Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation

Momburg, Frank; Ortı́z-Navarrete, Vianney; Neefjes, Jacques; Goulmy, Els; Wal, Yvonne van de; Spits, Hergen; Powis, Simon J.; Butcher, Geoffrey W.; Howard, Jonathan; Walden, Peter; Hämmerling, Günter J.

doi:10.1038/360174a0

Cited by 239 publications

(148 citation statements)

References 32 publications

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“…Such peptides also appear to be better substrates for the TAP transporter on the endoplasmic reticulum (34,35). Interestingly, mutant mice with a deletion of LMP2 have a significant defect in the presentation of certain antigens on MHC class I (25), and the lymphoblast mutant lacking LMP2 and -7 may have a decreased efficiency of antigen processing (33).…”

Section: Discussionmentioning

confidence: 99%

“…In related studies (36), we present evidence that the proteasome is the primary site for generation ofantigenic peptides. LMPs do not appear to be obligatory for antigen presentation, which can still occur in LMP-deficient 0.174 lymphoblasts (22,33). Proteasomes isolated from these cells still exhibit significant chymotryptic-like and tryptic-like activities (5).…”

Section: Discussionmentioning

confidence: 99%

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Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Gaczyńska

Rock

Spies

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

301

215

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Recent studies have implicated proteasomes in the generation ofthe antigenic peptides that are presented on major histocmpatibility complex class I molecules to T lymphocytes. Interferon y modifies the subunit composition of proteasomes and causes changes in their peptidase activities that should favor the production of peptides with hydrophobic or basic carboxyl -termini (i.e., the types found on major histocompatibility complex class I molecules). It has been proposed that these changesIn peptidase activity are due to incorporation into proteasomes of the major histocompatibility complex-encoded subunits.LM1P2 and -7, which are induced by interferon y. Here we show by gene transfection into lymphoblasts or HeLa cells that LMP7 Increases the capacity (V,) of 20S and 26S proteasomes to cleave peptides after hydrophobic and basic residues without, affecting hydrolysis after acidic residues. These changes depended on the amount of LMP7 subunits incorporated into proteasomes. Transfection ofLMP2 reduced cleavage of peptides after acidic residues, increased hydrolysis after basic residues, and did not affect the hydrophobic aciity. Since the activity of the total proteasome population changed after incorporation of only small amounts of LMP2 or -7, these subunits must cause major alterations in peptidase activity. Thus, their expression can account for the changes in proteasome activity induced by interferon 7, and these findings lend further support to the proposed roles of LMPs in altering the nature of the peptides generated for antigen presentation.An initial step in the presentation of intracellular and viral proteins to the immune system is their proteolytic degradation in the cytosol to 8-or 9-residue peptides (1, 2). The antigenic fragments are then taken up into the endoplasmic reticulum, where they associate with the major histocompatibility complex (MHC) class I molecules (3). These peptideprotein complexes are then transported to the cell surface for presentation to cytotoxic T cells (1,3). Recently, strong evidence has been obtained implicating the proteasome (multicatalytic proteinase complex) and the ATP-ubiquitindependent pathway (4) in the process of the generation of antigenic fragments (5-10).The 20S (700 kDa) proteasome particle functions as the proteolytic core of the 26S (1500 kDa) complex that degrades ubiquitin-conjugated proteins (10-13). This particle exhibits multiple peptidase activities, including three well-characterized activities that preferentially hydrolyze small peptides on the carboxyl side of hydrophobic, basic, or acidic residues (10-13). Moreover, these three activities are regulated by the cytokine interferon y (IFN-y), which is a potent stimulator of MHC class I antigen presentation. Treatment of cells with IFN-y does not affect the ability of 20S or 26S particles to degrade model proteins or ubiquitinated proteins but increases their capacity to cleave oligopeptides after hydrophobic and basic residues and reduces cleavage after acidic residues (5-7). These functiona...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Gaczyńska

Rock

Spies

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

301

215

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“…Cell Lines-T2, T2-TAP2 (expressing rat TAP2 only), T2-TAP1 (expressing rat TAP1 only), and T2 TAP1ϩ2 (expressing rat TAP1 and TAP2) cells (39,40) were maintained in RPMI 1640 medium with 5% fetal calf serum and 1.0 mg/ml G418 (Life Technologies, Inc.). * The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

Three-dimensional Structure of Transporter Associated with Antigen Processing (TAP) Obtained by Single Particle Image Analysis

Velarde¹,

Ford²,

Rosenberg³

et al. 2001

Journal of Biological Chemistry

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The transporter associated with antigen processing (TAP) is an ATP binding cassette transporter responsible for peptide translocation into the lumen of the endoplasmic reticulum for assembly with major histocompatibility complex class I molecules. Immunoaffinity-purified TAP particles comprising TAP1 and TAP2 polypeptides, and TAP2 particles alone were characterized after detergent solubilization and studied by electron microscopy. Projection structures of TAP1؉2 particles reveal a molecule ϳ10 nm across with a deeply staining central region, whereas TAP2 molecules are smaller in projection. A three-dimensional structure of TAP reveals it is isolated as a single heterodimeric complex, with the TAP1 and TAP2 subunits combining to create a central 3-nm-diameter pocket on the predicted endoplasmic reticulum-lumenal side. Its structural similarity to other ABC transporters demonstrates a common tertiary structure for this diverse family of membrane proteins. ATP binding cassette (ABC)1 transporters are ubiquitous and can form a significant component of an organism's genome, for example 2% in Escherichia coli (1). They are defined by the ABC domain, a nucleotide hydrolysis domain whose activity powers substrate transport (2), and are classically composed of at least four domains, two transmembrane domains (TMDs) and two cytoplasmic and soluble nucleotide binding domains (NBDs). These domains can be expressed together as a single modular polypeptide or separately.TAP is an ABC transporter expressed in the endoplasmic reticulum (ER), which supplies peptides from the cytosol for binding by MHC class I molecules (3-8). In the absence of TAP the assembly of MHC class I and, consequently, antigen presentation are both severely impaired (9 -12). TAP is expressed as two polypeptide subunits, TAP1 and TAP2, each composed of one TMD and one NBD, which together form the functional transporter. Models for the predicted structure of TAP have relied on the sequence-based prediction of multiple transmembrane-spanning regions in the TMDs (13), the location of point mutations or naturally occurring polymorphisms affecting peptide selection and transport (14 -17), or the cytosolic or ER lumenal orientation of TAPs engineered to contain reporter molecules (18,19). More recently, expression of truncated TAP polypeptides suggests interactions between both the TMDs and NBDs of . Nevertheless, no direct structural imaging of this crucial molecule to the development of cellular immune responses has yet been obtained.There is a need for structural studies of whole ABC transporters to permit insights into their overall structure and functions. Single particle image analysis has successfully been applied to molecules of comparable size to TAP (23-26). Furthermore, low resolution electron microscopic structures of the ABC transporters P-glycoprotein and MRP1 have been determined using a combination of single particle and crystallographic analysis (27,28). Recently a structure for MsbA, a prokaryotic half-ABC transporter, has been published, all...

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“…Although the possiblity that the MHC-encoded subunits are essential for these processes was eliminated (Arnold et al, 1992;Momburg et al, 1992), it was recently shown that mice with a targeted deletion of the genes Imp2 or lmp7 have reduced levels of MHC class I siirface expression and present endogenous peptides inefficiently (Fehling et al, 1994;Van Kaer et al, 1994). The composition of newly synthesized 20s proteasomes is modified by the interfon(1FN)-y-induced exchange of the subunits 6 and c to LMP7 and LMP2, respectively (Yang et al, 1992;Akiyama et al, 1994a,b;Fruh et al, 1994;Belich et al, 1994).…”

mentioning

confidence: 99%

Effects of Major‐Histocompatibility‐Complex‐Encoded Subunits on the Peptidase and Proteolytic Activities of Human 20S Proteasomes

Ehring

Meyer

Eckerskorn

et al. 1996

European Journal of Biochemistry

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The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitintagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20s proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20s proteasome can cledve insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20s proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.

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Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation

Cited by 239 publications

References 32 publications

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.

Three-dimensional Structure of Transporter Associated with Antigen Processing (TAP) Obtained by Single Particle Image Analysis

Effects of Major‐Histocompatibility‐Complex‐Encoded Subunits on the Peptidase and Proteolytic Activities of Human 20S Proteasomes

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