Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

NMR assignments of the DNA binding domain of Ml4 protein from Mesorhizobium loti

Ml4 protein from Mesorhizobium loti has a 58% sequence identity with the Ros protein from Agrobacterium tumefaciens that contains a prokaryotic Cys₂His₂ zinc finger domain. Interestingly, Ml4 is a zinc-lacking protein that does not contain the Cys₂His₂ motif and is able to bind the Ros DNA target sequence with high affinity. Here we report the ¹H, ¹⁵N and ¹³C NMR assignments of the Ml4 protein DNA binding domain (residue 52–151), as an important step toward elucidating at a molecular level how this prokaryotic domain can overcome the metal requirement for proper folding and DNA-binding activity.     

Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix

Antisera from some hamsters bearing adenovirus-induced tumors contain antibodies to an 11,000 M_r adenovirus-induced protein. In adenovirus-infected HeLa cells, this early viral protein was specifically associated with the nuclear matrix fraction. After two-dimensional gel electrophoresis, two forms of the 11,000 M_r protein at pI 5.6 and pI 5.4 were found. Only the pI 5.4 form of this protein was associated with the nuclear matrix fraction. Adenoviruses from groups A, B, C, D and E all produced an early viral protein (10,000 to 12,000 M_r) that reacted with group C antibody to the 11,000 M_r protein. To date, this is the only known early viral protein that is immunologically conserved in all of the human adenovirus groups.The positions of two methionine and seven leucine residues were determined by sequencing the first 35 amino acids from the N terminus of the adenovirus serotype 2 group C 11,000 M_r protein. The positions of these amino acid residues were compared to the adenovirus serotype 2 nucleotide sequence, which uniquely localized the structural gene of the 11,000 M_r protein to region E4, subregion 3 in type 2 adenovirus. A frameshift mutant, which contained a deletion of one base-pair in the structural gene of the 11,000 M_r protein, was isolated and mapped by marker rescue and nucleotide sequence analysis. This mutant failed to produce immunologically detectable 11,000 M_r protein. The mutant had a viable phenotype, producing normal levels of infectious virus in both HeLa cells and WI38 cells in culture. These experiments identify the first adenovirus early region 4 protein detected in virus-infected cells.     

Translational control of hepatic alpha2u globulin synthesis by growth hormone.

α_2u Globulin is a male rat liver protein under complex hormonal control which represents approximately 1% of hepatic protein synthesis in an adult male rat. Hypophysectomy completely abolishes the hepatic synthesis of this protein, and the reinduction of its synthesis can be effected by the simultaneous administration of glucocorticoid, androgen, thyroid hormone and pituitary growth hormone. Growth hormone is absolutely required for the synthesis of a normal level of α_2u globulin. It was found, however, that hepatic α_2u globulin mRNA can be raised to a normal level in hypophysectomized animals by administration of steroids and thyroid hormone alone; nevertheless, no detectable synthesis of the protein occurs in these animals. Administration of growth hormone to the hypophysectomized animals that had been pretreated with steroids and thyroid hormone results in the immediate synthesis of α_2u globulin protein with very little change in the level of α_2u globulin mRNA. In an intact male animal, α_2u globulin mRNA sequences are found to be preferentially associated with bound polysomes. By contrast, the untranslated α_2u globulin mRNA sequences that accumulate in the livers of hypophysectomized rats treated with steroids and thyroid hormone are found on free polysomes and in the supernatant (nonpolysomal) fractions. Administration of growth hormone to these animals effects a shift of α_2u globulin mRNA sequences to bound polysomes, concurrent with the induction of detectable synthesis of the protein. This effect of growth hormone in vivo can be mimicked by administration of high doses of insulin, indicating that this effect may be somatomedin-mediated. It thus appears that growth hormone induces hepatic α_2u globulin synthesis by way of a translational control mechanism.     

Properties of a peripheral 34 kDa protein in Synechococcus vulcanus Photosystem II particles. Its exchangeability with spinach 33 kDa protein in reconstitution of O2 evolution

Photosystem II (PS II) particles retaining a high rate of O₂ evolution were prepared from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland, and the composition and properties of their peripheral proteins were investigated. The following results were obtained. (1) The O₂-evolving PS II particles of S. vulcanus contained only one peripheral protein with a molecular mass of 34000 which corresponded to the 33 kDa protein in higher plant PS II particles, but no other peripheral proteins corresponding to the 24 and 16 kDa proteins of higher plant PS II particles. (2) The cyanobacterial peripheral 34 kDa protein was removed from the particles by 1 M CaCl₂-washing concomitant with total inactivation of O₂ evolution, and the inactivated O₂ evolution was reconstituted to 75% of the original activity by rebinding of this protein back to the washed particles. (3) The cyanobacterial peripheral 34 kDa protein rebound to CaCl₂-washed spinach PS II particles and restored O₂ evolution to an appreciable extent (28%). (4) The spinach peripheral 33 kDa protein rebound to CaCl₂-washed PS II particles of S. vulcanus and partially restored O₂ evolution (60%). These results suggested that the peripheral 34 kDa protein of S. vulcanus possesses the determinants for both binding and activity reconstitution identical with those of the peripheral 33 kDa protein of spinach.     

Mixed and uniform co-operativity of ligand binding to multisite proteins: the co-operativity types allowed by the adair equation and conditions for them

It is proved for the first time that the macroscopic co-operativity of binding to a protein with q binding sites may change signs over a single binding curve any number of times from 0 to (q-2), but no more than (q-2). n changes of sign of macroscopic co-operativity requires as a necessary condition at least n changes of sign of microscopic co-operativity, but this necessary condition is not a sufficient one. The necessary and sufficient condition that decides whether there are two changes of sign in a four-site protein is obtained. There are no changes when K₁K₃(K₂-K₄)+K₁K₂(K₃-K₂)+K₂K₃(K₄-K₃) is positive, and two changes when it is negative, presuming the above mentioned necessary conditions to be satisfied. The K's of this formula are the “intrinsic” per-site Adair constants. As a result, the conditions for all six co-operativity types possible with a four-site protein are now known.     

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of ?82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of ?110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O₂. The spectra of the CO and O₂ complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O₂-binding protein in C. vinosum are discussed.     

Yeast AEP3p Is an Accessory Factor in Initiation of Mitochondrial Translation

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA_f^Met). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA_f^Met, as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA_f^Met to translation initiation factor 2 (IF2). We hypothesize the existence of an accessory factor that assists mitochondrial IF2 (mIF2) in utilizing unformylated Met-tRNA_f^Met. This accessory factor might be unnecessary when formylated Met-tRNA_f^Met is present but becomes essential when only the unformylated species are available. Using a synthetic petite genetic screen in yeast, we identified a mutation in the AEP3 gene that caused a synthetic respiratory-defective phenotype together with Δfmt1. The same aep3 mutation also caused a synthetic respiratory defect in cells lacking formylated Met-tRNA_f^Met due to loss of the MIS1 gene that encodes the mitochondrial C₁-tetrahydrofolate synthase. The AEP3 gene encodes a peripheral mitochondrial inner membrane protein that stabilizes mitochondrially encoded ATP6/8 mRNA. Here we show that the AEP3 protein (Aep3p) physically interacts with yeast mIF2 both in vitro and in vivo and promotes the binding of unformylated initiator tRNA to yeast mIF2. We propose that Aep3p functions as an accessory initiation factor in mitochondrial protein synthesis.     

Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO₂-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO₂. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO₂ conditions. The activity of inorganic carbon (C_i) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO₂ was the main species of C_i transported and the activities of CO₂ and HCO₃⁻ transport increased when high CO₂-grown cells were exposed to low CO₂. We conclude that the 42-kilodalton protein is not directly involved in the C_i-accumulating mechanism of Synechococcus PCC 7942.     

The trafficking protein Tmed2/p24β1 is required for morphogenesis of the mouse embryo and placenta

During vesicular transport between the endoplasmic reticulum and the Golgi, members of the TMED/p24 protein family form hetero-oligomeric complexes that facilitate protein-cargo recognition as well as vesicle budding. In addition, they regulate each other's level of expression. Despite analyses of TMED/p24 protein distribution in mammalian cells, yeast, and C. elegans, little is known about the role of this family in vertebrate embryogenesis. We report the presence of a single point mutation in Tmed2/p24β₁ in a mutant mouse line, 99J, identified in an ENU mutagenesis screen for recessive developmental abnormalities. This mutation does not affect Tmed2/p24β₁ mRNA levels but results in loss of TMED2/p24β₁ protein. Prior to death at mid-gestation, 99J homozygous mutant embryos exhibit developmental delay, abnormal rostral-caudal elongation, randomized heart looping, and absence of the labyrinth layer of the placenta. We find that Tmed2/p24β₁ is normally expressed in tissues showing morphological defects in 99J mutant embryos and that these affected tissues lack the TMED2/p24β₁ oligomerization partners, TMED7/p24γ₃ and TMED10/p24δ₁. Our data reveal a requirement for TMED2/p24β₁ protein in the morphogenesis of the mouse embryo and placenta.     

A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa₂CysXaa₂₀CysXaa₂Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation     

Identification and characterization of PP2C phosphatase SjPtc1 in Schistosoma japonicum

Protein phosphorylation, regulated by protein kinases and protein phosphatases, is crucial for protein structure and function in eukaryotic organisms. Type 2C protein phosphatase (PP2C) belongs to the serine/threonine phosphatase family and its activities require the presence of a divalent magnesium or manganese ion. In the present study, a potential PP2C phosphatase (SjPtc1) was identified in Schistosoma japonicum. The SjPTC1 gene was found to be highly expressed in adult worms. A recombinant SjPtc1 protein showed typical PP2C phosphatase activity. Heterologous SjPTC1 expression reversed the sensitivity of yeast ptc1 null mutants toward H₂O₂, ZnCl₂, cisplatin, and rapamycin. Collectively, the results suggest that SjPtc1 may take part in the regulation of cellular responses to oxidative stress, DNA damage stress, and the TOR (target of rapamycin) signaling pathway.     

Physiological and antioxidant enzyme responses to acute and chronic exposure of Laeonereis acuta (Polychaeta, Nereididae) to copper

Chronic (14 days) and acute (48 h) copper effects on the antioxidant defense system and some physiological variables of Laeonereis acuta (Polychaeta, Nereididae) were evaluated. In both assays, two nominal copper concentrations (chronic: C₁=31.25 and C₂=62.50 μg/l; acute: A₁=250 and A₂=500 μg/l) and one control group (C_c and A_c=0 μg/l) were tested. End points analyzed were antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD; and glutathione S-transferase, GST), oxygen consumption, metahemoglobin concentration, and lipid peroxidation (LPO). In the chronic assay, CAT activity was significantly higher in worms exposed to both concentrations of copper tested (C₁=3.36±0.07 U CAT/mg protein; C₂=4.06 0.32 U CAT/mg protein) than in control worms (C_c=2.16±0.39 U CAT/mg protein). SOD activity was also increased in the two copper-exposed groups (C₁=16.85±4.22 U SOD/mg protein; C₂=38.19±4.31 U SOD/mg protein) than in control group (C_c=3.54±0.46 U SOD/mg protein). However, GST activity was increased only in worms exposed to the higher copper concentration (C₂=0.022±9.10⁻⁴ U GST/mg protein) when compared to the other groups tested (C_c=0.012±3.10⁻³ U GST/mg protein; C₁=0.016±9.10⁻⁴ U GST/mg protein). None of the physiological variables analyzed (oxygen consumption, metahemoglobin concentration, and lipid peroxidation) was affected by chronic copper exposure. In the acute assay, only GST activity was induced in worms exposed to copper. This induction was observed only in the A₁ group (0.027±2.10⁻³ U GST/mg protein) when compared to A_c (0.017±2.10⁻³ U GST/mg protein) or A₂ (0.016±7.10⁻⁴ U GST/mg protein) groups. On the other hand, lipid peroxidation was higher in A₂ (481.9±49.2 nmol CHP/g ww) than in control worms (A_c=337.9±25.0 nmol CHP/g ww). Oxygen consumption was higher in worms acutely exposed to the lower copper concentration tested (A₁=0.27±0.04 mg O₂/g ww/h) than in the higher concentration (A₂=0.14±0.01 mg O₂/g ww/h). Changes in the swimming behavior of copper-exposed animals in both assays and edemas in the body wall of worms acutely exposed to copper were also observed. Results suggest that copper exposure favors reactive oxygen species generation and that enzymatic defense system is induced under chronic exposure, preventing oxygen consumption changes and lipid peroxidation and metahemoglobin formation. However, in acutely exposed worms, in spite of a transient peak of GST activity, no induction of antioxidant enzymes occurs, leading to morphological and physiological changes.     

Specificity of nucleotide binding sites in isolated chloroplast coupling factor (CF1)

The binding of various nucleotides to chloroplast coupling factor CF₁ was studied by two dialysis techniques. It was found that the number of nucleoside diphosphate sites and their specificities for the base moiety is dependent on the magnesium concentration. In the presence of 50 μM added MgCl₂, the protein has a single strong site/mol protein with K_d = 0.5 μM for ADP and high specificity (K_d > 20 μM for ?ADP, GDP, CDP). In the presence of 5 mM MgCl₂, the protein has two independent tight ADP sites (K_d = 0.4 μM) of low specificity (K_d ≈ 0.8, 2, and 2 μrmM, respectively for ?ADP, GDP, and CDP). These results are compared with the specificity of the partial reactions for photophosphorylation.     

Myocardial KChIP2 Expression in Guinea Pig Resolves an Expanded Electrophysiologic Role

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K⁺ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K⁺ channel (I_to), is a Ca²⁺ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate I_to. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to I_to, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as I_Kr and I_Ks were unchanged, but was the result of enhanced L-type Ca²⁺ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered I_Na from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current I_to may mask more subtle effects of KChIP2. Evaluation in the guinea pig where I_to is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of I_to, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization.     

Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O₂⁻) production after a lag time. The rank order of efficiency of these PAs in triggering O₂⁻ production was PA_8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)]>PA_10:0 (1,2-didecanoyl-GP)>PA_6:0 (1,2-dicaproyl-GP)≫PA_12:0 (1,2-dilauroyl-GP). The O₂⁻ release from PMN stimulated with PA_10:0 or PA_12:0, but not with PA_6:0 or PA_8:0, was lowered by the addition of 1 mM extracellular Ca²⁺. Studies with various inhibitors showed that the mechanism of multiphasic O₂⁻ production induced by PA_8:0 depended on its concentration: 1 and 3 μM PA_8:0 induced O₂⁻ production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 μM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 μM TMB-8. Concentrations of 30, 60 and 100 μM PA first elicited transient O₂⁻ production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O₂⁻ production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [¹⁴C]PA_8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O₂⁻ production via three different mechanisms described above.