A Soluble ATP-Dependent Proteolytic System Is Responsible for Protein DegradationMethodist Research Institute, Clarian Health Partners and Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana Correspondence: Gary Zaloga, MD, Medical Director, Methodist Research Institute, 1812 N. Capitol Ave, Wile Hall, Room 120, Indianapolis, IN 26202. Electronic mail may be sent to gzaloga{at}clarian.org. Cachexia is defined as progressive wasting of body tissues, primarily muscle and adipose tissue. Cachexia is a major cause of morbidity and mortality in patients with a large variety of diseases that include cancer, infection, heart failure, chronic obstructive pulmonary disease, acquired immune deficiency syndrome (AIDS), arthritis, and surgery. In many forms of chronic illness, cachexia accounts for much of the increased morbidity and mortality. In addition to effects upon mortality and disease complications, cachexia is commonly associated with decreased quality of life that includes weakness, fatigue, and lack of interest in life-associated activities. Anorexia frequently accompanies cachexia and can contribute significantly to tissue loss (ie, anorexia-cachexia syndromes). We prefer to use the term cachexia to refer to tissue loss that occurs despite adequate nutrition intake so as to separate the true cachexia syndromes from those that result from anorexia alone. Cachexia that results from anorexia alone responds well to nutrition therapies. On the other hand, most forms of cachexia result from the underlying disease and respond poorly to current nutrition therapies.
The molecular etiologies of cachexia are complex and not well understood.
In muscle tissue, there is an imbalance between protein synthesis and
degradation that results in progressive tissue loss. Protein synthesis is
depressed, uptake of amino acids inhibited, and protein degradation increased.
The increase in protein degradation is linked to increased activity of the
ubiquitin-proteasome pathway. Various circulating factors (ie, tumor necrosis
factor-
Loss of adipose tissue results from an imbalance between lipid synthesis
and degradation. Lipoprotein lipase is the major enzyme responsible for
synthesis of lipids from triglycerides, whereas lipid hydrolysis is controlled
by hormone-sensitive lipase. Most cytokines inhibit lipoprotein lipase. On the
other hand, lipid hydrolysis is stimulated by tumor necrosis factor- Proteins within cells are continuously degraded to peptides and amino acids. This degradation is responsible for recycling nitrogen in the form of amino acids for synthesis of new proteins, curtailing the activity of proteins, and removing abnormal and potentially harmful proteins. Current research indicates that 3 major enzymatic systems are responsible for most cellular protein degradation: the ubiquitin-proteasome system, calcium-activated neutral proteases, and lysosomal enzymes. The lysosomal enzyme system primarily degrades extracellular proteins that are endocytosed by the cell. Degradation occurs within the lysosomes. Cytoplasmic degradation of intracellular proteins occurs via the proteasome and neutral protease enzymatic systems, with the proteasome being responsible for over 80% of protein degradation. These enzyme systems are activated by cytokines and other circulating factors. Current nutrition formulas fail to inhibit this activation. Thus continued proteolysis despite continued nutrient intake can result in progressive tissue loss and cachexia. In a landmark article, Etlinger and Goldberg1 (Figure 1) first reported the discovery of a soluble adenosine triphosphate (ATP)-dependent proteolytic enzyme system in 1977. Subsequently, Ciechanover et al2 reported a heat-stable polypeptide essential for the activity of the ATP-dependent proteolytic system in reticulocytes and called it ubiquitin. Today, we recognize this proteolytic system as the ubiquitin-proteasome proteolytic enzyme complex.3–6 Before the Etlinger and Goldberg1 publication, despite recognition of the physiologic importance of protein degradation, the responsible enzymes and degradation pathways were unknown. At the time, most researchers assumed that the lysosome was responsible for intracellular protein degradation.
In the early 1970s, it was known that (a) cell-free preparations usually failed to demonstrate the characteristics of intracellular proteolysis and (b) that inhibitors of energy metabolism inhibited protein degradation. Thus, protein degradation appeared to be an energy-dependent process. Some assumed that ATP was required to transport substrate into lysosomes. However, energy was also required to degrade protein in bacteria that lacked lysosomes, suggesting that the energy was not required for the lysosome.
In an attempt to develop a cell-free system that degrades protein and requires energy, Etlinger and Goldberg1 studied rapid degradation of abnormal proteins in reticulocytes. These preparations could be easily obtained, and the preparations synthesized primarily 1 protein, hemoglobin. In addition, the structures of normal and variant hemoglobins were known, allowing the researchers to study features of the protein that affects degradation. It was also known that incorporation of valine and lysine analogs into polypeptides or premature termination of polypeptides with puromycin in reticulocytes led to their rapid degradation. Etlinger and Goldberg obtained reticulocytes from rabbits and pulse labeled the proteins with 3H-leucine or 14C-leucine. Protein synthesis was also carried out in the presence of a valine or lysine analog or puromycin (to produce abnormal proteins). Cells were washed to remove extracellular radioactivity, and cell-free extracts were prepared from these cells. Protein breakdown was determined by the amount of acid-soluble radioactivity relative to that initially found in protein (acid-precipitable radioactivity). In reticulocytes, incorporation of the lysine and valine analogs produced abnormal proteins that were rapidly degraded. Inhibition of ATP production by incubating the cells without glucose or with 2,4-dinitrophenol (which prevents oxidative phosphorylation) markedly reduced proteolysis. However, the rate of degradation was not affected by inhibition of new protein synthesis with cycloheximide. The investigators next used cell-free extracts of reticulocytes. A highly active degradation system was found in the soluble dialyzed fraction of reticulocyte lysates when proteolysis was assessed in the presence of ATP. The analog proteins were rapidly hydrolyzed, whereas normal proteins were not. Thus, both the substrate and proteolytic system were found in the cell-free extracts. The cell-free extract resembled the intact cell because normal protein degradation was slow, whereas abnormal protein degradation (using amino acid analogs) was fast. ATP consistently stimulated the degradation of abnormal proteins. On the other hand, adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) failed to stimulate protein degradation, whereas adenosine diphosphate (ADP) had a minor effect. These data indicated that high-energy phosphates were required for protein degradation. ATP-dependent degradation was optimal in the neutral and slightly alkaline pH range. Little degradation occurred at acid pH, where most lysosomal enzymes are active. Thus, a new proteolytic system appeared to be responsible for most protein degradation in reticulocytes. Abnormal proteins (using amino acid analogs or puromycin) were degraded at much higher rates than normal proteins. Cell-free extracts hydrolyzed proteins at similar rates to intact cells. Chloromethylketones and sulfhydryl blocking reagents inhibited degradation in cells and extracts. A zinc chelator also inhibited protein breakdown. These results suggested the involvement of a sulfhydryl-dependent enzyme and metalloenzyme in the degradation process. Thus, the observations of this investigation indicated the presence of a novel proteolytic system that was responsible for the rapid degradation of abnormal proteins in reticulocytes. This novel system required energy, and results were consistent with participation of a sulfhydryl protease and metalloenzyme. This system was clearly different from lysosomal degradation. It was soluble rather than membrane-bound and had a pH optimum of 7.8 (higher than lysosomal enzymes). This system demonstrated specificity for abnormal proteins, which were degraded at much higher rates than normal proteins.
Numerous subsequent studies confirmed the presence of an energy-dependent nonlysosomal protein degradation system in muscle tissue and other tissues. This proteolytic enzyme system was later characterized and named the ubiquitin-proteasome proteolytic complex. For their discovery of ubiquitin-mediated protein degradation, Ciechanover, Hershko, and Rose were awarded the Nobel Prize in Chemistry in 2004.
Today, the ubiquitin-proteasome proteolytic enzyme complex is known to be the primary proteolytic system responsible for the hypercatabolism of acute illness and cachexia of chronic illness.5 Inhibitors to this protein complex are under study for prevention of cachexia.
Nutrition in Clinical Practice, Vol. 21, No. 1,
88-91 (2006)
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, interleukin-6, interleukin-1β, proteolysis inducing
factor) are felt to play etiologic roles in the development of cachexia
through induction of signaling pathways that result in tissue degradation.
Many of these factors activate the ubiquitin-proteasome pathway though
activation of the transcription factor nuclear factor-
β
(NF
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