Nancy A. Thornberry

Caspases: killer proteases

Caspases (cysteinyl aspartate-specific proteinases) mediate highly specific proteolytic cleavage events in dying cells, which collectively manifest the apoptotic phenotype. The key and central role that these enzymes play in a biochemical cell-suicide pathway has been conserved throughout the evolution of multicellular eukaryotes.

Human ICE/CED-3 Protease Nomenclature

It is now clear that members of the ICE/CED-3 protease family play key biological roles in inflammation and mammalian apoptosis. To date, ten homologs of human origin have been published (Figure 1Figure 1). The frenetic pace of identification of new homologs has led to inconsistent and multiple names for many of these enzymes. As a consequence, the general scientific community is finding it increasingly difficult to follow this provocative and rapidly moving field. In an effort to remedy this situation, several of us who have been involved in the identification and characterization of these enzymes have formed a committee, with the objective of proposing a nomenclature for the human members of this protease family that is sensible and easy to use. The purpose of this letter is to outline our recommendations.Figure 1Caspase Designations for Human Members of the ICE/CED3 Protease Family and Phylogenic Relationships among these ProteasesThe phylogenic relationships were determined using the PILEUP algorithm (version 8.1; gap weight = 3.0; gap length weight = 0.1) (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin 53711). This figure was contributed by Don Nicholson, Merck Frosst.View Large Image | View Hi-Res Image | Download PowerPoint SlideWe propose to use the trivial name “caspase” as a root for serial names for all family members. The selection of caspase was based on two catalytic properties of these enzymes. The “c” is intended to reflect a cysteine protease mechanism, and “aspase” refers to their ability to cleave after aspartic acid, the most distinctive catalytic feature of this protease family. To designate individual family members, caspase will be followed by an arabic numeral, which will be assigned based on its date of publication. Current assignments are shown in Figure 1Figure 1. The root name for the corresponding gene will be CASP.Each of these enzymes is synthesized as a proenzyme that is proteolytically activated to form a heterodimeric catalytic domain. Proenzymes will be referred to as pro-“enzyme name” (e.g. pro-caspase-1). Subunits of the heterodimer will be described by the enzyme name, followed by the correct molecular size of the subunit (e.g. caspase-1-p10, caspase-1-p20). In a general sense, subunits may be referred to as large and small. The N-terminal peptide that is removed during proteolytic activation of these proteases will be referred to as such.Splice variants will be given a small English character suffix, separated from the name of the protease by a slash, which will be assigned based on order of publication (e.g. caspase-1/a).Please consult one of the members of the committee prior to final publication of any new homolog, so that the appropriate number can be assigned. A new homolog is defined as a fully sequenced protein, cDNA, or gene that has a statistically significant relationship to the large and small subunits of one of the established family members. We strongly encourage all investigators of these proteases to adopt these nomenclature recommendations. It is only through compliance that we will achieve our goal of improving communication between scientists both inside and outside this exciting field.

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.

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.

Caspases: key mediators of apoptosis

Recent studies have established that members of the caspase protease family are essential components of a conserved cell death program. Insights into their biological roles, structure and mechanism are enabling investigators to begin to explore the therapeutic potential of caspase inhibition.

Chronic Inhibition of Dipeptidyl Peptidase-4 With a Sitagliptin Analog Preserves Pancreatic β-Cell Mass and Function in a Rodent Model of Type 2 Diabetes

Inhibitors of dipeptidyl peptidase-4 (DPP-4), a key regulator of the actions of incretin hormones, exert antihyperglycemic effects in type 2 diabetic patients. A major unanswered question concerns the potential ability of DPP-4 inhibition to have beneficial disease-modifying effects, specifically to attenuate loss of pancreatic β-cell mass and function. Here, we investigated the effects of a potent and selective DPP-4 inhibitor, an analog of sitagliptin (des-fluoro-sitagliptin), on glycemic control and pancreatic β-cell mass and function in a mouse model with defects in insulin sensitivity and secretion, namely high-fat diet (HFD) streptozotocin (STZ)-induced diabetic mice. Significant and dose-dependent correction of postprandial and fasting hyperglycemia, HbA1c, and plasma triglyceride and free fatty acid levels were observed in HFD/STZ mice following 2–3 months of chronic therapy. Treatment with des-fluoro-sitagliptin dose dependently increased the number of insulin-positive β-cells in islets, leading to the normalization of β-cell mass and β-cell–to–α-cell ratio. In addition, treatment of mice with des-fluoro-sitagliptin, but not glipizide, significantly increased islet insulin content and improved glucose-stimulated insulin secretion in isolated islets. These findings suggest that DPP-4 inhibitors may offer long-lasting efficacy in the treatment of type 2 diabetes by modifying the courses of the disease.

Mice lacking dipeptidyl peptidase IV are protected against obesity and insulin resistance

Dipeptidyl peptidase IV (DP-IV), a member of the prolyl oligopeptidase family of peptidases, is involved in the metabolic inactivation of a glucose-dependent insulinotropic hormone, glucagon-like peptide 1 (GLP-1), and other incretin hormones. Here, we investigated the impact of DP-IV deficiency on body weight control and insulin sensitivity in mice. Whereas WT mice displayed accelerated weight gain and hyperinsulinemia when fed a high-fat diet (HFD), mice lacking the gene encoding DP-IV (DP-IV-/-) are refractory to the development of obesity and hyperinsulinemia. Pair-feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to HFD-induced obesity in the DP-IV-/- mice. Ablation of DP-IV also is associated with elevated GLP-1 levels and improved metabolic control in these animals, resulting in improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced loss of β cell mass and hyperglycemia. Together, these observations suggest that chronic deletion of DP-IV gene has significant impact on body weight control and energy homeostasis, providing validation of DP-IV inhibition as a viable therapeutic option for the treatment of metabolic disorders related to diabetes and obesity.

Granzyme B Directly and Efficiently Cleaves Several Downstream Caspase Substrates: Implications for CTL-Induced Apoptosis

Caspase-mediated proteolysis of downstream substrates is a critical element of the execution pathway common to all forms of apoptosis studied to date. While this caspase-dependent pathway is activated during cytotoxic lymphocyte granule-induced cell death, recent studies have also provided evidence for caspase-independent pathways. However, the mechanisms mediating these additional pathways have not been defined. The current study demonstrates that DNA-PKcs and NuMA are directly and efficiently cleaved by granzyme B in vitro and in vivo, generating unique substrate fragments not observed during other forms of apoptosis. This direct, caspase-independent ability of granzyme B to cleave downstream death substrates constitutes an apoptotic effector mechanism that is insensitive to inhibitors of the signaling or execution components of the endogenous apoptotic cascade.

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist. RESEARCH DESIGN AND METHODS We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r−/− and Gcgr−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG. RESULTS Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR. CONCLUSIONS Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.