Heme Regulatory Motifs in Heme Oxygenase-2 Form a Thiol/Disulfide Redox Switch That Responds to the Cellular Redox State

Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.Heme oxygenase (HO³ ; EC 1.14.99.3) catalyzes the O₂- and NADPH-dependent conversion of heme to biliverdin, carbon monoxide (CO), and iron in a reaction that is coupled to cytochrome P450 reductase. Then, biliverdin reductase catalyzes the NADPH-dependent reduction of biliverdin to the antioxidant bilirubin. Several recent reviews on HO (1–5) and biliverdin reductase (6) are available. HO is present in organisms from bacteria to eukaryotes and, as the only known enzyme that can degrade heme, plays a critical role in heme and iron homeostasis.There are two major HO isoforms in mammals: inducible HO-1, which is ancient and widely distributed among organisms from bacteria to man, and constitutively expressed HO-2, which emerged 250 million years ago with the amniotes (7). HO-1 is found in most tissues and is highly expressed in spleen and liver (8). Conversely, HO-2 has a narrow tissue distribution, exhibiting high expression levels in the brain, testes, and carotid body (8, 9). Both HO-1 and HO-2 catalyze the NADPH- and cytochrome P450 reductase-dependent degradation of heme to CO, iron, and biliverdin, which is quickly reduced to bilirubin in the presence of biliverdin reductase (10). Controlling cellular heme concentrations is crucial because heme is required as a prosthetic group by regulatory and redox proteins, yet concentrations higher than 1 μm free heme are toxic (11). Thus, as the only mammalian proteins known to degrade heme, HOs play a key role in cellular heme homeostasis; furthermore, in vitro and in vivo studies of cellular and tissue injuries, such as oxidative stress and hemin-induced cytotoxicity, indicate that HO is cytoprotective (9).HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12, 13); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys²⁶⁵ in HRM1 and Cys²⁸² in HRM2⁴ (12, 14) (Fig. 1). It was shown recently that the HRMs in HO-2 do not bind heme per se but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15).Open in a separate windowFIGURE 1.Major structural regions in HO-1 and HO-2. His²⁵ in HO-1 or His⁴⁵ in HO-2 is the heme-binding ligand.The HRM has been proposed to constitute a heme-binding site (16, 17) that regulates key metabolic processes from bacteria to humans. The HRM consists of a conserved Cys-Pro core sequence that is usually flanked at the N terminus by basic amino acids and at the C terminus by a hydrophobic residue. HRM/heme interactions have been proposed to regulate the activity and/or stability of proteins that play central roles in respiration and oxidative damage (18, 19), coordination of protein synthesis and heme availability in reticulocytes (20, 21), and controlling iron and heme homeostasis (22–26). An important component of the last process is HO-2.Here, we demonstrate that the C-terminal HRMs, which form a thiol/disulfide redox switch between Cys²⁶⁵ and Cys²⁸², exhibit a redox potential that falls well within the ambient cellular redox potential. By expressing HO-2 in bacterial and human cells and trapping the thiols using the isotope-coded affinity tag (ICAT) technique, it was shown that the redox state of the C-terminal HRMs in growing cells responds to the cellular redox state. The disulfide state is favored under oxidative conditions, and the dithiol state is predominant under reducing conditions. Thus, the HRMs act as a molecular rheostat that responds to the ambient intracellular redox potential and, based on earlier studies (14), controls activity of HO-2 by regulating heme binding to the enzyme.     

Prediction of Proteins Putatively Involved in the Thiol: Disulfide Redox Metabolism of a Bacterium (Listeria): The CXXC Motif as Query Sequence

Thiol:disulfide redox metabolism (TDRM) is a central metabolic network in all living cells. However, numerous proteins with different biochemical functions and several structural domains are involved, making it not trivial to identify and annotate its constituents in sequenced genomes. We developed an uncomplicated approach to solve the problem using existing web-based tools and public databases with the gram-positive bacterium Listeria monocytogenes EGD-e as a model organism. A pattern search for the Cys-Xaa-Xaa-Cys (CXXC) motif - a hallmark of TDRM proteins - in the genome sequence of the bacterium yielded 156 proteins. After initial refinement by protein and domain analysis, 14 candidate proteins remained. Subsequent detailed analyses, supported by modeling of 3D structures and data integration yielded 6 thioredoxin-like proteins plus thioredoxin reductase, glutaredoxin, one redox-sensitive regulator, one peptide methionine reductase - all typical TDRM constituents - and three putative novel components of the TDRM. For all 14 proteins orthologues were found in other Listeria species. Homology searches and phylogenetic analyses showed that related proteins are present mainly in other Firmicutes. This fast approach required minimal resources. It is immediately applicable to any genome with appropriate modifications and should be practicable also for other conserved, functionally important amino acid motifs.     

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca²⁺ and voltage-activated K⁺ channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca²⁺-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K_d = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K_d = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.     

The Guanine-Nucleotide Exchange Factor SGEF Plays a Crucial Role in the Formation of Atherosclerosis

The passage of leukocytes across the endothelium and into arterial walls is a critical step in the development of atherosclerosis. Previously, we showed in vitro that the RhoG guanine nucleotide exchange factor SGEF (Arhgef26) contributes to the formation of ICAM-1-induced endothelial docking structures that facilitate leukocyte transendothelial migration. To further explore the in vivo role of this protein during inflammation, we generated SGEF-deficient mice. When crossed with ApoE null mice and fed a Western diet, mice lacking SGEF showed a significant decrease in the formation of atherosclerosis in multiple aortic areas. A fluorescent biosensor revealed local activation of RhoG around bead-clustered ICAM-1 in mouse aortic endothelial cells. Notably, this activation was decreased in cells from SGEF-deficient aortas compared to wild type. In addition, scanning electron microscopy of intimal surfaces of SGEF^−/− mouse aortas revealed reduced docking structures around beads that were coated with ICAM-1 antibody. Similarly, under conditions of flow, these beads adhered less stably to the luminal surface of carotid arteries from SGEF ^−/− mice. Taken together, these results show for the first time that a Rho-GEF, namely SGEF, contributes to the formation of atherosclerosis by promoting endothelial docking structures and thereby retention of leukocytes at athero-prone sites of inflammation experiencing high shear flow. SGEF may therefore provide a novel therapeutic target for inhibiting the development of atherosclerosis.     

Phosphoenolpyruvate Carboxylase in Arabidopsis Leaves Plays a Crucial Role in Carbon and Nitrogen Metabolism

Phosphoenolpyruvate carboxylase (PEPC) is a crucial enzyme that catalyzes an irreversible primary metabolic reaction in plants. Previous studies have used transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition to examine the role of PEPC in carbon and nitrogen metabolism. To date, the in vivo role of PEPC in carbon and nitrogen metabolism has not been analyzed in plants. In this study, we examined the role of PEPC in plants, demonstrating that PPC1 and PPC2 were highly expressed genes encoding PEPC in Arabidopsis (Arabidopsis thaliana) leaves and that PPC1 and PPC2 accounted for approximately 93% of total PEPC activity in the leaves. A double mutant, ppc1/ppc2, was constructed that exhibited a severe growth-arrest phenotype. The ppc1/ppc2 mutant accumulated more starch and sucrose than wild-type plants when seedlings were grown under normal conditions. Physiological and metabolic analysis revealed that decreased PEPC activity in the ppc1/ppc2 mutant greatly reduced the synthesis of malate and citrate and severely suppressed ammonium assimilation. Furthermore, nitrate levels in the ppc1/ppc2 mutant were significantly lower than those in wild-type plants due to the suppression of ammonium assimilation. Interestingly, starch and sucrose accumulation could be prevented and nitrate levels could be maintained by supplying the ppc1/ppc2 mutant with exogenous malate and glutamate, suggesting that low nitrogen status resulted in the alteration of carbon metabolism and prompted the accumulation of starch and sucrose in the ppc1/ppc2 mutant. Our results demonstrate that PEPC in leaves plays a crucial role in modulating the balance of carbon and nitrogen metabolism in Arabidopsis.Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is a crucial enzyme that functions in primary metabolism by irreversibly catalyzing the conversion of phosphoenolpyruvate (PEP) and HCO₃⁻ to oxaloacetate (OAA) and inorganic phosphate. PEPC is found in all plants, green algae, and cyanobacteria, and in most archaea and nonphotosynthetic bacteria, but not in animals or fungi (Chollet et al., 1996; O’Leary et al., 2011a). Several isoforms of PEPC are present in plants, including plant-type PEPCs and one bacterium-type PEPC (Sánchez and Cejudo, 2003; Sullivan et al., 2004; Mamedov et al., 2005; Gennidakis et al., 2007; Igawa et al., 2010). Arabidopsis (Arabidopsis thaliana) possesses three plant-type PEPC genes, AtPPC1, AtPPC2, and AtPPC3, and one bacterium-type PEPC gene, AtPPC4. Unlike plant-type PEPCs, bacterium-type PEPCs lack a seryl-phosphorylation domain near the N terminus, a typical domain conserved in plant-type PEPCs (Sánchez and Cejudo, 2003). Plant-type PEPCs form class 1 PEPCs, which exist as homotetramers. Recently, bacterium-type PEPCs have been reported to interact with plant-type PEPCs to form heterooctameric class 2 PEPCs in several species, including unicellular green algae (Selenastrum minutum), lily (Lilium longiflorum), and castor bean (Ricinus communis; O’Leary et al., 2011a).Because of the irreversible nature of the enzymatic reactions catalyzed by PEPC isoforms, they are strictly regulated by a variety of mechanisms. PEPC is an allosteric enzyme and is activated by its positive effector, Glc-6-P, and inhibited by its negative effectors, malate, Asp, and Glu (O’Leary et al., 2011a). Control by reversible phosphorylation is another important mechanism that regulates the activity of PEPC. In this reaction, phosphorylation catalyzed by PEPC kinase changes the sensitivity of PEPC to its allosteric effectors (Nimmo, 2003). In addition, monoubiquitination may also regulate plant-type PEPC activity (Uhrig et al., 2008). Recent research in castor oil seeds suggests that bacterium-type PEPC is a catalytic and regulatory subunit of class 2 PEPCs, as class 1 and class 2 PEPCs show significant differences in their sensitivity to allosteric inhibitors (O’Leary et al., 2009, 2011b).A number of studies on PEPC function have been performed in a variety of organisms (O’Leary et al., 2011a). The best described function of PEPC is in fixing photosynthetic CO₂ during C4 and Crassulacean acid metabolism photosynthesis. However, in most nonphotosynthetic tissues and the photosynthetic tissues of C3 plants, the fundamental function of PEPC is to anaplerotically replenish tricarboxylic acid cycle intermediates (Chollet et al., 1996). PEPC also functions in malate production in guard cells and legume root nodules (Chollet et al., 1996). A chloroplast-located PEPC isoform in rice (Oryza sativa) was recently found to be crucial for ammonium assimilation (Masumoto et al., 2010). In addition, previous work in Arabidopsis suggested that AtPPC4 might play a role in drought tolerance (Sánchez et al., 2006).Transgenic plants expressing ectopic PEPC forms with diminished feedback inhibition showed an increase in overall organic nitrogen content at the expense of starch and soluble sugars (Rademacher et al., 2002; Chen et al., 2004; Rolletschek et al., 2004). However, the in vivo function of PEPC in carbon and nitrogen metabolism has not been reported previously.To further investigate the function of PEPC in higher plants, we isolated and characterized mutants of Arabidopsis deficient in the expression of the PEPC-encoding genes PPC1 and PPC2. We demonstrated that PPC1 and PPC2 were the most highly expressed PEPC genes in the leaves. To further define their role, we produced a double mutant (ppc1/ppc2) deficient in the expression of the PPC1 and PPC2 genes. We then conducted a detailed molecular, biochemical, and physiological characterization of this double mutant.     

Topographic Heterogeneity Plays a Crucial Role for Grasshopper Diversity in a Southern African Megabiodiversity Hotspot

Topographic heterogeneity as a determinant of insect diversity pattern has been little studied. Responses of grasshopper assemblages to three hill sizes were assessed in the arid Succulent Karoo, South Africa. This area is one of the world’s 25 hotspots for conservation priorities. Small hills overall were more speciose than medium or large hills. There were also significantly higher densities of small-sized grasshoppers on small hills than on medium or large ones. The slopes of the three hill sizes did not differ significantly either in their species richness or abundance, and there was no significant difference in species richness between summits only of the three hill sizes. Patterns of grasshopper species dominance were markedly variable among sites, but with clear differences between small and larger hills, associated with vegetation characteristics. Vegetation cover and grass cover was less on the small hills. Grasshopper taxonomic groups varied among the three hill sizes, with small hills being taxonomically more diverse, supporting species from four families and nine subfamilies, while medium and large hills only supported Acrididae. It is concluded that topography has a remarkably strong effect on various aspects of grasshopper spatial heterogeneity and that small hills in particular are a major factor to consider in spatial conservation planning.     

Pyruvate Carboxylase Plays a Crucial Role in Carbon Metabolism of Extra- and Intracellularly Replicating Listeria monocytogenes

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the ¹³C isotopologue perturbation method in a defined minimal medium containing [U-¹³C₆]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-¹³C₆]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.Listeria monocytogenes is a human pathogen that can cause systemic infections, especially in immunocompromised people, with symptoms such as septicemia, (encephalo)meningitis, placentitis, and stillbirth. These Gram-positive bacteria are able to enter the cytosol of many mammalian cells after being taken up via normal or induced phagocytosis by professional phagocytes, mainly macrophages and dendritic cells, and nonphagocytic cells, such as epithelial cells, fibroblasts, and endothelial cells (1, 8, 13). While the virulence genes and their regulation (4, 21), as well as the encoded virulence factors (20, 22), necessary for the various steps of the intracellular replication cycle of L. monocytogenes have been extensively studied in the past few decades, there is still little information concerning the metabolic capacities and the metabolic adaptation processes (10) that enable these bacteria to efficiently replicate in the cytosol of their host cells.The information on listerial metabolism obtained from the genome sequence (7) suggests that these heterotrophic bacteria are capable of utilizing a variety of carbohydrates as carbon sources, since a large number of genes encoding phosphoenolpyruvate (PEP)-phosphotransferase systems (PTS) were identified. Furthermore, all genes encoding the enzymes necessary for the catabolism of glycerol and dihydroxyacetone are present in the L. monocytogenes genome (7, 11). This genomic information is in accord with data from previous and more recent physiological studies (11, 17, 24).Most genes encoding the enzymes for the major catabolic pathways, namely, glycolysis, the citrate cycle, and the pentose phosphate cycle, are present in L. monocytogenes. The citrate cycle, however, seems to be interrupted, since the genes encoding 2-oxoglutarate dehydrogenase have not been identified in all L. monocytogenes strains sequenced so far, including EGD-e (7), or in Listeria innocua strain Clip 11262. This enzymatic gap in the citrate cycle was recently confirmed by ¹³C isotopologue perturbation studies using uniformly ¹³C-labeled glucose. The results showed that two C₄ amino acids, aspartate and threonine, are generated in L. monocytogenes, predominantly from building blocks comprising one or three ¹³C atoms, respectively (2). These data suggested that oxaloacetate, the direct or indirect precursor of both amino acids, is generated by an anaplerotic reaction assembling precursors composed of one and three carbon atoms, respectively. This can be afforded by the carboxylation of pyruvate catalyzed by the ATP-dependent pyruvate carboxylase (PYC) encoded by pycA.The genes encoding the enzymes for most anabolic pathways, but not those for the biosynthesis of thiamine (vitamin B₁), riboflavin (vitamin B₂), biotin, and thiotic acid (lipoate), were also identified in L. monocytogenes. However, these bacteria grow efficiently in a mineral salt medium containing a suitable carbon source (e.g., glucose) and these four cofactors only when the amino acids cysteine, methionine, glutamine, arginine, valine, isoleucine, and leucine are also added (17). According to Tsai and Hodgson, strain 10403S requires only methionine and cysteine (24). The missing sulfate reductase in L. monocytogenes readily explains the strict requirement for cysteine/methionine as a sulfur source, while the missing nitrate reductase may be the reason for the stimulatory growth effect of glutamine and arginine as reduced nitrogen sources. However, the need for the three branched-chain amino acids (BCAA) valine, isoleucine, and leucine for efficient growth of L. monocytogenes EGD-e (references 17 and 24 and our unpublished results) is less obvious, since L. monocytogenes has the complete genetic set for synthesis of the BCAA, indicating the role of metabolic intermediates in listerial growth.The central precursor for the biosynthesis of the BCAA is pyruvate, which is channeled into their biosynthetic pathways either directly, via oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), or more indirectly via oxaloacetate (generated by pyruvate carboxylation) to aspartate and further to threonine. Thus, biosynthesis of the BCAA may compete with the PYC-mediated generation of oxaloacetate for the common substrate pyruvate. These data suggest that PYC may play an important role in the carbon metabolism of L. monocytogenes.To more precisely determine the significance of this anaplerotic enzyme for listerial metabolism and pathogenesis, we generated a mutant of L. monocytogenes EGD-e defective in pycA, the gene encoding PYC, and studied the replication of this mutant under different extra- and intracellular growth conditions. The results show that PYC indeed plays a crucial role in the intracellular replication of L. monocytogenes and hence in the infection process.     

Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus

The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.     

Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA⁺ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA⁺ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA⁺ bacteria exposed to ionizing radiation.     

The C-Terminal Portion of an Archaeal Toxin,aRelE, Plays a Crucial Role in Protein Synthesis Inhibition

Mutations of amino acids in the C-terminal region of an archaeal toxin, aRelE, from Pyrococcus horikoshii were characterized with respect to protein synthesis inhibitory activity and 70S ribosome-binding activity. The results suggest that basic residues at the C-terminal region in aRelE play a crucial role both in 70S ribosome binding and in protein synthesis inhibition activities.     

Distal C Terminus of CaV1.2 Channels Plays a Crucial Role in the Neural Differentiation of Dental Pulp Stem Cells

L-type voltage-dependent Ca_V1.2 channels play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. C-terminal cleavage of Ca_V1.2 channels was reported in several types of excitable cells, but its expression and possible roles in non-excitable cells is still not clear. The aim of this study was to determine whether distal C-terminal fragment of Ca_V1.2 channels is present in rat dental pulp stem cells and its possible role in the neural differentiation of rat dental pulp stem cells. We generated stable Ca_V1.2 knockdown cells via short hairpin RNA (shRNA). Rat dental pulp stem cells with deleted distal C-terminal of Ca_V1.2 channels lost the potential of differentiation to neural cells. Re-expression of distal C-terminal of Ca_V1.2 rescued the effect of knocking down the endogenous Ca_V1.2 on the neural differentiation of rat dental pulp stem cells, indicating that the distal C-terminal of Ca_V1.2 is required for neural differentiation of rat dental pulp stem cells. These results provide new insights into the role of voltage-gated Ca²⁺ channels in stem cells during differentiation.     

Drosophila Dyrk2 Plays a Role in the Development of the Visual System

The DYRKs (dual-specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that are associated with a number of neurological disorders, but whose biological targets are poorly understood. Drosophila encodes three Dyrks: minibrain/Dyrk1A, DmDyrk2, and DmDyrk3. Here we describe the creation and characterization of a DmDyrk2 null allele, DmDyrk2^1w17. We provide evidence that the smell impaired allele smi35A¹, is likely to encode DmDyrk2. We also demonstrate that DmDyrk2 is expressed late in the developing third antennal segment, an anatomical structure associated with smell. In addition, we find that DmDyrk2 is expressed in the morphogenetic furrow of the developing eye, that loss of DmDyrk2 in the eye produced a subtle but measurable defect, and that ectopic DmDyrk2 expression in the eye produced a strong rough eye phenotype characterized by increased secondary, tertiary and bristle interommatidial cells. This phenotype was dependent on DmDyrk2 kinase activity and was only manifest when expressed in post-mitotic non-neuronal progenitors. Together, these data indicate that DmDyrk2 is expressed in developing sensory systems, that it is required for the development of the visual system, and that the eye is a good model to identify DmDyrk2 targets.     

Modulation of Macrophage Polarization and HMGB1-TLR2/TLR4 Cascade Plays a Crucial Role for Cardiac Remodeling in Senescence-Accelerated Prone Mice

The aim of this study was to investigate the role of macrophage polarization in aging heart. Macrophage differentiation is pathogenically linked to many inflammatory and immune disorders. It is often preceded by myocardial inflammation, which is characterized by increased cardiac damage and pro-inflammatory cytokine levels. Therefore, we investigated the hypothesis that senescence accelerated-prone (SAMP8) mice cardiac tissue would develop macrophage polarization compared with senescence-resistant control (SAMR1) mice. Both SAMP8 and SAMR1 mice were sacrificed when they became six month old. We evaluated, histo-pathological changes and modifications in protein expression by Western blotting and immuno-histochemical staining for M1 and M2 macrophage markers, high mobility group protein (HMG)B1 and its cascade proteins, pro-inflammatory factors and inflammatory cytokines in cardiac tissue. We observed significant upregulation of HMGB1, toll-like receptor (TLR)2, TLR4, nuclear factor (NF)κB p65, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interferon (IFN)γ, interleukin (IL)-1β, IL-6 and M1 like macrophage specific marker cluster of differentiation (CD)68 expressions in SAMP8 heart. In contrast, M2 macrophage specific marker CD36, and IL-10 expressions were down-regulated in SAMP8 mice. The results from the study demonstrated that, HMGB1-TLR2/TLR4 signaling cascade and induction of phenotypic switching to M1 macrophage polarization in SAMP8 mice heart would be one of the possible reasons behind the cardiac dysfunction and thus it could become an important therapeutic target to improve the age related cardiac dysfunction.     

Intersubunit Disulfide Interactions Play a Critical Role in Maintaining the Thermostability of Glucose-6-phosphate Dehydrogenase from the Hyperthermophilic Bacterium Aquifex aeolicus

Proteins from thermophilic microorganisms are stabilized by various mechanisms to preserve their native folded states at higher temperatures. A thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus was expressed as a recombinant protein in Escherichia coli. The A. aeolicus G6PDH is a homodimer exhibiting remarkable thermostability (t_1/2=24 hr at 90°C). Based on homology modeling and upon comparison of its structure with human G6PDH, it was predicted that cysteine 184 of one subunit could form a disulfide bond with cysteine 352 of the other subunit resulting in reinforced intersubunit interactions that hold the dimer together. Site-directed mutagenesis was performed on tG6PDH to convert C184 and C352 to serines. The tG6PDH double mutant exhibited a dramatic decrease in the half-life from 24 hr to 3 hr at 90°C. The same decrease in half-life was also found when either C184 or C352 was mutated to serine. The result indicates that C184 and C352 may play a crucial role in strengthening the dimer interface through disulfide bond formation, thereby contributing to the thermal stability of the enzyme.     

Eicosapentaenoic Acid Plays a Beneficial Role in Membrane Organization and Cell Division of a Cold-Adapted Bacterium, Shewanella livingstonensis Ac10

Shewanella livingstonensis Ac10, a psychrotrophic gram-negative bacterium isolated from Antarctic seawater, produces eicosapentaenoic acid (EPA) as a component of phospholipids at low temperatures. EPA constitutes about 5% of the total fatty acids of cells grown at 4°C. We found that five genes, termed orf2, orf5, orf6, orf7, and orf8, are specifically required for the synthesis of EPA by targeted disruption of the respective genes. The mutants lacking EPA showed significant growth retardation at 4°C but not at 18°C. Supplementation of a synthetic phosphatidylethanolamine that contained EPA at the sn-2 position complemented the growth defect. The EPA-less mutant became filamentous, and multiple nucleoids were observed in a single cell at 4°C, indicating that the mutant has a defect in cell division. Electron microscopy of the cells by high-pressure freezing and freeze-substitution revealed abnormal intracellular membranes in the EPA-less mutant at 4°C. We also found that the amounts of several membrane proteins were affected by the depletion of EPA. While polyunsaturated fatty acids are often considered to increase the fluidity of the hydrophobic membrane core, diffusion of a small hydrophobic molecule, pyrene, in the cell membranes and large unilamellar vesicles prepared from the lipid extracts was very similar between the EPA-less mutant and the parental strain. These results suggest that EPA in S. livingstonensis Ac10 is not required for bulk bilayer fluidity but plays a beneficial role in membrane organization and cell division at low temperatures, possibly through specific interaction between EPA and proteins involved in these cellular processes.