Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

The structure of inosine-5'-monophosphate dehydrogenase (IMPDH) in complex with IMP and mycophenolic acid (MPA) has been determined by X-ray diffraction. IMPDH plays a central role in B and T lymphocyte replication. MPA is a potent IMPDH inhibitor and the active metabolite of an immunosuppressive drug recently approved for the treatment of allograft rejection. IMPDH comprises two domains: a core domain, which is an alpha/beta barrel and contains the active site, and a flanking domain. The complex, in combination with mutagenesis and kinetic data, provides a structural basis for understanding the mechanism of IMPDH activity and indicates that MPA inhibits IMPDH by acting as a replacement for the nicotinamide portion of the nicotinamide adenine dinucleotide cofactor and a catalytic water molecule.

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Interleukin-1␤-converting enzyme (ICE) is a novel cysteine protease responsible for the cleavage of pre-interleukin-1␤ (pre-IL-1␤) 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 P 1 aspartate residue. We have examined the kinetics of processing pre-IL-1␤ 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-1␤, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADPribose) 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 ؋ 10 ICE 1 is the prototypical member of a new family of mammalian cysteine proteases (the caspases) 2 that is distinct from cysteine proteases in the papain superfamily (1-3). The mutagenesis experiments and crystal structure reported by Wilson et al. (4) revealed a different active site geometry and catalytic mechanism for ICE than observed for papain. The structure of the ICE active site contains a Cys-His catalytic diad, and two Arg residues that confer high selectivity for peptidyl substrates with Asp residues at the P 1 position (Nterminal to the scissile bond) (4, 5). Although ICE has recently been reported to cleave other proteins in vitro (6, 7), it was identified from its essential role in processing the inactive 31-kDa precursor of interleukin-1␤ (pre-IL-1␤) to the mature 17-kDa cytokine (8).In 1993, Yuan et al. (9) reported the sequence of the Caenorhabditis elegans programmed cell death gene ced-3. This gene is 29% identical to human ICE. Due to the central role of the CED-3 protein in C. elegans apoptosis, Yuan and colleagues deduced that ICE or ICE homologs might play a similar role in mammalian apoptosis. Overexpression of ICE in rat fibroblast, mammalian COS cells, and neuronal cell lines demonstrated that this protease can indeed induce apoptosis (6, 10, 11). Subsequently, Kuida et al. (12) confirmed an in vivo role for ICE in Fas-mediated apoptosis by disruption of the murine ICE gene.A family of ICE-related proteases (the caspases) was discovered by searching human cDNA libraries for sequences homologous to 14). At present, at least 10 human homologs of ICE possessing cysteine protease activity have been identified. These homologs can be grouped by sequence similarity into three subfamilies. The most closely related homologs to ICE are TX (caspase-4, also denoted ICH-2 or ICErel II) (15-17) and TY (caspase-5, also denoted ICE rel III) (17, 18), which are 58 and 57% identical to ICE at the amino acid level. Another homolog, ICH-1 (caspase-2, corresponding to the murine Nedd-2 gene) (19,20), is 20% identical to ICE and belongs to a distinct subfamily. A third gr...

Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.