Rolle von Beclin-1 für die Autophagie-Induktion in B-Lymphozyten
Zusammenfassung der Projektergebnisse
Autophagy is a degradation process during which long-lived proteins and organelles are sequestered by double-membraned vesicles and delivered to lysosomes. The bona fide cytoprotective process of autophagy has been implicated in both the causation and prevention of certain pathologies, including cancer, neurodegeneration, and infections. B lymphocytes are a central component of adaptive immunity. The main effector function of B lymphocytes which are terminally differentiated is the production of soluble immunoglobulins. Autophagic processes contribute to central aspects of B cell physiology, e. g. development and antigen presentation. The Vps34/PI3K class III complex is central for the induction of autophagy. In mammals, the PI3K class III core complex at least consists of the catalytic subunit Vps34, the adaptor Vps15 (p150), and Beclin 1 (Atg6). Beclin 1 forms the scaffold for the recruitment of additional activators or repressors of the PI3K class III complex. In order to analyze the role of Beclin 1 for autophagy induction in B lymphocytes, Beclin 1-deficient chicken DT40 B lymphocytes were generated by targeted disruption of the two beclin 1 alleles. The established clones revealed a reduced proliferation rate as determined by phase contrast live cell imaging and MTT assay. Beclin 1-/- DT40 cells showed a high background cell death during cultivation and were also sensitized for B cell antigen receptor (BCR)-induced apoptosis. However, it appears that beclin 1-/- DT40 cells can still induce autophagy following thapsigargin treatment as determined by LC3 immunoblotting. The existence of a Beclin 1-independent autophagy pathway has to be confirmed and alternative pro-autophagic stimuli will be assessed. This cell line represents a valuable tool for the analysis of the crosstalk between apoptosis and autophagy, since the association between Bcl-2 and Beclin 1 could be verified by coimmunopurification experiments. Future studies will aim at the exact regulation of the Bcl- 2/Beclin 1 interaction, e.g. by post-translational modifications. The polarization between apoptosis and autophagy is of unique interest in B lymphocytes, since it has been previously reported that autophagy is specifically induced by apoptotic BCR signaling. Interestingly, beclin 1-/- DT40 cells are impaired in their general BCR signaling capacity, as observed by reduced tyrosine phosphorylation of central adaptor proteins such as SLP-65 and Dok-3, respectively. Based on this research project, our prospective work will aim at the mechanistic details of the signaling network regulating apoptosis and autophagy in B lymphocytes. Ultimately, our studies of these pathways will hopefully contribute to the understanding of B cell-dependent adaptive immunity, including B cell development, antigen presentation, and tolerance induction.
