The Roles And Mechanisms Of Endoplasmic Reticulum-resident E3 Ubiquitin Ligase Hrd1 In The Development And Activation Of B Cells

Hrd1, also called synoviolin, belongs to E3 ubiquitin ligase. It is situated in the ER along with Sel1, Derlin1 and Herp and other proteins as part of an ERAD complex. There are a hydrophilic six-transmembrane domain in its N-terminal, a RING-H2 domain and a proline-rich domain in C-terminal located in cytoplasm. While RING-H2 domain is the catalytic site of Hrd1 as a E3 ubquitin ligase, proline-rich domain may serve as the one that mediate protein-protein interaction through recruiting proteins to be ubiquitinated in cytoplasm.Early studies mainly found that Hrd1 is implicated in ER-associated protein degradation pathway by recognizing, ubiquitinating and retrotranslocating target proteins. This pathway is important for the maintenance of ER homeostasis and protein quality control by clearing the misfolded proteins promptly. But later studies found Hrd1 can do more than just ubiquitinate misfolded proteins and mediate their degradation: It can control many biologic processes in various cells through ubiquitinating non-misfolded proteins. For examples, in DCs it can prompt the antigen presentation function of DCs by targeting Blimp1 and in neurons it can prevent neuron degradation by ubiquitinating amyloid precursor protein. It has been reported that, in cell lines Hrd1 is able to ubiquitylate unassembled Igμ and non-glycoslated variant of the Igκ light chain which implies Hrd1 could targets these two molecules in physiological setting to regulate B cell development and immunity. In the present study, we adopted the B-cell specific deletion of Hrd1 mouse model to extensively investigate the function of Hrd1 in the development and differentiation of B cells.Aims: a) To analyze the effect of Hrd1 on B cell development and immunity systematically and comprehensively and its physiological/pathological meaning; b) To explore the underlying molecular mechanisms.Methods:To analyze composition, apoptosis and proliferation of B cell subsets in Hrd1-/- and WT mice; to stimulate BCR, CD40 co-stimulator or Toll-Like Receptor(TLR4) to activate mature B cells in vitro and to immunize mice with NP-KLH and NP-LPS to induce T cell dependent and T cell independent response, respectively in vivo, then analyze activation,apoptosis,proliferation and plasma cell differentiation of activated B cells by FACS and to detect the antibodies tiltreation by ELISA.; to investigate the underlying mechanisms using molecular and biological methods.Results: After analyzing B cell subsets at different stages of B cell development in bone marrow and spleen, we found compared to wild-type littermates, in bone marrow Hrd1-/- large pre-B cells increased significally while small pre-B and recirculating mature B cells decreased; in spleen total B cells decreased. By activating mature B cells in vitro or vivo, we found Hrd1-/- mature B cells had more apoptosis compared to those in wild type. Further study revealed that once Hrd1 is deleted, pre-BCR and Fas was accumulated in pro/pre B cells and activated mature B cells, respectively. Subsequent study showed that pre-BCR and Fas can be ubiquitinated by Hrd1 to enter into degradation pathway, so Hrd1 deficiency leads to their accumulation in cells.Significance: Our study demonstrates that Hrd1 controls two distinct checkpoints in B cell development and immunity by ubiquitinating pre-BCR complex and Fas, respectively. pre-BCR downregulation is required for the development of pre-B and subsequent B cell.It has been reported pre-BCR signal is able to downregulate pre-BCR by negative feedback method. However, other mechanisms for the regulation of pre-BCR are rarely reported. Here, for the first time, we come up with that Hrd1 is able to mediate pre-BCR down-regulation at posttranslational level through ubiquitination. Fas can adjust immune response and clear autoactive B through activation-induced cell death(AICD). Until now, even though there have been a lot of literatures reporting the regulation of Fas at transcriptional level, regulation after this level remains largely unknown. Here, we found Hrd1 can regulate Fas at posttranslational level in activated B cells.