Caspase Cleavage Sites in the Human Proteome: CaspDB, a Database of Predicted Substrates

<div><p>Caspases are enzymes belonging to a conserved family of <b><u>c</u>ysteine-dependent <u>asp</u>artic-specific prote<u>ases</u></b> that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The <i>in silico</i> predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.</p></div

Optimized parameter values for trained classifier.

<p>Optimized parameter values for trained classifier.</p

Quality measures of trained classifiers and comparison with publicly available prediction model [44].

<p>Abbreviations: TP – number of true positives, FN-false negatives, FP-false positives, TN-true negatives, ACC-accuracy, PRC-precision, SPC-specificity, MCC-Matthews correlation coefficient, Kappa-Kappa statistical value, RF-Random Forest method, NB- Naïve Bayes, J48-decision tree algorithm, SMO-Sequential Minimal Optimization.</p><p>Quality measures of trained classifiers and comparison with publicly available prediction model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110539#pone.0110539-Piippo1" target="_blank">[44]</a>.</p

Comparision of CaspDB and Cascleave 2.0 scores.

<p>(A) Probability score comparison of caspase-1 cleavage sites. (B) Caspase-8 cleavage sites. CaspDB and Cascleave 2.0 scores are marked in red and black, respectively.</p

Toolbox of Fluorescent Probes for Parallel Imaging Reveals Uneven Location of Serine Proteases in Neutrophils

Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that play roles in the control of cell-signaling pathways and defense against pathogens and whose imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end-stage cells with a short half-life and minimal ongoing protein synthesis. To gain insight into the regulation of NSP activity we have generated a small-molecule chemical toolbox consisting of activity-based probes with different fluorophore-detecting groups with minimal wavelength overlap and highly selective natural and unnatural amino acid recognition sequences. The key feature of these activity-based probes is the ability to use them for simultaneous observation and detection of all four individual NSPs by fluorescence microscopy, a feature never achieved in previous studies. Using these probes we demonstrate uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually excluded from each other, suggesting the existence of unknown granule-targeting mechanisms

Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

<div><p>Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.</p></div

Visualization of PK401 with purified NSP4 and all NSP’s.

<p>(A) NSP4 was treated with PK401 in a range from 1 to 2000nM. (B) 100nM of NE, PR3, CatG and NSP4 with or without 100nM of PK401. (A, B) Samples were denatured in SDS sample buffer, run in SDS/PAGE followed by membrane transfer. The blot was developed with fluorescently-tagged streptavidin and imaged by fluorescence scanning (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132818#pone.0132818.s001" target="_blank">S1 Text</a>).</p

Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).

<p>NI–no inhibition observed; K_m values relate to the substrate used for analysis,</p><p>* K_m for this substrate was above 100μM, the concentration used in the assay. AMC – 7-amino-4-methylcoumarin.</p><p>Inhibition rate constants of NSPs by Biot-Ahx-hCha-Phe(guan)-Oic-Arg^P(OPh)₂ (PK401).</p

Kinetic analysis of tetrapeptide substrates for NSP4.

<p>Results are shown as an average of a minimum of 2 separate experiments with S.D.</p

Scheme of the HyCoSuL P1 Arg library.

<p>The general library structure contains tetrapeptide derivatives with the sequence Ac-P4-X-X-Arg-ACC, Ac-X-P3-X-Arg-ACC, Ac-X-X-P2-Arg-ACC, where P4, P3 and P2 represents one of 120 fixed natural or unnatural amino acids and X represents an equimolar mixture of natural amino acids (omitting Cys and substituting Nle for Met) with ACC (7-amino-4-carbamoylmethylcoumarin) as a reporter group.</p