Peter-Michael Kloetzel

A Kinetic Model of Vertebrate 20S Proteasome Accounting for the Generation of Major Proteolytic Fragments from Oligomeric Peptide Substrates

There is now convincing evidence that the proteasome contributes to the generation of most of the peptides presented by major histocompatibility complex class I molecules. Here we present a model-based kinetic analysis of fragment patterns generated by the 20S proteasome from 20 to 40 residues long oligomeric substrates. The model consists of ordinary first-order differential equations describing the time evolution of the average probabilities with which fragments can be generated from a given initial substrate. First-order rate laws are used to describe the cleavage of peptide bonds and the release of peptides from the interior of the proteasome to the external space. Numerical estimates for the 27 unknown model parameters are determined across a set of five different proteins with known cleavage patterns. Testing the validity of the model by a jack knife procedure, about 80% of the observed fragments can be correctly identified, whereas the abundance of false-positive classifications is below 10%. From our theoretical approach, it is inferred that double-cleavage fragments of length 7-13 are predominantly cut out in C-N-order in that the C-terminus is generated first. This is due to striking differences in the further processing of the two fragments generated by the first cleavage. The upstream fragment exhibits a pronounced tendency to escape from second cleavage as indicated by a large release rate and a monotone exponential decline of peptide bond accessibility with increasing distance from the first scissile bond. In contrast, the release rate of the downstream fragment is about four orders of magnitude lower and the accessibility of peptide bonds shows a sharp peak in a distance of about nine residues from the first scissile bond. This finding strongly supports the idea that generation of fragments with well-defined lengths is favored in that temporary immobilization of the downstream fragment after the first cleavage renders it susceptible for a second cleavage.

20S proteasome biogenesis.

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.

20S proteasome from LMP7 knock out mice reveals altered proteolytic activities and cleavage site preferences

20S proteasomes of tissues from LMP7 knock out mice which show reduced MHC class I restricted antigen presentation were analyzed with regard to their subunit composition, peptide hydrolyzing activity and their ability to cleave a synthetic 25‐mer polypeptide. LMP7 deficiency results in an enhanced incorporation of subunit MB1 and in a 2–3.8‐fold increase in V max for the Suc‐LLVY‐MCA hydrolyzing activity. Since LMP7 deficiency also affects the cleavage site preference of 20S proteasomes the reduced MHC class I antigen presentation of LMP7 knock out mice is most likely due to an impairment in peptide generation.

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response.

Abstract The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing β-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.

Differential expression regulation of the alpha and beta subunits of the PA28 proteasome activator in mature dendritic cells.

Activation of dendritic cells (DC) by Th-dependent (CD40) or -independent (LPS, CpG, or immune complexes) agonistic stimuli strongly enhances the expression of the proteasome activator PA28αβ complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2180-188 in a PA28αβ-dependent manner. In contrast to other cell types, regulation of PA28αβ expression in DC after maturation was found to be IFN-γ independent. In the present study, we show that expression of PA28α and β subunits was differentially regulated. Firstly, PA28α expression is high in both immature and mature DC. In contrast, PA28β expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-κB site in the PA28β promoter, which is absent in the PA28α promoter, indicating regulation of PA28β expression by transcription factors of the NF-κB family. In addition, glycerol gradient analysis of DC lysates revealed elevated PA28αβ complex formation upon maturation. Thus, induction of PA28β expression allows proper PA28αβ complex formation, thereby enhancing proteasome activity in activated DC. Therefore, maturation of DC not only improves costimulation but also MHC class I processing. This mechanism enhances the CD8+ CTL (cross)-priming capacity of mature DC.

The effect of the interferon-γ-inducible processing machinery on the generation of a naturally tumor-associated human cytotoxic T lymphocyte epitope within a wild-type and mutant p53 sequence context

The human wild‐type (wt) p53.264–272 peptide is a universal tumor antigen and recognized by HLA‐A*0201 (A2.1)‐restricted CTL. Generation of this epitope by constitutive 20S proteasomes is prevented by a p53 R to H hotspot mutation at the C‐terminal flanking residueu2004273. We report on the impact of the interferon‐γ (IFN‐γ)‐inducible proteasomal activator PA28 (11S regulator) and the immunoproteasome on the in vitro and cellular processing of wt and mutant (mut)u2004p53 substrates. We found that production of the antigenic 264–272u2004peptide from wtu2004p53 by constitutive as well as immunoproteasomes is accelerated and amplified by the PA28 activator. PA28 and (immuno)proteasomes were not capable to reconvert the resistance of epitope release from mutu2004p53. Maximum and accelerated antigen production in vitro and on the cellular level required the IFN‐γ‐inducible interaction of immunoproteasomes and PA28. We conclude that efficient processing of p53.264272 from wtu2004p53 is governed by the proteasome/PA28 complex. These studies have important implications for p53‐specific cancer immunotherapy and demonstrate that the effects of the immunoproteasome and PA28 are influenced by the individual epitope and its flanking sequence context.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8+ T cells. Three IFN‐γ‐inducible catalytic proteasome (immuno)subunits as well as the IFN‐γ‐inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I‐presented antigenic peptides. To determine whether these IFN‐γ‐inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits β5i/LMP7 and β2i/MECL‐1. Analyses of MHC class I cell‐surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I‐binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8+ T‐cell responses to multiple dominant Influenza and Listeria‐derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I‐binding peptides; however, only the immunosubunit‐imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen‐specific CD8+ T‐cell responses.

The Role of the Ubiquitin-proteasome Pathway in MHC Class I Antigen Processing: Implications for Vaccine Design

Proteasomes are multisubunit enzyme complexes that reside in the cytoplasm and nucleus of eukaryotic cells. By selective protein degradation, proteasomes regulate many cellular processes including MHC class I antigen processing. Three constitutively expressed catalytic subunits are responsible for proteasome mediated proteolysis. These subunits are exchanged for three homologous subunits, the immunosubunits, in IFNgamma-exposed cells and in cells with specialized antigen presenting function. Both constitutive and immunoproteasomes degrade endogenous proteins into small peptide fragments that can bind to MHC class I molecules for presentation on the cell surface to cytotoxic T lymphocytes. However, immunoproteasomes seem to fulfill this function more efficiently. IFNgamma further induces the expression of a proteasome activator, PA28, which can also enhance antigenic peptide production by proteasomes. In this review, we will introduce the ubiquitin-proteasome system and summarize recent findings regarding the role of the IFNgamma-inducible proteasome subunits and proteasome regulators in antigen processing. We review the different ways by which tumors and viruses have been found to target the proteasome system to avoid MHC class I presentation of their antigens, and discuss recent progressions in the development of computer assisted approaches to predict CTL epitopes within larger protein sequences, based on proteasome cleavage specificity. The availability of such programs as well as a general insight into the proteasome mediated steps in MHC class I antigen processing provides us with a rational basis for the design of new antiviral and anticancer T cell vaccines.

The N-terminal Flanking Region of the TRP2360–368 Melanoma Antigen Determines Proteasome Activator PA28 Requirement for Epitope Liberation

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-γ-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A*0201 restricted melanoma antigen TRP2360–368 by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875–2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2288–296 epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2360–368 flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products.

Cross-Presentation of Synthetic Long Peptides by Human Dendritic Cells: A Process Dependent on ERAD Component p97/VCP but Not sec61 and/or Derlin-1

Antitumor vaccination using synthetic long peptides (SLP) is an additional therapeutic strategy currently under development. It aims to activate tumor-specific CD8+ CTL by professional APCs such as DCs. DCs can activate T lymphocytes by MHC class I presentation of exogenous antigens - a process referred to as “cross-presentation”. Until recently, the intracellular mechanisms involved in cross-presentation of soluble antigens have been unclear. Here, we characterize the cross-presentation pathway of SLP Melan-A16–40 containing the HLA-A2-restricted epitope26–35 (A27L) in human DCs. Using confocal microscopy and specific inhibitors, we show that SLP16–40 is rapidly taken up by DC and follows a classical TAP- and proteasome-dependent cross-presentation pathway. Our data support a role for the ER-associated degradation machinery (ERAD)-related protein p97/VCP in the transport of SLP16–40 from early endosomes to the cytoplasm but formally exclude both sec61 and Derlin-1 as possible retro-translocation channels for cross-presentation. In addition, we show that generation of the Melan-A26–35 peptide from the SLP16–40 was absolutely not influenced by the proteasome subunit composition in DC. Altogether, our findings propose a model for cross-presentation of SLP which tends to enlarge the repertoire of potential candidates for retro-translocation of exogenous antigens to the cytosol.