David J. Livingston

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.

Substrate and Inhibitor Specificity of Interleukin-1β-converting Enzyme and Related Caspases

Interleukin-1β-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1β (pre-IL-1β) to the mature cytokine and a member of a family of related proteases (the caspases) that includes the Caenorhabditis elegans cell death gene product, CED-3. In addition to their sequence homology, these cysteine proteases display an unusual substrate specificity for peptidyl sequences with a P1 aspartate residue. We have examined the kinetics of processing pre-IL-1β to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processes pre-IL-1β, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cleaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 × 106 M−1 s−1 for CPP32 to 1.0 × 103 M−1 s−1 for TX. In addition, we report kinetic constants for several reversible inhibitors and irreversible inactivators, which have been used to implicate one or more caspases in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (DEVD-CHO) is a potent inhibitor of CPP-32 with a Ki value of 0.5 nM, but is also potent as inhibitor of CMH-1 (Ki = 35 nM) and ICE (Ki = 15 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presented here reveals electrostatic interactions not present in the Ac-YVAD-CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A., Chambers, S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) Nature 370, 270-275), accounting for the surprising potency of this inhibitor against ICE.

In vitro and in vivo studies of ICE inhibitors

Interleukin‐1β‐converting enzyme (ICE) is a cysteine protease responsible for proteolytic activation of the biologically inactive interleukin‐1β precursor to the proinflammatory cytokine. ICE and homologous proteases also appear to mediate intracellular protein degradation during programmed cell death. Inhibition of ICE is a new antiinflammatory strategy being explored by the design of both reversible inhibitors and irreversible inactivators of the enzyme. Such compounds are capable of blocking release of interleukin‐1β from human monocytes. ICE inhibitors that cross react against multiple ICE homologs can also block apoptosis in diverse cell types. ICE inhibitors impart protection in vivo from endotoxin‐induced sepsis and collagen‐induced polyarthritis in rodent models. Further optimization of the current generation of peptidyl ICE inhibitors will be required to produce agents suitable for administration in chronic inflammatory and neurodegenerative diseases. J. Cell. Biochem. 64:19–26.

Kinetic Characterization of Human Immunodeficiency Virus Type-1 Protease-resistant Variants

Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.

The synthesis and evaluation of peptidyl aspartyl aldehydes as inhibitors of ice.

Abstract The tetrapeptide aldehyde Ac-Tyr-Val-Ala-AspH ( 1 , L-709,049) has been reported to be a potent reversible inhibitor of Interleukin-1β Converting Enzyme (ICE). We have prepared a series of analogs of 1 , in order to explore the active sige of ICE. The effects of truncation, methylation of the amide nitrogens and modification of the aldehyde group of 1 are presented.

In Vitro Selection and Characterization of VX-478 Resistant HIV-1 Variants

VX-478 (141W94), a potent inhibitor of HIV protease, is in late stage clinical trials for the treatment of HIV infection and AIDS. Resistant viruses were raised in vitro by passage of HIV-1IIIB in the presence of increasing concentrations of VX-478 and the related hydroxyethylamino sulfonamide inhibitor VB-11,328. By direct PCR analysis of selected viruses, a number of mutations were identified (L10F, M46I, I47V, I50V and I84V) in the protease gene. These mutations were introduced into recombinant HIV-1 protease and the mutant enzymes assayed against a panel of inhibitors of diverse chemical structure. For VX-478, significant increases in IC90 and Ki were observed for virus or protease, respectively, containing I50V single mutation or an M46I/I47V/I50V triple mutation. The mutant proteases were also characterized for their kinetic competence to process substrates representing cleavage sites of gag-pol viral polypeptide. The kinetic data were interpreted with the aid of molecular modeling to understand the effect of mutations on inhibitor binding and processing of the gag-pol polypeptide to generate infective virions.

Structure-based design of non-peptidic pyridone aldehydes as inhibitors of interleukin-1β converting enzyme

Abstract Pyridone derivatives, especially with 6-aryl substituents, have been shown to be useful P2-P3 peptidomimetic scaffolds for the design of potent inhibitors of ICE.

CONFORMATION OF FK506 IN X-RAY STRUCTURES OF ITS COMPLEXES WITH HUMAN RECOMBINANT FKBP12 MUTANTS

Abstract In the X-ray structure of the FK506 complex with an FKBP12 double-mutant (R42K+H87V), the ligand is seen to adopt a conformation in its effector domain region that is distinctly altered compared to that found in the complex structure with native FKBP12. Nonetheless, molecular dynamics simulations indicate that the FK506 conformations seen in the native and mutant complex structures are energetically equivalent. Our observations suggest caution in the application of drug design strategies for calcineurin-mediated immunosuppressants that are based on mimicry of the FK506 conformation seen in the structure of the ligand complex with native FKBP12.

A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

SummaryThe ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of 13C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.