Dita M. Rasper

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.

Caspase Cleavage of Gene Products Associated with Triplet Expansion Disorders Generates Truncated Fragments Containing the Polyglutamine Tract

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

Hsp60 accelerates the maturation of pro‐caspase‐3 by upstream activator proteases during apoptosis

The activation of caspases represents a critical step in the pathways leading to the biochemical and morphological changes that underlie apoptosis. Multiple pathways leading to caspase activation appear to exist and vary depending on the death‐inducing stimulus. We demonstrate that the activation of caspase‐3, in Jurkat cells stimulated to undergo apoptosis by a Fas‐independent pathway, is catalyzed by caspase‐6. Caspase‐6 was found to co‐purify with caspase‐3 as part of a multiprotein activation complex from extracts of camptothecin‐treated Jurkat cells. A biochemical analysis of the protein constituents of the activation complex showed that Hsp60 was also present. Furthermore, an interaction between Hsp60 and caspase‐3 could be demonstrated by co‐immunoprecipitation experiments using HeLa as well as Jurkat cell extracts. Using a reconstituted in vitro system, Hsp60 was able to substantially accelerate the maturation of procaspase‐3 by different upstream activator caspases and this effect was dependent on ATP hydrolysis. We propose that the ATP‐dependent ‘foldase’ activity of Hsp60 improves the vulnerability of pro‐caspase‐3 to proteolytic maturation by upstream caspases and that this represents an important regulatory event in apoptotic cell death.

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.

Caspase-7 Expanded Function and Intrinsic Expression Level Underlies Strain-Specific Brain Phenotype of Caspase-3-Null Mice

Caspase-3-deficient mice of the 129S1/SvImJ (129) strain show severe brain development defects resulting in brain overgrowth and perinatal lethality, whereas on the C57BL/6J (B6) background, these mice develop normally. We therefore sought to identify the strain-dependent ameliorating gene. We biochemically isolated caspase-7 from B6-caspase-3-null (Casp3-/-) tissues as being the enzyme with caspase-3-like properties and capability of performing a caspase-3 surrogate function, apoptotic DNA fragmentation. Moreover, we show that, in contrast to the human enzymes, mouse caspase-7 is as efficient as caspase-3 at cleaving and thus inactivating ICAD (inhibitor of caspase-activated DNase), the inhibitor of apoptotic DNA fragmentation. Low levels of caspase-7 expression and activation correlate with lack of DNA fragmentation in 129-Casp3-/- apoptotic precursor neurons, whereas B6-Casp3-/- cells, which can fragment their DNA, show higher levels of caspase-7 expression and activation. The amount of caspase-7 activation in apoptotic precursor neurons is independent of the presence of caspase-3. Together, our findings demonstrate for the first time a strong correlation between caspase-7 activity, normal brain development, and apoptotic DNA fragmentation in Casp3-/- mice.

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.

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin

Abstract. Huntingtin-interacting protein 1 (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1, suggesting a potential interaction in vivo that may influence the function of each respective protein.

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).