Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Abstract

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Figure 1

Labeling of subcellular fractions and purified proteasomes from EL-4 and adapted cells with covalent active site-directed affinity probes. (A) Twenty micrograms of each subcellular fraction was incubated with ¹²⁵I-NLVS or ¹²⁵I-YL₃VS for 2 h at 37°C, resolved by 12.5% SDS/PAGE, and visualized by autoradiography. The affinity probe labeling of proteasomal β subunits labeled is described elsewhere (23). (B) Lactacystin was incubated with intact EL-4 and adapted EL-4 cells for 12 h at 37°C, lysed, labeled with ¹²⁵I-YL₃VS, and analyzed by 12.5% SDS/PAGE and autoradiography. (C) Quantitative analysis of results in Fig. 3B. Bands representing the Z, X/Y/LMP-7, or LMP-2 subunits were quantified by using PhosphorImager analysis (imagequant, Molecular Dynamics). Mean values and standard deviations of three separate experiments are shown. (D) Purified 20S from EL-4 and adapted cells were incubated with ¹²⁵I-NLVS or ¹²⁵I-YL₃VS for 2 h at 37°C and visualized by silver staining the 12.5% SDS/PAGE and by autoradiography (E). Note: in proteasomes from adapted cells, LMP-7 is modified by NLVS and comigrates with LMP-8 (A, lane 2) (18).

Figure 2

Residual tryptic- and caspase-like activities in purified 20S from EL-4-adapted cells. Purified EL-4 20S incubated with (A) 20 μM LLVY-AMC, (C) 20 μM LLR-AMC, or (E) 100 μM YVAD-AMC. Purified EL-4 adapted 20S incubated with (B) 20 μM LLVY-AMC, (D) 20 μM LLR-AMC, or (F) 100 μM YVAD-AMC. Reactions were incubated with no inhibitor (●), 50 μM NLVS (○), 50 μM YL₃VS (□), 50 μM NLVS + 50 μM YL₃VS (▵), or 50 μM lactacystin (⋄). Note scale differences for panels.

Figure 3

Purified EL-4-adapted 20S have a reduced degradation rate compared with EL-4 20S. Degradation products of Sendai NP_317–338 peptide, incubated with purified EL-4 or EL-4-adapted 20S for specified times, were analyzed by reverse-phase HPLC and on-line MS (liquid chromatography-MS). (A) Reverse-phase HPLC chromatograms illustrate the formation of degradation products (boxes 1–5) generated by the EL-4 20S over indicated time intervals. The initial 21 residue peptide (S) and irrelevant peaks (*) are indicated. Mass spectra of each fragment are shown on the left. (B) The fragmentation products generated by the 20S from EL-4 (filled bars) and adapted cells (open bars) were identified by molecular mass in TIC chromatograms (data not shown) and quantified by their UV peak integration area.

Figure 4

Elevated levels of TPPII enable EL-4 cells to survive toxic concentrations of NLVS and process Ub-conjugated proteins. (A) Relative TPPII activity. FP-biotin binds TPPII at its active site in EL-4, TPPII transfectants, and adapted cells. The 60-kDa band is an unidentified polypeptide detected by Streptavidin-horseradish peroxidase. (B) Viability of EL-4, adapted, and TPPII transfectant cells treated with 0 (squares), 50 (rectangles), and 100 μM (circles) NLVS over 4 days as monitored by flow cytometry. (C) Western blot of accumulated poly-Ub-conjugated proteins using lysates from EL-4, TPPII transfectant, and adapted cells at 0, 4, 8, and 24 h after treatment with 50 μM NLVS assayed for an accumulation of polyubiquitinated proteins as described in Materials and Methods. Less polyubiquinated material is observed after 24 h, the result of partial cell death. One of 10 experiments is shown. (D) Pulse–chase analysis of polyubiquitinated material: control EL-4 (squares), adapted (circles), and TPPII transfectant cells (rectangles) were pulsed with [³⁵S]methionine and 50 μM NLVS and chased over time. Ub conjugates were analyzed by immunoprecipitation, SDS/PAGE, and autoradiography. Lanes were quantified by densitometric scan, and standard error mean was calculated. (E) NLVS is not a substrate for TPPII. EL-4 cells transfected with TPPII were either untreated or incubated with 50 μM NLVS for 24 h at 37°C. Lysates prepared from EL-4, TPPII transfectants, and adapted EL-4 cells were incubated with ¹²⁵I-NLVS as described above.

Figure 5

HPLC analysis and comparison of K^b-eluted radiolabeled peptides from control NLVS-treated EL-4 cells, TPPII transfectant, and adapted cells. Control EL-4 cells (A), cells treated with NLVS for 16 h (B), adapted cells (C), and TPPII transfectants treated with NLVS (D) were labeled with ³H-leucine and ³H-tyrosine for 6 h. Class I molecules (H-2K^b) were immunoprecipitated and bound peptides (solid lines) were eluted and separated by HPLC. Peptides loaded on HPLC were normalized for total cell incorporation of radiolabeled amino acids. Mock-precipitated material (dotted lines) refers to nonspecific background. Peptide standards included with each run showed elution times between runs to differ by less than a minute.