Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Crystal structure of a membrane stomatin-specific protease in complex with a substrate peptide

Membrane-bound proteases are involved in various regulatory functions. A previous report indicated that the N-terminal region of PH1510p (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511p. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis. Here, the crystal structure of 1510-N K138A in complex with a peptide substrate was determined at 2.25 ? resolution. In the structure, a 1510-N dimer binds to one peptide. The six central residues (VIVLML) of the peptide are hydrophobic and in a pseudopalindromic structure and therefore favorably fit into the hydrophobic active tunnel of the 1510-N dimer, although 1510-N degrades the substrate at only one point. A comparison with unliganded 1510-N K138A revealed that the binding of the substrate causes a large rotational and translational displacement between protomers and produces a tunnel suitable for binding the peptide. When the peptide binds, the flexible L2 loop of one protomer forms β-strands, whereas that of the other protomer remains in a loop form, indicating that one protomer binds to the peptide more tightly than the other protomer. The Ala138 residues of the two protomers are located very close together (the distance between the two Cβ atoms is 3.6 ?). Thus, in wild-type 1510-N, the close positioning of the catalytic Ser97 and Lys138 residues may be induced by electrostatic repulsion of the two Lys138 side chains of the protomers.     

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.     

Use of fluorescence-labelled cerebroside as a substrate for galactosylceramidase of human skin fibroblasts

Possible use of the fluorescently labelled cerebroside, 1-O-(beta-D-galactosyl)-2-N-[6-(2-antroyl)hexanoyl]-4-sphingeni n (Gal-A-sphingenin) as a substrate for galactosylceramidase (GC) from human skin fibroblasts was investigated. Studies involving TLC and fluorimetric methods revealed enzymatic splitting of Gal-A-sphingenin whose degree correlated with the amount of the enzyme and incubation time. Some kinetic parameters of GC were determined, using Gal-A-sphingenin as substrate. It was shown that the values of specific activity of GC (1.6 nmol/mg protein/hr) and Km (0.025 mM) for Gal-A-sphingenin agree well with the corresponding values obtained with the use of the galactocerebroside as a natural substrate. In experiments with mixtures of Gal-A-sphingening and galactocerebroside used as substrates in different molar ratios, it was shown that the enzyme splits each of the substrates with equal velocity. The results of experiments with the enzyme samples from skin fibroblasts of healthy individuals and of patients with GM1-gangliosidosis (GM1-beta-D-galactosidase deficiency) suggest that Gal-A-sphingenin is a specific substrate for GC.     

Triple-helical peptide analysis of collagenolytic protease activity

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 > MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.     

A male accessory gland peptide with protease inhibitory activity in Drosophila funebris

Male accessory glands of Drosophila funebris synthesize and secrete a peptide that shows a protease-inhibiting activity. Amino acid sequencing of the purified peptide revealed that the peptide consists of 63 amino acid residues. It is a serine protease inhibitor belonging to the pancreatic trypsin inhibitor (Kunitz) family. The inhibitory function and the kinetic characteristics of the inhibition have been examined with various substrates. The peptide possibly plays a role as an acrosin inhibitor involved in Drosophila reproduction.     

Detection of a very rapid first phase in complex formation of DnaK and peptide substrate

Complex formation of the Hsp70 chaperone DnaK with the fluorescence-labeled peptide ALLLSAPRR shows a very rapid first phase that has as yet not been observed with other peptides. This first phase is completed within the dead time (1–2 ms) of the stopped-flow instrument and corresponds to two thirds of the total increase in fluorescence. It occurs both in the presence and in the absence of ATP and is followed by a fast, a slow and a very slow step. These binding kinetics that are vastly different from those observed with other peptides might indicate the existence of a second substrate-binding site of DnaK.     

Disentanglement of protease substrate repertoires

Identification of protease substrates and detailed characterization of processed sites are essential for understanding the biological function of proteases. Because of inherent complexity reasons, this however remains a formidable analytical challenge, illustrated by the fact that the majority of the more than 500 human proteases are uncharacterized to date. Recently, in addition to conventional genetic and biochemical approaches, diverse quantitative peptide-centric proteomics approaches, some of which selectively recover N-terminal peptides, have emerged. These latter proteomic technologies in particular allow the identification of natural protease substrates and delineation of cleavage sites in a complex, natural background of thousands of different proteins. We here review current biochemical, genetic and proteomic methods for global analysis of substrates of proteases and discuss selected applications.     

Bicyclic peptide inhibitor reveals large contact interface with a protease target

From a large combinatorial library of chemically constrained bicyclic peptides we isolated a selective and potent (K(i) = 53 nM) inhibitor of human urokinase-type plasminogen activator (uPA) and crystallized the complex. This revealed an extended structure of the peptide with both peptide loops engaging the target to form a large interaction surface of 701 ?(2) with multiple hydrogen bonds and complementary charge interactions, explaining the high affinity and specificity of the inhibitor. The interface resembles that between two proteins and suggests that these constrained peptides have the potential to act as small protein mimics.     

The Arabidopsis AtSTE24 is a CAAX protease with broad substrate specificity

Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1 Delta ste24 Delta mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-Rac fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation.     

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.     

Flexible linkers leash the substrate binding domain of SspB to a peptide module that stabilizes delivery complexes with the AAA+ ClpXP protease

SspB dimers bind proteins bearing the ssrA-degradation tag and stimulate their degradation by the ClpXP protease. Here, E. coli SspB is shown to contain a dimeric substrate binding domain of 110-120 N-terminal residues, which binds ssrA-tagged substrates but does not stimulate their degradation. The C-terminal 40-50 residues of SspB are unstructured but are required for SspB to form substrate-delivery complexes with ClpXP. A synthetic peptide containing the 10 C-terminal residues of SspB binds ClpX, stimulates its ATPase activity, and prevents SspB-mediated delivery of GFP-ssrA for ClpXP degradation. This tripartite structure--an ssrA-tag binding and dimerization domain, a flexible linker, and a short peptide module that docks with ClpX--allows SspB to deliver tagged substrates to ClpXP without interfering with their denaturation or degradation.     

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase,a thermostable serine protease fromThermoactinomyces vulgaris

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with K_i values of 7×10^–8 M and 2×10^–7 M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.     

Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH

FtsH, a member of the AAA family of proteins, is the only membrane ATP-dependent protease universally conserved in prokaryotes, and the only essential ATP-dependent protease in Escherichia coli. We investigated the mechanism of degradation by FtsH. Other well-studied ATP-dependent proteases use ATP to unfold their substrates. In contrast, both in vitro and in vivo studies indicate that degradation by FtsH occurs efficiently only when the substrate is a protein of low intrinsic thermodynamic stability. Because FtsH lacks robust unfoldase activity, it is able to use the protein folding state of substrates as a criterion for degradation. This feature may be key to its role in the cell and account for its ubiquitous distribution among prokaryotic organisms.     

A microassay for the peptidase activity of enzymes utilizing ribonuclease S peptide as a substrate

A microassay for the peptidase activity of proteins obtained in minute amounts was devised. The method uses ribonuclease S peptide as a substrate. The substrate when cleaved is unable to reconstitute an active ribonuclease S complex. Therefore the loss in activity of the reconstituted complex is a measure of the peptidase activity. The method was previously tested with known peptidases such as clastase (9), chymotrypsin (8), and trypsin. In this work the peptidase activity of a protein related to a sperm-decapitating factor (1) is evidenced.