Abstract
Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.
Highlights
Ubiquitylation has been linked to all cellular processes and defects in ubiquitylation pathways can contribute to disease pathogenesis
We recently demonstrated that ASB2α transcripts are expressed in mouse primary immature DCs generated ex vivo, the so-called bone marrow-derived DCs (BMDCs), and down-regulated following BMDC activation[9]
MRNAs are expressed in mouse conventional DCs isolated from lymphoid or non-lymphoid tissues[21,22] and because mRNAs of the alpha isoform of ASB2 were expressed in mouse immature bone marrow- and spleen-derived DCs9, we evaluated the expression of ASB2α mRNAs in DCs purified from mouse spleens
Summary
Ubiquitylation has been linked to all cellular processes and defects in ubiquitylation pathways can contribute to disease pathogenesis. This had successfully lead to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit[12] In this context, we have previously developed a strategy for the identification of substrates of E3s that are targeted to proteasome degradation using cell lines induced to express an active or a defective ubiquitin ligase mutant of an E313. ASB2α E3 activity drives proteasome-mediated degradation of FLNs and can regulate integrin-dependent functions such as cell spreading, cell adhesion and initiation of cell migration of several cell lines[13,15,16,17,18]. We proposed and validated a label free quantitative proteomic approach to identify E3 substrates to be degraded in physiological relevant settings
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