Clonidine displacement from type 1 imidazoline receptor by p-aminobenzamidine,the prototype of trypsin-like serine protease inhibitors

p-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I1-R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [3H]clonidine displacement from I1-R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [3H]clonidine from I1-R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control.     

Conformational selection in trypsin-like proteases

For over four decades, two competing mechanisms of ligand recognition-conformational selection and induced-fit-have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics.     

Sensitive fluorescent determination of trypsin-like proteases

A new sensitive assay for trypsin-like proteases has been developed using as substrate protamine sulfate from herring with the amino terminal group blocked with dinitrofluorobenzene. Enzymatic hydrolysis liberated amino groups which were quantitated by measuring fluorescence after reaction with fluorescamine. Thrombin was capable of hydrolyzing this substrate at a concentration as low as 8.3 NIH units per ml. Amino acid analysis of the protamine suggests that thrombin is capable of hydrolyzing a peptide bond other than an arginyl-glycine bond. Inhibition of thrombin by n-acetylimidazole suggests a relationship between the clotting and proteolytic activities of thrombin.     

Synthesis and enzymatic evaluation of a P1 arginine aminocoumarin substrate library for trypsin-like serine proteases

A method for the solid-phase synthesis of P1 arginine containing peptides via attachment of the arginine side-chain guanidine group is described. This procedure is applied to the preparation of a tetrapeptide, P1 arginine aminocoumarin PS-SCL. This library was validated by using it to determine the P4-P2 specificity for thrombin and comparing the results to the known thrombin subsite specificity. This is the first reported example of a PS-SCL library containing a P1 arginine.     

Glycosylated trypsin-like proteases from earthworm Eisenia fetida

Although groups of earthworm proteases have been found by several laboratories, it is still unclear how many of the isolated trypsin-like fibrinolytic enzymes are in glycosylated form. Here, eight glycosylated fibrinolytic proteases (EfP-0-1, EfP-0-2, EfP-I-1, EfP-I-2, EfP-II-1, EfP-II-2, EfP-III-1 and EfP-III-2) were isolated from an earthworm species (Eisenia fetida) through a stepwise-purification procedure: ammonium sulfate precipitation, affinity chromatography on a Sepharose-4B column coupled with soybean trypsin inhibitor (SBTI), and ionic chromatography with a DEAE-Cellulose-52 column. Among the eight purified trypsin-like glyco-proteases, EfP-0-2 and EfP-II-2 were newly isolated isozymes. Glycoprotein staining of the proteases on native-PAGE with a Schiff's reagent (sodium meta-periodate) revealed that the eight proteases were glycoproteins. Measurements of the glycan content with sodium meta-periodate and glycoprotein-test reagent showed that these proteases had different carbohydrate contents. Dot-blotting assay with ConA suggested the oligosaccharides were composed of mannose residues.     

Sequential activation of caspases and serine proteases (serpases) during apoptosis

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TICK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP-reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Allostery in trypsin-like proteases suggests new therapeutic strategies

Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.     

Genomic overview of serine proteases

Serine proteases (SP) are peptidases with a uniquely activated serine residue in the substrate-binding pocket. They represent about 0.6% of all proteins in the human genome. SP are involved in many vital functions such as digestion, blood clotting, fibrinolysis, fertilization, and complement activation and are related to many diseases including cancer, arthritis, and emphysema. In this study, we performed a genomic analysis of human serine proteases utilizing different databases, primarily that of MEROPS. SP are distributed along all human chromosomes except 18 and Y with the highest density (23 genes) on chromosome 19. They are either randomly located within the genome or occur in clusters. We identified a number of SP clusters, the largest being the kallikrein cluster on chromosome 19q13.4 which is formed of 15 adjacent genes. Other clusters are located on chromosomes 19p13, 16p13, 14q11, 13q35, 11q22, and 7q35. Genes of each cluster tend to be of comparable sizes and to be transcribed in the same direction. The members of some clusters are sometimes functionally related, e.g., the involvement of many kallikreins in endocrine-related malignancies and the hematopoietic cluster on chromosome 14. It is hypothesized that members of some clusters are under common regulatory mechanisms and might be involved in cascade enzymatic pathways. Several functional domains are found in SP, which reflect their functional diversity. Membrane-type SP tend to cluster in 3 chromosomes and have some common structural domains. Several databases are available for screening, structural and functional analysis of serine proteases. With the near completion of the Human Genome Project, research will be more focused on the interactions between SP and their involvement in pathophysiological processes.     

The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved

We examined the influence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of differences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the effects on k_cat depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not affect the S2–S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1–P1 interaction over substrate binding in the S2–S4 sites.     

Electrostatic recognition in substrate binding to serine proteases

Serine proteases of the Chymotrypsin family are structurally very similar but have very different substrate preferences. This study investigates a set of 9 different proteases of this family comprising proteases that prefer substrates containing positively charged amino acids, negatively charged amino acids, and uncharged amino acids with varying degree of specificity. Here, we show that differences in electrostatic substrate preferences can be predicted reliably by electrostatic molecular interaction fields employing customized GRID probes. Thus, we are able to directly link protease structures to their electrostatic substrate preferences. Additionally, we present a new metric that measures similarities in substrate preferences focusing only on electrostatics. It efficiently compares these electrostatic substrate preferences between different proteases. This new metric can be interpreted as the electrostatic part of our previously developed substrate similarity metric. Consequently, we suggest, that substrate recognition in terms of electrostatics and shape complementarity are rather orthogonal aspects of substrate recognition. This is in line with a 2‐step mechanism of protein‐protein recognition suggested in the literature.     

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

Analgesia induced by certain tricyclic antidepressants has been largely used for decades, yet the mechanisms involved are incompletely understood. Starting from previously reported dual effects of amitriptyline on wild-type ENaC (Pena F, et al. J Pharm Pharmacol 54:1393-8: 2002), we extended our study to ASIC1a by performing a series of whole cell and single-channel recordings of proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min intervals, and amitriptyline (1-500 μM) was applied at a holding pH of 7.4 or 8.4 between pulses. Dose-response plots were fitted with dual Hill type functions, yielding a half-activatory constant of 0.3 μM and a half-inhibitory constant of 382 μ M at pH 7.4. At pH 8.4 both constants were shifted to higher values (0.5 and 444 μM, respectively). In whole-cell experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 μM and decreased it at 1 and 100 μM. Single-channel recordings were idealized and fitted using an 8-state linear connectivity model comprising four consecutive activation steps. Both amitriptyline (1 μM) and FMRF-amide (0.1 μM) increased the unitary current amplitude, and modified the opening and closing rates of the first gating mode. They also increased the transition rate from the second to the first gating mode, and the rate of final closure. The activatory effect of both compounds vanished after a mild trypsin pretreatment, suggesting the existence of activatory sites for FMRF-amide and amitriptyline in the outer vestibule of ASIC1a, which can be removed by exo- or endogenous serine-proteases.     

ASIC1a activation by amitriptyline and FMRF-amide is removed by serine proteases

Analgesia induced by certain tricyclic antidepressants has been largely used for decades, yet the mechanisms involved are incompletely understood. Starting from previously reported dual effects of amitriptyline on wild-type ENaC (Pena F, et al. J Pharm Pharmacol 54:1393-8: 2002), we extended our study to ASIC1a by performing a series of whole cell and single-channel recordings of proton-activated currents in HEK293 cells. Acid pulses were applied at 2 or 5 min intervals, and amitriptyline (1-500 microM) was applied at a holding pH of 7.4 or 8.4 between pulses. Dose-response plots were fitted with dual Hill type functions, yielding a half-activatory constant of 0.3 microM and a half-inhibitory constant of 382 microM at pH 7.4. At pH 8.4 both constants were shifted to higher values (0.5 and 444 microM, respectively). In whole-cell experiments, FMRF-amide increased the peak amplitude of ASIC1a transients at 0.1 microM and decreased it at 1 and 100 microM. Single-channel recordings were idealized and fitted using an 8-state linear connectivity model comprising four consecutive activation steps. Both amitriptyline (1 microM) and FMRF-amide (0.1 microM) increased the unitary current amplitude, and modified the opening and closing rates of the first gating mode. They also increased the transition rate from the second to the first gating mode, and the rate of final closure. The activatory effect of both compounds vanished after a mild trypsin pretreatment, suggesting the existence of activatory sites for FMRF-amide and amitriptyline in the outer vestibule of ASIC1a, which can be removed by exo- or endogenous serine-proteases.     

Thermodynamics of Na+ binding to coagulation serine proteases

The sodium binding to serine proteases triggers a conformational change in the proteins that enhances the catalytic activity of the enzymes. The interaction of the cation with the protein is mediated by the hydrogen-bonding network of water molecules that embed the Na+ site. We pointed out the crucial role of the insertion loop 186a-d and the I16-D194 ion pair in the stabilization of sodium binding pocket in thrombin. This paper contributes to better explain the molecular mechanism of sodium binding for different serine proteases leading to the identification of the structural changes necessary to engineer a functional Na+ site and regulate catalytic activity in serine proteases.     

Degradation of myofibrillar proteins by trypsin-like serine proteinases.

The effects of the Ca2+-activated cysteine proteinase, the rat trypsin-like serine proteinase and bovine trypsin on myofibrillar proteins from rabbit skeletal muscle are compared. 2. Myofibrils that had been treated at neutral pH with the Ca2+-dependent proteinase and with the rat enzyme were (a) analyzed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and (b) examined in the electron microscope. Treatment with each proteinase resulted in the loss of the Z-discs, but the rat enzyme caused much more extensive disruption of the ultrastructure and degraded more of the myofibrillar proteins. 3. Purified F-actin was almost totally resistant to the proteinases, whereas G-actin was degraded by the rat trypsin-like proteinase at a rate approx. 15 times faster than was obtained with bovine trypsin. 4. Similar results were obtained with alpha-actinin, whereas tropomyosin was degraded more readily by bovine trypsin than by the rat trypsin-like proteinase. 5. The implications of these findings for the non-lysosomal breakdown of myofibrillar proteins in vivo are considered.     

Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).     

Hydrolysis of polyesters by serine proteases

The substrate specificity of -chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(-caprolactone) (PCL). -Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to -chymotrypsin.     

The HtrA family of serine proteases

HtrA, also known as DegP and probably identical to the Do protease, is a heat shock-induced serine protease that is active in the periplasm of Escherichia coli . Homologues of HtrA have been described in a wide range of bacteria and in eukaryotes. Its chief role is to degrade misfolded proteins in the periplasm. Substrate recognition probably involves the recently described PDZ domains in the C-terminal half of HtrA and, we suspect, has much in common with the substrate recognition system of the tail-specific protease, Prc (which also possesses a PDZ domain). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes an alternative sigma factor, RpoE, an anti-sigma factor, RseA, a two-component regulatory system, CpxRA, and two phosphoprotein phosphatases, PrpA and PrpB. Mutations in the htrA genes of Salmonella , Brucella and Yersinia cause decreased survival in mice and/or macrophages, and htrA mutants can act as vaccines, as cloning hosts and as carriers of heterologous antigens.