Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors

Abstract

The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a 125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus. 125I-NIP-L3VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.

Figure 1

Synthesis (A) and mechanism of inhibition (B) of the peptide vinyl sulfones. (A) PyBOP, DIEA, CH₃NHOCH₃, CH₂Cl₂; (B) LAH, Et₂O (C) (EtO)₂P(O)CH₂S(O)₂CH₃, NaH; TsOH, Et₂O; (E) DCC, HOBT, DMF; (F) 20% K₂CO₃/MeOH; (G) PyBOP, DIEA, CH₂Cl₂; (H) TsOH, Et₂O (I) Biotin-NP ester, DMF, DIEA; (J). 4-Hydroxy-3-iodo-5-nitrophenylacetic acid, PyBOP, DIEA, CH₂Cl₂; (K) 4-hydroxy-3-nitrophenylacetic acid, PyBOP, DIEA, CH₂Cl₂; (L) Na¹²⁵I, Iodogen.

Figure 2

The peptide vinyl sulfones inhibit the proteasome in vitro. Purified preparations of 20S and 26S proteasomes were incubated with increasing concentrations of the three inhibitors [Z-L₃H, Z-L₃VS, and lactacystin either simultaneously with substrates (A)], or samples were preincubated for 1 hr (37°C) and diluted 10-fold prior to addition of substrates (B). Hydrolysis of substrates corresponding to the acidic (Cbz-LLE-βNA), basic (Boc-LRR-MCA), and hydrophobic (Suc-LLVY-MCA) activities of the proteasome were measured by fluorescence spectroscopy. K_assoc for the compound, Z-L₃VS, were determined for the three peptidase activities of purified 20S proteasomes (C). V represents velocity of the reaction at the time (t), and V_o is the velocity at time 0. K_assoc = ln(V/V_o)/[I] where [I] is the concentration of inhibitor.

Figure 3

The peptide vinyl sulfone, ¹²⁵I-NIP-L₃VS, labels multiple β subunits of the proteasome in total cell extracts and in purified proteasome preparations. Total cell extracts (T) and purified preparations of proteasomes (P) were incubated with ¹²⁵I-NIP-L₃-VS (1.8 × 10⁴ Bq/ml) at 37°C for 2 hr, resolved on a 12.5% polyacrylamide gel and visualized by silver staining, by autoradiography or by two-dimensional isoelectric focusing SDS/PAGE for rabbit muscle (A), S. cerevisiae (B), or T. acidophilum (C). For T. acidophilum, recombinant β subunits containing an NH₂-terminal threonine, serine, or alanine were compared, as indicated.

Figure 4

The peptide vinyl sulfones covalently modify the HslV gene product from the E. coli protease complex HslV/HslU. Purified preparations of HslV and HslU were incubated with ¹²⁵I-NIP-L₃VS (1.8 × 10⁴ Bq/ml) independently or as a 1:4 mixture (HslV:HslU) in the presence of 2 mM ATP (+ATP) or 5 unit/ml apyrase (−ATP).

Figure 5

The peptide vinyl sulfones covalently modify multiple β subunits of the proteasome in living cells including the γ-interferon-inducible subunits LMP-2 and LMP-7. (A) US11+, HOM-2, and T2 cells (1.5 × 10⁶ cells) were incubated with ¹²⁵I-NIP-L₃VS (1.8 × 10⁴ Bq/ml) for 2 hr at 37°C, washed several times, and lysed. Total cell extracts were resolved on a 12.5% polyacrylamide gel and visualized by autoradiography. Immunoprecipitations were performed on lysates from ¹²⁵I-NIP-L₃VS-labeled HOM-2 cells using mAbs recognizing MHC class I molecules (W6-32) or a proteasomal α subunit (α prot.) (B) HOM-2 cells were incubated with ¹²⁵I-NIP-L₃VS, lysed, and analyzed by autoradiography (Direct Load) or proteasomes were immunoprecipitated (I.P. Proteasome) as in A. Note the absence of a single labeled polypeptide (26 kDa) in the proteasome immunoprecipitation. A total cell lysate from ¹²⁵I-NIP-L₃VS labeled HOM-2 cells was also separated by two-dimensional nonequilibrium pH gradient gel electrophoresis/PAGE (11% polyacrylamide gel) and visualized by autoradiography. (C) T1 and T2 cells (1.5 × 10⁶ cells) were incubated with ¹²⁵I-NIP-L₃VS (1.8 × 10⁴ Bq/ml) for 2 hr at 37°C, lysed, resolved by two-dimensional nonequilibrium pH gradient gel electrophoresis/PAGE, and analyzed by autoradiography. Note the presence of labeled LMP-2 and LMP-7 in lysates from T1 cells only.

Figure 6

The peptide vinyl sulfones, the peptide aldehyde, and lactacystin compete for binding to all proteasome subunits modified by ¹²⁵I-NIP-L₃VS. (A) HOM-2 cells were incubated simultaneously with ¹²⁵I-NIP-L₃VS (1.8 × 10⁴ Bq/ml) and with increasing concentrations of NIP-L₃VS, Z-L₃VS, lactacystin, or with 500 μM of the control peptides Z-L₃OH, Z-L₃NH₂, or (D)Z-L₃VS for 2 hr at 37°C. Note the 26-kDa polypeptide shown in Fig. 5B remains resistant to competition by lactacystin (see ←). (B) HOM-2 cells were labeled with ¹²⁵I-NIP-L₃VS as described or with the cysteine protease inhibitor Cbz-¹²⁵I-Tyr-Ala-CN₂ for 2 hr at 37°C. Lysates were separated on a 12.5% polyacrylamide gel and analyzed by autoradiography. (C) HOM-2 cells were incubated simultaneously with Cbz-¹²⁵I-Try-Ala-CN₂ and increasing concentrations of Z-L₃H, NIP-L₃VS, and lactacystin as indicated for 2 hr at 37°C. Lysates were resolved on a 12.5% polyacrylamide gel and analyzed by autoradiography.

Figure 7

The peptide vinyl sulfones block proteasome function in vivo. US11+ cells were preincubated in methionine-free medium without (−) or with the indicated concentrations of Z-L₃H, Z-L₃VS, NIP-L₃VS, or lactacystin for 1 hr. Cells were pulse labeled for 10 min and chased for 20 min. Class I molecules were immunoprecipitated with rabbit anti-heavy chain serum, resolved on a 12.5% polyacrylamide gel, and analyzed by fluorography. Note the presence of a class I heavy chain band devoid of carbohydrates, which migrates faster than the glycosylated species, and accumulates in the presence of an active proteasome inhibitor. ZL₃OH, peptide alcohol.