Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.     

Actin bundling and polymerisation properties of eukaryotic elongation factor 1 alpha (eEF1A), histone H2A-H2B and lysozyme in vitro

Elongation factor 1 alpha (eEF1A) is a positively charged protein which has been shown to interact with the actin cytoskeleton. However, to date, a specific actin binding site within the eEF1A sequence has not been identified and the mechanism by which eEF1A interacts with actin remains unresolved. Many protein–protein interactions occur as a consequence of their physicochemical properties and actin bundle formation has been shown to result from non-specific electrostatic interaction with basic proteins. This study investigated interactions between actin, eEF1A and two other positively charged proteins which are not regarded as classic actin binding proteins (namely lysozyme and H2A–H2B) in order to compare their actin organising effects in vitro. For the first time using atomic force microscopy (AFM) we have been able to image the interaction of eEF1A with actin and the subsequent bundling of actin in vitro. Interestingly, we found that eEF1A dramatically increases the rate of polymerisation (45-fold above control levels). We also show for the first time that H2A–H2B has remarkably similar effects upon actin bundling (relative bundle size/number) and polymerisation (35-fold increase above control levels) as eEF1a. The presence of lysozyme resulted in bundles which were distinct from those formed due to eEF1A and H2A–H2B. Lysozyme also increased the rate of actin polymerisation above the control level (by 10-fold). Given the striking similarities between the actin bundling and polymerisation properties of eEF1A and H2A–H2B, our results hint that dimerisation and electrostatic binding may provide clues to the mechanism through which eEF1A-actin bundling occurs.     

Biochemical and morphogenic effects of the interaction between protein kinase C-epsilon and actin in vitro and in cultured NIH3T3 cells.

Protein kinase C-epsilon coordinately regulates changes in cell growth and shape. Cells overproducing protein kinase C-epsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions that are reminiscent of the morphology observed in ras-transformed fibroblasts. Here we report that the regulatory C1 domain contains an actin binding hexapeptide motif that is essential for the morphogenic effects of protein kinase C-epsilon in cultured NIH3T3 murine fibroblasts. The extension of elongate processes by protein kinase C-epsilon transformed fibroblasts appeared to be driven by a kinase-independent mechanism that required organized networks of both actin and microtubules. Flow cytometry of phalloidin-stained cells demonstrated that protein kinase C-epsilon significantly increased the cellular content of polymerized actin in NIH3T3 cells. Studies with a cell-free system suggest that protein kinase C-epsilon inhibits the in vitro disassembly of actin filaments, is capable of desequestering actin monomers from physiologically relevant concentrations of thymosin beta4, and increases the rate of actin filament elongation by decreasing the critical concentration of actin. Based on these and other observations, it is proposed that protein kinase C-epsilon may function as a terminal downstream effector in at least one of the signaling pathways that mitogens engage to initiate outgrowth of cellular protrusions.     

Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A^pro based on sequence comparison and homology modeling with human rhinovirus 2A^pro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A^pro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A^pro structure.     

Time evolution of noise induced oxidation in outer hair cells: Role of NAD(P)H and plasma membrane fluidity

Background

Noise exposure impairs outer hair cells (OHCs). The common basis for OHC dysfunction and loss by acoustic over-stimulation is represented by reactive oxygen species (ROS) overload that may affect the membrane structural organization through generation of lipid peroxidation.

Methods

Here we investigated in OHC different functional zones the mechanisms linking metabolic functional state (NAD(P)H intracellular distribution) to the generation of lipid peroxides and to the physical state of membranes by two photon fluorescence microscopy.

Results

In OHCs of control animals, a more oxidized NAD(P)H redox state is associated to a less fluid plasma membrane structure. Acoustic trauma induces a topologically differentiated NAD(P)H oxidation in OHC rows, which is damped between 1 and 6 h. Peroxidation occurs after ~ 4 h from noise insult, while ROS are produced in the first 0.2 h and damage cells for a period of time after noise exposure has ended (~ 7.5 h) when a decrease of fluidity of OHC plasma membrane occurs. OHCs belonging to inner rows, characterized by a lower metabolic activity with respect to other rows, show less severe metabolic impairment.

Conclusions

Our data indicate that plasma membrane fluidity is related to NAD(P)H redox state and lipid peroxidation in hair cells.

General Significance

Our results could pave the way for therapeutic intervention targeting the onset of redox umbalance.     

Identification and expression of an autosomal paralogue of ribosomal protein S4, X-linked,in mice: Potential involvement of testis-specific ribosomal proteins in translation and spermatogenesis

Recent studies have revealed heterogeneity in the structure of eukaryotic cytoplasmic ribosomes, including a difference in protein composition. It has been proposed that this heterogeneity, or the specialized ribosome, contributes to tissue development and homeostasis through selective mRNA translation, although this remains largely unclear. Our previous proteomic survey of rodent ribosomes found the testis-specific ribosomal proteins L10-like and L39-like, which are paralogues of the X-linked ribosomal proteins L10 and L39, respectively. We have hypothesized that the rodent testis provides a good model for examining the possible functional importance of ribosome heterogeneity. In the present study, a new paralogue of X-linked ribosomal protein S4 has been identified in the mouse testis. The gene encoding this paralogue was autosomal, intronless and expressed predominantly in the testis. It appeared that this paralogue was included in polysomes as a component of the ribosome. Although these properties were similar to those of the ribosomal proteins L10-like and L39-like, this S4 paralogue and L10-like showed partially different expression patterns in spermatogenic cells. These findings are discussed in relation to the unique evolution of genes encoding a paralogue of ribosomal protein S4 in mammals and to the significance of testis-specific paralogues of ribosomal proteins in active ribosomes during spermatogenesis.     

Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers

The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.     

Involvement of K+ channels and calcium-dependent pathways in the action of T3 on amino acid accumulation and membrane potential in Sertoli cells of immature rat testis

The purpose of this study was to investigate the involvement of calcium in K+ currents and its effects on amino acid accumulation and on the membrane potential regulated by tri-iodo-L-thyronine (T3) in Sertoli cells. Immature rat testes were pre-incubated for 30 min in Krebs-Ringer bicarbonate buffer and incubated for 60 min in the presence of [14C]methylaminoisobutyric acid with and without T3 or T4 (dose-response curve). Specific channel blockers or chelating agents were added at different concentrations during pre-incubation and incubation periods to study the basal amino acid accumulation and a selected concentration of each drug was chosen to analyze the influence on the stimulatory hormone action. All amino acid accumulation experiments were carried out in a Dubnoff metabolic incubator at 32 degrees C, pH 7.4 and gassed with O2:CO2 (95:5; v/v). Seminiferous tubules from immature Sertoli cell-enriched testes were used for the electrophysiology experiments. Intracellular recording of the Sertoli cells was carried out in a chamber perfused with KRb with/without T3, T4 or blockers and the membrane potential was monitored. We found that T3 and T4 stimulated alpha-[1-14C] methylaminoisobutyric acid accumulation in immature rat testes and induced a membrane hyperpolarization in Sertoli cells. The action of T3 on amino acid accumulation and on the hyperpolarizing effect was inhibited by the K(+)-ATP channel blocker tolbutamide as well as the voltage-dependent Ca2+ channel blocker verapamil. These results clearly demonstrate for the first time the existence of an ionic mechanism related to Ca2+ and K+ fluxes in the rapid, nongenomic action of T3.     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

Effect of sterol structure on ordered membrane domain (raft) stability in symmetric and asymmetric vesicles

Sterol structure influences liquid ordered domains in membranes, and the dependence of biological functions on sterol structure can help identify processes dependent on ordered domains. In this study we compared the effect of sterol structure on ordered domain formation in symmetric vesicles composed of mixtures of sphingomyelin, 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, and in asymmetric vesicles in which sphingomyelin was introduced into the outer leaflet of vesicles composed of DOPC and cholesterol. In most cases, sterol behavior was similar in symmetric and asymmetric vesicles, with ordered domains most strongly stabilized by 7-dehydrocholesterol (7DHC) and cholesterol, stabilized to a moderate degree by lanosterol, epicholesterol and desmosterol, and very little if at all by 4-cholesten-3-one. However, in asymmetric vesicles desmosterol stabilized ordered domain almost as well as cholesterol, and to a much greater degree than epicholesterol, so that the ability to support ordered domains decreased in the order 7-DHC > cholesterol > desmosterol > lanosterol > epicholesterol > 4-cholesten-3-one. This contrasts with values for intermediate stabilizing sterols in symmetric vesicles in which the ranking was cholesterol > lanosterol ~ desmosterol ~ epicholesterol or prior studies in which the ranking was cholesterol ~ epicholesterol > lanosterol ~ desmosterol. The reasons for these differences are discussed. Based on these results, we re-evaluated our prior studies in cells and conclude that endocytosis levels and bacterial uptake are even more closely correlated with the ability of sterols to form ordered domains than previously thought, and do not necessarily require that a sterol have a 3β-OH group.     

Structural flexibility and the positive charges are the key factors in bacterial cell selectivity and membrane penetration of peptoid-substituted analog of Piscidin 1

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic α-helical structure isolated from the mast cells of hybrid striped bass. In our previous study, we showed that Pis-1[PG] with a substitution of Pro⁸ for Gly⁸ in Pis-1 had higher bacterial cell selectivity than Pis-1. We designed peptoid residue-substituted peptide, Pis-1[NkG], in which Gly⁸ of Pis-1 was replaced with Nlys (Lys peptoid residue). Pis-1[NkG] had higher antibacterial activity and lower cytotoxicity against mammalian cells than Pis-1 and Pis-1[PG]. We determined the tertiary structure of Pis-1[PG] and Pis-1[NkG] in the presence of DPC micelles by NMR spectroscopy. Both peptides had a three-turn helix in the C-terminal region and a bent structure in the center. Pis-1[PG] has a rigid bent structure at Pro⁸ whereas Pis-1[NkG] existed as a dynamic equilibrium of two conformers with a flexible hinge structure at Nlys⁸. Depolarization of the membrane potential of Staphylococcus aureus and confocal laser-scanning microscopy study revealed that Pis-1[NkG] effectively penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas Pis-1[PG] did not penetrate the membrane but remained outside or on the cell surface. Introduction of a lysine peptoid at position 8 of Pis-1 provided conformational flexibility and increased the positive charge at the hinge region; both factors facilitated penetration of the bacterial cell membrane and conferred bacterial cell selectivity on Pis-1[NkG].     

Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice

Background

Membrane proteins constitute a major group of proteins and are of great significance as pharmaceutical targets, but underrepresented in the Protein Data Bank. Particular reasons are their low expression yields and the constant need for cautious and diligent handling in a sufficiently stable hydrophobic environment substituting for the native membrane. When it comes to protein crystallization, such an environment is often established by detergents.

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.