Formation and properties of S-protein complex with S-peptide-containing fusion protein

A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S-protein by RNase activity assays. The possibility of effective purification of S-peptide-containing proteins by affinity chromatography on an S-protein-Sepharose column has been demonstrated.     

A buried polar interaction can direct the relative orientation of helices in a coiled coil

Coiled coils consist of bundles of two or more alpha-helices that are aligned in a parallel or an antiparallel relative orientation. The designed peptides, Acid-p1 and Base-p1, associate in solution to form a parallel, heterodimeric two-stranded coiled coil [O'Shea, E. K., Lumb, K. J., and Kim, P. S. (1993) Curr. Biol. 3, 658]. The buried interface of this complex is formed by hydrophobic Leu residues, with the exception of an Asn residue from each strand that is positioned to engage in a buried polar interaction. Substitution of these buried Asn residues by Leu residues results in a loss of structural uniqueness, as evidenced by a lack of a particular helix orientation in the Acid-Base coiled-coil complex [Lumb, K. J., and Kim, P. S. (1995) Biochemistry 34, 8642]. Here, we alter the positions of the Asn residues in the Acid and Base peptides such that a buried polar interaction is only expected to occur when the helices are in an antiparallel orientation. The resulting peptides, Acid-a1 and Base-a1, associate to form a helical heterodimer, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. The helix orientation preference has been measured using covalently linked, disulfide-containing heterodimers in which the constituent peptides are constrained to interact in either a parallel or an antiparallel orientation. Although both the parallel and antiparallel heterodimers form stable, helical structures, the antiparallel heterodimer is the predominant species at equilibrium when the heterodimers are allowed to undergo thiol-disulfide exchange. In addition, the antiparallel heterodimer is more stable to chemical denaturation than the parallel counterpart by approximately 2.3 kcal/mol. These results demonstrate that a single buried polar interaction in the interface between the helices of a coiled coil is sufficient to determine the relative orientation of its constituent helices.     

Structural properties of the putative fusion peptide of fertilin, a protein active in sperm-egg fusion, upon interaction with the lipid bilayer

We recently demonstrated that a peptide representing the putative fusion domain of fertilin, a surface membrane protein of sperm involved in sperm-egg fusion, induces fusion of large unilamellar vesicles containing negatively charged lipids [Martin, I., and Ruysschaert, J. M. (1997) FEBS Lett. 405, 351-355]. In the present work, we demonstrate that increasing the concentration in negatively charged lipids strongly enhances the binding of the fertilin fusion peptide to the membrane, suggesting that electrostatic attractions play a crucial role in the binding process. While no significant change of the secondary structure content is observed by increasing the amounts of negatively charged lipids in the bilayer, the orientation of the alpha-helix changes from a parallel to an oblique orientation in the membrane. This topological change is confirmed by amide II hydrogen/deuterium exchange measurements that monitor the accessibility of the peptide to the water medium. Differential scanning calorimetry data also suggest that the fertilin fusion peptide lowers the bilayer to hexagonal phase transition temperature of model membranes composed of mixtures of dipalmitoleoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylserine and therefore promotes negative curvature in lipid vesicles. A comparison of the biophysical properties and the membrane-perturbing activities of fertilin and of viral fusion peptides is discussed in terms of sperm-egg fusion and virus cell fusion.     

Extremely fast folding of a very stable leucine zipper with a strengthened hydrophobic core and lacking electrostatic interactions between helices

The dimer interface of a leucine zipper involves hydrophobic as well as electrostatic interactions between the component helices. Here we ask how hydrophobic effects and electrostatic repulsion balance the rate of folding and thermodynamic stability of a designed dimeric leucine zipper formed by the acidic peptide A that contains four repeating sequence units, (abcdefg)4. The aliphatic a and d residues of peptide A were the same as in the GCN4 leucine zipper but the e and g positions were occupied by Glu, which prevented folding above pH 6 because of electrostatic repulsion. Leucine zipper A2 was formed by protonation of the e and g side chains with a sharp transition midpoint at pH 5.2. Folding could be described by a two-state transition from two unfolded random coil monomers to a coiled coil dimer. There was a linear relationship between the logarithm of the rate constants and the number of repulsive charges on the folded leucine zipper dimer. The same linear relationship applied to the free energy of unfolding and the number of repulsive charges at thermodynamic equilibrium. Fully protonated peptide A folded at a near diffusion-limited rate (kon = 3 x 10(8) M-1 s-1), and the free energy of folding was -55 kJ mol-1 at 25 degrees C. The present work shows that protonation of Glu in positions e and g increases both the folding rate and the stability of the leucine zipper in the absence of any interhelical electrostatic interactions. Protonated Glu is proposed to act like a nonpolar residue and to strengthen the hydrophobic core by folding back toward the core residues in the a and d positions. This effect adds more to the free energy of unfolding and to the rate of folding than maximizing the number of salt bridges across the helix interface in an electrostatically stabilized heterodimeric leucine zipper [Wendt, H., Leder, L., H?rm?, H., Jelesarov, I., Baici, A., and Bosshard, H. R. (1997) Biochemistry 36, 204-213].     

Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein

Immature airways are highly compliant compared to the adult, suggesting that trachea and bronchi from immature animals may be easily compressed. Although tracheal compression has been extensively studied, the effect of transmural pressure on occlusion of immature bronchi has been neglected. The transmural pressure at which the lumen closed was determined from the transmittance of pressure along the lumen of isolated bronchi from late-term foetal, 1 and 4 week old pigs. Bronchi from eight cases of sudden infant death syndrome (SIDS) were also studied. In several experiments, smooth muscle tone was produced either by electrical field stimulation or carbachol challenge, and the relationship between active muscle tone and resting transmural pressure was studied. Bronchi from foetal, 1 and 4 week old pigs were occluded by intraluminal pressures of -4, -5 and -24 cmH2O. SIDS bronchi closed at -11 cmH2O. Histological and endoscopic investigations showed that closure of the bronchi occurred along a plane and was not uniform along the bronchus. Carbachol precontraction increased the transmural pressure required to close bronchi by approximately 5 cmH2O. The relationship between muscle tone and resting pressure was the same in all age groups, except when transmural pressure was at or below closing pressure. Bronchi from immature animals and human infants are vulnerable to collapse by small changes in transmural pressure. Bronchial closure is partly dependent on smooth muscle tone, particularly in younger animals.     

Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.     

Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.     

Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.     

Partial purification and characterization of maize-leaf pyruvate, orthophosphate dikinase regulatory protein: a low-abundance, mesophyll-chloroplast stromal protein

We focused here on steady-state mRNA levels of genes involved in antioxidant defense, i.e., manganese superoxide dismutase and copper-zinc superoxide dismutase, and in cell proliferation, i.e., ornithine decarboxylase, c-jun, and glyceraldehyde-3-phosphate-dehydrogenase in whole-lung homogenates from Fischer 344 rats at 3 h to 20 d after exposure to crocridolite asbestos. Changes in gene expression were correlated with histopathologic findings, total and differential cell counts in bronchoalveolar lavage, and levels of hydroxyproline in lung. Dosage-dependent increases in mRNA levels of antioxidant enzymes and proliferation-related genes were observed. Differential cell counts revealed a dose-related infiltration of neutrophils that preceded elevations in gene expression. Neutrophil infiltration into lung and focal lesions of fibrosis as well as increased levels of hydroxyproline were observed only at high concentrations of asbestos. These results indicate that high airborne concentrations of asbestos cause molecular changes in lung that may be related to antioxidant defense and the triggering of cell proliferation, a feature of asbestosis and lung cancer.     

HRX leukemic fusion proteins form a heterocomplex with the leukemia-associated protein SET and protein phosphatase 2A

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene at chromosome locus 11q23, resulting in HRX fusion proteins. Using the yeast two-hybrid system, in vitro binding studies, and human cell culture coimmunoprecipitation experiments, we show here that a region of the HRX protein that is consistently retained in HRX leukemic fusion proteins interacts directly with SET, another protein implicated in leukemia. We have identified the binding sites on HRX for SET and show that these sequences are clustered near the A.T hooks that have been shown to bind DNA. We also show that carboxyl-terminal SET sequences, possibly the acidic tail of SET, bind to HRX. We have also found serine/threonine-specific protein phosphatase activity in anti-HRX coimmunoprecipitates. Using the phosphatase inhibitor okadaic acid and Western blotting, the phosphatase was identified as protein phosphatase 2A (PP2A). Mutation of a single amino acid in one of the SET binding sites of HRX resulted in lower amounts of both coimmunoprecipitated SET protein and coimmunoprecipitated PP2A. These results suggest that the leukemogenic effects of HRX fusion proteins may be related to interactions with SET and PP2A.     

Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit

Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.     

Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA-binding protein

Cyclin B is a regulatory subunit of maturation-promoting factor. In goldfish (Carassius auratus) oocytes, cyclin B is synthesized de novo after stimulation by 17alpha,20beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone). In this study, we examined goldfish oocyte proteins bound to cyclin B mRNA. Using oligo(dT)-cellulose affinity chromatography and northwestern blotting analysis, we identified a 54-kDa cyclin B mRNA-binding protein (p54). Southwestern blotting analysis showed the binding of p54 to the Y box DNA element (CTGATTGGCCAA), suggesting that p54 is a Y box protein in goldfish. We isolated two cDNA clones, GFYP1 and GFYP2, the latter of which encodes a germ-cell-specific Y box protein. An antibody against a GFYP2 protein recognized p54, suggesting that p54 is identical or highly similar to GFYP2 protein. This is also supported by the finding that a recombinant GFYP2 expressed in bacteria bound to both the Y box DNA element and the goldfish cyclin B mRNA synthesized in vitro. These results suggest that p54 is a germ-cell-specific Y box protein and is a potential masking protein of cyclin B mRNA in goldfish oocytes.     

TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion

We previously identified BET3 by its genetic interactions with BET1, a gene whose SNARE-like product acts in endoplasmic reticulum (ER)-to-Golgi transport. To gain insight into the function of Bet3p, we added three c-myc tags to its C-terminus and immunopurified this protein from a clarified detergent extract. Here we report that Bet3p is a member of a large complex ( approximately 800 kDa) that we call TRAPP (transport protein particle). We propose that TRAPP plays a key role in the targeting and/or fusion of ER-to-Golgi transport vesicles with their acceptor compartment. The localization of Bet3p to the cis-Golgi complex, as well as biochemical studies showing that Bet3p functions on this compartment, support this hypothesis. TRAPP contains at least nine other constituents, five of which have been identified and shown to be highly conserved novel proteins.     

Purification and activity of a wheat 9-kDa lipid transfer protein expressed in Escherichia coli as a fusion with the maltose binding protein

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.