203
Specificity of the proteasome and the TAP transporter
Stephan Uebel and Robert Tampé*
The generation of antigenic peptides and their transport across
the membrane of the endoplasmic reticulum for assembly with
MHC class I molecules are essential steps in antigen
presentation to cytotoxic T lymphocytes. Recent studies have
characterized the substrate specificities of the proteasome and
the transporter associated with antigen processing. It is
interesting to compare the specificity of this transporter to the
wide spectrum of peptides generated by the proteasome, to
the binding motifs of MHC class I molecules and in particular
to the principles of T cell recognition.
Addresses
Institut für Physiologische Chemie, Philipps-Universität Marburg,
Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany
*e-mail: [email protected]
Correspondence: Robert Tampé
Current Opinion in Immunology 1999, 11:203–208
http://biomednet.com/elecref/0952791501100203
© Elsevier Science Ltd ISSN 0952-7915
Abbreviations
CTL
cytotoxic T lymphocyte
ER
endoplasmic reticulum
hTAP
human TAP
interferon γ
IFN-g
P1
position 1
TAP
transporter associated with antigen processing
Introduction
Supplying peptides to the assembling MHC class I molecules for potential recognition by cytotoxic T lymphocytes
(CTLs) involves at least two steps: the first step is the
degradation of proteins (marked for degradation through
polyubiquitinylation) by the proteasome complex — a multisubunit, multicatalytic protease; the second step is the
transport of the peptides thus generated into the lumen of
the endoplasmic reticulum (ER), by the action of the transporter associated with antigen processing (TAP) (for reviews
see [1,2]). In the ER, the assembly of the class I heavy chain
and β2-microglobulin takes place in a tightly regulated fashion involving chaperones such as calnexin, calreticulin and
ERp57 as well as tapasin. The latter bridges class I to TAP
and provides the ER-retention signal for empty class I molecules (Figure 1). While the principles governing substrate
selection by MHC class I molecules are now well understood (for a review, see [3]) and also a binding motif for TAP
has recently been established (for a review, see [4]), the relevance of peptide generation and transport into the ER for
the selection of dominant and subdominant epitopes still
remains elusive. Some open questions remain: these regard
the selectivity and kinetics of peptide generation by the proteasome; the concentration of peptides available for
transport and the kinetics of such transport (and thus the
concentration of peptides available for loading onto class I in
the ER); and the involvement of other factors influencing
the availability of peptides in the respective compartments,
such as chaperones or other transporters. If peptide supply
to class I molecules should prove to be rate-limiting for antigen presentation, selectivity of this ‘funneling’ step could
be imprinted onto the pool of epitopes. This might explain
why only a small number of the potential class-I-binders
within a given protein elicit CTL responses. This review
will describe the advances in understanding the molecular
mechanisms underlying substrate recognition by the proteasome and the TAP, giving new insight into the coevolution
of major components of the MHC class I pathway and the
principles underlying epitope selection.
The proteasome: using an evolutionarily
conserved enzymatic principle for a
novel function
The proteasome complex is found in all three domains of
life, where it has a key function in the degradation and
turnover of proteins (reviewed in [5]). In mammals, the proteasome acquired an additional function and appears to
provide the major proteolytic machinery for generating the
pool of peptide epitopes that are loaded onto MHC class I
molecules (reviewed in [6,7]). Enzymatic inhibition or genetic knockouts reveal that most of the proteasomal genes are
essential for cell function. From comprehensive structural
and functional studies, it became clear that this multicatalytic enzyme complex uses the evolutionarily conserved
principle of self-compartmentalization to restrict and control
protein degradation in the cytosol. The barrel-shaped 20S
proteasome complex comprises 28 subunits which can be
classified into α- and β-types. These subunits are arranged
into four heptameric rings (α7–β7–β7–α7) which are stacked
to enclose three large cavities. The central cavity is formed
by β-subunits only, which harbor the first amino-terminal
Thr residue of each active subunit as the active site.
In Eukarya, the 20S proteasome represents the catalytic
core complex of an even larger 26S complex which functions in the ATP-dependent unfolding of ubiquitinylated
proteins (reviewed in [5]). Although the processing efficiency of 20S and 26S complexes might differ significantly
and the site of ubiquitinylation might also have some
impact on selectivity, the cleavage specificity of all larger
co-complexes is believed to be determined by the 20S core
complex. As described above, the eukaryotic 20S proteasomes contain two identical α7–β7 units although only three
of the seven β subunits in each β7 ring are active. From the
use of model substrates and inhibitors, as well as of proteasome mutants, it became apparent that these active
β subunits of the multicatalytic protease are responsible for
three distinct proteolytic activities according to the position 1 (P1) residue of the substrate: subunit β5
(corresponding to X/MB1/ε in humans), is responsible for
the chymotrypsin-like activity (for hydrophobic residues at
204
Immunological techniques
Figure 1
Pathway of antigen processing via MHC class I.
(a) Endogenous proteins are degraded in the
proteasome-dependent degradation pathway
and (b) peptides are transported into the ER
lumen by TAP dimers. Several molecules in the
ER have been implicated in the tightly regulated
folding, assembly and loading of MHC class I
molecules: (c) calnexin and ERp57;
(d) calreticulin and β2-microglobulin (β2m); and
(e) tapasin. A peptide-trimming activity may also
be present (not shown). (f) Stable
MHC–peptide complexes can leave the ER via
the Golgi compartment (g) to the cell surface,
potentially (h) for recognition by CTLs.
CTL
TCR
Peptide–class-I–β2m
CD8 (h)
Target
cell
(g)
(a)
Proteasome
complex
Golgi
Peptides
Protein
(b)
(f)
TAP
Class I
(e)
(d)
(c)
Tapasin
Calreticulin
β2 m
ERp57
Calnexin
MHC
class I
heavy
chain
ER
Translocon
Ribosome
Current Opinion in Immunology
P1); β2 (Z/α in humans) for the trypsin-like activity (basic
residues at P1); and β1 (Y/δ in humans) for the peptidylglutamylpeptide-hydrolyzing activity (acidic residues at P1).
Beside this minimal set of activities, additional proteolytic
components — for example the so-called ‘branched chain
amino acid preferring’ activity, cleaving after Leu — were
found. To a large extent, the specificity is determined by a
binding pocket which is modulated by residue 45 of each of
the active β subunits and by additional residues of the adjacent β-type subunit within the same ring [8].
Experimentally, it has been demonstrated that residues
other than P1 are involved in determining the substrate
cleavage site of mammalian proteasomes [9,10]. By using
wild-type and mutated yeast proteasomes, the cleavage
motif of the active β subunits was analyzed in detail from
peptide or protein digests [11••,12•]. It was found that only
the three active subunits are responsible for the cleavage
specificity, excluding the possibility of additional catalytic
sites. Interestingly, subunits β1 and β2 also cleave after
some hydrophobic residues — reflecting the ‘branched
chain amino acid preferring’ component. This preference
of the yeast proteasome to generate peptides with
hydrophobic residues (e.g. Leu) at the carboxyl terminus
indicates that the basic proteolytic activity alone might lead
to the generation of MHC class I ligands in vertebrates.
Interestingly, cleavage after residue Gly at P1 appears to be
disfavored by the yeast proteasome and a preference for
small and β-turn-promoting residues was seen in P1 [11••].
In vertebrates, this basic activity pattern becomes even more
complex, as IFN-γ induces a major structural reorganization
of the 20S proteasome. By co-ordinative replacement of the
three active subunits by β2i (MECL1), β1i (LMP2) and β5i
(LMP7) — the latter two encoded in the MHC region —
so-called ‘immunoproteasomes’ are formed (reviewed in [7]).
In addition, IFN-γ induces the formation of the PA28 activator complex, which may itself complex with proteasomes;
PA28 promotes processing of endogenous antigens [13]. The
identification of the proteasome as the central machinery for
epitope generation has brought up interesting questions
regarding the specificity of the proteasome and how its selectivity might be modulated by IFN-γ. The immunecell-specific subunits (β1i, β2i, β5i) are not essential for antigen presentation and from studies of various cell lines, or
mice expressing or lacking MHC-encoded proteasomal subunits, controversial results have been obtained. In
conclusion, the formation of immunoproteasomes may alter
the substrate specificity in such a way that the peptide repertoire is expanded. The direct or indirect generation — as
well as the stability — of various epitopes upon proteasomal
degradation have been studied in numerous in vitro or in vivo
assays and the relevance of the sequence context of the epitope and its flanking residues, as well as steric constraints
imposed by substrates containing (for example) Gly–Ala
repeats or Pro residues, has been pointed out [14–17]; however, the issue of epitope generation by the proteasome is
still controversial and little is known about the principles that
Specificity of the proteasome and the TAP transporter Uebel and Tampé
govern specificity beyond position P1 and that alter the
cleavage pattern of the mammalian proteasome complex
upon immune stimulation.
The proteasome complex generates fragments with a
Gaussian-like length distribution but with a preference for
peptides containing eight to eleven residues [9,18,19]. This
characteristic length distribution seems well preserved for all
proteasomes, even for mutant 20S proteasomes which carry
only one active subunit [11••]. This implies that the length
distribution is not determined by the distance between the
active sites, as it was originally proposed [19]. The observation that the prosequences of some inactive β subunits are
partially processed to match the length distribution of degradation products leads us to speculate that the substrate and
the prosequence might be similarly recognized. As can be
seen from the crystal structure, propeptides are bound at an
extended binding-cleft formed by the active subunit and its
adjacent subunit [8,20]. We would like to propose that this
binding site selects for polypeptides with a minimum length
of eight residues, in order to increase the lifetime of the
enzyme–substrate complex that favors cleavage at P1. This
model is also in agreement with the proposed enzyme mechanism in which, unlike conventional proteases, the
proteasome degrades proteins processively without release of
polypeptide intermediates [21]. Most recently, substrate
binding, processing and product release of the proteasome
complex could be followed in real-time by surface plasmon
resonance spectroscopy — leading to new insights into the
recognition process of the proteasome (IT Dorn, R Eschrich,
E Seemüller, R Guckenberger, R Tampé, unpublished data).
Peptide selection by the TAP transport complex
The heterodimeric TAP complex, composed of TAP1 and
TAP2, belongs to the ATP-binding cassette (ABC) superfamily of transporter proteins, which are involved in transport
of a wide variety of substrates across biological membranes.
Members of this family are found in Eukarya as well as in
Prokarya and include the multidrug-resistance P-glycoprotein
(MDR), the cystic fibrosis transmembrane conductance regulator (CFTR) and the oligopeptide transporter of Salmonella
typhimurium. Hydrophobicity profiles of these proteins suggest that they have two domains each, consisting of six to ten
membrane-spanning regions and two hydrophilic domains.
The designation of this protein family is derived from these
highly conserved domains, where the Walker motifs A and B
form a site for ATP-binding and hydrolysis — which is why
they are called nucleotide-binding domains.
The role of TAP as a peptide transporter has been shown
by restoration of peptide transport and class I surface
expression in cell lines that were previously TAP-deficient, by in vitro transport assays using semipermeabilized
cells and by heterologous expression of TAP in insect cells
or yeasts; moreover TAP-dependent transport could be
directly shown through trapping of translocated peptides,
carrying an N-glycosylation recognition sequence, in the
ER lumen after glycosylation by enzymes on the lumenal
205
side of the ER membrane [22,23]. By comparison of the
amount of N-glycosylated peptide that has accumulated in
the ER, this assay was used for the analysis of substrate
selection by TAP [24–27]. A second assay system directly
measures peptide–TAP interaction as peptide-binding
affinities and thus circumvents several problems associated
with the N-glycosylation-based assays; these are mainly
due to the kinetics of the glycosylation reaction and an efficient peptide export system in the ER [28–30,31••].
Conclusive results concerning the length selectivity of
TAP come from the use of randomized peptide libraries in
conjunction with competition binding assays [28] and from
transport assays using peptides that carry the N-glycosylation recognition sequence at one terminus and a Tyr
residue for radioiodination at the opposite terminus [32].
Peptides that are 8–16 amino acids in length seem to be
good substrates for TAP, with peptides of 9–12 residues
suited best, although transport could also be unequivocally shown for considerably longer peptides. For rat TAP,
where a functional polymorphism exists, conflicting results
have been reported: whereas one group finds that TAP
from the RT1a strain is more permissive towards longer
peptides than TAP from the RT1u strain or human TAP
(hTAP) [32], another report suggests the opposite [25].
When it comes to the side-chain specificity, the results
from both assay systems are in good agreement with
respect to the relative order of preferred residues. In fact,
when the same set of peptides has been tested in both systems, similar results have been obtained and the two assay
systems have been termed ‘functionally indistinguishable’
[33]. The major difference in the results, though, is the
absolute range of affinities covered by peptides. While
mouse TAP and rat TAP of the RT1u strain were found to
be clearly selective for hydrophobic carboxyl termini,
hTAP and rat RT1a -strain TAP have been dubbed rather
nonselective from the results of the N-glycosylation-based
assays [34]. In contrast, two independent studies — using
binding assays — have shown at least three orders of magnitude difference in peptide affinities for hTAP
[31••,35••]. Interestingly, the binding assays have shown
that the residues determining peptide affinity, in addition
to the carboxyl terminus, are not evenly distributed across
the peptide. Considerable selectivity is exerted by the
three amino-terminal residues, particularly by P2
(Figure 2). Thus, the peptide residues relevant for binding
to hTAP are the same ones that are used as anchors for
most MHC class I molecules. At least for the carboxyl terminus, preferences of hTAP and class I are well matched:
Phe, Leu, Arg, Tyr and Val (favored by hTAP) comprise
80% of the residues found as class I anchors [3]. Thus, it is
interesting to speculate that hTAP, in a process of coevolution with class I molecules, could ‘concentrate’ on
hydrophobic and basic carboxyl termini without limiting
the pool of peptides available for class I binding. In contrast, even the high-resolution binding assays — in
combination with combinatorial libraries — did not reveal
206
Immunological techniques
Figure 2
HLA-binding
motif
TCR
recognition motif
H
O
+
(a)
H
H
Sequence diversity
N
C
_
O
Length flexibile (8–16 aa)
(c) Side-chain
preferences
+
(b) Position
–
K,N,R R,I,Q W,Y
1
2
3
4
5
D,E,F
P,L
D,E,G
N
T
R
I
6
7
D,E
Peptide recognition principle of TAP.
(a) Selected sequences will contain HLAbinding motifs and diverse TCR recognition
sequences with variable numbers of amino
acids (aa). (b) Positions 1–9 of a peptide
sequence are indicated as an example (singleletter code is used for amino acids). Peptide
residues that are preferred (+) or disfavored
(–) by human TAP are given at the individual
positions as extracted from combinatorial
peptide libraries [31••]. Residues in bold show
the strongest effects. (c) The degree of TAP
selectivity for various side chains is given as
the variance of the stabilization factors and
(d) the relevance of the peptide backbone for
recognition by TAP derived from D-amino acid
libraries is indicated.
F,L,R,Y,V
8
9
D,E,N,S,G
(d) Relevance of
peptide backbone
Current Opinion in Immunology
any considerable selectivity at P5–P8 of peptides, which
strongly contribute to T cell recognition [31••].
An interesting effect has been noted for Pro at P2. It is the
strongest single destabilizing residue found, nearly completely abolishing binding to hTAP. Similar results were
obtained for murine TAP at peptide P3, suggesting that it
reflects an important principle [24]. Subsequent analysis
has shown that the effect of Pro results from interaction of
hTAP with the peptide backbone, mainly at P2 but also at
P1 and P3 [31••]. Thus, by holding on to the peptide
backbone, TAP could provide high affinity binding while
at the same time enlarging the pool of peptides used for
TCR recognition. Using peptide libraries containing Damino acids and Pro-containing sequences, this motif
could also be extended to longer peptides [31••,35••].
This raises the question of how the class I alleles, with Pro
as an anchor at P2, are supplied with peptides. Taking into
account that TAP also transports peptides longer than
those suited for class I binding, amino-terminal trimming
of transported peptides seems to play a key role.
Epitopes in the making
What is the physiological relevance of TAP-mediated substrate selection? TAP has seemingly coevolved (see [36])
with the other components of the class I pathway so that it
exerts minimal restriction on the pool of epitopes available
for recognition by the TCR, while providing high affinities
and thus high transport efficiencies at the presumably low
concentrations of free peptides in the cytosol. Still, even for
hTAP, some imprint of its selectivity on the peptides bound
to class I should be visible. In particular, a prominent influence of TAP would be expected at P2 and P3 of the peptides
naturally presented on class I. Unfortunately, no functional
polymorphism for hTAP and mouse TAP has been found
[34,37,38•] — a situation in contrast to that in the rat, where
RT1u-strain TAP selectivity leads to impaired intracellular
transport of class I molecules of the RT1a allele [36]; therefore, studies on the physiological relevance of peptide
selection of human and mouse TAP have, to date, relied on
circumstantial evidence. The clearest results come from a
study of hTAP where a disequilibrium was found between
predicted peptide affinities for known class-I binders, as
opposed to randomly picked sequences [35••]. Additionally,
the disequilibrium found was different for several class I alleles — again suggesting a role for trimming prior to loading
onto some alleles; this reflects the fact that HLA-A2 is often
found associated with signal-sequence-derived peptides.
From these results it appears unlikely that other additional
factors, such as chaperones, play a critical role in peptide
selection in the MHC class I pathway. This does not exclude
the fact that certain peptides bind to chaperones; however
each additional, specific binding event will restrict the peptide repertoire presented to CTLs. For example the binding
motif of Hsp70-like chaperons recognizing a specific pattern
of hydrophobic residues in the centre of the peptide is different from the binding motif of MHC class I and TAP
Specificity of the proteasome and the TAP transporter Uebel and Tampé
molecules and, if generalized, would drastically affect the
pool of peptides presented on MHC class I molecules.
Conclusions
The adaptive immune system has obviously acquired an
evolutionarily conserved, enzymatic principle to generate a
large pool of protein fragments with a defined length distribution imprinting some preference for generating
peptides with hydrophobic residues. Upon this given pool
of peptides, TAP has seemingly coevolved with other components of the class I pathway sharing a similar binding
motif; however to maximize (on one side) the efficiency of
the antigen-processing machinery and to minimize (on the
other) the restrictions on the pool of peptides for recognition by the TCR, some imprint of its selectivity should be
visible — assuming that peptide supply is rate-limiting
under physiological conditions. The influence should be
distinguishable for different class I alleles, since they vary
in their assembly and loading kinetics. Regulation of
retention time in the ER for a given peptide concentration
might thus be the true function of factors involved in
class I assembly and the generation of kinetically stable
MHC–peptide complexes, making some class I alleles
available for loading from alternative peptide sources (signal sequences, trimmed peptides, etc.). ‘Flooding’ of the
ER with peptides derived from highly expressed proteins
due to IFN-γ-dependent upregulation of subunits of the
proteasome complex and TAP (e.g. after viral infection)
might disturb this balance in such a way that these viral
sequences are predominantly presented to CTLs.
10. Bogyo M, Shin S, Mcmaster JS, Ploegh HL: Substrate-binding and
sequence preference of the proteasome revealed by active-sitedirected affinity probes. Chem Biol 1998, 5:307-320.
11. Nussbaum AK, Dick TP, Keilholz W, Schirle M, Stevanovic S, Dietz K,
•• Heinemeyer W, Groll M, Wolf DH, Huber R et al.: Cleavage motifs of
the yeast 20S proteasome beta-subunits deduced from digests
of enolase-1. Proc Natl Acad Sci USA 1998, 95:12504-12509.
A 45 kDa protein from yeast was degraded in vitro by purified wild-type and
mutant yeast 20S proteasomes. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments with an average length of approximately 8 amino acids.
A detailed analysis of the cleavages also allowed the identification of certain
amino acid characteristics in positions flanking the P1 residue.
12. Dick TP, Nussbaum AK, Deeg M, Heinemeyer W, Groll M, Schirle M,
•
Keilholz W, Stevanovic S, Wolf DH, Huber R et al.: Contribution of
proteasomal beta-subunits to the cleavage of peptide-substrates
analyzed with yeast mutants. J Biol Chem 1998,
273:25637-25646.
This study relates to [11••]; using wild-type and mutated yeast 20S proteasomes, the specificities and contributions of the different β subunits to the
degradation of fluorogenic substrates containing MHC class I ligands were
analyzed. A correlation between the contribution of the different subunits to
the cleavage of long peptide substrates was found.
13. Dick TP, Ruppert T, Groettrup M, Kloetzel PM, Kuehn L, Koszinowski UH,
Stevanovic S, Schild H, Rammensee HG: Coordinated dual cleavages
induced by the proteasome regulator PA28 lead to dominant MHC
ligands. Cell 1996, 86:253-262.
14. Shimbara N, Ogawa K, Hidaka Y, Nakajima H, Yamasaki N, Niwa S,
Tanahashi N, Tanaka K: Contribution of proline residue for efficient
production of MHC class-I ligands by proteasomes. J Biol Chem
1998, 273:23062-23071.
15. Sharipo A, Imreh M, Leonchiks A, Imreh S, Masucci MG: A minimal
glycine-alanine repeat prevents the interaction of ubiquitinated
i-kappa-b-alpha with the proteasome — a new mechanism for
selective-inhibition of proteolysis. Nat Med 1998, 4:939-944.
16. Niedermann G, Grimm R, Geier E, Maurer M, Realini C, Gartmann C,
Soll J, Omura S, Rechsteiner MC, Baumeister W, Eichmann K:
Potential immunocompetence of proteolytic fragments produced
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17.
References and recommended reading
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This study provides the binding motif for human TAP at high resolution using
complex peptide libraries. The use of libraries containing D-amino acids
allows the extension of the motif to different-length peptides and highlights
the relevance of the peptide backbone for binding to TAP, leading to a model
for the interaction of TAP and peptide at the molecular level.
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25:2170-2176.
35. Daniel S, Brusic V, Caillatzucman S, Petrovsky N, Harrison L, Riganelli D,
•• Sinigaglia F, Gallazzi F, Hammer J, van Endert PM: Relationship
between peptide selectivities of human transporters associated
with antigen-processing and HLA class-I molecules. J Immunol
1998, 161:617-624.
The authors present a matrix for predicting human TAP peptide-binding affinities using a polyalanine-based peptide. The interesting aspect of the study
is the comparison of TAP affinities for known class-I binders with randomly
selected sequences. The observed bias for the class-I binders represents
the first proof for the relevance of epitope selection by human TAP in vivo.
Unfortunately, the binding affinities for TAP that are used are predicted rather
than being experimental values.
36. Joly E, Lerolle AF, Gonzalez AL, Mehling B, Stevens J, Coadwell WJ,
Hunig T, Howard JC, Butcher GW: Coevolution of rat TAP
transporters and MHC class-I RT1-a molecules. Curr Biol 1998,
8:169-172.
37.
Schumacher TNM, Kantesaria DV, Serreze DV, Roopenian DC,
Ploegh HL: Transporters from H-2(b), H-2(d), H-2(s), H-2(k), and
H-2(g7) (NOD/lt) haplotype translocate similar sets of peptides.
Proc Natl Acad Sci USA 1994, 91:13004-13008.
38. Daniel S, Caillatzucman S, Hammer J, Bach JF, van Endert PM:
•
Absence of functional relevance of human transporter associated
with antigen-processing polymorphism for peptide selection.
J Immunol 1997, 159:2350-2357.
This is a systematic search for a functional polymorphism in human TAP.
Although a sensitive binding assay is used, no differences were observed for
the alleles tested.