Phosphorylated Nitrate Reductase and 14-3-3 Proteins1 : Site of Interaction, Effects of Ions, and Evidence for an AMP-Binding Site on 14-3-3 Proteins

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.

Interaction of Cryptochrome 1, Phytochrome, and Ion Fluxes in Blue-Light-Induced Shrinking of Arabidopsis Hypocotyl Protoplasts1

Protoplasts isolated from red-light-adapted Arabidopsis hypocotyls and incubated under red light exhibited rapid and transient shrinking within a period of 20 min in response to a blue-light pulse and following the onset of continuous blue light. Long-persisting shrinkage was also observed during continuous stimulation. Protoplasts from a hy4 mutant and the phytochrome-deficient phyA/phyB double mutant of Arabidopsis showed little response, whereas those from phyA and phyB mutants showed a partial response. It is concluded that the shrinking response itself is mediated by the HY4 gene product, cryptochrome 1, whereas the blue-light responsiveness is strictly controlled by phytochromes A and B, with a greater contribution by phytochrome B. It is shown further that the far-red-absorbing form of phytochrome (Pfr) was not required during or after, but was required before blue-light perception. Furthermore, a component that directly determines the blue-light responsiveness was generated by Pfr after a lag of 15 min over a 15-min period and decayed with similar kinetics after removal of Pfr by far-red light. The anion-channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid prevented the shrinking response. This result, together with those in the literature and the kinetic features of shrinking, suggests that anion channels are activated first, and outward-rectifying cation channels are subsequently activated, resulting in continued net effluxes of Cl− and K+. The postshrinking volume recovery is achieved by K+ and Cl− influxes, with contribution by the proton motive force. External Ca2+ has no role in shrinking and the recovery. The gradual swelling of protoplasts that prevails under background red light is shown to be a phytochrome-mediated response in which phytochrome A contributes more than phytochrome B.

Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

The β and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein–protein interactions with the β clamp. Thus, β interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

Nucleotide-specific interaction of Ran/TC4 with nuclear transport factors NTF2 and p97.

The use of permeabilized cell models to study nuclear protein import has led to the identification of cytosolic components of the import machinery, including the NLS receptor, p97, Ran/TC4, and nuclear transport factor 2 (NTF2). These proteins are required to reconstitute docking of transport ligand at the nuclear pore complex and subsequent translocation through the nuclear pore. However, a detailed molecular understanding of how these factors mediate protein import is lacking. Here we describe the results of solution and solid phase binding assays, which demonstrate that the small GTPase Ran/TC4 interacts directly with the cytosolic transport factors p97 and NTF2. By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively. These data together with previous studies lead us to suggest that the interaction of the GTP-bound form of Ran/TC4 with p97 is linked to an early step in the nuclear protein import pathway and that the association of the GDP-bound form of Ran/TC4 with NTF2 helps define vectorial transport.

Interaction of the protein import and folding machineries of the chloroplast.

We report the molecular cloning of import intermediate associated protein (IAP) 100, a 100-kDa protein of the chloroplast protein import machinery of peas. IAP100 contains two potential alpha-helical transmembrane segments and also behaves like an integral membrane protein. It was localized to the inner chloroplast envelope membrane. Immunoprecipitation experiments using monospecific anti-IAP100 antibodies and a nonionic detergent-generated chloroplast lysate gave the following results. (i) The four integral membrane proteins of the outer chloroplast import machinery were not coprecipitated with IAP100 indicating that the inner and outer membrane import machineries are not coupled in isolated chloroplasts. (ii) the major protein that coprecipitated with IAP100 was identified as stromal chaperonin 60 (cpn60); the association of IAP100 and cpn60 was specific and was abolished when immunoprecipitation was carried out in the presence of ATP. (iii) In a lysate from chloroplasts that had been preincubated for various lengths of time in an import reaction with radiolabeled precursor (pS) of the small subunit of Rubisco, we detected coimmunoprecipitation of IAP100, cpn60, and the imported mature form (S) of precursor. Relative to the time course of import, coprecipitation of S first increased and then decreased, consistent with a transient association of the newly imported S with the chaperonin bound to IAP100. These data suggest that IAP100 serves in recruiting chaperonin for folding of newly imported proteins.

Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.

Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, respectively. Both genes are approximately 16 kb and are comprised of 13 exons and 12 introns, the junctions of which conform to donor and acceptor site consensus sequences. Putative CAAT and TATA boxes are located, respectively, at positions -147 and -40 of the Npt2 gene and -143 and -51 of the NPT2 gene, relative to nucleotide 1 of the corresponding cDNAs. The translation initiation site is within exon 2 of both genes. The first 220 bp of the mouse and human promoter regions exhibit 72% identity. Two transcription start sites (at positions -9 and - 10 with respect to nucleotide 1 of NaPi-7 cDNA) and two polyadenylylation signals were identified in the Npt2 gene by primer extension, 5' and 3' rapid amplification of cDNA ends (RACE). A 484-bp 5' flanking region of the Npt2 gene, comprising the CAAT box, TATA box, and exon 1, was cloned upstream of a luciferase reporter gene; this construct significantly stimulated luciferase gene expression, relative to controls, when transiently transfected into OK cells, a renal cell line expressing type II Na+ -Pi cotransporter activity. The present data provide a basis for detailed analysis of cis and trans elements involved in the regulation of Npt2/NPT2 gene transcription and facilitate screening for mutations in the NPT2 gene in patients with autosomally inherited disorders of renal Pi reabsorption.

Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein.

Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids. Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms. Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms. Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties. Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes. COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro. When overexpressed in COS-1 cells, Nuc was found to be extracellularly released. However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80%. This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell. It also identifies Nuc as a potential regulator of COXs through this interaction.

Earthquake prediction: the interaction of public policy and science.

Earthquake prediction research has searched for both informational phenomena, those that provide information about earthquake hazards useful to the public, and causal phenomena, causally related to the physical processes governing failure on a fault, to improve our understanding of those processes. Neither informational nor causal phenomena are a subset of the other. I propose a classification of potential earthquake predictors of informational, causal, and predictive phenomena, where predictors are causal phenomena that provide more accurate assessments of the earthquake hazard than can be gotten from assuming a random distribution. Achieving higher, more accurate probabilities than a random distribution requires much more information about the precursor than just that it is causally related to the earthquake.

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements.

The Drosophila CF2II protein, which contains zinc fingers of the Cys2His2 type and recognizes an A+T-rich sequence, behaves in cell culture as an activator of a reporter chloramphenicol acetyltransferase gene. This activity depends on C-terminal but not N-terminal zinc fingers, as does in vitro DNA binding. By site-specific mutagenesis and binding site selection, we define the critical amino acid-base interactions. Mutations of single amino acid residues at the leading edge of the recognition helix are rarely neutral: many result in a slight change in affinity for the ideal DNA target site; some cause major loss of affinity; and others change specificity for as many as two bases in the target site. Compared to zinc fingers that recognize G+C-rich DNA, CF2II fingers appear to bind to A+T-rich DNA in a generally similar manner, but with additional flexibility and amino acid-base interactions. The results illustrate how zinc fingers may be evolving to recognize an unusually diverse set of DNA sequences.

Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.

Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.