Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases - the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time.

Intrinsic cleavage of receptor-interacting protein kinase-1 by caspase-6.

Necroptosis is a form of programmed cell death that occurs in the absence of caspase activation and depends on the activity of the receptor-interacting protein kinases. Inactivation of these kinases by caspase-mediated cleavage has been shown to be essential for successful embryonic development, survival and activation of certain cell types. The initiator of extrinsic apoptosis, caspase-8, which has a pro-death as well as a pro-life function, has been assigned this role. In the present study we demonstrate that caspase-6, an executioner caspase, performs this role during apoptosis induced through the intrinsic pathway. In addition, we demonstrate that in the absence of caspase activity, intrinsic triggers of apoptosis induce the receptor-interacting-kinase-1-dependent production of pro-inflammatory cytokines. We show that ubiquitously expressed caspase-6 has a supporting role in apoptosis by cleaving this kinase, thus preventing production of inflammatory cytokines as well as inhibiting the necroptotic pathway. These findings shed new light on the regulation of necroptosis as well as cell death in an inflammatory environment wherein cells receive both intrinsic and extrinsic death signals.

Enzymatically active single chain caspase-8 maintains T-cell survival during clonal expansion.

The extrinsic, or death receptor, pathway integrates apoptotic signals through the protease caspase-8 (casp8). Beyond cell death regulation, non-apoptotic functions of casp8 include its essential requirement for hematopoiesis and lymphocyte clonal expansion, and tempering of autophagy in T cells. However, the mechanistic basis for the control of these disparate cellular processes remains elusive. Here, we show that casp8-deficient T-cell survival was rescued by enzymatically active, but not inactive, casp8-expressing retroviruses. The casp8 catalytic induction in proliferating T cell occurred independent of extrinsic and intrinsic apoptotic-signaling cascades and did not induce casp8 proteolytic processing. Using a biotinylated probe selectively targeting enzymatically active caspases, catalytically active full-length casp8 was found in vivo in dividing T cells. A casp8 D387A processing mutant was able to rescue casp8-deficient T-cell proliferation, validating that casp8 self-processing is not required for its non-apoptotic function(s). Finally, casp8 activity was highest in CD8(+) T cells, the most rapidly proliferating subset. These results show that the catalytically competent form of casp8 is required for rapid T-cell proliferation in response to TCR ligation, but that processing of the caspase is only necessary to promote apoptosis.

Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC.

Drosophila Nedd2-like caspase (DRONC), an initiator caspase in Drosophila melanogaster and ortholog of human caspase-9, is cleaved during its activation in vitro and in vivo. We show that, in contrast to conclusions from previous studies, cleavage is neither necessary nor sufficient for DRONC activation. Instead, our data suggest that DRONC is activated by dimerization, a mechanism used by its counterparts in humans. Subsequent cleavage at Glu352 stabilizes the active dimer. Since cleavage is at a Glu residue, it has been proposed that DRONC is a dual Asp- and Glu-specific caspase. We used positional-scanning peptide libraries to define the P1-P4 peptide sequence preferences of DRONC, and show that it is indeed equally active on optimized tetrapeptides containing either Asp or Glu in P1. Furthermore, mutagenesis reveals that Asp and Glu residues are equally tolerated at the primary autoprocessing site of DRONC itself. However, when its specificity is tested on a natural substrate, the Drosophila executioner caspase DRICE, a clear preference for Asp emerges. The formerly proposed Glu preference is thus incorrect. DRONC does not differentiate between Asp and Glu in poor substrates, but prefers Asp when tested on a good substrate.

The mechanism of peptide-binding specificity of IAP BIR domains.

We describe the peptide-binding specificity of the baculoviral IAP repeat (BIR) domains of the human inhibitor of apoptosis (IAP) proteins, X-linked IAP, cellular IAP1 and neuronal apoptosis inhibitory protein (NAIP). Synthetic peptide libraries were used to profile each domain, and we distinguish two types of binding specificity, which we refer to as type II and type III BIR domains. Both types have a dominant selectivity for Ala in the first position of the four N-terminal residues of the peptide ligands, which constitute a core recognition motif. Our analysis allows us to define the signature of type III BIRs that demonstrate a preference for Pro in the third residue of the ligand, resembling the classic IAP-binding motif (IBM). The signature of the type II BIRs was similar to type III, but with a striking absence of specificity for Pro in the third position, suggesting that the definition of an IBM must be modified depending on the type of BIR in question. These findings explain how subtle changes in the peptide-binding groove of IAP BIR domains can significantly alter the target protein selectivity. Our analysis allows for prediction of BIR domain protein-binding preferences, provides a context for understanding the mechanism of peptide selection and heightens our knowledge of the specificity of IAP antagonists that are being developed as cancer therapeutics.

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways.

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.

Caspase substrates.

The relatively common occurrence of sequences within proteins that match the consensus substrate specificity of caspases in intracellular proteins suggests a multitude of substrates in vivo - somewhere in the order of several hundred in humans alone. Indeed, the list of proteins that are reported to be cleaved by caspases in vitro proliferates rapidly. However, only a few of these proteins have been rigorously established as biologically or pathologically relevant, bona fide substrates in vivo. Many of them probably simply represent 'innocent bystanders' or erroneous assignments. In this review we discuss concepts of caspase substrate recognition and specificity, give resources for the discovery and annotation of caspase substrates, and highlight some specific human or mouse proteins where there is strong evidence for biologic or pathologic relevance.

High-dose, short-term, anti-inflammatory treatment with dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-alpha-benzanthracene-induced mammary tumors in rats.

OBJECTIVE: To evaluate the effects of dexamethasone (DXM) alone or in combination with 5-fluorouracil (5-FU) on dimethyl-alpha-benzanthracene (DMBA)-induced mammary tumors in rats. MATERIAL AND METHODS: Female Sprague-Dawley rats were divided into 4 groups receiving: 1) saline (controls), 2) DXM (3 mg/kg), 3) 5-FU (1.5 mg/kg) and 4) DXM and 5-FU combined. The drugs were given i.p. every day for 4 days. Interstitial fluid pressure (Pif) and tumor growth were determined in all tumors on days 1, 5 and 7 using the "wick-in-the needle" technique and by external size measurements, respectively. Vessel density and inflammatory cell infiltration of tumor tissue were analyzed by immunohistochemistry. RESULTS: DXM treatment significantly retarded tumor growth and reduced Pif. Treatment with a combination of DXM and 5-FU reduced tumor size significantly more than any of the agents alone (p<0.01-0.001). Enhanced uptake of 5-FU by DXM treatment was demonstrated by microdialysis. There were no differences in the density of CD31-positive vessels after DXM or 5-FU treatment, but inflammatory cell infiltration of tumor tissue was significantly reduced after DXM treatment. CONCLUSIONS: Our data suggest that DXM may be beneficial as an adjuvant to chemotherapy in the treatment of mammary cancer by increasing the uptake of 5-FU in the tumor.

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (P) in rat skin.

AIM: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). METHODS: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. RESULTS: Platelet activating factor (50 microg mL(-1)) administered locally via the fibre, increased extravasation of radiolabelled 125I-HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 A, 56 A and 39 A after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P(if)) pressure after subcutaneous administration (50 microg mL(-1)). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 microg kg(-1)) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma-to-tissue clearance (E-alb) showed a significant increase after PAF (n = 7, P < 0.05). CONCLUSIONS: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P(if), indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.

Structural basis for the activation of human procaspase-7.

Caspases form a family of proteinases required for the initiation and execution phases of apoptosis. Distinct proapoptotic stimuli lead to activation of the initiator caspases-8 and -9, which in turn activate the common executioner caspases-3 and -7 by proteolytic cleavage. Whereas crystal structures of several active caspases have been reported, no three-dimensional structure of an uncleaved caspase zymogen is available so far. We have determined the 2.9-A crystal structure of recombinant human C285A procaspase-7 and have elucidated the activation mechanism of caspases. The overall fold of the homodimeric procaspase-7 resembles that of the active tetrameric caspase-7. Each monomer is organized in two structured subdomains connected by partially flexible linkers, which asymmetrically occupy and block the central cavity, a typical feature of active caspases. This blockage is incompatible with a functional substrate binding site/active site. After proteolytic cleavage within the flexible linkers, the newly formed chain termini leave the cavity and fold outward to form stable structures. These conformational changes are associated with the formation of an intact active-site cleft. Therefore, this mechanism represents a formerly unknown type of proteinase zymogen activation.

Dimer formation drives the activation of the cell death protease caspase 9.

A critical step in the induction of apoptosis is the activation of the apoptotic initiator caspase 9. We show that at its normal physiological concentration, caspase 9 is primarily an inactive monomer (zymogen), and that activity is associated with a dimeric species. At the high concentrations used for crystal formation, caspase 9 is dimeric, and the structure reveals two very different active-site conformations within each dimer. One site closely resembles the catalytically competent sites of other caspases, whereas in the second, expulsion of the "activation loop" disrupts the catalytic machinery. We propose that the inactive domain resembles monomeric caspase 9. Activation is induced by dimerization, with interactions at the dimer interface promoting reorientation of the activation loop. These observations support a model in which recruitment by Apaf-1 creates high local concentrations of caspase 9 to provide a pathway for dimer-induced activation.

Mechanism-based inactivation of caspases by the apoptotic suppressor p35.

Caspases play a crucial role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, one of which--p35--has so far only been found in baculoviruses. P35 has previously been shown to rapidly form essentially irreversible complexes with its target caspases in a process that is accompanied by peptide bond cleavage. To determine the protease-inhibitory pathway utilized by this very selective protease inhibitor, we have analyzed the thermodynamic and kinetic stability of the protein. We show that the conformation of p35 is stabilized following cleavage within its reactive site loop. An inactive catalytic mutant of caspase 3 is bound by p35, but much less avidly than the wild-type enzyme, indicating that the protease catalytic nucleophile is required for stable complex formation. The inhibited protease is trapped as a covalent adduct, most likely with its catalytic Cys esterified to the carbonyl carbon of the scissile peptide bond. Together these data reveal that p35 is a mechanism-based inactivator that has adopted an inhibitory device reminiscent of the widely distributed serpin family, despite a complete lack of sequence or structural homology.

Inhibition of distant caspase homologues by natural caspase inhibitors.

Caspases play an important role in the ability of animal cells to kill themselves by apoptosis. Caspase activity is regulated in vivo by members of three distinct protease inhibitor families, two of which, baculovirus p35 and members of the inhibitor of apoptosis (IAP) family, are thought to be caspase specific. However, caspases are members of the clan of cysteine proteases designated CD, which also includes animal and plant legumains, and the bacterial proteases clostripain, gingipain-R and gingipain-K. Since these proteases have been proposed to have a common mechanism and evolutionary origin, we hypothesized that the caspase inhibitors may also regulate these other proteases. We tested this hypothesis by examining the effect of the natural caspase inhibitors on other members of protease clan CD. The IAP family proteins were found to have only a slight inhibitory effect on gingipain-R. The cowpox viral cytokine-response modifier A (CrmA) serpin had no effect on any of the proteases tested but a single point mutation of CrmA (Asp-->Lys) resulted in strong inhibition of gingipain-K. More substantial, with respect to the hypothesis, was the strong inhibition of gingipain-K by wild-type p35. The site in p35, required for inhibition of gingipain-K, was mapped to Lys94, seven residues C-terminal to the caspase inhibitory site. Our data indicate that the virally encoded caspase inhibitors have adopted a mechanism that allows them to regulate disparate members of clan CD proteases.

Structural basis for the inhibition of caspase-3 by XIAP.

The molecular mechanism(s) that regulate apoptosis by caspase inhibition remain poorly understood. The main endogenous inhibitors are members of the IAP family and are exemplified by XIAP, which regulates the initiator caspase-9, and the executioner caspases-3 and -7. We report the crystal structure of the second BIR domain of XIAP (BIR2) in complex with caspase-3, at a resolution of 2.7 A, revealing the structural basis for inhibition. The inhibitor makes limited contacts through its BIR domain to the surface of the enzyme, and most contacts to caspase-3 originate from the N-terminal extension. This lies across the substrate binding cleft, but in reverse orientation compared to substrate binding. The mechanism of inhibition is due to a steric blockade prohibitive of substrate binding, and is distinct from the mechanism utilized by synthetic substrate analog inhibitors.