Caspase-like activity is associated with bacterial infection of the urine in urinary tract diseases.

Urinary tract infections (UTIs) are a serious public health problem. They can be caused by a number of pathogens, but the most common are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus. Bacterial infection is diagnosed by examining a urine sample. The presence of bacteria or white blood cells is determined under a microscope or a urine culture is performed. In this study, we used a panel of chromogenic substrates for the qualitative determination of specific enzyme activity in the urine of patients with confirmed bacterial infection and/or urinary tract disease. Healthy patients were used as a control group. It turned out that in the case of Escherichia coli infection, we observed the activity of the caspase subunit of the human 20S proteasome. We did not observe similar correlations for infections with other types of bacteria.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Ultrasensitive internally quenched substrates of human cathepsin L.

Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO2)-NH2 with high specificity constant (kcat/KM=2.6×10(7)M(-1)s(-1)) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.

Clinical Evaluation of Pathognomonic Salivary Protease Fingerprinting for Oral Disease Diagnosis.

Dental decay (Caries) and periodontal disease are globally prevalent diseases with significant clinical need for improved diagnosis. As mediators of dental disease-specific extracellular matrix degradation, proteases are promising analytes. We hypothesized that dysregulation of active proteases can be functionally linked to oral disease status and may be used for diagnosis. To address this, we examined a total of 52 patients with varying oral disease states, including healthy controls. Whole mouth saliva samples and caries biopsies were collected and subjected to analysis. Overall proteolytic and substrate specific activities were assessed using five multiplexed, fluorogenic peptides. Peptide cleavage was further described by inhibitors targeting matrix metalloproteases (MMPs) and cysteine, serine, calpain proteases (CSC). Proteolytic fingerprints, supported by supervised machine-learning analysis, were delineated by total proteolytic activity (PepE) and substrate preference combined with inhibition profiles. Caries and peridontitis showed increased enzymatic activities of MMPs with common (PepA) and divergent substrate cleavage patterns (PepE), suggesting different MMP contribution in particular disease states. Overall, sensitivity and specificity values of 84.6% and 90.0%, respectively, were attained. Thus, a combined analysis of protease derived individual and arrayed substrate cleavage rates in conjunction with inhibitor profiles may represent a sensitive and specific tool for oral disease detection.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes.

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate.

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.

Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus.

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72 kDa could be visualized after silver staining and Western blotting. Assays performed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profile very similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze the ACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE inhibitor lisinopril (Ki of 1.26 nM). However, in contrast to other ACE isoforms, crab-ACE presented a very particular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Other interesting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around 45°C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a better understanding of the biochemical function of this enzyme in invertebrates.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.