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Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits.

Griffin TA, Nandi D, Cruz M, Fehling HJ, Kaer LV, Monaco JJ, Colbert RA - J. Exp. Med. (1998)

Bottom Line: We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation.The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity.This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

View Article: PubMed Central - PubMed

Affiliation: William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

ABSTRACT
LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

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Relative levels of proteasome subunits in spleen cells from  B10 (wild type), LMP7−/−, and LMP2−/− mice. Lysates of mouse spleen  Con A–stimulated T cell blasts (1.5 × 106/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts  of proteasomes and total protein present in each sample.
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Figure 4: Relative levels of proteasome subunits in spleen cells from B10 (wild type), LMP7−/−, and LMP2−/− mice. Lysates of mouse spleen Con A–stimulated T cell blasts (1.5 × 106/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts of proteasomes and total protein present in each sample.

Mentions: Since T2 cells lack a large portion of the MHC class II region (27), they are deficient in not only LMP2 and LMP7, but also other genes involved in antigen processing (TAP1 and TAP2) and potentially other as yet undiscovered genes. Thus, we were interested in determining whether LMP7 is required for efficient LMP2 processing in other model systems, such as mice with targeted deletions of LMP2 or LMP7. Indeed, consistent results were obtained using Con A–stimulated T cell blasts from spleens of B10 (wild type), LMP7−/−, and LMP2−/− mice (Fig. 4). Immunoblots reflecting steady-state subunit levels demonstrated that all of the detectable LMP2 in wild-type cells was processed, whereas the majority of LMP2 in cells from LMP7−/− mice was unprocessed. Comparable to T2 transfectants, LMP2 deficiency had no effect on LMP7 processing. Interestingly, using an antibody against mouse MECL, we found that the level of processed MECL was dramatically reduced in the absence of either LMP7 or LMP2. Furthermore, unprocessed MECL accumulated in the absence of LMP7, similar to the accumulation of unprocessed LMP2 in these cells, indicating that absence of LMP7 results in inefficient processing of not only LMP2 but also MECL. Groettrup et al. have also noted that MECL incorporation into proteasomes is enhanced by overexpression of mouse LMP2 in transfected T2 cells, and LMP2 incorporation is enhanced by overexpression of MECL in transfected mouse B8 fibroblasts (29). The reduced level of MECL in cells from LMP2−/− mice is possibly a consequence of increased degradation of free MECL secondary to its inefficient incorporation into proteasomes in the absence of LMP2. Notably, reduced levels of each processed inducible subunit correlated with increased levels of each respective constitutive homologue. For example, the amount of delta was increased not only in the absence of LMP2 but also when LMP2 was processed inefficiently in cells from LMP7−/− mice. Similarly, the amount of Z was increased when processed MECL was reduced due to lack of either LMP7 or LMP2. The amount of X was increased only when LMP7 was absent.


Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits.

Griffin TA, Nandi D, Cruz M, Fehling HJ, Kaer LV, Monaco JJ, Colbert RA - J. Exp. Med. (1998)

Relative levels of proteasome subunits in spleen cells from  B10 (wild type), LMP7−/−, and LMP2−/− mice. Lysates of mouse spleen  Con A–stimulated T cell blasts (1.5 × 106/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts  of proteasomes and total protein present in each sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199179&req=5

Figure 4: Relative levels of proteasome subunits in spleen cells from B10 (wild type), LMP7−/−, and LMP2−/− mice. Lysates of mouse spleen Con A–stimulated T cell blasts (1.5 × 106/lane) were subjected to SDS-PAGE and specific proteasome subunits were visualized by immunoblotting. The C9 and TAP2 immunoblots demonstrate the relative amounts of proteasomes and total protein present in each sample.
Mentions: Since T2 cells lack a large portion of the MHC class II region (27), they are deficient in not only LMP2 and LMP7, but also other genes involved in antigen processing (TAP1 and TAP2) and potentially other as yet undiscovered genes. Thus, we were interested in determining whether LMP7 is required for efficient LMP2 processing in other model systems, such as mice with targeted deletions of LMP2 or LMP7. Indeed, consistent results were obtained using Con A–stimulated T cell blasts from spleens of B10 (wild type), LMP7−/−, and LMP2−/− mice (Fig. 4). Immunoblots reflecting steady-state subunit levels demonstrated that all of the detectable LMP2 in wild-type cells was processed, whereas the majority of LMP2 in cells from LMP7−/− mice was unprocessed. Comparable to T2 transfectants, LMP2 deficiency had no effect on LMP7 processing. Interestingly, using an antibody against mouse MECL, we found that the level of processed MECL was dramatically reduced in the absence of either LMP7 or LMP2. Furthermore, unprocessed MECL accumulated in the absence of LMP7, similar to the accumulation of unprocessed LMP2 in these cells, indicating that absence of LMP7 results in inefficient processing of not only LMP2 but also MECL. Groettrup et al. have also noted that MECL incorporation into proteasomes is enhanced by overexpression of mouse LMP2 in transfected T2 cells, and LMP2 incorporation is enhanced by overexpression of MECL in transfected mouse B8 fibroblasts (29). The reduced level of MECL in cells from LMP2−/− mice is possibly a consequence of increased degradation of free MECL secondary to its inefficient incorporation into proteasomes in the absence of LMP2. Notably, reduced levels of each processed inducible subunit correlated with increased levels of each respective constitutive homologue. For example, the amount of delta was increased not only in the absence of LMP2 but also when LMP2 was processed inefficiently in cells from LMP7−/− mice. Similarly, the amount of Z was increased when processed MECL was reduced due to lack of either LMP7 or LMP2. The amount of X was increased only when LMP7 was absent.

Bottom Line: We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation.The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity.This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

View Article: PubMed Central - PubMed

Affiliation: William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA.

ABSTRACT
LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

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