Direct interaction and cooperative role of tumor suppressor p16 with band 3 (AE1)

By using the C-terminal 112-residue of band 3 to screen the K562 cDNA library, we find that the p16 interacts with band 3, which was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with increased anion transport activity in 293t cells. Moreover, expression of endogenous p16 in 293t cells was increased at 24 and 36 h after transfection with band 3. Our findings provide a novel regulation pathway for both band 3 and p16.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Role of the Transmembrane Potential in the Membrane Proton Leak

The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨ_m) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨ_m (similar to ΔΨ_m in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨ_m. The application of ΔΨ_m up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨ_m and the increase of FA membrane concentration bring about the significant exponential G_m increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA⁻ remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨ_m more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨ_m may be key in mitochondrial respiration and metabolism.     

Host peroxisomal properties are not restored to normal after treatment of visceral leishmaniasis with sodium antimony gluconate

Reason for post-kala-azar dermal leishmaniasis (PKDL) is yet to be established. Earlier it was observed that morphology and biochemical properties of host peroxisomes were impaired during Leishmania infection. As peroxisome is known to be involved in various metabolic pathways to monitor normal function of the host cells, it is essential that Leishmania-induced dysfunction of this organelle should totally be repaired during treatment of visceral leishmaniasis (VL). In this paper it has been shown that resumption of normal peroxisomal function could not be attained when one of the existing drugs sodium antimony gluconate (SAG) was used for chemotherapy against VL. Although Leishmania parasite was found to be completely eliminated from host liver and spleen after SAG treatment, normal activities of peroxisomal catalase and superoxide dismutase could not be restored. Also unusual peptides were found to be present due to abnormal proteolytic cleavage of proteins. It is proposed that peroxisomal disorder which exists even after successful chemotherapy of VL may be figured out as one of the possible reasons to develop PKDL. It may also be pointed out that continued effect of peroxisomal disorder even after complete treatment of this parasitic disease may also lead to genetic disorders not yet been explored in post-kala-azar patients.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

Activation of alternative oxidase and uncoupling protein lowers hydrogen peroxide formation in amoeba Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii were used to determine the role of two energy-dissipating systems, i.e., a free fatty acid (FFA)-activated, purine nucleotide-inhibited uncoupling protein (AcUCP) and a FFA-insensitive, purine nucleotide-activated ubiquinol alternative oxidase (AcAOX), in decreasing reactive oxygen species production in unicellular organisms. It is shown that the activation of AcUCP by externally added FFA resulted in a strong decrease in H2O2 production, whilst the inhibition of the FFA acid-induced AcUCP activity by GDP or addition of bovine serum albumin (BSA) enhanced production of H2O2. Similarly, the activation of antimycin-resistant AcAOX-mediated respiration by GMP significantly lowered H2O2 production, while inhibition of the oxidase by benzohydroxamate cancelled the GMP-induced effect on H2O2 production. When active together, both energy-dissipating systems revealed a cumulative effect on decreasing H2O2 formation. The results suggest that protection against mitochondrial oxidative stress may be a physiological role of AOX and UCP in unicellulars, such as A. castellanii.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC < POPC < DOPC, which have 0, 1, and 2 cis double bonds, respectively.     

Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for the presence of an amino acid racemase in plants

We demonstrated several kinds of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of L- and D-alanine. The K(m) and V(max) values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K(eq) value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.     

Thermal activation energy for bidirectional movement of actin along bipolar tracks of myosin filaments

Previous in vitro motility assays using bipolar myosin thick filaments demonstrated that actin filaments were capable of moving in both directions along the myosin filament tracks. The movements; however, were slower in the direction leading away from the central bare zone than towards it. To understand the mechanism underlying these different direction-dependent motilities, we have examined the effects of temperature on the velocities of the bidirectional movements along reconstituted myosin filaments. Activation energies of the movements were determined by Arrhenius plots at high and low concentrations of ATP. As a result, the thermal activation energy of the movement away from the central bare zone was significantly higher than that of the movement toward the zone. Given that the backward movement away from the central bare zone would cause the myosin heads to be constrained and the stiffness of the cross-bridges to increase, these results suggest that elastic energy required for the cross-bridge transition is supplied by thermal fluctuations.     

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.     

A high-throughput assay for measurement of multidrug resistance protein-mediated transport of leukotriene C4 into membrane vesicles

This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50nM [3H]leukotriene C(4) (LTC(4)) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5'-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D(4) antagonist, MK 571, with a k(i) of 0.67 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/min/mg protein. The properties of MRP1-mediated transport of LTC(4) are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.     

Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca²⁺, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca²⁺-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca²⁺-induced changes in cTnI-cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC-cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca²⁺ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca²⁺ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI.     

PAC-1 Activates Procaspase-3 in Vitro through Relief of Zinc-Mediated Inhibition

The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key “executioner” caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.     

Synthesis of a glycolipid for studying mechanisms of mitochondrial uncoupling proteins

Glucose-O-omega-palmitic acid is an amphipathic molecule that is useful as a tool for studying the mechanism of mitochondrial uncoupling proteins. The synthesis of this glycolipid is described herein. The study of the reaction of a series of glycosyl donors with appropriate acceptors derived from 16-hydroxyhexadecanoic acid showed that a glycosyl trichloroacetimidate donor was more efficient than thioglycoside, glycosyl halide and glycosyl acetate donors for synthesis of this glycolipid.     

Interaction of spin-labeled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector

The osteoclast variant of the vacuolar H⁺-ATPase (V-ATPase) is a potential therapeutic target for combating the excessive bone resorption that is involved in osteoporosis. The most potent in a series of synthetic inhibitors based on 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienamide (INDOL0) has demonstrated specificity for the osteoclast enzyme, over other V-ATPases. Interaction of two nitroxide spin-labeled derivatives (INDOL6 and INDOL5) with the V-ATPase is studied here by using the transport-active 16-kDa proteolipid analog of subunit c from the hepatopancreas of Nephrops norvegicus, in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Analogous experiments are also performed with vacuolar membranes from Saccharomyces cerevisiae, in which subunit c of the V-ATPase is replaced functionally by the Nephrops 16-kDa proteolipid. The INDOL5 derivative is designed to optimize detection of interaction with the V-ATPase by EPR. In membranous preparations of the Nephrops 16-kDa proteolipid, the EPR spectra of INDOL5 contain a motionally restricted component that arises from direct association of the indolyl inhibitor with the transmembrane domain of the proteolipid subunit c. A similar, but considerably smaller, motionally restricted population is detected in the EPR spectra of the INDOL6 derivative in vacuolar membranes, in addition to the larger population from INDOL6 in the fluid bilayer regions of the membrane. The potent classical V-ATPase inhibitor concanamycin A at high concentrations induces motional restriction of INDOL5, which masks the spectral effects of displacement at lower concentrations of concanamycin A. The INDOL6 derivative, which is closest to the parent INDOL0 inhibitor, displays limited subtype specificity for the osteoclast V-ATPase, with an IC₅₀ in the 10-nanomolar range.     

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis.