alpha/beta-type small, acid-soluble spore proteins (SASP) of Bacillus subtilis bind to DNA and alter its conformation, topology, and photochemistry, and thereby spore resistance to UV light. Three mutations have been introduced into the B. subtilis sspC gene, which codes for the alpha/beta-type wild-type SASP, SspCwt. One mutation (SspCTyr) was a conservative change, as residue 29 (Leu) was changed to Tyr, an amino acid found at this position in other alpha/beta-type SASP. The other mutations changed residues conserved in all alpha/beta-type SASP. In one (SspCAla), residue 52 (Gly) was changed to Ala; in the second (SspCGln), residue 57 (Lys) was changed to Gln. The effects of the wild-type and mutant SspC on DNA properties were examined in vivo in B. subtilis spores and Escherichia coli as well as in vitro with use of purified protein. Both SspCwt and SspCTyr interacted similarly with DNA in vivo and in vitro, restoring much UV resistance to spores lacking major alpha/beta-type SASP, causing a large increase in plasmid negative supercoiling, and altering DNA UV photochemistry from cell type to spore type. In contrast, SspCAla had no detectable effect on DNA properties in vivo or in vitro, while SspCGln had effects intermediate between those of SspCAla and SspCwt. Strikingly, neither SspCAla nor SspCGln bound well to DNA in vitro. These results confirm the importance of the conserved primary sequence of alpha/beta-type SASP in the ability of these proteins to bind to spore DNA and cause spore UV resistance.

Factor VIII binds to phospholipid membranes and to von Willebrand factor (vWf) via its second C domain, which has lectin homology. The crystal structure of the C2 domain has prompted a model in which membrane binding is mediated by two hydrophobic spikes, each composed of a pair of residues displayed on a beta-hairpin turn, and also by net positive charge and specific interactions with phospho-l-serine. To test this model, we prepared 16 factor VIII mutants in which single or multiple amino acids were changed to alanine. Mutants at Arg(2215), Arg(2220), Lys(2227), Lys(2249), Gln(2213), Asn(2217), and Phe(2196)/Thr(2197) had specific activities that were >70% of the wild type. Mutants at Arg(2209), Lys(2227), Trp(2313), and Arg(2320) were degraded within the cell. Hydrophobic spike mutants at Met(2199)/Phe(2200), Leu(2251)/Leu(2252), and Met(2199)/Phe(2200)/Leu(2251)/Leu(2252) (4-Ala) exhibited 43, 59, and 91% reduction in specific activity in the activated partial thromboplastin time assay. In a phospholipid-limiting factor Xa activation assay, these mutants had a 65, 85, and 96% reduction in specific activity. Equilibrium binding of fluorescent, sonicated phospholipid vesicles to mutants immobilized on Superose beads was measured by flow cytometry. The affinities for phospholipid were reduced approximately 20-, 30-, and >35-fold for 2199/2200, 2251/2252, and 4-Ala, respectively. A dimeric form of mature vWf bound to immobilized factor VIII and the same mutants, but the affinities of the mutants were reduced approximately 5-, 10-, and >20-fold, respectively. In a competition, solution phase enzyme-linked immunosorbent assay, plasma vWf bound factor VIII and the same mutants with the affinities for the mutants reduced >5-, >5-, and >50-fold, respectively. We conclude that the two hydrophobic spikes are constituents of both the phospholipid-binding and vWf-binding motifs. In plasma, vWf apparently binds the inherently sticky membrane-binding motif, preventing nonspecific interactions.

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68->>Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.

Proteolysis, as well as protein synthesis, is a major process that contributes to the body protein turnover. Despite the huge variety of proteases in the body, there are very few proteolytic systems contributing to the complete hydrolysis of proteins to amino acids. The autophagic-lysosomal pathway is responsible for bulk proteolysis, whereas the ubiquitin-proteasome pathway plays a significant role in the fine control of the degradation of specific proteins. Both systems can produce free amino acids as a final product, but only the autophagy system is physiologically controlled by plasma amino acids. Recently, the study of amino acids as regulators of macromolecular turnover has been focused on for their signal transduction mechanism. In autophagic proteolysis, several amino acids have a direct regulatory potential: Leu, Gln, Tyr, Phe, Pro, Met, Trp and His in the liver, and Leu in the skeletal muscle. These amino acids are recognized at the plasma membrane, indicating the possible existence of an amino acid receptor/sensor for their recognition and subsequent intracellular signaling. Another line of evidence has emerged that protein kinase cascades such as mTOR, Erk, eIF2alpha etc. may be involved in the regulation of autophagy, and that amino acids, in combination with insulin, may exert their effects through these pathways. From the viewpoint of amino acid safety, the contribution of proteolysis to possible adverse effects caused by excessive amino acid intake is not clear. At present, there is one report that excess glutamine at 10-fold the plasma level has an abnormal inhibitory effect on hepatic proteolysis, due to a lysosomotropic toxicity of ammonia derived from glutamine degradation. Whether this may lead to an adverse effect in humans remains to be clarified.

In order to obtain an HCC-selective drug delivery system, a novel functional lipid, which is cleaved by the protease activity of matrix metalloproteinase-2 (MMP-2), was developed. The amino group of dioleoylphosphatidylethanolamine (DOPE) was conjugated with PEGylated MMP-2 substrate peptide (Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln), and MMP-2-cleavable PEG-Peptide-DOPE (PEG-PD) was synthesized. When PEG-PD was incorporated in galactosylated liposomes (Gal-PEG-PD-liposomes), we expected that Gal-PEG-PD-liposomes would not be taken up by normal hepatocytes due to the steric hindrance effect, but would be activated around HCC cells by secreted MMPs. In the pretreatment by hMMP2 (1, 5, and 10mug/ml), an hMMP2 concentration-dependent higher uptake of Gal-PEG-PD-liposomes was observed in HepG2 cells, suggesting PEG-PD cleavage. In the presence of an excess of galactose, the uptake of Gal-PEG-PD-liposomes with hMMP2 was significantly inhibited, suggesting asialoglycoprotein receptor-mediated uptake of Gal-PEG-PD-liposomes following the PEG-PD cleavage. Pretreatment of Gal-PEG-PD-liposomes with the conditioned medium of B16BL6, which contained secreted MMPs, enhanced the binding to HepG2 cells, as in the case of hMMP-2 treatment. Moreover, the cytotoxicity of N(4)-octadecyl-1-beta-d-arabinofuranosylcytosine (NOAC) incorporated Gal-PEG-PD-liposomes was enhanced by hMMPs (5mug/ml) and its cytotoxicity was significantly reduced by the presence of an excess of galactose in HepG2 cells. In conclusion, Gal-PEG-PD-liposomes were successfully developed for novel HCC-selective targeting.

Two variants of the IL-4R alpha-chain (IL-4Ralpha) gene have been recently identified in association with different atopic disorders. To clarify the etiological relationship between the two variants, we analyzed responsiveness to IL-4 of transfectants with four kinds of IL-4Ralpha carrying either Val or Ile at 50 and either Gln or Arg at 551. The substitution of Ile for Val augmented STAT6 activation, proliferation, and transcription activity of the Iepsilon promoter by IL-4, whereas that of Arg for Gln did not change these IL-4 signals. Arg551 was not associated with atopic asthma in the Japanese population. CD23 expression and IgE synthesis by IL-4 were augmented in Ile50-bearing PBMC, compared with those bearing Val50. Taken together, substitution of Arg551 does not enhance the IL-4 signal for generation of germline epsilon transcript, whereas the substitution of Ile50 contributes to enhancement of IgE synthesis.

Reduced lipolysis in hormone-sensitive lipase-deficient mice is associated with impaired glucose-stimulated insulin secretion (GSIS), suggesting that endogenous beta-cell lipid stores provide signaling molecules for insulin release. Measurements of lipolysis and triglyceride (TG) lipase activity in islets from HSL(-/-) mice indicated the presence of other TG lipase(s) in the beta-cell. Using real time-quantitative PCR, adipose triglyceride lipase (ATGL) was found to be the most abundant TG lipase in rat islets and INS832/13 cells. To assess its role in insulin secretion, ATGL expression was decreased in INS832/13 cells (ATGL-knockdown (KD)) by small hairpin RNA. ATGL-KD increased the esterification of free fatty acid (FFA) into TG. ATGL-KD cells showed decreased glucose- or Gln + Leu-induced insulin release, as well as reduced response to KCl or palmitate at high, but not low, glucose. The K(ATP)-independent/amplification pathway of GSIS was considerably reduced in ATGL-KD cells. ATGL(-/-) mice were hypoinsulinemic and hypoglycemic and showed decreased plasma TG and FFAs. A hyperglycemic clamp revealed increased insulin sensitivity and decreased GSIS and arginine-induced insulin secretion in ATGL(-/-) mice. Accordingly, isolated islets from ATGL(-/-) mice showed reduced insulin secretion in response to glucose, glucose + palmitate, and KCl. Islet TG content and FFA esterification into TG were increased by 2-fold in ATGL(-/-) islets, but glucose usage and oxidation were unaltered. The results demonstrate the importance of ATGL and intracellular lipid signaling for fuel- and non-fuel-induced insulin secretion.

Influenza viruses of the H3N2 subtype similar to Aichi/2/68 and Victoria/3/75 persist in pigs many years after their antigenic counterparts have disappeared from humans (Shortridge et al. (1977). Science 19, 1454-1455). To provide information on the mechanism of conservation of these influenza viruses in pigs, the hemagglutinin (HA) of four isolates from swine derived from Taiwan and Southern China were analyzed antigenically and genetically. The reactivity pattern of these viruses with a panel of monoclonal antibodies indicates that the HAs of these swine viruses were antigenically closely related to duck H3 and early human H3 viruses. Sequence analysis of the H3 genes from three swine viruses revealed that the swine H3 genes are more closely related to the duck genes than to early human H3 virus (A/Aichi/2/68). The degree of sequence homology of these genes is extremely high (more than 96.5%). Furthermore, the deduced amino acid sequence of the three swine HAs at residues 226 to 228 in the proposed receptor-binding site is Gln-Ser-Gly and is common with the majority of avian influenza viruses. These findings indicate that these H3 viruses may have been introduced into pigs from ducks. The HA gene of the fourth swine influenza virus from Southern China was genetically equally related to avian and early human H3 strains although the sequence through the receptor-binding pocket (226-228) was typical of a human H3 virus, suggesting that either this swine HA gene was derived from ducks or an early human H3 virus was introduced into the pig population where the virus accumulated substantial mutations. The present strains revealed genetic heterogeneity of swine H3 influenza viruses in nature.

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

The System L transporter facilitates cellular import of large neutral amino acids (AAs) such as Leu, a potent activator of the intracellular target of rapamycin (TOR) pathway, which signals for cell growth. System L is an AA exchanger, proposed to accumulate certain AAs by coupling to dissipation of concentration gradient(s) of exchange substrates generated by secondary active AA transporters such as System A (SNAT2). We addressed the hypothesis that this type of coupling (termed tertiary active transport) acts as an indirect mechanism to extend the range of AA stimulating TOR to those transported by both Systems A and L (e.g., Gln) through downstream enhancement of Leu accumulation. System A overexpression enabled Xenopus oocytes to accumulate substrate AAs (notably Ser, Gln, Ala, Pro, Met; totaling 2.6 nmol/oocyte) from medium containing a physiological AA mixture at plasma concentrations. Net accumulation of System L (4F2hc-xLAT1) substrates from this medium by System L-overexpressing oocytes was increased by 90% (from 0.7 to 1.35 nmol/oocyte; mainly Leu, Ile) when Systems A and L were coexpressed, coincident with a decline in accumulation of specific System A substrates (Gln, Ser, Met), as expected if the latter were also System L substrates and functional coupling of the transport Systems occurred. AA flux coupling was confirmed as trans-stimulation of Leu influx in System L-expressing oocytes by Gln injection (0.5 nmol/oocyte). The observed changes in Leu accumulation are sufficient to activate the TOR pathway in oocytes, although intracellular AA metabolism limits the potential for AA accumulation by tertiary active transport in this system.

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source-independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the DeltameaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.

Peptides derived from tryptic hydrolysate of jumbo squid (Dosidicus gigas) skin gelatin were assessed for their antioxidant properties in different in vitro assay systems. The hydrolysate itself exhibited a strong lipid peroxidation inhibition and it was much higher than that of natural antioxidant, alpha-tocopherol. In addition, it could scavenge highly active free radicals in oxidative systems, in the order of hydroxyl and carbon-centered radicals. Two representative peptides with comparatively higher antioxidant potency were purified and characterized as Phe-Asp-Ser-Gly-Pro-Ala-Gly-Val-Leu (880.18 Da) and Asn-Gly-Pro-Leu-Gln-Ala-Gly-Gln-Pro-Gly-Glu-Arg (1241.59 Da). Furthermore, viability of radical-mediated oxidation-induced human lung fibroblasts was enhanced following the treatment of two peptides. However it did not exhibit substantial ion chelation, and we presumed that the observed radical scavenging potency of these peptides play a vital role for their strong antioxidant activity. Based on our results we suggest that hydrophobic amino acids present in peptide sequences contributed greatly for observed antioxidant activities.

In view of its critical role in influenza A virus (IAV) tropism and pathogenesis, we evaluated the receptor binding properties of HA proteins of the closely related swine and new pandemic human IAVs. We generated recombinant soluble trimeric H1 ectodomains of several IAVs and analyzed their sialic acid binding properties using fetuin-binding and glycan array analysis. The results show that closely related swine and new pandemic H1 proteins differ dramatically in their ability to bind these receptors. Although new pandemic H1 protein exhibited hardly any binding, swine H1 bound efficiently to a number of α2-6-linked sialyl glycans. The responsible amino acids were identified by analyzing chimeric H1 proteins and by performing systematic site-directed mutagenesis of swine and new pandemic human H1 proteins. The difference was found to map to residues at positions 200 and 227. Although substitution of either residue significantly affected the binding phenotype, substitution of both was found to act synergistically and reverse the phenotype almost completely. Modeling of the T200A and E227A substitutions into the crystal structure of the new pandemic human H1 protein revealed the loss of potential hydrogen bond formation with Gln(191), which is part of the 190-loop of the receptor binding site, and with the penultimate galactose, respectively. Thus, a residue not belonging to the receptor binding site may affect the interaction of HA with its receptor. Interestingly, whereas alanine at position 200 is found in most new pandemic human viruses, the residue at position 227 in these viruses is invariably a glutamic acid.

Cerebrovascular amyloid beta-protein (Abeta) deposition is a pathological feature of several related disorders including Alzheimer disease and hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). HCHWA-D is caused by a point mutation in the gene that encodes the Abeta precursor and results in a Glu ->> Gln substitution at position 22 of Abeta. In comparison to Alzheimer disease, the cerebrovascular Abeta deposition in HCHWA-D is generally more severe, often resulting in intracerebral hemorrhage when patients reach 50 years of age. We recently reported that Abeta(1-42), but not the shorter Abeta(1-40) induces pathologic responses in cultured human leptomeningeal smooth muscle cells including cellular degeneration that is accompanied by a marked increase in the levels of cellular Abeta precursor and soluble Abeta peptide. In the present study, we show that the HCHWA-D mutation converts the normally nonpathologic Abeta(1-40) into a highly pathologic form of the peptide for cultured human leptomeningeal smooth muscle cells. These findings suggest that these altered functional properties of HCHWA-D mutated Abeta may contribute to the early and often severe cerebrovascular pathology that is the hallmark of this disorder.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ((38)SFNGGPQNTFEEFPLSDIE(56)) and PAR4 ((51)KSSDKPNPR downward arrow GYPGKFCANDSDTLELPASSQA(81), downward arrow = site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment (47)FEEFPLSDIE(56). Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d approximately 10 A with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.

Myosin-binding protein C (MyBP-C) binds to myosin with two binding sites, one close to the N terminus and the other at the C terminus. Here we present the solution structure of one part of the N-terminal binding site, the third immunoglobulin domain of the cardiac isoform of human MyBP-C (cC2) together with a model of its interaction with myosin. Domain cC2 has the beta-sandwich structure expected from a member of the immunoglobulin fold. The C-terminal part of the structure of cC2 is very closely related to telokin, the myosin binding fragment of myosin light chain kinase. Domain cC2 also contains two cysteines on neighboring strands F and G, which would be able to form a disulfide bridge in a similar position as in telokin. Using NMR spectroscopy and isothermal titration calorimetry we demonstrate that cC2 alone binds to a fragment of myosin, S2Delta, with low affinity (kD = 1.1 mM) but exhibits a highly specific binding site. This consists of the C-terminal surface of the C'CFGA' beta-sheet, which includes Glu(301), a residue mutated to Gln in the disease familial hypertrophic cardiomyopathy. The binding site on S2 was identified by a combination of NMR binding experiments of cC2 with S2Delta containing the cardiomyopathy-linked mutation R870H and molecular modeling. This mutation lowers the binding affinity and changes the arrangement of side chains at the interface. Our model of the cC2-S2Delta complex gives a first glimpse of details of the MyBP-C-myosin interaction. Using this model we suggest that most key interactions are between polar amino acids, explaining why the mutations E301Q in cC2 and R870H in S2Delta could be involved in cardiomyopathy. We expect that this model will stimulate future research to further refine the details of this interaction and their importance for cardiomyopathy.

Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly(143)-Gly(160) followed by the removal of a Val(161)-Ala(162) dipeptide at the N terminus of the C-terminal 16-kDa TRAP subunit. Cathepsin L initially released a shorter Gln(151)-Gly(160) peptide and completed processing at Ser(145) or Gly(143) at the C terminus of the N-terminal 23-kDa TRAP subunit and at Arg(163) at the N terminus of the C-terminal 16-kDa TRAP subunit. Mutation of Ser(145) to Ala partly mimicked the effect of proteolysis on catalytic activity, identifying Ser(145) as well as Asp(146) (Funhoff, E. G., Ljusberg, J., Wang, Y., Andersson, G., and Averill, B. A. (2001) Biochemistry 40, 11614-11622) as repressive amino acids of the loop region to maintain the TRAP enzyme in a catalytically latent state. The C-terminal sequence of TRAP isolated from rat bone was consistent with cathepsin K-mediated processing in vivo. Moreover, cathepsin K, but not cathepsin L, co-localized with TRAP in osteoclast-resorptive compartments, supporting a role for cathepsin K in the extracellular processing of monomeric TRAP in the resorption lacuna.

The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA(3) receptor has been proposed to be selectively activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA(1) and LPA(2) showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA sn-1 regioisomer. To identify residues involved in ligand recognition of LPA(3), we developed and validated computational models of LPA(3) complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 of LPA(3) form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA(3) underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC(50) of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.

Otoconia are biominerals within the utricle and saccule of the inner ear that are critical for the perception of gravity and linear acceleration. The classical mouse mutant tilted (tlt) and a new allele, mergulhador (mlh), are recessive mutations that affect balance by impairing otoconial morphogenesis without causing collateral deafness. The mechanisms governing otoconial biosynthesis are not known. Here we show that tlt and mlh are mutant alleles of a novel gene (Otopetrin 1, Otop1), encoding a multi-transmembrane domain protein that is expressed in the macula of the developing otocyst. Both mutants carry single point mutations leading to non-conservative amino acid substitutions that affect two putative transmembrane (TM) domains (tlt, Ala(151)->>Glu in TM3; mlh, Leu(408)->>Gln in TM8). Otop1 and Otop1-like paralogues, Otop2 and Otop3, define a new gene family with homology to the C. elegans and D. melanoganster DUF270 genes.

OBJECTIVE

A novel [(68)Ga]-labeled DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 peptide (BAY86-7548) having high affinity to bombesin receptor subtype II to detect primary and metastatic prostate carcinoma using positron emission tomography/computed tomography (PET/CT) was synthesized and evaluated for prostate cancer.

METHODS

In this first human study with BAY86-7548, 14 men scheduled for radical prostatectomy (n = 11) or with biochemical recurrence after surgery or hormonal therapy (n = 3) were enrolled. The patients received an intravenous injection of BAY86-7548 followed by over 60-minute dynamic imaging of prostate gland (n = 10) and/or subsequent whole-body imaging (n = 14). The visual assessment of PET/CT images included evaluation of intraprostatic (12 subsextants) and pelvic nodal uptake of BAY86-7548 in 11 surgical patients and detection of potential metastatic foci in all patients. In patients with biochemical recurrence, results were compared with those of either [(11)C]-acetate (n = 2) or [(18)F]-fluoromethylcholine (n = 1) PET/CT.

RESULTS

We found a sensitivity, specificity, and accuracy of 88%, 81% and 83%, respectively, for detection of primary PCa and sensitivity of 70% for metastatic lymph nodes using histology as gold standard. BAY86-7548 correctly detected local recurrence in prostate bed and showed nodal relapse in accordance with [(11)C]-acetate PET/CT in 2 patients with biochemical relapse. In the third hormone refractory patient, BAY86-7548 failed to show multiple bone metastases evident on [(18)F]-fluoromethylcholine PET/CT.

CONCLUSIONS

BAY86-7548 PET/CT is a promising molecular imaging technique for detecting intraprostatic prostate cancer.

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)->>Gln (alpha-hOgg1-Gln(46)) and Arg(154)->>His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll beta-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core beta-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4- and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. We show that the two molecules bind to the EphA4 ligand-binding domain with K(d) values of 20.4 and 26.4 microm, respectively. NMR heteronuclear single quantum coherence titrations revealed that upon binding, both molecules significantly perturb EphA4 residues Ile(31)-Met(32) in the D-E loop, Gln(43) in the E beta-strand, and Ile(131)-Gly(132) in the J-K loop. Molecular docking shows that they can occupy a cavity in the high affinity ephrin binding channel of EphA4 in a similar manner, by interacting mainly with the EphA4 residues in the E strand and D-E and J-K loops. However, many of the interactions observed in Eph receptor-ephrin complexes are absent, which is consistent with the small size of the two molecules and may account for their relatively weak binding affinity. Thus, our studies provide the first published structure of the ligand-binding domain of an EphA receptor of the A subclass. Furthermore, the results demonstrate that the high affinity ephrin binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.

Cell culture-adapted laboratory strains of Sindbis virus (SB) exhibit efficient initial attachment to cell surface heparan sulfate (HS) receptors. In contrast, non-cell-adapted strains, such as the SB consensus sequence virus TR339, interact weakly with HS and cell surfaces. Regardless of their HS binding phenotype, most SB strains do not cause fatal disease in adult mice, whether inoculated subcutaneously (s.c.) or intracranially (i.c.). However, laboratory strains of SB can be rendered neurovirulent for adult mice by introduction of a glutamine (Gln)-to-histidine (His) mutation at position 55 of the E2 envelope glycoprotein. In the current work, we have determined that E2 His 55-containing viruses require a second-site mutation (Glu to Lys) at E2 position 70 that confers efficient HS binding in order to exhibit virulence for adult mice and that virulence is correlated with very high infectivity for many cell types. Furthermore, introduction of E2 Lys 70 or certain other HS-binding mutations alone also increased morbidity and/or mortality over that of TR339 for older mice inoculated i.c. However, all viruses containing single HS-binding mutations were attenuated in s.c. inoculated suckling mice in comparison with TR339. These results suggest that HS binding may attenuate viral disease that is dependent on high-titer viremia; however, efficient cell attachment through HS binding can increase virulence, presumably through enhancing the replication of SB within specific host tissues such as the brain.