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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33414166

RESUMO

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

2.
FEBS Lett ; 592(1): 112-121, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237230

RESUMO

Focal adhesion (FA) proteins, kindlin-2 and integrin-linked kinase (ILK), regulate cell adhesion and migration. ILK interacts with and promotes kindlin-2 targeting to FAs. Leu353 and Leu357 in kindlin-2 have been reported to be important for the interaction between kindlin-2 and ILK. However, the binding interface between kindlin-2 and ILK remains unclear. Using molecular modeling and molecular dynamics simulations, we show that Asp344, Asp352, and Thr356 in kindlin-2 and Arg243 and Arg334 in ILK kinase domain (KD) are important in kindlin-2/ILK complex formation. Mutations that disrupt these interactions abrogate kindlin-2 and ILK colocalization in HeLa cells. The interactions are direct based on data from pull-down assays using purified recombinant kindlin-2 F2-pleckstrin homology and ILK KDs. These data provide additional insights into the binding interface between kindlin-2 and ILK.


Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Adesão Celular/genética , Adesão Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Células HeLa , Humanos , Proteínas de Membrana/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática
3.
Proteins ; 82(11): 3194-209, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25212695

RESUMO

Copper-Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo-SOD1 aggregates may be due to their ability to cause defects within cell membranes by co-aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane-bound copper chaperones. But how apo-SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc-binding loops in apo-SOD1 dimers play a critical role in the binding of 1-octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo-SOD1 monomer also associates with phospholipid bilayer via its zinc-binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane-bound copper chaperone. The orientation also suggests how membrane-bound monomers could act as seeds for membrane-associated SOD1 aggregation.


Assuntos
Lipídeos/química , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , 1-Octanol/química , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Dimiristoilfosfatidilcolina/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Eletricidade Estática , Superóxido Dismutase-1 , Água/química , Zinco/metabolismo
4.
Biomaterials ; 35(27): 7750-61, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24954734

RESUMO

The topography of the extracellular microenvironment influences cell morphology, provides conduct guidance and directs cell differentiation. Aspect ratio and dimension of topography have been shown to affect cell behaviours, but the ability and mechanism of depth-sensing is not clearly understood. We showed that murine neural progenitor cells (mNPCs) can sense the depth of the micro-gratings. Neurite elongation, alignment and neuronal differentiation were observed to increase with grating depth. We proposed a mechanism for depth-sensing by growing neurites: filopodial adhesion in the growth cones favour elongation but the bending rigidity of the neurite cytoskeleton resists it. Thus, perpendicular extension on deeper grooves is unfavourable as neurites need to bend over a larger angle. A quantitative model was developed and its prediction of neurite growth on gratings fit well with the experimental data. The results indicated that mNPC fate can be directed by appropriately designed patterned surfaces.


Assuntos
Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dimetilpolisiloxanos/farmacologia , Neuritos/metabolismo , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Hipocampo/citologia , Camundongos , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura
5.
Proteins ; 79(7): 2203-13, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21557324

RESUMO

Integrins are transmembrane (TM) proteins that mediate bidirectional mechanical signaling between the extracellular matrix and the cellular cytoskeletal network. Each integrin molecule consists of non-covalently associated α- and ß-subunits, with each subunit consisting of a large ectodomain, a single-pass TM helix, and a short cytoplasmic tail. Previously we found evidence for a polar interaction (hydrogen bond) in the outer membrane clasp (OMC) of the leukocyte integrin αLß2 TMs that is absent in the platelet integrin αIIß3 OMC. Here, we compare the self-assembly dynamics of αLß2 and αIIß3 TM helices in a model membrane using coarse-grained molecular dynamics simulations. We found that although αIIß3 TM helices associate more easily, packing is suboptimal. In contrast, αLß2 TM helices achieve close-to-optimal packing. This suggests that αLß2 TM packing is more specific, possibly due to the interhelix hydrogen bond. Theoretical association free energy profiles show a deeper minimum at a smaller helix-helix separation for αLß2 compared with αIIß3. The αIIß3 profile is also more rugged with energetic barriers whereas that of αLß2 is almost without barriers. Disruption of the interhelix hydrogen bond in αLß2 via the ß2T686G mutation results in poorer association and a similar profile as αIIß3. The OMC polar interaction in αLß2 thus plays a significant role in the packing of the TM helices.


Assuntos
Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/metabolismo , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Termodinâmica
6.
J Mol Biol ; 398(4): 569-83, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20338181

RESUMO

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of alpha and beta subunits. Each subunit contains a single alpha-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of alphabeta TM packing. The leukocyte integrin alpha L beta 2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of alpha L beta 2 TMs is consistent with that of the integrin alpha IIb beta 3 TMs. However, molecular dynamics simulations of alpha L beta 2 TMs in lipids predicted a polar interaction involving the side chains of alpha L Ser1071 and beta2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled alpha L beta 2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of alpha L beta 2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of beta2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated alpha L beta 2, alpha M beta 2, and alpha X beta 2 in 293T transfectants. We also show that the expression of mutant beta2 Thr686Gly in beta2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1 alpha treatment as compared to wild-type beta2-expressing cells. These two TM polar residues are totally conserved in other members of the beta2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of alpha L beta 2 function.


Assuntos
Antígeno-1 Associado à Função Linfocitária/metabolismo , Mapeamento de Interação de Proteínas , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Humanos , Antígeno-1 Associado à Função Linfocitária/genética , Lipídeos de Membrana/metabolismo , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Alinhamento de Sequência
7.
J Mol Graph Model ; 28(6): 548-54, 2010 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-20044289

RESUMO

Bacterial flagellum is a nano-scale motility device constructed by self-assembly. During construction of the cell-exterior filament (the 'propeller'), subunit proteins (called flagellin) are thought to be exported through the hollow flagellum to the growing filament tip in an unfolded state. To gain insight into the unfolded state preceding any force-spectroscopy experiments on flagellin, we employed force-probe molecular dynamics simulations. Two schemes to attain an unfolded state suitable for efficient transport were examined: (i) stretching flagellin along its length; (ii) unzipping flagellin from its adjacently placed termini. Atomic-level unfolding pathways and the mechanical efforts involved under each scheme were obtained for the four-domain flagellin from S. typhimurium. Flagellin appeared stiffer and required larger unfolding forces when stretched as the relative sliding of beta-strands require the breaking of multiple hydrogen bonds at once. In contrast, unzipping requires lower unfolding forces as it mainly involves unraveling beta-sheets by breaking hydrogen bonds one by one.


Assuntos
Flagelos/química , Flagelina/química , Flagelina/metabolismo , Simulação de Dinâmica Molecular , Dobramento de Proteína , Salmonella typhimurium/química , Fenômenos Biomecânicos , Estrutura Secundária de Proteína
8.
Biophys J ; 94(10): 3858-71, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18263660

RESUMO

Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin in aqueous solution as an attempt to gain atomic-level insight into the refolding process. Each molecule comprises two filament-core domains {D0, D1} and two hypervariable-region domains {D2, D3}. D2 can be separated into subdomains D2a and D2b. We observed a similar unfolding order of the domains as reported in experimental thermal denaturation. D2a and D3 exhibited high thermal stability and contained persistent three-stranded beta-sheets in the denatured state which could serve as folding cores to guide refolding. A recent mutagenesis study on flagellin stability seems to suggest the importance of the folding cores. Using crude size estimates, our data suggests that the chamber might be large enough for either denatured hypervariable-region domains or filament-core domains, but not whole flagellin; this implicates a two-staged refolding process.


Assuntos
Flagelos/química , Flagelina/química , Flagelina/ultraestrutura , Modelos Químicos , Modelos Moleculares , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/ultraestrutura , Simulação por Computador , Movimento (Física) , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína
9.
Bioinform Biol Insights ; 2: 25-45, 2008 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19812764

RESUMO

In this review, we summarize the progress on coarse-grained elastic network models (CG-ENMs) in the past decade. Theories were formulated to allow study of conformational dynamics in time/space frames of biological interest. Several highlighted models and their underlined hypotheses are introduced in physical depth. Important ENM offshoots, motivated to reproduce experimental data as well as to address the slow-mode-encoded configurational transitions, are also introduced. With the theoretical developments, computational cost is significantly reduced due to simplified potentials and coarse-grained schemes. Accumulating wealth of data suggest that ENMs agree equally well with experiment in describing equilibrium dynamics despite their distinct potentials and levels of coarse-graining. They however do differ in the slowest motional components that are essential to address large conformational changes of functional significance. The difference stems from the dissimilar curvatures of the harmonic energy wells described for each model. We also provide our views on the predictability of 'open to close' (open-->close) transitions of biomolecules on the basis of conformational selection theory. Lastly, we address the limitations of the ENM formalism which are partially alleviated by the complementary CG-MD approach, to be introduced in the second paper of this two-part series.

10.
Bioinform Biol Insights ; 2: 171-85, 2008 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-19812774

RESUMO

Molecular dynamics (MD) simulation has remained the most indispensable tool in studying equilibrium/non-equilibrium conformational dynamics since its advent 30 years ago. With advances in spectroscopy accompanying solved biocomplexes in growing sizes, sampling their dynamics that occur at biologically interesting spatial/temporal scales becomes computationally intractable; this motivated the use of coarse-grained (CG) approaches. CG-MD models are used to study folding and conformational transitions in reduced resolution and can employ enlarged time steps due to the absence of some of the fastest motions in the system. The Boltzmann-Inversion technique, heavily used in parameterizing these models, provides a smoothed-out effective potential on which molecular conformation evolves at a faster pace thus stretching simulations into tens of microseconds. As a result, a complete catalytic cycle of HIV-1 protease or the assembly of lipid-protein mixtures could be investigated by CG-MD to gain biological insights. In this review, we survey the theories developed in recent years, which are categorized into Folding-based and Molecular-Mechanics-based. In addition, physical bases in the selection of CG beads/time-step, the choice of effective potentials, representation of solvent, and restoration of molecular representations back to their atomic details are systematically discussed.

11.
Proteins ; 67(4): 868-85, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17380484

RESUMO

Molecular dynamics simulations reveal that the hydrophobic cavity in human cytokine Interleukin-1beta is hydrated and can dynamically accommodate between one and four water molecules. These waters have residence times >> 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813-1817). The waters also display high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103-108). The water molecules hydrate the hydrophobic cavity preferentially as complex clusters. These clusters result from a combination of hydrogen bonds between the waters and stabilizing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of approximately approximately 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A/ps (relative to 6 A/ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hydrated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, together with recent observations based on mutagenesis by Heidary et al. (J Mol Biol 2005; 353:1187-1198) that hydrogen bond networks couple motions across long distances in interleukin-1beta, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermodynamically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Interleucina-1beta/química , Água/química , Sequência de Aminoácidos , Animais , Simulação por Computador , Sequência Conservada , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oxigênio/química , Estrutura Terciária de Proteína , Alinhamento de Sequência
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