Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2

We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes

Of the three major classes of ligand-gated ion channels, nicotinic receptors and ionotropic glutamate receptors are known to be organized as pentamers and tetramers, respectively. The architecture of the third class, P2X receptors, is under debate, although evidence for a trimeric assembly is accumulating. Here we provide biochemical evidence that in addition to the rapidly desensitising P2X1 and P2X3 receptors, the slowly desensitising subtypes P2X2, P2X4, and P2X5 are trimers of identical subunits. Similar (heteromeric) P2X subunits also formed trimers, as shown for co-expressed P2X1 and P2X2 subunits, which assembled efficiently to a P2X1+2 receptor that was exported to the plasma membrane. In contrast, P2X6 subunits, which are incapable of forming functional homomeric channels in Xenopus oocytes, were retained in the ER as apparent tetramers and high molecular mass aggregates. Altogether, we conclude from these data that a trimeric architecture is the structural hallmark of functional homomeric and heteromeric P2X receptors.     

Synthesis and antimetabolite properties of 5-substituted 2'-deoxyuridines. Anomeric 5-(2-aminohexafluoroprop-2-yl)- and 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine

Alkylation of 2,4-bis-O-(trimethylsilyl)uracil with hexafluoroacetone trifluoroacetylimine gave 5-(2-trifluoroacelylaminohexafluoroprop-2-yl)uracil, which was transformed by alkaline hydrolysis to 5-(2-aminohexafluoroprop-2-yl)uracil. The latter was glycosytated with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-ribofyranosyl chloride by means of various modifications of the silyl method leading to the predominant formation of beta-deoxynucleoside; after deacylation 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil was obtained. Interaction of silylated 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)uracil with acylgalogenose gave anomeric O-substitutet deoxynucleosides, which were deblocked to give 5-(2-trifluoroacetylaminohexafluoroprop-2-yl)-2'-deoxyuridine and corresponding alpha-anomer. Alkaline hydrolysis of N-trifluoroacetyl group in both individual anomers produced 1-(2-deoxy-alpha-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ur acil and the abovementioned beta-anomer. Of all compounds synthesised only 1-(2-deoxy-beta-D-ribofuranosyl)-5-(2-aminohexafluoroprop-2-yl)ura cil has a moderate inhibitory effect on replication of vaccinia virus in vitro.     

GTPase-deficient G alpha i2 oncogene gip2 inhibits adenylylcyclase and attenuates receptor-stimulated phospholipase A2 activity

The GTPase activity of a G protein alpha subunit functions as a timer to control the lifetime of the activated conformation of the protein. Expression of the GTPase-deficient Gi2 alpha subunit oncogene, gip2 (alpha i2Q205L), in Chinese hamster ovary cells inhibited the stimulation of adenylylcyclase and altered the calcium regulation of the Gi2-phospholipase A2 (PLA2) effector complex. The phenotypic consequence of the activated alpha i2 mutant on hormonal stimulation of PLA2 varied depending on the cytoplasmic calcium transient elicited by different Gi2-linked receptors. The stimulation of PLA2 by thrombin, which mobilized calcium only from internal stores, was markedly attenuated in gip2-expressing cells. In contrast, the attenuation of the PLA2 response to ATP, a purinergic agonist which mobilizes calcium from both extracellular space and internal stores, was significantly less than that observed for thrombin. Ionomycin, a calcium ionophore, stimulated PLA2 activity in clones which expressed gip2 to a level similar to that observed in wild-type Chinese hamster ovary cells. Thus, the dominant GTPase-deficient gip2 polypeptide will constitutively inhibit adenylylcyclase but differentially modulate enzymes regulated by calcium and coupled to Gi2.     

Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2.

The manganese peroxidase produced by Phanerochaete chrysosporium, which catalyzes the oxidation of Mn(2+) to Mn(3+), is easily inactivated by the hydrogen peroxide (H2O2) presented in the reaction. We attempted to increase H2O2 resistance by the conformational stabilization around the H2O2-binding pocket. Based on its structural model, engineering of oxidizable Met273 located near the pocket to a non-oxidizable Leu showed a great improvement. Furthermore, after treatment at 1 mM H2O2 where the wild-type is completely inactivated, full activity can be retained by engineering the Asn81, which might have conformational changes due to the environment of the pocket, to a non-bulky and non-oxidizable Ser.     

Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae

Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Short synthesis of 2-methoxyestradiol and 2-hydroxyestradiol

The estrogen metabolite 2-methoxyestradiol was synthesized from estradiol bis-THP-ether which was 2-hydroxylated using the superbase LIDAKOR, trimethyl borate, and H(2)O(2), then methylated and deprotected to obtain 2-methoxyestradiol in three steps and 61% yield. 2-Hydroxyestradiol was obtained by deprotecting the 2-hydroxyestradiol bis-THP-ether from the first step.     

Computational determination of binding modes of 2-acetoxyphenylhept-2-ynyl sulfide to cyclooxygenase-2

Abstract

2-Acetoxyphenylhept-2-ynyl sulfide (APHS) is a potent covalent inhibitor with cyclooxygenase-2 (COX-2) selectivity. However, no crystal structure for APHS?COX-2 complex has been reported. In this work, we have extensively studied the binding modes and interactions between APHS and COX-2. Molecular docking followed by MD simulations identified a stable and reactive binding mode, of which the calculated binding free energy was in good agreement with the experimental reports. Furthermore, binding modes of six analogs of APHS were also analyzed to study the effects on binding affinity of the triple bond, heteroatom and length of alkyl chain. The findings help to understand the action mechanisms of APHS and explain why it is more potent than the analogs, which might be useful in the design of new compounds with higher inhibitory activity to COX-2.

Communicated by Ramaswamy H. Sarma     

Ca2+ regulation of thyroid NADPH-dependent H2O2 generation

A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s).     

Preparation of 2''-thio-2''-deoxycytidine 2'':3''-phosphorothioate.

The synthesis of a novel ribonucleotide analog 2'-thio-2' deoxycytidine 2':3'-O,S-phosphorothioate is described. In the first step, 2,2'-anhydro 1-beta-D-arabinosylcytosine was thiophosphorylated by the action of dithiophosphate, a process which gave predominantly the 3'-O-phosphorothioate isomer. An intramolecular displacement reaction led to the formation of the title compound. Structure and reactivity of this thioanalog differ substantially from 2':3'-CMP.     

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.     

Hemoglobin-based O2 carrier O2 affinity and capillary inlet pO2 are important factors that influence O2 transport in a capillary

Hemopure (Biopure; Cambridge, MA) and PolyHeme (Northfield Laboratories; Evanston, IL) are two acellular hemoglobin-based O2 carriers (HBOCs) currently in phase III clinical trials for use as red blood cell substitutes. The most common adverse side effect that these HBOCs exhibit is increased vasoconstriction. Autoregulatory theory has been presented as a possible explanation for this physiological effect, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding arterioles, thereby eliciting vasoconstriction. In this paper, we wanted to investigate HBOC oxygenation of tissue surrounding a capillary, which is the smallest element of the circulatory system. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (synthesized by our group and others) and human red blood cells (hRBCs) has been simulated using a Krogh tissue cylinder model (KTCM) comprising a capillary surrounded by a capillary membrane and skeletal muscle tissue in cylindrical coordinates with specified tissue O2 consumption rates and Michaelis-Menten kinetics. In this study, the total hemoglobin (hRBCs and HBOCs) concentration was kept constant. The HBOCs studied possessed O2 affinities that were higher and lower compared to hRBCs (P50's spanned 5-55 mmHg), and the equilibrium binding/release of oxygen to/from the HBOCs was modeled using the Adair equation. At normoxic inlet pO2's, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC. Additionally, we studied the change in the O2 equilibrium curve of HBOCs with different O2 affinities over a wide range of inlet pO2's and found that changing the inlet pO2 greatly affected which HBOC, having a unique O2 affinity, best delivered O2 to the surrounding tissue. The analysis of oxygen transport presented could lead to a better prediction of which acellular HBOC is best suited for a specific transfusion application that many times depends on the capillary inlet pO2 tension.     

植物ABA-H2O2介导的气孔关闭

本文综述了脱落酸作为根源信号物质经由木质部被传递到叶片,经重新分配再与脱落酸受体结合,然后刺激气孔开放因子,调节烟酰胺腺嘌呤二核苷酸磷酸氧化酶等关键酶活性产生过氧化氢,过氧化氢可使胞质碱化并刺激钙离子通道使钙离子内流,活化阴离子通道使阴离子外流,最终导致气孔关闭的一系列过程。该过程涉及到的因子包括：脱落酸受体、气孔开放因子、磷脂酰环己六醇、分裂原激活蛋白激酶、烟酰胺腺嘌呤二核苷酸磷酸氧化酶、Ca^（2＋）、pH、一氧化氮等。     

Cytoplasmic Retention of Protein Phosphatase 2A Inhibitor 2 (I2PP2A) Induces Alzheimer-like Abnormal Hyperphosphorylation of Tau

Abnormal hyperphosphorylation of Tau leads to the formation of neurofibrillary tangles, a hallmark of Alzheimer disease (AD), and related tauopathies. The phosphorylation of Tau is regulated by protein phosphatase 2A (PP2A), which in turn is modulated by endogenous inhibitor 2 (I₂^PP2A). In AD brain, I₂^PP2A is translocated from neuronal nucleus to cytoplasm, where it inhibits PP2A activity and promotes abnormal phosphorylation of Tau. Here we describe the identification of a potential nuclear localization signal (NLS) in the C-terminal region of I₂^PP2A containing a conserved basic motif, ¹⁷⁹RKR¹⁸¹, which is sufficient for directing its nuclear localization. The current study further presents an inducible cell model (Tet-Off system) of AD-type abnormal hyperphosphorylation of Tau by expressing I₂^PP2A in which the NLS was inactivated by ¹⁷⁹RKR¹⁸¹ → AAA along with ¹⁶⁸KR¹⁶⁹ → AA mutations. In this model, the mutant NLS (mNLS)-I₂^PP2A (I₂^PP2AAA-AAA) was retained in the cell cytoplasm, where it physically interacted with PP2A and inhibited its activity. Inhibition of PP2A was associated with the abnormal hyperphosphorylation of Tau, which resulted in microtubule network instability and neurite outgrowth impairment. Expression of mNLS-I₂^PP2A activated CAMKII and GSK-3β, which are Tau kinases regulated by PP2A. The immunoprecipitation experiments showed the direct interaction of I₂^PP2A with PP2A and GSK-3β but not with CAMKII. Thus, the cell model provides insights into the nature of the potential NLS and the mechanistic relationship between I₂^PP2A-induced inhibition of PP2A and hyperphosphorylation of Tau that can be utilized to develop drugs preventing Tau pathology.     

SNT-2 interacts with ERK2 and negatively regulates ERK2 signaling in response to EGF stimulation

The control of cellular responses with fibroblast growth factors and neurotrophins is mediated through membrane-linked docking proteins, SNT (suc1-binding neurotrophic target)-1/FRS2alpha and SNT-2/FRS2beta. ERK1/2 are members of the mitogen-activated protein kinase family that regulate diverse cellular activities in response to various stimuli. Here, we demonstrate that SNT-2 does not become tyrosine phosphorylated significantly in response to EGF but forms a complex with ERK2 via the region of 186-252 amino acid residues, and the complex formation is enhanced upon EGF stimulation. SNT-2 downregulates ERK2 phosphorylation, suppresses and delays ERK2 nuclear accumulation which occurs following EGF stimulation. In contrast, the mutant SNT-2 which carries deletion of 186-252 amino acids and lacks ERK2 binding does not have these effects. These observations suggest that SNT-2 negatively regulates ERK2 signaling activated via EGF stimulation through direct binding to ERK2.