Biomedical subjects

Peter-M Kloetzel

Publications and source records attributed to Peter-M Kloetzel.

18 recordsLinked to original sources

PubMed2006

Circulating proteasome levels are an independent prognostic factor for survival in multiple myeloma.

The proteasome is a proteolytic complex for intracellular degradation of ubiquitinated proteins which are involved in cell-cycle regulation and apoptosis. A constitutively increased proteasome activity has been found in myeloma cells. We studied circulating proteasome levels and their prognostic significance in sera of 50 control subjects, 20 persons with monoclonal gammopathies of undetermined significance (MGUS), and 141 previously untreated patients with multiple myeloma (MM) by an anti-20S proteasome enzyme-linked immunoabsorbent assay (ELISA). Serum proteasome concentrations were significantly elevated in MM compared with controls (P < .001), in MM versus MGUS (P = .03), and in active (n = 101) versus smoldering (n = 40) MM (P < .001). In patients with active MM, there was a significant (P < .001) decrease from pretreatment to post-treatment proteasome concentrations in responders to chemotherapy, but not in nonresponders. Circulating proteasome levels were identified as a prognostic factor for overall survival in the univariate (P < .001 log-rank test) and in the multivariate (hazard ratio, 4.38) survival analysis in patients with active MM. We demonstrate for the first time that increased serum proteasome concentrations correlate with advanced disease and are an independent prognostic factor in MM.

Female↗

PubMed2006

Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.

IFN-gamma is known as the initial and primary inducer of immunoproteasomes during viral infections. We now report that type I IFN induced the transcription and translation of immunoproteasome subunits, their incorporation into the proteasome complex, and the generation of an immunoproteasome-dependent CD8 T cell epitope in vitro and provide in vivo evidence that this mechanism occurs prior to IFN-gamma responses at the site of viral infection. Type I IFN-mediated generation of immunoproteasomes was initiated by either poly(I:C) or HCV RNA in human hepatoma cells and was inhibited by neutralization of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in immunoproteasome subunit mRNA preceded intrahepatic IFN-gamma responses by several weeks, instead coinciding with intrahepatic type I IFN responses. Thus, viral RNA-induced innate immune responses regulate the antigen-processing machinery, which occurs prior to the detection of IFN-gamma at the site of infection. This mechanism may contribute to the high effectiveness (95%) of type I IFN-based therapies if administered early during HCV infection.

Acute Disease↗

PubMed2006

Combined effect of proteasome and calpain inhibition on cisplatin-resistant human melanoma cells.

Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.

Acrylates↗

PubMed2006

Tumor cell lines expressing the proteasome subunit isoform LMP7E1 exhibit immunoproteasome deficiency.

The immune system can recognize antigenic peptides derived from tumors by their presentation on MHC class I complexes to CTLs. Immunoproteasomes (i20S) can substantially enhance the MHC class I peptide repertoire, making down-regulation of i20S an important strategy of tumor cells in manipulating immune surveillance. Here, we report that human cancer cells express the nonfunctional immunosubunit-variant LMP7E1, in addition to, or instead of LMP7E2, in response to IFN-gamma. This preferential expression of LMP7E1 and the consequent down-regulation of LMP7E2 results in i20S deficiency. The molecular explanation for this phenomenon is the incapacity of LMP7E1 to interact efficiently with the proteasome maturation protein, which regularly recruits LMP7E2 into nascent i20S precursor complexes. In contrast to previous reports, i20S formation in these cancer cells cannot be restored by IFN-gamma treatment. However, expression of LMP7E2 in these cells restores the i20S-deficient phenotype. Thus, our data describe a novel mechanism that contributes to the process of oncogenesis.

Antigens, Neoplasm↗

PubMed2005

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 PA28alphabeta complex. Upon activation of DC, increased MHC class I presentation occurred of the melanocyte-associated epitope tyrosinase-related protein 2(180-188) in a PA28alphabeta-dependent manner. In contrast to other cell types, regulation of PA28alphabeta expression in DC after maturation was found to be IFN-gamma independent. In the present study, we show that expression of PA28alpha and beta subunits was differentially regulated. Firstly, PA28alpha expression is high in both immature and mature DC. In contrast, PA28beta expression is low in immature DC and strongly increased in mature DC. Secondly, we show the presence of a functional NF-kappaB site in the PA28beta promoter, which is absent in the PA28alpha promoter, indicating regulation of PA28beta expression by transcription factors of the NF-kappaB family. In addition, glycerol gradient analysis of DC lysates revealed elevated PA28alphabeta complex formation upon maturation. Thus, induction of PA28beta expression allows proper PA28alphabeta 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.

Animals↗

PubMed2005

IFN-gamma-induced immune adaptation of the proteasome system is an accelerated and transient response.

Peptide generation by the proteasome is rate-limiting in MHC class I-restricted antigen presentation in response to IFN-gamma. IFN-gamma-induced de novo formation of immunoproteasomes, therefore, essentially supports the rapid adjustment of the mammalian immune system. Here, we report that the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal beta5i subunit low molecular weight protein 7 (LMP7) has a key position in this immune adaptive program. IFN-gamma-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both beta5 subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, our data reveal that immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes. In consequence, our studies demonstrate that the cytokine-induced rapid immune adaptation of the proteasome system is a tightly regulated and transient response allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required.

Apoptosis↗

PubMed2005

Interferon-gamma, the functional plasticity of the ubiquitin-proteasome system, and MHC class I antigen processing.

The proteasome system is a central component of a cascade of proteolytic processing steps required to generate antigenic peptides presented at the cell surface to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The nascent protein pool or DRiPs (defective ribosomal products) appear to represent an important source for MHC class I epitopes. Owing to the destructive activities of aminopeptidases in the cytosol, at most 1% of the peptides generated by the ubiquitin-proteasome system seems to be made available to the immune system. Interferon-gamma (IFN-gamma) helps to override these limitations by the formation of immunoproteasomes, the activator complex PA28, and the induction of several aminopeptidases. Both immunoproteasomes and PA28 use cleavage sites already used by constitutive proteasomes but with altered and in some cases dramatically enhanced frequency. Therefore, two proteolytic cascades appear to have evolved to provide MHC class I epitopes. The 'constitutive proteolytic cascade' is designed to efficiently degrade proteins to single amino acid residues, allowing only a small percentage of peptides to be presented at the cell surface. In contrast, the IFN-gamma-controlled proteolytic cascade generates larger amounts of appropriate antigenic peptides, assuring more peptides to overcome the proteolytic restrictions of the constitutive system, thereby enhancing MHC class I antigen presentation.

Animals↗

PubMed2004

The proteasome and MHC class I antigen processing.

By generating peptides from intracellular antigens, which are then presented to T cells, the ubiquitin/26S proteasome system plays a central role in the cellular immune response. Under the control of interferon-gamma the proteolytic properties of the proteasome are adapted to the requirements of the immune system. Interferon-gamma induces the formation of immunoproteasomes and the synthesis of the proteasome activator PA28. Both alter the proteolytic properties of the proteasome complex and enhance proteasomal function in antigen presentation. Thus, a combination of several of regulatory events tunes the proteasome system for maximal efficiency in the generation of MHC class I antigens.

Animals↗

PubMed2004

Hepatitis C virus mutation affects proteasomal epitope processing.

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Amino Acid Sequence↗

PubMed2003

Human immunodeficiency virus-1 Tat protein interacts with distinct proteasomal alpha and beta subunits.

The human immunodeficiency virus-1 (HIV-1) Tat protein was previously reported to compete the association of PA28 regulator with the alpha rings of the 20S proteasome and to inhibit its peptidase activity. However, the distinct interaction sites within the proteasome complex remained to be determined. Here we show that HIV-1 Tat binds to alpha4 and alpha7, six beta subunits of the constitutive 20S proteasome and the interferon-gamma-inducible subunits beta2i and beta5i. A Tat-proteasome interaction can also be demonstrated in vivo and leads to inhibition of proteasomal activity. This indicates that Tat can modulate or interfere with cellular proteasome function by specific interaction with distinct proteasomal subunits.

Cell Line↗

PubMed2003

Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes.

The 26 S proteasome is a high molecular mass proteinase complex that is built by at least 32 different protein subunits. Such protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation. However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell. Here, we show that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activity induce a transient and concerted up-regulation of all mammalian 26 S proteasome subunit mRNAs. Proteasome inhibition in combination with inhibition of transcription revealed that the observed up-regulation is mediated by coordinated transcriptional activation of the proteasome genes and not by post-transcriptional events. Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of proteasomes. This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP. Thus, our experiments present the first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that there exists an autoregulatory feedback mechanism that allows the compensation of reduced proteasome activity.

Animals↗

PubMed2003

An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope.

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.

Aminopeptidases↗

PubMed2003

MAPPP: MHC class I antigenic peptide processing prediction.

MAPPP is a bioinformatics tool for the prediction of potential antigenic epitopes presented on the cell surface by major histocompatibility complex class I (MHC I) molecules to CD8 positive T lymphocytes. It combines existing predictions for proteasomal cleavage with peptide anchoring to MHC I molecules.

Algorithms↗

PubMed2002

PI31 is a modulator of proteasome formation and antigen processing.

Regulation of the proteasome system, which is responsible for the generation of most MHC class I-bound peptides, occurs through the interaction of the 20S proteasome with several regulatory proteins. One of these is PI31, which acts in vitro as an inhibitor of proteasome activity. Here, we demonstrate that, rather than inhibiting proteasome function, PI31 acts as a selective modulator of the proteasome-mediated steps in MHC class I antigen processing. Overexpression of PI31 in mouse embryonic cells has no impact on proteasome-mediated proteolysis. Instead, PI31, which localizes at the nuclear envelope/endoplasmic reticulum membrane, selectively interferes with the maturation of immunoproteasome precursor complexes. Consequently, overexpression of PI31 abrogates MHC class I presentation of an immunoproteasome-dependent cytotoxic T lymphocyte epitope and reduces the surface MHC class I levels on IFN-gamma-treated mouse embryonic cells. Thus, PI31 represents a cellular regulator of proteasome formation and of proteasome-mediated antigen processing.

Adjuvants, Immunologic↗

PubMed2002

Expression of the proteasome activator PA28 rescues the presentation of a cytotoxic T lymphocyte epitope on melanoma cells.

The proteasome system represents a major source of HLA class I- presented peptides exposed to CTLs. Stimulation of cells with IFN-gamma instantly induces the expression of the proteasome immunosubunits as well as the proteasome activator PA28. These proteins have been shown to optimize class I antigen presentation of several viral CTL epitopes; however, their contribution to tumor antigen processing remains poorly understood. Here, we analyzed the generation of an HLA-A*0201-presented epitope derived from the melanoma antigen tyrosinase-related protein 2 (TRP2). Melanoma cells that lacked the IFN-gamma-inducible proteasome activator PA28 and immunoproteasomes did not display the TRP2(360-368) epitope to specific CTLs. Our experiments demonstrate that epitope presentation correlated with the presence of PA28 and could be completely rescued by restoration of PA28 expression. In vitro digestion of TRP2 polypeptides with 20S proteasomes confirmed that PA28 is essential for epitope liberation. Thus, our experiments indicate that PA28 provides the threshold for CTL recognition of this epitope. Importantly, processing of a second TRP2-derived epitope, TRP2(288-296), was diminished in IFN-gamma-treated cells, even in the absence of immunoproteasome up-regulation. Therefore, the reported IFN-gamma-induced self-regulation of epitopes may not necessarily be a consequence of immunoproteasomes as suggested previously.

Amino Acid Sequence↗

PubMed2002

Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity.

Adoptive transfer of cross-reactive HSP60-specific CD8(+) T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8(+) T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of alpha and beta chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8(+) T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.

Amino Acid Sequence↗

PubMed2002

The role of the proteasome activator PA28 in MHC class I antigen processing.

The proteasome system is the major source for the generation of viral antigens and tumor antigens presented by major histocompatibility complex class I (MHC class I) molecules. A specific feature of the proteasomal antigen processing machinery is that five of its components are inducible by IFN-gamma. Two of these are the alpha and beta subunits of the proteasome activator PA28. Our results show that PA28 selectively up-regulates the presentation of viral MHC class I epitopes and that down regulation PA28 in tumor cells results in impaired presentation of a human TRP2 tumor antigen.

Animals↗

PubMed2002

Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases.

OBJECTIVE: The 20S proteasome plays a leading immunologic role in the cytosolic generation of MHC class I restricted antigens, and it represents an abundant antigen in several autoimmune diseases. To investigate the effects of autoimmune inflammatory and perioperative traumatic cellular damage, we determined qualitative and quantitative properties of released proteasomes (circulating proteasomes, cProteasomes) from serum samples of patients with a variety of autoimmune diseases. METHODS: cProteasomes were analyzed from serum samples of 314 patients with several systemic and organ-specific autoimmune diseases and 85 healthy controls. The concentrations of cProteasomes were determined by sandwich ELISA using a monoclonal and a polyclonal proteasome-specific antibody. Followup analyses were performed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) as well as in patients with myasthenia gravis undergoing thoracoscopic thymectomy. RESULTS: Strongly increased levels of cProteasomes (> 1000 ng/ml) were detected in samples obtained from patients with autoimmune myositis, SLE, primary Sjögren's syndrome, RA, and autoimmune hepatitis. Significant differences were observed in the mean values of cProteasomes comparing systemic with organ-specific autoimmune diseases. Followup analyses revealed a close correlation of cProteasome with the autoimmune process as well as cellular damage. Moreover, cProteasomes were isolated in intact and native as well as in degraded or dissociated forms from the serum samples. The immuno-subunit LMP7 was found to be incorporated in the circulating protease complex. CONCLUSION: Levels of cProteasomes are markedly elevated in patients with systemic autoimmune diseases, apparently correlating with disease activity. The cProteasomes represent novel sensitive markers of the autoimmune inflammatory processes and/or reflect the magnitude of cellular damage.

Arthritis, Rheumatoid↗