Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.

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Spinach NADPH:ferredoxin oxidoreductase (EC 1.6.7.1) catalyzes the NADPH-dependent reduction of the anthracyclines daunomycin, aclacinomycin A, and nogalamycin and their respective 7-deoxyanthracyclinones. Under anaerobic conditions, the endogenous rate of O2 reduction by NADPH catalyzed by ferredoxin reductase (0.12 s-1 at pH 7.4) is augmented by the anthracyclines and 7-deoxyanthracyclinones. The catalytic constants are approximately equivalent for this augmentation for all substrates (approximate V of 2 s-1 and KM of 75 microM). Both O2- and H2O2 are made. Under anaerobic conditions, anthracycline reduction catalyzed by ferredoxin reductase results in the elimination of the C-7 substituent to provide a quinone methide intermediate. Following tautomerization by C-7 protonation, 7-deoxyanthracyclinones are obtained. Under appropriate conditions these may be further reduced to the 7-deoxyanthracyclinone hydroquinones. For daunomycin, the quinone methide is formed rapidly after reduction and is easily monitored at 600 nm. It may react with electrophiles other than H+, as demonstrated by its competitive trapping by p-carboxybenzaldehyde. It may also react with nucleophiles, as demonstrated by its competitive trapping by N-acetylcysteine. For aclacinomycin, quinone methide formation is also rapid although no distinct transient near 600 nm occurs. In addition to protonation, it reacts with itself providing the 7,7'-dimer. With ethyl xanthate as a thiolate nucleophile, adducts derived from addition to C-7 are obtained. For nogalamycin, quinone methide formation is not rapid. Nogalamycin is reduced to its hydroquinone, which slowly converts in a first-order process [k = (1.2 +/- 0.2) X 10(-3) s-1, pH 8.0, 30 degrees C] to the quinone methide, which is then quenched by protonation. Spinach ferredoxin in its reduced form is chemically competent for anthracycline reduction. Its effect on both the aerobic and anaerobic reactions catalyzed by ferredoxin reductase is to increase severalfold the overall velocity for anthracycline reduction. In conclusion, the aerobic reaction pathways for the anthracyclines as mediated by ferredoxin reductase are remarkably similar, while the anaerobic reactions are remarkably different. If these anthracyclines exert their antitumor activity by a common anaerobic pathway, it is most likely that the pathway is determined by the properties of the anthracycline as complexed to its in vivo target. The behavior of ferredoxin further suggests that not only low-potential flavin centers but also iron-sulfur centers should be regarded as important loci for anthracycline reductive activation.

No abstract

In the alpha subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake alpha-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind alpha-bungarotoxin (alpha-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for alpha-BTX. Using synthetic peptides corresponding to the complete AChR alpha subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for alpha-BTX binding between amino acid residues 181 and 200 [Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R. Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230; McLane, K. E., Wu, X., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265, 1537-1544]. To elucidate the structural requirements for alpha-BTX binding, we initially used nonconservative single amino acid substitution analogues of the parental alpha(181-200) sequence, and we found that residues at positions 188-190 (VYY), and 192-194 (CCP) and several flanking residues seemed to be involved in alpha-BTX binding [Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, A., & Maelicke, A. (1991) Biochemistry 30, 2575-2584]. In the present study, amino acid residues previously found to affect alpha-BTX binding were replaced by different conservative single amino acid substitutions, in order to determine the nature of the amino acid side-chain interactions with alpha-BTX. Whereas V188 could be replaced by Ile or Thr with minor effects on alpha-BTX binding, substitution of Phe, His, or Thr for Y189 and Y190 resulted in large to moderate decreases in alpha-BTX binding. Similarly, alpha-BTX binding activity was intolerant to substitutions of C192 or C193 with Ser, His, or Val. Structural changes of the peptide alpha(181-200) induced by substitution of P194 or P197 with two adjacent Gly residues, and insertion of a Gly between C192 and C193, were also incompatible with alpha-BTX binding. Conservative substitutions of other aliphatic and aromatic residues resulted in only minor effects on alpha-BTX binding, as did replacements of K185 and D195 that changed or maintained the charge distribution of peptide alpha (181-200). The recognition site for alpha-BTX formed by the prototope alpha(181-200), therefore, involves important interactions with Y189, Y190, C192, and C193 that are highly specific to the amino acid residue at that position.(ABSTRACT TRUNCATED AT 400 WORDS)

The apo chicken egg white riboflavin binding protein complexes several anthracycline antitumor antibiotics and their metabolites. The Kd value for three important anthracycline glycosides (adriamycin, daunomycin, aclacinomycin A) is approximately identical at 0.5 micro M. The anthracycline occupies the flavin binding site in this complex, having its D-B rings overlaying the region normally occupied by the riboflavin A-C rings, respectively. The glycoside of the anthracycline, attached to C-7 of the A ring, is exposed to the solvent; consequently, the binding protein discriminates poorly between anthracycline A ring geometric isomers. Anthracyclinones, metabolites lacking the C-7 glycoside, are bound about 10-fold more tightly. The basis for the occupancy by anthracyclines of this flavin binding site is a steric homology (both ligands contain planar, linear conjugated rings) permitting the energetically favorable displacement of water from the hydrophobic pocket. The binding protein-anthracycline complex has been used for anaerobic aqueous redox titrations. Dithionite reduction of the anthracycline glycoside provides, in a two-electron process, the 7-deoxyanthracyclinone, via reductive elimination. The bound 7-deoxyanthracyclinone is then further two-electron reduced to the bound hydroquinone. Semiquinone radical intermediates are observed transiently; these are neither stable in solution nor complexed to the binding protein. Oxidation of the hydroquinone is accomplished by several reagents (oxygen, hydrogen peroxide, ferricyanide, cytochrome c). In the case of 7-deoxydaunomycinone hydroquinone, a mixture of two products is produced upon oxidation; these are the chromo-7-deoxydaunomycinone (identical with the first 2-e- reduction intermediate) and its leuco isomer, 8-acetyl-7,8,9,10-tetrahydro-5,8,12-trihydroxy-1-methoxy-6,11-naphthacenedione (iso-7-deoxydaunomycinone). These studies provide useful information concerning the redox properties of the anthracyclines and suggest that these antitumor antibiotics may be capable of functioning as riboflavin antagonists in vivo.