There is compelling evidence that members of the caspase (interleukin-1␤ converting enzyme/CED-3) family of cysteine proteases and the cytotoxic lymphocytederived 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 and Caenorhabditis 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.

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 M. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 ؋ 10 2 M ؊1 s ؊1 for caspase-2 to 2.8 ؋ 10 M ؊1s ؊1 for caspase-1. The results obtained with peptidebased inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (K i < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.Members of the caspase family of cysteine proteases, which at present includes 11 homologues of human origin, are important mediators of both inflammation, where they are involved in the production of several inflammatory cytokines, and apoptosis, where they participate in signaling and effector pathways (for review, see Ref. 1). The evidence for the central role of these enzymes in both of these biological processes was initially obtained using potent peptide-based and macromolecular inhibitors. For example, the finding that Ac-YVAD-CHO, a potent inhibitor of caspase-1, prevented the release of interleukin-1␤ (IL-1␤) 1 from monocytes, suggested that this enzyme was, in fact, the pro-IL-1␤-processing enzyme (2). This was later confirmed with the description of caspase-1-deficient mice, which are defective in the production of this cytokine (3,4). Similarly, the observation that apoptosis could be attenuated by the cowpox serpin CrmA, also known to be a potent caspase-1 inhibitor, provided the first compelling evidence that caspases play an important role in mammalian cell death (5). This has recently been confirmed by several studies, including the description of caspase-3-deficient mice, which have a striking defect in the programmed cell deaths that occur during neuronal development (6).Studies using these and other caspase inhibitors continue to be an important component of the repertoire of scientists investigating these complex biological processes in whole cells and in vivo. Regarding the latter, there are numerous recent re...

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.cholesterol ͉ intestinal brush border membranes

Three new toxins from the venom of the scorpion Leiurus quinquestriatus var. hebraeus have been identified on the basis of their ability to block the Shaker K+ channel. These toxins have been purified using HPLC techniques and characterized as 38 amino acid peptides by mass spectroscopy, amino acid analysis, and sequence determination. Their chemical identity was confirmed by producing fully functional synthetic toxins using recombinant methods. These peptides are potent inhibitors of the Shaker K+ channel (Kd < 1 nM) as well as the mammalian homologues of Shaker. They are related to other previously described K+ channel toxins, but form a new subclass within the larger family of K+ channel inhibitors derived from scorpion venom. We have named these toxins agitoxin 1, 2, and 3, respectively.

Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands.

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 k cat /K m 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, Ca 2+ ) 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.

Potassium channels comprise groups of diverse proteins which can be distinguished according to each member's biophysical properties. Some types of K+ channels are blocked with high affinity by specific peptidyl toxins. Three toxins, charybdotoxin, iberiotoxin, and noxiustoxin, which display a high degree of homology in their primary amino acid sequences, have been purified to homogeneity from scorpion venom. While charybdotoxin and noxiustoxin are known to inhibit more than one class of channel (i.e., several Ca(2+)-activated and voltage-dependent K+ channels), iberiotoxin appears to be a selective blocker of the high-conductance, Ca(2+)-activated K+ channel that is present in muscle and neuroendocrine tissue. A distinct class of small-conductance Ca(2+)-activated K+ channel is blocked by two other toxins, apamin and leiurotoxin-1, that share no sequence homology with each other. A family of homologous toxins, the dendrotoxins, have been purified from venom of various related species of snakes. These toxins inhibit several inactivating voltage-dependent K+ channels. Although molecular biology approaches have been employed to identify and characterize several species of voltage-gated K+ channels, toxins directed against a particular channel can still be useful in defining the physiological role of that channel in a particular tissue. In addition, for those K+ channels which are not yet successfully probed by molecular biology techniques, toxins can be used as biochemical tools with which to purify the target protein of interest.

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsinlike activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.