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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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Effect of LMP7 on processing of LMP2 in transfected T2 cells.  Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and  LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1  (7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse  subunits and cross-react with human subunits. Immunoprecipitates from  5 × 106 cells were loaded per lane. The C3 immunoblot demonstrates the  relative amounts of proteasomes in each sample.
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Figure 1: Effect of LMP7 on processing of LMP2 in transfected T2 cells. Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1 (7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse subunits and cross-react with human subunits. Immunoprecipitates from 5 × 106 cells were loaded per lane. The C3 immunoblot demonstrates the relative amounts of proteasomes in each sample.

Mentions: T2 is a human lymphoblastoid cell line with a single copy of chromosome 6, which has a deletion in the MHC class II region that includes the genes for LMP2 and LMP7 (27, 28). We observed that proteasomes from T2 cells transfected with LMP2 (T2.LMP2) contained primarily unprocessed LMP2 (pre-LMP2) (Fig. 1). The relative amount of pre-LMP2 decreased when cells were overgrown; however, we always detected more of the precursor than the mature form (data not shown), suggesting that processing of LMP2 was inefficient in these cells. A similar observation has been made using .174 lymphoblasts (which also lack LMP2 and LMP7) transfected with LMP2 (16). To determine whether LMP7 could mediate efficient processing of LMP2, T2 cells were transfected with both LMP2 and LMP7 (T2.LMP2/LMP7). Proteasomes from these double transfectants contained predominantly processed LMP2 (Fig. 1), indicating that LMP7 can indeed enhance the efficiency of LMP2 processing. In contrast, there was no effect of LMP2 on the efficiency of LMP7 processing, as proteasomes from both double transfectants as well as T2 cells transfected with LMP7 alone (T2.LMP7) contained primarily processed LMP7. Immunoblotting with polyclonal antisera raised against human LMP2 and LMP7 produced identical results but greater background staining (data not shown). Consequently, all of the anti-LMP2 and anti-LMP7 immunoblots presented in this report use antisera raised against mouse subunits. Recently, Groettrup et al. did not observe that efficient LMP2 processing required LMP7 in transfected T2 cells (29). Their results may differ from ours because they used an expression vector (pSG5) that leads to significant overexpression of LMP2, and they used mouse LMP2 and LMP7 in this human cell line.


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)

Effect of LMP7 on processing of LMP2 in transfected T2 cells.  Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and  LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1  (7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse  subunits and cross-react with human subunits. Immunoprecipitates from  5 × 106 cells were loaded per lane. The C3 immunoblot demonstrates the  relative amounts of proteasomes in each sample.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199179&req=5

Figure 1: Effect of LMP7 on processing of LMP2 in transfected T2 cells. Proteasomes were immunoprecipitated with MCP21 from lysates (postnuclear supernatants) of T2 cells transfected with LMP2 (2), LMP2 and LMP7 (2 + 7 ), LMP2 and LMP7E1 (2 + 7E1), LMP7 (7), and LMP7E1 (7E1). Specific subunits were visualized by immunoblotting after SDS-PAGE. Anti-LMP2 and anti-LMP7 antisera were raised against mouse subunits and cross-react with human subunits. Immunoprecipitates from 5 × 106 cells were loaded per lane. The C3 immunoblot demonstrates the relative amounts of proteasomes in each sample.
Mentions: T2 is a human lymphoblastoid cell line with a single copy of chromosome 6, which has a deletion in the MHC class II region that includes the genes for LMP2 and LMP7 (27, 28). We observed that proteasomes from T2 cells transfected with LMP2 (T2.LMP2) contained primarily unprocessed LMP2 (pre-LMP2) (Fig. 1). The relative amount of pre-LMP2 decreased when cells were overgrown; however, we always detected more of the precursor than the mature form (data not shown), suggesting that processing of LMP2 was inefficient in these cells. A similar observation has been made using .174 lymphoblasts (which also lack LMP2 and LMP7) transfected with LMP2 (16). To determine whether LMP7 could mediate efficient processing of LMP2, T2 cells were transfected with both LMP2 and LMP7 (T2.LMP2/LMP7). Proteasomes from these double transfectants contained predominantly processed LMP2 (Fig. 1), indicating that LMP7 can indeed enhance the efficiency of LMP2 processing. In contrast, there was no effect of LMP2 on the efficiency of LMP7 processing, as proteasomes from both double transfectants as well as T2 cells transfected with LMP7 alone (T2.LMP7) contained primarily processed LMP7. Immunoblotting with polyclonal antisera raised against human LMP2 and LMP7 produced identical results but greater background staining (data not shown). Consequently, all of the anti-LMP2 and anti-LMP7 immunoblots presented in this report use antisera raised against mouse subunits. Recently, Groettrup et al. did not observe that efficient LMP2 processing required LMP7 in transfected T2 cells (29). Their results may differ from ours because they used an expression vector (pSG5) that leads to significant overexpression of LMP2, and they used mouse LMP2 and LMP7 in this human cell line.

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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