β2 Subunit Propeptides Influence Cooperative Proteasome Assembly

Abstract

Vertebrate proteasomes are structurally heterogeneous, consisting of both "constitutive" (or "standard") proteasomes and "immunoproteasomes." Constitutive proteasomes contain three ubiquitously expressed catalytic subunits, Delta (␤1), Z (␤2), and X (␤5), whereas immunoproteasomes contain three interferon-␥-inducible catalytic subunits, LMP2 (␤1i), MECL (␤2i), and LMP7 (␤5i). We recently have demonstrated that proteasome assembly is biased to promote immunoproteasome homogeneity when both types of catalytic subunits are expressed in the same cell. This cooperative assembly is due in part to differences between the LMP7 (␤5i) and X (␤5) propeptides. In the current study we demonstrate that differences between the MECL (␤2i) and Z (␤2) propeptides also influence cooperative assembly. Specifically, replacing the MECL propeptide with that of Z enables MECL incorporation into otherwise constitutive (Delta ؉ /X ؉ ) proteasomes and facilitates X incorporation into otherwise immunoproteasomes (MECL ؉ / LMP2 ؉ ). We also show, using MECL ؊/؊ mice, that LMP2 incorporation does not require MECL, in contrast with previous suggestions that their incorporation is mutually codependent. These results enable us to refine our model for cooperative proteasome assembly by determining which combinations of inducible and constitutive subunits are favored over others, and we propose a mechanism for how propeptides mediate cooperative assembly.Eukaryotic proteasomes are an integral component of ubiquitin-mediated protein degradation, which plays a major role in the turnover of cytoplasmic and nuclear proteins (1-5). By virtue of their role in protein metabolism, proteasomes are involved in a number of cellular processes, including cell cycle control, cellular stress responses, intracellular signaling, and major histocompatibility complex class I antigen processing (6). Immunoproteasomes are a specialized subset of vertebrate proteasomes that contain three interferon-␥-inducible catalytic subunits, LMP2 (␤1i), MECL (␤2i), and LMP7 (␤5i) (7,8).Immunoproteasomes are thought to possess enhanced capability for generating major histocompatibility complex class Ibinding peptides with basic or hydrophobic C termini as compared with constitutive proteasomes, which contain three constitutively synthesized catalytic subunits, Delta (␤1), Z (␤2), and X (␤5) (9 -14).The 20 S catalytic proteasome core is comprised of 28 subunits arranged in four stacked seven-member rings (15-17). Each outer ring contains seven different non-catalytic ␣-type subunits, ␣1-␣7, and each inner ring contains seven different ␤-type subunits, ␤1-␤7 (18), at least three of which are catalytic (␤1 or ␤1i, ␤2 or ␤2i, and ␤5 or ␤5i). The N-terminal proteolytic active sites are on the inner surface of the ␤ rings, whereas the C termini of ␤ subunits are on the outer surface of proteasomes (19 -21), enabling us to use C-terminal "epitope tags" to immunoprecipitate and track specific subunits because these tags do not appear to interfere with proteasome structure or catalytic act...

Heat Shock Up-Regulates lmp2 and lmp7 and Enhances Presentation of Immunoproteasome-Dependent Epitopes

“…Nevertheless, mixed proteasome populations exist in vivo (25). Importantly, the mecl1 subunit is not required for the efficient incorporation of lmp2 or lmp7 (29). Presentation of the NP 118 -126 L d -restricted epitope is enhanced in cells expressing three immunosubunits or in cells coexpressing mecl1 and lmp2 (30).…”

The proteasome immunosubunits, PA28 and ER‐aminopeptidase 1 protect melanoma cells from efficient MART‐126‐35‐specific T‐cell recognition

“…Additionally, low basal β1i/LMP2 level allowed for the incorporation of β2i/MECL-1 into proteasome complexes derived from HeLa A2+/β2i and HeLa A2+/β5i/β2i cells (Supporting Information Fig. 1A) [25][26][27][28]. Therefore, the HeLa A2+ transfectants represent a useful tool to dissect the role of the different immunosubunits in MART-1 26-35 epitope processing.…”

“…In vitro studies employing high-affinity MART-1-specific CD8 + T-cell clones demonstrated that some melanoma cell lines, despite expression of MART-1 and the corresponding HLA allele, were only barely recognized by specific T cells [3]. While different variants of the MART-1 epitope (including the MART-1 [26][27][28][29][30][31][32][33][34][35] 10-mer and the MART-1 27-35 9-mer) have been identified, only the 9-mer epitope can be eluted in significant amounts from the cell surface of melanoma cells [4]. This suggests that mechanisms related to antigen processing interfere with efficient generation of the 10-mer MART-1 [26][27][28][29][30][31][32][33][34][35] epitope in tumor cells.…”

“…It has been demonstrated that MART-1 [26][27][28][29][30][31][32][33][34][35] epitope processing is controlled by the proteasome, the major proteolytic machinery of the cytosol [5]. The standard proteasome (SP) contains the catalytic subunits β1, β2, and β5, whereas exposure of cells to IFN-γ enhances expression and incorporation of the immunosubunits β1i/LMP2 (LMP, low molecular weight protein), β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), and β5i/LMP7 into nascent proteasome complexes.…”

“…These data show that ERAP1/2 negatively contributes to MART-1 26-35 epitope generation and that in particular, ERAP1 is responsible for the impairment of epitope presentation by IFN-γ. To study the underlying molecular mechanism, we next performed in vitro trimming experiments using recombinant ERAPs in combination with the N-terminally extended 14-mer epitope precursor peptide MART-1 [22][23][24][25][26][27][28][29][30][31][32][33][34][35] . As shown by reversed-phase HPLC analyses, ERAP1 efficiently trimmed the MART-1 22-35 precursor peptide.…”