Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta.

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Incorporation of non-proteolytic proteins by murine alpha2-macroglobulin.

Human alpha2-macroglobulin is a tetrameric glycoprotein with a molecular weight of 718 kDa that is present in human plasma at high concentrations. Murine alpha2-macroglobulin is homologous to human alpha2-macroglobulin but it undergoes post-translational cleavage in the subunits. Each subunit of alpha2-macroglobulin contains a thiolester which can be cleaved by small nucleophiles. In human alpha2-macroglobulin this results in a conformational change to a receptor-recognized form and a change in the electrophoretic mobility. Recent work has demonstrated that this process is reversible and during this reversal non-proteolytic proteins can become covalently trapped within the human alpha2-macroglobulin molecule. The present study further investigates this observation and examines the question whether reversal of thiolester cleavage occurs in mouse alpha2-macroglobulin. Previous studies suggest that small nucleophiles only partially convert mouse alpha2-macroglobulin to a receptor-recognized form. We demonstrate here that under appropriate conditions, mouse alpha2-macroglobulin is fully converted by NH3. We also demonstrate that despite structural and kinetic differences between human and mouse alpha2-macroglobulin, both molecules are able to incorporate non-proteolytic ligands in a similar manner. This leads us to propose a general model of ligand incorporation via nucleophilic exchange in multimeric alpha-macroglobulins.

Interaction of subtilisins with serpins.

Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that alpha 1 proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and alpha 1 antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 degrees C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.

A sensitive homologous radioimmunoassay for human relaxin-2 (h-RLX-2) based on antibodies characterized by epitope mapping studies.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

Interdependency of the binding subsites in subtilisin.

Subtilisins are endopeptidases with an extended binding cleft comprising at least eight subsites, and kinetic studies have revealed that subsites distant from the scissile bond are important in determining the substrate preference of the enzymes. With the subtilisin enzyme Savinase, the interdependency of the individual Sn-Pn interactions has been investigated. It was found that the contributions from each subsite interaction to kcat/KM are not always additive. Such interdependency was also observed between subsites which are remote from each other. With a series of substrates covering S6 to S'4 of Savinase, it was observed that favorable amino acids in P1 or, more significantly, P4 of the substrate shield adverse effects of less favorable amino acids at other positions. Thus, an upper limit of kcat/KM was observed, suggesting a limit on the amount of substrate interaction energy which can be converted into transition-state stabilization. Furthermore, with substrates in which all positions had been optimized, an upper limit of kcat/KM (approximately 2 x 10(9) min-1 M-1) was seen, both for a substrate with a high kcat and for one with a low KM. These results emphasize that the design of optimal substrates or substrate-derived inhibitors for endopeptidases preferably should be based on subsite mappings where interdependent substrate-subsite interactions have been eliminated.

Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching.

Subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites. Interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes. The development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the active sites of subtilisin BPN' from Bacillus amyloliquefaciens and Savinase from Bacillus lentus. For each enzyme, the eight positions S5-S'3 were characterized by determination of kcat/KM values for the hydrolysis of substrates in which the amino acids were systematically varied. The results emphasize that in both subtilisin BPN' and Savinase interactions between substrate and S4 and S1 are very important. However, it is apparent that interactions between other subsites and the substrate exert a significant influence on the substrate preference. The results are rationalized on the basis of the structural data available for the two enzymes.

Nonproteolytic incorporation of protein ligands into human alpha 2-macroglobulin: implications for the binding mechanism of alpha 2-macroglobulin.

alpha 2-Macroglobulin (alpha 2M) is a complex tetrameric protein of 718 kDa. In native alpha 2M, each of the four subunits contains a thiol ester between the side chains of Cys949 and Gln952. Cleavage of the thiol ester with small nucleophiles destabilizes the native conformation and causes a major conformational change in alpha 2M, which leads to exposure of receptor binding sites and a change in electrophoretic mobility. Recently it has been shown that nucleophilic cleavage of the four thiol esters in alpha 2M is a reversible process with energy requirements dependent on the nucleophile [Grøn, H., Thøgersen, I. B., Enghild, J. J., and Pizzo, S. V. (1996) Biochem. J. 318, 539-545]. The present study is a further investigation of the properties of alpha 2M with cleaved thiol esters and the potential for incorporation of protein ligands at the site of the thiol ester. The thiol ester in alpha 2M was cleaved by NH3. After removal of excess NH3, the alpha 2M derivative was incubated with excess protein ligand (hen egg lysozyme or bovine insulin) at 23, 37, or 50 degreesC, leading to covalent incorporation of the ligands in alpha 2M as analyzed by SDS-PAGE, gel filtration, and centrifugal microfiltration. Receptor binding studies and native pore-limit PAGE confirmed that the alpha 2M derivatives with ligand incorporated remained in the receptor-recognized, "fast" migrating conformation. This is the first demonstration of nonproteolytic, covalent incorporation of protein ligands into receptor-recognized alpha 2M.

Peptides as tools in drug discovery.

Drug discovery in the post-genomics era provides enormous opportunities as well as new challenges. The discovery of effective therapeutics will require choosing the right targets, building robust sensitive assays, executing an efficient screening program and judicious follow-up on promising active compounds. These processes require the development and adoption of new tools and techniques that will allow the rapid and efficient movement of interesting targets through a discovery program. This review will describe how peptide ligands targeted to protein functional sites can be used to validate a site on a candidate target protein, aid in a structure-based design program and format robust, sensitive high-throughput screens to identify compounds that modulate the function of the target protein. Combining these approaches in an integrated discovery program creates a new paradigm that allows for rapid, parallel flow of multiple targets from target choice through lead identification.

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.

Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human alpha 2-macroglobulin, rat alpha 1-inhibitor-3 and chemically modified derivatives.

The alpha-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal beta-cysteinyl-gamma-glutamyl thiol ester bonds are an important structural feature of most alpha-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human alpha 2-macroglobulin (alpha 2M) and monomeric rat alpha 1-inhibitor-3 (alpha 1I3). When employing NH3 as the nucleophile, the thiol ester in alpha 1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, alpha 2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between alpha 2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for alpha 2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native alpha 2M is restored when the proteinase-inhibitory capacity is recovered.

The potential role of alpha 2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis.

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M). Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3). alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta.

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs alpha and beta and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER alpha and ER beta ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.