Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 9(1): 1892, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29760438

RESUMO

Actin polymerization powers key cellular processes, including motility, morphogenesis, and endocytosis. The actin turnover cycle depends critically on "re-charging" of ADP-actin monomers with ATP, but whether this reaction requires dedicated proteins in cells, and the underlying mechanism, have remained elusive. Here we report that nucleotide exchange catalyzed by the ubiquitous cytoskeletal regulator cyclase-associated protein (CAP) is critical for actin-based processes in vivo. We determine the structure of the CAP-actin complex, which reveals that nucleotide exchange occurs in a compact, sandwich-like complex formed between the dimeric actin-binding domain of CAP and two ADP-actin monomers. In the crystal structure, the C-terminal tail of CAP associates with the nucleotide-sensing region of actin, and this interaction is required for rapid re-charging of actin by both yeast and mammalian CAPs. These data uncover the conserved structural basis and biological role of protein-catalyzed re-charging of actin monomers.


Assuntos
Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Difosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Transporte/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Camundongos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
2.
J Biol Chem ; 292(33): 13566-13583, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28642367

RESUMO

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Deleção de Genes , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína
3.
Proteins ; 84(7): 948-56, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27028786

RESUMO

The actin capping protein (CP) binds to actin filaments to block further elongation. The capping activity is inhibited by proteins V-1 and CARMIL interacting with CP via steric and allosteric mechanisms, respectively. The crystal structures of free CP, CP/V-1, and CP/CARMIL complexes suggest that the binding of CARMIL alters the flexibility of CP rather than the overall structure of CP, and this is an allosteric inhibition mechanism. Here, we performed molecular dynamics (MD) simulations of CP in the free form, and in complex with CARMIL or V-1. The resulting trajectories were analyzed exhaustively using Motion Tree, which identifies various rigid-body motions ranging from small local motions to large domain motions. After enumerating all the motions, CP flexibilities with different ligands were characterized by a list of frequencies for 20 dominant rigid-body motions, some of which were not identified in previous studies. The comparative analysis highlights the influence of the binding of the CARMIL peptide to CP flexibility. In free CP and the CP/V-1 complex, domain motions around a large crevice between the N-stalk and the CP-S domain occur frequently. The CARMIL peptide binds the crevice and suppresses the motions effectively. In addition, the binding of the CARMIL peptide enhances and alters local motions around the pocket that participates in V-1 binding. These newly identified motions are likely to suppress the binding of V-1 to CP. The observed changes in CP motion provide insights that describe the mechanism of allosteric regulation by CARMIL through modulating CP flexibility. Proteins 2016; 84:948-956. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Regulação Alostérica , Animais , Proteína de Capeamento de Actina CapZ/química , Proteína de Capeamento de Actina CapZ/metabolismo , Galinhas , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas
4.
Nat Commun ; 6: 8730, 2015 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-26564775

RESUMO

Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas/metabolismo , Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Humanos , Cinética , Ligação Proteica , Proteínas/química , Proteínas/genética , Coelhos
5.
FEBS Lett ; 589(20 Pt B): 3085-9, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26348398

RESUMO

The effects of toxofilin (an actin binding protein of Toxoplasma gondii) on G-actin was studied with spectroscopy techniques. Fluorescence anisotropy measurements proved that G-actin and toxofilin interact with 2:1 stoichiometry. The affinity of toxofilin to actin was also determined with a fluorescence anisotropy assay. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of toxofilin. The results can be explained by the shift of the nucleotide binding cleft into a closed conformational state. Differential scanning calorimetry measurements revealed that actin monomers become thermodynamically more stable due to the binding of toxofilin.


Assuntos
Proteínas de Capeamento de Actina/química , Actinas/química , Proteínas de Protozoários/química , Termodinâmica , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Algoritmos , Animais , Sítios de Ligação/genética , Ligação Competitiva , Varredura Diferencial de Calorimetria , Polarização de Fluorescência , Temperatura Alta , Cinética , Modelos Químicos , Músculo Esquelético/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Ligação Proteica , Desnaturação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura de Transição
6.
PLoS One ; 9(5): e96326, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24788460

RESUMO

The actin-Capping Protein heterodimer, composed of the α and ß subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and ß decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and ß subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.


Assuntos
Proteínas de Capeamento de Actina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Multimerização Proteica/genética , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Actinas/metabolismo , Junções Aderentes/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Western Blotting , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
7.
Biophys J ; 106(3): 526-34, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24507593

RESUMO

The actin cortex has a well-documented ability to rapidly remodel and flow while maintaining long-range connectivity, but how this is achieved remains poorly understood. Here, we use computer simulations to explore how stress relaxation in cross-linked actin networks subjected to extensional stress depends on the interplay between network architecture and turnover. We characterize a regime in which a network response is nonaffine and stress relaxation is governed by the continuous dissipation of elastic energy via cyclic formation, elongation, and turnover of tension-bearing elements. Within this regime, for a wide range of network parameters, we observe a constant deformation (creep) rate that is linearly proportional to the rate of filament turnover, leading to a constant effective viscosity that is inversely proportional to turnover rate. Significantly, we observe a biphasic dependence of the creep rate on applied stress: below a critical stress threshold, the creep rate increases linearly with applied stress; above that threshold, the creep rate becomes independent of applied stress. We show that this biphasic stress dependence can be understood in terms of the nonlinear force-extension behavior of individual force-transmitting network elements. These results have important implications for understanding the origins and control of viscous flows both in the cortex of living cells and in other polymer networks.


Assuntos
Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/química , Actinas/química , Simulação de Dinâmica Molecular , Polimerização , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Elasticidade , Humanos , Viscosidade
8.
Oncogene ; 33(16): 2027-39, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-23644660

RESUMO

The Src family kinases c-Src, and its downstream effectors, the Rho family of small GTPases RhoA and Jun N-terminal kinase (JNK) have a significant role in tumorigenesis. In this report, using the Drosophila wing disc epithelium as a model system, we demonstrate that the actin-Capping Protein (CP) αß heterodimer, which regulates actin filament (F-actin) polymerization, limits Src-induced apoptosis or tissue overgrowth by restricting JNK activation. We show that overexpressing Src64B drives JNK-independent loss of epithelial integrity and JNK-dependent apoptosis via Btk29A, p120ctn and Rho1. However, when cells are kept alive with the Caspase inhibitor P35, JNK acts as a potent inducer of proliferation via activation of the Yorkie oncogene. Reducing CP levels direct apoptosis of overgrowing Src64B-overexpressing tissues. Conversely, overexpressing capping protein inhibits Src64B and Rho1, but not Rac1-induced JNK signaling. CP requires the actin-binding domain of the α-subunit to limit Src64B-induced apoptosis, arguing that the control of F-actin mediates this effect. In turn, JNK directs F-actin accumulation. Moreover, overexpressing capping protein also prevents apoptosis induced by ectopic JNK expression. Our data are consistent with a model in which the control of F-actin by CP limits Src-induced apoptosis or tissue overgrowth by acting downstream of Btk29A, p120ctn and Rho1, but upstream of JNK. In turn, JNK may counteract the effect of CP on F-actin, providing a positive feedback, which amplifies JNK activation. We propose that cytoskeletal changes triggered by misregulation of F-actin modulators may have a significant role in Src-mediated malignant phenotypes during the early stages of cellular transformation.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Actinas/genética , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/genética , Western Blotting , Cateninas/genética , Cateninas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epitélio/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Discos Imaginais/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Microscopia Confocal , Mutação , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Asas de Animais/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
9.
Nat Struct Mol Biol ; 20(9): 1069-76, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23912276

RESUMO

Proteins containing repeats of the WASP homology 2 (WH2) actin-binding module are multifunctional regulators of actin nucleation and assembly. The bacterial effector VopF in Vibrio cholerae, like VopL in Vibrio parahaemolyticus, is a unique homodimer of three WH2 motifs linked by a C-terminal dimerization domain. We show that only the first and third WH2 domains of VopF bind G-actin in a non-nucleating, sequestered conformation. Moreover, dimeric WH2 domains in VopF give rise to unprecedented regulation of actin assembly. Specifically, two WH2 domains on opposite protomers of VopF direct filament assembly from actin or profilin-actin by binding terminal subunits and uncapping capping protein from barbed ends by a new mechanism. Thus, VopF does not nucleate filaments by capping a pointed-end F-actin hexamer. These properties may contribute to VopF pathogenicity, and they show how dimeric WH2 peptides may mediate processive filament growth.


Assuntos
Actinas/química , Actinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vibrio cholerae/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Animais , Proteínas de Bactérias/genética , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vibrio cholerae/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
10.
Mol Biol Cell ; 24(19): 3047-55, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23904264

RESUMO

The regulation of free barbed ends is central to the control of dynamic actin assembly and actin-based motility in cells. Capping protein (CP) is known to regulate barbed ends and control actin assembly in cells. The CARMIL family of proteins can bind and inhibit CP in vitro, but the physiological significance of the interaction of CARMIL with CP in cells is poorly understood. Mammalian cells lacking CARMIL1 have defects in lamellipodia, macropinocytosis, cell migration, and Rac1 activation. Here we investigate the physiological significance of the CARMIL1-CP interaction, using a point mutant with a well-defined biochemical defect. We find that the CARMIL1-CP interaction is essential for the assembly of lamellipodia, the formation of ruffles, and the process of macropinocytosis. In contrast, the interaction of CARMIL1 with CP shows little to no importance for other functions of CARMIL1, including localization of CARMIL1 to the membrane, activation of Rac1, and cell migration. One implication is that lamellipodia are only marginally important for cell migration in a wound-healing model. The results also suggest that the ability of CARMIL1 to inhibit CP in cells may be regulated.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas de Capeamento de Actina/química , Motivos de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Humanos , Proteínas dos Microfilamentos , Pinocitose/genética , Mutação Puntual , Ligação Proteica , Pseudópodes/metabolismo , Pseudópodes/patologia
11.
Science ; 339(6118): 452-6, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23239625

RESUMO

Actin and spectrin play important roles in neurons, but their organization in axons and dendrites remains unclear. We used stochastic optical reconstruction microscopy to study the organization of actin, spectrin, and associated proteins in neurons. Actin formed ringlike structures that wrapped around the circumference of axons and were evenly spaced along axonal shafts with a periodicity of ~180 to 190 nanometers. This periodic structure was not observed in dendrites, which instead contained long actin filaments running along dendritic shafts. Adducin, an actin-capping protein, colocalized with the actin rings. Spectrin exhibited periodic structures alternating with those of actin and adducin, and the distance between adjacent actin-adducin rings was comparable to the length of a spectrin tetramer. Sodium channels in axons were distributed in a periodic pattern coordinated with the underlying actin-spectrin-based cytoskeleton.


Assuntos
Actinas/ultraestrutura , Axônios/química , Axônios/ultraestrutura , Proteínas de Ligação a Calmodulina/ultraestrutura , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Espectrina/ultraestrutura , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/ultraestrutura , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Animais , Proteínas de Ligação a Calmodulina/química , Células Cultivadas , Dendritos/química , Dendritos/ultraestrutura , Hipocampo/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência/métodos , Neurônios/química , Neurônios/ultraestrutura , Multimerização Proteica , Ratos , Ratos Wistar , Canais de Sódio/química , Canais de Sódio/ultraestrutura , Espectrina/química
12.
PLoS Comput Biol ; 8(11): e1002765, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23133367

RESUMO

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP(2)). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP(2)/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP-membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.


Assuntos
Proteínas de Capeamento de Actina/antagonistas & inibidores , Proteínas de Capeamento de Actina/química , Ácidos Fosfatídicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Aviárias/antagonistas & inibidores , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Biologia Computacional , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ácidos Fosfatídicos/química , Fosfatos de Fosfatidilinositol/química , Filogenia , Ligação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade
13.
BMC Struct Biol ; 12: 12, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22657106

RESUMO

BACKGROUND: Capping protein (CP), also known as CapZ in muscle cells and Cap32/34 in Dictyostelium discoideum, plays a major role in regulating actin filament dynamics. CP is a ubiquitously expressed heterodimer comprising an α- and ß-subunit. It tightly binds to the fast growing end of actin filaments, thereby functioning as a "cap" by blocking the addition and loss of actin subunits. Vertebrates contain two somatic variants of CP, one being primarily found at the cell periphery of non-muscle tissues while the other is mainly localized at the Z-discs of skeletal muscles. RESULTS: To elucidate structural and functional differences between cytoplasmic and sarcomercic CP variants, we have solved the atomic structure of Cap32/34 (32=ß- and 34=α-subunit) from the cellular slime mold Dictyostelium at 2.2 Å resolution and compared it to that of chicken muscle CapZ. The two homologs display a similar overall arrangement including the attached α-subunit C-terminus (α-tentacle) and the flexible ß-tentacle. Nevertheless, the structures exhibit marked differences suggesting considerable structural flexibility within the α-subunit. In the α-subunit we observed a bending motion of the ß-sheet region located opposite to the position of the C-terminal ß-tentacle towards the antiparallel helices that interconnect the heterodimer. Recently, a two domain twisting attributed mainly to the ß-subunit has been reported. At the hinge of these two domains Cap32/34 contains an elongated and highly flexible loop, which has been reported to be important for the interaction of cytoplasmic CP with actin and might contribute to the more dynamic actin-binding of cytoplasmic compared to sarcomeric CP (CapZ). CONCLUSIONS: The structure of Cap32/34 from Dictyostelium discoideum allowed a detailed analysis and comparison between the cytoplasmic and sarcomeric variants of CP. Significant structural flexibility could particularly be found within the α-subunit, a loop region in the ß-subunit, and the surface of the α-globule where the amino acid differences between the cytoplasmic and sarcomeric mammalian CP are located. Hence, the crystal structure of Cap32/34 raises the possibility of different binding behaviours of the CP variants toward the barbed end of actin filaments, a feature, which might have arisen from adaptation to different environments.


Assuntos
Proteínas de Capeamento de Actina/química , Sequência Conservada , Citoplasma/metabolismo , Dictyostelium/química , Proteínas dos Microfilamentos/química , Músculos/metabolismo , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína de Capeamento de Actina CapZ/química , Galinhas , Cristalografia por Raios X , Lipídeos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência
14.
J Biol Chem ; 287(19): 15251-62, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22411988

RESUMO

Capping protein (CP) controls the polymerization of actin filaments by capping their barbed ends. In lamellipodia, CP dissociates from the actin cytoskeleton rapidly, suggesting the possible existence of an uncapping factor, for which the protein CARMIL (capping protein, Arp2/3 and myosin-I linker) is a candidate. CARMIL binds to CP via two motifs. One, the CP interaction (CPI) motif, is found in a number of unrelated proteins; the other motif is unique to CARMILs, the CARMIL-specific interaction motif. A 115-aa CARMIL fragment of CARMIL with both motifs, termed the CP-binding region (CBR), binds to CP with high affinity, inhibits capping, and causes uncapping. We wanted to understand the structural basis for this function. We used a collection of mutants affecting the actin-binding surface of CP to test the possibility of a steric-blocking model, which remained open because a region of CBR was not resolved in the CBR/CP co-crystal structure. The CP actin-binding mutants bound CBR normally. In addition, a CBR mutant with all residues of the unresolved region changed showed nearly normal binding to CP. Having ruled out a steric blocking model, we tested an allosteric model with molecular dynamics. We found that CBR binding induces changes in the conformation of the actin-binding surface of CP. In addition, ∼30-aa truncations on the actin-binding surface of CP decreased the affinity of CBR for CP. Thus, CARMIL promotes uncapping by binding to a freely accessible site on CP bound to a filament barbed end and inducing a change in the conformation of the actin-binding surface of CP.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Proteínas dos Microfilamentos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície
15.
Proteins ; 80(4): 1066-77, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22253039

RESUMO

Capping protein (CP) is important for the regulation of actin polymerization. CP binds to the barbed end of the actin filament and prevents actin polymerization. This interaction is modulated through competitive binding by regulatory proteins such as myotrophin (V-1) and the capping protein interacting (CPI) motif from CARMIL. The binding site of myotrophin overlaps with the region of CP that binds to the barbed end of actin filament, whereas CPI binds at a distant site. The binding of CPI to the myotrophin-CP complex dissociates myotrophin from CP. Detailed multicopy molecular dynamics simulations suggest that the binding of CPI shifts the conformational equilibria of CP away from states that favor myotrophin binding. This shift is underpinned by allosteric effects where CPI inhibits CP through suppression of flexibility and disruption of concerted motions that appear to mediate myotrophin binding. Accompanying these effects are changes in electrostatic interactions, notably those involving residue K142ß, which appears to play a critical role in regulating flexibility. In addition, accessibility of the site on CP for binding the key hydrophobic residue W8 of myotrophin is modulated by CPI. These results provide insights into the modulation of CP by CPI and myotrophin and indicate the mechanism by which CPI drives the dissociation of the myotrophin-CP complex.


Assuntos
Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/química , Simulação por Computador , Peptídeos e Proteínas de Sinalização Intercelular/química , Regulação Alostérica , Sítios de Ligação , Sequência Conservada , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Polimerização , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Eletricidade Estática , Especificidade por Substrato
16.
Phys Biol ; 8(3): 035005, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21572169

RESUMO

The actin capping protein (CP) tightly binds to the barbed end of actin filaments to block further elongation. The ß-tentacle in CP is an important region that ensures stable interaction with actin filaments. CARMIL inhibits the interaction of CP with actin filaments via the C-terminal portion containing the CP-binding motif, located in an intrinsically disordered region. We have proposed an allosteric inhibition model in which CARMIL suppresses CP by the population shift mechanism. Here, we solved a crystal structure of CP in complex with a CARMIL-derived peptide, CA32. The new structure clearly represents the α-helical form of the ß-tentacle that was invisible in other CP/CARMIL peptide complex structures. In addition, we exhaustively performed a normal mode analysis with the elastic network model on all available crystal structures of the CP/CARMIL peptide complexes, including the new structure. We concluded that the CP-binding motif is necessary and sufficient for altering the fluctuation of CP, which is essential for attenuating the barbed-end-capping activity along the population shift mechanism. The roles and functions of the ß-tentacle and the CP-binding motif are discussed in terms of their intrinsically disordered nature.


Assuntos
Proteínas de Capeamento de Actina/antagonistas & inibidores , Proteínas de Capeamento de Actina/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Capeamento de Actina/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica
17.
Mol Biol Cell ; 22(14): 2541-50, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21613547

RESUMO

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin "comet tails," using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-P(i) filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.


Assuntos
Citoesqueleto de Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Destrina/química , Proteínas de Capeamento de Actina/química , Animais , Bovinos , Movimento Celular/fisiologia , Recuperação de Fluorescência Após Fotodegradação , Cinética , Microesferas , Coelhos
18.
J Mol Biol ; 404(5): 794-802, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20969875

RESUMO

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the ß-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Animais , Cinética , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica
19.
PLoS Biol ; 8(7): e1000416, 2010 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-20625546

RESUMO

The actin capping protein (CP) tightly binds to the barbed end of actin filaments, thus playing a key role in actin-based lamellipodial dynamics. V-1 and CARMIL proteins directly bind to CP and inhibit the filament capping activity of CP. V-1 completely inhibits CP from interacting with the barbed end, whereas CARMIL proteins act on the barbed end-bound CP and facilitate its dissociation from the filament (called uncapping activity). Previous studies have revealed the striking functional differences between the two regulators. However, the molecular mechanisms describing how these proteins inhibit CP remains poorly understood. Here we present the crystal structures of CP complexed with V-1 and with peptides derived from the CP-binding motif of CARMIL proteins (CARMIL, CD2AP, and CKIP-1). V-1 directly interacts with the primary actin binding surface of CP, the C-terminal region of the alpha-subunit. Unexpectedly, the structures clearly revealed the conformational flexibility of CP, which can be attributed to a twisting movement between the two domains. CARMIL peptides in an extended conformation interact simultaneously with the two CP domains. In contrast to V-1, the peptides do not directly compete with the barbed end for the binding surface on CP. Biochemical assays revealed that the peptides suppress the interaction between CP and V-1, despite the two inhibitors not competing for the same binding site on CP. Furthermore, a computational analysis using the elastic network model indicates that the interaction of the peptides alters the intrinsic fluctuations of CP. Our results demonstrate that V-1 completely sequesters CP from the barbed end by simple steric hindrance. By contrast, CARMIL proteins allosterically inhibit CP, which appears to be a prerequisite for the uncapping activity. Our data suggest that CARMIL proteins down-regulate CP by affecting its conformational dynamics. This conceptually new mechanism of CP inhibition provides a structural basis for the regulation of the barbed end elongation in cells.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Proteínas de Capeamento de Actina/química , Regulação Alostérica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Galinhas , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Células PC12 , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos
20.
J Biol Chem ; 285(37): 29014-26, 2010 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-20630878

RESUMO

Capping protein (CP) is a ubiquitously expressed, 62-kDa heterodimer that binds the barbed end of the actin filament with approximately 0.1 nm affinity to prevent further monomer addition. CARMIL is a multidomain protein, present from protozoa to mammals, that binds CP and is important for normal actin dynamics in vivo. The CARMIL CP binding site resides in its CAH3 domain (CARMIL homology domain 3) located at or near the protein's C terminus. CAH3 binds CP with approximately 1 nm affinity, resulting in a complex with weak capping activity (30-200 nm). Solution assays and single-molecule imaging show that CAH3 binds CP already present on the barbed end, causing a 300-fold increase in the dissociation rate of CP from the end (i.e. uncapping). Here we used nuclear magnetic resonance (NMR) to define the molecular interaction between the minimal CAH3 domain (CAH3a/b) of mouse CARMIL-1 and CP. Specifically, we show that the highly basic CAH3a subdomain is required for the high affinity interaction of CAH3 with a complementary "acidic groove" on CP opposite its actin-binding surface. This CAH3a-CP interaction orients the CAH3b subdomain, which we show is also required for potent anti-CP activity, directly adjacent to the basic patch of CP, shown previously to be required for CP association to and high affinity interaction with the barbed end. The importance of specific residue interactions between CP and CAH3a/b was confirmed by site-directed mutagenesis of both proteins. Together, these results offer a mechanistic explanation for the barbed end uncapping activity of CARMIL, and they identify the basic patch on CP as a crucial regulatory site.


Assuntos
Proteínas de Capeamento de Actina/química , Proteínas de Transporte/química , Proteínas de Capeamento de Actina/genética , Proteínas de Capeamento de Actina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Camundongos , Proteínas dos Microfilamentos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Propriedades de Superfície
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA