Global Mapping of the Topography and Magnitude of Proteolytic Events in Apoptosis

Dix, Melissa M.; Simón, Gabriel; Cravatt, Benjamin F.

doi:10.1016/j.cell.2008.06.038

Cited by 335 publications

(399 citation statements)

References 52 publications

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“…Recently, an approach combining gel fractionation, MS, and an innovative bioinformatic analysis has been developed to identify protease substrates during apoptosis (37). This method determines the identity of proteins that are cleaved and is not designed to identify the specific cleavage sites.…”

Section: Discussionmentioning

confidence: 99%

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Shin

Jaffrey

2009

Proc. Natl. Acad. Sci. U.S.A.

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Proteolysis has major roles in diverse biologic processes and regulates the activity, localization, and intracellular levels of proteins. Linking signaling pathways and physiologic processes to specific proteolytic processing events is a major challenge in signal transduction research. Here, we describe N-CLAP (N-terminalomics by chemical labeling of the ␣-amine of proteins), a general approach for profiling protein N-termini and identifying protein cleavage sites during cellular signaling. In N-CLAP, simple and readily available reagents are used to selectively affinity label the ␣-amine that characterizes the protein N terminus over the more highly abundant -amine on lysine residues. Protein cleavage sites are deduced by identifying the corresponding N-CLAP peptides, which are derived from the N-termini of proteins, including the N-termini of the newly formed polypeptide products of proteolytic cleavage. Through selective affinity purification and tandem mass spectrometry analysis of 278 N-CLAP peptides, we characterized proteolytic cleavage events associated with methionine aminopeptidases and signal peptide peptidases, as well as proteins that are proteolytically cleaved after cisplatin-induced apoptosis. Many of the protein cleavage sites that are elicited during apoptotic signaling are consistent with caspase-dependent cleavage. These data demonstrate the utility of N-CLAP for proteomic profiling of protein cleavage sites that are generated during cellular signaling.caspase ͉ N-CLAP ͉ N-terminalomics ͉ tandem mass spectrometry

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Section: Discussionmentioning

confidence: 99%

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Shin

Jaffrey

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Because calpain substrates were identified by nonsystematic means, the number of different proteins in mammalian neurons that are cleaved by calpains is certain to be significantly larger than 100. The known substrates of activated mammalian caspases are nearly 1000 different proteins [177][178][179][180] (Fig. 4).…”

Section: Varshavskymentioning

confidence: 99%

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

Varshavsky

2012

Protein Science

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Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca 21 fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca 21 -activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca 21 transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca 21 alone or Ca 21 and its ionophore (Erickson et al., Science 1978;199:1219-1221 Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep.

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“…While many intrinsic and extrinsic stimuli can initiate apoptosis, these ultimately converge on the activation of a related family of aspartate-specific cysteine proteases, the caspases, that execute widespread proteolysis and induce noninflammatory death (1). We and others have surveyed N termini that occur in apoptotic cells and collectively reported more than 1,000 caspase-derived cleavages (2)(3)(4)(5). This explosion of proteomic data has defined a vast array of caspase substrates proteolyzed during apoptosis.…”

mentioning

confidence: 99%

Global kinetic analysis of proteolysis via quantitative targeted proteomics

Agard

Mahrus

Trinidad

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

124

152

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Mass spectrometry-based proteomics is a powerful tool for identifying hundreds to thousands of posttranslational modifications in complex mixtures. However, it remains enormously challenging to simultaneously assess the intrinsic catalytic efficiencies (k cat ∕K M ) of these modifications in the context of their natural interactors. Such fundamental enzymological constants are key to determining substrate specificity and for establishing the timing and importance of cellular signaling. Here, we report the use of selected reaction monitoring (SRM) for tracking proteolysis induced by human apoptotic caspases-3, -7, -8, and -9 in lysates and living cells. By following the appearance of the cleaved peptides in lysate as a function of time, we were able to determine hundreds of catalytic efficiencies in parallel. Remarkably, we find the rates of substrate hydrolysis for individual caspases vary greater than 500-fold indicating a sequential process. Moreover, the rank-order of substrate cutting is similar in apoptotic cells, suggesting that cellular structures do not dramatically alter substrate accessibility. Comparisons of extrinsic (TRAIL) and intrinsic (staurosporine) inducers of apoptosis revealed similar substrate profiles, suggesting the final proteolytic demolitions proceed by similarly ordered plans. Certain biological processes were rapidly targeted by the caspases, including multiple components of the endocyotic pathway and miRNA processing machinery. We believe this massively parallel and quantitative label-free approach to obtaining basic enzymological constants will facilitate the study of proteolysis and other posttranslational modifications in complex mixtures.apoptosis | caspase | enzymology | mass spectrometry | selected reaction monitoring A poptosis is a form of programmed cell death that serves to eliminate unnecessary, infected, or tumorigenic cells from eukaryotic organisms. While many intrinsic and extrinsic stimuli can initiate apoptosis, these ultimately converge on the activation of a related family of aspartate-specific cysteine proteases, the caspases, that execute widespread proteolysis and induce noninflammatory death (1). We and others have surveyed N termini that occur in apoptotic cells and collectively reported more than 1,000 caspase-derived cleavages (2-5). This explosion of proteomic data has defined a vast array of caspase substrates proteolyzed during apoptosis. While these data identify caspase targets, and in some cases the sites of proteolysis, they fail to reveal the relative rates of cleavage, a parameter necessary to establish the order of proteolytic events and their importance in extracts and intact cells.The recent application of selected reaction monitoring (SRM) methods, traditionally used for metabolite identification, to proteomic studies has enabled the simultaneous label-free quantification of hundreds of peptides (6, 7). Our development of a N-terminal enrichment platform (3) is ideally suited to the application of SRM to apoptotic proteolysis. Using this platfor...

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Global Mapping of the Topography and Magnitude of Proteolytic Events in Apoptosis

Cited by 335 publications

References 52 publications

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Global profiling of protease cleavage sites by chemoselective labeling of protein N-termini

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

Global kinetic analysis of proteolysis via quantitative targeted proteomics

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