Proteomic profiling of metalloprotease activities with cocktails of active-site probes

Abstract

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.

Figure 1

Functional proteomic analysis of metalloprotease activities using active site-directed chemical probes. (a) General structure of the alkyne-tagged hydroxamate-benzophenone (HxBPyne) probe library for the proteome profiling of metalloprotease activities. The structures of the R₁ and R₂ substituents are shown and named based on the amino acid that they mimic (R₁ sublibrary, 15-23; Trp-R₂ sublibrary, 11-14; R₂ sublibrary, 8-10). (b) Application of HxBPyne probes to proteomes and analysis of results by gel-based (upper) or MudPIT-based (lower) ABPP. In gel-based ABPP, individual HxBPyne-treated proteomes are reacted with an azide-rhodamine (Rh) reporter tag under CC conditions and separated by SDS-PAGE, and labeled metalloproteases are visualized by in-gel fluorescence scanning. In ABPP-MudPIT, proteomes are treated with a cocktail of HxBPyne probes and reacted with an azide-biotin (B) reporter tag under CC conditions. Probe-labeled metalloproteases are then captured on avidin beads, digested with trypsin and analyzed by multidimensional LC-MS.

Figure 2

Profiling metalloprotease activities in proteomes with the HxBPyne probe library. (a) Concentration-dependent labeling profiles for representative HxBPyne probes added to mouse liver proteome (grayscale image of fluorescent gels showing probe-proteome reactions conjugated by CC with a Rh-azide reporter tag). Highly distinct proteome labeling profiles are seen for individual probes. Metalloprotease targets showing different probe selectivities were isolated and identified in separate reactions using biotin-azide tags and avidin chromatography-LC-MS procedures, as described previously.(b) Concentration dependence for reactivity of metalloproteases with their corresponding optimal probes. (c) Recombinant expression and HxBPyne labeling profiles of PSAP and ALAP. Both recombinant metalloproteases show probe selectivity profiles similar to those of their native counterparts (a). The LysR2 reactive protein observed at low levels in the mock lane is most likely endogenous PSAP.

Figure 3

Profiling MMP activities by gel-based ABPP. (a) Comparison of the reactivity of MMPs with the original Rh-tagged HxBP probe and its alkyne counterpart (LeuR₂ HxBPyne probe). MMPs generally show much stronger labeling with the alkyne probe. (b) Sensitivity of detection of MMPs by LeuR₂ HxBPyne probe. Human recombinant MMPs were added at the indicated concentrations into mouse liver proteome and the samples treated with 1 mM of LeuR₂ HxBPyne probe. Detection limits was defined as signals that were 200% of the background fluorescence signals observed in lanes lacking added MMPs.

Figure 4

Profiling metalloprotease activities by ABPP-MudPIT. (a) Sensitivity of detection of MMPs by ABPP-MudPIT. Human recombinant MMPs were added at the indicated concentrations to mouse liver proteome and the samples were treated with 100 nM of the LeuR₂ HxBPyne probe and analyzed by ABPP-MudPIT as shown in Figure 1b. Relative amounts of MMP activities were estimated by spectral counting.(b) Endogenous metalloprotease activities identified by ABPP-MudPIT in human cancer cell lines. For a and b, C indicates control reactions run in the presence of 100 × HxBPane probes to compete the specific labeling of metalloproteases by HxBPyne probes. (c) Endogenous metalloprotease activities identified by ABPP-MudPIT that are at significantly different levels in invasive (MUM-2B) and noninvasive (MUM-2C) cancer lines. All data shown were statistically significant (P < 0.05) between comparison groups (for a and b, probe-treated versus C groups; for c, MUM-2B versus MUM-2C groups) as determined by Student’s t-test or Mann-Whitney test. Data represent the average values ± s.e.m. of three independent experiments.

Figure 5

Characterization of ADAM targets of the HxBPyne library. (a) HxBPyne labeling of recombinant ADAM10 and ADAM17 (4 mg ml^-1 in a background of 1 mg ml^-1 mouse liver proteome), where red boxes designate the HxBPyne probe with strongest labeling (LeuR2 and AspR1 for ADAM10 and ADAM17, respectively). (b) HxBPyne labeling of ADAM10 and ADAM17 was blocked by 100 × HxBPane probes (C reactions). (c) The AspR1 probe inhibits ADAM17 activity, as determined with a fluorescent substrate assay. Data represent the average values ± s.e.m. of three independent experiments.

Figure 6

Location of targets of HxBPyne library on a phylogenic tree of the metalloprotease superfamily. With the exception of the carboxypeptidases, members from all major subfamilies of metalloproteases were targeted by the HxBPyne library. The tree was constructed from the peptidase domains of the complete human members of the metalloprotease clan, as designated by the MEROPS database (205 total predicted members, 150 putative active members that possess conserved catalytic residues). A pairwise alignment was generated using ClustalW and the phylogenic tree constructed using the neighbor-joining method. Major branches of the tree were designated by different colors, labeled by name, where appropriate, and displayed in hyperbolic space using the Hypertree program.