The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2

Oncoprotein Mdm2 is a master negative regulator of the tumor suppressor p53 and has been recently shown to regulate the ubiquitination of beta-arrestin 2, an important adapter and scaffold in signaling of G-protein-coupled receptors (GPCRs). However, whether beta-arrestin 2 has any effect on the function of Mdm2 is still unclear. Our current results demonstrated that the binding of Mdm2 to beta-arrestin 2 was significantly enhanced by stimulation of GPCRs. Activation of GPCRs led to formation of a ternary complex of Mdm2, beta-arrestin 2, and GPCRs and thus recruited Mdm2 to GPCRs at plasma membrane. Moreover, the binding of beta-arrestin 2 to Mdm2 suppressed the self-ubiquitination of Mdm2 and consequently reduced the Mdm2-mediated p53 degradation and ubiquitination. Further experiments revealed that overexpression of beta-arrestin 2 enhanced the p53-mediated apoptosis while suppression of endogenous beta-arrestin 2 expression by RNA interference technology considerably attenuated the p53-mediated apoptosis. Our study thus suggests that beta-arrestin 2 may serve as a cross-talk linker between GPCR and p53 signaling pathways.     

The reaction of hemin with H2O2

The kinetics of formation of the dominant intermediate (CII) formed between hemin and H2O2 has been studied by the stopped-flow method. CII is preceded by a precursor (CI) for which a steady state is established at an early stage of the reaction. The formation of CI from hemin and H2O2 causes only a marginal change in the optical absorbance (A). The transition CI----CII is accompanied by a substantial decrease of A in the Soret region. Relevant rate constants (or combinations of them) and the molar absorption coefficients of the intermediates at 400 nm have been determined. The absorption spectrum of CII in the Soret region has been evaluated. Aspects of the catalysis of decomposition of H2O2 by hemin in relation to the Fe3+ ion and catalase are discussed.     

Structural and energetic aspects of Grb2-SH2 domain-swapping

The SH2 domain of growth factor receptor-bound protein 2 (Grb2) has been the focus of numerous studies, primarily because of the important roles it plays in signal transduction. More recently, it has emerged as a useful protein to study the consequences of ligand preorganization upon energetics and structure in protein-ligand interactions. The Grb2-SH2 domain is known to form a domain-swapped dimer, and as part of our investigations toward correlating structure and energetics in biological systems, we examined the effects that domain-swapping dimerization of the Grb2-SH2 domain had upon ligand binding affinities. Isothermal titration calorimetry was performed using Grb2-SH2 in both its monomeric and domain-swapped dimeric forms and a phosphorylated tripeptide AcNH-pTyr-Val-Asn-NH(2) that is similar to the Shc sequence recognized by Grb2-SH2 in vivo. The two binding sites of domain-swapped dimer exhibited a 4- and a 13-fold reduction in ligand affinity compared to monomer. Crystal structures of peptide-bound and uncomplexed forms of Grb2-SH2 domain-swapped dimer were obtained and reveal that the orientation of residues V122, V123, and R142 may influence the conformation of W121, an amino acid that is believed to play an important role in Grb2-SH2 ligand sequence specificity. These findings suggest that domain-swapping of Grb2-SH2 not only results in a lower affinity for a Shc-derived ligand, but it may also affect ligand specificity.     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.     

Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling

Excessive generation of reactive oxygen species (ROS) has been implicated in the pathogenesis of many diseases, including atherosclerosis, hypertension, and vascular complications of diabetes. However, the precise mechanisms by which ROS contribute to the development of these diseases are not fully characterized. Hydrogen peroxide (H₂O₂), a ROS, has been shown to activate several signaling protein kinases, such as extracellular signal-regulated kinase (ERK)1/2 and protein kinase B (PKB) in different cell types, notably in vascular smooth muscle cells. Because these pathways regulate cellular mitogenesis, migration, proliferation, survival, and death responses, their aberrant activtion has been suggested to be a potential mechanism of ROS-induced pathologies. The upstream elements responsible for H₂O₂-induced ERK1/2 and PKB activation remain poorly characterized, but a potential role of receptor and nonreceptor protein tyrosine kinases (PTKs) as triggers that initiate such events has been postulated. Therefore, the aim of this review is to highlight the involvement of receptor and nonreceptor PTKs in modulating H₂O₂-induced ERK1/2 and PKB signaling.     

Comparison of the reactivity of oxaliplatin, pt(diaminocyclohexane)Cl2 and pt(diaminocyclohexane1)(OH2)2(2+) with guanosine and L-methionine

The initial rates of reactivity of oxaliplatin, its metabolites Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) with guanosine and L-met in water, NaCl and phosphate were compared. Versus guanosine, the most reactive molecule was Pt(dach)(OH2)2(2+), about 40 fold that of oxaliplatin, the least reactive was Pt(dach)Cl2, Versus L-met, Pt(dach)(OH2)2(2+), was also the most reactive species but only about 2 fold more reactive than Pt(dach)Cl2 and oxaliplatin. Pt(dach)(OH2)2(2+) was approximately 3 fold less reactive versus methionine than guanosine whereas oxaliplatin and Pt(dach)Cl2 were about seven fold more reactive versus methionine than guanosine. Thus, the three platinum compounds oxaliplatin, Pt(dach)Cl2 and Pt(dach)(OH2)2(2+) react with L-met but only the Pt(dach)(OH2)2(2+) has a high reactivity with guanosine. Oxaliplatin, which is stable in water, has to be transformed in the presence of chloride in chloro-derivatives which are aquated to become active particularly versus guanosine. These data demonstrate that oxaliplatin has similarities with cisplatin in terms of chloride versus water coordination and in terms of dependence on chloride concentration for transformations.     

Frequency of the atypical aldehyde dehydrogenase-2 gene (ALDH2(2)) in Japanese and Caucasians.

All Caucasians have two major aldehyde dehydrogenase isozymes--i.e., the cytosolic ALDH1 and the mitochondrial ALDH2-while approximately 50% of Orientals are atypical and lack the catalytically active ALDH2 in their tissues. The atypical ALDH2(2) gene has a nucleotide base change and produces the defective ALDH2(2) protein, which has a Glu----Lys substitution at the 14th position from the COOH-terminal (Yoshida et al. 1984; Hsu et al. 1985). With the use of a pair of synthetic oligonucleotides-one complementary to the usual ALDH1(2) and the other complementary to the atypical ALDH2(2)-genotypes of 49 unrelated Japanese individuals and 12 Caucasians were determined. The frequency of the atypical ALDH2(2) allele was found to be .35 in the Japanese samples examined. The atypical ALDH2(2) gene was not found in the Caucasians.     

The synthesis of diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride

The N-trifluoroacetyl- and N-tetrachlorophthaloyl-protected bromide of D-glucosamine has been used for the first time as a glycosyl donor for the glycosylation of diosgenin [(25R)-spirost-5-en-3beta-ol]. Both 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-beta-D-glucopy ranoside and 1,3,4,6-tetra-O-acetyl-2-deoxy-2-tetrachlorophthalimido-alpha,beta -D-glucopyranoside were transformed into the appropriate glycosyl bromides. These reacted with diosgenin under mild conditions, using silver triflate as a promoter, and gave the corresponding protected diosgenyl glycosides. Each was deprotected to give diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride. The structures of the new glycosides were established by 1H NMR spectroscopy.     

Characterization of Angiotensin Converting Enzyme-2 (ACE2) in Human Urine

Angiotensin converting enzyme-2 (ACE2) is a recently described membrane-bound carboxypeptidase identified by its homology to ACE, the enzyme responsible for the formation of the potent vasoconstrictor angiotensin II (Ang II). ACE2 inactivates Ang II and is thus thought to act in a counter-regulatory fashion to ACE. ACE2 is highly expressed in epithelial cells of distal renal tubules, and recent evidence indicates that expression is increased in a range of renal diseases. A soluble form of ACE, generated by proteolytic cleavage of the membrane-bound form, has been shown to be present in urine; although evidence for a similar release of ACE2 has been reported in cell culture, it is not yet known whether this occurs in vivo. The present study has identified ACE2 in human urine, both by a sensitive fluorescence-based activity assay and by Western immunoblot. Levels of ACE2 were surprisingly higher than ACE, which may reflect preferential targeting of the enzyme to the luminal surface of the renal epithelium. Future studies will determine whether increased expression of ACE2 in renal diseases are reflected in higher urinary levels of this novel enzyme.Australian Peptide Conference Issue.     

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     

Crystal structures of O-acetylated 2-acylamino-2-deoxy-D-galactose derivatives

The X-ray structures of 1,3,4,6-tetra-O-acetyl-2-deoxy-alpha-D-galactopyranoside derivatives with four different 2-(acylamino) substituents have been determined with Mo K(alpha) radiation at 123 K. The structure of the 2-acetylamino derivative and of its acyl-homologs with a 2-(propanoylamino)-, 2-(butanoylamino)-, and 2-(2-methyl-propanoylamino)-group crystallized in the monoclinic space group C2. The pyranose unit of all compounds has the usual 4C(1) shape. The different orientations of the 6-O-acetyl-groups are discussed. Conformations of the acylamino-group are compared to those found in the crystal structure of N-acetyl-alpha-D-galactosamine.     

PGH2 degradation pathway catalyzed by GSH-heme complex bound microsomal prostaglandin E2 synthase type 2: the first example of a dual-function enzyme

Prostaglandin E2 synthase (PGES) catalyzes the isomerization of PGH2 to PGE2. PGES type 2 (mPGES-2) is a membrane-associated enzyme, whose N-terminal section is apparently inserted into the lipid bilayer. Both intact and N-terminal truncated enzymes have been isolated and have similar catalytic activity. The recombinant N-terminal truncated enzyme purified from Escherichia coli HB101 grown in LB medium containing delta-aminolevulinate and Fe(NO3)3 has a red color, while the same enzyme purified from the same E. coli grown in minimal medium has no color. The red-colored enzyme has been characterized by mass, fluorescence, and EPR spectroscopies and X-ray crystallography. The enzyme is found to contain bound glutathione (GSH) and heme. GSH binds to the active site with six H-bonds, while a heme is complexed with bound GSH forming a S-Fe coordination bond with no polar interaction with mPGES-2. There is a large open space between the heme and the protein, where a PGH2 might be able to bind. The heme dissociation constant is 0.53 microM, indicating that mPGES-2 has relatively strong heme affinity. Indeed, expression of mPGES-2 in E. coli stimulates heme biosynthesis. Although mPGES-2 has been reported to be a GSH-independent PGES, the crystal structure and sequence analysis indicate that mPGES-2 is a GSH-binding protein. The GSH-heme complex-bound enzyme (mPGES-2h) catalyzes formation of 12(S)-hydroxy-5(Z),8(E),10(E)-heptadecatrienoic acid and malondialdehyde from PGH2, but not formation of PGE2. The following kinetic parameters at 37 degrees C were determined: KM = 56 microM, kcat = 63 s-1, and kcat/KM = 1.1 x 10(6) M-1 s-1. They suggest that mPGES-2h has significant catalytic activity for PGH2 degradation. It is possible that both GSH-heme complex-free and -bound enzymes are present in the same tissues. mPGES-2 in heme-rich liver is most likely to become the form of mPGES-2h and might be involved in degradation reactions similar to that of cytochrome P450. Since mPGES-2 is an isomerase and mPGES-2h is a lyase, mPGES-2 cannot simply be classified into one of six classes set by the International Union of Biochemistry and Molecular Biology.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.     

CTTNBP2, but not CTTNBP2NL,regulates dendritic spinogenesis and synaptic distribution of the striatin–PP2A complex

Cortactin-binding protein 2 (CTTNBP2) interacts with cortactin to regulate cortactin mobility and control dendritic spine formation. CTTNBP2 has also been associated with autistic spectrum disorder. The regulation of dendritic spinogenesis could explain the association of CTTNBP2 with autism. Sequence comparison has indicated that CTTNBP2 N-terminal–like protein (CTTNBP2NL) is a CTTNBP2 homologue. To confirm the specific effect of CTTNBP2 on dendritic spinogenesis, here we investigate whether CTTNBP2NL has a similar function to CTTNBP2. Although both CTTNBP2 and CTTNBP2NL interact with cortactin, CTTNBP2NL is associated with stress fibers, whereas CTTNBP2 is distributed to the cortex and intracellular puncta. We also provide evidence that CTTNBP2, but not CTTNBP2NL, is predominantly expressed in the brain. CTTNBP2NL does not show any activity in the regulation of dendritic spinogenesis. In addition to spine morphology, CTTNBP2 is also found to regulate the synaptic distribution of striatin and zinedin (the regulatory B subunits of protein phosphatase 2A [PP2A]), which interact with CTTNBP2NL in HEK293 cells. The association between CTTNBP2 and striatin/zinedin suggests that CTTNBP2 targets the PP2A complex to dendritic spines. Thus we propose that the interactions of CTTNBP2 and cortactin and the PP2A complex regulate spine morphogenesis and synaptic signaling.