Since the rate at which a cell grows depends on a net balance between protein synthesis and protein breakdown, the mechanisms and control of both processes are of great biomedical importance. Over the years, much has been learned about the protein synthetic process but the notion still remains that the degradation of proteins occurs primarily in lysosomes by acid hydrolases. However, recent evidence indicates that a nonlysosomal degradative process, one which requires ATP, may catalyze the rate-limiting step in the breakdown of both abnormal and short- lived normal proteins in mammalian tissue. A cytoplasmic ATP- dependent protease, which we first isolated from reticulocytes and more recently from skeletal muscle and liver, is involved in the degradation of proteins conjugated to ubiquitin and may function to prevent the accumulation of such proteins in these cells. The structure and mechanism of this novel class of enzymes has not been investigated. To help clarify the role of the ATP-dependent degradative pathway in protein breakdown and propose to 1) purify the ATP- dependent proteases from muscle and identify their physiological substrates and inhibitors. 2) The purified enzymes will be used to produce polyclonal antibodies for an ELISA assay to monitor levels of the enzyme during periods of rapid muscle loss (e.g. denervation, starvation, disuse, infection, and cancer). 3) Antibodies to ubiquitin will identify which subcellular compartments and components of the cell contain ubiquitin- protein conjugates. 4) Characterization of the purified ATP- dependent proteases will help identify specific inhibitors to this proteolytic pathway and will also provide a tool or investigating the mechanism of action of these proteases. 5) By in vitro physiological techniques, we hope to clarify the relative importance of the ATP-dependent proteases and the other proteolytic system in muscle with inhibitors to these pathways. We will test whether rates of protein breakdown correlate with levels of a ATP-dependent proteases and the extent of ubiquitination. 6) mRNAs which code for the ATP-dependent proteases will be isolated and used to study the biosynthesis and the regulation of these proteases at the genetic level. cDNA clones will be isolated using synthetic oligonucleotides as well as antibodies to obtain hybridization probes. It will then be possible to measure whether changes in enzyme levels under different physiological conditions are due to regulation of the steady-state mRNA levels or to changes in gene copy.
| Tiao, G; Fagan, J; Roegner, V et al. (1996) Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest 97:339-48 |
| Tiao, G; Fagan, J M; Samuels, N et al. (1994) Sepsis stimulates nonlysosomal, energy-dependent proteolysis and increases ubiquitin mRNA levels in rat skeletal muscle. J Clin Invest 94:2255-64 |
