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Complex formation between the human papilloma virus type-16 E7 protein (HPV-16 E7) and the retinoblastoma suppressor protein (pRB) is believed to be important in the process of cellular transformation that leads to cervical carcinoma. Utilizing an in vitro solution assay as well as a plate binding assay that measures the association between HPV-16 E7 and pRB proteins, we have examined a series of synthetic HPV-16 E7 peptides. HPV-16 E7 peptides which lie between amino acid residues 14 and 32 were found to be potent inhibitors of E7/pRB binding. The minimal peptide structure that possessed full antagonist activity was N-acetyl-E7-(21-29)-peptide amide. This peptide inhibited 100% of E7/pRB binding and exhibited an IC50 of 40 nM in the plate binding assay. A purified beta-galactosidase-E7 fusion protein exhibited an IC50 of 2 nM in the same assay. These results suggest that other regions of the E7 molecule in addition to amino acids 21-29 may contributed to E7/pRB interaction. Analysis of E7-(20-29)-peptides containing single amino acid substitutions suggests that Cys24, Tyr23, Tyr25, Asp21, and Glu26 are important residues for maintaining maximal antagonist activity. This series of peptides should prove useful in analyzing the biological consequences of E7/pRB binding in HPV-infected cells.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

Gastrin releasing peptide (GRP) is a peptide hormone containing 27 amino acids which is structurally analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated in the pathophysiology of small cell lung cancer. Previous work has demonstrated that the modified C terminus of GRP, N-acetyl-GRP-20-27, exerts full agonist activity in a variety of assay systems. However, no systematic comparison of binding of GRP fragments to its receptor and mitogenic potency has been reported. To investigate whether smaller GRP fragments could bind to the GRP receptor without stimulating mitogenesis, we performed binding inhibition and thymidine uptake assays with Swiss 3T3 fibroblasts. These studies were facilitated by the development of a novel tritiated GRP-based radioligand, [3H-Phe15] GRP-15-27, which exhibits enhanced chemical stability compared to iodinated GRP derivatives. We examined a series of C-terminal GRP fragments, from the pentapeptide to the octapeptide, with both N-acetyl and free amine moieties at the N terminus. N-Acetylated derivatives were more potent than their primary amine counterparts in both assays. Deletion of N-terminal residues from GRP-20-27 resulted in significant loss of potency in both assays: the EC50 values of N-acetyl-GRP-21-27 were 10(2)-fold higher than N-acetyl-GRP-20-27, those of N-acetyl-GRP-22-27 were 10(4)-fold higher, and N-acetyl-GRP-23-27 showed minimal activity at concentrations below 100 microM. These results suggest that 1) both His20 and Trp21 play an important role in binding of GRP to the receptor, and 2) for this series of N-terminal deletions, binding to the receptor and mitogenic activity are tightly coupled.

Semisynthetic analogues of insulin were prepared from derivatives of desoctapeptide-(B23-30)-insulin (DOI). A1, B1-(Boc)2-DOI (di-Boc-DOI) was converted to A1, B1-(Boc)2-DOI-B22-phenylhydrazide (di-Boc-DOI-NHNH-C6H5) by the trypsin-catalyzed addition of phenylhydrazine in aqueous organic solvents at pH 6.5 [Canova-Davis, E., & Carpenter, F. H. (1981) Biochemistry 20, 7053-7058]. Treatment of di-Boc-DOI-NHNH-C6H5 with BNPS-skatole produced the phenyldiimide. The latter was coupled with a variety of protected peptides that, after removal of protecting groups, yielded the following compounds whose biological activities were compared to that of insulin in binding, in stimulation of hexose transport (), and in the stimulation of lipogenesis [)), in terms of percent of insulin activity, all in the isolated epididymal fat cell: di-Boc-DOI 0.2, (0.1), [0.2]; di-Boc-DOI-NHNH-C6H5 0.5, (0.2), [0.5]; DOI 0.2, (0.2), [0.1]; DOI-(Gly)B23 0.2, (0.2), [0.1]; DOI-(Gly-Phe)B23-24 6.3, (6.3), [8.0]; DOI-(Gly-Phe-Phe)B23-25 17.0, (25.6), [24.7]; DOI-(Gly-Phe-Phe-Tyr)B23-26 59.0, (50.0), [69.0]. The semisynthetic derivatives represent a stepwise readdition of the aromatic residues near the C terminus of the B chain. A given analogue demonstrated comparable activity in all three biological assays. The results indicate that the stepwise addition of aromatic residues to the B-chain C terminus of DOI produces an increase in insulin-like activity. The biological activity of DOI-(Gly-Phe-Phe-Tyr)B23-26, the derivative in which the aromatic region has been completely reassembled, is the same order of magnitude as that of insulin.

Human tumor necrosis factor (hTNF) mediates a variety of biologic activities, which are dependent on the attachment of hTNF to cell-surface receptors. To identify regions of the hTNF protein involved in binding hTNF to its receptor, we prepared five synthetic peptides [hTNF-(1-15), hTNF-(1-31), hTNF-(65-79), hTNF-(98-111), and hTNF-(124-141)] and two hydroxylamine cleavage fragments [hTNF-(1-39) and hTNF-(40-157)] of hTNF. The hTNF-synthetic peptides and hTNF fragments were tested in hTNF receptor binding assays and in two biologic assays: cytolysis of tumor cells and suppression of lipoprotein lipase in adipocytes. Neither the synthetic peptides nor hTNF fragments were active agonists or antagonists in these assays. The synthetic peptides were also conjugated to thyroglobulin, and peptide-specific antisera were raised. All five peptide-thyroglobulin conjugates induced antibody responses to the immunizing peptide and to hTNF. Each antiserum was tested for antagonist activity in hTNF binding assays. Only antisera raised against hTNF-(1-15) or hTNF-(1-31) and antisera against whole hTNF blocked binding. IgGs purified from these three antisera also block hTNF-induced cytolysis and lipoprotein lipase suppression. We conclude that antibodies that recognize the N-terminus of hTNF block the attachment of hTNF to its cellular receptor and inhibit the biologic effects of hTNF.

Múllerian Inhibiting Substance (MIS) is a testicular hormone that promotes involution of the Múllerian duct during embryogenesis. The Múllerian duct gives rise to adult female reproductive ducts including the fallopian tubes, uterus, and upper vagina. Thus, testicular MIS ensures the regression of female sex organ primordia. Partially purified bovine MIS was reported to inhibit proliferation of tumor cells derived from human gynecological cancers. These observations suggest that MIS might be an effective anticancer agent for some human tumors. Recombinant human MIS (rHu-MIS) has recently become available. To assess the antiproliferative activity of rHu-MIS, we examined its effects on 11 ovarian, six endometrial, and two nongynecological human tumor cell lines. rHu-MIS had no effect on proliferation of these cell lines in five independent assays. Forty-three primary human tumor explants were also examined in human tumor colony forming assays, gel-supported primary culture assays, and subrenal capsule assays. rHu-MIS significantly inhibited the growth of five of these tumors including four ovarian and one small cell lung cancer explant. The four ovarian cancer responses include three of 13 (23%) explants tested in human tumor colony-forming assays and one of eight (12.5%) explants tested in gel-supported primary culture assays. We conclude that rHu-MIS may have antiproliferative activity against some human ovarian cancers.

Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.

Factors affecting aspartimide formation, such as protecting groups, acidity, basicity, and temperature, were studied using the model tetrapeptide, Glu-Asp-Gly-Thr. The aspartyl carboxyl side chain in this tetrapeptide was either free or protected as a benzyl or cyclohexyl ester. Our results showed that the cyclohexyl ester led to far less aspartimide formation during acidic or tertiary amine treatment than the corresponding benzyl ester. The rate constants of aspartimide formation in HF-anisole (9:1, v/v) for the tetrapeptide protected as the benzyl ester were found to be 6.2 x 10(-6) and 73.6 x 10(-6) s-1 at -15 degrees and 0 degrees C respectively. These values were about three times faster than the corresponding free- or cyclohexyl ester-protected tetrapeptide. Little difference was seen when the studies were carried out at room temperature. The cyclohexyl protected tetrapeptide gave only 0.3% aspartimide in diisopropylethylamine treatment in 24 h, a 170-fold reduction of imide formation when compared with the benzyl protected tetrapeptide. Thus, using the cyclohexyl ester for aspartyl protection, our studies showed aspartimide formation could be significantly reduced to less than 2% under standard peptide synthesis conditions. Furthermore, with these model peptides, the mechanism of acid catalyzed aspartimide was studied in a range of HF concentrations. In dilute HF cleavage conditions (HF:dimethylsulfide 1:3, v/v), the mechanism was found to be of the AAC2 type, with the rate of aspartimide formation increasing very slowly with increasing acid concentration. In concentrated HF solutions (HF greater than 70% by volume), the rate of aspartimide formation increased rapidly with the increase in acid concentration. However, from model studies, the mechanism of aspartimide formation in concentrated HF was AAC2 rather than AAC1.

Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.

The genome of varicella-zoster virus (VZV) encodes three major glycoproteins, two (gpI and gpII) having been mapped and sequenced, which carry epitopes capable of eliciting neutralizing antibodies. The product of the third major glycoprotein gene (gpIII) was purified, and seven consecutive amino acids at its N-terminus were identified. A degenerate pool of oligonucleotides based upon this sequence was used as a probe to localize the gpIII gene to the HindIII B fragment of the VZV genome. An analysis of the DNA sequence from this region revealed an open reading frame (ORF) encoding 841 amino acids. Rabbit antisera against three synthetic peptides derived from the putative gpIII gene recognized a protein which comigrated with gpIII in Western blots and immunoprecipitation analysis. Preclearing with a monoclonal antibody to gpIII specifically abolished immunoprecipitation of this protein. Also a polypeptide translated from mRNA selected by the putative gpIII gene could be immunoprecipitated by the anti-peptide sera. Therefore, we conclude that gpIII is encoded by the identified ORF in HindIII B. In addition, gpIII is implicated as essential for the cell-to-cell spread of VZV.

To understand the relationship between growth factor-induced mitogenesis and spontaneous cell transformation, a clonal isolate of epidermal growth factor (EGF)-responsive NRK cells was passed in vitro until morphologically transformed variants arose. Subclones of EGF responsive (Cl-3) and EGF nonresponsive (Cl-10) NRK cells were isolated. Cl-3 cells grew as flat, contact-inhibited monolayers, while Cl-10 cells grew as rounded or spindle-shaped cells that formed dense foci. Cl-10 cells formed colonies in soft agar more efficiently (p less than 0.01) and formed larger tumors in nude mice (p less than 0.05) than Cl-3 cells. Cl-3 cells exhibited a sixfold increase in DNA synthesis in response to 1.0 nM EGF. Cl-10 cells did not increase DNA synthesis on exposure to 100 nM EGF. These different responses to EGF occurred despite similar numbers of receptors and similar receptor.binding affinities for EGF (Cl-3: 7000 receptors, Kd = 0.67 nM; Cl-10: 8000 receptors, Kd = 0.72 nM). No evidence of transforming growth factor-alpha was detected in either of these cell lines using Northern blots, Western blots, or biologic assays. We conclude that NRK cells which undergo spontaneous morphologic transformation and exhibit enhanced anchorage-independent growth lose their mitogenic response to EGF.