Transporter Associated With Antigen Processing Biology Essay

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A family of 49 different members in humans1, ABC transporters play a vital role in the normal functioning of almost all cells. They are evolutionary highly conserved ATPases, classified into 7 subfamilies by their sequence homology in their ABC (ATP-Binding Cassette) region into 7 subfamilies, ABC-A to ABC-G2. The majority are involved in the transportation of a large variety of substrates across a lipid bilayer intra or extra-cellularly (exceptions here include the soluble RNAse L inhibitor ABCE13). The TAP transporter (involved in the MHC class 1 processing pathway4) is a heterodimer consisting of two proteins, TAP1 and TAP23 forming 2 members of the 11 making up the ABCB subfamily (ABCB2 and ABCB3 respectively)2. TAP1 and TAP2 each consist of a Nucleotide Binding Domain (NBD) containing the conserved ABC region typical of ABC proteins, and a Transmembrane Domain (TMB) 4. Together they have been shown to be necessary and sufficient for peptide transport into the ER from the cytosol6,7. Through this essay I will attempt to give the structural features and mechanisms of TAP which are typical of ABC transporters including any additional unique features, then offer insight on its biological function in health and in disease.

The NBD of TAP1 and TAP2 consist of 259 and 232 amino acids at the C terminus end3 out of a total of 748 and 686 respectively4. The structures of the NBDs found in TAP are typical for those found in other ABC transporter NBDs which is not surprising given the entire family shares 25% sequence homology in this domain (regardless of prokaryotic or eukaryotic origin)3. The characteristic motifs found in all ABC proteins are predictably found in TAP - the walker A (GX4GKS/T) and B (4xhydrophobic amino acids followed by D) motifs and the C loop (ABC signature motif - LSGGQ)8. Other conserved loop motifs exist but only contain a single common amino acid (e.g. Q, P, H, or G)9. The overall fold consists of 2 'arms', an F1-ATPase-like arm1 (containing the walker A and B motifs) and a more flexible α-helical arm2 (containing the C loop) which has been indicated in signalling10. See Fig.139 for diagram.

Variations nevertheless do exist, even within the conserved motifs. The TAP2 C-loop in humans has the sequence LAAGQ (as opposed to LSGGQ in other ABC proteins)3. In-fact the fourth G residue is the only conserved amino acid in this motif for TAP2 proteins across all orders3. This residue is involved in forming a hydrogen bond to the ɣ-phosphate of ATP11. The role of this difference between TAP1 and TAP2 C-loops has as yet not been made clear, though evidence is present showing that the second position S or A present in all known TAP sequences influences the ATP hydrolysis rate and peptide transport3. Other distinct features are a G residue in place of the H residue in the TAP1 H-loop, and a D residue in place of an E residue downstream of the walker B motif also in TAP13. Variations in these residues give heterogeneity between TAP1 and TAP2, and may account for their difference in ATPase activity3.

The TMD of TAP1 and TAP2 consist of the first 488 and 453 amino acids from the N terminus respectively3. This region shows very little homology between different ABC family members in both numbers of TM regions and in sequence (e.g. MsbA of E.coli has 6 TM helices (TMs)12, BtuCD also from E.coli has 10 TMs13 and even human TAP1 and TAP2 TMDs differ by 60% in sequence4). This variation is thought to be due to the difference in the substrate size and shape, as this is their binding domain3. Ten TMs are proposed for TAP1 and nine for TAP214, though it has been shown that only six from each play a role in translocation of peptides (as in Pgp - another ABC protein in the same subfamily)3. The others which form the N-terminal final stretch, are thought to be essential for the binding of tapasin (another protein involved in MHC 1 processing), and are not essential for TAP targeting to the ER, or dimerization of the complex15.

ATP binding and peptide binding to TAP have been shown to be independent of one-another16, and the proposed mechanism of action is similar to that of other ABC transporters. Once the nucleotides are bound, a closed form of the NBD dimer is formed in two stages - a fast nucleotide association, then a slow conformational change8 involving one fourth of all TAP amino acids17 (dubbed the 'power stroke'3). This is like a molecular switch which activates ATPase activity3 and also correlates with peptide binding (peptides which are too bulky bind TAP but do not allow ATP hydrolysis)18. Two ATP molecules are required for the transport cycle, the binding pockets for which are formed by both NBD subunits4 (as opposed to one binding pocket for one ATP on each NBD) although it has been found that they are hydrolysed sequentially8 and that hydrolysis by TAP1 may not be essential19. A second conformational change occurs after ATP binding as the lateral membrane mobility of TAP decreases with bound peptide and nucleotide20. Studies have indicated that ATP hydrolysis only occurs once the peptide has been transferred across the membrane21 suggesting its role as resetting the transporter for further cycles3. Exactly how the peptide is moved through the TM pore is yet to be resolved. Fig.23 shows a proposed model, though other models can't be disregarded.

The biological role of TAP shall now be discussed. TAP forms an essential role in the MHC class 1 processing pathway, which allows the presentation of endogenous antigen peptides on the outside of the cell to passing cytotoxic (CD8+) T-cells22. This pathway is constitutively observed in almost all nucleated cells but is up regulated in the presence of the cytokine IFN-É£4. If the antigen peptide displayed is from a 'non-self' or foreign protein (e.g. peptide fragment from a mycobacterium or a viral protein), the cell is lysed or made to undergo apoptosis4. Peptides found to be optimal for binding to TAP are of length 8-16 amino acids long23, and are bound at the TMD (both TAP1 and TAP2 are involved in the peptide binding region)24 by 'anchor' residues - basic or hydrophobic amino acids at the end of the C-terminus and specific first 3 amino acids from the N-terminus give the affinity25. Since the T-cell receptor only recognizes the amino acids from positions 3 to 826, there is no restriction of peptides in the pool which can be presented.

Although TAP can independently transport peptides across membranes4, it forms part of a multi-component Peptide Loading Complex (PLC)4 found on the ER and cis-golgi membranes27. It consists of one TAP heterodimer with 4 tapasin molecules, 4 MHC 1 heavy chains28 (bound temporarily to β2Microglobulin (β2M) - an invariant light chain) with the chaperone calreticulin and the oxidoreductase ERp5723. Tapasin is a major protein in the complex binding the N-terminus end TMs of TAP1 and TAP215 connecting it to the recipient of the transferred peptide, MHC 1/β2M29. It has also been found to stabilize TAP and the MHC/β2M as well optimisation of the peptides ('peptide editing')3. The TAP protein itself forms a link for peptide fragments prepared in the cytosol to the ER part of the presentation pathway. See Fig.3 for diagram.

A defect in the transportation of peptides into the ER will inevitably lead to a decrease in the expression of MHC class 1, as without the peptide, the MHC 1 molecule is unstable and so is degraded by cellular proteases3. This will lead to an impairment of immune response to viral invaders and tumor-associated peptide antigens. Three levels of disruption can lead to a loss of TAP function. The first is as a consequence of a mutation in the gene for either TAP1 or TAP2 (proving the need for the heterodimer) leading to the immunodeficiency disorder Bare Lymphocyte Syndrome (BLS) type 1. Secondly, there can be malfunction in the regulatory mechanisms of transcription of TAP1 or 2, occurring in some tumors and by some viral proteins. Thirdly, the function of TAP can be impeded via post-translational means by inhibitory proteins from viruses.3

BLS is a rare autosomal recessive disease of which 3 types exist30. Those with type 1 are the only ones to survive to adulthood, although patients eventually die from progressive lung damage3. The mutation can be on either the TAP1 or TAP2 gene and is most often a non-sense mutation3,31, though people with mutations in the TAP2 gene often show no symptoms32 at all perhaps due to the fact that if the mutation was in TAP1, there would be hardly any of this subunit being produced and the TAP2 being made would be degraded immediately after production as this is very unstable without TAP114; whereas with a mutation in TAP2, there will be TAP1 being produced and kept, as this is relatively more stable on its own14.

It has been documented that many tumors go undetected from circulating cytotoxic T-cells, due to a down-regulation of MHC 1 protein14 or a mutant protein being produced33. For example it has been found that a point mutation in the P-loop of TAP1 from a lung cancer cell line (R659Q) reduced the cells ability to mediate surface expression of MHC 133, as ATP hydrolysis or binding is affected4. Another mechanism of down-regulation of MHC 1 by tumor cells is thought to be through the inactive p53 tumor suppressor protein which normally induces TAP1 expression34, leading to a decrease in the number of functional TAP complexes in the cell. These flaws can often be made right through the application of IFN-É£, which gives rise to mechanisms up regulating TAP expression14.

Slow replicating DNA viruses have developed several mechanisms to avoid detection by the host immune system, often using several strategies in parallel3. One target of these strategies is the TAP protein. The group E adenovirus produces a protein called E3/19K3. This binds to the TAP complex as well as the MHC1/β2m, but not at the same time (unlike tapasin)35. This leads to an unstable MHC 1 leading to its degradation35, and therefore loss of MHC 1 on the cell surface. The herpes virus family have also developed means to target TAP, for example the herpes simplex virus produces a protein called ICP47 which interferes directly with TAP36. ICP47 associates with the ER membrane adopting an α-helical structure37, then binds TAP to block peptide binding (but not ATP binding)3. It also has a destabilizing effect on the TAP1/2 dimer38.

The similarities between peptide binding specificities of MCH 1 and TAP transporter, as well as the ability of T-cells to recognize only the middle section of the peptide so the pool of antigens that can be recognized is not restricted strongly suggests a co-evolution of all three proteins. Though viruses have also evolved ways to evade detection by the immune system through interfering with the processing pathway, they have been invaluable tools in research to determine various aspects in the pathway. This knowledge can potentially lead us to novel targets for pharmacological therapy, to restore our immune system's ability to recognize viral infections or to suppress the immune system where needed. Research however is still ongoing, with several aspects of TAP yet to be discovered, for example the structure and mechanism of the TM pore or the stoichiometry of the entire TAP transport mechanism. These are only two of several aspects yet to be discovered, though as time passes I am confident our knowledge will improve in this accelerating field.