C and use of recombinant proteins have been effective in studying its roles in contractile function and disease. Surprisingly, while the N-terminus of cMyBP-C is important to regulate myofilament binding and contains many HCM mutations, an incorrect sequence, lacking the N-terminal 8 amino acids has been used in many studies. OBJECTIVES: To determine the N-terminal cMyBP-C sequences in ventricles and investigate the roles of species-specific differences in cMyBP-C on myofilament binding. METHODS AND RESULTS: We determined cMyBP-C sequences in mouse and human by inspecting available sequence databases. N-terminal differences were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cosedimentation assays with actin or myosin were used to examine binding in mouse, human and chimeric fusion proteins of cMyBP-C. Time-resolved FRET (TR-FRET) with site-directed probes on cMyBP-C was employed to measure structural dynamics. LC-MS/MS supported the sequencing data that mouse cMyBP-C contains an eight-residue N-terminal extension (NTE) not found in human. Cosedimentation assays revealed that cardiac myosin binding was strongly influenced by the presence of the NTE, which reduced binding by 60%. 75% more human C0-C2 than mouse bound to myosin. Actin binding of mouse C0-C2 was not affected by the NTE. 50% more human C0-C2 than mouse bound to actin. TR-FRET indicates that the NTE did not significantly affect structural dynamics across domains C0 and C1. CONCLUSIONS: Our functional results are consistent with the idea that cardiac myosin binding of N-terminal cMyBP-C is reduced in the mouse protein due to the presence of the NTE, which is proposed to interfere with myosin regulatory light chain (RLC) binding. The NTE is a critical component of mouse cMyBP-C, and should be considered in extrapolation of studies to cMyBP-C and HCM mechanisms in human.


Assuntos
Proteínas de Transporte/metabolismo , Miofibrilas/metabolismo , Actinas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cromatografia Líquida , Humanos , Camundongos , Miosinas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem
7.
Phys Biol ; 13(6): 066005, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27848929

RESUMO

Cysteine residues (Cys) in the membrane proximal region are common post-translational modification (PTM) sites in transmembrane proteins. Herein, the effects of a highly conserved membrane proximal α-subunit Cys1368 on the diffusion properties of αPS2CßPS integrins are reported. Sequence alignment shows that this cysteine is palmitoylated in human α3 and α6 integrin subunits. Replacing Cys1368 in wild-type integrins with valine (Val1368) putatively blocks a PTM site and alters integrins' ligand binding and diffusion characteristics. Both fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) diffusion measurements show Val1368 integrins are more mobile compared to wild-type integrins. Approximately 33% and 8% more Val1368 integrins are mobile as measured by FRAP and SPT, respectively. The mobile Val1368 integrins also exhibit less time-dependent diffusion, as measured by FRAP. Tandem mass spectrometry data suggest that Cys1368 contains a redox or palmitoylation PTM in αPS2CßPS integrins. This membrane proximal Cys may play an important role in the diffusion of other alpha subunits that contain this conserved residue.


Assuntos
Cisteína/metabolismo , Cadeias alfa de Integrinas/química , Cadeias alfa de Integrinas/genética , Substituição de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bioquímica/métodos , Cisteína/química , Difusão , Drosophila/genética , Proteínas de Drosophila/química , Recuperação de Fluorescência Após Fotodegradação/métodos , Cadeias alfa de Integrinas/metabolismo , Proteínas Luminescentes/genética , Modificação Traducional de Proteínas , Valina/química , Valina/metabolismo
8.
J Gen Physiol ; 155(3)2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36633587

RESUMO

Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z' factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.


Assuntos
Cálcio , Troponina C , Humanos , Animais , Suínos , Cálcio/metabolismo , Fluorescência , Troponina C/metabolismo , Miocárdio/metabolismo , Contração Miocárdica/fisiologia
9.
bioRxiv ; 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-37066417

RESUMO

Cardiac MyBP-C (cMyBP-C) interacts with actin-myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces binding of cMyBP-C to actin or myosin, is often decreased in heart failure (HF) patients, and is cardioprotective in model systems for HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL), and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethyl rhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding and/or structural changes in the protein complex. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fskC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish feasibility for discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

10.
J Biol Chem ; 286(35): 30981-30993, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21757698

RESUMO

We examined over 50 mutations in the Drosophila ßPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the ß tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of ßPS and αPS2C reveal different effects on ligand binding from those observed for αIIbß3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the ßPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.


Assuntos
Proteínas de Drosophila/genética , Matriz Extracelular/metabolismo , Cadeias alfa de Integrinas/genética , Cadeias beta de Integrinas/genética , Mutação , Alelos , Animais , Adesão Celular , Drosophila , Humanos , Ligação de Hidrogênio , Ligantes , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas/química , Talina/metabolismo
11.
Dev Biol ; 340(2): 504-17, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20152825

RESUMO

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Genitália Masculina/fisiologia , Asas de Animais/fisiologia , Animais , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Proteínas da Matriz Extracelular/genética , Imuno-Histoquímica , Hibridização In Situ , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Larva/genética , Larva/metabolismo , Ligantes , Masculino , Morfogênese/genética , Mutação , Transgenes , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
12.
J Biol Chem ; 285(3): 1841-9, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19917607

RESUMO

Integrin alphaIIbbeta3 affinity regulation by talin binding to the cytoplasmic tail of beta3 is a generally accepted model for explaining activation of this integrin in Chinese hamster ovary cells and human platelets. Most of the evidence for this model comes from the use of multivalent ligands. This raises the possibility that the activation being measured is that of increased clustering of the integrin rather than affinity. Using a newly developed assay that probes integrins on the surface of cells with only monovalent ligands prior to fixation, I do not find increases in affinity of alphaIIbbeta3 integrins by talin head fragments in Chinese hamster ovary cells, nor do I observe affinity increases in human platelets stimulated with thrombin. Binding to a multivalent ligand does increase in both of these cases. This assay does report affinity increases induced by either Mn(2+), a cytoplasmic domain mutant (D723R) in the cytoplasmic domain of beta3, or preincubation with a peptide ligand. These results reconcile the previously observed differences between talin effects on integrin activation in Drosophila and vertebrate systems and suggest new models for talin regulation of integrin activity in human platelets.


Assuntos
Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática , Humanos , Imunoglobulina M/metabolismo , Ligantes , Camundongos , Estrutura Terciária de Proteína , Talina/química , Talina/metabolismo , Trombina/metabolismo
13.
J Gen Physiol ; 153(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33600558

RESUMO

Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin-binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0-C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0-C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin-tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0-C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0-C2 phosphorylation and altered binding. Lastly, C0-C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.


Assuntos
Citoesqueleto de Actina , Actinas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Humanos , Ligação Proteica
14.
Mol Biol Cell ; 18(10): 4190-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17699602

RESUMO

The control of gene expression by the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) requires its translocation into the nucleus. In Drosophila S2 cells nuclear accumulation of diphospho-ERK (dpERK) is greatly reduced by interfering double-stranded RNA against Drosophila importin-7 (DIM-7) or by the expression of integrin mutants, either during active cell spreading or after stimulation by insulin. In both cases, total ERK phosphorylation (on Westerns) is not significantly affected, and ERK accumulates in a perinuclear ring. Tyrosine phosphorylation of DIM-7 is reduced in cells expressing integrin mutants, indicating a mechanistic link between these components. DIM-7 and integrins localize to the same actin-containing peripheral regions in spreading cells, but DIM-7 is not concentrated in paxillin-positive focal contacts or stable focal adhesions. The Corkscrew (SHP-2) tyrosine phosphatase binds DIM-7, and Corkscrew is required for the cortical localization of DIM-7. These data suggest a model in which ERK phosphorylation must be spatially coupled to integrin-mediated DIM-7 activation to make a complex that can be imported efficiently. Moreover, dpERK nuclear import can be restored in DIM-7-deficient cells by Xenopus Importin-7, demonstrating that ERK import is an evolutionarily conserved function of this protein.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cadeias alfa de Integrinas/metabolismo , Carioferinas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Adesões Focais/efeitos dos fármacos , Insulina/farmacologia , Modelos Biológicos , Mutação/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Xenopus
15.
Mol Biol Cell ; 15(8): 3829-40, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15194810

RESUMO

We conducted a genetic screen for mutations in myospheroid, the gene encoding the Drosophila betaPS integrin subunit, and identified point mutants in all of the structural domains of the protein. Surprisingly, we find that mutations in very strongly conserved residues will often allow sufficient integrin function to support the development of adult animals, including mutations in the ADMIDAS site and in a cytoplasmic NPXY motif. Many mutations in the I-like domain reduce integrin expression specifically when betaPS is combined with activating alphaPS2 cytoplasmic mutations, indicating that integrins in the extended conformation are unstable relative to the inactive, bent heterodimers. Interestingly, the screen has identified alleles that show gain-of-function characteristics in cell culture, but have negative effects on animal development or viability. This is illustrated by the allele mys(b58); available structural models suggest that the molecular lesion of mys(b58), V409>D, should promote the "open" conformation of the beta subunit I-like domain. This expectation is supported by the finding that alphaPS2betaPS (V409>D) promotes adhesion and spreading of S2 cells more effectively than does wild-type alphaPS2betaPS, even when betaPS is paired with alphaPS2 containing activating cytoplasmic mutations. Finally, comparisons with the sequence of human beta8 suggest that evolution has targeted the "mys(b58)" residue as a means of affecting integrin activity.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Integrinas/química , Integrinas/metabolismo , Alelos , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Adesão Celular/genética , Movimento Celular/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Epitélio/química , Testes Genéticos , Cadeias alfa de Integrinas , Cadeias beta de Integrinas/análise , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/metabolismo , Integrinas/genética , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína
16.
Mol Biol Cell ; 13(4): 1352-65, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11950944

RESUMO

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.


Assuntos
Citoplasma/metabolismo , Proteínas de Drosophila , Drosophila/embriologia , Integrinas/química , Alelos , Sequência de Aminoácidos , Animais , Células Cultivadas , Genes Dominantes , Genótipo , Cadeias alfa de Integrinas , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Fenótipo , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Asas de Animais/embriologia
17.
Genetics ; 162(1): 285-96, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12242240

RESUMO

The Drosophila PS1 and PS2 integrins are required to maintain the connection between the dorsal and ventral wing epithelia. If alphaPS subunits are inappropriately expressed during early pupariation, the epithelia separate, causing a wing blister. Two lines of evidence indicate that this apparent loss-of-function phenotype is not a dominant negative effect, but is due to inappropriate expression of functional integrins: wing blisters are not generated efficiently by misexpression of loss-of-function alphaPS2 subunits with mutations that inhibit ligand binding, and gain-of-function, hyperactivated mutant alphaPS2 proteins cause blistering at expression levels well below those required by wild-type proteins. A genetic screen for dominant suppressors of wing blisters generated null alleles of a gene named moleskin, which encodes the protein DIM-7. DIM-7, a Drosophila homolog of vertebrate importin-7, has recently been shown to bind the SHP-2 tyrosine phosphatase homolog Corkscrew and to be important in the nuclear translocation of activated D-ERK. Consistent with this latter finding, homozygous mutant clones of moleskin fail to grow in the wing. Genetic tests suggest that the moleskin suppression of wing blisters is not directly related to inhibition of D-ERK nuclear import. These data are discussed with respect to the possible regulation of integrin function by cytoplasmic ERK.


Assuntos
Drosophila/genética , Integrinas/genética , Carioferinas/genética , Animais , Núcleo Celular/metabolismo , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes Supressores , Carioferinas/fisiologia , Transporte Proteico , Receptores Citoplasmáticos e Nucleares
18.
Clin Cancer Res ; 18(9): 2429-35, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22415315

RESUMO

Abnormal Hedgehog (Hh) pathway activity has been reported in many cancers, including basal cell carcinomas, medulloblastomas, rhabdomyosarcomas, glioblastomas, and breast and prostate cancers. For this reason, the Hh pathway is a flourishing area for development of anticancer drugs such as Hh ligand antagonists (e.g., 5E1 and robotnikinin), Smo inhibitors (e.g., GDC-0449 and IPI-926), and Gli transcriptional activity inhibitors (e.g., GANT58 and GANT61). It is now clear that primary cilia are required for activation of the Hh pathway in normal vertebrate cells. It is in the primary cilium that both positive and negative effectors of the Hh pathway are processed by posttranslational modifications. In many cancers, preliminary results suggest that primary cilia are lost. As drugs that inhibit different steps of the Hh pathway are developed, it will be important to consider how these drugs will function in the context of primary cilia in the tumor environment. Here, we discuss why some of the Hh inhibitors may be ineffective if primary cilia are lost on cancer cells. Understanding the relationships between clinical inhibitors of the Hh pathway and the presence or absence of primary cilia may turn out to be critical for targeting these therapeutics to the correct population of patients and improving their efficacy. Further work is needed in this area to maximize the potential of these exciting therapeutic targets.


Assuntos
Cílios/fisiologia , Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Animais , Antineoplásicos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Processamento de Proteína Pós-Traducional/efeitos dos fármacos
19.
Mol Biol Cell ; 19(8): 3589-98, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18508915

RESUMO

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.


Assuntos
Drosophila/metabolismo , Regulação da Expressão Gênica , Integrinas/metabolismo , Talina/química , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Citometria de Fluxo , Modelos Biológicos , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA
20.
Anal Chem ; 79(8): 3142-7, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17346031

RESUMO

A method for measuring the microclustering of a class of cell surface receptors called integrins is reported. Integrins are proteins involved in bidirectional signaling across the cell membrane and are important in cell adhesion, growth, and survival. Their activity is regulated by changes in protein conformation and protein clustering. The developed in vivo clustering assay uses fluorescence resonance energy transfer (FRET) and has the benefit of requiring a single cloning step to generate FRET donors and acceptors that can be used to measure the clustering of a series of integrin mutants. The FRET reporters contain extracellular donor or acceptor fluorescent protein attached to native integrin cytoplasmic and transmembrane domains, and these are expressed along with wild-type or mutant integrins. Expression of the FRET reporters has no affect on the ligand binding properties of coexpressed integrins. FRET values are calculated for cell lines spreading on ligand coated surfaces, and these values are independent of fluorescent protein expression. No FRET is observed in cell lines expressing the reporters in the absence of integrins. Integrin-dependent FRET values increase approximately 2-3-fold when the integrins contain mutations that result in increased ligand binding affinities.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Integrinas/análise , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células Cultivadas , Clonagem Molecular , Drosophila , Integrinas/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Plasmídeos/química , Plasmídeos/genética
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