8536d_ch08_185-199 8/2/02 10: 08 AM Page 190 mac79 Mac 79: 45_BWFpldsby et al./Immunology 5e: 190 PART I1 Generation of B-Cell and T-Cell Response MHC molecules. Association of viral antigen with class I(a) MHC molecules required replication of the influenza virus E-amino group ol and viral protein synthesis within the target cells; association lysine side chain with class II did not. These findings suggested that the pep- tides presented by class I and class II MHC molecules are trafficked through separate intracellular compartments; class I MHC molecules interact with peptides derived from cy- NH2 tosolic degradation of endogenously synthesized proteins, ②=三② class II molecules with peptides derived from endocytic degradation of exogenous antigens. The next two sections examine these two pathways in detail enzyme subuni Endogenous Antigens The Cytosolic Pathway ② In eukaryotic cells, protein levels are carefully regulated Every protein is subject to continuous turnover and is de- Protein Proteasome graded at a rate that is generally expressed in terms of its ha life.Some proteins(e.g, transcription factors, cyclins, and FIGURE 8-5 Cytosolic proteolytic system for degradation of intra- metabolic enzymes)have very short half-lives; dena- cellular proteins. (a) Proteins to be degraded are often covalently tured, misfolded, or otherwise abnormal proteins also are de linked to a small protein called ubiquitin. In this reaction, which re- graded rapidly. The pathway by which endogenous antigens quires ATP, a ubiquinating enzyme complex links several ubiquitin are degraded for presentation with class I MHC molecules molecules to a lysine-amino group near the amino terminus of the utilizes the same pathways involved in the normal turnover protein. (6) Degradation of ubiquitin-protein complexes occurs of intracellular proteins peptides Proteasomes are large cylindrical particles whose subunits catalyze cleavage of peptide bo Peptides for Presentation Are Generated by Protease Complexes Called Proteasomes residues. As described in Chapter 7, peptides that bind to Intracellular proteins are degraded into short peptides by a cy- class I MHC molecules terminate almost exclusively with hy- tosolic proteolytic system present in all cells. Those proteins drophobic or basic residues targeted for proteolysis often have a small protein, called ubiquitin, attached to them(Figure 8-5a). Ubiquitin-protein mplex called a proteasome. Each proteasome is a large to the Rough Endoplasmic Reticulumo/ conjugates can be degraded by a multifunctional protease Peptides Are Transported from the Cytosol lindrical particle consisting of four rings of pro- tein subunits with a central channel of diameter 10-50 A. Insight into the role that peptide transport, the delivery of A proteasome can cleave peptide bonds between 2 or 3 peptides to the MHC molecule, plays in the cytosolic pro- different amino acid combinations in an ATP-dependent cessing pathway came from studies of cell lines with defects process(Figure 8-5b). Degradation of ubiquitin-protein in peptide presentation by class I MHC molecules. One such omplexes is thought to occur within the central hollow of mutant cell line, called RMA-S, expresses about 5% of the proteasome. normal levels of class i mhc molecules on its membrane. Al Experimental evidence indicates that the immune system though RMA-S cells synthesize normal levels of class I o tilizes this general pathway of protein degradation to chains and B2-microglobulin, neither molecule appears on produce small peptides for presentation with class I MHC the membrane. a clue to the mutation in the RMA-S cell line molecules. The proteasomes involved in antigen processing was the discovery by A. Townsend and his colleagues that include two subunits encoded within the MHC gene cluster,"feeding "these cells peptides restored their level of mem LMP2 and LMP7, and a third non-MHC protein, LMP10 brane-associated class I MHC molecules to normal. These (also called MECL-1) All three are induced by increased lev- investigators suggested that peptides might be required to els of the T-cell cytokine IFN-Y. The peptidase activities of stabilize the interaction between the class I a chain and proteasomes containing LMP2, LMP7, and LMP10 preferen- B2-microglobulin. The ability to restore expression of class ally generate peptides that bind to MHC class I molecules. I MHC molecules on the membrane by feeding the cells Such proteasomes, for example, show increased hydrolysis predigested peptides suggested that the RMA-S cell line of peptide bonds that follow basic and/or hydrophobic might have a defect in peptide transport.MHC molecules. Association of viral antigen with class I MHC molecules required replication of the influenza virus and viral protein synthesis within the target cells; association with class II did not. These findings suggested that the pep￾tides presented by class I and class II MHC molecules are trafficked through separate intracellular compartments; class I MHC molecules interact with peptides derived from cy￾tosolic degradation of endogenously synthesized proteins, class II molecules with peptides derived from endocytic degradation of exogenous antigens. The next two sections examine these two pathways in detail. Endogenous Antigens: The Cytosolic Pathway In eukaryotic cells, protein levels are carefully regulated. Every protein is subject to continuous turnover and is de￾graded at a rate that is generally expressed in terms of its half￾life. Some proteins (e.g., transcription factors, cyclins, and key metabolic enzymes) have very short half-lives; dena￾tured, misfolded, or otherwise abnormal proteins also are de￾graded rapidly. The pathway by which endogenous antigens are degraded for presentation with class I MHC molecules utilizes the same pathways involved in the normal turnover of intracellular proteins. Peptides for Presentation Are Generated by Protease Complexes Called Proteasomes Intracellular proteins are degraded into short peptides by a cy￾tosolic proteolytic system present in all cells. Those proteins targeted for proteolysis often have a small protein, called ubiquitin, attached to them (Figure 8-5a). Ubiquitin-protein conjugates can be degraded by a multifunctional protease complex called a proteasome. Each proteasome is a large (26S), cylindrical particle consisting of four rings of pro￾tein subunits with a central channel of diameter 10–50 Å. A proteasome can cleave peptide bonds between 2 or 3 different amino acid combinations in an ATP-dependent process (Figure 8-5b). Degradation of ubiquitin-protein complexes is thought to occur within the central hollow of the proteasome. Experimental evidence indicates that the immune system utilizes this general pathway of protein degradation to produce small peptides for presentation with class I MHC molecules. The proteasomes involved in antigen processing include two subunits encoded within the MHC gene cluster, LMP2 and LMP7, and a third non-MHC protein, LMP10 (also called MECL-1). All three are induced by increased lev￾els of the T-cell cytokine IFN-. The peptidase activities of proteasomes containing LMP2, LMP7, and LMP10 preferen￾tially generate peptides that bind to MHC class I molecules. Such proteasomes, for example, show increased hydrolysis of peptide bonds that follow basic and/or hydrophobic residues. As described in Chapter 7, peptides that bind to class I MHC molecules terminate almost exclusively with hy￾drophobic or basic residues. Peptides Are Transported from the Cytosol to the Rough Endoplasmic Reticulum Insight into the role that peptide transport, the delivery of peptides to the MHC molecule, plays in the cytosolic pro￾cessing pathway came from studies of cell lines with defects in peptide presentation by class I MHC molecules. One such mutant cell line, called RMA-S, expresses about 5% of the normal levels of class I MHC molecules on its membrane. Al￾though RMA-S cells synthesize normal levels of class I  chains and 2-microglobulin, neither molecule appears on the membrane. A clue to the mutation in the RMA-S cell line was the discovery by A. Townsend and his colleagues that “feeding” these cells peptides restored their level of mem￾brane-associated class I MHC molecules to normal. These investigators suggested that peptides might be required to stabilize the interaction between the class I  chain and 2-microglobulin. The ability to restore expression of class I MHC molecules on the membrane by feeding the cells predigested peptides suggested that the RMA-S cell line might have a defect in peptide transport. 190 PART II Generation of B-Cell and T-Cell Responses COOH H2N NH C O Ubiquitin (b) COOH NH2 (a) ε-amino group on lysine side chain COOH H2N NH C O Ubiquitin NH2 Ubiquinating enzyme complex + ubiquitin ATP AMP + PPi Protein Proteasome Peptides Proteolytic enzyme subunit FIGURE 8-5 Cytosolic proteolytic system for degradation of intra￾cellular proteins. (a) Proteins to be degraded are often covalently linked to a small protein called ubiquitin. In this reaction, which re￾quires ATP, a ubiquinating enzyme complex links several ubiquitin molecules to a lysine-amino group near the amino terminus of the protein. (b) Degradation of ubiquitin-protein complexes occurs within the central channel of proteasomes, generating a variety of peptides. Proteasomes are large cylindrical particles whose subunits catalyze cleavage of peptide bonds. 8536d_ch08_185-199 8/2/02 10:08 AM Page 190 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: