Protein Degradation in Health and Disease (Progress in Molecular and Subcellular Biology)
Moreover, liver cells of patients with alcoholic liver disease exhibit an accumulation of specific undegraded ubiquitin—protein conjugates called cytokeratin filaments. These proteins form microscopic structures, Mallory bodies, whose presence indicates that proteasome function is suppressed. The formation of neuronal bodies also is related to reduced proteasome function.
When these abnormal molecules are attached to proteins, they cannot be removed as easily as normal ubiquitin, thereby slowing down the degradation of the attached protein see figure 4. Thus, the mechanisms underlying this novel molecular change remain to be determined.
Intracellular Proteolytic Systems
The left panel shows the normal ubiquitin—proteasome pathway, in which the cytokeratin protein is modified by the addition of several ubiquitin molecules and then degraded in the proteasome. Modified from McPhaul et al. For example, proteasome activity declines by up to 43 percent in certain animal models in which the animals are continuously fed alcohol, achieving blood alcohol levels that are two to three times the legal intoxicating level in humans Donohue et al. Although these alcohol levels exceed the alcohol concentration of 0.
Thus, the alcohol levels used with experimental animals, while high, are in line with those recorded in humans. Similarly, researchers have found that when cultured liver cells that metabolize alcohol are exposed to comparably high alcohol concentrations, proteasome activity declines Osna et al. Lower alcohol levels i.
The alcohol—related decrease in proteasome activity appears to be linked to the activities of two enzymes involved in alcohol metabolism—alcohol dehydrogenase and cytochrome P 2E1 CYP2E1. Both of these enzymes convert alcohol into acetaldehyde, a toxic and reactive substance. In addition, CYP2E1 generates highly reactive oxygen species that can inactivate proteins and contribute to liver damage for more information on reactive oxygen species and their effects, see the article by Wu and Cederbaum in this issue. Chronic alcohol consumption can elevate the levels of i.
Under these conditions, when alcohol concentrations in the blood and liver reach a certain level, the alcohol can interact with CYP2E1, causing the enzyme to become resistant to degradation by the proteasome. These elevated CYP2E1 levels can lead to excessive generation of reactive oxygen species, which in turn can inactivate the proteasome Bardag—Gorce et al. The resulting suppression of proteasome activity can result in reduced cell viability through various mechanisms described in the following sections also see Donohue Excess production of reactive oxygen species and reactive molecules is one of the factors contributing to a harmful cellular state called oxidative stress.
Alcohol consumption additionally contributes to oxidative stress by depleting the levels of the intracellular molecule glutathione, which acts as an antioxidant—that is, it neutralizes many of the radicals generated by CYP2E1. Therefore, alcohol contributes to oxidative stress through several mechanisms, including increased production of oxygen radicals and reduced antioxidant levels, thereby exacerbating proteasome dysfunction.
Alcohol can cause a specific form of cell death called apoptosis. The proteasome plays a critical role in regulating apoptosis and ensuring cell survival by degrading proteins that can induce apoptosis. Accordingly, suppression of proteasome function with specific inhibitors can cause cell death.
Alcohol—induced proteasome dysfunction also may contribute to apoptosis because proteasomes normally degrade certain proteins in the mitochondrial membrane that promote apoptosis i. Therefore, one can speculate that if alcohol suppresses proteasome function, these pro—apoptotic factors could accumulate in the mitochondria and enhance liver cell apoptosis.
Long—term alcohol consumption can cause inflammation of the liver tissue and liver cell death, leading eventually to liver injury i. Under conditions of oxidative stress, however, a series of reactions occur that lead to the ubiquitylation of this inhibitor protein. As described previously, the oxidative stress leading to this process can result from alcohol consumption. On the other hand, oxidative stress can inhibit proteasome function, as mentioned in the previous section. Researchers do not yet know exactly how these two different effects of alcohol on the proteasome system can be explained; however, it appears that after long—term i.
Excessive alcohol consumption can impair proteolysis mediated by the ubiquitin—proteasome system through several mechanisms. First, alcohol partially inactivates the proteasomes, presumably as a result of oxidative stress—related inactivation of the enzymes. This alcohol—induced impairment of proteasome function may have profound ramifications for cell viability. For example, inhibition of proteasome activity can result in the accumulation of modified, potentially toxic proteins in cells and can cause tissue inflammation as well as premature cell death by apoptosis.
The third major proteolytic system affected by alcohol consists of a family of intracellular proteinases called calpains, which require calcium for their activities. Several molecular forms of the calpains exist, but the two major ones are calpain I and calpain II. The various calpains can be distinguished by the amounts of calcium required for their activities—calpain I needs less calcium for its activity than calpain II. The calpains are believed to be involved in several physiological processes, including the maturation and processing of certain enzymes and the breakdown of proteins associated with the cytoskeleton—a complex array of proteins that gives cells their shapes and enables them to contract and divide.
Because calpains are responsible for the proteolysis of cytoskeletal proteins, investigators have suggested that calpain activity is involved in modulating cell structure in both normal and pathological states. For example, the calpains may have a major role in nerve cell development. Effects of Alcohol Consumption on the Calpains. Studies of how alcohol consumption affects the calpains have largely been restricted to the brain. Like the liver, the brain is significantly affected by excessive alcohol consumption, which ultimately can result in alcohol—related nerve cell degeneration.
Recent studies examining the effects of alcohol consumption on calpain activity in the brain demonstrated that the activity of these enzymes is elevated in the brains of alcohol—fed animals compared with untreated animals Rajgopal and Vemuri Furthermore, the brains of alcohol—fed animals had higher levels of the degradation products of a cytoskeletal protein called spectrin, which is degraded by calpains.
Researchers believe that the activation of calpain in this instance results from an alcohol—induced increase in calcium concentrations within the nerve cells Rajgopal and Vemuri Thus, in contrast to the lysosomal and ubiquitin—proteasome systems, which are inhibited by chronic alcohol consumption, the activity of the calpain system is enhanced by chronic alcohol consumption.
Excessive or untimely protein degradation, however, can be just as harmful to the organism as reduced protein degradation. This article has described the effects of alcohol consumption on the functions of three major proteolytic pathways that regulate the quantity and the types of proteins inside cells. Through a variety of mechanisms, alcohol significantly influences each of these proteolytic pathways, interfering with their normal functions.
All of these changes, however, can lead to the same end results: Although researchers have learned much about alcohol and its effects on proteolytic systems, just as many issues remain to be explored. For example, the identity of the molecules that inhibit lysosome assembly and cause proteasome inhibition has not yet been determined. Finally, researchers should investigate whether these proteolytic systems can be employed as therapeutic targets. For example, the proteasome, which is inhibited by a large number of agents, also can be activated by several small molecules, including some naturally occurring lipids Dahlmann et al.
Consequently, the administration of these compounds to alcohol—treated animals or cells may conceivably reactivate partially inactivated proteasomes and thus restore normal protein degradation after alcohol exposure. The effect of ethanol—induced cytochrome PE1 on the inhibition of proteasome activity by alcohol.
Biochemical and Biophysical Research Communications Annual Review of Biophysics and Biomolecular Structure When lysosomes get old. Experiments in Gerontology In vitro activation of the 20S proteasome. The proteasome, a novel protease regulated by multiple mechanisms.
Peptides that are released by the proteasome only exist in the cell for seconds, because they are quickly digested into amino acids by the abundant cytosolic endopeptidases and aminopeptidases. The amino acids can be reutilized to synthesize new proteins or metabolized, yielding energy 48 , Although the proteasome principally catalyzes the complete hydrolysis of cell proteins, in a few cases, the 26S proteasome degrades proteins only partially, yielding a biologically active fragment. In its central chamber, the proteasome contains six proteolytic sites: Two cleave preferentially after hydrophobic amino acids, two cleave preferentially after basic residues, and two cleave after acidic ones 39 , This exception seems to be important in the pathogenesis of certain neurodegenerative diseases e.
Apparently, the glutamines are poorly degraded and accumulate as toxic, intracellular inclusions. The active sites in the proteasome cleave peptide bonds by a unique mechanism; peptide bonds are cleaved by the hydroxyl group on a critical threonine residue Because the proteolytic mechanism is novel, highly specific inhibitors of the active sites have been synthesized or microorganisms have produced them One such synthetic inhibitor, Bortezomib Velcade, PS has emerged as an important new anticancer drug.
Bortezomib has been approved by the US Food and Drug Administration and is widely used for the treatment of multiple myeloma; clinical trials against various other cancers are under active investigation 47 , However, inhibition of the proteasome was found to induce apoptosis, especially in neoplastic cells and transplanted tumors With the backing of the National Cancer Institute, these agents went into human trials against various cancers, and the special sensitivity of myeloma cells became evident in phase I trials Surprising, these agent have therapeutic efficacy, even when protein degradation by the proteasome in cancer cells is only partially compromised.
In addition, malignant plasma cells produce exceptionally large amounts of abnormal Ig that are degraded by the ER-associated degradation—proteasome pathway see section titled The Proteasome and Immune Surveillance. However, promising responses have been obtained in patients with other hematologic malignancies, and Bortezomib in combination with other chemotherapeutic agents is being tested against other malignancies in clinical trials. Surprising, proteasome inhibitors also have benefits in animal models of strokes.
The compounds seem to reduce postischemia adhesion of cells and reperfusion injury. Human trials of the compounds in treatment of strokes soon will be undertaken. Notably, these applications initially were not recognized and probably could not have been predicted. In short, the development of proteasome inhibitors that exhibit several biologic properties emphasizes the enormous benefits that are emerging from basic biochemical research.
Besides essential roles in regulating cell growth and metabolism and in the elimination of misfolded proteins, the UPP serves a critical role as an information-gathering mechanism for the immune system 2 , 47 , These peptides enable circulating lymphocytes to screen for foreign proteins in the extracellular and intracellular environments. These lymphocytes are activated to produce antibodies Figure 4. The generation and presentation of antigenic peptides.
Intracellular proteins are degraded by the UPP and imported into the endoplasmic reticulum, where they are processed and lead to binding to MHC class I receptors. CD8-positive T cells respond to the antigen. Extracellular proteins that are endocytosed and degraded to peptides in lysosomes are processed to bind to MHC class II receptors.
CD4-positive T cells respond to these antigens. The continual breakdown of intracellular proteins as may arise during viral or bacterial infection or with cancer allows the immune system to screen for non-native proteins within cells 2 , 53 , Although the great majority of peptides that are generated by the proteasome during breakdown of intracellular proteins are digested rapidly to free amino acids 49 , 55 , some escape this fate and are transported into the ER. When non-native epitopes e. This critical role of the proteasome in antigen presentation was demonstrated first through the use of proteasome inhibitors These inhibitors proved invaluable in the elucidation of this immunologic process 2 , To bind to most MHC class I molecules, peptides have to be eight to nine residues long.
The products that are used as antigenic precursors not only are the eight- to nine-residue peptides but also the longer ones with additional amino acids on their amino termini. They are taken up into the ER by a specific peptide transport system, called transporter associated with antigen processing TAP. Within the ER is a newly discovered aminopeptidase, endoplasmic reticulum aminopeptidase 1 ERAP1 , which has the unusual capacity to trim extra amino acids off the longer precursors and then stops at eight to nine residues, the precise length for binding to MHC molecules 53 , 57 , In inflammatory conditions, the immune cells e.
The immunoproteasomes differ because they contain three novel peptidase subunits in place of the proteases that normally are present.
Protein Degradation in Health and Disease
These specialized subunits exhibit different specificities that enable them to cleave proteins differently so that more of the products have the correct features for processing to peptides that are capable of binding to MHC class I molecules 54 , It binds to one end of the 20S proteasome and forms a hybrid 26S particle that has a 19S complex at the other end These hybrid particles degrade ubiquitinated proteins at normal rates but cleave them differently, generating an even higher fraction of peptides that are capable of serving as antigenic precursors.
Together, these changes stimulate the host defense. However, a number of viruses have evolved sophisticated mechanisms to escape immune detection by inhibiting the uptake of proteasome products into the ER by the TAP transporter or by promoting the degradation of MHC class I molecules This competition between immune defenses and viruses demonstrates that the immune systems in higher vertebrates have evolved in terms of modifications of the UPP that otherwise is highly conserved in organisms from yeast to human.
These alterations in proteasome structure and the changes in the proteolytic pathway allow efficient antigen presentation and, therefore, the basal recognition of infected or neoplastic cells and their rapid elimination under a variety of conditions. The maintenance of tissue mass and body protein stores in normal adults occurs when cells achieve a balance between the rates of synthesis and degradation of cell proteins.
In uremic patients, protein stores frequently are depressed: Serum albumin is low, and there is weight loss largely as a result of loss of muscle mass 61 , In experimental animals and humans with uremia, overall rates of protein synthesis generally are unchanged, whereas rates of protein degradation tend to increase 65 , Because the rates of protein turnover in cells are very high 3.
Most of this acceleration of protein degradation in muscle in disease states occurs via a programmed activation of the UPP 66 , Recent studies in rodent models have established that accelerated muscle protein catabolism that is induced by uremia involves similar cellular mechanisms that cause muscle wasting in a variety of other catabolic conditions, such as cancer cachexia, starvation, insulin deficiency, or sepsis 1.
Atrophying muscles from such animals show accelerated proteolysis via the UPP, higher levels of mRNA that encode certain components of this proteolytic system, and a similar pattern of changes both increases or decreases in the expression of approximately atrophy-related genes, also termed atrogenes 35 Table 1. In humans with these conditions, there also is evidence for activation of the UPP in muscle e. In these catabolic states, the increase in mRNA levels for these atrophy-related genes in muscle occurs through increased gene transcription 35 , 66 , 71 , As part of this common transcriptional program, the expression of various growth-related genes decreases in the atrophying tissues Therefore, multiple transcriptional factors seem to change coordinately to bring about the loss of muscle mass.
The strongest evidence for activation of the UPP muscles of animals that undergo atrophy as a result of uremia or other catabolic diseases is that when studied in vitro , muscles from these models exhibit increased proteolysis that can be blocked by inhibitors of the proteasome 66 , 71 , Biochemical adaptations found in atrophying skeletal muscle a. For these reasons, muscle wasting represents a specific, carefully orchestrated response that is triggered by various stimuli in various pathologic conditions e.
In fasting and presumably in other disease states, an identifiable function of accelerated proteolysis is to mobilize amino acids from dispensable muscle proteins to provide the organism with precursors for hepatic gluconeogenesis or for new protein synthesis 1. However, these losses can have deleterious effects with time, especially in renal failure, in which disposal of nitrogenous waste is compromised, and they accumulate to cause symptoms and metabolic problems. Because the UPP serves many essential functions in cell regulation and homeostasis, its activation in these states must be highly selective and precisely regulated to avoid the unwanted removal of muscle proteins that are essential for cell function in muscle and other organs.
Whereas atrophy seems to affect all muscle cell components, contractile proteins are lost differentially. The cellular content of E3s varies among tissues and physiologic states. Two Ub ligases, atrogin-1 also known as MAFbx and MuRF-1, are specific constituents of muscle; their expression increases dramatically eight- to fold in catabolic states, and they play a critical role in mediating the loss of muscle protein Figure 5.
In mice that lack the genes for atrogin-1 or MuRF-1, muscles grow normally, but if they are denervated, then the rate of muscle atrophy is reduced Muscles of these knockout mice also show reduced atrophy upon fasting. In wild-type animals that are subjected to muscle denervation or disuse, expression of atrogin-1 and MuRF-1 rises quickly just when muscle atrophy is most rapid, and in cultured muscle cells, the content of atrogin-1 mRNA correlates tightly with rates of protein breakdown 75 — The balance between protein synthesis and degradation determines whether muscles hypertrophy or atrophy.
This allows FoxO to stimulate the expression of the E3 enzymes atrogin-1 and muscle ring finger-1 MuRF-1 and protein degradation. Studies in cultured skeletal muscle cells and in transgenic mice that overexpress atrogin-1 in heart muscle have suggested that myoD and calcineurin A might be substrates for ubiquitination by atrogin-1 38 , It is unlikely that degradation of myoD by atrogin-1 will explain the mechanism of muscle atrophy, because myoD is critical for the differentiation of muscle cells during development, whereas atrogin-1 is expressed in fully differentiated muscle only during wasting.
It is not known whether calcineurin A is a target of atrogin-1 in skeletal muscle, but in terms of maintaining muscle mass, calcineurin signaling pathways seem to affect fiber type more than fiber size MuRF-1 was found to interact with glucocorticoid modulatory element binding protein-1, a glucocorticoid-modulated nuclear protein that regulates transcriptional responses It is intriguing that glucocorticoids are potent activators of muscle protein breakdown and that MuRF-1 expression is increased in the presence of glucocorticoids.
MuRF-1 has been found to associate with and conjugate Ub to troponin I in cardiac muscle; it is not known whether MuRF-1 serves a similar function in skeletal muscle Because muscle atrophy that is caused by uremia and other catabolic diseases requires activation of UPP generally and atrogin-1 and MuRF-1 specifically 35 , 82 , efforts are being pursued to reduce the activities of the Ub proteasome pathway or to prevent the induction or activation of these E3s as means of slowing muscle wasting.
It is not known how the evidence of inflammation that frequently is present in patients with kidney disease affects muscle protein degradation Myofibrillar proteins, which comprise approximately two thirds of the protein in muscle, require additional mechanisms for their degradation in addition to the UPP.
Other steps are necessary because the UPP readily degrades the main components of the myofibril actin, myosin, troponin, or tropomyosin. These same proteins are digested only very slowly by the UPP when they are present as complexes or in intact myofibrils Recent findings suggest that these proteins initially are cleaved by cytosolic proteases, which promote myofibrillar disassembly to facilitate degradation of constituent proteins by the proteasome. Because another protease must breakdown the complex structure of muscle to provide substrates for the UPP, we tested whether caspases may play this critical initial role.
We found that caspase-3 cleaves actomyosin in vitro and in cultured muscle cells to produce and yields substrates that are degraded rapidly by the UPP as well as a kD C-terminal fragment of actin that accumulates in the insoluble fraction of the cell The same processes occur in the muscle of animals with uremia, diabetes, and angiotensin II—induced hypertension, and in humans, a similar kD actin fragment is found in muscle of patients with muscle atrophy as a result of disuse from the pain of osteodystrophy and related to either hemodialysis treatment or burn injury 92 — Another family of proteases, the calpains, also has been suggested as a mechanism that catalyzes the initial cleavage of myofibrillar proteins in atrophy.
Calpains are calcium-dependent cysteine proteases that have been suggested to play roles in several disorders, especially muscular dystrophy. Although inhibition of the calcium-activated proteases in muscles from rodents with uremia or several other types of atrophy has not been found to block the increase in protein degradation or the degradation of myofibrillar proteins or the accumulation of the kD actin fragment in muscle cells 92 , the transgenic expression of calpastatin, an endogenous calpain inhibitor, can reduce denervation atrophy and progression of muscular dystrophy in mice Chronic kidney disease is associated with several physiologic complications that can trigger the UPP to breakdown muscle protein, metabolic acidosis, decreased insulin action, increased glucocorticoids, high levels of angiotensin II, and inflammation 66 , 71 , 93 , These complications seem to be related to and function in concert to bring about protein loss.
- Nestbautrieb: Die schaurig-schönste Zeit des Lebens (German Edition).
- Riding for the Brand: Stories!
- Michele Reboud-Ravaux's Protein Degradation in Health and Disease (Progress in PDF.
- Die Positiven (German Edition).
- Mrs. Skaggs Husbands and Other Stories;
- Protein Degradation by the Ubiquitin–Proteasome Pathway in Normal and Disease States.
For example, metabolic acidosis stimulates muscle protein breakdown by the UPP, but it also increases glucocorticoid production and causes insulin resistance in muscle. Either of these clinical problems can activate the UPP and the breakdown of muscle protein 72 , 90 , 98 — Glucocorticoids also suppress insulin sensitivity, and insulin deficiency or insulin resistance increases production of these hormones 72 , , Finally, the sensitivity of muscle to catabolic signals, such as glucocorticoids, rises with inactivity.
Angiotensin II infusion into rodents causes both anorexia and muscle protein losses by mechanisms that depend on glucocorticoids Last, inflammatory mediators suppress insulin responses, and various conditions that are associated with inflammation e. As noted, activation of the UPP in various types of muscle wasting and the coordinated changes in the expression of a set of genes in muscle suggest that these catabolic states activate a common cellular signaling pathway IGF-1 is released by the liver and mediates the anabolic effects of the growth hormone, but it also is an autocrine factor that is released by muscle after exercise.
Rapid atrophy, however, also requires enhanced proteolysis and induction of the E3 atrogin-1 and MuRF-1, leading somehow to enhanced protein degradation in muscle cells 75 , 76 , These recent results show that activation of protein degradation and induction of these E3 enzymes in atrophying muscles also result from a decrease in activated Akt i. One of the targets of activated Akt is the forkhead family of the transcription factors FoxO1, 3, and 4 ; when they are not phosphorylated, they migrate into the nucleus and catalyze the transcription of atrogin-1 75 , 76 , This pathway seems to catalyze the degradation of particular cellular components, especially mitochondria.
The influence of Akt on the other critical E3 Ub ligase, MuRF-1, is somewhat less clear, but decreased phosphorylation of Akt was found to increase MuRF-1 and atrogin-1 transcription after glucocorticoid treatment or denervation In the various conditions that are associated with muscle atrophy, both transcription factors seem to contribute to muscle wasting, but their relative importance remains to be resolved.
This would decrease the breakdown of the complex structure of muscle proteins. In insulin-deficient rats that exhibit accelerated muscle protein degradation, we found that the proapoptotic factor Bax is activated, leading to the release of cytochrome C from mitochondria, which in turn activates caspase-3 and increases production of the kD actin fragment Similar changes were seen in cultured muscle cells with genetic or pharmacologic inhibition of PI3-K activity. Together, these recent developments provide evidence that the activation of muscle protein loss in kidney disease and other catabolic conditions occurs through a common signaling pathway that alters transcription of key enzymes that modulate protein synthesis and degradation in complementary ways to cause muscle wasting.
One of the key endocrine factors that are essential for these catabolic responses is glucocorticoids.
These hormones also exert permissive effects that activate the UPP when other catabolic signals are present. For example, activation of muscle proteolysis does not occur in adrenalectomized animals that are starved or treated with NH 4 Cl to induce metabolic acidosis or made insulin deficient unless the animals are given a physiologic dose of glucocorticoids 72 , , Similarly, the increase in muscle proteolysis that is induced by angiotensin II or sepsis can be blocked by inhibiting the glucocorticoid receptor It is important to note that in these experiments, the same physiologic levels of cortisol did not stimulate muscle protein degradation unless the animals also were acidotic or made insulin deficient; normal animals require higher, pharmacologic doses to cause muscle wasting.
When glucose is needed, an increase in glucocorticoids mobilizes amino acids from muscle protein. At the same time, these hormones induce gluconeogenic enzymes in liver that catalyze conversion of the amino acids to glucose and urea. In this brief review of the UPP, we have discussed how the complex series of biochemical reactions within this pathway act to tag and degrade proteins. Because the UPP is responsible for the turnover of so many different cellular proteins, there are critical mechanisms that regulate its function precisely. We have emphasized the importance of the UPP in the turnover of transport proteins, in presenting antigens to the immune system, and how uremia activates the UPP to cause muscle wasting because these functions are of special interest to nephrology.
However, the UPP also plays important roles in the regulation of other cellular functions, ranging from the control of the cell cycle to activities that promote cancer. Indeed, inhibitors of proteasome activity, the final component of the pathway, have emerged as novel chemotherapeutic agents. Involvement in such a wide range of functions explains why the Nobel Prize in Chemistry was awarded for the discovery of Ub and its role in orchestrating cellular protein turnover. Published online ahead of print. Publication date available at www. Skip to main content. You have access Restricted Access.
Lecker , Alfred L. Goldberg and William E. Ubiquitin Proteasome Pathway During the past two decades, the UPP has taken center stage in our understanding of the control of protein turnover Figure 1. Rapid Removal of Proteins Unlike most regulatory mechanisms, protein degradation is inherently irreversible. Regulation of Gene Transcription Ub conjugation affects transcription by multiple mechanisms