Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions, We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation, General serine-threonine phosphatase inhibitors such sodium fluoride, or beta-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I-1 or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains, These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

Synthetic studies of the HIV-1 protease inhibitive didemnaketals: Stereocontrolled synthetic approach to the key mother spiroketals

[GRAPHICS] The stereocontrolled synthesis of (2S,4R,6R,8S,10S,1'R,1"R)-2(acetylhydroxymethyl)-4, 10-dimethyl-8(isopropenylhydroxymethyl)-1, 7-dioxaspiro[5,5]-undecane (4a) and its C1"-epimer (4b), the key mother spiroketals of the HIV-1 protease inhibitive didemnaketals from the ascidian Didemnum sp., has been carried out through multisteps from the natural (R)-(+)-pulegone, which involved the diastereoselective construction of four chiral carbon centers(C-2, C-6, C-8, and C-1') by intramolecular chiral induce.

Solution structures by H-1 NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by H-1-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 Angstrom and 0.66 Angstrom, respectively. The structures consist of two short antiparallel beta -strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta -strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides. (C) 2001 Academic Press.

Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus-transformed B-cell lines - A potential therapy for posttransplant lymphoproliferative disease

Background. Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. Method. This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABRA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. Results. In vitro treatment of lymphoblastoid cell lines with ABRA showed that they were effectively killed by low doses of the drug (ID50 2-5 mug/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitt's lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. Conclusion. These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

Structure and function of plant toxins (with emphasis on cystine knot toxins)

Plant toxins are substances produced and secreted by plants to defend themselves against predators. In a broad sense, this includes all substances that have a toxic effect on targeted organisms, whether they are microbes, other plants, insects, or higher animals. Plant toxins have a diverse range of structures, from small organic molecules through to proteins. This review gives an overview of the various classes of plant toxins but focuses on an interesting class of protein-based plant toxins containing a cystine knot motif. This structural motif confers exceptional stability on proteins containing it and is associated with a wide range of biological activities. The biological activities and structural stability offer many potential applications in the pharmaceutical and agricultural fields. One particularly exciting prospect is in the use of protein-based plant toxins as molecular scaffolds for displaying pharmaceutically important bioactivities. Future applications of plant toxins are likely to involve genetic engineering techniques and molecular pharming approaches.

beta-strand mimicking macrocyclic amino acids: Templates for protease inhibitors with antiviral activity

New amino acids are reported in which component macrocycles are constrained to mimic tripeptides locked in a beta-strand conformation. The novel amino acids involve macrocycles functionalized with both an N- and a C-terminus enabling addition of appendages at either end to modify receptor affinity, selectivity, or membrane permeability. We show that the cycles herein are effective templates within inhibitors of HIV-1 protease. Eleven compounds originating from such bifunctionalized cyclic templates are potent inhibitors of HIV-1 protease (Ki 0.3-50 nM; pH 6.5, I = 0.1 M). Unlike normal peptides comprising amino acids, five of these macrocycle-containing compounds are potent antiviral agents with sub-micromolar potencies (IC50 170-900 nM) against HIV-1 replication in human MT2 cells. The most active antiviral agents are the most lipophilic, with calculated values of LogD(6.5) greater than or equal to 4. All molecules have a conformationally constrained 17-membered macrocyclic ring that has been shown to structurally mimic a tripeptide segment (Xaa)-(Val/Ile)-(Phe/Tyr) of a peptide substrate in the extended conformation. The presence of two trans amide bonds and a para-substituted aromatic ring prevents intramolecular hydrogen bonds and fixes the macrocycle in the extended conformation. Similarly constrained macrocycles may be useful templates for the creation of inhibitors for the many other proteins and proteases that recognize peptide beta-strands.

Thermolysin activates equine lamellar hoof matrix metalloproteinases

Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinse-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact tested on a calibrated force transducer, but when treated with an NIMP activator, developed in-vitro laminitis, separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated NIP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and batimastat. These findings suggest that thermolysin-mediated gelatinase activation is not dependent on membrane-bound matrix metalloproteinase (MT-MMP) activation, providing further evidence that bacteria can produce potent MMP activators that probably facilitate host invasion. (C) 2002 Harcourt Publishers Ltd.

Effects of the vasopeptidase inhibitor, omapatrilat, in 723 patients with coronary heart disease

Introduction Among individuals with a history of myocardial infarction (MI), higher levels of blood pressure (BP) are associated with increased long-term risks of death from coronary heart disease. Treatment with a BP-lowering regimen, based on omapatrilat may result in greater clinical benefits than treatment with a regimen based on a regular angiotensin-converting enzyme (ACE) inhibitor because of more favourable effects on the renin-angiotensin-aldosterone system. Methods Seven hundred and twenty-three clinically stable patients with a history of MI or unstable angina, and a mean entry BP of 134/77 mmHg, were randomised to six months treatment with omapatrilat 40 mg, omapatrilat 20 mg, or matching placebo. Results After six months, mean BP levels (systolic/diastolic) in the omapatrilat 40 mg group were reduced by 4.3/ 2.9 mmHg (95% confidence interval 1.3 to 7.2/1.2 to 4.6). Mean BP levels in the omapatrilat 20 mg group were reduced by 4.6/1.0 mmHg (1.6 to 7.6/-0.7 to 2.6) in comparison with the placebo group. Both doses of omapatrilat also produced significant decreases in plasma ACE activity and significant increases in levels of plasma renin activity, atrial natriuretic peptide, endothelin and homocysteine (p

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.