Dataset Information

MARCH1 ubiquitination of MHC II generates a branched K63/K11 ubiquitin chain.

ABSTRACT: Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination, with poly-ubiquitination (Ub) of MHC II directing it away from the plasma membrane. The MHC II poly-Ub chain that dictates the fate and function of ubiquitinated MHC II has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary dendritic cells (DCs) and B cells. Analysis was conducted for endogenous H2-A and H2-E molecules immunoprecipitated from primary cells. We show that the ubiquitination of both I-A and I-E expressed by DCs and B cells is dependent on the E3 Ub ligase MARCH1. Comprehensive Ub chain analysis reveals MARCH1-dependent poly-Ub associated with MHC II is composed of Ub chains with branched K11/K63 linkages.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): B Cell, Dendritic Cell

SUBMITTER: Laura Dagley

LAB HEAD: Prof. Justine D. Mintern

PROVIDER: PXD063057 | Pride | 2025-12-01

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	P3248_standard1_1_01_8288.d.zip	Other
	P3248_standard2_1_01_8287.d.zip	Other
	P3248_standard3_1_01_8286.d.zip	Other
	P3248_standard4_1_01_8285.d.zip	Other
	P3248_standard5_1_01_8284.d.zip	Other

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Publications

Major histocompatibility class II in murine antigen presenting cells is modified with a branched K63 and K11-linked ubiquitin chain.

Liu Haiyin H Francis Lauren L Dagley Laura F LF Cobbold Simon A SA Webb Andrew I AI Komander David D Villadangos Jose A JA Mintern Justine D JD

Scientific reports 20251125 1

Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination. Poly-ubiquitination (Ub) of MHC II directs it away from the plasma membrane and is a critical determinant of MHC II turnover. The MHC II Ub chain has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary murine antigen presenting cells; conventional dendritic cells (cDCs) and B cells. Analysis was co ...[more]

PMID: 41290815

Similar Datasets

Project description:Purpose: Production of broad-spectrum antibodies against T cell-dependent antigens requires marginal zone (MZ) B cells to present antigenic peptides complexed to MHC II. Here we describe the trogocytic acquisition of these complexes from dendritic cells (DCs). Complement C3 (C3) binds to MHC II on murine and human DCs. Recognition of C3 by MZ B cell complement receptor 2 triggers trogocytosis of DC plasma membrane and its receptors, which become exposed on the MZ B cells. Excessive trogocytosis is deleterious but prevented by membrane ubiquitin ligase MARCH1, which limits the number of MHC II–C3 complexes displayed on the DC surface. MHC II molecules are thus receptors for constitutively activated and potentially harmful C3, an activity that enables acquisition of DC-like functions by trogocytic MZ B cells. Methods: mRNA profiles of splenic cDC1s, B cells and CD11cintCD24+CD8int cells of wild type (WT) and March1-/- mice mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. All 100-base-pair single-end reads were mapped to the reference mouse genome (GRCm38/mm10) using STAR aligner, and gene-wise counts were obtained using Subread package. Differential expression analysis of RNA sequencingdata was carried out using the edgeR and limma-voom packages in Bioconductor. Results: Using an optimized data analysis workflow, we mapped about 14 million sequence reads per sample to the mouse genome (build mm10) and identified 16,182 genes in the different cell types from WT and March1−/− mice.Transcriptomic profiling revealed over 6500 genes differentially expressed between cDC1s and B cells. Principle component analyses revealed very few differences between WT and March1−/− cDC1s, and between WT and March1−/− B cells. Notably, the transcriptome of CD11cintCD24+CD8int cells was similar to those of WT or March1−/− B cells, indicating they belonged to the B cell lineage. Conclusions: In our study we have described that MHC II ubiquitination by MARCH1 plays two important roles: first, to enhance the removal of surface MHC II–C3 complexes on all APCs; and second, to limit MZ B cell trogocytosis and elimination of cDCs. It is conceivable these two functions, more than the regulation of MHC II antigen presentation, have been the major drivers for the conservation of MHC II ubiquitination through evolution.

Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.