The interleukin 1beta converting enzyme (ICE) family plays a pivotal role in programmed cell death and has been implicated in stroke and neurodegenerative diseases. During reperfusion after filamentous middle cerebral artery occlusion, ICE-like cleavage products and tissue immunoreactive interleukin 1beta (IL-1beta) levels increased in ischemic mouse brain. Ischemic injury decreased after intracerebroventricular injections of ICE-like protease inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.FMK), acetyl-Tyr-Val-Ala-Asp-chloromethylketone, or a relatively selective inhibitor of CPP32-like caspases, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone, but not a cathepsin B inhibitor, N-benzyloxycarbonyl-Phe-Ala-fluoromethylketone. z-VAD.FMK decreased ICE-like cleavage products and tissue immunoreactive IL-1beta levels in ischemic mouse brain and reduced tissue damage when administered to rats as well. Only z-VAD.FMK and acetyl-Tyr-Val-Ala-Asp-chloromethylketone reduced brain swelling, and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone did not attenuate the ischemia-induced increase in tissue IL-1beta levels. The three cysteine protease inhibitors significantly improved behavioral deficits, thereby showing that functional recovery of ischemic neuronal tissue can follow blockade of enzymes associated with apoptotic cell death. Finally, we examined the effect of z-VAD.FMK on excitotoxicity and found that it protected against alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-induced or to a lesser extent N-methyl-D-aspartate-induced excitotoxic brain damage. Thus, ICE-like and CPP32-like caspases contribute to mechanisms of cell death in ischemic and excitotoxic brain injury and provide therapeutic targets for stroke and neurodegenerative brain damage.

During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 (alpha M beta 2, CD11b/CD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097-1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.

Filamentous hemagglutinin is a surface-associated adherence protein of Bordetella pertussis, which is a component of some new acellular pertussis vaccines. The nucleotide sequence of an open reading frame that encompasses the filamentous hemagglutinin structural gene, fhaB, suggests that proteolytic processing is necessary to generate the mature 220-kDa filamentous hemagglutinin product. An Arg-Gly-Asp (RGD) tripeptide is found within filamentous hemagglutinin that may be involved in its adherence properties. An internal in-frame deletion in fhaB, encompassing the RGD region, causes loss of B. pertussis-binding to ciliated eukaryotic cells, confirming a potential role for this protein in host-cell binding and infection.

An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

Oxidative stress, manifested by protein oxidation, lipid peroxidation, DNA oxidation and 3-nitrotyrosine formation, among other indices, is observed in Alzheimer's disease (AD) brain. Amyloid beta-peptide (1-42) [Abeta(1-42)] may be central to the pathogenesis of AD. Our laboratory and others have implicated Abeta(1-42)-induced free radical oxidative stress in the neurodegeneration observed in AD brain. This paper reviews some of these studies from our laboratory. Recently, we showed both in-vitro and in-vivo that methionine residue 35 (Met-35) of Abeta(1-42) was critical to its oxidative stress and neurotoxic properties. Because the C-terminal region of Abeta(1-42) is helical, and invoking the i + 4 rule of helices, we hypothesized that the carboxyl oxygen of lle-31, known to be within a van der Waals distance of the S atom of Met-35, would interact with the latter. This interaction could alter the susceptibility for oxidation of Met-35, i.e. free radical formation. Consistent with this hypothesis, substitution of lle-31 by the helix-breaking amino acid, proline, completely abrogated the oxidative stress and neurotoxic properties of Abeta(1-42). Removal of the Met-35 residue from the lipid bilayer by substitution of the negatively charged Asp for Gly-37 abrogated oxidative stress and neurotoxic properties of Abeta(1-42). The free radical scavenger vitamin E prevented A(beta (1-42)-induced ROS formation, protein oxidation, lipid peroxidation, and neurotoxicity in hippocampal neurons, consistent with our model for Abeta-associated free radical oxidative stress induced neurodegeneration in AD. ApoE, allele 4, is a risk factor for AD. Synaptosomes from apoE knock-out mice are more vulnerable to Abeta-induced oxidative stress (protein oxidation, lipid peroxidation, and ROS generation) than are those from wild-type mice. We also studied synaptosomes from allele-specific human apoE knock-in mice. Brain membranes from human apoE4 mice have greater vulnerability to Abeta(1-42)-induced oxidative stress than brain membranes from apoE2 or E3, assessed by the same indices, consistent with the notion of a coupling of the oxidative environment in AD brain and increased risk of developing this disorder. Using immunoprecipitation of proteins from AD and control brain obtained no longer than 4h PMI, selective oxidized proteins were identified in the AD brain. Creatine kinase (CK) and beta-actin have increased carbonyl groups, an index of protein oxidation, and Glt-1, the principal glutamate transporter, has increased binding of the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE). Abeta inhibits CK and causes lipid peroxidation, leading to HNE formation. Implications of these findings relate to decreased energy utilization, altered assembly of cytoskeletal proteins, and increased excitotoxicity to neurons by glutamate, all reported for AD. Other oxidatively modified proteins have been identified in AD brain by proteomics analysis, and these oxidatively-modified proteins may be related to increased excitotoxicity (glutamine synthetase), aberrant proteasomal degradation of damaged or aggregated proteins (ubiquitin C-terminal hydrolase L-1), altered energy production (alpha-enolase), and diminished growth cone elongation and directionality (dihydropyrimindase-related protein 2). Taken together, these studies outlined above suggest that Met-35 is key to the oxidative stress and neurotoxic properties of Abeta(1-42) and may help explain the apoE allele dependence on risk for AD, some of the functional and structural alterations in AD brain, and strongly support a causative role of Abeta(1-42)-induced oxidative stress and neurodegeneration in AD.

The molecular action of leptomycin B (LMB), an agent inducing arrest of the eukaryotic cell cycle at G1 and G2 phases, was investigated by analyzing an LMB resistance gene of Schizosaccharomyces pombe. A genomic library of an LMB-resistant mutant was screened for LMB resistance, and a DNA fragment containing an open reading frame (ORF) of 1078 amino acids was cloned on a multicopy vector. The plasmid was found to confer drug resistance specifically to LMB. Nucleotide sequencing revealed that the ORF was a mutant gene for the essential nuclear protein crm1, which had been reported to complement a cold-sensitive mutation causing deformed nuclear morphology. The gene product named crm1-N1 had two amino acid replacements (Gly-503 to Asp and Met-546 to Ile). Two allelic mutants of crm1 (crm1-809 and crm1-119) were found to be hypersensitive and resistant, respectively, to LMB. Nuclear morphology of the cold-sensitive crm1-809 mutant at the restrictive temperature was almost the same as that of the wild-type cells treated with LMB. Furthermore, a low concentration of LMB induced the intracellular accumulation of a 25-kDa protein in the wild-type cells, which was immunologically identical to the protein accumulating in the crm1-809 mutant cells. These results strongly suggest that LMB primarily inhibits the function of the crm1 gene which is required for maintaining higher order chromosome structures, correct gene expression, and cell growth in the fission yeast.

As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.

Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

The extracellular matrix (ECM) of bone and dentin contains several non-collagenous proteins. One category of non-collagenous protein is termed the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family, that includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE). These polyanionic SIBLING proteins are believed to play key biological roles in the mineralization of bone and dentin. Although the specific mechanisms involved in controlling bone and dentin formation are still unknown, it is clear that some functions of the SIBLING family members are dependent on the nature and extent of post-translational modifications (PTMs), such as phosphorylation, glycosylation, and proteolytic processing, since these PTMs would have significant effects on their structure. OPN and BSP are present in the ECM of bone and dentin as full-length forms, whereas amino acid sequencing indicates that DMP1 and DSPP exist as proteolytically processed fragments that result from scission of X-Asp bonds. We hypothesized that the processing of DMP1 and DSPP is catalyzed by the PHEX enzyme, since this protein, an endopeptidase that is predominantly expressed in bone and tooth, has a strong preference for cleavage at the NH2-terminus of aspartyl residue. We envision that the proteolytic processing of DMP1 and DSPP may be an activation process that plays a significant, crucial role in osteogenesis and dentinogenesis, and that a failure in this processing would cause defective mineralization in bone and dentin, as observed in X-linked hypophosphatemic rickets.

BACKGROUND

Metabolic perturbations arising from malignant transformation have not been systematically characterized in human lung cancers in situ. Stable isotope resolved metabolomic analysis (SIRM) enables functional analysis of gene dysregulations in lung cancer. To this purpose, metabolic changes were investigated by infusing uniformly labeled 13C-glucose into human lung cancer patients, followed by resection and processing of paired non-cancerous lung and non small cell carcinoma tissues. NMR and GC-MS were used for 13C-isotopomer-based metabolomic analysis of the extracts of tissues and blood plasma.

RESULTS

Many primary metabolites were consistently found at higher levels in lung cancer tissues than their surrounding non-cancerous tissues. 13C-enrichment in lactate, Ala, succinate, Glu, Asp, and citrate was also higher in the tumors, suggesting more active glycolysis and Krebs cycle in the tumor tissues. Particularly notable were the enhanced production of the Asp isotopomer with three 13C-labeled carbons and the buildup of 13C-2,3-Glu isotopomer in lung tumor tissues. This is consistent with the transformations of glucose into Asp or Glu via glycolysis, anaplerotic pyruvate carboxylation (PC), and the Krebs cycle. PC activation in tumor tissues was also shown by an increased level of pyruvate carboxylase mRNA and protein.

CONCLUSIONS

PC activation - revealed here for the first time in human subjects - may be important for replenishing the Krebs cycle intermediates which can be diverted to lipid, protein, and nucleic acid biosynthesis to fulfill the high anabolic demands for growth in lung tumor tissues. We hypothesize that this is an important event in non-small cell lung cancer and possibly in other tumor development.

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.

An extracellular protease of Staphylococcus aureus, strain V8, previously shown to cleave specifically the peptide bonds on the carboxyl-terminal side of either aspartate or glutamate residues in phosphate buffer (pH 7.8) hydrolyzes only glutamoyl bonds in either ammonium bicarbonate (pH 7.8) or ammonium acetate (pH 4.0). Of all aspartoyl bonds tested, only the Asp-Gly linkage is cleaved at a detectable rate. The staphylococcal protease hydrolyzes all of the seventeen different glutamoyl bonds studied, although those involving hydrophobic aminoacid residues with bulky side chains are cleaved at a lower rate.

The Group IV phospholipase A2 family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A2 (cPLA2) alpha, cPLA2beta, cPLA2gamma, cPLA2delta, cPLA2epsilon and cPLA2zeta. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA2s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA2s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA2alpha. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA2, PLA1, lysophospholipase and transacylase activities). cPLA2alpha, the most extensively studied Group IV PLA2, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA2 and lysophospholipase activities and is the only PLA2 that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA2alpha activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA2s suggests they may have distinct mechanisms of regulation and functional roles.

Hormones produced by adipose tissue play a critical role in the regulation of energy intake, energy expenditure, and lipid and carbohydrate metabolism. This review will address the biology, actions, and regulation of three adipocyte hormones-leptin, acylation stimulating protein (ASP), and adiponectin-with an emphasis on the most recent literature. The main biological role of leptin appears to be adaptation to reduced energy availability rather than prevention of obesity. In addition to the well-known consequences of absolute leptin deficiency, subjects with heterozygous leptin gene mutations have low circulating leptin levels and increased body adiposity. Leptin treatment dramatically improves metabolic abnormalities (insulin resistance and hyperlipidemia) in patients with relative leptin deficiency due to lipoatrophy. Leptin production is primarily regulated by insulin-induced changes of adipocyte metabolism. Dietary fat and fructose, which do not increase insulin secretion, lead to reduced leptin production, suggesting a mechanism for high-fat/high-sugar diets to increase energy intake and weight gain. ASP increases the efficiency of triacylglycerol synthesis in adipocytes leading to enhanced postprandial lipid clearance. In mice, ASP deficiency results in reduced body fat, obesity resistance, and improved insulin sensitivity. Adiponectin production is stimulated by thiazolidinedione agonists of peroxisome proliferator-activated receptor-gamma and may contribute to increased insulin sensitivity. Adiponectin and leptin cotreatment normalizes insulin action in lipoatrophic insulin-resistant animals. These effects may be mediated by AMP kinase-induced fat oxidation, leading to reduced intramyocellular and liver triglyceride content. The production of all three hormones is influenced by nutritional status. These hormones, the pathways controlling their production, and their receptors are promising targets for managing obesity, hyperlipidemia, and insulin resistance.

The human organic anion transporting polypeptide-C (OATP-C) (gene SLC21A6) is a liver-specific transporter importantly involved in the hepatocellular uptake of a variety of endogenous and foreign chemicals. In this study, we demonstrate the presence of multiple functionally relevant single-nucleotide polymorphisms (SNPs) in OATP-C in a population of African- and European-Americans. Moreover, examination of 14 nonsynonymous polymorphisms indicated that genotypic frequencies were dependent on race. Functional assessment of 16 OATP-C alleles in vitro revealed that several variants exhibited markedly reduced uptake of the OATP-C substrates estrone sulfate and estradiol 17beta-d-glucuronide. Specifically, alterations in transport were associated with SNPs that introduce amino acid changes within the transmembrane-spanning domains (T217C (Phe-73 ->> Leu), T245C (Val-82 ->> Ala), T521C (Val-174 ->> Ala), and T1058C (Ile-353 ->> Thr)) and also with those that modify extracellular loop 5 (A1294G (Asn-432 ->> Asp), A1385G (Asp-462 ->> Gly), and A1463C (Gly-488 ->> Ala)). Cell surface biotinylation experiments indicated that the altered transport activity of some OATP-C variants was due, in part, to decreased plasma membrane expression. Given the relatively high genotypic frequency of the T521C (14%) transition in European-Americans and the G1463C (9%) transversion in African-Americans, SNPs in OATP-C may represent a heretofore unrecognized factor influencing drug disposition.

Fas is a type-I membrane protein that transduces an apoptotic signal. Binding of Fas ligand or agonistic anti-Fas antibody to Fas kills the cells by apoptosis. Studies in the nematode Caenorhabditis elegans have suggested that proteases such as interleukin-1 beta-converting enzyme (ICE) or the product of the C. elegans cell-death gene ced-3 are involved in apoptotic signal transduction. The activity of ICE can be inhibited by the product of crmA, a cytokine-response modifier gene encoded by cowpox virus. We report here that expression of crmA inhibits cytotoxicity induced by anti-Fas antibody or tumour necrosis factor (TNF). We have found a specific ICE inhibitor tetrapeptide (acetyl-Tyr-Val-Ala-Asp-chloromethylketone) that also prevents apoptosis induced by anti-Fas antibody. These results suggest an involvement of an ICE-like protease in Fas-mediated apoptosis and TNF-induced cytotoxicity.

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21-amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr(168) to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr(168)-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

The c-kit proto-oncogene encodes a receptor tyrosine kinase. Binding of c-kit ligand, stem cell factor (SCF) to c-kit receptor (c-kitR) is known to activate c-kitR tyrosine kinase, thereby leading to autophosphorylation of c-kitR on tyrosine and to association of c-kitR with substrates such as phosphatidylinositol 3-kinase (PI3K). In a human mast cell leukemia cell line HMC-1, c-kitR was found to be constitutively phosphorylated on tyrosine, activated, and associated with PI3K without the addition of SCF. The expression of SCF mRNA transcript in HMC-1 cells was not detectable by means of PCR after reverse transcription (RT-PCR) analysis, suggesting that the constitutive activation of c-kitR was ligand independent. Sequencing of whole coding region of c-kit cDNA revealed that c-kit genes of HMC-1 cells were composed of a normal, wild-type allele and a mutant allele with two point mutations resulting in intracellular amino acid substitutions of Gly-560 for Val and Val-816 for Asp. Amino acid sequences in the regions of the two mutations are completely conserved in all of mouse, rat, and human c-kit. In order to determine the causal role of these mutations in the constitutive activation, murine c-kit mutants encoding Gly-559 and/or Val-814, corresponding to human Gly-560 and/or Val-816, were constructed by site-directed mutagenesis and expressed in a human embryonic kidney cell line, 293T cells. In the transfected cells, both c-kitR (Gly-559, Val-814) and c-kitR (Val-814) were abundantly phosphorylated on tyrosine and activated in immune complex kinase reaction in the absence of SCF, whereas tyrosine phosphorylation and activation of c-kitR (Gly-559) or wild-type c-kitR was modest or little, respectively. These results suggest that conversion of Asp-816 to Val in human c-kitR may be an activating mutation and responsible for the constitutive activation of c-kitR in HMC-1 cells.