Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Wejda, Magdalena; Impens, Francis; Takahashi, Nozomi; Damme, Petra Van; Gevaert, Kris; Vandenabeele, Peter

doi:10.1074/jbc.m112.384552

Cited by 40 publications

(48 citation statements)

References 76 publications

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“…In general, less than 3% of aspartic acid cleavage sites are detected in the absence of exogenous caspase, suggesting little caspase activation. Several small-scale studies have previously identified a total of 37 and 68 substrates for caspase-2 and caspase-6, respectively (11,22,23). More than half of the previously reported caspase-2 substrates were found in our dataset (19 of the 37), and we identified 13 of 61 caspase-6 substrates reported in the CutDB database (cutdb.burnham.org/).…”

Section: Resultsmentioning

confidence: 66%

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Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Julien

Zhuang

Wiita

et al. 2016

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

110

106

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Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (k cat /K M ) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.N-terminomics | caspase | proteolysis | proteomics | apoptosis C aspases are cysteine-class proteases that typically cleave after aspartic acid and play critical roles in cellular remodeling during development, cell differentiation, inflammation, and cell death (reviewed in refs. 1-3). There are 12 caspases in humans alone, which have been classically grouped on the basis of sequence homology, domain architecture, and cell biology as inflammatory (caspase-1, caspase-4, caspase-5, and caspase-11), apoptotic initiators (caspase-2, caspase-8, caspase-9, and caspase-10), or executioners (caspase-3, caspase-6, and caspase-7) (4, 5). Identifying the natural protein substrates for caspases can provide important insights into their cellular roles. A series of large-scale proteomic studies from several laboratories has collectively identified nearly 2,000 native proteins cleaved after an aspartic acid during apoptosis of human cells (recently reviewed in refs. 6-8). By adding individual caspases to extracts, it has been possible to begin to identify caspasespecific substrates, ranging from just a few, in the case of caspase-4, caspase-5, and caspase-9 (9), to several hundred for caspase-1, caspase-3, caspase-7, and caspase-8 (10, 11). Some of these substrates overlap, but others are unique to specific caspase...

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Section: Resultsmentioning

confidence: 66%

“…[6][7][8]. By adding individual caspases to extracts, it has been possible to begin to identify caspasespecific substrates, ranging from just a few, in the case of caspase-4, caspase-5, and caspase-9 (9), to several hundred for caspase-1, caspase-3, caspase-7, and caspase-8 (10,11). Some of these substrates overlap, but others are unique to specific caspases.…”

mentioning

confidence: 99%

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Julien

Zhuang

Wiita

et al. 2016

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

110

106

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Section: -5mentioning

confidence: 99%

“…[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage. [13][14][15][16][17] Using the subtiligasebased N-terminomics approach, we identified more than 1700 aspartate cleavage events distributed among about 1200 different protein substrates upon induction of apoptosis across seven human cell lines (http://wellslab.ucsf.edu/degrabase/).…”

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confidence: 99%

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

et al. 2016

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Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity. Human caspases are a family of 12 homologous intracellular proteases known for driving cellular state changes such as apoptosis and differentiation, as well as inflammatory responses. Caspases are cysteine-class proteases named for their signature ability to cleave after aspartate residues, or P1 is aspartate using the Schetchter and Berger notation. 1,2Synthetic peptide profiling for purified caspases show distinctive subsite preferences extending from P1 to P4 or P5. 3-5Sequence conservation and signal pathway analyses have further grouped the proteases into apoptotic initiators (−2, − 8, − 9 and − 10), apoptotic executioners (−3, − 6 and − 7), regulators of inflammation (−1, − 4, − 5 and − 12) and keratinocyte differentiation − 14).The past decade has seen a significant advancement in the use of LC-MS to define the spectrum of natural proteins cleaved by caspases in cells. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] In addition to providing unbiased information about which proteins are cleaved, in some cases, these experiments locate the precise sites and quantify the rates of cleavage.13-17 Using the subtiligasebased N-terminomics approach, we identified more than 1700 aspartate cleavage eve...

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“…This progressive cleavage of rPaPARP may be explained by the presence of 25 arginine and 62 lysine residues representing potential cleavage sites of metacaspases. It is conceivable that there is a preference of metacaspases recognizing specific amino acids upstream of the P1 position of their substrate as it is characteristic for mammalian caspases (72). The amino acid sequences DEVD, YVAD, or VAD are known to be common cleavage sites for aspartate-specific caspases (8) but not for arginine-and lysine-specific metacaspases.…”

Section: Discussionmentioning

confidence: 99%

Poly(ADP-Ribose) Polymerase Is a Substrate Recognized by Two Metacaspases of Podospora anserina

Strobel

Osiewacz

2013

Eukaryot Cell

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The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.A poptosis is a type of programmed cell death (PCD) that is fundamental in removing unneeded cells from the body during development of multicellular organisms. In addition, in mammals, apoptosis plays a key role in removing severely damaged cells which are at risk of transforming into cancer cells. In unicellular and multicellular lower eukaryotes, like the yeast Saccharomyces cerevisiae, or in filamentous fungi, the role of PCD is less clear and leads to death of the whole organism or parts of it. Previously, it has been suggested that under natural conditions under which individuals compete for the available nutrients, PCD guarantees the conservation of the species via providing nutrients and other factors for survival of younger cells (1, 2).Although differing in complexity, basic components and reactions associated with PCD are known to be conserved from lower eukaryotes to mammals. For instance, DNA fragmentation, the release of apoptotic factors from mitochondria, and the activation of specific cysteine proteases are hallmarks of apoptosis in all eukaryotic systems (3, 4). In fungi, plants, and protozoa, metacaspases were identified as sequence homologs of mammalian caspases (5). Caspases and metacaspases share structural similarities such as a common caspase hemoglobinase fold and conserved catalytic cysteine and histidine residues. Metacaspases can be subdivided into type I and type II analogs to mammalian initiator and effector caspases, respectively. The characteristic amino-terminal domain of type I met...

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Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Cited by 40 publications

References 76 publications

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues

Poly(ADP-Ribose) Polymerase Is a Substrate Recognized by Two Metacaspases of Podospora anserina

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