Krithi C Sundaram: 20.109 Module 3 Assignment Wiki

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Module 3 Assignment: Home

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work in progress

stuff may be in phrases only - if they dont make sense, please ASK ME!!!

background

IF1 - the protein that inhibits the F1 moiety of ATp synthase

WBlot and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F-1 (IF1), a small protein that binds the F-1 moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath.

found likely role of IF1 in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events.

IF1: either overexpressed by transient transfection or knocked down using small interfering RNA (siRNA).


The putative IMV (isolated myelin vesicles) ATP synthase is functional in also in ATP hydrolysis. ATP hydrolysis observed in IMV was progressively decreased with the lowering of pH levels (7; 6.8 and 6.5) and was abolished below pH 6.8.

Any condition that favors the loss of the driving force for ATP synthesis (the electrochemical proton gradient), i.e., absence of oxygen or respiratory substrates or presence of an uncoupler of oxidative phosphorylation produces an inversion of the nanomotor so that the F1 moiety catalyzes the hydrolysis of ATP.

To preserve the cellular ATP pool from the hydrolysis by F1 ATPase, mitochondria contain IF1 dimers that bind the F1 moiety when the cellular pH decreases. This pH-dependent mechanism is consistent with the acidification of cellular pH, due to lactic acid production, when anaerobic glycolysis is the only ATP source. IF1 action is more potent against the Fo-F1 ATP synthase complex than against the soluble F1 domain. Our data are consistent with the presence of IF1 in IMV and with the reports indicating that the binding of IF1 to the Fo-F1 ATP synthase requires an optimal pH of 6.7 and depends on the pH decrease. Immunofluorescence analysis carried out on optical nerve sections co-localized IF1 with MBP, a typical myelin sheath protein.

Several studies suggest that IF1 plays an important role in diseases that involve ATP hydrolysis by the Fo-F1 ATP synthase. For example in myocardial ischemia, a pathology depending on an insufficient supply of oxygen to miocardiocytes. In severe forms of ischemia, blood flow is so impaired that ATP production through oxidative phosphorylation is significantly reduced. ATP production from anaerobic glycolysis can only maintain ionic homeostasis for short periods of time. So, under ischemic conditions, FoF1- ATP synthase becomes a potent ATP hydrolytic enzyme, which would cause as much as 50–80% of ATP hydrolysis in several ischemic events. This indiscriminate ATP dissipation is prevented by the reversible binding of IF1 to the ATPase, which is rapidly reverted when the oxygen levels are restored. It is tempting to presume that the presence of IF1 in myelin sheath serves to block the ATP hydrolysis by Fo-F1 ATP synthase when the oxygen supply decreased, as during cerebral ischemia events. So, in central nervous system ischemic episodes IF1 would operate to avoid ATP depletion in myelin sheath.


When mitochondrial function is compromised and the mitochondrial membrane potential (Dcm) falls below a threshold, the F1Fo-ATP synthase can reverse, hydrolysing ATP to pump protons out of the mitochondrial matrix. Although this activity can deplete ATP and precipitate cell death, it is limited by the mitochondrial protein IF1, an endogenous F1Fo-ATPase inhibitor. IF1, therefore, preserves ATP at the expense of Dcm. Despite a wealth of detailed knowledge on the biochemistry of the interaction of IF1 and the F1Fo-ATPase, little is known about its physiological activity. Emerging research suggests that IF1 has a wider ranging impact on mitochondrial structure and function than previously thought.


Project

project: to study expression of IF1 in myelin

(we can figure out which specific part we're working on later - this is the "overview" of the entire sequence

  • Find the genes responsible for producing IF1 in myelin
  • Overexpression/ gene knockout - see results
  • Simultaneously, we study binding of IF1 to ATP synthase, SELEX-type selection of best binders
  • Characterize the best binders
  • Modify genes/ drugs to cause production of the improved IF1 (better binder w/above characteristics)
  • In vivo trials
  • Clinical trials


references

  • Silvia Ravera et al. Characterization of Myelin Sheath FoF1-ATP Synthase and its Regulation by IF1. Cell Biochem Biophys (2011) 59:63–70
  • Michelangelo Campanella, Nadeene Parker, Choon Hong Tan, Andrew M. Hall and Michael R. Duchen. IF1: setting the pace of the F1Fo-ATP synthase. Trends Biochem Sci 2009.
  • Michelangelo Campanella et al. Regulation of Mitochondrial Structure and Function by the F1Fo-ATPase Inhibitor Protein, IF1. Cell Metabolism 2008