Global mapping of the topography and magnitude of proteolytic events in apoptosis

Abstract

Proteolysis is a key regulatory process that promotes the (in)activation, translocation, and/or degradation of proteins. As such, there is considerable interest in methods to comprehensively characterize proteolytic pathways in biological systems. Here, we describe a robust and versatile proteomic platform that enables direct visualization of the topography and magnitude of proteolytic events on a global scale. We use this method to generate a proteome-wide map of proteolytic events induced by the intrinsic apoptotic pathway. This profile contained 91 characterized caspase substrates as well as 170 additional proteins not previously known to be cleaved during apoptosis. Surprisingly, the vast majority of proteolyzed proteins, regardless of the extent of cleavage, yielded persistent fragments that correspond to discrete protein domains, suggesting that the generation of active effector proteins may be a principal function of apoptotic proteolytic cascades.

Figure 1. General methodological features of PROTOMAP

Proteomes from control and experimental systems are separated by 1D SDS-PAGE and the gel lanes cut into bands at fixed intervals. Bands are digested with trypsin to release peptides that are analyzed by 1D reverse-phase LC-MS/MS. The resulting proteomic data are integrated into peptographs, which plot, in the left panel, sequence-coverage for a given protein in the horizontal dimension (N- to C-terminus, left to right) versus SDS-PAGE migration in the vertical dimension [high to low molecular weight (MW), top to bottom]. In the right panel, the peptograph also displays average spectral counts for each protein in each gel band. Proteins that undergo proteolytic cleavage are identified by shifts in migration from higher (parental, red) to lower (fragments, blue) MW species in control versus experimental systems. The sequence coverage shown in the peptograph provides a topographical map of the protein fragments that persist following proteolysis. The magnitude of proteolysis is estimated by comparing spectral counts of the parental protein in control versus experimental proteomes.

Figure 2. Established markers of apoptotic proteolytic pathways visualized by PROTOMAP

Apoptosis was induced in Jurkat T-cells by treatment with staurosporine (STS) for 4 hrs, which produced DNA fragmentation (A) and caspase-3 cleavage (B). Cleavage of caspase-3 is evident from the peptograph (B, right panels), which identified peptides for the 32 kDa pro-form of this protease in control cells (red signals, bands 15 and 16), and peptides for the 12 and 17 kDa activated forms in STS-treated cells (blue signals, bands 21 and 22). Peptides detected in both samples are shown in purple. (C) Cleavage of ROCK1 (parent species, 158 kDa) to 130 and 28 kDa fragments. (D) and (E) Cleavage of PARP1 in STS-treated cells. Parental PARP1 was found in the particulate fraction of control cells (D) and, upon induction of apoptosis, was detected in the soluble fraction as a series of cleaved fragments (E). The asterisk in the PARP1 western blot of the particular fraction likely corresponds to antibody cross-reactivity with a background protein, since no spectral counts for PARP1 were detected in the corresponding gel band. Notably, a number of fragments of PARP1 that are not observed by immunoblotting were evident from the peptographs. This likely reflects the restricted epitopes recognized by anti-PARP1 antibodies. (F) Peptographs for representative members of the COPS family, showing selective cleavage of COPS6 in apoptotic cells, as previously demonstrated (Correia et al., 2007). Peptographs for all nine members of the COPS family can be found in Supplemental Figure 2. Spectral count data are represented as the mean +/− SEM for four independent experiments.

Figure 3. Global analysis of cleaved proteins in apoptotic cells

(A) A total of 261 predicted cleaved proteins were identified by PROTOMAP in apoptotic cells, 91 (35%) of which corresponded to established caspase substrates and/or proteins known to be proteolyzed during apoptosis. The remaining 170 proteins (65%) were not previously known to be cleaved during apoptosis. (B) Comparison of the spectral count values for previously known versus unknown cleaved proteins in apoptotic cells. Note that proteins with high spectral count values were predominantly from the former group. (C) and (D) Examples of predicted cleaved proteins that were confirmed by western blotting. In the case of JMJD1B (C), an implicit caspase cleavage sequence at amino acids 817–823 (tandem DLSD) could be identified that resided in between the persistent 75 kDa N- and 100–125 kDa C-terminal fragments. In the case of MAP2K2 (D), the explicit caspase cleavage site was identified by PROTOMAP at residue D284. The asterisk in the MAP2K2 western blot likely corresponds to antibody cross-reactivity with a background protein, as no spectral counts were detected for MAP2K2 in the corresponding gel band. Spectral count data are represented as the mean +/− SEM for four independent experiments.

Figure 4. Estimation of the magnitude of protein cleavage events in apoptotic cells

(A) Predicted cleaved proteins were divided into three general classes: I) near-complete (class I, < 20% spectral counts remaining in STS-treated cells), moderate (class II, 20–80% spectral counts remaining in STS-treated cells), and minor (class III, > 80% spectral counts remaining in STS-treated cells) degradation of the parent species. (B) and (C) Western blot confirmation that class I proteins PAK2 (B) and Vimentin (C) underwent near-complete degradation in apoptotic cells. (D) and (E) Western blot confirmation that the class II proteins TRIM28 (D) and MAP2K1 (E) were only partially degraded in apoptotic cells. The bar graph to the right of each peptograph represents total spectral counts detected for the parental species in control (red) and apoptotic (blue) samples. Spectral count data are represented as the mean +/− SEM for four independent experiments.

Figure 5. Visualization of the topography of protein cleavage events in apoptotic cells

(A) The vast majority (> 95%) of cleaved proteins generated at least one persistent fragment in apoptotic cells. Essentially all possible topographical classes of fragments were observed: 1) N-terminal (25%) 2) C-terminal (13%), 3) N- & C-terminal (21%), and 4) internal (23%). “Unclear” refers to cleaved proteins with fragments that could not be obviously assigned to one of the other four fragment classes. (B)–(E) Peptographs for representative examples of N-terminal (B), C-terminal (C), N- and C- terminal (D), and multiple/internal (E) fragments. Predicted domain structures for each protein are shown below each peptograph to highlight the fact that most persistent fragments map to functional protein domains. Spectral count data are represented as the mean +/− SEM for four independent experiments.

Figure 6. Time-course analysis of proteolytic events in apoptotic cells

(A) Rates of degradation of proteins could be divided into two general categories, rapid and slow, based on the quantity of parental species remaining at the 4 hr time-point following STS treatment (< 50% versus > 50% parental protein remaining, respectively). The time-course of degradation is shown for all proteins in these two categories, with the global averages indicated by dark lines. (B) Global analysis of the temporal stability of persistent fragments. Most proteins displayed fragments that were highly persistent, being detected at either all three time-points (C) or two consecutive time-points (D). A small fraction of proteins displayed transient persistent fragments (E) that were detected exclusively at a single time-point. See Supplementary Table 3 for a complete list of time-course profiles for cleaved proteins. Spectral count data are represented as the mean +/− SEM for three independent experiments.

Figure 7. Mapping precise sites of caspase-mediated proteolysis

(A) Peptograph for the cleaved protein U2AF2 showing the locations of a peptide from the parent species that spans the exact site of cleavage (B), as well as two half-tryptic peptides from persistent C-terminal and N-terminal fragments (C and D, respectively). For each spectrum dominant diagnostic y- and b-ions are identified. All ions are in the +1 charge state unless otherwise indicated. (E) Additional representative examples of explicit caspase cleavage sites identified by PROTOMAP that map to the ends of N- or C-terminal persistent fragments (AP2A2 and UBE2O, respectively). (F) Representative examples of established caspase cleavage sites that can be implicitly assigned by PROTOMAP (DNM2 and SATB1, respectively). Spectral count data are represented as the mean +/− SEM for four independent experiments.