Protein post-translational modification by poly(ADP-ribose) (PAR) is implicated in a growing number of biological regulatory events associated with DNA damage responses, chromatin remodeling, and transcriptional regulation. Hyperproduction of free PAR polymers is linked to cellular commitment to apoptosis and is suggested to play a role in human diseases in which poly(ADP-ribose) polymerase-1 (PARP-1) activity is increased, including neurodegeneration, cardiovascular disease, and stroke. Binding of free PAR polymers to mitochondrial apoptosis-inducing factor (AIF) releases AIF from the mitochondrial inner membrane to effect large-scale degradation of nuclear DNA and cell death, a pathway termed "parthanatos." It is unclear how PAR binding enables apoptotic cleavage of AIF's mitochondrial transmembrane anchor to allow escape from mitochondria. My preliminary data indicate that PAR binding to AIF causes exposure of a putative calpain-I recruitment (PEST) motif, and I propose that PAR binding promotes AIF release by increasing association with calpain-1 protease. I will investigate this mechanism for PAR-stimulated release of AIF from mitochondria by (1) characterizing structural remodeling of AIF by PAR polymers and (2) evaluating the relevance of AIF's PEST motif in stimulating AIF proteolysis by calpain-1.
Aim 1 will establish the affinity and specificity of interaction between AIF and PAR chains, delineate the binding interface between AIF and PAR ligands using x-ray crystallography, and characterize macromolecular remodeling of AIF in the presence of PAR using small- angle x-ray scattering (SAXS).
Aim 2 will determine whether the PEST motif participates in calpain-1 cleavage of AIF by measuring cleavage rates of PEST mutants using a time-resolved FRET assay. AIF mutants defective in PAR binding or calpain-I processing will be tested in Aim 3 for their ability to undergo mitochondrial release and nuclear translocation in damaged cells or to exit isolated mitochondria upon exposure to PAR chains. Outcomes from this study will inform the development of small molecule tools for probing disease states associated with PAR hyperproduction and provide critical insight into how other caspase-independent death pathways potentially regulate AIF release.
Hyperproduction of poly(ADP-ribose) (PAR) chains is phenomenologically linked to mitochondrial release of apoptosis-inducing factor (AIF) and subsequent cell death in neurodegenerative disorders, cardiovascular disease, and brain injury. This study will establish the molecular mechanism by which PAR ligands regulate AIF release from mitochondria. Knowledge of this mechanism will enable development of small molecules to probe aberrant activation of this pathway and ultimately provide therapeutic intervention in human diseases involving these mechanisms.
|Brosey, Chris A; Ho, Chris; Long, Winnie Z et al. (2016) Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor. Structure 24:2067-2079|