The Endogenous Pathway of Antigen Processing and Presentation

The cytosol of a typical eukaryotic cell is a very busy place. The level of each type of protein there is carefully regulated, at least partly by controlling their rates of synthesis and degradation. The half-life of a protein, or time required for that protein to reach one-half its concentration, varies widely but can range from minutes to days (or longer, in a few cases). Some proteins, like those involved in formation of the nucleus, tend to have long half-lives, while others, such as transcription factors, cyclins, and key metabolic enzymes, typically have very short half-lives. Denatured, misfolded, or otherwise abnormal proteins also are degraded rapidly. In particular, defective ribosomal products (called DRiPs), which can come from premature translation termination, protein misfolding, or defects in multisubunit protein assembly, constitute a rather large subset of protein products in the cytosol. The consequence of this steady turnover of both normal and defective proteins is a constant pool of proteins and their fragments that are no longer needed by the cell. While many will be reduced to their constituent amino acids and recycled, some persist in the cytosol as peptides. The cell constantly samples from this pool of peptides and presents some fragments on the plasma membrane in association with MHC class I molecules, where cells of the immune system can likewise sample these peptides to keep a lookout for foreign proteins lurking inside of host cells. The pathway by which these endogenous peptides are generated for presentation with MHC class I molecules utilizes mechanisms similar to those involved in the normal turnover of intracellular proteins.

Peptides Are Generated by Protease Complexes Called Proteasomes

Intracellular proteins are degraded into short peptides by the proteasome, a cytosolic proteolytic system present in all cells (Figure 7-13a). The large (20S) proteasome is composed of multiple α and β subunits arranged in concentric rings; the α subunits make up the top and bottom rings while the β subunits construct the middle two rings. There are a total of 14 β subunits arrayed in this barrel-like structure of symmetrical rings.

FIGURE 7-13 Proteolytic system for degradation of intracellular proteins. (a) Endogenous proteins in all cells are targeted for degradation by ubiquitin conjugation. These proteins are degraded by the 26S proteasome complex, which includes the 20S constitutive proteasome and a 19S regulator, generating a pool of peptides for MHC class I loading. In activated APCs, several proteins in the constitutive proteasome (β1, β2, and β5) are replaced by proteins encoded by the LMP genes (β1i, β2i, and β5i). (b) This generates an immunoproteasome with increased efficiency for creating unique peptides with a proclivity for assembly with MHC class I molecules.

Part a shows a cytosolic protein pool above two components labeled as the constitutive proteasome and an Immunoproteasome. Both these proteasomes have a 19 S regulator. The constitutive proteasome shows a cluster of rounded cells arranged over a cylindrical surface, representing the beta 1, beta 2 and beta 5 proteins. The Immunoproteasome has the beta 1i, beta 2i and beta 5i proteins. Two downward arrows from the constitutive proteasome and an Immunoproteasome point toward a peptide pool in part b.

Part b shows two overlapping ovals containing smaller peptide strands, representing peptides generated by the constitutive proteasome only and Peptides generated by the immunoproteasome only. The overlapping region represents peptides generated by both. The peptides generated by the constitutive proteasome only and the peptides generated by both lead to M H C class 1 antigen presentation on a somatic cell. The peptides generated by both and the peptides generated by the immunoproteasome only lead to M H C class 1 antigen presentation on a dendritic cell.

We know that many proteins are targeted for proteolysis when a small protein called ubiquitin is attached to them. These ubiquitin-protein conjugates enter the proteasome complex, consisting of the 20S base and an attached 19S regulatory component, through a narrow channel at the 19S end. The proteasome complex cleaves peptide bonds in an ATP-dependent process. Degradation of ubiquitin-protein complexes is thought to occur within the central hollow core of the proteasome.

The immune system adds a twist to the general pathway of protein degradation to specifically produce small peptides optimized for binding to MHC class I molecules. In addition to the standard 20S proteasomes resident in all cells, a distinct proteasome of the same size, the immunoproteasome, can be found in pAPCs and some infected cells. It has the same basic structure as the traditional proteasome with some unique subunit substitutions. In most cells, these new subunits are not constitutively expressed like the other components of the proteasome but are induced by exposure to certain cytokines, such as IFN-γ or TNF. LMP2 and LMP7, genes that are located within the class II region (see Figure 7-7) and are responsive to these cytokines, encode replacement catalytic protein subunits that convert standard proteasomes into immunoproteasomes, increasing the efficiency with which cytosolic proteins are cleaved into peptide fragments that specifically bind to MHC class I molecules (Figure 7-13b). In fact, a subset of the peptides created in the presence of immunoproteosomes is not found in cells lacking these structures. The half life of an immunoproteasome is shorter than that of a standard proteasome, possibly because the increased level of protein degradation in its presence may have negative consequences beyond the targeting of infected cells. It is possible that in some cases autoimmunity results from increased processing of self-proteins in cells with high levels of immunoproteasomes.

Key Concept:

• While all cells have constitutive proteasomes involved in the processing and presentation of cytosolic proteins in MHC class I molecules, infected cells or activated APCs can temporarily express immunoproteasomes, which generate peptide fragments that are optimized for MHC class I binding.

Peptides Are Transported from the Cytosol to the Rough Endoplasmic Reticulum

Insight into the cytosolic or endogenous processing pathway came from studies of cell lines with defects in peptide presentation by MHC class I molecules. One such mutant cell line, called RMA-S, expresses about 5% of the normal levels of MHC class I molecules on its membrane. Although RMA-S cells synthesize normal levels of class I α chain and β₂-microglobulin, few MHC class I complexes appear on the membrane. A clue to the mutation in the RMA-S cell line was the discovery, by Townsend and colleagues, that “feeding” peptides to these cells restored the level of membrane-associated MHC class I molecules to normal. These investigators suggested that peptides might be required to stabilize the interaction between the class I α chain and β₂-microglobulin. The ability to restore expression of MHC class I molecules on the membrane by supplying the cells with predigested peptides suggested that the RMA-S cell line might have a defect in peptide processing or transport.

Subsequent experiments showed that the defect in the RMA-S cell line occurs in the protein that transports peptides from the cytoplasm into the rough endoplasmic reticulum (RER), where the transmembrane MHC class I molecules are synthesized. When RMA-S cells were transfected with a functional gene encoding this transporter protein, the cells began to express class I molecules on the membrane at near normal levels. The transporter protein, designated TAP (transporter associated with antigen processing), is an ER-membrane-spanning heterodimer consisting of two proteins: TAP1 and TAP2 (Figure 7-14a). In addition to their transmembrane segments, the TAP1 and TAP2 proteins each have a domain projecting into the lumen of the RER and an ATP-binding domain that projects into the cytosol. Both TAP1 and TAP2 belong to the family of ATP-binding cassette proteins found in the membranes of many cells, including bacteria. This family of proteins mediates ATP-dependent transport of amino acids, sugars, ions, and peptides across membranes.

FIGURE 7-14 TAP (transporter associated with antigen processing). (a) Schematic diagram of TAP, a heterodimer anchored in the membrane of the rough endoplasmic reticulum (RER). The two chains are encoded by TAP1 and TAP2. The cytosolic domain in each TAP subunit contains an ATP-binding site, and peptide transport depends on the hydrolysis of ATP. (b) In the cytosol, ubiquitinated proteins are directed to a constitutive or immunoproteasome, where they are digested into peptide fragments. These peptides are translocated by TAP into the RER lumen, where, in a process mediated by several other proteins, they will associate with MHC class I molecules.

Part a shows two chains labeled TAP1 and TAP2 each binded to an A T P molecule and anchored into the R E R lumen through the double walled R E R membrane. The A T P binding site of the two chains are in the cytosol.

Part b shows a protein strand approaching an immuno proteasome resulting in the formation of peptides which are further converted into amino acids. The peptides are also directed toward the hetrodimeric T A P chains, anchored on the R E R membrane, and are translocate into the R E R lumen. A text within the R E R lumen reads, peptide ready to be loaded onto M H C class 1 molecule. A curved arrow above the R E R membrane indicates the action of A T P on the A T P molecule and the P subscript i molecule.

Peptides generated in the cytosol by the proteasome are translocated by TAP into the RER by a process that requires the hydrolysis of ATP (Figure 7-14b). TAP has affinity for peptides containing 8 to 16 amino acids. The optimal peptide length for final association with MHC class I is nine amino acids. It was later discovered that longer peptides can also bind but are trimmed by enzymes present in the lumen of the ER, such as ERAP (endoplasmic reticulum aminopeptidase). In addition, TAP appears to favor peptides with hydrophobic or basic carboxyl-terminal amino acids, the preferred anchor residues for MHC class I molecules (see Figure 7-4). Thus, TAP is pre-optimized to transport peptides that are likely to interact with MHC class I molecules. The TAP and LMP genes (encoding immunoproteosome components) map within the MHC class II region. A few different allelic forms of these genes exist within the population and can account for modest differences in antigen processing and MHC class I presentation, some of which have been linked to disorders such as psoriasis, a skin disease with autoimmune features. TAP deficiencies can lead to more severe disease syndromes, many of which share aspects of both immune deficiency (poor immune responses) and autoimmunity (overactive immune responses to self).

Key Concept:

• A transmembrane protein in the RER called TAP (transporter associated with antigen processing) is required to transport peptides of approximately the right size (that have been cleaved by cytosolic proteasomes/immunoproteasomes) into the lumen of the RER, where they can associate with newly formed MHC class I molecules.

Chaperones Aid Peptide Assembly with MHC Class I Molecules

Like other proteins destined for the plasma membrane, the α chain and β₂-microglobulin components of the MHC class I molecule are synthesized on ribosomes on the RER. Assembly of these components into a stable MHC class I molecular complex that can exit the RER requires the presence of a peptide in the binding groove of the class I molecule. The assembly process involves several steps and includes the participation of molecular chaperones that facilitate the folding of polypeptides.

The first molecular chaperone involved in MHC class I assembly is calnexin, a resident membrane protein of the ER. ERp57, a protein with enzymatic activity, and calnexin associate with the class I α chain and promote its folding (Figure 7-15). When β₂-microglobulin binds to the α chain, calnexin is released and the class I-ERp57 complex associates with the chaperones calreticulin and tapasin. Tapasin (TAP-associated protein) brings the TAP transporter into proximity with the class I molecule and allows it to acquire an antigenic peptide. The TAP protein then promotes peptide capture by the class I molecule before the peptides are exposed to the luminal environment of the RER. In 2000 the tapasin-related protein, TAPBPR (for TAP-binding protein related), was found to bind MHC class I molecules much like tapasin does. However, TAPBPR appears to be more than just an analogue of tapasin. This conclusion comes from the observation that while tapasin overexpression in cells leads to increased class I surface expression, TAPBPR overexpression has the opposite effect, resulting in decreased MHC class I surface expression. The final role for TAPBR in the endogenous pathway is yet to be resolved.

FIGURE 7-15 Assembly and stabilization of MHC class I molecules. Within the rough endoplasmic reticulum (RER) membrane, a newly synthesized class I α chain associates with calnexin (a molecular chaperone) and ERp57 until β₂-microglobulin binds to the α chain. The binding of β₂-microglobulin releases calnexin and allows binding to calreticulin and to tapasin, which is associated with the peptide transporter TAP. This association creates a protein loading complex (PLC) that promotes binding of an antigenic peptide. Antigens in the RER can be further processed via aminopeptidases such as ERAP, producing fragments ideally suited for binding to class I. Peptide association stabilizes the class I molecule–peptide complex, allowing it to be transported from the RER to the plasma membrane.

An E R p 57 molecule associates with an M H C class 1 alpha chain and Calnexin to form a complex molecule that binds beta 2-microglobulin, releasing tapasin, upon which Calreticulin enters the process toward the TAP chains. A Calreticulin-tapasin�associated MHC class 1 molecule (P L C) next to the TAP chains is directed toward the M H C class 1 molecule, which then exits the R E R to move toward the cytosol. In a parallel process, a protein strand is directed toward an (Immuno) proteasome molecule within the cytosol below, resulting in the formation of smaller peptides, which are ingested into the TAP chains and are directed toward the E R A P. The peptides from E R A P are also directed toward the M H C class 1 molecule in the R E R lumen. An E R p 57 molecule, a Calreticulin molecule and a Tapasin molecule remain within the R E R lumen.

Some ER peptides are too long to bind efficiently to class I molecules, and exoproteases can act on these proteins. One ER aminopeptidase, ERAP1, removes the amino-terminal residue from peptides to achieve optimum class I binding size (see Figure 7-15). In fact, ERAP1 has little affinity for peptides shorter than eight amino acids in length. As a consequence of productive peptide binding, the class I molecule displays increased stability and can dissociate from the complex with calreticulin, tapasin, and ERp57 (also known as the peptide loading complex). The class I molecule can then exit from the RER, proceeding to the Golgi complex and exocytic vesicles, before ultimately reaching the cell surface.

Key Concept:

• In the RER, chaperone proteins and proteases assist with the loading and processing of peptide fragments as they associate with MHC class I molecules, stabilizing this protein complex and allowing peptide-loaded MHC class I molecules to move out of the RER and toward the cell surface.