ATP Synthase Regulation

Exploring Regulatory Pathways of ATP Synthase

Background

 * Myocardial ischemia and acidosis are two common examples in which cells exhibit positive feedback responses to depolarization during respiratory inhibition. This protects cells from ischemic cell death by preserving ATP under anoxic conditions.


 * It is highly conserved among aerobic species and cells just like its cognate subunits in F1 ATPase, and has many interesting properties:
 * IF1 inhibits ATPase at pH 6.7 to preserve ATP in the cell at the expense of the membrane potential
 * In experiments when respiration is inhibited by CN-, protons link into the membrane and lower the pH
 * proton-pumping ATPase responds by sustaining a new steady state potential when IF1 is suppressed
 * ATP hydrolysis is downregulated when IF1 is overexpressed, which collapses the membrane potential while preserving intracellular ATP levels


 * The relative F1 β-subunit:IF1 expression levels correspond to a cellular responses to respiratory inhibition, which is determined by a cell’s metabolic properties. For example the relative ratios between neurons and astrocytes are consistent with respectively greater glycolytic and oxidative functions.
 * the ratio of IF1: β in different tissues may have various functional roles that can be explored
 * The differences are characterized by their responses to CN- treatment:
 * In astrocytes, potential drops and is maintained at a new steady state, but completely collapses in Neurons.
 * IF1 expression promotes ATPase dimerization and increased density of mitochondrial cristae
 * dimerization is also concomitant with cristae formation
 * binds 1:2 to β-subunits
 * Some suggest that dimerization improves efficiency by increasing proton flux
 *  IF1’s role in resting cell physiology are relatively unexplored 

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 * ''' The fact that overexpression can influence ATP consumption or maintenance of membrane potential means that there is “room for increased functional interaction between recombinant IF1 and ATPase”

Methods

 * Explore the resting state function of IF1 by modulating it's inhibitory activity (up and down regulation) in a way that allows for direct measurements of IF1 activity and resting state potential of the membranes.


 * Bason, J. V.; Runswick, M. J.; Fearnley, I. M.; Walker, J. E. Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase J. Mol. Biol. 2011, 406, 443-453.
 * The most recent profile of IF1 inhibitory domains


 * Cabezon, E.; Butler, P. J.; Runswick, M. J.; Walker, J. E. Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH J. Biol. Chem. 2000, 275, 25460-25464.
 * A model for pH-sensitive modulation of inhibitor activity
 * We will modify the dimerization potential of IF1 proteins by aptamer selection or mutagenesis, to obtain a library of Inhibitors with varying capacity for homo-dimerization as a function of pH.


 * Once we've characterized the pH responses of a representative group of inhibitors, we will measure the redox potential of NADH at multiple pH levels, which we can map to various degrees of ATPase activity.
 * From this data, we can determine the resting redox state of the cell in the absence of inhibition, and observe the change in membrane potential in response IF1 activation during respiratory inhibition.


 * Additionally, we may search for other targets of IF1 in cells during normal metabolic function.

More Background
ATP Synthase Specs:

(11) Muench, S. P.; Trinick, J.; Harrison, M. A. Structural divergence of the rotary ATPases Q. Rev. Biophys. 2011, 1-46.

(12) Nakamoto, R. K.; Baylis Scanlon, J. A.; Al-Shawi, M. K. The rotary mechanism of the ATP synthase Arch. Biochem. Biophys. 2008, 476, 43-50.

(13) Oster, G.; Wang, H. Reverse engineering a protein: the mechanochemistry of ATP synthase. Biochim. Biophys. Acta 2000, 1458, 482-510.

SELEX approach:

(14) Ellington, A. D.; Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346, 818-822.

(15)Niles, J. C.; Marletta, M. A. Utilizing RNA aptamers to probe a physiologically important heme-regulated cellular network. ACS Chem. Biol. 2006, 1, 515-524.