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

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

Optimized parameter values for trained classifier.

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

ATP levels in neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), repectively. ATP levels were determined with a luciferase-based assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Membrane potential of isolated neutrophil mitochondria.

<p>Isolated neutrophil mitochondria were analysed by flow cytometry after staining with Mitotracker Green, to differentiate intact mitochondria from cellular debris, and TMRM to determine Δψ_m. A) Scatter plot displaying intact mitochondria, defined as Mitotracker-Green-positive events (FITC channel) with high side scatter in Q2. B) Intact mitochondria gained TMRM staining (Δψ_m, PE channel) after incubation with mitochondrial substrates. The complex-I substrates glutamate/malate (green) and the complex-II substrate succinate (yellow) induced a lower Δψ_m than glycerol phosphate (blue). Graphs are representative of three independent experiments. C) Graphic representation of the pooled experiments. Relative Δψ_m is expressed as a percentage of the mean fluorescence intensity (MFI) in the PE channel of the mitochondria population (Q2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002013#pone-0002013-g007" target="_blank">Figure 7A</a>). The MFI of glycerol phosphate (GlycP) treated mitochondria was set at 100% while the MFIs of the untreated (C-), glutamate/malate (Glut/Mal) and succinate (Succ) treated cells were expressed as a percentage of this value. The average original value for C- was 1701±498 (SEM), for Glut/Mal 4442±2689 (SEM), for Succ 5481±3106 (SEM) and for GlycP 5838±3139 (SEM). Bars represent the means (±SEM) of three independent experiments.</p

HL-60 cells loose supercomplex organisation and acquire neutrophil-like properties during differentiation.

<p>HL-60 cells were differentiated to neutrophil-like cells in 9 days. To demonstrate that HL-60 cells gain a neutrophil-like morphology and abilities during differentiation, the expression of the neutrophil markers gp91^PHOX and EMR3 was determined by flowcytometry (A) and their ability to produce a respiratory burst was determined by an Amplex Red assay (B). On day 9 of differentiation, approximately 60% of the cells stained positive for both neutrophil markers, while responding to all three stimuli (PMA, fMLP and the combination of PAF and fMLP) by producing a respiratory burst. Plots are representative of four independent experiments. C) Supercomplex organisation in HL-60 mitochondria before (Day 0) and after (Day 9) differentiation. Respiratory chain complexes were detected on Western blots of 2D-native/reduced SDS-PAGE gels with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots are representative of four independent experiments. During differentiation, HL-60 cells gain the metabolic properties of neutrophils (D and E). Undifferentiated (▪,□) and differentiated (•,○) HL-60 cells were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the complex-I inhibitor rotenone (D) or the complex-III inhibitor antimycin A (E) as indicated. Afterwards, ATP (left panels) and lactate (right panels) levels were determined. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Supercomplex organisation of OXPHOS complexes in PBMC and neutrophils.

<p>A–B) Mitochondria isolated from PBMC (A) and neutrophils (B) were solubilized in digitonin, and 200 µg of mitochondrial protein was separated by BN-PAGE. Strips from the first dimension were excised and used for second dimension SDS-(12%)PAGE, transferred to PVDF membrane and immuno-detected with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots are representative of three independent experiments. High molecular weight purified proteins were used as molecular mass markers in the first dimension BN-PAGE: thyroglobulin, 669 kDa; ferritin monomer, 440 kDa; catalase 232 kDa; lactate dehydrogenase, 140 kDa.</p

Mitochondrial content and respiratory chain enzyme activity in neutrophils, PBMC and HL-60 cells.

<p>A) Neutrophils (PMN) contain significantly less mitochondria as expressed per unit citrate synthase activity. Data represent the mean (±SD) of 13 (PMN), 6 (HL-60) or 16 (PBMC) different experiments performed in duplicate. B) Neutrophils retain full complex II and V activity while the activity of the remaining complexes is significantly reduced. Data represent the mean (±SD) of 10 different experiments performed in duplicate. Values were corrected for mitochondrial protein content and citrate synthase activity. C) Crucial subunits of the respiratory chain can be detected in neutrophil lysates on Western blot. 20 µg of mitochondrial protein was separated by SDS-PAGE and immuno-detected with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots exposed for 15 sec and 5 minutes are representative of three independent experiments.</p

Lactate levels produced by neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), respectively. Lactate levels were determined with an enzymatic assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p