Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.     

DNA sequence motifs which direct adeno-associated virus site-specific integration in a model system

The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288-11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966-7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.     

Proteolytic degradation routes for turkey beta 1-adrenoceptor probed with antipeptide antibodies against the N-terminal sequence of the receptor

Anti-peptide antibodies, raised against the N-terminal sequence (amino acids 2-10) of the turkey beta 1-adrenoceptor [Yarden et al., Proc. Natl. Acad. Sci. USA (1986) 83, 6795-6799] recognized the 50 kDa- but not the 40 kDa-form of the receptor, thus confirming the previous assumption that the N-terminus of the 50 kDa form is lost during its conversion to the 40 kDa-form [Jür beta, R., Hekman, M. & Helmreich, E.J.M. (1985) Biochemistry 24, 3349-3354]. By in situ proteolysis small amounts of receptor fragments were formed, which could be recognized by the N-terminus specific antibody. Therefore, although the production of the stable 40 kDa receptor species by proteolytic removal of a portion of the N-terminal appears to be the predominant route, there exists an additional pathway of degradation which must involve the initial cleavage of the carboxyl terminal.     

c[D-pro-Pro-D-pro-N-methyl-Ala] adopts a rigid conformation that serves as a scaffold to mimic reverse-turns

Naturally occurring cyclic tetrapeptides (CTPs) such as tentoxin (Halloin et al., Plant Physiol 1970, 45, 310-314; Saad, Phytopathology 1970, 60, 415-418), ampicidin (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147), HC-toxin (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447), and trapoxin (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328; Itazaki et al., J Antibiot (Tokyo) 1990, 43, 1524-1532) have a wide range of biological activity and potential use ranging from herbicides (Walton, Proc Natl Acad Sci USA 1987, 84, 8444-8447; Judson, J Agric Food Chem 1987, 35, 451-456) to therapeutics (Loiseau, Biopolymers 2003, 69, 363-385) for malaria (Darkin-Rattray, Proc Natl Acad Sci USA 1996, 93, 13143-13147) and cancer (Yoshida and Sugita, Jpn J Cancer Res 1992, 83, 324-328). To elucidate scaffolds that have few low-energy conformations and could serve as semirigid reverse-turn mimetics, the flexibility of CTPs was determined computationally. Four analogs of cyclic tetraproline c[Pro-pro-Pro-pro] with alternating L- and D-prolines, namely c[pro-Pro-pro-NMe-Ala], c[pip-Pro-pip-Pro], c[pro-Pip-pro-Pro], and c[Ala-Pro-pip-Pro] were synthesized and characterized by NOESY NMR. Both molecular mechanics and Density Functional Theory quantum calculations found these head-to-tail CTPs to be constrained to one or two relatively stable conformations. NMR structures, while not always yielding the same lowest energy conformation as expected by in silico predictions, confirmed only one or two highly populated solution conformations for all four peptides examined. c[pro-Pro-pro-NMe-Ala] was shown to have a single all trans-amide bond conformation from both in silico predictions and NMR characterization, and to be a reverse-turn mimetic by overlapping four Calpha-Cbeta bonds with those for approximately 6.5% (Tran, J Comput Aided Mol Des 2005, 19, 551-566) of reverse-turns in the Protein Data Bank PDB with a RMSD of 0.57 A.     

Purification and characterization of PrbA,a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens)

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.     

Endoplasmic reticulum degradation of a subunit of the asialoglycoprotein receptor in vitro. Vesicular transport from endoplasmic reticulum is unnecessary.

The H2a subunit of the human asialoglycoprotein receptor is rapidly degraded from the endoplasmic reticulum (ER) when expressed in CHO15B cells. We have reconstituted ER degradation of H2a in semipermeable cells. At least the initial step in degradation (a proteolytic cleavage inhibited by N alpha-p-tosyl-L-lysine chloromethyl ketone and L-1-tosylamido-2-phenylethyl chloromethyl ketone) can occur in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate or in the absence of ATP and postnuclear supernatant, conditions that do not allow vesicular transport of subunit H1 from the ER to the Golgi. We conclude that vesicular transport from the ER is not required for ER degradation of H2a to occur and thus that it takes place in the ER itself.     

Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure.

Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.     

Purification and characterization of protease Re, a cytoplasmic endoprotease in Escherichia coli.

Protease Re, a new cytoplasmic endoprotease in Escherichia coli, was purified to homogeneity by conventional procedures, using [3H]casein as the substrate. The enzyme consists of a single polypeptide of 82,000 molecular weight. It is maximally active between pH 7 and 8.5 and is independent of ATP. It has a pI of 6.8 and a Km of 10.8 microM for casein. Since diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride inhibited this enzyme, it appears to be a serine protease. Protease Re was sensitive to inhibition by L-1-tosylamido-2-phenylethylchloromethylketone but not to that by 1-chloro-3-tosylamido-7-aminoheptanone, thiol-blocking reagents, chelating agents, or various peptide aldehydes. Re also degraded [125I]globin, [125I]glucagon, and 125I-labeled denatured bovine serum albumin to acid-soluble products (generally oligopeptides of greater than 1,500 daltons), but it showed no activity against serum albumin, growth hormone, insulin, or a variety of fluorometric peptide substrates. It also hydrolyzed oxidatively inactivated glutamine synthetase (generated by ascorbate, oxygen, and iron) four- to fivefold more rapidly than the native protein. Protease Re appears to be identical to the proteolytic enzyme isolated by Roseman and Levine (J. Biol. Chem. 262:2101-2110, 1987) by its ability to degrade selectively oxidatively damaged glutamine synthetase in vivo. Its role in intracellular protein breakdown is uncertain.     

Lysosomal Chat Maintains the Balance

The original idea that each protein follows a particular proteolytic pathway for its degradation is no longer supported. Instead, different proteolytic systems can simultaneously contribute to the degradation of a particular protein, or they can alternate in this task depending, for the most part, on the cellular conditions. It is thus reasonable to expect that some level of communication exists among different proteolytic systems to orchestrate these coordinated activities. Direct cross-talk between two forms of autophagy, macroautophagy and chaperone-mediated autophagy (CMA) has been recently demonstrated. Cells respond to blockage of CMA by upregulating macroautophagy. Although macroautophagy cannot completely substitute for the lack of CMA, the partial redundancy between both pathways allows some level of compensation, enough to maintain protein degradation and preserve cell homeostasis. Understanding the cross-talk among different autophagic pathways and with other proteolytic systems is important to predict the type of compensatory mechanisms that could be elicited in response to failure of one of these systems, and to understand the consequences that manipulating one of these pathways for therapeutic purposes could have on the activity of the other pathways.

Addendum to:

Consequences of the Selective Blockage of Chaperone-Mediated Autophagy

A.C. Massey, S. Kaushik, G. Sovak, R. Kiffin and A.M. Cuervo

Proc Natl Acad Sci USA 2006; 103:5805-10     

Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin. Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation.

We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8258-8262). While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult. We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH. We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor.     

Glucose-induced degradation of yeast fructose-1,6-bisphosphatase requires additional triggering events besides protein phosphorylation

Glucose addition to yeast cells stimulates a cAMP overshoot with concomitant activation of cAMP-dependent protein kinase, which in turn rapidly phosphorylates fructose-1,6-bisphosphatase. The phosphorylated enzyme subsequently undergoes a slow proteolytic breakdown. Also, it has been proposed that phosphorylation represents the mechanism that initiates proteolysis. Here we present experiments carried out on a yeast mutant defective in adenylate cyclase [(1982) Proc. Natl. Acad. Sci. USA 79, 2355-2359] in which extracellular cAMP triggers full enzyme phosphorylation but a scanty proteolysis, whereas glucose plus cAMP provoke both phosphorylation and complete proteolytic breakdown. Thus, besides a glucose-induced cAMP peak, which results in enzyme phosphorylation, other effects evoked by the sugar are indispensable for its proteolytic degradation.     

Identification of the cysteine residue of beta-tubulin alkylated by the antimitotic agent 2,4-dichlorobenzyl thiocyanate, facilitated by separation of the protein subunits of tubulin by hydrophobic column chromatography

The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., & Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with beta-tubulin [Bai, R., Duanmu, C., & Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of beta-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate that cysteine-239 of beta-tubulin is essential for microtubule assembly. Although alpha-tubulin is alkylated almost as well as beta-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), beta-tubulin is alkylated about 25 times as extensively as alpha-tubulin, almost exclusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of beta-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, alpha- and beta-tubulin.     

Characterization of bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase from aspen.

Bispecific O-methyltransferase (OMT, EC 2.1.1.68) which catalyses the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid was purified to homogeneity from the developing secondary xylem of aspen (Populus tremuloides). The enzyme was purified by conventional techniques and affinity chromatography on S-adenosyl-L-homocysteine agarose using substrate elution. The enzyme has a M(r) of 40,000 as determined by SDS-PAGE. Amino acid sequences of three peptides produced from a proteolytic digest of bispecific OMT were obtained by automated Edman degradation. Polyclonal antibodies raised against the purified OMT reacted strongly to OMT in both purified and unpurified fractions in western blots. Addition of an equal concentration of anti-OMT IgG to crude extract protein inhibited OMT activity by more than 70%.     

Partial purification of pepsin from turkey proventriculus

In the present study, turkey proventriculus (stomach) was mixed with 10% NaCl (1:2 w/v) and extracted by centrifugation to produce crude extract. The partial purification of the extract was carried out by using Sephadex G-75 resin in gel filtration column chromatography. Crude extract (CE) and fractions obtained by gel filtration were analysed for milk-clotting activity (MCA), protein content, proteolytic activity (PA), and SDS-PAGE electrophoresis. The first 7 fractions did not have any activities; the fraction of 9, 10 and 11 had the most milk-clotting and proteolytic activities. Electrophoretic patterns proved that further purification steps should be applied for better results.     

Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli

In order to determine if the major acidic phospholipids of Escherichia coli are essential to the organism, we constructed a null allele (pgsA30) of the pgsA gene thus rendering the organism incapable of synthesizing phosphatidylglycerol or cardiolipin. In strains carrying the pgsA30 allele cell viability, synthesis of gene product and the ability to synthesize the two major acidic phospholipids were dependent on the presence of a functional copy of the pgsA gene carried on a plasmid which was temperature-sensitive for replication. Growth ceased at the temperature restrictive for plasmid replication when the acidic phospholipid content dropped to about 10% of wild type levels which is slightly higher than the level reported in cells carrying the pgsA3 allele in a genetic background derived from strain SD12; the latter cells, which are capable of synthesizing low levels of acidic phospholipids, were previously shown to have no abnormal growth phenotype (Miyazaki, C., Kuroda, M., Ohta, A., and Shibuya, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7530-7534). The pgsA30 allele, unlike the pgsA3 allele, could not support growth in strain SD12. Neither allele could support growth in two other independently derived strains of E. coli. Therefore, there is a direct dependence of cell viability on a functional pgsA gene product. Strain SD12 appears to contain a suppressor which allows cells with a reduced capability to synthesize acidic phospholipid (pgsA3 allele) to grow, but cannot support growth in cells with a complete lack of synthetic capability (pgsA30 allele).     

Immunological and biochemical studies on the relationship between two actin-binding proteins, phosphofructokinase and gelsolin

Phosphofructokinase and gelsolin-like proteins coexist in many muscle and non-muscle tissues. They are both actin-binding proteins, and some of their biochemical parameters are remarkably similar. In a previous report [Füchtbauer, A., Jockusch, B. M., Leberer, E. & Pette, D. (1986) Proc. Natl Acad. Sci. USA 83, 9502-9506] it was shown that phosphofructokinase preparations contained actin-filament-severin activities characteristic for gelsolin. Therefore, we investigated a possible relationship between these proteins with respect to their actin-binding properties. Immunoblotting experiments with specific and non-cross-reacting antibodies to both proteins revealed two distinct polypeptides with slightly different molecular mass in SDS-PAGE of crude extracts from rabbit skeletal muscle, indicating that phosphofructokinase and gelsolin are not identical. An actin-filament-severing activity as well as the component detected by anti-gelsolin were found to copurify with phosphofructokinase during its preparation. However, the presumptive gelsolin was completely eliminated after a heat-denaturation step leaving the phosphofructokinase activity unaffected. Purified phosphofructokinase had no effects on the polymer state of preformed actin filaments. Unlike gelsolin, phosphofructokinase did not promote nucleation of actin polymerization but delayed the nucleation step. We therefore conclude that phosphofructokinase and gelsolin are functionally and structurally distinct proteins.     

Tryptic and chymotryptic cleavage sites in sequence of alpha-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney

Localization of selective proteolytic splits in alpha-subunit of (Na+ + K+)-ATPase is important for understanding the mechanism of active Na+,K+-transport. Proteolytic fragments of alpha-subunit from pig kidney were purified by chromatography in NaDodSO4 on TSK 3000 SW columns. NH2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of alpha-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691-695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490-1495). The primary chymotryptic split in the E1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E1-form is located between Lys-30 and Glu-31 (Bond 2). In the E2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH2-terminal for E1-E2 transition. Protonation of His-13 facilitates transition from E1- to E2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na+ and the shift in poise of E1-E2 equilibrium in direction of E1-forms. The NH2-terminal sequence in renal alpha-subunit is not conserved in alpha + from rat neurolemma or in alpha-subunit from Torpedo or brine shrimp. A regulatory function of the NH2-terminal part of the alpha-subunit may thus be a unique feature of the alpha-subunit in (Na+ + K+)-ATPase from mammalian kidney.     

In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.

The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins.