GUJHS. 2008 Dec; Vol. 5, No. 2.
Mohammad Ali1, Emma Furino2, Naomi Leland2, Allan Angerio, PhD1
Department of Human Science1, Department of Nursing2
School of Nursing and Health Studies
Proteasomes are complexes in the body essential to the degradation of abnormal proteins, and are thus, naturally tied with several disease processes. A single complex consists of two end-caps and two enzymatic rings, stacked in a barrel-shaped manner to facilitate their form with peptidase functionality. Abnormal proteins in the body, often precursors to disease, are first tagged to be degraded by the protein ubiquitin before they can be recognized by the proteasome cap. Once recognized, the ubiquitin-proteasome system, or UPS, ultimately decomposes the abnormal proteins into chains of amino acids as they move along the internal core. The promotion and utility of UPS plays an essential role in disease processes such as neurodegenerative disorders, cancers, and prion-related illnesses. Neurodegenerative diseases are characterized by clumps of accumulated abnormal proteins that are unable to be broken down by the UPS. Similarly, prion-related diseases employ a mechanism that triggers abnormal aggregation of proteins, impairing the proteolytic activity of the UPS. Within the context of cancers, proteasome promotion and inhibition both play instrumental roles in the clinical prognosis. Potent viruses, such as HPV, have adapted ways to “fool” proteasomes into destroying the body’s own benefactors, tumor suppressor proteins, thereby prevailing over the cell’s proteolytic defenses. The extent of the faculties of the UPS and its roles in treatment and prevention of diseases, specifically entailing those mentioned, remain the foci of contemporary biomedical research.
Introduction: The Proteasome’s Function in the Body
The proteasome is a protein complex that bears the histological significance of numerous protein-related diseases. Among these are neurodegenerative disorders, prion diseases, and cancers. Ideally, the proteasome serves to degrade and break down various forms of aberrant proteins that may be toxic to the body. Paramount to this degradation is the proteasome-ubiquitin pathway.
The proteasome complex consists of four rings, which are stacked into a cylindrical shape with a hollow core. Each ring is composed of seven protein subunits. The outer two rings, or “caps,” are made of alpha-type subunits and function as a selective barrier for substances entering and leaving the core of the proteasome. The inner two rings are made of beta-type subunits and provide the active sites for protein degradation. In human proteasomes, there are six distinct active sites. A typical sequence of protein degradation is that ubiquitin marks the protein, the protein unfolds, enters the proteasome, and interacts with the active sites to break down into oligopeptides, which can then escape the proteasome complex (see Figure 1) (1).
For years, protein degradation was thought to occur only by lysosomes. But a recent discovery has revealed the truth behind protein degradation within a cell: that proteasomes and ubiquitin actually work together in a system to tag and destroy abnormal proteins. The first step in the process requires for there to be an abnormal protein, and proteins may be labeled abnormal for various reasons. There are several reasons a protein may be destined for degradation within the context of everyday metabolic activities inside the cell. The cell may produce proteins that have misfolded or contain errors in the peptide sequence, and it is beneficial and necessary to the health of the cell that these proteins be degraded. Some of them do not fold properly, like in insulin. Some proteins may be incomplete strands due to an error in RNA splicing. Some of this could be caused by transcription and translation errors, but the exact reasons are still unknown.
One thing that is known is that in general, the cell is a very inhospitable environment for proteins, giving way for a multitude of errors. For example, the temperature of the cell is of 37°C or higher, which can easily denature a protein (2). Denatured proteins may have a hydrophobic area, which marks them to be broken down. Proteins that are less stable, for example, proteins with an exposed amino acid sequence that is unusually prone to hydrolysis, are more likely to be tagged for degradation (2). Some proteins, such as cyclins, commonly contain a destruction sequence in order for them to be sent to the UPS (1). There are many other molecules in the cell that a protein may attempt to react with, causing oxidation, deamination, glycation, or nitrosylation. There are other enzymes within cells, such as proteases or kinases, which may modify the protein. The salt concentration and fatty acid concentration may also denature or dissociate the proteins (2). With all of these possibilities for error, it is clear how a protein may be altered very quickly following its synthesis. Up to 80% of the proteins in a cell may be abnormal and they must be destroyed before they can cause harm to the cell (3).
In disease processes, the ubiquitin tagging mechanism malfunctions, and defective proteins are not degraded by the proteasome. If ubiquitin receptor proteins do not first unfold and refold defective proteins, the proteins are tagged with ubiquitin and sent to the proteasome for degradation. The ubiquitin-protein ligase is often defective during diseases processes, and the proteasome has no indication that the proteins need to be degraded. This can lead to an accumulation of defective protein aggregates, which raise the toxicity of the cell and may lead to rapid cell death (4). In some cases, one of these insoluble aggregates will block the proteasome from performing its function, which also leads to a build-up of defective proteins (4). In a third situation, aging proteasomes may simply slow down in sporadic diseases, which also produces a build-up of protein aggregates (5).
Ubiquitin is a small protein that recognizes these abnormal proteins and “tags” them in a process called ubiquitination. There are three major steps to ubiquitination, which is carried out by E1, E2, and E3 structures (4). In the first step, the ubiquitin must be activated. An E1 ubiquitin-activating enzyme requires energy in the form of ATP. This enzyme creates a thioester linkage between the ubiquitin’s C terminal carboxyl group and the cysteine sulfhydryl group of E1. The second step is transfer. The ubiquitin must be transferred from E1 to E2, a ubiquitin-conjugating enzyme. It attaches to the active site in a reaction called transthioesterification. In the final step, hundreds of E3 enzymes (ubiquitin-protein ligases) recognize the amino acid sequences and detect abnormalities (4). The E3 can also recognize a protein than did not fold properly or is damaged in some way. This would make the protein a target. Ubiquitin molecules then attach and make polyubiquitin chains on the protein. This chain is the “tag” that targets the protein for destruction (see Figure 2).
Following polyubiquitination, the ubiquitous-protein complex is subject to degradation by the proteasome. To accomplish this catabolic task, however, the proteasome must be able to recognize the polyubiquitin chain of at least four ubiquitin monomers attached to the defective protein (6). It is hypothesized that the alpha 19S caps of the 26S human proteasome recognize ubiquitin-like and ubiquitin-associated domains on ubiquitin receptor proteins. The ubiquitin-like domains carry an amine-terminus, which is a free amine group attached to the terminal amino acid in a polypeptide chain. This allows them to be recognized by the 19S proteasome caps, since “the E3 ubiquitin ligase E3ρα/Ubr1 [are the molecules] responsible for targeting [amine-terminus] substrates” (7). The ubiquitin-associated domains, on the other hand, bind ubiquitin by triple helix bundles (8).
Once attached to the 19S alpha subunit, the polyubiquintinated protein must undergo de-ubiquitination and become unfolded. The former allows not only for ubiquitin to be recycled in the cell, but it also eases the process of translocation, which involves moving the protein from 19S pore to the enzymatic 20S portion of the proteasome. It has been observed that, to unfold a defective protein, energy obtained from the hydrolysis of ATP is required. The protein is then engulfed past the two outer rings and travels into the center of the barrel. There, protease digests the protein into small peptides, amino acid sequences usually 3-24 in length. It is unknown how the proteasome determines the amino acid length, but it is generally thought to be a characteristic linked to the substrate, rather than the proteasome.
Although proteasomes are essential parts to the maintenance of a eukaryotic cell, they are the proteins responsible for many of the neurodegenerative diseases of today. To understand how the process may cause problems for the cell, first, one must understand how the ubiquitin-proteasome system (UPS) functions correctly.
The UPS and Neurodegeneration and Proteinopathy
So far, this explanation has explored how the ubiquitin-proteasome system works in a biologically ideal environment. However, this pathway does not always work or is prohibited from working properly as in neurodegenerative diseases or proteinopathy diseases. Protein aggregation, or the clumping of abnormal proteins, is directly related to these neurodegenerative diseases, but whether it is a byproduct or a cause of the disease is still unknown. Proteins that are prone to aggregation cause massive clumps of abnormal protein that the UPS is unable to breakdown, putting stress on the system. The proteins continue to accumulate and the stressed UPS cannot break them down, so the problem turns into a viscous cycle. Researchers have tested and proven that the UPS does not break down cells with large aggregates of proteins. The system becomes powerless. One possibility is that the tangled protein does partially enter the proteasome, but cannot be fully pulled in. Since the proteasomes cannot digest the protein or release it, they are forced to remain inactive. In Alzheimer’s disease, for example, cells are found to have large clumps of tangled proteins (4). These proteins and the proteasomes in the cell cannot contribute to the cell. It is possible that the excess proteins are what destroy the neurons that categorize these diseases. Another example is seen while looking at Huntington’s disease. Here, polyglutamine expansion mutation is the cause. This disease correlates with polyglutamine tract length, and tract length correlates with aggregation tendency. Thus, it may be inferred that the propensity for aggregation may be a major factor in the causes of Huntington’s disease and diseases like it. In many of these proteinopathies, mutations may be caused by toxicity levels, which may be caused by or correlate with the tendency for proteins to aggregate.
One theory on what causes neurodegeneration can be explained by analyzing the effects of mammalian mutations on the UPS (4). One such example is with E3 ligase, in which there is a loss-of-function mutation, which can lead to Parkinson’s disease. Another mutation is a heterozygous mutation in the DNA, which codes for ubiquitin carboxy-terminal hydrolase. This theory has not been absolutely confirmed through studies, but there is a confirmed pattern that families with Parkinson’s disease often have this gene mutation (4). The role of the carboxy-terminal hydrolase is related to intraneuronal aggregates, but the exact function is still unclear. What is known is that this mutation is definitely associated with loss of function. Yet another mutation, that in CDC48/p97, is found to be responsible for diseases such as Paget’s disease and other dementias. In these diseases, the mutation is responsible for aggregates in the cytoplasm and nucleus in muscles and brain tissue (4).
Macroautophagy, or simply autophagy, is the metabolic breakdown of cellular material through the use of lysosomes (4). This process is well-regulated and mediated by the cell, but if it does not work properly, major problems may occur for the cell. One of these problems is the formation of inclusions. When this gene does not function properly, intraneuronal aggregates form, followed by neurodegeneration.
There are still many facets of neurodegenerative diseases that remain unknown as a result of the variety of factors involved. Although these relationships are still under research, neurodegenerative diseases are related in some way with the tendency for proteins to aggregate and render toxicity. As evidenced, the role of proteasomes is synonymous with health and disease.
By the same token, encephalopathic diseases caused by proteinaceous infectious particle virions, or prions, present with fatal nervous system degradation, including severe loss of neurons, gliosis, and spongiform appearance (9). Prion-related diseases have typically appeared as short endemic bursts rather than lingering diseases. In the United Kingdom, for example, nearly 150 people succumbed to a lethal “variant of Creutzfeldt-Jakob disease, acquired through ingestion of prion-containing beef” (9). Though the neurodegenerative diseases such as Alzheimer’s and prion-related encephalopathic diseases are quite similar in a phenotypic approach, the underlying mechanisms by which the latter strikes are indubitably unique.
The mechanisms that prion diseases generally employ to bypass defensive proteasomes are pernicious and irreversible, to say the very least. The toxic protein virus, when transmitted to an otherwise healthy individual, invades cells to modify “normal soluble, monomeric cell constituents” named PrPc into PrPsc which is “insoluble, [resistant] to proteases…has more amyloid forming Beta-sheet structures, and forms larger aggregates” than the protein otherwise would (9). These abnormal proteins clump at an alarming rate, and play a direct role in the nervous system. Unfortunately, the link between aggregated toxins and degenerated neurons is still an unresolved matter. As would be expected, the clinical picture with which prion diseases present has much more apparent, speedy, and fatal effects than most neurodegenerative diseases. Nevertheless, it is widely accepted that the role of the ubiquitin-proteasome mechanism is synonymous with several different types of diseases.
Proteasomes’ Role in Cancer
Proteasomes also play a role in the disease processes of cancer. The role of proteasomes is complicated by apparent pro- and anti- apoptotic effects of in cell growth and development. As previously described, proteasomes are highly effective in their destruction of proteins. Apoptosis, defined as a genetically determined process of cell self-destruction that is marked by the fragmentation of nuclear DNA, is activated either by the presence of a stimulus or by the removal of a stimulus or suppressing agent (7). Apoptosis requires each cell to carry the proteins necessary for its death. An ubiquitin-proteasome system helps prevent premature cell death by destroying proapoptotic proteins. Control of proapoptotic proteins ensures the prevention of abnormal activation of cell apoptosis (10). Unfortunately, these processes can be used by viruses to their own advantage. One such case is the potency and prevalence of Human Papillomaviruses. HPV is linked to genital warts, as well as anal and cervical cancers. These cancerous growths, under normal conditions, would be blocked by p53, a tumor suppressor. However, the gene product E6 of HPV targets the p53 protein for degradation by the ubiquitin-proteasome system (11). This is accomplished by making a protein that binds simultaneously to p53 and an E3 protein (responsible for marking proteins as targets for ubiquitination). The ubiquitination of p53 leads to its degradation by the proteasome (12). An additional factor, E6-AP (associated protein), was also proven necessary for the E6-dependent ubiquitination of p53. E6-AP, an ubiquitin-ligase, suggests a cascade pathway including E1-E2-E3 necessary for ubiquitination(13).
In addition, proteasomes, or rather the inhibition of proteasome complexes, play an instrumental role in the induction of apoptosis. These effects are found in rapidly growing cells. In response to apoptotic stimuli, three subunits of 19S are cleaved by caspases and are, therefore, unable to destroy ubiquitinated substrates. Ubiquitinated proteins accumulate and lead to an increase in the levels of proapoptotic proteins, resulting in not only a critical threshold for caspase activation, but also intensification the apoptotic signal (10). For this reason, the use of proteasome inhibitors in the promotion of cell apoptosis plays a pivotal role in the treatment of cancers (14).
In a clinical setting, cancer treatment involves inhibition of the UPS by medications that interact with the proteasome complex. However, the functions of these medications counteract treatments for neurodegenerative diseases, as proteasome inhibition can significantly affect proteinopathies. To combat this obstacle, proteasome-inhibiting medications have been designed to avoid the entering into nervous system (see Figure 3).
The Future of Proteasome Research
With numerous frontiers still unexplored, the target of ongoing research on proteasomes is ultimately to find stronger ties between the UPS pathway and various diseases. Among these are the three specific proteinopathies mentioned, as well as a span of other maladies such as cataract formations, cardiac diseases, and immunodeficiencies. With recent advancements in the field of cellular biology, specially encompassing proteasomal pathways, comes an optimistic outlook towards new and more effective treatment for many illnesses that ail mankind.
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