Role of ubiquitination in the regulation of intracellular MHC class I traffic and in cross-presentation – MARCHxIRAP
The goal of the MHC I antigen processing pathway is to load MHC I molecules with thousands of different peptides for display on the cell surface. This goal is accomplished by merging two distinct pathways: one for generating peptide-receptive MHC I molecules in the endoplasmic reticulum and another for generating antigenic peptides in the cytoplasm. Initial antigen degradation by cytosolic proteasome requires additional N-terminal peptide trimming, mediated by ER resident aminopeptidases. To obtain efficient CD8+ effector T cells responses, however, naïve CTLs require priming by dendritic cells (DCs). In the case of pathogens that do not infect DCs, or for molecular determinants not expressed by these cells, an alternative presentation pathway called “cross-presentation” is required for internalized antigen-bearing cells and circulating antigens to gain access to the processing and loading MHC I machinery. Although much attention has been given to the encounter of the MHC I loading machinery with the internalized antigens, little information is available on the regulation of MHC I transport to endocytic compartments and on the antigenic peptides processing associated with such compartments.
A novel family of E3 ubiquitin ligases, termed MARCH (for membrane-associated RING-CH proteins) and their role in the regulation of MHC molecules in human DCs. Team 1 has discovered the role of MARCH 9 on the control of an uncharacterized MHC I access routes to a subtype of syntaxin 6-positive endosomes. Team 2 has demonstrated that in DCs, the insulin-regulated aminopeptidase (IRAP) is localized to an endosomal compartment, which contains both Rab 14 and syntaxin 6. Interestingly IRAP interacts with MHC class I molecules and was found, by team 2, to be necessary for cross-presentation. The two teams propose to associate their competence to address the spatio-temporal coordination of cross-presentation and understand how DCs synchronize MHC I presentation and pathogen sensing.
Cross-presentation strongly depends on endosomal organelle dynamic and its regulation is directly linked to intracellular traffic plasticity in response to pathogen detection. Little information is available on the regulation of MHC I transport to endocytic compartments and on the antigenic peptides processing associated with such compartments. We postulate that the previously unsuspected ubiquitination of MHC I molecules and the likely involvement of MARCH 9 in this process is key for MHC I and IRAP traffic through a specialized subset of endocytic. Our work focus on the biochemical, cell biological and immunological consequences of these processes mostly in human cells and is organized in five main tasks. 1) Project coordination. 2) The ubiquitination of MHC I and IRAP and its regulation by MARCH IX. 3) The impact of MARCH IX on the transport, localization and function of MHC I and IRAP. 4) The physiological relevance of ubiquitination and MARCH IX for cross-presentation. 5) Role of IRAP and MARCH IX in cross-presentation of antigens internalized through receptors versus phagocytosis In these different tasks, we address specifically the following questions: 1) Is MHC I molecule ubiquitination regulated in DCs? 2) Which residues are ubiquitinated and how does this affect MHC I transport? 3) What is the function of MARCH IX in this process? 4) Is IRAP ubiquitinated and does MHC I ubiquitination affects MHC I/IRAP interactions? 5) Is the transport of IRAP affected by MHC I and MARCH IX? 6) What is the nature of the STX6 positive compartment containing MHC I and IRAP? 7) Is the transport of MHC I and IRAP different in DCs sub-types? 8) Is cross-presentation dependent on MHC I ubiquitination and MARCH IX? We focus mostly on human cells (MoDCs), which are more amenable to manipulation (e.g. using RNAi) and we investigate the role of MARCH IX in knock-out mice which are available to the coordinator’s team.
We are planning to carry-on the project as planned, with an emphasis on the physiological consequences of MARCH9 deletion on cross-presentation using different antigen presentation assays both with mouse and human T cells.
Revues à comité de lecture 1. TLR-dependent regulation of MHC transport is orchestrated by MARCH E3 ligase-dependent ubiquitination in antigen presenting cells. Gatti E. Molecular Immunology (2012) 51, 1.
2. De Gassart A., De Angelis-Rigotti F., Pierre P. and Gatti E. Molecular Biology of the Cell (2012) 22S
Ouvrages ou chapitres d’ouvrage 1. MHC ubiquitination. De Gassart A., De Angelis Rigotti F., and Gatti E. “Methods in Antigen Processing and Presentation approaches” in Methods in Molecular Biology (2012) in press.
Communications (conférence) 1. Workshop on immunobiology of dendritic cells Cargèse, France - October 19- 22, 2011 - Ubiquitination and MARCH E3 ubiquitin ligases regulate the intracellular trafic of antigen presenting MHC molécules. Oral presentation.
2. Institut Curie, Paris, France - May 4th, 2011. Antigen présentation and ubiquitination. Invited talk
3.
Immune surveillance by CD8+ T cells is one of the key mechanisms for detecting and eliminating abnormal cells, including those infected with viruses or bacteria and tumor cells. CD8+ T cells probe the repertoire of peptide/MHC class I complexes on the target cell surface for novel peptides that indicate expression of foreign or abnormal gene products. The essence of the MHC I antigen processing pathway is to load MHC I molecules with thousands of different peptides for display on the cell surface. This goal is accomplished by merging two distinct pathways: one for generating peptide-receptive MHC I molecules in the endoplasmic reticulum (ER) and another for generating antigenic peptides in the cytoplasm. The two pathways merge when cytoplasmic peptides are translocated into the ER by the transporter associated with antigen processing (TAP) and made available to peptide-receptive MHC I molecules. Initial antigen degradation by cytosolic proteasome complexes frequently requires additional N-terminal peptide trimming, mediated by ER resident aminopeptidases (ERAPs). To obtain efficient CD8+ effector T cells responses, however, naïve CTLs require priming by dendritic cells (DCs). In the case of pathogens that do not infect DCs, or for molecular determinants not expressed by these cells, an alternative presentation pathway called “cross-presentation” is required for internalized antigen-bearing cells and circulating antigens to gain access to the processing and loading MHC I machinery. Cross-presentation strongly depends on endosomal organelle dynamic and its regulation is directly linked to intracellular traffic plasticity in response to pathogen detection. Although much attention has been given to the encounter of the MHC I loading machinery with the internalized antigens, little information is available on the regulation of MHC I transport to endocytic compartments and on the antigenic peptides processing associated with such compartments.
Team 1 has recently characterized a novel family of ten E3 enzymes, termed MARCH (for membrane-associated RING-CH proteins) and their role in the regulation of MHC molecules in human DCs. They have discovered the role of the MARCH IX ligase on the control of a previously uncharacterized MHC I access routes to the cell surface, which transit through a subtype of syntaxin 6-positive endosomes. Team 2 has demonstrated that in DCs, the insulin-regulated aminopeptidase (IRAP) is localized to an endosomal storage compartment, which in DCs may qualify as a specialized cross-presentation compartment containing both Rab 14 and syntaxin 6. Interestingly IRAP interacts with MHC class I molecules and was found, by team 2, to be necessary for cross-presentation. The two teams propose to associate their competence and non-overlapping expertise to address the spatio-temporal coordination of cross-presentation and understand how DCs synchronize MHC I repertoire presentation and pathogen sensing. The role of ubiquitination and MARCH ligases in regulating the encounter of MHC I molecules and IRAP to allow exogenous peptide exchange and final trimming will be at the center of the project. A combination of cell biology and immunological approaches will be developed to decipher this novel step of MHC I-restricted antigen presentation in DCs. They will attempt to demonstrate the existence of a MARCH IX-dependent cross-presentation pathway, which could control the access of neosynthesized MHC I complexes and IRAP to a type of subtype of endosomes, which will be further characterized.
Project coordination
Evelina GATTI (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA) – gatti@ciml.univ-mrs.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V
Help of the ANR 380,000 euros
Beginning and duration of the scientific project:
- 36 Months
