The role of Sel1L-Hrd1 ER Associated Degradation in B cell development and function.
The Endoplasmic Reticulum (ER) serves as the site for protein synthesis, folding, maturation and modification in the cell. In mammalian cells, approximately one-third of the newly synthesized proteins enter the ER in an unfolded state. Despite various folding machineries in the ER, protein folding is inherently error prone and often results in the generation of irreparable misfolded proteins which disrupts ER homeostasis. ER-associated degradation (ERAD) is a principle quality-control mechanism responsible for restoring ER homeostasis by targeting misfolded proteins in the ER for cytosolic proteasomal degradation. The best characterized ERAD machinery in mammals is the highly conserved Sel1L-Hrd1 complex, consisting of E3 ubiquitin ligase Hrd1 and its adaptor protein Sel1L. Although studies using tissue specific knockout mice, have provided insights into the regulatory role of ERAD in highly secretory cell types such as adipocytes and intestinal epithelium, the physiological importance of Sel1L-Hrd1 ERAD in cell differentiation and development has not been explored to date. To this end, we chose to investigate B cell development as a model system, as it requires tightly orchestrated expression of many growth factors and cell surface-associated proteins for different stages of development. We generated B cell specific Sel1L knockout mice driven by CD19-Cre (Sel1LCD19). Here, we report that B cell development, which requires a sequential progression of differentiation in the bone marrow, was blocked at the large pre-B cell stage in Sel1LCD19 mice. We show that ERAD manages an early checkpoint in B cell development by selectively targeting the pre-BCR complex for proteasomal degradation, hence terminating pre-BCR signaling. In the absence of Sel1L-Hrd1 ERAD, pre-BCR accumulates at the cell surface, leading to persistent pre-BCR signaling and cell cycling. Strikingly, our study shows that pre-BCR complex, but not BCR complex, as an endogenous protein substrate of ERAD in B cells. To further understand the physiological importance of ERAD in B cell function, we examined the mucosal immunity of Sel1LCD19 mice. While IgG/IgM secretion in Sel1LCD19 mice is comparable to the wild type mice, the IgA secretion level was significantly reduced. Surprisingly, Sel1LCD19 mice exhibited exacerbated inflammatory response to dextran sodium sulfate (DSS)-induced colitis. Interestingly, this colitogenic phenotype was transmissible only between littermates bred from Sel1LCD19 dam. Collectively, the works described in this thesis provide novel insight into the physiological importance of ERAD in B cell development. Further studies based on this dissertation may provide important mechanistic insight into protein complex substrate selection by ERAD.
Immunology; B cell development; Colitis; ERAD; IgA; PreBCR; Sel1L-Hrd1
August, Avery; Appleton, Judith Ann
PHD of Immunology&Infectious Disease
Doctor of Philosophy
dissertation or thesis