The Mitochondrial Protein Translocation Motor: Structural Conservation between the Human and Yeast Tim14/Pam18-Tim16/Pam16 co-Chaperones

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing Saccharomyces cerevisiae as a model system. Recently, several mammalian homologues of the mitochondrial motor proteins were identified. Of particular interest for us is the human Tim14/Pam18-Tim16/Pam16 complex. We chose a structural approach in order to examine the evolutionary conservation between yeast Tim14/Pam18-Tim16/Pam16 proteins and their human homologues. For this purpose, we examined the structural properties of the purified human proteins and their interaction with their yeast homologues, in vitro. Our results show that the soluble domains of the human Tim14/Pam18 and Tim16/Pam16 proteins interact with their yeast counterparts, forming heterodimeric complexes and that these complexes interact with yeast mtHsp70.     

Lights,Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.     

Determination of the Initiating Capability of Detonators Containing TKX‐50, MAD‐X1, PETNC,DAAF, RDX,HMX or PETN as a Base Charge,by Underwater Explosion Test

A comprehensive investigation to determine the initiation power of detonators containing as a base charge the novel explosives: dihydroxylammonium 5,5′‐bis(tetrazolate‐1N‐oxide) – TKX‐50, dihydroxylammonium 5,5′‐bis(3‐nitro‐1,2,4‐triazolate‐1N‐oxide) – MAD‐X1, pentaerythritol tetranitrocarbamate – PETNC and 3,3′‐diamino‐4,4′‐azoxyfurazan – DAAF in comparison with RDX, HMX and PETN was undertaken. In order to estimate the initiation power of the detonators, the underwater initiating capability test was used. The total energy as a sum of the primary shock wave energy and the bubble gas energy was determined for each of these explosives, by measuring the overpressure of the shock waves generated in water. Moreover, the complete synthesis for novel explosives is presented. The thermal behavior of the explosives was investigated using DSC (differential scanning calorimetry). The gas phase absolute molar enthalpies at 298 K and 10⁵ Pa were calculated theoretically using the modified complete basis set method (CBS‐4M; M referring to the use of minimal population localization) with the Gaussian 09 software. Gas phase standard molar enthalpies of formation (ΔH_f°_(g)) at 298 K were computed using the atomization energy method. Standard molar enthalpies of formation (▵H_(s)°) were calculated using ΔH_f°_(g) and the standard molar enthalpies of sublimation by applying Trouton’s rule. The Chapman‐Jouguet (CJ) characteristics based on calculated ▵H_(s)° values were computed using the EXPLO5 V6.01 thermochemical computer code. For the calculations the theoretical maximum densities and densities obtained during the experiments presented in this work were used.     

Crystal Structure of a Hidden Protein,YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa,with and without an Acrylamide Adduct

As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.     

Kinetics and Equilibrium Studies for the Removal of Cobalt,Manganese, and Silver in Ethanol using Dowex 50W-x8 Cation Exchange Resin

Organic solvents such as ethanol, find a wide range of applications in fuel, pharmaceutical industries, food industries, and paint formulations, among others. The removal of Ag(I), Co(II), and Mn(II) ions in ethanol by cation exchange resin, Dowex 50W-x8, was investigated. The adsorption characteristics of metal ions onto Dowex 50W-x8 resin were described by Langmuir isotherms. The maximum sorption exchange capacities at 298 K were obtained as 47.4 mg g^?1, 52.6 mg g^?1, and 58.5 mg g^?1 for Ag(I), Co(II), and Mn(II), respectively. The data was also fitted to Temkin and Dubinin-Radushkevich adsorption isotherm models to evaluate other adsorption properties. The ion exchange of silver, cobalt, and manganese on cation exchange resin followed pseudo-second-order kinetics, and the intraparticle diffusion was rate-determining step. The thermodynamic parameters indicated that the sorption of metal ions onto Dowex 50W-x8 resin was spontaneous (negative ΔG°) and endothermic in nature (positive ΔH°) implying that the adsorption capacity increased with increasing temperature. The resin can be regenerated by eluting metal ions with 3.0 mol L^?1 HNO₃ followed by washing it with 10 mL of Millipore water and 10 mL of 2.0 M NaOH, respectively. The proposed method was applied for metal ion removal in real ethanol samples.     

A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus) microplus Embryos

The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (P(i)) and this activation was much greater using polyP(3) than polyP(15). After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP(3) was 10 times stronger than that for polyP(15). Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5'-triphosphate synthesis in hard tick embryos.     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.     

Versatile pattern generation of periodic,high aspect ratio Si nanostructure arrays with sub-50-nm resolution on a wafer scale

We report on a method of fabricating variable patterns of periodic, high aspect ratio silicon nanostructures with sub-50-nm resolution on a wafer scale. The approach marries step-and-repeat nanoimprint lithography (NIL) and metal-catalyzed electroless etching (MCEE), enabling near perfectly ordered Si nanostructure arrays of user-defined patterns to be controllably and rapidly generated on a wafer scale. Periodic features possessing circular, hexagonal, and rectangular cross-sections with lateral dimensions down to sub-50 nm, in hexagonal or square array configurations and high array packing densities up to 5.13 × 10⁷ structures/mm² not achievable by conventional UV photolithography are fabricated using this top-down approach. By suitably tuning the duration of catalytic etching, variable aspect ratio Si nanostructures can be formed. As the etched Si pattern depends largely on the NIL mould which is patterned by electron beam lithography (EBL), the technique can be used to form patterns not possible with self-assembly methods, nanosphere, and interference lithography for replication on a wafer scale. Good chemical resistance of the nanoimprinted mask and adhesion to the Si substrate facilitate good pattern transfer and preserve the smooth top surface morphology of the Si nanostructures as shown in TEM. This approach is suitable for generating Si nanostructures of controlled dimensions and patterns, with high aspect ratio on a wafer level suitable for semiconductor device production.     

Loss of DIAPH3, a Formin Family Protein,Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells

Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.     

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm−2 to ≃0.1μm−2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.     

Mellein, a Trail Pheromone Component of the Ant Lasius fuliginosus

3,4-Dihydro-8-hydroxy-3-methylisocoumarin (mellein) and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one are among the volatile constituents identified from the hindgut of the formicine ant Lasius (Dendrolasius) fuliginosus Mellein induces trail-following behavior in worker ants of this species and evokes electrophysiological responses from their antennae. The trail-following activity released by (R)-(–)-mellein is significantly higher than that elicited by its (S)-(+) antipode, or the racemic mixture. The above-mentioned pyranone is found also in the honeydew of aphids, the customary diet of this ant species. The pyranone also evokes some trail-following behavior, but its activity is far less pronounced than that of mellein. Apparently, extra pheromone components are present in the hindgut, since the activity of these two constituents, either individually or as a mixture, does not completely account for the total activity released by a hindgut extract containing similar amounts of these two compounds.     

Impact of Environmental Risk Factors on Mitochondrial Dysfunction,Neuroinflammation, Protein Misfolding,and Oxidative Stress in the Etiopathogenesis of Parkinson’s Disease

As a prevalent progressive neurodegenerative disorder, Parkinson’s disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.     

Identification of a Sex Pheromone Component of the Raspberry Budmoth, Heterocrossa rubophaga

The female-produced sex pheromone of the New Zealand raspberry budmoth, Heterocrossa rubophaga, was investigated. Gas chromatographic–mass spectrometric analyses revealed the ketone, (Z)-12–nonadecen9–one (Z12–19–9–one). This compound had previously been found in extracts of the sex pheromone gland of the only other carposine moth for which a sex pheromone has been identified, Carposina niponensis, although its effect on the behavior of C. niponensis males had not been established. Field trapping trials in berry fruit gardens showed this compound to elicit high catches of male H. rubophaga, with the catch appearing to plateau (and perhaps decrease) above a dosage of 300 g on a red rubber septum. In an analysis of an extract of female H. rubophaga sex pheromone glands, there was a suggestion that the homologous (Z)-7–eicosen-11–one, the known sex pheromone component of C. niponensis, was also present. However this could not be established unequivocally and, in a field trial, addition of a small amount of this compound to Z12–19–9–one resulted in no significant increase in trap catch relative to traps baited with Z12–19–9–one alone.     

Bee Venom Melittin Disintegrates the Respiration of Mitochondria in Healthy Cells and Lymphoblasts,and Induces the Formation of Non-Bilayer Structures in Model Inner Mitochondrial Membranes

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O₂ consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O₂ consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin’s cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by ³¹P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.     

Identification of a Sex Pheromone Component for the Blueberry Leafminer, Caloptilia porphyretica

Coupled gas chromatographic-electroantennographic detection (GC-EAD) of both gland extracts and effluvial collections from female blueberry leafminer, Caloptilia porphyretica Braun (Lepidoptera: Gracillariidae), showed that females produced a single EAD-active compound. The amount of the compound collected from virgin female C. porphyretica was below GC and mass spectrometry (MS) detection thresholds, even with highly concentrated gland extracts (approximately 150 female equivalent). (E)-11-Hexadecenal (E11-16:Ald) was determined to be a sex pheromone component mainly by comparison of retention times with authentic standards on both polar and nonpolar capillary columns, microreaction-GC-EAD analyses, and field trapping tests. GC-EAD experiments showed that synthetic E11-16:Ald exhibited extraordinarily high electrophysiological activity, stimulating significant male antennal responses at as low as 10 fg. Traps baited with E11-16:Ald alone were attractive to males. Addition of 1 or 3% of its geometric isomer, Z11-16:Ald, to E11-16:Ald did not significantly increase trap captures, but an inhibitory effect was observed at the 10% level. The influence of two kinds of rubber septa on attraction was also evaluated. Male moth captures were higher in traps baited with red rubber septa than with gray rubber septa at 30-300-microg doses. Monitoring of adult flight activity with 3-microg doses of E11-16:Ald indicated at least three distinct flight periods throughout the 2003 season.     

Effect of Dieckol, a Component of Ecklonia cava, on the Promotion of Hair Growth

This study was conducted to evaluate the effect of Ecklonia cava, a marine alga native to Jeju Island in Korea, on the promotion of hair growth. When vibrissa follicles were cultured in the presence of E. cava enzymatic extract (which contains more than 35% of dieckol) for 21 days, E. cava enzymatic extract increased hair-fiber length. In addition, after topical application of the 0.5% E. cava enzymatic extract onto the back of C57BL/6 mice, anagen progression of the hair-shaft was induced. The treatment with E. cava enzymatic extract resulted in the proliferation of immortalized vibrissa dermal papilla cells (DPC). Especially, dieckol, among the isolated compounds from the E. cava enzymatic extract, showed activity that increased the proliferation of DPC. When NIH3T3 fibroblasts were treated with the E. cava enzymatic extract and the isolated compounds from the E. cava enzymatic extract, the E. cava enzymatic extract increased the proliferation of NIH3T3 fibroblasts, but the isolated compounds such as eckol, dieckol, phloroglucinol and triphlorethol-A did not affect the proliferation of NIH3T3 fibroblasts. On the other hand, the E. cava enzymatic extract and dieckol significantly inhibited 5α-reductase activity. These results suggest that dieckol from E. cava can stimulate hair growth by the proliferation of DPC and/or the inhibition of 5α-reductase activity.