Inhibition of Proteasome Activities and Subunit-Specific Amino-Terminal Threonine Modification by Lactacystin

Abstract

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.

Interaction with cyclin-dependent kinases and PCNA modulates proteasome-dependent degradation of p21

“…Similarly, E-64d, another inhibitor of calpains and cathepsins, had no detectable e ect on p21 expression in DLD1 transfectants. In contrast, lactacystin and MG132, two highly speci®c inhibitors of proteasomal activity (Fenteany et al, 1995;Palombella et al, 1994) were able to upregulate p21 levels. On longer exposures of the Western blots, higher molecular weight forms of p21 were clearly detected in cells treated with LLnL ( Figure 1b).…”

Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53

“…Another, credible possibility that should be evaluated is the extent to which synthetic peptide aldehydes, as compared to the Streptomyces metabolite Lactacystin, inhibit the catalytic threonine nucleophile activity of the 26S proteasome in whole cells. This is an appropriate consideration since ALLnL and ALLnM are competitive reversible inhibitors of proteasomal activity (Rock et al, 1994) while lactacystin is an irreversible inhibitor (Fenteany et al, 1995). Moreover, in the crystal structure of the yeast 20S proteasome complex, the peptide aldehyde ALLnL can be found bound to all three active site threonines in the subunits b1, b2 and b5 (for a review see Ciechanover and Schwartz, 1998).…”

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

“…Blot membrane was probed with 1340 antibody ubiquitinated forms of proteins thus targeted for degradation by the proteasome, such as b-catenin (Orford et al, 1997;Aberle et al, 1997). HeLa cells were treated with the speci®c proteasome inhibitor lactacystin (Fenteany et al, 1995), and with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal or calpain inhibitor I (ALLN), which is known to inhibit proteasome-mediated proteolysis, but also non-proteosomal thiol proteases such as calpains and cathepsins (Coux et al, 1996). As controls, we also treated cells with N-acetyl-Leu-Leu-methional or calpain inhibitor II (ALLM), a structurally related peptide aldehyde and a potent inhibitor of calpains and cathepsins that does not inhibit the proteolytic activity of the proteasome (Rock et al, 1994).…”