IFN-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response

Abstract

Peptide generation by the proteasome is rate-limiting in MHC class I-restricted antigen presentation in response to IFN-gamma. IFN-gamma-induced de novo formation of immunoproteasomes, therefore, essentially supports the rapid adjustment of the mammalian immune system. Here, we report that the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal beta5i subunit low molecular weight protein 7 (LMP7) has a key position in this immune adaptive program. IFN-gamma-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both beta5 subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, our data reveal that immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes. In consequence, our studies demonstrate that the cytokine-induced rapid immune adaptation of the proteasome system is a tightly regulated and transient response allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required.

Fig. 1.

Increased POMP levels after IFN-γ stimulation correlate with the absence of immunosubunits. (a) POMP mRNA levels are increased by 24-h IFN-γ stimulation (+) in different human cell lines analyzed by Northern blotting. Ethidium bromide-stained 28S rRNA bands are shown as an internal control (Top). Cellular POMP levels did not reflect mRNA levels, as shown by Western blot analysis of total lysates by using a POMP-specific antibody (Middle). Induction of POMP synthesis in response to IFN-γ as visualized by immunprecipitation of radio-labeled POMP from protein extracts of pulsed HeLa cells (Bottom). (b) Expression of immunosubunits reciprocally correlates with the cellular amount of POMP. Western blot analysis of the proforms (p) and the matured forms (m) of LMP2 (Upper) and LMP7 (Lower) in total cell lysates of different human cell lines in the presence (+) or absence (-) of IFN-γ.

Fig. 2.

i20S formation is accelerated and independent of IFN-γ signaling. (a) Turnover of precursor complexes and incorporated POMP is faster in the presence (+) than in the absence (-) of IFN-γ in metabolically labeled HeLa cells (Left). Turnover of i20S precursor complexes of labeled T2 LMP2 + 7 cells is accelerated in comparison to constitutive precursors of T2 independent of the cytokine signal (Right). Precursor complexes were specifically immunoprecipitated from total cell lysates at the different chase times indicated. Quantification of autoradiograms and calculated half-lives are shown (Lower). (b) For visualization of faster individual LMP7 subunit maturation compared with β5, subunits were immunoprecipitated from radiolabeled HeLa cell lysates (-/+ IFN-γ) at different chase times. The quantifications of POMP, proteasome precursors, or β5 proprotein turnover by phosphoimaging represent the mean of at least two independent experiments.

Fig. 3.

i20S are less stable than c20S independent of IFN-γ. c20S (T2 cells) or i20S (T2 LMP2 + 7 cells) were specifically immunoprecipitated at different time points during the chase period in the absence (Upper) or presence of 24-h IFN-γ (Lower). The diagram shows percent pixel density of time-point zero with trend lines. The calculated half-lives of 133 h for c20S and 27 h for i20S are the mean of five independent experiments. A representative experiment is shown.

Fig. 4.

Rapid turnover of POMP requires the presence of active LMP7. (a) The presence of LMP7 affected POMP stability as analyzed by Western blots of POMP, LMP2, and LMP7 in cell lysates of different T2 cell lines, stably expressing LMP7 and/or LMP2. The proforms (pi, p) and the matured forms (m) of the subunits LMP7 and LMP2 are indicated. (b) POMP stability is restored by expression of inactive variants of LMP7. Western blot analyses of POMP and LMP7 in cell lysates of T2 cell lines stably expressing LMP7 without the propeptide (ΔpLMP7) and the active-site mutation LMP7T1A. (c) POMP turnover in T2 is lower than in T2 LMP2 + 7 cells independent of the IFN-γ signal. Cells were metabolically labeled, and POMP was immunoprecipitated from total cell lysates at the different chase times indicated.

Fig. 5.

POMP interacts directly with LMP7. (a) Yeast two-hybrid assay for interaction of POMP with the proforms of β5 (Left) or LMP7 (Right). Interaction is shown by selection for His-prototrophy and β-galactosidase expression as detected by using activation-domain POMP fusion (AD-POMP) and the binding domain fused to the β5 subunit (BD-β5 or BD-LMP7) or vice versa. (b) Schematic representation of untagged β subunits and His-6-tagged POMP (His-tag = triangle). Both proforms of the human β5 subunits (1, β5; or 2, LMP7) bind to His-6-POMP, whereas the A. fulgidus β subunit did not bind (3, AFβ; specificity control). (c) The N-terminal His-6-fusions of human β5 subunits or their chimeras and the factor Xa site are schematically illustrated. (Upper) Input controls (12% input). (Lower) His-tagged proforms (1 and 4), β5 subunits without propeptides (2 and 5), as well as chimeric β subunits containing a human propeptide and the β subunit of A. fulgidus (3, β5-pro AFβ; 6, LMP7-pro AFβ) pulled down untagged POMP. Untagged POMP did not bind to the nickel beads (7, negative control)

Fig. 6.

Silencing of POMP gene expression by RNA interference results in a decrease of proteasomes. (a) Transfection of siRNA targeting POMP (POMPsi) silenced POMP expression in the presence or absence of IFN-γ (+/-) but not mock transfection of cells (control). GAPDH levels served as loading control (Upper). Silencing of POMP led to a strong reduction of incorporated β5 and β5i/LMP7 and immunoprecipitated 20S proteasome complexes (IP of 20S; Lower). GAPDH levels represent 15% input. (b) Prolonged knockdown of POMP (POMPsi) up to 48 h caused the induction of apoptosis as measured by caspase 3/7 activity in the presence (IFNg) or absence (co) of IFN-γ.(c) POMP depletion caused a decrease in MHC class I surface expression as measured by HLA class I fluorescence staining of mock (black line) or POMPsi RNA-transfected cells (gray line) after IFN-γ stimulation. Background staining with the second antibody only (2nd ab; IgG1) is shown (Left). Mean fluorescence levels are indicated as geometric (Geo) mean.