Publication Date

2013-05-24

Availability

Open access

Embargo Period

2013-05-24

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2013-05-07

First Committee Member

Amjad Farooq

Second Committee Member

Thomas K. Harris

Third Committee Member

Sapna Deo

Abstract

The BclXL apoptotic repressor, a member of the B-cell lymphoma 2 family of proteins, plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a wide array of biophysical methods, I investigate the molecular basis of action of BclXL. Briefly, I provide evidence that BclXL bears intrinsic propensity to oligomerize in solution. Importantly, such oligomerization of BclXL is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans fashion, whereby the TM domain of one monomer occupies the hydrophobic groove within the other monomer and vice versa. Of particular interest is the observation that acidic pH promotes the assembly of BclXL into a higher-order megadalton aggregate with a plume-like appearance and harboring structural features characteristic of a molten globule. Moreover, BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils at elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely alpha-helical fold into a predominantly beta-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL aggregates in solution display diminished affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL aggregates likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, my study sheds light on the propensity of BclXL to aggregate in solution, particularly under acidic conditions and at elevated temperatures—the physical factors that mimic cellular stress—thus bearing important consequences on its mechanism of action in gauging the apoptotic fate of cells in human health and disease.

Keywords

BclXL; apoptosis; oligomerization; molten globule; amyloid fibrils; stress

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