Adenosine Metabolism and Protein Homeostasis

Protein quality control (PQC), or the removal of damaged or misfolded proteins is critical for cell function and viability. Accumulation of misfolded proteins is a feature of heart failure as well as neurodegenerative diseases such as Alzheimers and Parkinsons disease. Adenosine is a well known cardioprotective agent which is produced continuously but increases dramatically under conditions in which energy supply does not meet energy demand. Recently, we identified a novel link between adenosine metabolism and protein quality control. Inside the cell, adenosine is either phosphorylated by adenosine kinase (ADK) or degraded by adenosine deaminase. We have evidence indicating that genetic disruption of ADK in the mouse heart impairs protein quality control. Mice deficient in ADK accumulate undegraded cardiac proteins under basal conditions and exhibit worse hypertrophy and heart failure under stress conditions. Acute pharmacological inhibition of ADK also rapidly alters ubiquitin-like modifications in the heart. Our findings suggest ADK mediates a novel link between energy status (as reported by adenosine) and protein quality control. The goal of this proposal is to define the mechanism(s) through which adenosine and ADK regulate cardiomyocyte protein quality control. Our working hypothesis is that: intracellular adenosine metabolism by ADK regulates ubiquitin-like modification processes important for cardiomyocyte protein catabolism. To test our hypothesis, we will measure changes in ubiquitin-like modifications and the activity of the two main proteolytic pathways (proteasome and autophagy) in wild type and ADK deficient mouse hearts using biochemical assays and GFP reporter mice. We will also assess how pharmacological manipulation of adenosine metabolism impacts PQC and cardiac function under stress conditions. ATP and adenine nucleotides will be measured in wild type and ADK deficient hearts to examine the role of energy status in PQC defects in ADK deficient mice. In cultured cardiomyocytes, we will use fluorescent reporters of proteolytic pathways (autophagy and proteasome), fluorescent ATP/AMP FRET probes, and manipulation of ADK activity to determine how ADK and adenosine influence protein clearance pathways during hypertrophic stress and energetic stress. Because ADK is located in the nucleus, we will genetically manipulate intracellular adenosine and AMP metabolic pathways in different subcellular compartments to determine how compartmentalized adenosine and AMP metabolism influences protein quality control. Previously, most adenosine research focused on the effects of cell membrane adenosine receptor activation. Our findings suggest a novel protective role of adenosine metabolism by ADK inside the cell that appears important for protein quality control. Because intracellular adenosine is increased by ATP turnover, these studies may provide novel insight on cross-talk between ATP utilization and protein quality control. This could have important implications for basic understanding of cell physiology, as well as conditions associated with toxic protein aggregates, such as heart failure and neurodegenerative disease.