GPS autoproteolysis is required for CD97 to up-regulate the expression of N-cadherin that promotes homotypic cell-cell aggregation

Most adhesion-class G protein-coupled receptors (adhesion-GPCRs) undergo a novel self-catalytic cleavage at the GPCR proteolysis site (GPS) to form a hetero-dimeric complex containing the extracellular and seven-span transmembrane subunits. However, little is known about the role of GPS auto-proteolysis in the function of adhesion-GPCRs. Here we show that GPS cleavage is essential for the homotypic cell aggregation promoted by CD97 receptor, a leukocyte-restricted adhesion-GPCR often aberrantly expressed in carcinomas. We find that CD97 does not mediate cell aggregation directly. Instead, expression of the wild type – but not the GPS cleavage-deficient CD97 up-regulates the expression of N-cadherin, leading to Ca⁺⁺-dependent cell–cell aggregation. Our results provide a clear evidence for the role of GPS proteolytic modification in the cellular function of adhesion-GPCRs.     

Over-expression,Purification, and Characterization of Recombinant Human Arylamine <Emphasis Type="Italic">N</Emphasis>-Acetyltransferase 1

Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 l of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNAT1 and hamster rNAT2. With both NATs, the second order rate constants (k_cat/K_mb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k _cat/K_mb3410 M^-1 s^-1 ) than for human rNAT1 (k_cat/K_mb 169.4 M^-1 s^-1 ). p-aminobenzoylglutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K_I values in 50–300 range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo.     

A thrombin inhibitor from the gut of <Emphasis Type="Italic">Boophilus microplus</Emphasis> ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin–Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.     

Characterization of the CaMKKβ-AMPK signaling complex

The AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis, and is a potential target for treatment of metabolic diseases as well as cancer. AMPK can be phosphorylated and activated by the tumor suppressor LKB1 or the Ca²⁺/CaM-dependent protein kinase kinase β (CaMKKβ). We previously identified a physical complex between CaMKKβ and AMPK (Anderson, K. A., Ribar, T. J., Lin, F., Noeldner, P. K., Green, M. F., Muehlbauer, M. J., Witters, L. A., Kemp, B. E., and Means, A. R. (2008) Cell Metabolism 7, 377–388). Here we expand our analysis of the CaMKKβ–AMPK signaling complex and show that whereas CaMKKβ can form a complex with and activate AMPK, CaMKKα cannot. In addition, we show that CaMKKβ and AMPK associate through their kinase domains, and CaMKKβ must be in an active conformation in order to bind AMPK but not to associate with an alternative substrate, Ca²⁺/Calmodulin-dependent protein kinase IV (CaMKIV). Our results demonstrate that CaMKKβ and AMPK form a unique signaling complex. This raises the possibility that the CaMKKβ–AMPK complex can be specifically targeted by small molecule drugs to treat disease.     

Synthesis,structure, and structure-activity relationships of divalent thrombin inhibitors containing an α-keto-amide transition-state mimetic

A new class of divalent thrombin inhibitors is described that contains an α-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the α-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their α-keto-amide counterparts. The most potent cv-keto-amide inhibitor, CVS995, with a K_i = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.     

The Bacillus anthracis arylamine N-acetyltransferase ((BACAN)NAT1) that inactivates sulfamethoxazole, reveals unusual structural features compared with the other NAT isoenzymes

Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that biotransform arylamine drugs. The Bacillus anthracis (BACAN)NAT1 enzyme affords increased resistance to the antibiotic sulfamethoxazole through its acetylation. We report the structure of (BACAN)NAT1. Unexpectedly, endogenous coenzymeA was present in the active site. The structure suggests that, contrary to the other prokaryotic NATs, (BACAN)NAT1 possesses a 14-residue insertion equivalent to the “mammalian insertion”, a structural feature considered unique to mammalian NATs. Moreover, (BACAN)NAT1 structure shows marked differences in the mode of binding and location of coenzymeA when compared to the other NATs. This suggests that the mechanisms of cofactor recognition by NATs is more diverse than expected and supports the cofactor-binding site as being a unique subsite to target in drug design against bacterial NATs.     

Phytochemical and Bioactivity Studies on Constituents of the Leaves of Vitex Quinata

A phytochemical investigation of the leaves of Vitex quinata (Lour.) F.N. Williams (Verbenaceae), guided by a cytotoxicity assay against the MCF-7 human breast cancer cell line, led to the isolation of a new δ-truxinate derivative (1) and a new phytonoic acid derivative (2), together with 12 known compounds. The structures of the new compounds were determined by spectroscopic methods as dimethyl 3,4,3′,4′-tetrahydroxy-δ-truxinate (1) and methyl 10R-methoxy-12-oxo-9(13),16E-phytodienoate (2), respectively. In a cytotoxicity assay, (S)-5-hydroxy-7,4′-dimethoxyflavanone (3) was found to be the sole active principle, with ED₅₀ values of 1.1–6.7 μM, respectively, when tested against a panel of three human cancer cells. Methyl 3,4,5-O-tricaffeoyl quinate (4) showed activity in an enzyme-based ELISA NF-κB p65 assay, with an ED₅₀ value of 10.3 μM.     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Structure of the E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) from Thermus thermophilus

Isoprenoids are biosynthesized via the mevalonate or the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways the latter being used by most pathogenic bacteria, some parasitic protozoa, plant plastids, but not by animals. We determined the X-ray structure of the homodimeric [4Fe–4S] cluster carrying E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate synthase (GcpE) of Thermus thermophilus which catalyzes the penultimate reaction of the MEP pathway and is therefore an attractive target for drug development. The [4Fe–4S] cluster ligated to three cysteines and one glutamate is encapsulated at the intersubunit interface. The substrate binding site lies in front of an (αβ)₈ barrel. The great [4Fe–4S] cluster-substrate distance implicates large-scale domain rearrangements during the reaction cycle.

Structured summary

gcpEbinds to gcpE by x-ray crystallography (View interaction)     

Electron-donating para-methoxy converts a benzamide-isoquinoline derivative into a highly Sigma-2 receptor selective ligand

The sigma-2 (σ2) receptor has been suggested to be a promising target for pharmacological interventions to curb tumor progression. Development of σ2-specific ligands, however, has been hindered by lack of understanding of molecular determinants that underlie selective ligand-σ2 interactions. Here we have explored effects of electron donating and withdrawing groups on ligand selectivity for the σ2 versus σ1 receptor using new benzamide-isoquinoline derivatives. The electron-donating methoxy group increased but the electron-withdrawing nitro group decreased σ2 affinity. In particular, an extra methoxy added to the para-position (5e) of the benzamide phenyl ring of 5f dramatically improved (631 fold) the σ2 selectivity relative to the σ1 receptor. This para-position provided a sensitive site for effective manipulation of the sigma receptor subtype selectivity using either the methoxy or nitro substituent. Our study provides a useful guide for further improving the σ2-over-σ1 selectivity of new ligands.     

Identification of an artificial peptide motif that binds and stabilizes reduced human DJ-1

Although the precise biochemical function of DJ-1 remains unclear, it has been found to exert cytoprotective activity against oxidative stress. Cys106 is central to this function since it has a distinctly low pK_a rendering it extremely susceptible for oxidation. This characteristic, however, also poses a severe hindrance to obtain reduced DJ-1 for in vitro investigation. We have developed an approach to produce recombinant human DJ-1 in its reduced form as a bona fide basis for exploring the redox capacities of the protein. We solved the crystal structure of this DJ-1 at 1.56 Å resolution, allowing us to capture Cys106 in the reduced state for the first time. The dimeric structure reveals one molecule of DJ-1 in its reduced state while the other exhibits the characteristics of a mono-oxygenated cysteine. Comparison with previous structures indicates the absence of redox dependent global conformational changes in DJ-1. The capture of reduced Cys106 is facilitated by stabilization within the putative active site achieved through a glutamate side chain. This side chain is provided by a crystallographic neighbor as part of a ‘Leu–Glu’ motif, which was added to the C-terminus of DJ-1. In the structure this motif binds DJ-1 in close proximity to Cys106 through extended hydrophilic and hydrophobic interactions depicting a distinct binding pocket, which can serve as a basis for compound development targeting DJ-1.     

The extended catalysis of glutathione transferase

Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ? [E]. As glutathione transferase lowers the pK_a of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed.     

基于Ti4+-IMAC富集的分枝菌酸小杆菌深度覆盖磷酸化蛋白质组研究

【目的】本研究以分枝菌酸小杆菌(Mycolicibacterium smegmatis)为研究对象,探索适于原核微生物理想的磷酸化富集方法。【方法】我们比较了二氧化钛(TiO₂)、Fe³⁺-NTA和Ti⁴⁺螯合在磷酸酯修饰的固相微球(Ti⁴⁺-IMAC) 3种不同富集方法磷酸化肽段的富集效率,并用不同分辨率的质谱仪评估富集稳定性。【结果】Ti⁴⁺-IMAC富集效率最高,磷酸化位点数是TiO₂或Fe³⁺-NTA方法的7倍以上;TiO₂和Fe³⁺-NTA方法富集到的磷酸化位点数相差不大,与已报道的用TiO₂方法富集的磷酸化位点数目接近。Ti⁴⁺-IMAC富集结果稳定性很好,高分辨率Lumos质谱仪鉴定到的磷酸化位点数是Velos的2.6倍。【结论】本研究较高效地实现了分枝菌酸小杆菌磷酸化事件的鉴定,共鉴定到2 280个磷酸化蛋白、10 880个磷酸化肽段及4 433个可信磷酸化位点,有望用于其他微生物的磷酸化蛋白质组学研究。     

Metabolism of <Emphasis Type="Italic">N</Emphasis>-Methyl, <Emphasis Type="Italic">N</Emphasis>-propargylphenylethylamine: Studies with Human Liver Microsomes and cDNA Expressed Cytochrome P450 (CYP) Enzymes

Summary 1. We used an in vitro screening procedure and studies with individual human liver microsomes and cDNA-expressed CYP enzymes to investigate the metabolism of the putative neuroprotective drug N-methyl,N-propargyl-2-phenylethylamine (MPPE) to N-methylphenylethylamine (N-methylPEA) and N-propargylphenylethylamine (N-propargylPEA). 2. An electron-capture gas chromatographic procedure previously developed in our laboratories was used to measure the quantities of N-methylPEA and N-propargylPEA formed in the experiments with a single donor human liver microsome panel and cDNA expressed single CYP enzyme systems. The data were fitted to nonlinear regressions using Prism to determine kinetic constants. The results from a fluorogenic screen determined which cDNA-expressed single CYP enzymes were investigated. 3. CYP2B6, CYP2C19, and CYP2D6 all contributed to the formation of N-methylPEA, while only CYP2B6 catalyzed the formation of N-propargylPEA. The K _M and V _max values for N-propargylPEA formation were 290 ± 70 μM and 139±16 ng/mL/min. The values for formation of N-methylPEA were not determined from these experiments due to the complexity of fitting the data to a three-variable equation, but data on the time course of N-methylPEA formation are presented. 4. Catabolism of MPPE to N-methylPEA and N-propargylPEA is catalyzed by CYP enzymes. CYP2B6, 2C19 and 2D6 all contribute to the depropargylation of the parent compound, but only CYP2B6 also catalyzes demethylation. CYP2C19 was found to be the most active with respect to generation of N-methylPEA.     

Mild caloric restriction up-regulates the expression of prohibitin: a proteome study

The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)₄Lφ motif (X: variable, φ: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the ⁵⁹⁷SDVVL⁶⁰¹ sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence ⁵⁷²YDREFLL⁵⁷⁸. The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor

The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (−)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K_i values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k_cat regulation. Surprisingly, the T11A mutant had a decreased GSH K_m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.