Direct expression of recombinant activated human protein C, a serine protease

Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.     

Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide

The gene encoding the secreted 53-kDa metalloprotease (protease B) and the 5' end of the gene encoding the secreted 55-kDa metalloprotease (protease C) of the Gram-negative bacterium Erwinia chrysanthemi have been sequenced. The predicted sequences of the two proteases do not have typical signal sequences at their NH2 termini. Both proteases are synthesized as inactive higher molecular weight precursors (zymogens proB and proC) which are secreted into the external medium where divalent cation-mediated activation occurs. The activation of proB occurs with a t1/2 of less than 5 min at 37 degrees C in Luria broth medium, whereas that of proC occurs with a t1/2 of about 150 min. The NH2 termini of purified proteases B, proB, and C were sequenced. ProB starts at the initiator methionine whereas B and C start, respectively, at residues +16 and +18 of the sequence deduced from the nucleotide sequence. A short NH2-terminal extension is therefore removed during the activation process, most likely by an autocatalytic mechanism. Protease B shows a high degree of sequence homology with the secreted 50-kDa metalloprotease of Serratia marcescens, which also lacks a signal peptide and for which an inactive higher molecular weight form has been reported.     

Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.     

Activation and Inhibition of Cerebral Prolidase

Purified of prolidase from calf brain (acetone and [NH4]2SO4 fractionation) separated this enzyme from proteases, leucine aminopeptidase, master dipeptidase, and Gly-Gly dipeptidase. Prolidase was tested with peptidase and protease inhibitors, used at higher levels (35 times or more) than their ID50 for peptidases and proteases. Bacitracin, leupeptin, chymostatin, and antipain had no effect; pepstatin slightly increased activity, and only bestatin was inhibitory. Antibiotics that affect protein synthesis did not inhibit prolidase. Peptides with proline at the NH2 end activated prolidase, whereas those with proline at the carboxyl end inhibited it. Di, tri, and tetra-Pro peptides increased prolidase activity. Thyrotropin-releasing hormone had no effect on prolidase; its analog Pro-His-Pro-NH2 gave high activation and decreased the Km from 20 mM to 1.54 mM. Pro-peptide inhibitors and activators were not themselves split by prolidase. The results indicate influences of specific peptides, for both inhibition and activation, on prolidase activity.     

Novel proteases: common themes and surprising features

Proteases perform a wide variety of functions, inside and outside cells, regulating many biological processes. Recent years have witnessed a number of significant advances in the structural biology of proteases, including aspects of intracellular protein and peptide degradation by self-compartmentalizing proteases, activation of proteases in proteolytic cascades of regulatory pathways, and mechanisms of microbial proteases in pathogenicity.     

Secretion, processing and activation of bacterial extracellular proteases

Many different bacteria secrete proteases into the culture medium. Extracellular proteases produced by Gram-positive bacteria are secreted by a signal-peptide-dependent pathway and have a propeptide located between the signal peptide and the mature protein. Many extracellular proteases synthesized by Gram-negative bacteria are also produced as precursors with a signal peptide. However, at least two species of Gram-negative bacteria secrete one or more proteases via a novel signal-peptide-independent route. Most proteases secreted by Gram-negative bacteria also have a propeptide whose length and location vary according to the protease. Specific features of protease secretion pathways and the mechanisms of protease activation are discussed with particular reference to some of the best-characterized extracellular proteases produced by Gram-positive and Gram-negative bacteria.     

7S Nerve growth factor alpha and gamma subunits are closely related proteins

The polypeptide composition and partial amino acid sequence of the 7S nerve growth factor (NGF) alpha subunit have been determined. Residues in 76 unique positions corresponding to 35% of the molecule were identified. The sequence shows that the NGF alpha subunit is closely related to the NGF gamma subunit and thus a member of the same protein family as the serine proteases. This finding is unexpected since the NGF alpha subunit is devoid of detectable protease activity. However, the NGF alpha subunit differs in one important respect from the NGF gamma subunit and related serine proteases. The highly conserved amino-terminal activation cleavage structure, common to most serine proteases, has been deleted, and an uncleaved activation peptide remains attached to the amino terminus of the mature NGF alpha subunit. It is suggested that this feature is causally related to the apparent lack of proteolytic activity.     

Proteases involved in long-term potentiation

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.     

Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Peptide substrates rapidly modulate expression of dipeptide and oligopeptide permeases in Candida albicans

Previous studies showed that peptide transport activity in Candida albicans was completely repressed by NH4+, and that growth on amino acids as sole nitrogen source stimulated transport to a basal level. Here we show that addition of peptide mixtures to culture media gives a further 5-fold increase in transport of dipeptides and oligopeptides; the effect is specific for peptide transport, amino acid uptake being unaffected. Presence of peptides but not amino acids overrides NH4+ repression of peptide transport. Step-up activation of transport activity, caused by addition of peptides to incubation media, and step-down inhibition that accompanies removal of peptides, occurs rapidly (within 30 min at 28 degrees C). Step-up is independent of de novo protein synthesis. This substrate-induced regulation is compatible with a rapid, reversible activation of plasma membrane-bound peptide permease(s), or a mechanism of endocytosis involving a cycle of insertion and retrieval of preformed permease components. These results are considered in relation to the expression of peptide permeases in vivo, and the development of synthetic anticandidal peptide carrier prodrugs designed to exploit these systems.     

Emerging role of rhomboid family proteins in mammalian biology and disease

From proteases that cleave peptide bonds in the plane of the membrane, rhomboids have evolved into a heterogeneous superfamily with a wide range of different mechanistic properties. In mammals 14 family members have been annotated based on a shared conserved membrane-integral rhomboid core domain, including intramembrane serine proteases and diverse proteolytically inactive homologues. While the function of rhomboid proteases is the proteolytic release of membrane-tethered factors, rhomboid pseudoproteases including iRhoms and derlins interact with their clients without cleaving them. It has become evident that specific recognition of membrane protein substrates and clients by the rhomboid fold reflects a spectrum of cellular functions ranging from growth factor activation, trafficking control to membrane protein degradation. This review summarizes recent progress on rhomboid family proteins in the mammalian secretory pathway and raises the question whether they can be seen as new drug targets for inflammatory diseases and cancer. This article is part of a special issue entitled: Intramembrane Proteases.     

Neurotrophic factors regulate cathepsin S in macrophages and microglia: A role in the degradation of myelin basic protein and amyloid beta peptide.

BACKGROUND: Cathepsin S is a member of the family of cysteine lysosomal proteases preferentially expressed in macrophages and microglia and is active after prolonged incubation in neutral pH. Upon activation of macrophages by a number of inflammatory mediators, there is an increase in secreted cathepsin S activity accompanied by a decrease in cellular cathepsin S activity and protein content, as well as a decrease in cathepsin S mRNA. The decrease in cathepsin S mRNA and protein at the cellular level is in contrast to the response observed in some in vivo scenarios. MATERIALS AND METHODS: We investigated the effect of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), two growth factors present during cell injury and inflammation but not known to activate macrophages and microglia, on the expression of cathepsin S, cathepsin B, and cathepsin L mRNAs in these cells, and on cathepsin S activity. We then tested the ability of cathepsin S to degrade myelin basic protein, and amyloid beta peptide at both acidic and neutral pH. RESULTS: Basic FGF and NGF treatment of macrophages and microglia significantly increased the levels of cathepsin S, B, and L mRNAs (2- to 5-fold). Basic FGF also increased cathepsin S activity intra- and extracellularly. Recombinant human cathepsin S was able to degrade myelin basic protein and monomeric and dimeric amyloid beta peptide at both acidic and neutral pH, as well as to process human amyloid precursor protein generating amyloidogenic fragments. CONCLUSIONS: These data suggest that bFGF and NGF may be the molecular signals that positively regulate the expression and activity of cysteine lysosomal proteases (cathepsin S in particular) in macrophages and microglia in vivo, and that there is an interplay between these factors and the activators of inflammation. Disruption of the balance between these two categories of signals may underlie the pathological changes that involve cysteine proteases. http://link.springer-ny.com/link/service/journals/00020/bibs /5n5p334. html     

Interaction of the protein C activation peptide with platelets

The peptide NH(2)-DTEDQEDQVDPR-COOH is released during activation of protein C zymogen. We measured the effect of a synthetic peptide with an amino acid sequence similar to that of the natural peptide on platelets from healthy individuals using platelet aggregometry. We found that this synthetic peptide inhibits platelet aggregation induced by thrombin; furthermore, it diminishes mobilization of intraplatelet calcium. Molecular docking showed weak interaction between the synthetic peptide and thrombin. Our findings suggest that this synthetic peptide may interact with a receptor located on the platelet cell membrane.     

Structural relationship between "glutamic acid" and "lysine" forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.

Urokinase digestion of maleinated plasminogen results in cleavage of the single peptide bond Arg-68-Met-69, which is one of the bonds normally cleaved during the first step of the activation procedure. The inactive intermediate compound formed in this way was subjected to NH2-terminal amino acid sequence analysis, which clearly demonstrates the structural relationship between the forms of plasminogen with different NH2-terminal amino acids. It is thus shown that lysine-78 and valine-79 in the "glutamic acid" plasminogen actually are the NH2-terminal amino acids in "lysine" and "valine" plasminogen respectively. The forms with glutamic acid in NH2-terminal position are called plasminogen A, while all other forms lacking the NH2-terminal part of the molecule and which can be activated in a single step are called plasminogen B. By affinity chromatographic studies of the NH2-terminal activation peptide on insolubilized plasminogen B, it was demonstrated that this peptide has specific affinity for plasminogen B. It was also shown that this noncovalent interaction is broken by 6-aminohexanoic acid in two concentration. The tryptic heptapeptide (Ala-Phe-Gln-Tyr-His-Ser-Lys) which occupies the positions number 45 to 51 in the NH2-terminal activation peptide (as well as in the intact plasminogen molecule) is importance for the conformational state of the plasminogen molecule.     

A comparative study on the NH2-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins

Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.     

The mechanism of activation of rabbit plasminogen by urokinase.

The data presented in this paper show that when rabbit plasminogen is activated to plasmin by urokinase at least two peptide bonds are cleaved in the process. Urokinase first cleaves an internal peptide bond in plasminogen, leading to two-chain disulfide-linked plasmin molecule. The plasmin heavy chain of molecular weight 66,000 to 69,000 possesses an NH2-terminal amino acid sequence identical with the original plasminogen (molecular weight 88,000 to 92,000). The plasmin light chain of molecular weight 24,000 to 26,000 is known to be derived from the COOH-terminal portion of plasminogen. The plasmin generated during the activation of plasminogen is capable, by a feedback process, of cleaving a peptide of molecular weight 6,000 to 8,000 from the NH2 terminus of the heavy chain, producing a proteolytically modified heavy chain of molecular weight 58,000 to 62,000. Plasmin also can cleave this same peptide from the original plasminogen, yielding an altered plasminogen of molecular weight 82,000 to 86,000. This plasmin-altered plasminogen and the plasmin heavy chain derived from it by urokinase activation process NH2-terminal amino acid sequences which are identical with each other and with the plasminolytic product of the original plasmin heavy chain. These studies support a mechanism of activation of plasminogen by urokinase which involves loss of a peptide located on the NH2 terminus of plasminogen. However, these same results show that this NH2-terminal peptide need not be released from rabbit plasminogen prior to the cleavage of the internal peptide bond which leads to the two-chain plasmin molecule. Furthermore, these studies show that urokinase cannot remove this peptide from either the original rabbit plasminogen molecule or from the heavy chain of the initial plasmin formed.     

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.