Oligopeptide inhibition of class I ribonucleotide reductases

Class I ribonucleotide reductases (RRs), which are well-recognized targets for cancer chemotherapeutic and antiviral agents, are composed of two different subunits, R1 and R2, and are inhibited by oligopeptides corresponding to the C-terminus of R2, which compete with R2 for binding to R1. These peptides specifically inhibit the RRs from which they are derived, and closely homologous RRs, but do not inhibit less homologous RRs. Here we review results obtained for oligopeptide inhibition of RRs from several sources, including related x-ray, NMR, and modeling results. The most extensive studies have been performed on herpes simplex virus-RR (HSV-RR) and mammalian-RR (mRR). A common model fits the data obtained for both enzymes, in which the C-terminal residue of the oligopeptide (Leu for HSV-RR, Phe for mRR) binds with high specificity to a narrow and deep hydrophobic subsite, and two or more hydrophobic groups at the N-terminal portion of the peptide bind to a broad and shallow second hydrophobic subsite. The studies have led to the development of highly potent and specific inhibitors of HSV-RR and promising inhibitors of mRR, and indicate possible directions for the development of inhibitors of bacterial and fungal RRs.     

Interaction of heparin with synthetic peptides corresponding to the C-terminal domain of intestinal mucins

Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind³H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, givingK _d values of 10^–7 to 10^–8 m. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of³H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin-binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they formin vivo, are thus likely to be confined to the ER and/or Golgi compartments.     

Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction: kinetic analysis of inhibition studies

The active complex of Escherichia coli ribonucleotide reductase comprises two dissociable, nonidentical homodimeric proteins, B1 and B2. When B2 is the varied component, the reductase activity is competitively inhibited by synthetic peptides of varying lengths corresponding to the C-terminus of protein B2. This finding provides the first evidence that the C-terminal peptides and protein B2 share the same binding domain on protein B1. Our data also show that two molecules of peptide can bind to protein B1 with equal affinity. Similar inhibition constants (18 microM) were obtained for peptides containing the C-terminal 20, 30, and 37 residues. When the invariant residue Tyr 356 was omitted, a 2-fold decrease in peptide inhibitory ability was observed. A small peptide, lacking the last 11 residues, had virtually no inhibitory potency. These results, coupled with our previous observations that truncated protein B2, in which one or both polypeptide chains are missing approximately 24 C-terminal residues, had considerably lower or no affinity for B1, suggest that the C-terminal regions are the major determinants in the B1-B2 interaction. In the Appendix, two methods for treatment of kinetic situations pertinent to the ribonucleotide reductase system are presented. One method deals with the determination of kinetic parameters for two components present at comparable levels; the other is concerned with the differentiation of linear and nonlinear competitive inhibition involving the binding of two inhibitor molecules. Both methods should find application to other similar cases.     

Inhibition of human cytomegalovirus DNA polymerase by C-terminal peptides from the UL54 subunit

In common with other herpesviruses, the human cytomegalovirus (HCMV) DNA polymerase contains a catalytic subunit (Pol or UL54) and an accessory protein (UL44) that is thought to increase the processivity of the enzyme. The observation that antisense inhibition of UL44 synthesis in HCMV-infected cells strongly inhibits viral DNA replication, together with the structural similarity predicted for the herpesvirus processivity subunits, highlights the importance of the accessory protein for virus growth and raises the possibility that the UL54/UL44 interaction might be a valid target for antiviral drugs. To investigate this possibility, overlapping peptides spanning residues 1161 to 1242 of UL54 were synthesized and tested for inhibition of the interaction between purified UL54 and UL44 proteins. A peptide, LPRRLHLEPAFLPYSVKAHECC, corresponding to residues 1221 to 1242 at the very C terminus of UL54, disrupted both the physical interaction between the two proteins and specifically inhibited the stimulation of UL54 by UL44. A mutant peptide lacking the two carboxy-terminal cysteines was markedly less inhibitory, suggesting a role for these residues in the UL54/UL44 interaction. Circular dichroism spectroscopy indicated that the UL54 C-terminal peptide can adopt a partially alpha-helical structure. Taken together, these results indicate that the two subunits of HCMV DNA polymerase most likely interact in a way which is analogous to that of the two subunits of herpes simplex virus DNA polymerase, even though there is no sequence homology in the binding site, and suggest that the UL54 peptide, or derivatives thereof, could form the basis for developing a new class of anti-HCMV inhibitors that act by disrupting the UL54/UL44 interaction.     

Mechanism-based inhibition of ribonucleotide reductases: new mechanistic considerations and promising biological applications.

Ribonucleotide reductases (RNRs) perform the de novo biosynthesis of 2'-deoxynucleoside 5'-(di or tri)phosphates. Inhibition of RNRs removes a crucial source of genetic components and enhances the probability of salvage incorporation of analogues into DNA. Several laboratories have clarified aspects of the reaction cascades initiated by generation of substrate nucleotide C3' free radicals by RNRs. New considerations for radical-mediated mechanism-based inhibition and biological applications are discussed.     

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.     

Regulation of the ribonucleotide reductases in Lactococcus lactis subsp. cremoris

Lactococcus lactis has two essential ribonucleotide reductases for DNA biosynthesis and repair which are affected in the presence or absence of oxygen. Expression of glutaredoxin like protein (NrdH), the hydrogen donor for ribonucleotide reductase, was found to be regulated by the FNR like proteins (FlpA and FlpB). Proteomics study demonstrated that expression level of NrdH significantly decreased in the flpA and flpAB deletion mutants. The nrdH gene is located in an nrdHIEF operon and encoding the NrdEF ribonucleotide reductase, which is active under aerobic and anaerobic conditions. Regulation of expression of the nrdHIEF operons was investigated using beta-galactosidase as a reporter gene. The 588 bp fragment containing the nrdH promoter and gene cloned into the pORI vector immediately upstream of a promoterless lacZ gene. Constructed plasmid was transferred into wild type (MG1363), single mutant (flpA orflpB) and double mutant (flpAB). Aerobically, nrdH promoter activity is 15-fold higher than anaerobic expression.     

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.     

Secondary structure and structure-activity relationships of peptides corresponding to the subunit interface of herpes simplex virus DNA polymerase

The interaction of the catalytic subunit of herpes simplex virus DNA polymerase with the processivity subunit, UL42, is essential for viral replication and is thus a potential target for antiviral drug discovery. We have previously reported that a peptide analogous to the C-terminal 36 residues of the catalytic subunit, which are necessary and sufficient for its interaction with UL42, forms a monomeric structure with partial alpha-helical character. This peptide and one analogous to the C-terminal 18 residues specifically inhibit UL42-dependent long chain DNA synthesis. Using multidimensional (1)H nuclear magnetic resonance spectroscopy, we have found that the 36-residue peptide contains partially ordered N- and C-terminal alpha-helices separated by a less ordered region. A series of "alanine scan" peptides derived from the C-terminal 18 residues of the catalytic subunit were tested for their ability to inhibit long-chain DNA synthesis and by circular dichroism for secondary structure. The results identify structural aspects and specific side chains that appear to be crucial for interacting with UL42. These findings may aid in the rational design of new drugs for the treatment of herpesvirus infections.     

Different susceptibility of human immunodeficiency virus type 1 to Env gp41-derived synthetic peptides corresponding to the C-terminal heptad repeat region

Two functional domains, alpha-helical heptad repeat 1 (HR-1) and HR-2, located in the N-terminal and C-terminal regions of human immunodeficiency virus type 1 (HIV-1) Env gp41, respectively, play an important role in the fusion process. Synthetic 34-amino-acid peptide that contains the HR-2 region, named C34, has been shown to inhibit the HIV-1 fusion process. Here, we prepared six representative peptides (C34-B1, -B2, -A, -C1, -C2, and -E from subtypes B, A, C, and E, respectively) according to the sequences from the HIV sequence database of Los Alamos. All the C34 peptides had lower ability to inhibit the primary isolates (subtypes B and CRF01_AE) than subtype B laboratory strain LAI. On the other hand, the L-2 cell clone, isolated from persistently LAI-infected MT-4 cells (MT-4/LAI), showed unique C34 peptide sensitivities. L-2 virus has the same sequences at HR-1 and HR-2 regions as LAI, but showed higher syncytia formation activity than LAI. Interestingly, the sensitivity of L-2 was higher to C34-B2 and -A but slightly lower to C34-C1 at higher concentrations than MT-4/LAI, while C34-B1, -C2, and -E showed similar activity against both viruses. Thus, in addition to the sequences of the C34 peptide as well as of the HR-1 and HR-2 regions in target viruses used for fusion assays, the fusion inhibitory activities of C34 peptides seem to be affected by viral factor(s) other than the gp41 alpha-helical heptad repeats.     

Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor.

The nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2-(329-337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5-fold. Nx-acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac-VZV R2-(298-306) exhibited a 15-fold higher potency on reductase inhibition than HSV R2-(329-337). The degradation process of HSV R2-(329-337) was partially inhibited by amastatin, bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2-(332-337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2-(329-337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2-(329-337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.     

C-terminal sequencing by mass spectrometry: application to gelatine-derived proline-rich peptides

Protonated peptides derived from proline‐rich proteins (PRP) are often difficult to sequence by standard collision‐induced dissociation (CID) mass spectrometry (MS) due to preferential amide bond cleavage N‐terminal to proline. In connection with bovine spongiform encephalopathy regulations, proteolytic products derived from the PRP collagen have been suggested as markers for contamination of animal feedstuffs with processed animal protein (Fernandez Ocaña, M. et al., Analyst 2004, 129, 111–115). Herein, we report the identification of these marker peptides using the strategy of C‐terminal sequencing by CID MS from their sodium and lithium adducts. Upon fragmentation a new cationized peptide was produced that is one C‐terminal amino acid shorter in length. This dissociation pathway allowed for the facile identification of the C‐terminal residue by matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry. Each newly formed cationized peptide was further fragmented by up to seven stages of electrospray ionization ion trap MS. Proline‐rich C‐terminal sequence tags were established which permitted successful database identification of collagen alpha type I proteins.     

Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein

PKI-(5-24)-amide is a 20-residue peptide with the sequence, Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-A la-Ile-His- Asp24-NH2, that corresponds to the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992). Amino acid residues in PKI-(5-24)-amide responsible for the potent inhibition (Ki = 2.3 nM) of the catalytic subunit of protein kinase were further investigated using deletion and substitution analogs of the synthetic peptide. Residues 5, 23, and 24 were not required for activity since the 17-residue PKI-(6-22)-amide retained full potency. Sequential removal of the first seven amino acids from the NH2 terminus of PKI-(5-24)-amide caused a progressive 50-fold loss of inhibitory potency. In contrast, substitution of either Thr6, Asp9, or Ile11 with alanine, or Ala8 by leucine, in PKI-(5-22)-amide produced less than 3-fold decreases in potency. Of the 2 aromatic residues in PKI-(5-22)-amide, the individual substitution of Phe10 and Tyr7 by alanine caused, respectively, 90- and 5-fold decreases in inhibitory potency, demonstrating important roles for each. This NH2-terminal portion of the peptide is believed to contain a significant portion of alpha-helix. Many recognition or structural determinants are also essential in the COOH-terminal portion of PKI-(5-22)-amide. In addition to the basic subsite provided by the three arginines, several other of the residues are critical for full inhibitory potency. Substitution of Ile22 by glycine in either PKI-(5-22)-amide or PKI-(14-22)-amide lowered the inhibitory potency by 150- and 50-fold, respectively. Separate replacement of Gly17 or Asn20, in either PKI-(5-22)-amide or PKI-(14-22)-amide, caused 7-15-fold decreases in potency. Substitution of both Gly17 and Asn20 together (in PKI-(14-22)-amide) produced a synergistic loss of inhibitory activity. [Leu13,Ile14]PKI-(5-22)-amide, a doubly substituted analog exhibited a 42-fold increase in Ki value. We conclude that Ser13 and/or Gly14, Gly17, Asn20, and Ile22 each contribute important features to the binding of these inhibitory peptides to the protein kinase, either by providing recognition determinants, inducing structure, and/or allowing essential peptide backbone flexibility.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allosteric control of three B12-dependent (class II) ribonucleotide reductases. Implications for the evolution of ribonucleotide reduction

Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes.