Figure 1.
Biochemical protease interactions represented by graph theory.
Proteases influence cleavage of substrates both directly and also indirectly through cleavage of other proteases and inhibitors. Protease interactions as represented biochemically (left) and by graph theory (right). Proteases are green or blue, inhibitors are red, and other substrates are grey. Examples of protease interactions (cleavage and inhibition events) are outlined on the left: (i) In the simplest case, a protease directly cleaves a substrate, as indicated by the presence of proteolytic activity, with no further interactions. A protease can also indirectly influence cleavages by cleaving another protease for (ii) zymogen activation [76], (iii) catalytic domain removal, or (iv) exosite domain removal [77]. This will increase (ii), decrease (iii), or alter (iv) [78] the activity of the affected protease and thereby influence the cleavage of its substrates. (v) If a protease inhibitor is present, the protease does not cleave substrates. (vi) An inhibitor can be cleaved and inactivated by another protease [13],[79], which leads to increased cleavage of substrates by its cognate protease. Proteases also compensate for loss of function of other proteases or complement their activity by (vii) cleaving the same substrate at the same site or (viii) substrate cleavage by one protease can depend on prior cleavage by another protease at a different site. By graph theory of protease interactions (right), all proteins are nodes. Proteases (P) are represented as green or blue circles, inhibitors (I) as red diamond shapes, and substrates (S) are grey squares or rectangles. An edge from protein A to protein B signifies a direct regulatory influence from A on B. Such a regulatory effect could either be a cleavage or inhibition, resulting in higher, lower, altered, or unchanged activity of the target.
Table 1.
Human and mouse proteolytic networks created from all annotated proteases, inhibitors, and substrates.
Figure 2.
Reachability in network examples and the human and murine protease webs.
Connectivity in the protease networks as measured by the reachability distribution of nodes in the network. (A) Reachability in three theoretical model networks: In an unconnected network without edges, each node has a reachability of 1. In a strongly connected network, where each node can reach each other node, the reachability of each node is the sum of nodes. In a hierarchical, cascade-like network (apoptosis cascade taken from KEGG [80]), reachability values are high for upstream regulators and decrease as one descends the cascade towards the downstream effector proteins. For each protein, the corresponding reachability values are shown on the right. Proteases are represented as green circles and inhibitors as red diamonds. Edges are cleavages (green, with arrow head) and inhibitions (red, with “T” head). Although these two types of edges have biologically distinct interpretations, the implication for the graph model and reachability is identical. (B) Reachability values of nodes in a theoretical hierarchical cascade (cascade), unconnected (single), or strongly connected (circle) networks shown in (A). Reachability is plotted as an inverse cumulative function of the percentage of nodes, which can reach a given minimum number of nodes in the corresponding network. (C) Inverse cumulative function of reachability values of the largest connected components of the human protease web (255 nodes, red line) and the mouse protease web (187 nodes, blue line). Reachability is plotted as the inverse cumulative function of the percentage of nodes that can reach a given minimum percentage of nodes in the corresponding network. (D) Histogram of the path length of all shortest paths in the human network comprised of a total of 24,166 paths.
Table 2.
Proteins comprising the human and mouse protease webs.
Figure 3.
The largest connected component of the human protease web.
The structure of the core of the human protease web is comprised of 255 connected proteases and inhibitors that form the largest connected component. Proteins are designated by their UniProt gene names. Proteases are circles and inhibitors are diamonds. Nodes are color-shaded according to their betweenness. All nodes are positioned from top to bottom by decreasing reachability, which is indicated by the depth of shade of the green background. Edges are cleavages (with arrow head) or inhibitions (“T” head). Nodes of known protease cascades are labeled and marked by dashed circles. The figure is rendered in high resolution for click to zoom.
Table 3.
Reachability values of nodes in the human protease web.
Figure 4.
Interactions between protease groups in the human protease web.
(A) Click-to-zoom figure of detailed connections between pathways and protease groups in the strongly connected component of the network. The network presented is limited to proteases (no inhibitors) with a reachability of 153 from Figure 3. Nodes are proteases and edges are cleavages. Proteases are designated by their UniProt gene names. (B) Interactions between classes of proteases and their inhibitors. Nodes are classes of proteins: classes of proteases are green circles; classes of protease inhibitors are red diamonds. The size of the nodes represents the number of proteins in each class as exemplified with groups of 10, 50, and 100 nodes in the legend. Protein classification: “M” are metallo, “S” are serine, “C” are cysteine, “A” are aspartate, and “T” are threonine proteases (as classified in MEROPS) or the corresponding inhibitors (as annotated in neXtProt). “B” are broad-spectrum inhibitors that are annotated to inhibit more than one class of protease and include A2M, serpin B4, serpin B9, PZP, histidine-rich glycoprotein, ovostatin homolog 1, and reversion-inducing cysteine-rich protein with Kazal motifs. Edges are cleavages (green, with arrow head) or inhibitions (red, with “T” head). Thickness of edges corresponds to the number of cleavages or inhibitions between the classes as exemplified with edges corresponding to 10, 50, or 100 interactions in the legend.
Figure 5.
Reachability in the human protease web after various perturbations.
Reachability of the largest connected component of the protease web (shown in Figures 2C and 3) after various perturbations. Reachability is plotted as the inverse cumulative function of the percentage of nodes that can reach a given minimum number of nodes in the corresponding network. (A) Reachability in the high confidence network comprised of nodes annotated as having physiological relevance. The reachability distribution of the original network (“orig,” red solid line as also shown in Figure 2C) is compared to networks where edges were removed to create a high confidence network (“hc,” black dashed line) and the high confidence network plus inhibitors (“hc+i,” black solid line). (B) Reachability before (“orig,” red line) and after (“inh rm,” black line) removing edges, reflecting cleavages of inhibitors. Cleavage edges were removed if (i) the inhibitor is annotated to be a serine protease inhibitor and the protease is a serine or cysteine protease or (ii) the inhibitor is A2M or PZP. (C) Reachability after removal of six nodes from the original network (PLG, alpha-1-antitrypsin, A2M, CTSL1, alpha-1-antichymotrypsin, and KLK4). The reachability after removing these six nodes (“6 rm,” black solid line) is compared to the reachability distribution of the original network (“orig,” red line) and to six networks representing each possible combination of keeping one of the six nodes and removing the other five (“5 rm,” black dotted lines), each showing much smaller reduction in reachability. (D) Reachability after removal of random edges. The reachability in the original network (“orig,” red line) compared to networks where 10%, 20%, 30%, or 40% of edges were removed at random. In each case, random edge deletion was carried out 200 times and the worst AUC value was selected for plotting.
Figure 6.
Reachability in tissue-specific protease webs.
(A) Beanplot of reachability distributions in the largest connected components of 23 human tissue-specific networks based on gene expression in the corresponding tissues and the original protease web reachability distributions. Overlaid is a scatterplot of the precise values of each node. Numbers in parentheses refer to the size of the network. (B) Inverse cumulative distribution plot of reachability values for skin (dashed black line), spleen (black solid line), liver (dotted black line), and original network (red solid line). Reachability is plotted as an inverse cumulative function of the percentage of nodes that can reach a given minimum percentage of nodes in the corresponding network.
Figure 7.
Proteases and their inhibitors involved in multiple, discreet biological processes.
(A) A matrix showing the annotation of proteases and inhibitors with selected, protease-specific biological processes based on Gene Ontology [61]. Proteins annotated with more than one term are displayed. (B) A subnetwork of the protease web connecting TIMP1 to coagulation: TIMP1 (UniProt: P01033) inhibits MMP10 (UniProt: P09238) and MMP1 (UniProt: P03956), which both cleave and activate MMP9 (UniProt: P14780), which cleaves PLG (UniProt: P00747). Similarly, MMP1 and MMP9 cleave and inactivate serpin A1 (UniProt: P01009), which is an inhibitor of PLG.
Figure 8.
Protease web affects validation in vivo.
(A) Tris-Tricine 15% SDS-PAGE and MALDI-TOF mass spectrometry analyses of LIX cleavage following incubation with wild-type (WT) or MMP8-deficient (KO) murine polymorphonuclear leukocytes (PMNs) for up to 3 h after PMA stimulation to release PMN proteases to the culture medium. (B) Sequence of the N- and C-terminal regions of LIX with cleavage sites by PMN MMP8 and the unknown protease (?) annotated. (C) Tris-Tricine SDS-PAGE analysis of LIX cleavage by PMNs after addition of protease inhibitors: AEBSF, 2-aminoethyl benzenesulfonyl fluoride hydrochloride; α1-PI, α1-proteinase inhibitor; SLPI, secreted leukocyte proteinase inhibitor. (D) LIX cleavage by murine (m) MMP8 and murine neutrophil elastase (mNE) analyzed by 15% Tris-Tricine SDS-PAGE analysis and MALDI-ToF mass spectrometry. E:S, enzyme-to-substrate ratio; “Marker,” molecular weight markers as indicated. (E) Network effects on LIX cleavage. Proteases are green, inhibitors red, and other substrate proteins are grey. Edges are cleavages (green, with arrow head) or inhibitions (red, with “T” head). (F) MMP8 cleavage of α1-proteinase inhibitor (α1-PI). The serine protease inhibitor α1-PI was incubated with MMP8 for 16 h at 37°C in 50 mM Tris, 200 mM NaCl, 5 mM CaCl2, pH 7.4 containing 1 mM APMA. The enzyme-to-substrate (E:S) ratio ranged from 1∶5 to 1∶5,000 (w:w). Reactions were visualized on a 10% SDS-PAGE (silver stained). Below, a time course of MMP8 cleavage of α1-PI at 1∶50 (w:w) E:S ratio. (G) Bronchioalveolar lavage of mice stimulated with LPS. WT, wild-type mouse; KO, MMP8 knockout mouse. LPS (2 µg) was instilled in the lungs of female mice, and after 48 h, the mice were sacrificed and the lungs lavaged with PBS. Cell-free bronchioalveolar lavage from three mice was pooled and concentrated by acetone precipitation. α1-PI detection was with Alexa-conjugated antibodies (Molecular Probes) on the LiCOR Odyssey. (H) Numbers of PMNs in the bronchioalveolar lavage after LPS stimulation with (n = 3) and without (n = 3) instillation of GW311616 (GW), a specific neutrophil elastase inhibitor.