Abstract
Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves the sequestration of cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositolâ3âkinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiledâcoil domain (CCD). However, the last 16 CCD residues, composing an âoverlap helixâ (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the Câterminal βâÎą repeated, autophagyâspecific domain (BARAD). Here, using circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and smallâangle Xâray scattering (SAXS), we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the twoâdomain CCDâBARAD. We also used cellular assays demonstrating that mutation of these OHâinterface residues abrogates starvationâinduced upâregulation of autophagy, but does not affect basal autophagy. Lastly, we have obtained crystals of the CCDâBARAD that we are optimizing for Xâray diffraction in order to obtain an atomicâresolution, threeâdimensional structure of the homodimer. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains, i.e. the CCD or the BARAD. Our findings have important implications for the relative stability of autophagyâinactive and autophagyâactive BECN1 complexes.Support or Funding InformationThis work was supported by NIH grants RO3 NS090939 and R15 GM122035 (S.S.) and R15 GM113227 (C.C.); a National Science Foundation grant MCBâ1413525 (S.S.); a North Dakota State University Graduate School doctoral dissertation award for K.G.; and an ND EPSCoR doctoral dissertation award for K.G. Work performed at BioâCAT was supported by NIH NIGMS 9P41 GM103622 and use of the Pilatus 3 1M detector funded by NIH NIGMS 1S10OD018090â01. This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEâAC02â06CH11357. The authors also acknowledge the NDSU Core Biology Facility (funded by NIH grant P30 GM103332â01) for access to tissue culture facilities and the NDSU Advanced Imaging and Microscopy Core Laboratory for access to microscopy and imaging equipment. We also thank Dr. Pawel Borowicz for assistance with imaging and puncta quantification method development.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have