Biochimica et Biophysica Acta (BBA) - General Subjects

Published by Elsevier
Online ISSN: 0304-4165
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Article
Our previous study finds that miR-143 is differentially expressed in rat uteri between pre-receptive and receptive phase via microRNA (miRNA) microarray analysis. However, it is still not clear whether miR-143 play roles during embryo implantation in rat. In the study, we found that the expression level of miR-143 on gestation day 5-8 (g.d. 5-8) was higher than on g.d.3-4 in uteri of pregnant rat. MiR-143 was mainly localized in the superficial stroma/primary decidual zone, luminal and glandular epithelium. The expression of miR-143 was not significantly influenced by pseudopregnancy, but the activation of delayed implantation and experimentally induced decidualization significantly promoted miR-143 expression. Over-expression of miR-143 in human endometrial stromal cells (ESCs) inhibited cell proliferation, migration and invasion Knockdown of miR-143 promoted cell proliferation and invasion. The results of recombinant luciferase reporters showed that miR-143 could bind to the 3'-untranslated region (UTR) of leukemia inhibitory factor receptor (Lifr) to inhibit Lifr translation. These data demonstrate that uterine miR-143 is important for the successful pregnancy, especially during the process of blastocyst implantation. Copyright © 2014. Published by Elsevier B.V.
 
Article
Antimicrobial agents, with different pore-formation mechanisms, may be differently influenced by alteration of the dipolar electric field of a lipid membrane. By using electrophysiological measurements on reconstituted lipid membranes, we used alamethicin, melittin and magainin to report on how controlled manipulation of the membrane dipole potential by the styrylpyridinium dye RH 421 affects the kinetic and transport features of peptides within membranes. Our data demonstrate that the increase of the membrane dipole potential caused by RH 421 decreases the activity and single-channel conductance of alamethicin. Surprisingly, we found that RH 421 increases the activity of melittin and magainin, suggesting that RH 421 may contribute via electrostatic repulsions, among others, to an increase in the monolayer spontaneous curvature of the membrane. We propose that RH 421-induced dipole potential and membrane elasticity changes alter the peptide-induced channel dynamics, and the prevalence of one mechanism over the other for particular classes of peptides is dictated by the electrical and mechanical interactions which rule the pore-formation mechanism of such peptides. These results point to a novel paradigm in which electrical and mechanical effects promoted by chemicals which preferentially alter the electrostatics of the membrane, may be employed to help distinguish among various pore-formation mechanisms of membrane-permeabilizing peptides.
 
Article
The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to E. coli Endo IV, His-83 is believed to coordinate one of three Zn(2+) ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn(2+) ions involved in enzymatic catalysis. The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. We consider His-83 properly coordinates the active site Zn(2+) ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn(2+) ions. Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents. Copyright © 2015. Published by Elsevier B.V.
 
Instability of Nt-R298S 
Crystal model and monomer-dimer equilibrium measurement of Nt A. Side-view of the Nt dimer, B. Front view of the Nt dimer. C. Monomer-dimer equilibrium study of the Nt. The crystal models are oriented so that the membrane is above and the cytoplasm is below. Helixes are colored purple, β-strands are green, and loops are yellow or green. In A, the solvent extended loop of the Nt is highlighted in green and is the most notable structural difference between Nt of NBCe1 and Nt of AE1 [20]. In B, the dimer is held by two-interlocking arms from each subunit. In C, the Nt is revealed to actually be in equilibrium, by the drop off in molecular mass as a function of concentration. Three concentration gradients were performed, one beginning at ~ 3 mg/ml, at ~0.3 mg/ml, and another at ~0.03 mg/ml. These are independent experiments, and the data overlays and lines up within an error of 5%, and also are corroborated by the SEC-MALS experiments in Fig 2. 
Putative conduits in Nt core The figure shows side & top views of the conduits (blue plumes) into the transmembrane domain. The conduits are lined by electrostatic residues (e.g. R298) that make up binding pockets. See text for a description of the transport pathways. 
Rescue of trafficking at low temperature 
Schematic mechanism of how R298S leads to abnormal aggregation 
Article
Band keratopathy, an ocular disease that is characterized by hypercalcemia and opaque bands across the cornea, has been associated with kidney disease. Type-II renal tubular acidosis (RTA), a condition in which the kidneys fail to recover bicarbonate (HCO3-) in the proximal tubule of the nephron, results in HCO3- wastage in the urine and low blood pH. The development of these diseases is associated with autosomal-recessive mutations in the Na(+)-coupled HCO3- cotransporter NBCe1-A located at the basolateral membranes of either cell type. We provide insight into the devastating R298S mutation found in type-II RTA-afflicted individuals using confocal-microscopy imaging of fluorescently-tagged NBCe1-A and NBCe1-A-R298S molecules expressed in human corneal endothelial and proximal tubule cells and from in-depth biophysical studies of their cytoplasmic N-terminal domains (Nt and Nt-R298S), including Nt crystal structure, melting-temperature, and homodimer dissociation constant (KD) analyses. We illuminate and rescue trafficking defects of the R298S mutation of NBCe1-A. The KD for Nt monomer-dimer equilibrium is established. The KD for Nt-R298S is significantly higher, but immeasurable due to environmental factors (pH, temperature, concentration) that result in dimer instability leading to precipitation. The crystal structure of Nt-dimer shows that R298 is part of a putative substrate conduit and resides near the dimer interface held together by hydrogen-bond networks. The R298S is a temperature-sensitive mutation in Nt that results in instability of the colloidal system leading to abnormal aggregation. Our findings provide new perspectives to the aberrant mechanism of certain ocular pathologies and type-II RTA associated with the R298S mutation. Copyright © 2015. Published by Elsevier B.V.
 
Article
Human alpha(1)-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug-AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via pi-pi stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the beta-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.
 
Article
Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.
 
Article
Naturally occurring single mutants, I56T, F57I, W64R and D67H of lysozyme in human, have been known to form abnormal protein aggregates (amyloid fibrils) and to accumulate in several organs, including the liver, spleen and kidney, resulting in familial systemic amyloidosis. These human pathogenic lysozyme variants are considered to raise subtle conformational changes compared to the wild type. Here we examined the effects of the aberrant mutant lysozymes I56T, F57I, W64R and D67H, each of which possesses a point mutation in its molecule, on a cultured human cell line, HEK293, in which the genes were individually integrated and overexpressed. Western blot analyses showed lesser amounts of these variant proteins in the medium compared to the wild type, but they were abundant in the cell pellets, indicating that the modified lysozyme proteins were scarcely secreted into the medium but were retained in the cells. Immunocytochemistry revealed that these proteins resided in restricted regions which were stained by an endoplasmic reticulum (ER) marker. Moreover, the overexpression of the mutant lysozymes were accompanied by marked increases in XBP-1s and GRP78/BiP, which are downstream agents of the IRE1α signaling pathway responding to the unfolded protein response (UPR) upon ER stress. RNAi for the mutant lysozymes' expression greatly suppressed the increases of these agents. Our results suggest that the accumulation of pathogenic lysozymes in the ER caused ER stress and the UPR response mainly via the IRE1α pathway. Copyright © 2015. Published by Elsevier B.V.
 
Article
Electroporation is a method of choice to transform living cells. The ability of electroporation to transfer small or large chemicals across the lipid bilayer membrane of eukaryotic cells or gram-negative bacteria relies on the formation of transient pores across the membrane. To exist these pores rely on an insulator (the bilayer membrane) and the presence of a potential difference on either of the membrane mediated by an external electric field. In gram-positive bacteria however the wall is not an insulator but pores can still form when an electric field is applied. Past works have shown that the electrostatic charge of teichoic acids, a major wall component; sensitizes the wall to pore formation when an external electric field is applied. These results suggest that teichoic acids mediate the formation of defects in the wall of gram-positive bacteria. We model the electrostatic repulsion between teichoic acids embedded in the bacterial wall composed of peptidoglycan when an electric field is applied. The repulsion between teichoic acids gives rise to a stress pressure that is able to rupture the wall when a threshold value has been reached. The size of such small defects can diverge leading to the formation of pores. It is demonstrated herein that for a bonding energy of about ~1-10kBT between peptidoglycan monomers an intra-wall pressure of about ~5-120kBT/nm(3) generates spherical defects of radius ~0.1-1nm diverging in size to create pores. The electrostatic cavitation of the bacterial wall theory has the potential to highlight the role of teichoic acids in the formation pores, providing a new step in the understanding of electroporation in gram-positive bacteria without requiring the use of an insulator. Copyright © 2014. Published by Elsevier B.V.
 
Article
Voltage-gated Na(+) channels (Nav) are responsible for the initiation and conduction of neuronal and muscle action potentials. Nav gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms. Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Nav1.4 D1S5-S6 linker modulate Nav gating. All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data. The data indicate that sialic acids attached to both N- and O-glycans residing within the Nav1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar. Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that environmental, metabolic, and/or congenital changes in glycosylation that impact sugar substrate levels, could lead, potentially, to changes in Nav sialylation and gating that would modulate AP waveforms and conduction. Copyright © 2014 Elsevier B.V. All rights reserved.
 
Article
Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4μg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15μM), but not DNA gyrase in S. aureus, which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. DTBN from Nuphar japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTNB will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug. Copyright © 2015. Published by Elsevier B.V.
 
Article
Background: The effect of in vivo treatment with ursolic acid (UA) on glycemia in hyperglycemic rats and its mechanism of action on muscle were studied. Methods: The UA effects on glycemia, glycogen, LDH, calcium and on insulin levels were evaluated after glucose tolerance curve. The β-cells were evaluated through the transmission electron microscopy. UA mechanism of action was studied on muscles through the glucose uptake with/without specific insulin signaling inhibitors. The nuclear effect of UA and the GLUT4 expression on muscle were studied using thymidine, GLUT4 immunocontent, immunofluorescence and RT-PCR. Results: UA presented a potent antihyperglycemic effect, increased insulin vesicle translocation, insulin secretion and augmented glycogen content. Also, UA stimulates the glucose uptake through the involvement of the classical insulin signaling related to the GLUT4 translocation to the plasma membrane as well as the GLUT4 synthesis. These were characterized by increasing the GLUT4 mRNA expression, the activation of DNA transcription, the expression of GLUT4 and its presence at plasma membrane. Also, the modulation of calcium, phospholipase C, protein kinase C and PKCaM II is mandatory for the full stimulatory effect of UA on glucose uptake. UA did not change the serum LDH and serum calcium balance. Conclusions: The antihyperglycemic role of UA is mediated through insulin secretion and insulinomimetic effect on glucose uptake, synthesis and translocation of GLUT4 by a mechanism of cross-talk between calcium and protein kinases. General significance: UA is a potential anti-diabetic agent with pharmacological properties for insulin resistance and diabetes therapy.
 
Article
The oligomeric state of the human DNMT3A is functionally important and cancer cells are known to undergo changes in pH (intracellular). Light scattering, gel filtration, and fluorescence anisotropy. Also, methylation and processivity assays. Physiologically relevant changes in pH result in changes in DNMT3A oligomer composition which have dramatic consequences on DNMT3A function. The pH changes which occur within cancer cells alter the oligomeric state and function of DNMT3A which could contribute to changes in genomic DNA methylation observed in vivo. Copyright © 2015. Published by Elsevier B.V.
 
Article
Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.
 
Article
Hypoxia-inducible factor-1α (HIF-1α) is an important transcription factor that modulates cellular responses to hypoxia and also plays critical roles in cancer progression. Recently, somatic mutations and decreased copy number of mitochondrial DNA (mtDNA) were detected in hepatocellular carcinoma (HCC). These mutations were shown to have the potential to cause mitochondrial dysfunction. However, the effects and mechanisms of mitochondrial dysfunction on HIF-1α function are not fully understood. This study aims to explore the underlying mechanism by which mitochondrial dysfunction regulates HIF-1α expression. Human hepatoma HepG2 cells were treated with various mitochondrial respiration inhibitors and an uncoupler, respectively, and the mRNA and protein expressions as well as transactivation activity of HIF-1α were determined. The role of AMP-activated protein kinase (AMPK) was further analyzed by compound C and AMPK knock-down. Treatments of mitochondrial inhibitors and an uncoupler respectively reduced both the protein level and transactivation activity of HIF-1α in HepG2 cells under normoxia or hypoxia. The mitochondrial dysfunction-repressed HIF-1α protein synthesis was associated with decreased phosphorylations of p70(S6K) and 4E-BP-1. Moreover, mitochondrial dysfunction decreased intracellular ATP content and elevated the phosphorylation of AMPK. Treatments with compound C, an AMPK inhibitor, and knock-down of AMPK partially rescued the mitochondrial dysfunction-repressed HIF-1α expression. Mitochondrial dysfunctions resulted in reduced HIF-1α protein synthesis through AMPK-dependent manner in HepG2 cells. Our results provided a mechanism for communication from mitochondria to the nucleus through AMPK-HIF-1α. Mitochondrial function is important for HIF-1α expression in cancer progression.
 
Article
The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venoms peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species venoms. The peptide fractions from B. g. gabonica, B. nasicornis, B. g. rhinoceros and B. arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules GENERAL SIGNIFICANCES: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension. Copyright © 2015. Published by Elsevier B.V.
 
Article
In the aging process, mitochondrial function gradually declines with an increase of mutations in mitochondrial DNA (mtDNA) in tissue cells. Some of the aging-associated mtDNA mutations have been shown to result in not only inefficient generation of ATP but also increased production of reactive oxygen species (ROS) such as superoxide anions (O2-) and hydrogen peroxide (H2O2) in the mitochondria of aging tissues. Extensive studies have revealed that such an increase of oxidative stress is a contributory factor for alterations in the expression and activities of antioxidant enzymes and increased oxidative damage to DNA, RNA, proteins, and lipids in tissues and cultured cells from elderly subjects. Recently, we observed that gene expression of several proteins and enzymes related to iron metabolism is altered and that aconitase is extremely susceptible to oxidative damage in senescent skin fibroblasts and in cybrids harboring aging-associated A8344G mutation of mtDNA. Of great importance is the perturbation at the protein and activity levels of several enzymes containing iron-sulfur clusters in skin fibroblasts of elderly subjects. Taken together, these findings suggest that cellular response to oxidative stress and oxidative damage elicited by mitochondrial dysfunction and/or mtDNA mutations plays an important role in human aging.
 
Article
Pin1 is an intracellular signaling molecule which plays a critical but opposite role in the pathogenesis of Alzheimer's disease (AD) and many human cancers. We review the structure and function of the Pin1 enzyme, the diverse roles it plays in cycling cells and neurons, the epidemiologic evidence for the inverse association between cancer and AD, and the potential therapeutic implications of Pin1-based therapies. Pin1 is a unique enzyme that has effects the function of target proteins by "twisting" them into different shapes. Cycling cells use Pin1 to help coordinate cell division. It is over-expressed and/or activated by multiple mechanisms in many common human cancers, and acts on multiple signal pathways to promote tumorigenesis. Inhibition of Pin1 in animal models has profound anti-tumor effects. In contrast, Pin1 is down-regulated or inactivated by multiple mechanisms in AD brains. The absence of Pin1 impairs tau function and amyloid precursor protein processing, leading to tangle- and amyloid-related pathologies and neurodegeneration in an age-dependent manner, resembling human AD. We have developed cis and trans conformation-specific antibodies to provide the first direct evidence that tau exists in distinct cis and trans conformations and that Pin1 accelerates its cis to trans conversion, thereby protecting against tangle formation in AD. Available studies on Pin1 suggest that cancer and AD may share biological pathways that are deregulated in different directions. Pin1 biology opens exciting preventive and therapeutic horizons for both cancer and neurodegeneration. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. Copyright © 2015. Published by Elsevier B.V.
 
Analysis of body weight and blood glucose level. (A) Body weight; (B) blood glucose level. Data = Means ± SEM (n = 9 for control, n = 10 for DM, n = 8 for Andro). ***P b 0.001 compared to control.
Andro inhibited retinal angiogenesis in STZ-induced PDR mice and attenuated BRB breakdown in STZ-induced NPDR mice. (A) Immunofluorescence staining of retinas with CD31 in STZ-induced PDR mice, and the enlarged partial view of CD31-stained retina was showed (original magnification ×100). (a. Control, b. DM, c. DM + Andro 10 mg/kg). (B) Quantitative results of CD31-stained retinal vessels. Data = Means ± SEM (n = 4). ***P b 0.001 compared to control; ### P b 0.001 compared to DM without Andro. (C) BRB breakdown was detected by using Evans blue leakage assay in STZ-induced NPDR mice. Data = Means ± SEM (n = 6). ***P b 0.001 compared to control; # P b 0.05 compared to DM without Andro.
Effects of Andro on VEGF expression in STZ-induced PDR mice. (A) Serum content of VEGF. Data = Means ± SEM (n = 6). (B) VEGF content in vitreous cavity. Data = Means ± SEM (n = 8 for control, n = 8 for DM, n = 6 for Andro). (C) Retinal mRNA expression of VEGF and its receptors including FLT-1 and KDR. Data = Means ± SEM (n = 7 for control, n = 8 for DM, n = 6 for Andro). *P b 0.05, **P b 0.01, ***P b 0.001 compared to control; # P b 0.05, ## P b 0.01, ### P b 0.001 compared to DM without Andro.
Effect of Andro on NF-κB signaling pathway in STZ-induced NPDR mice. (A) Andro inhibited the nuclear translocation of NF-κBp65 protein in retinas. Results represent at least three repeated experiments. (B) Quantitative densitometric analysis of NF-κBp65 in cytoplasm and nucleus. (C) Retinal expression of phosphorylated NF-κBp65, IκB, IKK. Phosphorylated NFκBp65, IκB, IKK are detected by immunoblotting using specific antibodies. Results represent at least three repeated experiments. (D) Quantitative densitometric analysis of phosphorylated NF-κBp65, IκB, IKK. (E) Retinal mRNA expression of TNF-α, IL-1β, IL-6. (F) Serum contents of TNF-α, IL-1β, IL-6. Data are expressed as means ± SEM (n = 9 for control, n = 11 for DM, n = 10 for Andro). *P b 0.05, **P b 0.01, ***P b 0.001 compared to control; # P b 0.05, ## P b 0.01, ### P b 0.001 compared to DM without Andro.
Effect of Andro on Egr1 signaling pathway in STZ-induced NPDR mice. (A) Andro inhibited the nuclear translocation of Egr1 protein in retinas. Results represent at least three repeated experiments. (B) Quantitative densitometric analysis of Egr1 in cytoplasm and nucleus. (C) Retinal mRNA expression of Egr1, serpine1, TF. (D) Serum contents of serpine1 and TF. Data are expressed as means ± SEM (n = 9 for control, n = 11 for DM, n = 10 for Andro). *P b 0.05, **P b 0.01, ***P b 0.001 compared to control; ## P b 0.01, ### P b 0.001 compared to DM without Andro.
Article
Andrographolide (Andro) is the main compound distributed in medicinal herb Andrographis paniculata. This study aims to observe the amelioration of Andro on streptozotocin (STZ)-induced diabetic retinopathy (DR) in mice. STZ-induced non-proliferative DR (NPDR) for 2months and proliferative DR (PDR) for 5month in C57BL/6 mice were used in this study, respectively. Retinal vessels were observed by immunofluorescence staining for cluster of differentiation 31 (CD31). Evans blue permeation assay was used to detect the breakdown of blood-retinal barrier (BRB). Real-time PCR and immune-blot were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β. Retinal immunofluorescence staining with CD31 showed that Andro reduced the increased retinal vessels in STZ-induced PDR mice. Evans blue permeation results demonstrated that Andro attenuated the breakdown of BRB in STZ-induced NPDR mice. In STZ-induced PDR mice, Andro decreased the increased vascular endothelial growth factor (VEGF) in serum and vitreous cavity, and reduced the increased retinal mRNA expression of VEGF and its receptors. In STZ-induced NPDR mice, Andro abrogated the nuclear translocation of nuclear factor κB (NF-κB) p65 and early growth response-1 (Egr-1), and reduced the increased phospho-NF-κBp65, -inhibitor of kappa B (IκB), and -IκB kinase (IKK). Andro also decreased the increased serum and retinal mRNA expression of TNF-α, IL-6, IL-1β, serpine1, and tissue factor (TF). Andro ameliorates DR via attenuating retinal angiogenesis and inflammation, and VEGF, NF-κB, and Egr1 signaling pathways all play important roles in this process. Copyright © 2015. Published by Elsevier B.V.
 
Article
Poly(amidoamine) (PAMAM) dendrimers are widely used biomedical polymers, which are extensively applied in drug delivery, gene delivery, contrast agent, etc. In these biomedical applications, the bio-safety of the PAMAM dendrimers is a critical issue, which affects not only their toxicity to the host but also the expected in vivo biofunctions of the materials. To clarify the bio-safety of PAMAM dendrimers, the effects of generation 5 PAMAM dendrimers with amine, hydroxyl or carboxyl groups on immune molecules were explored in this work. Specifically, the effect of the PAMAM dendrimers on the secondary structure and conformation of immune molecule γ-globulin was studied by using ultraviolet-visible, fluorescence, and circular dichroism spectroscopies. The effect of the PAMAM dendrimers on complement activation was determined by enzyme-linked immunosorbent assay. Further, the effect of the PAMAM dendrimers on antigen-antibody reaction was studied by using human red blood cell agglutination assay. The results showed that, the PAMAM dendrimers could affect the secondary structure and conformation of γ-globulin, and inhibited complement activation. Generation 5 PAMAM dendrimer with carboxyl group at 10mg/mL impaired red blood cell (RBC) antigen-antibody reaction. From these results, the effects of the PAMAM dendrimers on immune molecules depend on their bulk structure and surface groups. This work provides important information for the immunocompatibility evaluation, preclinical design, and clinical applications of PAMAM dendrimers. Copyright © 2014 Elsevier B.V. All rights reserved.
 
Article
We previously demonstrated that though the human SAA1 gene shows no typical STAT3 response element (STAT3-RE) in its promoter region, STAT3 and the nuclear factor (NF-κB) p65 first form a complex following interleukin IL-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with a region downstream of the NF-κB RE in the SAA1 promoter. In this study, we employed a computational approach based on indirect read outs of protein-DNA contacts to identify a set of candidates for non-consensus STAT3 transcription factor binding sites (TFBSs). The binding of STAT3 to one of the predicted non-consensus TFBSs was experimentally confirmed through a dual luciferase assay and DNA affinity chromatography. The present study defines a novel STAT3 non-consensus TFBS at nt -75/-66 downstream of the NF-κB RE in the SAA1 promoter region that is required for NF-κB p65 and STAT3 to activate SAA1 transcription in human HepG2 liver cells. Our analysis builds upon the current understanding of STAT3 function, suggesting a wider array of mechanisms of STAT3 function in inflammatory response, and provides a useful framework for investigating novel TF-target associations with potential therapeutic implications.
 
Article
While oxygen limitation can be extremely damaging for many animals, some vertebrates have perfected anaerobic survival. Freshwater turtles belonging to the Trachemys and Chrysemys genera, for example, can survive many weeks without oxygen, and as such are commonly used as model animals for vertebrate anoxia tolerance. In the present review we discuss the recent advances made in understanding the biochemical and molecular nature of natural anoxia tolerance of freshwater turtles. Research in recent years has shown that activation of several important pathways occurs in response to anoxia in turtles, including those that function in the stress response, cell cycle arrest, inhibition of gene expression and metabolism. These likely contribute to anoxia tolerance in turtle tissues by minimizing cell damage in response to anoxia, as well as facilitating metabolic rate depression. The research discussed in the present review contributes to the understanding of how freshwater turtles can survive without oxygen for prolonged periods of time. This could also improve understanding of the molecular nature of hypoxic/ischemic injuries in mammalian tissues and suggest potential ways to avoid these. Copyright © 2015. Published by Elsevier B.V.
 
Article
An alternative to conventional antibiotics is needed to fight against emerging multiple drug resistant pathogenic bacteria. In this endeavor, the effect of silver nanoparticle (Ag-NP) has been studied quantitatively on two common pathogenic bacteria Escherichia coli and Staphylococcus aureus, and the growth curves were modeled. The effect of Ag-NP on bacterial growth kinetics was studied by measuring the optical density, and was fitted by non-linear regression using the Logistic and modified Gompertz models. Scanning Electron Microscopy and fluorescence microscopy were used to study the morphological changes of the bacterial cells. Generation of reactive oxygen species for Ag-NP treated cells were measured by fluorescence emission spectra. The modified Gompertz model, incorporating cell death, fits the observed data better than the Logistic model. With increasing concentration of Ag-NP, the growth kinetics of both bacteria shows a decline in growth rate with simultaneous enhancement of death rate constants. The duration of the lag phase was found to increase with Ag-NP concentration. SEM showed morphological changes, while fluorescence microscopy using DAPI showed compaction of DNA for Ag-NP-treated bacterial cells. E. coli was found to be more susceptible to Ag-NP as compared to S. aureus. The modified Gompertz model, using a death term, was found to be useful in explaining the non-monotonic nature of the growth curve. The modified Gompertz model derived here is of general nature and can be used to study any microbial growth kinetics under the influence of antimicrobial agents. Copyright © 2014 Elsevier B.V. All rights reserved.
 
Article
Stem cell research has received increasing attention due to their invaluable potentials in the clinical applications to cure degenerative diseases, genetic disorders and even cancers. A great number of studies have been conducted with an aim to elucidate the molecular mechanisms involved in the regulation of self-renewal of stem cells and the mysterious circuits guiding them to differentiate into all kinds of progenies that can replenish the cell pools. However, little effort has been made in studying the metabolic aspects of stem cells. Mitochondria play essential roles in mammalian cells in the generation of ATP, Ca(2+) homeostasis, compartmentalization of biosynthetic pathways and execution of apoptosis. Considering the metabolic roles of mitochondria, they must be also critical in stem cells. This review is primarily focused on the biogenesis and bioenergetic function of mitochondria in the differentiation process and metabolic features of stem cells. In addition, the involvement of reactive oxygen species and hypoxic signals in the regulation of stem cell pluripotency and differentiation is also discussed.
 
Article
The apopto-phagocytic gene expression patterns during clearance of dying cells in the retina and the effect of triamcinolone (TC) upon these processes has relevance to development of age-related macular degeneration (AMD). ARPE-19 cells and primary human retinal pigment epithelium (hRPE) were induced to undergo cell death by anoikis and the clearance of these cells by living hRPE/ARPE-19 or human monocyte-derived macrophages (HMDMs) in the presence or absence of TC was quantified by flow cytometry. TaqMan low-density gene expression array determining known markers of phagocytosis and loss-of-function studies on selected apopto-phagocytic genes was carried out in HMDMs engulfing anoikic cells. The glucocorticoid TC had a profound phagocytosis-enhancing effect on HMDMs engulfing anoikic ARPE-19 or hRPE cells, causing a selective upregulation of the Mer tyrosine kinase (MERTK) receptor, while decreasing the expression of the AXL receptor tyrosine kinase and thrombospondin-1 (THSB-1). The key role of the MERTK could be demonstrated in HMDMs engulfing dying cells using gene silencing as well as blocking antibodies. Similar pathways were found upregulated in living ARPE-19 engulfing anoicic ARPE-19 cells. Gas6 treatment enhanced phagocytosis in TC treated HMDMs. Specific agonists of the Mertk receptor may have a potential role as phagocytosis enhancers in the retina and serve as future targets for AMD therapy. The use of Gas6 as enhancer of retinal phagocytosis via the MerTK receptor, alone or in combination with other specific ligands of the tyrosine kinase receptors' family may have a potential role in AMD therapy. Copyright © 2014. Published by Elsevier B.V.
 
Article
Common deletion (CD) 4977 bp of mitochondrial DNA (mtDNA) disrupt specifically mitochondrial complex I, IV and V on the electron transport chain (ETC) and is closely associated with wide spectrums of clinical manifestations. To quantitatively investigate how CD-induced ETC defect alters mitochondrial reactive oxygen species (mROS) generation as well as down stream apoptotic signaling, we employed an established array of human CD cytoplasmic hybrids (cybrids) harboring 0%-80% of CD. Pathological effects of CD on the mitochondria were visualized at single cell level by the application of fluorescent probes coupled with conventional and multiphoton imaging microscopy. Intriguingly, we observed CD-augmented mROS generation omitted "threshold effect". CD-augmented mROS generation was associated with depolarized mitochondrial membrane potential (DeltaPsi(m)). Upon oxidative stress, the amount of CD-augmented mROS generation was greatly enhanced to cause pathological apoptotic deterioration including opening of the mitochondrial permeability transition, cytochrome c release, phosphatidylserine externalization and DNA fragmentation. In addition, heterogeneous mitochondrial dysfunctions were found in cybrids containing 80% of CD (D cybrids), i.e., low sensitive-D (LS-D, roughly 80%) and a super sensitive-D (SS-D, 20%). As compared to LS-D, SS-D had higher resting mROS level but slightly hyperpolarized DeltaPsi(m). Upon H2O2 treatment, much faster generation of mROS was observed which induced a faster depolarization of DeltaPsi(m) and later apoptotic deterioration in SS-D. We proposed a dose-dependent, feed-forward and self-accelerating vicious cycle of mROS production might be initiated in CD-induced ETC defect without threshold effect. As CD-augmented mROS generation is obligated to cause an enhanced pathological apoptosis, precise detection of CD-augmented mROS generation and their degree of heterogeneity in single cells may serve as sensitive pathological indexes for early diagnosis, prognosis and treatment of CD-associated diseases.
 
Article
One of the primary plant mechanisms protecting leaf cells against enhanced atmospheric ozone is the accumulation of polyamines, generally observed as an increase in putrescine level, and in particular its bound form to thylakoid membranes. Ozone-sensitive plants of tobacco (cultivar Bel W3) in contrast to ozone-tolerant Bel B, are not able to increase their endogenous thylakoid membrane-bound putrescine when they are exposed to an atmosphere with enhanced ozone concentration, resulting in reduction of their photosynthetic rates and consequently reduction in plant biomass formation. In comparison to the tolerant cultivar Bel B, a prolongation of ozone exposure thus can lead to typical visible symptoms (necrotic spots) in leaves of the sensitive plant. Exogenously manipulated increase of the cellular putrescine levels of the ozone-sensitive Bel W3 is sufficient to revert these effects, whereas a reduction in endogenous putrescine levels of the tolerant cultivar Bel B renders them sensitive to ozone treatment. The results of this work reveal a regulator role for polyamines in adaptation of the photosynthetic apparatus and consequently to its protection in an environment polluted by ozone.
 
Primers used in this study
Article
Background Cytochrome bd oxidase, existing widely in bacteria, produces a proton motive force by the vectorial charge transfer of protons and more importantly, endows bacteria with a number of vitally important physiological functions, such as enhancing tolerance to various stresses. Although extensively studied as a CydA-CydB two-subunit complex for decades, the complex in certain groups of bacteria is recently found to in fact consist of an additional subunit, which is functionally essential. Methods We investigated the assembly of the CydA-CydB complex using BiFC. We investigated function of CydX using mutational analysis. Results and Conclusions CydX, a 38-amino-acid inner-membrane protein, is associated with the CydA-CydB complex in Shewanella oneidensis, a facultative anaerobe renowned for its respiratory versatility. It is clear that CydX is neither required for the in vivo assembly of the CydA-CydB complex nor relies on the complex for its translocation and integration into the membrane. The N-terminal segment (1-25 amino acid residues) and short periplasmic overhang of CydX, with respect to functionality, are important whereas the remaining C-terminal segment is rather flexible. Based on these findings, we postulate that CydX may function by positioning and stabilizing the prosthetic hemes, especially heme d in the CydA-CydB complex although a role of participating in catalytic reaction is not excluded. General Significance The work provides novel insights into our understanding of the small subunit of the cytochrome bd oxidase.
 
Article
The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins. We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods. All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)2 form represents the most favorable architecture for parasite P-proteins. The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1-P2 heterodimeric intermediate. On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role. Copyright © 2014 Elsevier B.V. All rights reserved.
 
Article
During protein folding reactions toward the native structure, short-lived intermediate states can be populated. Such intermediates expose hydrophobic patches and can self-associate leading to non-productive protein misfolding. A major focus of current research is the characterization of short-lived intermediates and how molecular chaperones enable productive folding. Real-time NMR spectroscopy, together with the development of advanced methods, is reviewed here and the potential these methods have to characterize intermediate states as well as interactions with molecular chaperone proteins at single-residue resolution is highlighted. Various chaperone interactions can guide the protein folding reaction and thus are important for protein structure formation, stability, and activity of their substrates. Chaperone-assisted protein folding, characterization of intermediates, and their molecular interactions using real-time NMR spectroscopy will be discussed. Additionally, recent advances in NMR methods employed for characterization of high-energy intermediates will be discussed. Real-time NMR combines high-resolution with kinetic information of protein reactions, which can be employed not only for protein folding studies and the characterization of folding intermediates but also to investigate the molecular mechanisms of assisted protein folding. Real-time NMR spectroscopy remains an effective tool to reveal structural details about the interaction between chaperones and transient intermediates. Methodologically, it provides in-depth understanding of how kinetic intermediates and their thermodynamics contribute to the protein folding reaction. This review summarizes the most recent advances in this field. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets. Copyright © 2014. Published by Elsevier B.V.
 
Article
Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available. Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry. The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues. Our results imply more specific functions and interactions between the proteins of this family than previously assumed. Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases-the laboratory mouse. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.
 
Article
Background: Atrial fibrillation (AF) is the most common arrhythmia in clinical practice and is a potential cause of thromboembolic events. AF induces significant changes in the electrophysiological properties of atrial myocytes and causes alterations in the structure, metabolism, and function of the atrial tissue. The molecular basis for the development of structural atrial remodeling of fibrillating human atria is still not fully understood. However, increased production of reactive oxygen or nitrogen species (ROS/RNS) and the activation of specific redox-sensitive signaling pathways observed both in patients with and animal models of AF are supposed to contribute to development, progression and self-perpetuation of AF. Scope of review: The present review summarizes the sources and targets of ROS/RNS in the setting of AF and focuses on key redox-sensitive signaling pathways that are implicated in the pathogenesis of AF and function either to aggravate or protect from disease. Major conclusions: NADPH oxidases and various mitochondrial monooxygenases are major sources of ROS during AF. Besides direct oxidative modification of e.g. ion channels and ion handling proteins that are crucially involved in action potential generation and duration, AF leads to the reversible activation of redox-sensitive signaling pathways mediated by activation of redox-regulated proteins including Nrf2, NF-κB, and CaMKII. Both processes are recognized to contribute to the formation of a substrate for AF and, thus, to increase AF inducibility and duration. General significance: AF is a prevalent disease and due to the current demographic developments its socio-economic relevance will further increase. Improving our understanding of the role that ROS and redox-related (patho)-mechanisms play in the development and progression of AF may allow the development of a targeted therapy for AF that surpasses the efficacy of previous general anti-oxidative strategies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.
 
Article
Background: The self-renewal ability and pluripotent differentiation potential of stem cells hold great promise for regenerative medicine. Many studies focus on the lineage-specific differentiation and expansion of stem cells, but little is known about the regulation of glycolysis and mitochondrial biogenesis and function during these processes. Recent studies have demonstrated a strong correlation between cellular metabolism and the pluripotency and differentiation potential of stem cells, which indicates the importance of bioenergetic function in the regulation of stem cell physiology. Scope of review: We summarize recent findings in the control of stem cell competence through the regulation of bioenergetic function in embryonic, hematopoietic, mesenchymal, and induced pluripotent stem cells, and discuss the up-to-date understanding of the molecular mechanisms involved in these biological processes. Major conclusions: It is believed that the metabolic signatures are highly correlated with the stemness status (high glycolytic flux) and differentiation potential (mitochondrial function) of stem cells. Besides, mitochondrial rejuvenation has been observed to participate in the reprogramming process. General significance: Understanding the metabolic regulation of stem cells will have great value in the characterization and isolation of stem cells with better differentiation potential. It also provides novel strategies of metabolic manipulation to increase the efficiency of cellular reprogramming. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.
 
Article
Molecular dynamics has emerged as an important research methodology covering systems to the level of millions of atoms. However, insufficient sampling often limits its application. The limitation is due to rough energy landscapes, with many local minima separated by high-energy barriers, which govern the biomolecular motion. In the past few decades methods have been developed that address the sampling problem, such as replica-exchange molecular dynamics, metadynamics and simulated annealing. Here we present an overview over theses sampling methods in an attempt to shed light on which should be selected depending on the type of system property studied. Enhanced sampling methods have been employed for a broad range of biological systems and the choice of a suitable method is connected to biological and physical characteristics of the system, in particular system size. While metadynamics and replica-exchange molecular dynamics are the most adopted sampling methods to study biomolecular dynamics, simulated annealing is well suited to characterize very flexible systems. The use of annealing methods for a long time was restricted to simulation of small proteins; however, a variant of the method, generalized simulated annealing, can be employed at a relatively low computational cost to large macromolecular complexes. Molecular dynamics trajectories frequently do not reach all relevant conformational substates, for example those connected with biological function, a problem that can be addressed by employing enhanced sampling algorithms. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014 Elsevier B.V. All rights reserved.
 
Article
Background: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch. Methods: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure. Results: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family. Conclusions: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family. General significance: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.
 
Article
The number of high-resolution structures of pharmacologically relevant membrane proteins has been strongly increasing. This makes computing relative affinities of chemically similar compounds binding to a membrane protein possible in order to guide decision making in drug design. However, the preparation step of such calculations is time-consuming and complex. We extended the free energy workflow tool FEW, available in AMBER, towards facilitating the setup of molecular dynamics simulations with explicit membrane, and the setup and execution of effective binding energy calculations according to a 1-trajectory implicit solvent / implicit membrane MM-PBSA approach for multiple ligands binding to the same membrane protein. We validated the implemented protocol initially on two model systems, a sodium ion in the presence of an implicit membrane slab and a proton traversing the M2 proton-channel of the influenza A virus. For the latter, we found a good agreement for several important events along the proton pathway with those obtained in a recent computational study. Finally, we performed a case study on effective binding energy calculations for a set of inhibitors binding to the M2 proton-channel. From the case study, we estimate a considerable speed up in the setup and analysis times for implicit solvent / implicit membrane MM-PBSA calculations by the extended version of FEW compared to a manual preparation. General Significance Together with the overall runtime and the analysis results, this suggests that such type of calculations can be valuable in later stages of drug design projects on membrane proteins. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.
 
Article
Somatic mutation in mitochondrial DNA (mtDNA) has been proposed to contribute to initiation and progression of human cancer. In our previous study, high frequency of somatic mutations was found in the D-loop region of mtDNA of gastric cancers. However, it is unclear whether somatic mutations occur in the coding region of mtDNA of gastric cancers. Using DNA sequencing, we studied 31 gastric cancer specimens and corresponding non-cancerous stomach tissues. Moreover, a human gastric cancer SC-M1 cell line was treated with oligomycin to induce mitochondrial dysfunction. Cisplatin sensitivity and cell migration were analyzed. We identified eight somatic mutations in the coding region of mtDNAs of seven gastric cancer samples (7/31, 22.6%). Patients with somatic mutations in the entire mtDNA of gastric cancers did not show significant association with their clinicopathologic features. Among the eight somatic mutations, five point mutations (G3697A, G4996A, G9986A, C12405T and T13015C) are homoplasmic and three mutations (5895delC, 7472insC and 12418insA) are heteroplasmic. Four (4/8, 50%) of these somatic mutations result in amino acid substitutions in the highly conserved regions of mtDNA, which potentially lead to mitochondrial dysfunction. In addition, in vitro experiments in SC-M1 cells revealed that oligomycin-induced mitochondrial dysfunction promoted resistance to cisplatin and enhanced cell migration. N-acetyl cysteine was effective in the prevention of the oligomycin-enhanced migration, which suggests that reactive oxygen species generated by defective mitochondria may be involved in the enhanced migration of SC-M1 cells. Our results suggest that somatic mtDNA mutations and mitochondrial dysfunction may play an important role in the malignant progression of gastric cancer.
 
Article
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected. We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity. The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acids 400-404. The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo. General Significance This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged. Copyright © 2014. Published by Elsevier B.V.
 
Article
Within cells, there is a narrow concentration threshold that governs whether reactive oxygen species (ROS) induce toxicity or act as second messengers. We discuss current understanding of how ROS arise, facilitate cell signaling, cause toxicities and disease related to abnormal cell differentiation and those (primarily) sulfur based pathways that provide nucleophilicity to offset these effects. Cellular redox homeostasis mediates a plethora of cellular pathways that determine life and death events. For example, ROS intersect with GSH based enzyme pathways to influence cell differentiation, a process integral to normal hematopoiesis, but also affecting a number of diverse cell differentiation related human diseases. Recent attempts to manage such pathologies have focused on intervening in some of these pathways, with the consequence that differentiation therapy targeting redox homeostasis has provided a platform for drug discovery and development. The balance between electrophilic oxidative stress and protective biomolecular nucleophiles predisposes the evolution of modern life forms. Imbalances of the two can produce aberrant redox homeostasis with resultant pathologies. Understanding the pathways involved provides opportunities to consider interventional strategies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2014. Published by Elsevier B.V.
 
Article
CFTR plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date. The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing RNAs. Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by the lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the JNK or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant NAC inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR. These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells. The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers. Copyright © 2015 Elsevier B.V. All rights reserved.
 
Top-cited authors
Shelly C Lu
  • Cedars-Sinai Medical Center
Fermon Gibson
  • City University of New York City - Lehman College
Tim Hardingham
  • The University of Manchester
Hiroshi Kitagawa
  • Kobe Pharmaceutical University
David J Ellar
  • University of Cambridge