Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 80
Filtrar
Mais filtros

Bases de dados
Tipo de documento
Intervalo de ano de publicação
1.
Nat Rev Mol Cell Biol ; 15(10): 677-89, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25207437

RESUMO

Capping protein (CP) binds the fast growing barbed end of the actin filament and regulates actin assembly by blocking the addition and loss of actin subunits. Recent studies provide new insights into how CP and barbed-end capping are regulated. Filament elongation factors, such as formins and ENA/VASP (enabled/vasodilator-stimulated phosphoprotein), indirectly regulate CP by competing with CP for binding to the barbed end, whereas other molecules, including V-1 and phospholipids, directly bind to CP and sterically block its interaction with the filament. In addition, a diverse and unrelated group of proteins interact with CP through a conserved 'capping protein interaction' (CPI) motif. These proteins, including CARMIL (capping protein, ARP2/3 and myosin I linker), CD2AP (CD2-associated protein) and the WASH (WASP and SCAR homologue) complex subunit FAM21, recruit CP to specific subcellular locations and modulate its actin-capping activity via allosteric effects.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Capeamento de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a DNA/fisiologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/fisiologia , Modelos Moleculares , Fosfatos de Fosfatidilinositol/química , Ligação Proteica , Conformação Proteica
2.
Nature ; 552(7684): 214-218, 2017 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-29239354

RESUMO

Progress towards the integration of technology into living organisms requires electrical power sources that are biocompatible, mechanically flexible, and able to harness the chemical energy available inside biological systems. Conventional batteries were not designed with these criteria in mind. The electric organ of the knifefish Electrophorus electricus (commonly known as the electric eel) is, however, an example of an electrical power source that operates within biological constraints while featuring power characteristics that include peak potential differences of 600 volts and currents of 1 ampere. Here we introduce an electric-eel-inspired power concept that uses gradients of ions between miniature polyacrylamide hydrogel compartments bounded by a repeating sequence of cation- and anion-selective hydrogel membranes. The system uses a scalable stacking or folding geometry that generates 110 volts at open circuit or 27 milliwatts per square metre per gel cell upon simultaneous, self-registered mechanical contact activation of thousands of gel compartments in series while circumventing power dissipation before contact. Unlike typical batteries, these systems are soft, flexible, transparent, and potentially biocompatible. These characteristics suggest that artificial electric organs could be used to power next-generation implant materials such as pacemakers, implantable sensors, or prosthetic devices in hybrids of living and non-living systems.


Assuntos
Órgãos Artificiais , Biomimética/métodos , Fontes de Energia Elétrica , Electrophorus , Hidrogéis/química , Animais , Órgão Elétrico/fisiologia , Electrophorus/fisiologia , Microfluídica/instrumentação , Impressão Tridimensional/instrumentação , Próteses e Implantes
3.
Biophys J ; 119(9): 1800-1810, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33080221

RESUMO

The regulation of actin is key for controlled cellular function. Filaments are regulated by actin-binding proteins, but the nucleotide state of actin is also an important factor. From extended molecular dynamics simulations, we find that both nucleotide states of the actin monomer have significantly less twist than their crystal structures and that the ATP monomer is flatter than the ADP form. We also find that the filament's pointed end is flatter than the remainder of the filament and has a conformation distinct from G-actin, meaning that incoming monomers would need to undergo isomerization that would weaken the affinity and slow polymerization. Conversely, the barbed end of the filament takes on a conformation nearly identical to the ATP monomer, enhancing ATP G-actin's ability to polymerize as compared with ADP G-actin. The thermodynamic penalty imposed by differences in isomerization for the ATP and ADP growth at the barbed end exactly matches experimental results.


Assuntos
Actinas , Proteínas dos Microfilamentos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Difosfato de Adenosina , Trifosfato de Adenosina , Cinética , Proteínas dos Microfilamentos/metabolismo , Polimerização , Conformação Proteica
4.
Anal Chem ; 90(3): 1635-1642, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29266927

RESUMO

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a C-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 103 times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays.


Assuntos
Técnicas Biossensoriais/métodos , Fluoresceínas/análise , Gramicidina/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Anticorpos Monoclonais/imunologia , Fluoresceínas/síntese química , Fluoresceínas/química , Gramicidina/análogos & derivados , Gramicidina/síntese química , Haptenos/imunologia , Limite de Detecção , Bicamadas Lipídicas/química , Nanoporos
5.
Dev Biol ; 409(2): 406-19, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26658218

RESUMO

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.


Assuntos
Mutação/genética , Sistema Nervoso/embriologia , Tubulina (Proteína)/genética , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos/patologia , Epistasia Genética , Feminino , Testes Genéticos , Masculino , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Dados de Sequência Molecular , Atividade Motora , Placa Motora/patologia , Neurônios Motores/patologia , Sistema Nervoso/patologia , Junção Neuromuscular/patologia , Fenótipo , Polimerização , Multimerização Proteica , Estabilidade Proteica , Saccharomyces cerevisiae/metabolismo , Sinapses/patologia , Tubulina (Proteína)/química
6.
Chemistry ; 23(28): 6757-6762, 2017 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-28370726

RESUMO

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.


Assuntos
Eucariotos/metabolismo , Lipídeos/química , Lipossomos/química , Soro/química , Archaea/metabolismo , Linhagem Celular , Colesterol/química , Eucariotos/química , Humanos , Íons/química , Lipídeos/síntese química , Lipossomos/metabolismo , Microscopia de Fluorescência , Simulação de Dinâmica Molecular
7.
Biophys J ; 111(2): 323-332, 2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27463135

RESUMO

Point mutations in γ-cytoplasmic actin have been shown to result in autosomal-dominant, nonsyndromic, early-onset deafness. Two mutations at the same site, K118M and K118N, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from K113 to T126, and mutations that alter the position, dynamics, and/or biochemistry of this helix can result in a wide range of pathologies. Using a combination of computational and experimental studies, both employing yeast actin, we find that these mutations at K118 result in changes in the structure and dynamics of the DNase-I loop, alterations in the structure of the H73 loop as well as the side-chain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. Interestingly, in the case of K118N, the twist of the monomer is nearly identical to that of the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, these results indicate that mutations at this site give rise to a series of small changes that can be tolerated in vivo but result in misregulation of actin assembly and dynamics.


Assuntos
Actinas/genética , Actinas/metabolismo , Surdez/genética , Mutação Puntual , Acrilamida/química , Actinas/química , Regulação Alostérica/genética , Simulação de Dinâmica Molecular , Nucleotídeos/metabolismo , Fosfatos/metabolismo , Conformação Proteica em alfa-Hélice
8.
Biophys J ; 110(11): 2430-2440, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27276261

RESUMO

This work explores the proton/hydroxide permeability (PH+/OH-) of membranes that were made of synthetic extremophile-inspired phospholipids with systematically varied structural elements. A fluorescence-based permeability assay was optimized to determine the effects on the PH+/OH- through liposome membranes with variations in the following lipid attributes: transmembrane tethering, tether length, and the presence of isoprenoid methyl groups on one or both lipid tails. All permeability assays were performed in the presence of a low concentration of valinomycin (10 nM) to prevent buildup of a membrane potential without artificially increasing the measured PH+/OH-. Surprisingly, the presence of a transmembrane tether did not impact PH+/OH- at room temperature. Among tethered lipid monolayers, PH+/OH- increased with increasing tether length if the number of carbons in the untethered acyl tail was constant. Untethered lipids with two isoprenoid methyl tails led to lower PH+/OH- values than lipids with only one or no isoprenoid tails. Molecular dynamics simulations revealed a strong positive correlation between the probability of observing water molecules in the hydrophobic core of these lipid membranes and their proton permeability. We propose that water penetration as revealed by molecular dynamics may provide a general strategy for predicting proton permeability through various lipid membranes without the need for experimentation.


Assuntos
Hidróxidos/química , Lipossomos/química , Lipídeos de Membrana/química , Prótons , Lipossomas Unilamelares/química , Archaea/química , Materiais Biomiméticos/química , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ionóforos/química , Potenciais da Membrana , Metacrilatos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Permeabilidade , Valinomicina/química , Água/química
9.
Q Rev Biophys ; 47(3): 221-48, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25124728

RESUMO

The past decade has witnessed significant developments in molecular biology techniques, fluorescent labeling, and super-resolution microscopy, and together these advances have vastly increased our quantitative understanding of the cell. This detailed knowledge has concomitantly opened the door for biophysical modeling on a cellular scale. There have been comprehensive models produced describing many processes such as motility, transport, gene regulation, and chemotaxis. However, in this review we focus on a specific set of phenomena, namely cell polarization, F-actin waves, and cytokinesis. In each case, we compare and contrast various published models, highlight the relevant aspects of the biology, and provide a sense of the direction in which the field is moving.


Assuntos
Divisão Celular , Modelos Biológicos , Actinas/metabolismo , Animais , Movimento Celular , Citocinese , Humanos
10.
Biochim Biophys Acta ; 1848(2): 643-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25450342

RESUMO

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (Ps) of passive diffusion and rate constants of active transport (kT) by P-gp as a result of different experimental conditions. The Ps value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the kT value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in kT values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.


Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Hidrogéis/química , Modelos Estatísticos , Proteolipídeos/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Animais , Ligação Competitiva , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Difusão , Corantes Fluorescentes , Humanos , Cinética , Lepidópteros/química , Técnicas de Patch-Clamp , Ligação Proteica , Proteolipídeos/ultraestrutura , Rodamina 123 , Sefarose/química , Transgenes , Verapamil/farmacologia
11.
J Biol Chem ; 288(35): 25254-25264, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23843458

RESUMO

The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.


Assuntos
Simulação de Dinâmica Molecular , Multimerização Proteica , Receptor ErbB-2/química , Receptor ErbB-3/química , Animais , Humanos , Espectrometria de Massas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Células Sf9 , Spodoptera
12.
Proc Natl Acad Sci U S A ; 108(15): 6151-6, 2011 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-21444821

RESUMO

Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Evolução Molecular , Giardia lamblia/metabolismo , Giardíase/microbiologia , Proteínas dos Microfilamentos/metabolismo , Actinas/genética , Animais , Células COS , Chlorocebus aethiops , Cistos/microbiologia , Técnicas de Silenciamento de Genes , Giardia lamblia/patogenicidade , Humanos , Intestinos/microbiologia , Coelhos
13.
JCI Insight ; 9(13)2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38815134

RESUMO

The nonphysiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels affect therapeutic response by performing drug screening in human plasma-like medium. We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that recently failed in phase III clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response and how incorporation of human-specific physiological nutrient medium might help identify compounds whose efficacy could be influenced in humans.


Assuntos
Glicina , Sulfonas , Ácido Úrico , Humanos , Ácido Úrico/metabolismo , Glicina/farmacologia , Glicina/análogos & derivados , Sulfonas/farmacologia , Meios de Cultura , Avaliação Pré-Clínica de Medicamentos/métodos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia
14.
Biophys J ; 104(7): 1517-28, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23561528

RESUMO

Microtubules are cytoskeletal filaments responsible for cell morphology and intracellular organization. Their dynamical and mechanical properties are regulated through the nucleotide state of the tubulin dimers and the binding of drugs and/or microtubule-associated proteins. Interestingly, microtubule-stabilizing factors have differential effects on microtubule mechanics, but whether stabilizers have cumulative effects on mechanics or whether one effect dominates another is not clear. This is especially important for the chemotherapeutic drug Taxol, an important anticancer agent and the only known stabilizer that reduces the rigidity of microtubules. First, we ask whether Taxol will combine additively with another stabilizer or whether one stabilizer will dominate another. We call microtubules in the presence of Taxol and another stabilizer, doubly stabilized. Second, since Taxol is often added to a number of cell types for therapeutic purposes, it is important from a biomedical perspective to understand how Taxol added to these systems affects the mechanical properties in treated cells. To address these questions, we use the method of freely fluctuating filaments with our recently developed analysis technique of bootstrapping to determine the distribution of persistence lengths of a large population of microtubules treated with different stabilizers, including Taxol, guanosine-5' [(α, ß)-methyleno] triphosphate, guanosine-5'-O-(3-thiotriphosphate), tau, and MAP4. We find that combinations of these stabilizers have novel effects on the mechanical properties of microtubules.


Assuntos
Fenômenos Mecânicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Animais , Fenômenos Biomecânicos , Análise de Fourier , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Distribuição Normal , Paclitaxel/farmacologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Suínos , Temperatura , Proteínas tau/química , Proteínas tau/metabolismo
15.
Biophys J ; 105(1): 146-53, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23823233

RESUMO

The membrane-active enzyme phospholipase D (PLD) catalyzes the hydrolysis of the phosphodiester bond in phospholipids and plays a critical role in cell signaling. This catalytic reaction proceeds on lipid-water interfaces and is an example of heterogeneous catalysis in biology. Recently we showed that planar lipid bilayers, a previously unexplored model membrane for these kinetic studies, can be used for monitoring interfacial catalytic reactions under well-defined experimental conditions with chemical and electrical access to both sides of the lipid membrane. Employing an assay that relies on the conductance of the pore-forming peptide gramicidin A to monitor PLD activity, the work presented here reveals the kinetics of hydrolysis of long-chain phosphatidylcholine lipids in situ. We have developed an extension of a basic kinetic model for interfacial catalysis that includes product activation and substrate depletion. This model describes the kinetic behavior very well and reveals two kinetic parameters, the specificity constant and the interfacial quality constant. This approach results in a simple and general model to account for product accumulation in interfacial enzyme kinetics.


Assuntos
Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Fosfolipase D/metabolismo , Gramicidina/metabolismo , Cinética
16.
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
17.
PLoS Pathog ; 7(10): e1002280, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21998582

RESUMO

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.


Assuntos
Citoesqueleto de Actina/fisiologia , Movimento Celular , Evolução Molecular , Plasmodium/fisiologia , Toxoplasma/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Substituição de Aminoácidos , Animais , Sítios de Ligação , Depsipeptídeos/farmacologia , Regulação da Expressão Gênica , Modelos Moleculares , Parasitos/metabolismo , Parasitos/fisiologia , Faloidina/farmacologia , Filogenia , Plasmídeos , Plasmodium/metabolismo , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo
18.
J Nat Prod ; 76(9): 1565-72, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-24020843

RESUMO

Apicomplexan parasites such as Toxoplasma gondii rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of T. gondii in vitro. Structural modeling of chondramide A (2) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (2b-k) with substitutions in the ß-tyrosine moiety blocked parasite growth on host cell monolayers with EC50 values that ranged from 0.3 to 1.3 µM. In vitro polymerization assays using highly purified recombinant actin from T. gondii verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites.


Assuntos
Citoesqueleto de Actina/fisiologia , Depsipeptídeos/síntese química , Depsipeptídeos/farmacologia , Proteínas de Protozoários/metabolismo , Toxoplasma/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Sítios de Ligação , Depsipeptídeos/química , Proteínas dos Microfilamentos/efeitos dos fármacos , Proteínas dos Microfilamentos/metabolismo , Toxoplasma/metabolismo
19.
Proc Natl Acad Sci U S A ; 107(18): 8159-64, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20404198

RESUMO

The Arp2/3 complex polymerizes new actin filaments from the sides of existing filaments, forming Y-branched networks that are critical for actin-mediated force generation. Binding of the Arp2/3 complex to the sides of actin filaments is therefore central to its actin-nucleating and branching activities. Although a model of the Arp2/3 complex in filament branches has been proposed based on electron microscopy, this model has not been validated using independent approaches, and the functional importance of predicted actin-binding residues has not been extensively tested. Using a combination of molecular dynamics and protein-protein docking simulations, we derived an independent structural model of the interaction between two subunits of the Arp2/3 complex that are key to actin binding, ARPC2 and ARPC4, and the side of an actin filament. This model agreed remarkably well with the previous results from electron microscopy. Complementary mutagenesis experiments revealed numerous residues in ARPC2 and ARPC4 that were required for the biochemical activity of the entire complex. Functionally critical residues clustered together and defined a surface that was predicted by protein-protein docking to be buried in the interaction with actin. Moreover, key residues at this interface were crucial for actin nucleation and Y-branching, high-affinity F-actin binding, and Y-branch stability, demonstrating that the affinity of Arp2/3 complex for F actin independently modulates branch formation and stability. Our results highlight the utility of combining computational and experimental approaches to study protein-protein interactions and provide a basis for further elucidating the role of F-actin binding in Arp2/3 complex activation and function.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/química , Proteína 3 Relacionada a Actina/química , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína
20.
Nat Commun ; 14(1): 4318, 2023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37463892

RESUMO

Fundamental to all living organisms and living soft matter are emergent processes in which the reorganization of individual constituents at the nanoscale drives group-level movements and shape changes at the macroscale over time. However, light-induced degradation of fluorophores, photobleaching, is a significant problem in extended bioimaging in life science. Here, we report opening a long-time investigation window by nonbleaching phase intensity nanoscope: PINE. We accomplish phase-intensity separation such that nanoprobe distributions are distinguished by an integrated phase-intensity multilayer thin film (polyvinyl alcohol/liquid crystal). We overcame a physical limit to resolve sub-10 nm cellular architectures, and achieve the first dynamic imaging of nanoscopic reorganization over 250 h using PINE. We discover nanoscopic rearrangements synchronized with the emergence of group-level movements and shape changes at the macroscale according to a set of interaction rules with importance in cellular and soft matter reorganization, self-organization, and pattern formation.


Assuntos
Nanotecnologia , Imagem Óptica , Fotodegradação , Corantes Fluorescentes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA