John P. Vaillancourt

A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B FUNCTIONAL RELATIONSHIPS ESTABLISHED FOR KEY MEDIATORS OF APOPTOSIS

There is compelling evidence that members of the caspase (interleukin-1β converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocyte-derived serine protease granzyme B play essential roles in mammalian apoptosis. Here we use a novel method employing a positional scanning substrate combinatorial library to rigorously define their individual specificities. The results divide these proteases into three distinct groups and suggest that several have redundant functions. The specificity of caspases 2, 3, and 7 andCaenorhabditis elegans CED-3 (DEXD) suggests that all of these enzymes function to incapacitate essential homeostatic pathways during the effector phase of apoptosis. In contrast, the optimal sequence for caspases 6, 8, and 9 and granzyme B ((I/L/V)EXD) resembles activation sites in effector caspase proenzymes, consistent with a role for these enzymes as upstream components in a proteolytic cascade that amplifies the death signal.

Involvement of Caspases in Proteolytic Cleavage of Alzheimer’s Amyloid-β Precursor Protein and Amyloidogenic Aβ Peptide Formation

Abstract The amyloid-β precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated amyloid-β (Aβ) peptide formation. The predominant site of caspase-mediated proteolysis is within the cytoplasmic tail of APP, and cleavage at this site occurs in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury. Caspase-3 is the predominant caspase involved in APP cleavage, consistent with its marked elevation in dying neurons of Alzheimers disease brains and colocalization of its APP cleavage product with Aβ in senile plaques. Caspases thus appear to play a dual role in proteolytic processing of APP and the resulting propensity for Aβ peptide formation, as well as in the ultimate apoptotic death of neurons in Alzheimers disease.

Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms

Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10). Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has been proposed as a mediator of apoptosis induced by endoplasmic reticulum stress including amyloid-β cytotoxicity, suggesting that it might contribute to the pathogenesis of Alzheimers disease. Here we show that a single nucleotide polymorphism in caspase-12 in humans results in the synthesis of either a truncated protein (Csp12-S) or a full-length caspase proenzyme (Csp12-L). The read-through single nucleotide polymorphism encoding Csp12-L is confined to populations of African descent and confers hypo-responsiveness to lipopolysaccharide-stimulated cytokine production in ex vivo whole blood, but has no significant effect on apoptotic sensitivity. In a preliminary study, we find that the frequency of the Csp12-L allele is increased in African American individuals with severe sepsis. Thus, Csp12-L attenuates the inflammatory and innate immune response to endotoxins and in doing so may constitute a risk factor for developing sepsis.

PURIFICATION AND CATALYTIC PROPERTIES OF HUMAN CASPASE FAMILY MEMBERS

Members of the caspase family of cysteine proteases are known to be key mediators of mammalian inflammation and apoptosis. To better understand the catalytic properties of these enzymes, and to facilitate the identification of selective inhibitors, we have systematically purified and biochemically characterized ten homologues of human origin (caspases 1–10). The method used for production of most of these enzymes involves folding of active enzymes from their constituent subunits which are expressed separately in E. coli, followed by ion exchange chromatography. In cases where it was not possible to use this method (caspase-6 and -10), the enzymes were instead expressed as soluble proteins in E. coli, and partially purified by ion exchange chromatography. Based on the optimal tetrapeptide recognition motif for each enzyme, substrates with the general structure Ac-XEXD-AMC were used to develop continuous fluorometric assays. In some cases, enzymes with virtually identical tetrapeptide specificities have kcat/Km values for fluorogenic substrates that differ by more than 1000-fold. Using these assays, we have investigated the effects of a variety of environmental factors (e.g. pH, NaCl, Ca2+) on the activities of these enzymes. Some of these variables have a profound effect on the rate of catalysis, a finding that may have important biological implications.

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Confinement of caspase-12 proteolytic activity to autoprocessing

Caspase-12 is a dominant-negative regulator of caspase-1 (IL-1β-converting enzyme) and an attenuator of cytokine responsiveness to septic infections. This molecular role for caspase-12 appears to be akin to the role of cFLIP in regulating caspase-8 in the extrinsic cell death pathway; however, unlike cFLIP/Usurpin, we demonstrate here that caspase-12 is catalytically competent. To examine these catalytic properties, rat caspase-12 was cloned, and the recombinant enzyme was used to examine the cleavage of macromolecular and synthetic fluorogenic substrates. Although caspase-12 could mediate autoproteolytic maturation of its own proenzyme, in both cis and trans, it was not able to cleave any other polypeptide substrate, including other caspase proenzymes, apoptotic substrates, cytokine precursors, or proteins in the endoplasmic reticulum that normally undergo caspase-mediated proteolysis. The dearth of potential substrates for caspase-12 also was confirmed by whole-cell diagonal-gel analysis. Autolytic cleavage within the caspase-12 proenzyme was mapped to a single site at the large–small subunit junction, ATAD319, and this motif was recognized by caspase-12 when incorporated into synthetic fluorogenic substrates. The specific activity of caspase-12 with these substates was several orders of magnitude lower than caspases-1 and -3, highlighting its relative catalytic paucity. In intact cells, caspase-12 autoproteolysis occurred in the inhibitory complex containing caspase-1. We propose that the proteolytic activity of caspase-12 is confined to its own proenzyme and that autocleavage within the caspase-1 complex may be a means for temporal limitation of the inhibitory effects of caspase-12 on proinflammatory cytokine maturation.

High-frequency transposition for determining antibacterial mode of action

Connecting bacterial growth inhibitors to molecular targets at the whole-cell level is a major impediment to antibacterial development. Herein we report the design of a highly efficient and versatile bacteriophage-based mariner transposon delivery system in Staphylococcus aureus for determining inhibitor mode of action. Using bacteriophage-mediated delivery of concatameric minitransposon cassettes, we generated nonclonal transposant libraries with genome-wide insertion-site coverage in either laboratory or methicillin-resistant strain backgrounds and screened for drug resistance in situ on a single agar plate in one step. A gradient of gene-target expression levels, along with a correspondingly diverse assortment of drug-resistant phenotypes, was achieved by fitting the transposon cassette with a suite of outward-facing promoters. Using a panel of antibiotics, we demonstrate the ability to unveil not only an inhibitors molecular target but also its route of cellular entry, efflux susceptibility and other off-target resistance mechanisms.

A Caspase Active Site Probe Reveals High Fractional Inhibition Needed to Block DNA Fragmentation

Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, αII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [125I]M808, which acts as a caspase active site probe. [125I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [125I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.

GENOMIC ORGANIZATION OF THE HUMAN CASPASE-9 GENE ON CHROMOSOME 1P36.1-P36.3

Department of Medical Genetics, and Centre for Molecular Medicine and Therapeutics, University of British Columbia, 980 West 28th Avenue, Vancouver, British Columbia V5Z 4H4, Canada NeuroGenes, International Cooperative Research Project, Japan Science and Technology Corporation, Tokai University School of Medicine, Isehara , Kanagawa 259-1193, Japan Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Point Claire-Dorval, Quebec H9R 4P8, Canada Department of Genetics, The Hospital of Sick Children, Toronto, Ontario M5G 1X8, Canada Department of Pathology, The Hospital of Sick Children, Toronto, Ontario M5G 1X8, Canada Department of Molecular Neuroscience, Molecular Medicine Research Center, The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa 259-1193, Japan

Localization of the cell death genes CPP32 and Mch-2 to human chromosome 4q.

Programmed cell death, manifested as apoptosis, is a deliberate and systematic means of cell suicide characterized by several distinct biochemical and morphological changes including cell shrinkage, membrane blebbing, and chromatin condensation (Wyllie et al. 1980). While this mechanism for self-destruction can be triggered by different pathogenic stimuli including infectious agents, it can also occur physiologically to halt the spread of neighboring cells or to make room for new cell types. Thus, apoptosis plays an important role during normal development, helping to maintain the delicate balance between cell death and survival. However, this balance can go astray, resulting in disease. For instance, excessive apoptosis has been implicated in neurodegenerative diseases and ischemic damage, while insufficient apoptosis has been postulated to occur in cancers and autoimmune diseases (reviewed in Nicholson 1996). In the nematode Caenorhabditis elegans, 131 cells die during normal development by apoptosis (Hengartner and Horovitz 1994). This process is under the control of several genes including Ced-3, which encodes a key cell death protease that is absolutely necessary for apoptosis (Hengartner and Horovitz 1994). CPP32/apopain appears to be a key mammalian Ced-3 homolog acting early in the cell death pathway. Relative to other mammalian cysteine proteases, it shares a high level of homology with Ced-3 (Frenandes-Alnemri et al. 1994). Moreover, it is specifically responsible for cleavage and inactivation of key homeostatic proteins during apoptosis. These proteins include poly (ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair particularly in response to environmental stress (Nicholson et al. 1995; Tewari et al. 1995). In addition, the catalytic subunit of DNAdependent protein kinase (DNA-PKcs), an enzyme essential for repair of DNA double-stranded breaks (Casciola-Rosen et al., 1995), and the U1-70 kDa small ribonucleoprotein (CasciolaRosen et al. 1994), which is necessary for RNA splicing, are also involved. Huntingtin, the gene product for the gene associated with Huntingtons Disease, is also cleared by apopain (Goldberg et al. 1996). Over-expression of CPP32 in vitro leads to apoptosis, which can be blocked by a specific peptide aldehyde inhibitor of CPP32 (Nicholson et al. 1995). However, no mutations in CPP32 have been shown to underlie any disease, perhaps owing to either functional redundancy in this enzyme family or to embryonic lethality. Using fluorescence in situ hybridization (FISH; Lichter et al. 1990) of a genomic clone isolated from a P1 library (Ioannou et al. 1994), we have mapped CPP32 to the tip of the long arm of human Chr 4 (Fig. 1) and have further refined its localization against a YAC contig from this region spanning at least 2 megabases (Mb).