The DegraBase: A Database of Proteolysis in Healthy and Apoptotic Human Cells

Abstract

Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the ␣-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique ␣-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (ϳ10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (ϳ25% of total), and finally, other endoproteolytic cuts (ϳ65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells. Molecular & Cellular

The Novel Caspase-3 Substrate Gap43 is Involved in AMPA Receptor Endocytosis and Long-Term Depression

“…Although earlier studies have identified Ͼ1000 putative caspase substrates in nonneuronal cells (20,21), the identification of neuronal caspase-3 substrates, and their possible involvement in LTD, had yet to be established. To our knowledge, ours is the first systematic study of caspase-3 substrates in neurons.…”

“…Of these 56 proteins, 19 (18 human orthologs) are reported on the CAspase Substrate dataBAse Homepage (CASBAH, http:// bioinf.gen.tcd.ie/casbah/), and 43 are also in the recently established DegraBase (http://wellslab.ucsf.edu/degrabase/ index.htm) derived from human samples analyzed with the same methods used in this study (20,21). We analyzed the P4 to P1Ј motif composition of putative caspase-3 substrates identified from STS treated neurons.…”

“…Of these, 37 proteins (human and a single rat orthologs) were not previously reported in the CASBAH database (20), and 13 (human orthologs) are not included in the DegraBase data set compiled using the subtiligase methodology in non-neuronal tissue (21). Using complimentary methods, we further confirmed that, both in vivo and in vitro, caspase-3 cleaves three of these candidate substrates: growth associated protein 43 (Gap43), drebrin (Dbn1), and brain acid soluble protein 1 (BASP1).…”

“…Identification of specific proteins cleaved by caspases is therefore key to understanding the mechanisms mediating their biological functions. Several proteomic approaches, developed specifically for this purpose, have led to the identification of more than 2000 caspase cleavage sites (17)(18)(19)(20)(21)(22). In particular, the recently developed subtiligasebased method for isolating proteolytic products has led to the identification of Ͼ1,000 putative caspase substrates in human samples (21,23,24).…”

“…Several proteomic approaches, developed specifically for this purpose, have led to the identification of more than 2000 caspase cleavage sites (17)(18)(19)(20)(21)(22). In particular, the recently developed subtiligasebased method for isolating proteolytic products has led to the identification of Ͼ1,000 putative caspase substrates in human samples (21,23,24). Subtiligase is an engineered peptide ligase that ligates esterified peptides onto the N termini of proteins or peptides through free ␣-amines (25).…”