High-throughput fluorescence polarization method for identification of FKBP12 ligands.

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.

2-Aryl-2,2-difluoroacetamide FKBP12 ligands: synthesis and X-ray structural studies.

[structure: see text] 2-Aryl-2,2-difluoroacetamido-proline and pipecolate esters are high affinity FKBP12 ligands whose rotamase inhibitory activity is comparable to that seen for the corresponding ketoamides. X-ray structural studies suggest that the fluorine atoms participate in discrete interactions with the Phe36 phenyl ring and the Tyr26 hydroxyl group, with the latter resembling a moderate-to-weak hydrogen bond.

Fluoresceinated FKBP12 ligands for a high-throughput fluorescence polarization assay.

Several fluoresceinated FKBP12 ligands have been prepared for a high-throughput fluorescence polarization assay. K(i)s for FKBP12 rotamase inhibition by these ligands range from 1.3 microM to 32 nM, and their design is based on X-ray crystal structures of FKBP12 complexed with known immunophilin ligands.

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides.

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.

The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors: involvement of nonproteasomal cytosolic proteases in antigen processing?

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.

Ubiquitination of protein kinase C-alpha and degradation by the proteasome.

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.

Herbimycin A induces the 20 S proteasome- and ubiquitin-dependent degradation of receptor tyrosine kinases.

Herbimycin A is an ansamycin antibiotic isolated as an agent that reverses morphological transformation induced by v-src. Although herbimycin A is widely used as a tool for inhibiting multiple tyrosine protein kinases and tyrosine kinase-activated signal transduction, its mechanism of action is not well defined and includes a decrease in both tyrosine kinase protein levels and activity (Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Cancer Res. 49, 780-785). We now show that herbimycin A induces a profound decrease in the total cellular activity of transmembrane tyrosine kinase receptors, such as insulin-like growth factor, insulin, and epidermal growth factor receptors. A substantial proportion of the in vivo inhibition could be explained by an increase in the rate of degradation. The enhanced degradation of insulin-like growth factor-insulin receptor was prevented by inhibitors of the 20S proteasome, whereas neither lysosomotropic agents nor general serine- and cysteine-protease inhibitors were active in preventing receptor degradation induced by herbimycin A. Moreover, in a temperature-sensitive mutant cell line defective in the E1-catalyzed activation of ubiquitin, herbimycin A treatment at the restrictive temperature did not result in the degradation of insulin receptor. These results suggest that herbimycin A represents a novel class of drug that targets the degradation of tyrosine kinases by the 20S proteasome. The ubiquitin dependence of this process indicates that this degradation of tyrosine kinases might involve the 20S proteasome as the proteolytic core of the ubiquitin-dependent 26S protease.

Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes.

Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.

Evidence that the nature of amino acid residues in the P3 position directs substrates to distinct catalytic sites of the pituitary multicatalytic proteinase complex (proteasome).

Cleavage of bonds after neutral amino acids by the multicatalytic proteinase complex (MPC) has been recently shown to be catalyzed by at least three distinct components [Orlowski, M., Cardozo, C., & Michaud, C. (1993) Biochemistry 32, 1563-1572]. One component, designated as chymotrypsin-like (ChT-L), cleaves peptide bonds on the carboxyl side of hydrophobic residues and is also active toward peptidyl-arylamide bonds. A second component, designated as branched-chain amino acid preferring (BrAAP), and a third component, designated as small neutral amino acid preferring (SNAAP), cleave preferentially bonds on the carboxyl side of branched-chain amino acids and between small neutral amino acids, respectively. Evidence indicates that the BrAAP component is a major factor responsible for degradation of protein by the MPC. The purpose of the present study was to identify the structural requirements that determine the involvement of these components in cleavage of peptides after different neutral amino acids. A series of substrates was synthesized with the aim of probing the role of residues beyond those flanking the scissile bond in directing substrates to defined catalytic sites. The data indicate that a proline or glycine residue in the P3 position directs the substrate to the catalytic site of the BrAAP component provided that a branched-chain amino acid is present in the P1 position. A proline residue in P3 is also important for involvement of the SNAAP component in substrate degradation. The presence of this residue interferes with substrate binding to the catalytic site of the ChT-L activity, even in the presence of a phenylalanine residue in the P1 position.(ABSTRACT TRUNCATED AT 250 WORDS)

A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium was used to develop an overexpression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PPi from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRTase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydrolysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP <--> NAMN + PPi from its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysis. However, in contrast to other ATP-utilizing enzymes, when all ATP is hydrolyzed the unfavorable product/substrate ratio collapses. ATP/ADP exchange results suggest that the overall reaction involves a phosphoenzyme (E-P) arising from E.ATP. Km values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PPi stimulated the ATPase activity of the enzyme to Vmax values, suggesting that PPi formation during catalysis provides a trigger for cleavage of the putative E-P in the overall reaction and regenerates the low affinity form of the enzyme. A model is presented in which alternation of high and low affinity forms of NAPRTase provides a "steady-state" coupling between ATP hydrolysis and NAMN formation.

Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)

A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.

Cloning and nucleic acid sequence of the Salmonella typhimurium pncB gene and structure of nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium, encoding nicotinate phosphoribosyltransferase (NAPRTase), was cloned on a 4.7-kb Sau3A fragment. The gene contains a 1,200-bp open reading frame coding for a 400-residue protein. Amino acid sequencing of the amino-terminal and two interior peptides of the purified protein confirmed the deduced sequence and revealed that the amino-terminal methionine residue was removed, giving a 399-residue mature protein of Mr 45,512. No signal sequence was observed in the predicted NAPRTase primary structure, suggesting that the enzyme is not periplasmic. The protein does not demonstrate clear sequence similarity to the other seven phosphoribosyltransferases of known primary structure and frustrates attempts to define a consensus 5-phosphoribosyl-1-pyrophosphate-binding region. The NAPRTase reaction is ATP stimulated, and the protein contains a carboxy-terminal sequence diagnostic of an ATP-binding site. An inverted repeat of the sequence TAAACAA observed in the proposed promoter region of pncB is also present in the promoter of nadA, which, like pncB, is also regulated by the NadR (NadI) repressor. The sequence may thus define an NadR repressor-binding site.

Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium.

The chemical mechanism of the phosphoribosyltransferases (PRTases), although largely unknown, may proceed either via a concerted direct-transfer mechanism or with a two-step mechanism involving a carboxonium-like intermediate. To study this question, we have cloned the Salmonella typhimurium pyrE gene, coding for the enzyme orotate phosphoribosyltransferase (EC 2.2.4.10, OPRTase), and developed a bacterial strain that overproduces the enzyme, which we have purified to homogeneity. Initial velocity and product inhibition studies indicated that S. typhimurium OPRTase follows a random sequential kinetic mechanism. This result was further confirmed by equilibrium isotope exchange studies on two substrate-product pairs, PRPP-PPi and OMP-orotate. In addition, the rates of the individual equilibrium isotope exchanges allowed us to conclude that PPi release and PRPP release were the rate-determining steps in the forward and reverse reactions, respectively. Although partial reactions between the two substrate-product pairs, PRPP-PPi and OMP-orotate, were observed, further studies revealed that these exchanges were a result of contaminations. Our results are significant in that S. typhimurium OPRTase, like most PRTases but in contrast to its yeast homologue, follows sequential kinetics. The artifactual partial isotope exchanges found in this work may have implications for similar prior work on the yeast enzyme. In view of the careful isotope effect studies of Parsons and co-workers [Goitein, R.K., Chelsky, D., & Parsons, S.M. (1978) J. Biol. Chem. 253, 2963-2971] and the results obtained by us, we propose that PRTases may involve a direct-transfer mechanism but with low bond order to the leaving pyrophosphate moiety and attacking base.