l-Asparaginyl and l-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing l-isoaspartyl and d-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein l-isoaspartate(d-aspartate) O-methyltransferase (EC 184.108.40.206). The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal l-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues. To define the physiological relevance of this protein-repair pathway and to determine whether deficient protein repair would cause central nervous system dysfunction, we used gene targeting in mouse embryonic stem cells to generate protein l-isoaspartate(d-aspartate) O-methyltransferase-deficient mice. Analyses of tissues from methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain...
“Natural” polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor α) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common “public” epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.
Group I allergens are the major allergens of grass pollen, but their biological function is unknown. These proteins are shown here to be structurally related to expansins, which are able to induce extension (creep) of plant cell walls. Extracts of maize pollen possess potent expansin-like activity, as measured in wall extension and wall stress-relaxation assays. This activity is selective for grass cell walls and is, at least partly, due to the action of maize group I allergens. We propose that group I allergens facilitate invasion of the pollen tube into the maternal tissues by loosening the cell walls of the grass stigma and style. Additionally, the presence of related mRNAs in vegetative tissues of rice, Arabidopsis, and soybean implies that allergen homologs may function to loosen walls in growing vegetative tissues as well.
Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.
The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the transducins, is a key component in transducing the responses to compounds that humans equate with bitter and sweet. Rod transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein βγ subunits to provide the receptor-interacting heterotrimer. The taste receptor–gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin. Finally...
Rational protein design is an emerging approach for testing general theories of protein chemistry through the creation of new structures and functions. Here we present the first successful introduction by rational design of a [Fe4S4] cuboidal cluster into the hydrophobic core of Escherichia coli thioredoxin, a protein normally devoid of metal centers. Cuboidal [Fe4S4] is one of the stable forms of self-assembled iron–sulfur clusters that are thought to represent some of the earliest evolved biological redox centers. [Fe4S4] clusters have been recruited for use in a variety of proteins whose functions are central to many of the major biochemical processes ranging from simple soluble electron-transfer agents, to membrane-bound components of electron-transfer chains, to electron reservoirs in complex metalloenzymes such as nitrogenase. By situating an [Fe4S4] cluster into a protein environment not previously adapted by evolution we can explore the factors by which their activity is modulated by the protein matrix.
The special elongation factor SelB of Escherichia coli promotes selenocysteine incorporation into formate dehydrogenases. This is thought to be achieved through simultaneous binding to selenocysteyl-tRNASec and, in the case of formate dehydrogenase H, to an fdhF mRNA hairpin structure 3′ adjacent to the UGA selenocysteine codon. By in vitro selection, novel RNA sequences (“aptamers”), which can interact tightly and specifically with SelB, were isolated from an RNA library. The library was comprised of mutagenized variants of the wild-type fdhF mRNA hairpin. One-half of the selected sequences contained the apical stem–loop of the fdhF mRNA hairpin highly conserved. Some of the aptamers showed deviations in the primary sequence within this region of the wild-type fdhF hairpin motif while still binding with high affinity to SelB. Binding studies performed with truncated versions of SelB revealed that aptamers binding to different sites on the protein have been selected. To dissect SelB binding to the fdhF hairpin from the overall biological function of this complex, four selected aptamers were analyzed in vivo for UGA readthrough in a lacZ fusion construct. Among these, one promoted UGA readthrough in vivo. Three of the aptamers...
Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet—which is also a widespread motif involved in cell-to-cell adhesion—can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs...
Solution at 2.5-Å resolution of the three-dimensional structure of H-2Ld with a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (β2m) and peptide. Consistent with the biological data that show an unusually weak association of Ld with β2m, a novel orientation of the α1/α2 domains of Ld relative to β2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ld antigen-binding cleft determine the unique motif of peptides that it binds. Ld has no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ld and its peptide antigen. The unique structural interactions of Ld with β2m and peptide could make Ld more suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.
A method is proposed for selecting and aligning images of single biological particles to obtain high-resolution structural information by cryoelectron microscopy. The particles will be labeled with multiple heavy atom clusters to permit the precise determination of particle locations and relative orientations even when imaged close to focus with a low electron dose, conditions optimal for recording high-resolution detail. Heavy atom clusters should also allow selection of images free from many kinds of defects, including specimen movement and particle inhomogeneity. Heavy atom clusters may be introduced in a general way by the construction of “adaptor” molecules based on single-chain Fv antibody fragments, consisting of a constant framework region engineered for optimal cluster binding and a variable antigen binding region selected for a specific target. The success of the method depends on the mobility of the heavy atom cluster on the particle, on the precision to which clusters can be located in an image, and on the sufficiency of cluster projections alone to orient and select particles for averaging. The necessary computational algorithms were developed and implemented in simulations that address the feasibility of the method.
Activation energies for the individual steps of secretory and viral fusion are reported to be large [Oberhauser, A. F., Monck, J. R. & Fernandez, J. M. (1992) Biophys. J. 61, 800–809; Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990) Biochemistry 29, 1303–1308]. Understanding the cause for these large activation energies is crucial to defining the mechanisms of these two types of biological membrane fusion. We showed recently that the fusion of protein-free model lipid bilayers mimics the sequence of steps observed during secretory and viral fusion, suggesting that these processes may involve common lipid, rather than protein, rearrangements. To test for this possibility, we determined the activation energies for the three steps that we were able to distinguish as contributing to the fusion of protein-free model lipid bilayers. Activation energies for lipid rearrangements associated with formation of the reversible first intermediate, with conversion of this to a semi-stable second intermediate, and with irreversible fusion pore formation were 37 kcal/mol, 27 kcal/mol, and 22 kcal/mol, respectively. The first and last of these were comparable to the activation energies observed for membrane lipid exchange (42 kcal/mol) during viral fusion and for the rate of fusion pore opening during secretory granule release (23 kcal/mol). This striking similarity suggests strongly that the basic molecular processes involved in secretory and viral fusion involve a set of lipid molecule rearrangements that also are involved in model membrane fusion.
Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4Rα/IL-2Rγ (class I IL-4R), whereas endothelial cells express another type, IL-4Rα/IL-13Rα (class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2Rγ. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/R121E retained physical interaction with IL-2Rγ but not IL-13Rα; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activities in vitro that suggest utility in the treatment of certain autoimmune diseases.
Although phosphorylation of Thr-197 in the activation loop of the catalytic subunit of cAMP-dependent protein kinase (PKA) is an essential step for its proper biological function, the kinase responsible for this reaction in vivo has remained elusive. Using nonphosphorylated recombinant catalytic subunit as a substrate, we have shown that the phosphoinositide-dependent protein kinase, PDK1, expressed in 293 cells, phosphorylates and activates the catalytic subunit of PKA. The phosphorylation of PKA by PDK1 is rapid and is insensitive to PKI, the highly specific heat-stable protein kinase inhibitor. A mutant form of the catalytic subunit where Thr-197 was replaced with Asp was not a substrate for PDK1. In addition, phosphorylation of the catalytic subunit can be monitored immunochemically by using antibodies that recognize Thr-197 phosphorylated enzyme but not unphosphorylated enzyme or the Thr197Asp mutant. PDK1, or one of its homologs, is thus a likely candidate for the in vivo PKA kinase that phosphorylates Thr-197. This finding opens a new dimension in our thinking about this ubiquitous protein kinase and how it is regulated in the cell.
Rotating waves (rotors) of cellular activity were observed in nonconfluent cultures of embryonic chick heart cells by using a macroscopic imaging system that detected fluorescence from intracellular Ca2+. Unlike previous observations of rotors or spiral waves in other systems, the rotors did not persist but exhibited a repetitive pattern of spontaneous onset and offset leading to a bursting rhythm. Similar dynamics were observed in a cellular automaton model of excitable media that incorporates spontaneous initiation of activity, and a decrease of excitability as a consequence of rapid activity (fatigue). These results provide a mechanism for bursting dynamics in normal and pathological biological processes.
As the oxidation of low density lipoprotein (LDL) lipids may be a key event in atherogenesis, there is interest in antioxidants as potential anti-atherogenic compounds. Here we report that α-tocopheryl hydroquinone (α-TQH2) strongly inhibited or completely prevented the (per)oxidation of ubiquinol-10 (CoQ10H2), α-tocopherol (α-TOH), and both surface and core lipids in LDL exposed to either aqueous or lipophilic peroxyl radicals, Cu2+, soybean lipoxygenase, or the transition metal-containing Ham’s F-10 medium in the absence or presence of human monocyte-derived macrophages. The antioxidant activity of α-TQH2 was superior to that of several other lipophilic hydroquinones, including endogenous CoQ10H2, which is regarded as LDL’s first line of antioxidant defence. At least three independent activities contributed to the antioxidant action of α-TQH2. First, α-TQH2 readily associated with LDL and instantaneously reduced the lipoprotein’s ubiquinone-10 to CoQ10H2, thereby maintaining this antioxidant in its active form. Second, α-TQH2 directly intercepted aqueous peroxyl radicals, as indicated by the increased rate of its consumption with increasing rates of radical production, independent of LDL’s content of CoQ10H2 and α-TOH. Third...
Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.
The Ski oncogene has dramatic effects on the differentiation of several different cell types. It induces the differentiation of quail embryo cells into myoblasts and arrests the differentiation of chicken hematopoietic cells. The mechanism that Ski uses to carry out these disparate biological activities is unknown. However, we were struck by the similarity of these effects to those of certain members of the nuclear hormone receptor family. Both Ski and the thyroid hormone receptor-derived oncogene v-ErbA can arrest the differentiation of avian erythroblasts, and v-Ski-transformed avian multipotent progenitor cells resemble murine hematopoietic cells that express a dominant-negative form of the retinoic acid receptor, RARα. In this paper, we have tested the hypothesis that v-Ski and its cellular homologue c-Ski exert their effects by interfering with nuclear hormone receptor-induced transcription. We demonstrate that Ski associates with the RAR complex and can repress transcription from a retinoic acid response element. The physiological significance of this finding is demonstrated by the ability of high concentrations of a RARα-specific ligand to abolish v-Ski-induced transformation of the multipotent progenitors. These results strongly suggest that the ability of Ski to alter cell differentiation is caused in part by the modulation of RAR signaling pathways.
Approximately 2 μmol of a novel prokaryotic pheromone, involved in starvation-induced aggregation and formation of fruiting bodies by the myxobacterium Stigmatella aurantiaca, were isolated by a large-scale elution procedure. The pheromone was purified by HPLC, and high-resolution MS, IR, 1H-NMR, and 13C-NMR were used to identify the active substance as the hydroxy ketone 2,5,8-trimethyl-8-hydroxy-nonan-4-one, which has been named stigmolone. The analysis was complicated by a solvent-dependent equilibrium between stigmolone and the cyclic enol-ether 3,4-dihydro-2,2,5-trimethyl-6-(2-methylpropyl)-2H-pyran formed by intramolecular nucleophilic attack of the 8-OH group at the ketone C4 followed by loss of H2O. Both compounds were synthesized chemically, and their structures were confirmed by NMR analysis. Natural and synthetic stigmolone have the same biological activity at ca. 1 nM concentration.
The model of the human neurocognitive architecture proposed by evolutionary psychologists is based on the presumption that the demands of hunter-gatherer life generated a vast array of cognitive adaptations. Here we present an alternative model. We argue that the problems inherent in the biological markets of ancestral hominids and their mammalian predecessors would have required an adaptively flexible, on-line information-processing system, and would have driven the evolution of a functionally plastic neural substrate, the neocortex, rather than a confederation of evolutionarily prespecified social cognitive adaptations. In alignment with recent neuroscientific evidence, we suggest that human cognitive processes result from the activation of constructed cortical representational networks, which reflect probabilistic relationships between sensory inputs, behavioral responses, and adaptive outcomes. The developmental construction and experiential modification of these networks are mediated by subcortical circuitries that are responsive to the life history regulatory system. As a consequence, these networks are intrinsically adaptively constrained. The theoretical and research implications of this alternative evolutionary model are discussed.
We have developed an approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities. For this, we have applied directly matrix-assisted laser desorption/ionization MS to hemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular masses of immune-induced molecules and allowed the detection, in the molecular range of 1.5–11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides, and four correspond to already characterized antimicrobial peptides. We have further analyzed the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response. We also describe a methodology combining matrix-assisted laser desorption ionization time-of-flight MS, HPLC, and Edman degradation, which yielded the peptide sequence of three of the DIMs. Finally, molecular cloning and Northern blot analyses revealed that one of the DIMs is produced as a prepropeptide and is inducible on a bacterial challenge.