A novel glutamine–RNA interaction identified by screening libraries in mammalian cells

The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These results, together with the structure of a Rev peptide-Rev response element complex, suggest that the carboxamide groups of glutamine or asparagine are well-suited to hydrogen bond to G-A base pairs and begin to establish an RNA recognition code for the arginine-rich motif. The screening approach may provide a relatively general method for screening expression libraries in mammalian cells.

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ1I. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its Ka value is 0.20 × 106 M−1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Expressed protein ligation: A general method for protein engineering

A protein semisynthesis method—expressed protein ligation—is described that involves the chemoselective addition of a peptide to a recombinant protein. This method was used to ligate a phosphotyrosine peptide to the C terminus of the protein tyrosine kinase C-terminal Src kinase (Csk). By intercepting a thioester generated in the recombinant protein with an N-terminal cysteine containing synthetic peptide, near quantitative chemical ligation of the peptide to the protein was achieved. The semisynthetic tail-phosphorylated Csk showed evidence of an intramolecular phosphotyrosine-Src homology 2 interaction and an unexpected increase in catalytic phosphoryl transfer efficiency toward a physiologically relevant substrate compared with the non-tail-phosphorylated control. This work illustrates that expressed protein ligation is a simple and powerful new method in protein engineering to introduce sequences of unnatural amino acids, posttranslational modifications, and biophysical probes into proteins of any size.

A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the Escherichia coli cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.

Analysis of a peptide hormone–receptor interaction in the yeast two-hybrid system

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a β-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 → His, Glu-9 → Val, Arg-37 → Gly, and Met-59 → Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 → Phe and Ile-43 → Phe), and two gave no detectable signal (Leu-14 → Arg and Glu-46 → Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 → Pro) that allowed for a strong IGF-1–receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.

Design of fast enzymes by optimizing interaction potential in active site

The diffusional encounter between substrate and enzyme, and hence catalytic efficiency, can be enhanced by mutating charged residues on the surface of the enzyme. In this paper we present a simple method for screening such mutations. This is based on our earlier result that electrostatic enhancement of the enzyme-substrate binding rate constant can be accounted for just by the interaction potential within the active site. Assuming that catalytic and structural integrity is maintained, the catalytic efficiency can be optimized by surface charge mutations which lead to stronger interaction potential within the active site. Application of the screening method on superoxide dismutase shows that only charge mutations close to the active site will have practical effect on the catalytic efficiency. This rationalizes a large number of findings obtained in previous simulation and experimental studies.

Interaction of the Doa4 Deubiquitinating Enzyme with the Yeast 26S Proteasome

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin–proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

Identification of Osteopontin as a Novel Ligand for the Integrin α8β1 and Potential Roles for This Integrin–Ligand Interaction in Kidney Morphogenesis

Epithelio–mesenchymal interactions during kidney organogenesis are disrupted in integrin α8β1-deficient mice. However, the known ligands for integrin α8β1—fibronectin, vitronectin, and tenascin-C—are not appropriately localized to mediate all α8β1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for α8β1. We have coexpressed the extracellular domains of the mouse α8 and β1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the α8β1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with α8β1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In “far Western” blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to α8β1-AP. Cell adhesion assays using K562 cells expressing α8β1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin α8β1 may help regulate kidney development and other morphogenetic processes.

A regulator of G protein signaling interaction surface linked to effector specificity

Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the α subunits (Gα) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual Gα, and GTP hydrolysis rates of different Gαs can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-Gα binding interface and extends to include additional functionally important residues on the surface. One of these is within helix α3, two are in α5, and three are in the loop connecting α5 and α6. A cluster of surface residues on Gα previously identified by ET, and composed predominantly of residues from the switch III region and helix α3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the γ subunit of Gtα's effector, cGMP phosphodiesterase (PDEγ). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-Gα complex. Sequence variations in these residues correlate with PDEγ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.

Gene-experience interaction alters the cholinergic septohippocampal pathway of mice

Spatial learning requires the septohippocampal pathway. The interaction of learning experience with gene products to modulate the function of a pathway may underlie use-dependent plasticity. The regulated release of nerve growth factor (NGF) from hippocampal cultures and hippocampus, as well as its actions on cholinergic septal neurons, suggest it as a candidate protein to interact with a learning experience. A method was used to evaluate NGF gene-experience interaction on the septohippocampal neural circuitry in mice. The method permits brain region-specific expression of a new gene by using a two-component approach: a virus vector directing expression of cre recombinase; and transgenic mice carrying genomic recombination substrates rendered transcriptionally inactive by a “floxed” stop cassette. Cre recombinase vector delivery into transgenic mouse hippocampus resulted in recombination in 30% of infected cells and the expression of a new gene in those cells. To examine the interaction of the NGF gene and experience, adult mice carrying a NGF transgene with a floxed stop cassette (NGFXAT) received a cre recombinase vector to produce localized unilateral hippocampal NGF gene expression, so-called “activated” mice. Activated and control nonactivated NGFXAT mice were subjected to different experiences: repeated spatial learning, repeated rote performance, or standard vivarium housing. Latency, the time to complete the learning task, declined in the repeated spatial learning groups. The measurement of interaction between NGF gene expression and experience on the septohippocampal circuitry was assessed by counting retrogradely labeled basal forebrain cholinergic neurons projecting to the hippocampal site of NGF gene activation. Comparison of all NGF activated groups revealed a graded effect of experience on the septohippocampal pathway, with the largest change occurring in activated mice provided with repeated learning experience. These data demonstrate that plasticity of the adult spatial learning circuitry can be robustly modulated by experience-dependent interactions with a specific hippocampal gene product.

Does a neuroimmune interaction contribute to the genesis of painful peripheral neuropathies?

Painful peripheral neuropathies are precipitated by nerve injury from disease or trauma. All such injuries will be accompanied by an inflammatory reaction, a neuritis, that will mobilize the immune system. The role of the inflammation itself is difficult to determine in the presence of structural damage to the nerve. A method has been devised to produce a focal neuritis in the rat sciatic nerve that involves no more than trivial structural damage to the nerve. This experimental focal neuritis produces neuropathic pain sensations (heat- and mechano-hyperalgesia, and cold- and mechano-allodynia) in the ipsilateral hind paw. The abnormal pain sensations begin in 1–2 days and last for 4–6 days, with a subsequent return to normal. These results suggest that there is a neuroimmune interaction that occurs at the outset of nerve injury (and perhaps episodically over time in slow developing conditions like diabetic neuropathy) that produces neuropathic pain. The short duration of the phenomena suggest that they may prime the system for more slowly developing mechanisms of abnormal pain (e.g., ectopic discharge in axotomized primary afferent neurons) that underlie the chronic phase of painful neuropathy.

A maximum likelihood algorithm for genome mapping of cytogenetic loci from meiotic configuration data.

Frequencies of meiotic configurations in cytogenetic stocks are dependent on chiasma frequencies in segments defined by centromeres, breakpoints, and telomeres. The expectation maximization algorithm is proposed as a general method to perform maximum likelihood estimations of the chiasma frequencies in the intervals between such locations. The estimates can be translated via mapping functions into genetic maps of cytogenetic landmarks. One set of observational data was analyzed to exemplify application of these methods, results of which were largely concordant with other comparable data. The method was also tested by Monte Carlo simulation of frequencies of meiotic configurations from a monotelodisomic translocation heterozygote, assuming six different sample sizes. The estimate averages were always close to the values given initially to the parameters. The maximum likelihood estimation procedures can be extended readily to other kinds of cytogenetic stocks and allow the pooling of diverse cytogenetic data to collectively estimate lengths of segments, arms, and chromosomes.