Program :    Biomedical Research and Countermeasures	Ground Research
Element :    Physiology
Effect of Unloading on Protein Content in Skeletal Muscle: Regulation of the Ubiquitin-Proteasome System
Principal Investigator:
Alfred L. Goldberg, Ph.D. Department of Cell Biology Building C-Room 415 Harvard Medical School 240 Longwood Avenue Boston, MA 02115-5730	Phone: (617) 432-1855 Email: alfred_goldberg@hms.harvard.edu Fax: (617) 232-0173 Congressional District: MA-8
Co-Investigator(s):
Lecker, Stewart H. Beth Israel Deaconess Medical Center/Harvard Medical School
Monitoring Center: NSBRI	Solicitation: NSBRI
Initial Funding Date: 1997	Expiration: 2000
Students Funded Under Research: 0	Post-Doctoral Associates: 2
Joint Agency Participation:
Task Description:
Prior research had indicated that the muscle wasting seen in rat muscle upon denervation and in various systemic disease states (including diabetes, hyperthyroidism, sepsis, and cancer) is due primarily to enhanced protein breakdown. A variety of biochemical observations in these atrophying muscles had suggested an activation scheme for the proteolytic system that involves ubiquitin and the proteasome. In this process, proteins are tagged for destruction by being linked to a chain of ubiquitin molecules, which marks them for degradation by the 26S proteasome complex. We have now shown, using extracts of rat muscles, that rates of conjugation of cell proteins to ubiquitin are accelerated in atrophying muslces from septic, tumor-bearing, hyperthyroid, and diabetic animals, and similar studies are in progress with muscles atrophying due to hind-limb suspension. Cells contain multiple enzymes for ubiquitin-conjugation, and we have made the surprising finding that the enzymes responsible for most of this activity in normal muscle extracts are those comprising the N-end rule pathway. Moreover, the increased ubiquitination of proteins in atrophying muscles was due mainly to enhanced activity of these enzymes. On the other hand, in endocrine states where muscle protin breakdown is suppressed, this ubiquitination system is reduced. These findings are totally unanticipated, since this ubiquitination system is not of major importance in most cells and had previosly been believed to function only in the degradation of unusual types of abnormal proteins. These observations raise many new biochemical questions that have special relevance to the mechanisms of muscle atrophy. Previous studies clearly show that the slow (type I) myosin heavy chain (MHC) and other contractile protein isoforms are lost as part of the atrophy process that occurs in antigravity muscles of mammals (rodents) in response to spaceflight and/or the ground-based model of hindlimb unloading. However, relatively little is known concerning the regulation of the degradation process at the subcellular/molecular level. The primary objective of this investigation is to use the rodent hindlimb suspension model in order to examine the process of protein degradation of muscle proteins in general, and of type I MHC in particular, during atrophy. Using biochemical approaches, we shall examine the intrinsic level of activity of the ubiquitin-proteasome system, including changes in conjugating enzyme activity including E1, E2, and E3, as well as determine ubiquitin and ubiquitin-protein conjugate levels, and proteolytic activity of the proteasome complex in these muscles.
Muscle cell extracts prepared from hindlimb suspended rats were used to test whether ubiquitin conjugation by the "N-end rule pathway" is also increased in disuse atrophy. One week after hindlimb suspension, there was a 2- to 3-fold increase in the overall rate of Ub conjugation to endogenous proteins in the soleus muscle. This enhanced ubiquitin conjugation was inhibited by a "dominat negative" form of E214k. These findings argue that the disuse atrophy in space personnel involves a similar activation of protein ubiquitination as we have found in many other types of muscle wasting. Further experiments will be necessary to test whether similar effects occur in other muscles and to define the time course of these responses. One of the primary goals of this work has been to understand how hormones are able to retard protein breakdown in muscle. The lack of insulin and of IGF-1, seen in fasting and untreated diabetes, is associated with muscle wasting, which is due mainly to enhanced protein breakdown in skeletal muscle. The anabolic effects of insulin and its homolog, IGF-1, are also of interest because disuse and glucocorticoids are known to make skeletal muscle resstant to insulin. Recent investigations have suggested that this increased proteolysis is due to a general activation of the Ub-proteasome pathway. Accordingly, incubated muscles from streptocotocin-treated rats showed 40-60% greater rates of protein breakdown which could be reduced to control levels upon incubation with the proteasome inhibitor, MG132. To clarify the mechanisms that activate muscle proteolysis and by which insulin may retard protein breakdown, we tested whether this acceleration of protein degradation in diabetes is associated with an increased rate of Ub conjugation. Muscle extracts from streptozotocin-induced diabetic rats showed a 2-fold higher content of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of 125I-Ub to endogenous muscle proteins. The enhanced ubiquitination appeared to specifically involve the Ub-conjugating enzymes, E214k and E3a, which comprise the N-end rule pathway. A model substrate of this pathway, a-lactalbumin, was also ubinquitinated faster in the extracts from diabetic rats. Furthermore, a dominant negative mutant form of E214k inhibited this increase in rates of ubiquitination. Thus, in muscles from insulin- deficient animals, Ub conjugation by the N-end rule pathway is stimulated, as we also found in the muscle wasting seen in disuse, sepsis, cachexia, and hyperthyrodism. These findings support our working hypothesis that excessive proteolysis and muscle atrophy induced by diverse physiological or pathological stimuli occurs by a common cellular mechanism. The two components of the "N-end" rule pathway, E214k and E3a, appeared to be rate-limiting for Ub conjugation. Adding small amounts of either E214k or E3a to normal muscle extracts stimulated rates of Ub conjugation, and the addition of both together had a larger effect on Ub conjugation than either component alone. These findings suggest that small increases in the muscle's content of E214k and E3a enhance Ub conjgation in diabetic rats. To test this idea, we compared mRNA and protein levels of E1, E214k, and E3a in these muscles. Both E214k and E3a mRNA were elevated 2-fold in the muscles from diabetic rats, while mRNA for E1 did not change. However, no signficant increase in the amount of E214k or Ea could be detected by measurement of enzymatic activity or by immunoassay using a polyclonal antibody that we prepared against the N-terminus of this protein (closed and expressed in E. coli). The simplest interpretation of these findgs is that small increases in E214k and E3a lead to accelerated Ub conjugation and protein degradation in muscles of insulin-deficient (or insulin-resistant) animals (although additional adaptations may also contribute to the increased ubiquitination). Most of our studies of muscle wasting have focused on its overall rate of protein degradation in the muscles, but for understanding of regulatory mechanisms, it would be more informative to follow the breakdown of a specific short-lived muscle protein with use and disuse (e.g., hindlimb suspension). We therefore have initiated some studies of the regulation of calmodulin breakdown. Calmodulin appeared to be a good protein to study because its structure is well- characterized, and because it undergoes a marked conformational change upon binding of Ca2+ that enables it to interact with and alter the activity of many cell proteins. Following microinjection of calmodulin into oocytes, it was degraded by the proteasome, as shown by sensitivity to lactacystin. Purified rabbit muscle proteasomes rapidly degrade calmodulin, but addition of Ca2+ at saturating levels (above 100 mM) prevented proteasomal digestion and degradation in cell extracts. Surprisingly, Ca2+ promoted Ub conjugation to this molecule, although it retarded proteasome-mediated proteolysis. Thus in the case of calmdulin, enhancing Ub conjugation does not lead to enhanced degradation. Dr. Susan Hamilton's laboratory (Baylor University) has been studying mutants in calmodulin that alter its Ca2+-binding. She has provided us with these mutants. Preliminary studies indicate a reduced affinity for Ca2+ leads to more rapid breakdown of the calmodulin
Beyond its potential benefit to the space program and for understanding the biology of weightlessness, this research into the control of protein degradation in muscle may have benefit for understanding and potentially treating a variety of human diseases. The problem of muscle atrophy with disuse is certainly not restricted to space personnel. Wasting of body musculature very similar to that occurring in astronauts is seen in most bed-ridden patients, whether due to chronic disease, surgery, or traumatic injury. With most neuromuscular diseases and limb immobilization, muscle wasting also occurs, and also resembles that seen in the microgravity environment of space, and constitutes a major problem. Moreover, our recent research clearly indicates that there is a common cellular mechanism activating protein breakdown in skeletal muscle following disuse and denervation in the same fashion as that which occurs during many other serious systemic diseases, including cancer, sepsis, certain endocrine disorders (untreated diabetes, hyperthyroidism), and AIDS. All of these conditions are associated with slow muscle wasting, which is caused in large part by excessive protein degradation, as shown by studies in experimental animals. A final goal of this research is to find pharmacological agents (specifically, inhibitors of the ubiquitin- proteasome pathway) that may be able to selectively retard this excessive protein breakdown in space personnel. Obviously, such agents, if effective and safe, would also be very useful in treatment or rehabilitation of patients suffering from these other forms of muscle wasting. In addition, muscle atrophy is a major concern to populations of individuals that are either in advanced aging profiles or are leading a relatively inactive life style. While the wasting process is carried out over years and even decades in these individuals, the flight-based and ground-based models of muscle atrophy involving human and animal subjects exposed to unloading or non-weight bearing states are quite rapid. Thus, both ground-based and flight-based research using animals provides a model for studying the mechanisms of the atrophy response as well as a means to establish mechanisms to either prevent or slow the wasting process.

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