RESUMO
By tying peptide fragments originally distant in parental proteins, the proteasome can generate spliced peptides that are recognized by CTL. This occurs by transpeptidation involving a peptide-acyl-enzyme intermediate and another peptide fragment present in the catalytic chamber. Four main subtypes of proteasomes exist: the standard proteasome (SP), the immunoproteasome, and intermediate proteasomes ß1-ß2-ß5i (single intermediate proteasome) and ß1i-ß2-ß5i (double intermediate proteasome). In this study, we use a tandem mass tag-quantification approach to study the production of six spliced human antigenic peptides by the four proteasome subtypes. Peptides fibroblast growth factor-5172-176/217-220, tyrosinase368-373/336-340, and gp10040-42/47-52 are better produced by the SP than the other proteasome subtypes. The peptides SP110296-301/286-289, gp100195-202/191or192, and gp10047-52/40-42 are better produced by the immunoproteasome and double intermediate proteasome. The current model of proteasome-catalyzed peptide splicing suggests that the production of a spliced peptide depends on the abundance of the peptide splicing partners. Surprisingly, we found that despite the fact that reciprocal peptides RTK_QLYPEW (gp10040-42/47-52) and QLYPEW_RTK (gp10047-52/40-42) are composed of identical splicing partners, their production varies differently according to the proteasome subtype. These differences were maintained after in vitro digestions involving identical amounts of the splicing fragments. Our results indicate that the amount of splicing partner is not the only factor driving peptide splicing and suggest that peptide splicing efficiency also relies on other factors, such as the affinity of the C-terminal splice reactant for the primed binding site of the catalytic subunit.
Assuntos
Peptídeos , Complexo de Endopeptidases do Proteassoma , Antígenos/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Splicing de RNARESUMO
The proteasome is the major protease responsible for the production of antigenic peptides recognized by CD8+ cytolytic T cells (CTL). These peptides, generally 8-10 amino acids long, are presented at the cell surface by major histocompatibility complex (MHC) class I molecules. Originally, these peptides were believed to be solely derived from linear fragments of proteins, but this concept was challenged several years ago by the isolation of anti-tumor CTL that recognized spliced peptides, i.e. peptides composed of fragments distant in the parental protein. The splicing process was shown to occur in the proteasome through a transpeptidation reaction involving an acyl-enzyme intermediate. Here, we review the steps that led to the discovery of spliced peptides as well as the recent advances that uncover the unexpected importance of spliced peptides in the composition of the MHC class I repertoire.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Modelos Biológicos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína , Animais , Biocatálise , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Membrana Celular/metabolismo , Biologia Computacional , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Multimerização Proteica , Proteômica/métodos , Proteômica/tendências , Propriedades de SuperfícieRESUMO
Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes ß5i or ß1i-ß5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.
Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/metabolismo , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Anticorpos Imobilizados/química , Linhagem Celular , Ensaios Enzimáticos/instrumentação , Desenho de Equipamento , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/metabolismo , Corantes Fluorescentes/química , Humanos , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Peptídeos/química , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Inibidores de Proteassoma/farmacologia , Especificidade por SubstratoRESUMO
Four proteasome subtypes are commonly present in mammalian tissues: standard proteasomes, which contain the standard catalytic subunits ß1, ß2 and ß5; immunoproteasomes containing the immuno-subunits ß1i, ß2i and ß5i; and two intermediate proteasomes, containing a mix of standard and immuno-subunits. Recent studies revealed the expression of two tissue-specific proteasome subtypes in cortical thymic epithelial cells and in testes: thymoproteasomes and spermatoproteasomes. In this review, we describe the mechanisms that enable the ATP- and ubiquitin-dependent as well as the ATP- and ubiquitin-independent degradation of proteins by the proteasome. We focus on understanding the role of the different proteasome subtypes in maintaining protein homeostasis in normal physiological conditions through the ATP- and ubiquitin-dependent degradation of proteins. Additionally, we discuss the role of each proteasome subtype in the ATP- and ubiquitin-independent degradation of disordered proteins. We also discuss the role of the proteasome in the generation of peptides presented by MHC class I molecules and the implication of having different proteasome subtypes for the peptide repertoire presented at the cell surface. Finally, we discuss the role of the immunoproteasome in immune cells and its modulation as a potential therapy for autoimmune diseases.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Trifosfato de Adenosina , Animais , Citoplasma/metabolismo , Antígenos de Histocompatibilidade Classe I , Mamíferos/metabolismo , Peptídeos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (ß1-ß2-ß5), the immunoproteasome (ß1i-ß2i-ß5i) and the two intermediate proteasomes (ß1-ß2-ß5i and ß1i-ß2-ß5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three ß5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, ß5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ß5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HEK293 , Humanos , Oxirredução , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
CD8+ cytolytic T lymphocytes are essential players of anti-tumor immune responses. On tumors, they recognize peptides of about 8-to-10 amino acids that generally result from the degradation of cellular proteins by the proteasome. Until a decade ago, these peptides were thought to solely correspond to linear fragments of proteins that were liberated after the hydrolysis of the peptide bonds located at their extremities. However, several examples of peptides containing two fragments originally distant in the protein sequence challenged this concept and demonstrated that proteasome could also splice peptides together by creating a new peptide bond between two distant fragments. Unexpectedly, peptide splicing emerges as an essential way to increase the peptide repertoire diversity as these spliced peptides were shown to represent up to 25% of the peptides presented on a cell by MHC class I. Here, we review the different steps that led to the discovery of peptide splicing by the proteasome as well as the lightening offered by the recent progresses of mass spectrometry and bioinformatics in the analysis of the spliced peptide repertoire.
Assuntos
Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/genética , Splicing de RNA/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Splicing de RNA/imunologiaRESUMO
Cancer immunotherapy has recently emerged as a forefront strategy to fight cancer. Key players in antitumor responses are CD8+ cytolytic T lymphocytes (CTLs) that can detect tumor cells that carry antigens, in other words, small peptides bound to surface major histocompatibility complex (MHC) class I molecules. The success and safety of cancer immunotherapy strategies depends on the nature of the antigens recognized by the targeted T cells, their strict tumor specificity, and whether tumors and antigen-presenting cells can efficiently process the peptide. We review here the nature of the tumor antigens and their potential for the development of immunotherapeutic strategies. We also discuss the importance of proteasome in the production of these peptides in the context of immunotherapy and therapeutic cancer vaccines.
Assuntos
Neoplasias/imunologia , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Humanos , Imunoterapia , Neoplasias/terapia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
We recently developed a new assay to measure proteasome activity in vitro (CAPA for capture proteasome assay) [1], based on proteasome capture on an antibody-coated plate. When used with lysates originating from cells expressing either standard proteasome, immunoproteasome or intermediate proteasomes ß5i or ß1i-ß5i, this assay allows the individual monitoring of the chymotrypsin-like, trypsin-like and caspase-like activities of the corresponding proteasome subtypes. The efficiency and specificity of four proteasome inhibitors were studied using the CAPA assay, demonstrating the potential of this assay for the development of subtype-specific proteasome inhibitors.