Isoform-specific localization of A-RAF in mitochondria.

RAF kinase is a family of isoforms including A-RAF, B-RAF, and C-RAF. Despite the important role of RAF in cell growth and proliferation, little evidence exists for isoform-specific function of RAF family members. Using Western analysis and immunogold labeling, A-RAF was selectively localized in highly purified rat liver mitochondria. Two novel human proteins, which interact specifically with A-RAF, were identified, and the full-length sequences are reported. These proteins, referred to as hTOM and hTIM, are similar to components of mitochondrial outer and inner membrane protein-import receptors from lower organisms, implicating their involvement in the mitochondrial transport of A-RAF. hTOM contains multiple tetratricopeptide repeat (TPR) domains, which function in protein-protein interactions. TPR domains are frequently present in proteins involved in cellular transport systems. In contrast, protein 14-3-3, an abundant cytosolic protein that participates in many facets of signal transduction, was found to interact with C-RAF but not with A-RAF N-terminal domain. This information is discussed in view of the important role of mitochondria in cellular functions involving energy balance, proliferation, and apoptosis and the potential role of A-RAF in regulating these systems.

Understanding the cellular uptake of phosphopeptides.

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells--a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

C5a receptor antagonists.

The anaphylatoxin C5a is an extremely potent proinflammatory peptide produced during activation of the complement system. The structure of C5a includes a core region (N-terminal residues 1-63) consisting of four, antiparallel alpha-helices held together by three disulfide linkages and a structured C-terminal tail (residues 64-74). The C5a receptor belongs to the large class of seven transmembrane, G-protein-linked receptors. C5a appears to interact with its receptor at two sites: the C5a core binds to the receptor s N-terminal extracellular domain while C5a s tail binds the receptor near Arg206, near the membrane surface of transmembrane helix V. C5a receptors are concentrated on blood granulocytes (neutrophils, eosinophils, and basophils) and tissue inflammatory cells (macrophages, mast cells, microglia); thus the main effects of C5a are manifest as inflammation. Additionally, C5a receptors are also present, albeit in lower concentrations, on non-myeloid cells, e.g. endothelial and smooth muscle cells where they may further influence inflammatory reactions such as blood cell emigration and tissue edema. C5a has been implicated in myriad disorders, both acute and chronic; therefore a C5a receptor antagonist is predicted to have utility as a therapeutic agent. Unfortunately, few specific C5a receptor antagonists have been reported, and only two have demonstrated activity in vivo. Furthermore, those reported are peptidic and hence have limited application therapeutically. The current state of C5a receptor antagonists is discussed as well as the potential for their use against various human disorders. A model of C5a receptor dimerization is presented to account for the high potency of the disulfide antagonist C5aRAD.

Mapping the subsite preferences of protein tyrosine phosphatase PTP-1B using combinatorial chemistry approaches.

Protein tyrosine phosphatases (PTPases) are important regulators of signal transduction systems, but the specificity of their action is largely unexplored. We have approached this problem by attempting to map the subsite preferences of these enzymes using combinatorial chemistry approaches. Protein-tyrosine peptidomimetics containing nonhydrolyzable phosphotyrosine analogues bind to PTPases with high affinity and act as competitive inhibitors of phosphatase activity. Human PTP-1B, a PTPase implicated to play an important role in the regulation of growth factor signal transduction pathways, was used to screen a synthetic combinatorial library containing malonyltyrosine as a phosphotyrosine mimic. Using two cross-validating combinatorial chemistry screening approaches, one using an iterative method and the other employing library affinity selection-mass spectrometric detection, peptides with high affinity for PTP-1B were identified and subsite preferences were detailed by quantitatively comparing residues of different character. Consistent with previous observations, acidic residues were preferred in subsites X-3 and X-2. In contrast, aromatic substitutions were clearly preferred at the X-1 subsite. This information supports the concept that this class of enzymes may have high substrate specificity as dictated by the sequence proximal to the phosphorylation site. The results are discussed with regards to the use of combinatorial techniques in order to elucidate the interplay between enzyme subsites.

The RAF family: an expanding network of post-translational controls and protein-protein interactions.

Protein kinase RAF is strategically located in the "Ras-MAP-kinase signal transduction pathway", a principle system which transmits signals from growth factor receptors to the nucleus, resulting in cell proliferation. Growth factor responses are mediated in part by activation of Ras, which in turn activates RAF to phosphorylate MEK, its downstream substrate. MEK activates MAP-kinase to influence nuclear events. It is clear, however, that a network of signals other than those carried by Ras plays a role in RAF regulation. These orthogonal influences are mediated by: serine/threonine kinases, tyrosine kinases, and protein-protein interactions. As a further complication to the RAF network, three isoforms of RAF have been established which have divergent N-terminal regulatory domains. Whereas these divergent regulatory domains implicate isoform-specific functions, no clear evidence or hypothesis for distinct functions for individual isoforms has been presented. Recently, "isoform-specific protein interactions" have been identified among numerous proteins interacting with RAF. These studies may serve to delineate independent functions for RAF isoforms.

Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Exposure of human vascular smooth muscle cells to Raf-1 antisense oligodeoxynucleotides: cellular responses and pharmacodynamic implications.

To characterize the pharmacodynamic properties of CGP 69846A/ISIS 5132, an antisense oligodeoxynucleotide directed against the mitogenic signal transducer Raf-1 kinase, we investigated the elicited biological responses in human coronary artery smooth muscle cells. Cell exposure to CGP 69846A resulted in a reversible time- and concentration-dependent down-regulation of cellular Raf-1 gene expression and, ultimately, inhibition of cell cycle progression. The highest potencies of this compound to reduce Raf-1 mRNA and protein levels were observed after 24 and 48 hr of cell exposure, respectively, with corresponding IC50 values of approximately 100 and approximately 300 nM. Proliferation was inhibited with an IC50 value of approximately 300 nM after 72 hr. We interpreted the recovery rate of Raf-1 mRNA after cell exposure to antisense ODNs as the half-life (t1/2 approximately 50 hr) of active intracellular CGP 69846A in our cell culture system. The endogenous Raf-1 turnover half-life of approximately 30 hr, as assessed by monitoring metabolically labeled Raf-1 protein, correlated kinetically with the antisense-induced protein decay rate (50% decay in approximately 33 hr), indicating that the efficiency of CGP 69846A in decreasing Raf-1 protein levels was rate-limited by the endogenous protein turnover rate. The pharmacodynamic effects of CGP 69846A antisense ODNs are therefore limited by the duration of its intracellular activity rather than by its ability to transiently decrease mRNA levels. Local steady state exposure to CGP 69846A may represent a new approach to prevent the transition of quiescent vascular smooth muscle cells into the pathologically hyperproliferating cells seen after angioplasty.

Structural definition of the C5a C terminus by two-dimensional nuclear magnetic resonance spectroscopy.

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)-C5a, determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an alpha-helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel alpha-helical bundle. This C-terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor.

Characterization, crystallization and preliminary crystallographic analysis of human recombinant cyclooxygenase-2.

Purified recombinant membrane apoprotein cyclooxygenase-2 (COX-2) has been reconstituted with heme and characterized. The holoprotein has been crystallized in complex with the selective inhibitor CGP 28238 [6-(2,4-difluorophenoxy)-5- methylsulfonylamino-l-indanone] by the sitting-drop method of vapor diffusion using polyethylene glycol 2000 monomethyl ether as precipitant, in the presence of the nonionic detergent beta-octylglucoside. The crystals are orthorhombic, belonging to the space group P2(1)2(1)2 with cell dimensions a = 209.56, b = 71.28 and c = 93.82 A, and diffract to 2.5 A resolution. The asymmetric unit contains two COX-2 monomers, as confirmed by the molecular replacement solution and in agreement with the dimeric structure of the detergent-solubilized protein found with dynamic light scattering and size-exclusion chromatography. Structural work is in progress.

Molecular and cellular characterization of baboon C-Raf as a target for antiproliferative effects of antisense oligonucleotides.

C-Raf is a an essential member of the growth factor-ras pathway and a target for intervention strategies aimed at blocking cell proliferative responses. Excessive smooth muscle proliferation is considered one cause of the arterial closure in restenosis. Because of the similarity to the human cardiovascular system, a useful current animal model of the disease is a baboon model. As a foundation for animal studies employing antisense oligonucleotides, efforts were made to characterize the molecular and cellular biology of the baboon system. The nucleotide sequence of baboon c-raf cDNA was determined. Antisense phosphorothioate oligonucleotides specific to the 3'-UTR of c-raf mRNA from human and baboon were compared using primary baboon smooth muscle cells in culture. A particular human antisense oligonucleotide, referred to as ISIS 5132, was different by only 2 of 20 bases from the baboon sequence. The corresponding baboon antisense oligonucleotide ISIS 12959, however, was markedly more effective to inhibit c-raf mRNA, protein production, and DNA synthesis, and the results attest to the species specificity of the approach. After antisense treatment, c-raf mRNA levels dropped rapidly, whereas protein levels decreased with a half-life of roughly 24-48 hours, consistent with the antiproliferative effects. The data are discussed with regard to the profile of protein-protein interactions made by C-Raf and with the view that the baboon system closely parallels the human one at the signal transduction level. As this work progressed, a baboon cDNA homolog of a human c-raf-2 pseudogene was isolated, sequenced, and shown to be transcribed into mRNA.

Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.

Synthetic combinatorial libraries have proven to be a valuable source of diverse structures useful for large-scale biochemical screening. Their use has greatly facilitated the study of protein-protein interactions. We have developed a practical technique for screening such libraries by integrating affinity chromatography selection with electrospray ionization mass spectrometric detection, referred to as library affinity selection-mass spectrometry (LAS-MS). The process allows for rapid and efficient screening of solution phase libraries and provides detailed information such as the relative affinities of substrates. The method is generally applicable to include nonpeptide libraries; moreover, electrospray tandem mass spectrometry (ES-MS/MS) yields sequence-specific identification of individual components without the need for chemical tags. This technique is demonstrated using the Src homology 2 (SH2) domain of phosphatidylinositol 3-kinase (PI 3-kinase). The critical importance of methionine in the position +3 (relative to the phosphotyrosine position) is demonstrated in a library built with a phosphotyrosine mimic, (phosphonodifluoromethyl)phenylalanine. The described method has broad applicability to combinatorial library screening.

Phospholipase D regulation by a physical interaction with the actin-binding protein gelsolin.

Increases in intracellular phosphatidic acid levels caused by receptor- mediated activation of phospholipase D (PLD) have been implicated in many signal transduction pathways leading to cellular activation. PLD is known to be regulated by several means, including tyrosine kinase activity, increases in Ca2+, receptor-coupled G proteins, small GTP binding proteins, ceramide metabolisms, and protein kinase C. We have investigated a additional regulatory effect on PLD activity involving nucleoside triphosphates (NTPs). A NTP binding protein copurifies with LPD activity from rabbit brains using a GTP-agarose affinity column, and this protein stimulates PLD activity only in the absence of NPTs. The NTP effect is reversible and labile, and the binding protein is separable from the PLD activity by heparin-agarose chromatography. We identified this protein as the actin- binding protein gelsolin by amino acid sequencing following peptide mapping. This finding was verified by the co-immunoprecipitation of gelsolin and PLD activity as well as by the reconstitution of gelsolin- dependent nucleotide sensitive PLD activity by the addition of purified gelsolin-free PLD. Our data indicate that actin rearrangements and PLD signaling are coordinately regulated through the physical association between PLD and gelsolin and that this interaction may also serve to amplify both PLD signaling and actin reorganization.

Comparison of recombinant cyclooxygenase-2 to native isoforms: aspirin labeling of the active site.

The search for isoform-specific enzyme inhibitors has been the focus of much recent research effort. Towards this goal, human recombinant cyclooxygenase-2 (EC 1.14.99.1, prostaglandin H synthase) was expressed in insect cells and purified to > 98% purity. Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX-2 and COX-1. After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX-1 and corresponded to the enzyme dimer as measured with size-exclusion chromatography. Peptide mapping via Lys-C protease identified a site of N-linked glycosylation and the aspirin covalent modification site. In the presence of heme, aspirin-specifically acetylated Ser-516. The enzyme will be suitable for biophysical studies and may lead to isoform-specific enzyme inhibitors.

Stabilization of C5a receptor--G-protein interactions through ligand binding.

Binding of biotin-C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin-agarose affinity chromatography resulted in the isolation of a receptor complex with associated G-proteins. In contrast, when receptor was detergent-solubilized in the absence of C5a and purified by affinity chromatography with Affigel-C5a, G-proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor--G-protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor-effector interactions. When biotin-C5a-ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both Gialpha2 and Gialpha3 subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor-transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems.

Radioiodination of transforming growth factor-beta (TGF-beta) in a modified Bolton-Hunter reaction system.

We have optimized the use of the Bolton-Hunter reagent to prepare 125I-labeled transforming growth factor-beta (TGF-beta). Conditions were developed to obtain monovalent modification of human-recombinant TGF-beta 2 (hrTGF-beta 2) at a basic pH necessary for efficient protein acylation (> or = 26% of theoretical) while obviating the problems of TGF-beta aggregation/precipitation. The purified Bolton-Hunter labeled hrTGF-beta 2 had a specific activity of 1.8-2.1 microCi/pmol, and the 125I label was fully acid-precipitable. [125I]hrTGF-beta 2 was electrophoretically indistinguishable from unlabeled starting material and displayed full immunoreactivity with polyclonal anti-TGF-beta 2 antibody. Both hrTGF-beta 2 and Bolton-Hunter-labeled [125I]hrTGF-beta 2 inhibited the growth of mink lung epithelial cells with equal efficacy. These data validate a modified conjugation-iodination method for TGF-beta and invite general use of the Bolton-Hunter reagent for iodination of other TGF-beta isoforms and peptides similarly susceptible to precipitation/aggregation under standard Bolton-Hunter incubation conditions.

Characterization of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes.

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

Binding of glucocorticoid receptors to model DNA response elements.

DNA sequences were synthesized that contained the consensus 15-base pair glucocorticoid receptor binding site linked to flanking sequences of various lengths. Binding of these synthetic oligomers to glucocorticoid receptor, employing a reconstituted binding system with purified components, indicated that a minimal size of approximately 45 base pairs was necessary to bind the receptor optimally. Sequences containing multiple receptor binding sites competed more effectively for binding. These findings are consistent with recent demonstrations that multiple control elements act synergistically to affect transcriptional control by glucocorticoids and confirm that regions flanking the consensus GRE binding site are instrumental in optimizing binding interactions.

Substrate phosphorylation can inhibit proteolysis by trypsin-like enzymes.

The effect of substrate phosphorylation on the susceptibility to proteolytic cleavage by trypsin-like enzymes was investigated using the model heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly, a peptide representing the endogenous phosphorylation site of pyruvate kinase. Phosphorylation of Ser 5 altered the kinetics of proteolysis by two proteases, trypsin and rat plasma kallikrein, both of which cleaved between Arg 3 and Ala 4. In the case of trypsin, phosphorylation decreased the rate of cleavage 47-fold. In the case of rat plasma kallikrein, phosphorylation decreased proteolysis 13-fold. Phosphorylation resulted in an apparent redirection of the preferential site from Arg 3 to Arg 2. Because sequences analogous to this model peptide are commonly found in exposed domains of globular proteins, and since these regions are susceptible to both phosphorylation and protease attack, the results indicate that substrate phosphorylation may selectively influence protein processing and turnover.

Structure-activity relationships for glycine-extended peptides and the alpha-amidating enzyme derived from medullary thyroid CA-77 cells.

A peptidyl alpha-amidating enzyme has been partially purified from conditioned medium derived from cultured medullary thyroid CA-77 cells. The interactions of this enzyme with a series of tripeptides, pentapeptides, and mature glycine-extended prohormones has now been studied using a competition assay that features the enzymatic alpha-amidation of N-dansyl-Tyr-Val-Gly. While a peptide C-terminal glycine was obligatory for tight binding to the alpha-amidating enzyme, other peptide structural elements modulated the interaction. Thus, a greater than 1300-fold range in apparent inhibitor constants was observed by substitution at the -1 (penultimate) position in a C-terminal glycine-containing tripeptide with each of the 20 common L-amino acids. Peptide inhibitory potency decreased through the following amino acid groupings: sulfur containing greater than aromatic greater than or equal to histidine greater than nonpolar greater than polar greater than glycine greater than charged. This pattern was qualitatively dissimilar to that observed for a more limited series of substitutions at the -2 position, demonstrating the positional selectivity of these structural requirements. The structure-activity relationships observed with the tripeptides at the -1 position were consistent with the apparent inhibitor constants obtained for a collection of prohormones and their pentapeptide mimics. Finally, selected prohormones and their pentapeptide mimics were equipotent inhibitors, demonstrating that the peptide structural elements important for alpha-amidating enzyme recognition are located entirely within the C-terminal pentapeptide segment.