Heat-induced fibrillation of BclXL apoptotic repressor

Abstract

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease.

Keywords: 1,1′,2,2′-Tetraoleoyl cardiolipin (1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol); 1,2-Dihexanoyl-sn-glycero-3-phosphocholine; 20-mer peptide derived from the BH3 domain of Bid; 8-Anilinonaphthalene-1-sulfonate; ALS; ANS; Amyloid fibrils; Analytical light scattering; B-cell lymphoma 2; B-cell lymphoma extra large; BH1; BH2; BH3; BH3-interacting domain (death agonist); BH4; Bad; Bak; Bax; Bcl2; Bcl2 (homologous) antagonist/killer; Bcl2 homology 1 (domain); Bcl2 homology 2 (domain); Bcl2 homology 3 (domain); Bcl2 homology 4 (domain); Bcl2-associated X (protein); Bcl2-associated death (promoter); BclXL; BclXL homodimer in which the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa in a domain-swapped trans-fashion; BclXL_transTM; Bid; Bid_BH3; CD; Circular dichroism; DHPC; DLS; Dynamic light scattering; FM; Fluorescence microscopy; ITC; Irreversible aggregation; Isothermal titration calorimetry; Kinetic trap; LIC; Ligation-independent cloning; MD; MM; MOM; Membrane insertion; Molecular dynamics; Molecular modeling; Myr; SEC; SLS; SSF; Size-exclusion chromatography; Static light scattering; Steady-state fluorescence; TMAO; TOCL; ThT; Thioflavin T; mitochondrial outer membrane; myricetin; trimethylamine N-oxide; α–β structural transition.