Truncation of the caspase-related subunit (Gpi8p) of Saccharomyces cerevisiae GPI transamidase: dimerization revealed

Eukaryotic proteins can be post-translationally modified with a glycosylphosphatidylinositol (GPI) membrane anchor. This modification reaction is catalyzed by GPI transamidase (GPI-T), a multimeric, membrane-bound enzyme. Gpi8p, an essential component of GPI-T, shares low sequence similarity with caspases and contains all or part of the enzyme's active site [U. Meyer, M. Benghezal, I. Imhof, A. Conzelmann, Biochemistry 39 (2000) 3461-3471]. Structural predictions suggest that the soluble portion of Gpi8p is divided into two domains: a caspase-like domain that contains the active site machinery and a second, smaller domain of unknown function. Based on these predictions, we evaluated a soluble truncation of Gpi8p (Gpi8(23-306)). Dimerization was investigated due to the known proclivity of caspases to homodimerize; a Gpi8(23-306) homodimer was detected by native gel and confirmed by mass spectrometry and N-terminal sequencing. Mutations at the putative caspase-like dimerization interface disrupted dimer formation. When combined, these results demonstrate an organizational similarity between Gpi8p and caspases.     

Identification of three high molecular mass cysteine proteinases from rat skeletal muscle

Three cysteine proteinases were isolated from the post-myofibrillar fraction of rat skeletal muscle. Proteinase I preferentially hydrolyzes Z-Phe—Arg-NMec with pH optimum at 8–9. The enzyme activity is stabilized by ATP against thermal inactivation. Proteinase II and III were not resolved by anion-exchange chromatography, by affinity chromatography on Arginine—Sepharose or by gel filtration. Proteinase II, splitting Bz-Val---Gly---Arg-NMec optimally at pH 10–10.5, is inactivated by ATP, whereas Proteinase III, hydrolyzing Suc-Ala---Ala---Phe-NMec at pH 7–7.5 is not affected by the nucleotide. The molecular mass of proteinase I is about 750 000 and that of proteinase II and III is about 650 000, as determined by gel filtration.     

Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Cathepsins J and K: high molecular weight cysteine proteinases from human tissues

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.     

The cysteine proteinases of the pineapple plant.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.     

New protein inhibitors of cysteine proteinases in human saliva and salivary glands

New protein inhibitors of cysteine proteinases were found in human saliva and salivary glands. The inhibitory potency present in saliva against ficin is about 30% of that in serum: 1 ml of saliva gives 100% inhibition of 1 nmol of ficin. The same amount of saliva causes no inhibition of 1 nmol of trypsin. The salivary inhibitors occur as multiple forms with different isoelectric points (pI of 4.5-4.7, 5.8, 6.8, 7.8 and 8.2) and different molecular masses of approximately 16, 11, 10, 9.5 and 9 kDa. The inhibitor forms having molecular masses of less than 11 kDa have not yet been described by other authors. The salivary inhibitors have a high thermal stability and a high stability both in alkaline and acidic solutions.     

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.     

Plant cysteine proteinases: evaluation of the pharmacological activity

Cysteine proteinases are involved in virtually every aspect of plant physiology and development. They play a role in development, senescence, programmed cell death, storage and mobilization of germinal proteins, and in response to various types of environmental stress. In this review, we focus on a group of plant defensive enzymes occurring in germinal tissue of Caricaceae. These enzymes elicit a protective response in the unripe fruit after physical stress. We propose that these enzymes follow a strategy similar to mammalian serine proteinases involved in blood clotting and wound healing. We show evidence for the pharmacological role of plant cysteine proteinases in mammalian wound healing, immunomodulation, digestive conditions, and neoplastic alterations.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Structural characterization of the papaya cysteine proteinases at low pH

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.     

The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship

Background  

Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins.     

Darwinian aspects of molecular evolution at sublinear propagation rates

Mean fitness is non-decreasing in the symmetry sector of the frequency trajectory followed in competitive replication at sublinear propagation rates (parabolic time course). This sector contains the pairwise symmetric distribution of species frequencies and its neighboring states, and represents at least half the possible states of an evolving sublinear system. States in the non-symmetry sector produce a negative rate of change in mean fitness. The heterogeneous steady state attained in a finite sublinear system is destabilized by formation of a variant with above-threshold fitness. Evolution in the post-steady-state interval elevates the fitness threshold for coexistence. Contrary to the proposition that ‘parabolic growth invariably results in the survival of all competing species’, only species with sufficient fitness to avoid subthreshold fitness survive. An erratum to this article is available at .     

Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin

The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The immunophilin ligand GPI1046 protects neurons from the lethal effects of the HIV-1 proteins gp120 and Tat by modulating endoplasmic reticulum calcium load

The dysfunction and death of neuronal cells is thought to underlie the cognitive manifestations of human immunodeficiency virus (HIV)-associated neurological disorders. Although HIV-infected patients are living longer owing to the effectiveness of anti-retroviral therapies, the number of patients developing neurological disorders is on the rise. Thus, there is an escalating need for effective therapies to preserve cognitive function in HIV-infected patients. Using HIV-protein-induced neurotoxicity as a model system, we tested the effectiveness of a non-immunosuppressive immunophilin ligand to attenuate gp120 and Tat-induced modification of neuronal function. The immunophilin ligand GPI1046 attenuated endoplasmic reticulum (ER) calcium release induced by gp120 and Tat and protected neurons from the lethal effect of these neurotoxic HIV proteins. Both inositol 1,4,5 trisphosphate (IP(3)) and ryanodine-sensitive ER calcium release was attenuated by pre-incubation with GPI1046. Using the sarco/endoplasmic reticulum calcium pump inhibitor thapsigargin to release ER calcium, we determined that GPI1046 reduced the total ER calcium load. These findings suggest that non-immunosuppressive immunophilin ligands may be useful neuroprotective drugs in HIV dementia.     

Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane-permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane-permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of β-glucuronised, and N-acetyle-β-glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does-dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.