RESUMO
NK-2, a peptide derived from a cationic core region of NK-lysin, has emerged as a promising candidate for new antibiotics. In contrast to classical antibiotics, antimicrobial peptides target bacterial membranes and disintegrate the membrane by forming the transmembrane pores. However, complete understanding of the precise mechanisms of cellular apoptosis and molecular basis of membrane selectivity is still in dispute. In the present study, we have shown that NK-2 forms trans-membrane pores on negatively charged phospholipid membranes using phase contrast microscopy. As bacteria mimicking membranes, we have chosen large unilamellar vesicles (LUV) and giant unilamellar vesicles (GUV) composed of negatively charged phospholipid, dioleoyl phosphatidyl glycerol (DOPG) and neutral phospholipid, dioleoyl phophatidylcholine (DOPC). Leakage of internal fluid of giant unilamellar vesicles (GUV), leading to decrease in intensity in the halo region of phase contrast micrographs, suggests the formation of transmembrane pores. No such reduction of intensity in the halo region of DOPC was observed, indicating, neutral vesicles does not exhibit pores. Rate constant reckoned from the decaying intensity in the halo region was found to be 0.007 s-1. Further, significant interaction of NK-2 with anionic membranes has been envisaged from zeta potential and dynamic light scattering. Binding free energy and other interaction parameters have been delineated using theoretical ansatz. A proliferation of average Size of anionic LUV on increasing NK-2 concentration indicates membrane-membrane interaction leading to peptide induced large aggregates of vesicles.
Assuntos
Fosfolipídeos , Lipossomas Unilamelares , Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Antibacterianos/farmacologia , Peptídeos Antimicrobianos , Peptídeos/química , Fosfatidilcolinas/química , Fosfolipídeos/química , Lipossomas Unilamelares/químicaRESUMO
Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, amyotrophic lateral sclerosis (ALS). Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid-induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association-mediated toxic aggregation and survival time scale after ALS diagnosis.
Amyotrophic lateral sclerosis, or ALS, is an incurable neurodegenerative disease in which a person slowly loses specialized nerve cells that control voluntary movement. It is not fully understood what causes this fatal disease. However, it is suspected that clumps, or aggregates, of a protein called SOD1 in nerve cells may play a crucial role. More than 140 mutations in the gene for SOD1 have been linked to ALS, with varying degrees of severity. But it is still unclear how these mutations cause SOD1 aggregation or how different mutations influence the survival rate of the disease. The protein SOD1 contains a copper ion and a zinc ion, and it is possible that mutations that affect how these two ions bind to SOD1 influences the severity of the disease. To investigate this, Sannigrahi, Chowdhury, Das et al. genetically engineered mutants of the SOD1 protein which each contain only one metal ion. Experiments on these mutated proteins showed that the copper ion is responsible for the protein's role in neutralizing harmful reactive molecules, while the zinc ion stabilizes the protein against aggregation. Sannigrahi et al. found that when the zinc ion was removed, the SOD1 protein attached to a structure inside the cell called the mitochondria and formed toxic aggregates. Sannigrahi et al. then used these observations to build a computational model that incorporated different mutations that have been previously associated with ALS. The model suggests that mutations close to the site where zinc binds to the SOD1 protein increase disease severity and shorten survival time after diagnosis. This model was then experimentally validated using two disease variants of ALS that have mutations close to the sites where zinc or copper binds. These findings still need to be tested in animals and humans to see if these mechanisms hold true in a multicellular organism. This discovery could help design new ALS treatments that target the zinc binding site on SOD1 or disrupt the protein's interactions with the mitochondria.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Membrana Celular/enzimologia , Neurônios/enzimologia , Superóxido Dismutase-1/metabolismo , Zinco/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/patologia , Cobre/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neurônios/patologia , Agregados Proteicos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Superóxido Dismutase-1/genéticaRESUMO
Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (MÏ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and MÏ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing MÏ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and MÏ cell death.
Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/metabolismo , Proteínas de Bactérias/química , Morte Celular , Membrana Celular/microbiologia , Membrana Celular/patologia , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/patologia , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
We report the formation of crack patterns in drying films of Laponite-NaCl solution. Crack patterns that develop upon drying aqueous Laponite-NaCl solution change drastically as the amount of NaCl is varied in the solution. In this work, we have investigated the effect of NaCl on drying films of aqueous solution of Laponite under two conditions: (i) when the film is bounded by a wall, as in Petri dish experiments and (ii) when the film does not have any boundary, as in experiments with droplets. In order to obtain insights into the effect of the substrate, the experiments have been done with two different substrates of different hydrophobicities, polypropylene and glass. The formation of crack patterns has been explained on the basis of the wetting and spreading properties of the solution on these substrates and the effect of salt on colloidal aggregation. In this work, we have shown that the presence of salt in aqueous Laponite solution can induce crack patterns depending on the nature of the substrate. Another important aspect of this work is the role of NaCl in crack inhibition in desiccating films of aqueous Laponite, in the presence of static electric field. This effect can be utilized to suppress undesirable crack formation in many applications.
RESUMO
KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.