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The natural product lactacystin exerts its cellular antiproliferative effects through a mechanism involving acylation and inhibition of the proteasome, a cytosolic proteinase complex that is an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In vitro, lactacystin does not react with the proteasome; rather, it undergoes a spontaneous conversion (lactonization) to the active proteasome inhibitor, clasto-lactacystin beta-lactone. We show here that when the beta-lactone is added to mammalian cells in culture, it rapidly enters the cells, where it can react with the sulfhydryl of glutathione to form a thioester adduct that is both structurally and functionally analogous to lactacystin. We call this adduct lactathione, and like lactacystin, it does not react with the proteasome, but can undergo lactonization to yield back the active beta-lactone. We have studied the kinetics of this reaction under appropriate in vitro conditions as well as the kinetics of lactathione accumulation and proteasome inhibition in cells treated with lactacystin or beta-lactone. The results indicate that only the beta-lactone (not lactacystin) can enter cells and suggest that the formation of lactathione serves to concentrate the inhibitor inside cells, providing a reservoir for prolonged release of the active beta-lactone.

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces differentiation in a murine neuroblastoma cell line. The cellular target of lactacystin is the 20 S proteasome, also known as the multicatalytic proteinase complex, an essential component of the ubiquitin-proteasome pathway for intracellular protein degradation. In aqueous solution at pH 8, lactacystin undergoes spontaneous hydrolysis to yield N-acetyl-L-cysteine and the inactive lactacystin analog, clasto-lactacystin dihydroxy acid. We have studied the mechanism of lactacystin hydrolysis under these conditions and found that it proceeds exclusively through the intermediacy of the active lactacystin analog, clasto-lactacystin beta-lactone. Conditions that stabilize lactacystin (and thus prevent the transient accumulation of the intermediate beta-lactone) negate the ability of lactacystin to inactivate the proteasome. Together these findings suggest that lactacystin acts as a precursor for clasto-lactacystin beta-lactone and that the latter is the sole species that interacts with the proteasome.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent, factor Xa (fXa)-dependent small protein inhibitor of factor VIIa-tissue factor (fVIIa.TF), which binds to a site on fXa that is distinct from the catalytic center (exo-site). In the present study, the role of other fX derivatives in presenting rNAPc2 to fVIIa.TF is investigated. Catalytically active and active site blocked fXa, as well as a plasma-derived and an activation-resistant mutant of zymogen fX bound to rNAPc2 with comparable affinities (K(D) = 1-10 nm), and similarly supported the inhibition of fVIIa.TF (K(i)* = approximately 10 pm). The roles of phospholipid membrane composition in the inhibition of fVIIa.TF by rNAPc2 were investigated using TF that was either detergent-solubilized (TF(S)), or reconstituted into membranes, containing phosphatidylcholine (TF(PC)) or a mixture of phosphatidylcholine and phosphatidylserine (TF(PCPS)). In the absence of the fX derivative, inhibition of fVIIa.TF was similar for all three conditions (K(i) approximately 1 microm), whereas the addition of the fX derivative increased the respective inhibition by 35-, 150-, or 100,000-fold for TF(S), TF(PC), and TF(PCPS). The removal of the gamma-carboxyglutamic acid-containing domain from the fX derivative did not affect the binding to rNAPc2, but abolished the effect of factor Xa as a scaffold for the inhibition of fVIIa.TF by rNAPc2. The overall anticoagulant potency of rNAPc2, therefore, results from a coordinated recognition of an exo-site on fX/fXa and of the active site of fVIIa, both of which are properly positioned in the ternary fVIIa.TF.fX(a) complex assembled on an appropriate phospholipid surface.

We have developed an assay to continuously monitor the branched amino acid preferring peptidase (BrAAP) activity of the proteasome. This assay is based on the hydrolysis of the fluorogenic peptide, Abz-Gly-Pro-Ala-Leu-Ala-Nba (Abz is 2-aminobenzoyl and Nba is 4-nitrobenzylamide) which is cleaved exclusively at the Leu-Ala bond by the 20S proteasome with a kc/Km value of 13 000 M-1 s-1. Hydrolysis of this peptide is accompanied by an increase in fluorescence intensity (lambda ex = 340 nm, lambda em = 415 nm) due to release of the internally quenched 2-aminobenzoyl fluorescence that accompanies diffusion apart of the hydrolysis products, Abz-Gly-Pro-Ala-Leu and Ala-Nba. Using this assay, we examined inhibition of the BrAAP activity of the proteasome by a series of tripeptide aldehydes, Z-Leu-Leu-Xaa-H. When Xaa = Phe, (p-Cl)Phe, and Trp we observe biphasic or partial inhibition of the BrAAP activity. In contrast, when Xaa = Nva and Leu, simple inhibition kinetics are observed and allow us to calculate Ki values of 120 nM and 12 nM, respectively. The inhibitors that exhibit simple inhibition kinetics for BrAAP activity are also approximately equipotent for inhibition of the chymotrypsin-like (ChT-L) and peptidyl-glutamyl peptide hydrolyzing (PGPH) activities, dissociation constants varying by less than 25-fold, whereas the inhibitors that exhibit biphasic inhibition kinetics for BrAAP activity are300-fold more potent for inhibiting ChT-L activity than for PGPH activity. Inactivation of the BrAAP activity of the proteasome by clasto-lactacystin beta-lactone is also biphasic. beta-Lactone inactivates approximately 60% of the BrAAP activity rapidly, with kinetics indistinguishable from its inactivation of the chymotrypsin-like activity. The remaining 40% of the BrAAP activity is inactivated by beta-lactone at a 50-fold slower rate, with kinetics indistinguishable from its inactivation of the PGPH activity. These results suggest a mechanism in which hydrolysis of Abz-Gly-Pro-Ala-Leu-Ala-Nba (i.e., BrAAP activity) occurs at two different active sites in the 20S proteasome, and that these two active sites are the same ones that catalyze the previously described ChT-L and PGPH activities.