Tore 1
Grant Tore
Dr. Ely
Biology 303
1 November 2014
Inhibition of the 26S Proteasome
Among the most popular topics of modern biology, since it was first discovered in the 17th century, was the cell. Although renowned for its nature as the most basic building block of all forms of life, the cell had been discovered to be more and more complex than it was ever thought to be. Much of this complexity can be attributed to the numerous and uniquely functional proteins within each and every cell. However, with such a vast array of proteins and the functions they perform, it was not surprising that there is ample room for mistakes to be made. Due to the commonality of such errors in protein assembly and function, cells developed many mechanisms to maintain their protein homeostasis and overall efficiency. These cellular apparatuses became a popular area of study because of the many diseases and conditions associated with their malfunctioning.
In order for cells to be the efficient machines that they are, it was vital that they maintain homeostasis and not be wasteful in their routine functioning. Most of the time, this was the case, but there were often instances when even the slightest changes could cause abnormalities in the life of a cell. Some of the most common, yet destructive, changes that occurred in a cell wereerrors associated with the production or folding of a protein. Due to the fact that protein function matched its folded shape, failure to attain the intended shape could result in the implication of diseases as serious as Parkinson’s, mad cow, Alzheimer’s and many others (“protein.” 2005). Being so reliant on properly working proteins, it was necessary for cells to develop systems that maintain such conditions. Modern research had shown that of these cellular mechanisms, the ubiquitin proteasome pathway was the most significant proteolytic pathway in eukaryotic cells because of its regulation and selective degradation of proteins (Zhu et al., 2013).
Within the ubiquitin proteasome pathway, the biggest component is the 26S proteasome, shown in Figure 1.The 26S proteasome itself is composed of a 20S catalytic core and a pair of 19S regulatory subunits (Baumeisteret al., 1998). The 20S proteasome is a barrel-like structure made of two outer α-type rings and two inner β-type rings. The α-rings serve as a gate through which the selected proteins enter and exit the proteasome, while the β-rings are involved in the actual degradation ("26s proteasome." 2014). Each 19S particleon the ends of the proteasome contain both a base, made of ATPase subunits, and a lid, made of non-ATPase subunits. However, even in its complete form, the 26S proteasome is useless without another key substrate.
The other necessary component of the ubiquitin proteasome pathway was of course the ubiquitin. Ubiquitin itself is a regulatory protein that can affect the structure and function of other intercellular proteins in many ways. The process of adding ubiquitin to a protein, commonly known as ubiquitination, involves steps of activation, conjugation, and ligation ("Ubiquitin." 2014). Depending on the function of the ubiquitination, the ubiquitin can be attached either as a single protein or a chain. With respect to the ubiquitin proteasome pathway, the addition of a five member ubiquitin chain to a protein is the molecular signal for the degradation of that protein.
The next question researchers sought to answer was the one concerning how a cell determined which proteins or molecules would be marked for degradation by the ubiquitin proteasome pathway. In previous research, it was found that among the most abundant proteins in cells was a molecular chaperone, Hsp90. In general, molecular chaperones work to recognize non-native molecular structures within the cell to prevent their buildup and allow their conversion to the correct formation (Frydman, 2001). Then, once a cell recognized that a protein was improperly folded or assembled, it marked that protein with ubiquitin for the degradation pathway (Sherman and Goldberg, 2001). Researchers have also looked into the array of functions that the Hsp90 chaperone completes in general, and it was found that its most pivotal role dealt with the folding of proteins (Richter and Buchner, 2001). Knowing these functions of the Hsp90 chaperone and the 26S proteasome, biologists have most recently examined the relationship between this chaperone and the assembly and maintenance of the proteasome.
In the study conducted by Imai et al., researchers worked with two Hsp90 species in yeasts, known as Hsp82 and Hsc82, which are the equivalents of those found in mammals. Before trying to observe the relationship between the Hsp90 and the 26S proteasome directly, they first examined the effect of thermal stress on just the functional proteasome. It was known prior to this test that the functional proteasome could be classified further into three subsections including the free 20S proteasome, or C, and the two regulatory particles on either one or both sides, R2C and RC, respectively (Glickman et al., 1998). After proper preparation, the wild-type cells were incubated at 50° C for 20 minutes and then returned to normal conditions, which were at 25° C. The results of this thermal stress were then examined through polyacrylamide gel electrophoresis (PAGE) and western blotting analyses shown in Figure 2A. These analyses showed lower levels of both the R2C and RC forms and increased levels of the free 20S proteasome after the heat shock. It was only until nearly 6 hours later that the cells recovered the native levels of the complete 26S proteasome.
After observing the effect of the heat shock on the 26S proteasome by itself, Imai et al. (2003) began to look into the relationship between the 26S proteasome and the Hsp90 chaperone. Using the same stress temperatures and conditions as before, they then overexpressed Hsp90 in the cells. Once again, they used both the PAGE and western blotting to analyze the results of this overexpression shown in Figure 2B. Interestingly enough, although disassembly of the 26S proteasome still occurred, they found that the overexpression of Hsp90 partially suppressed the amount of disassembly.
Looking for the possible connection between the Hsp90 and the 26S proteasome, Imai et al. (2003) further explored their effect on each other. After observing overexpression of Hsp90, they then explored what would happen to the 26S proteasome when the chaperone was inactivated. By doing so, they hoped to better pinpoint the mechanism for how Hsp90 helped to protect the 26S proteasome from being disassembled. To do so, they used mutant yeast cells, known as hsp82-4 cells, which would have defective Hsp90 under heat-sensitive conditions. When the WT and hsp82-4 cells were put through both the permissive and non-permissive temperatures (25°C and 37°C), it was observed that the signals of the RC and R2C positions decreased only in the mutant cells. This suggested that inactivation of the Hsp90 in the heat-sensitive mutant cells caused the disassembly of the 26S proteasome into its components(Figure 3). They also added SDS, an activator of the latent 20S proteasome. With this addition, the PAGE activity analysis also showed that the loss of Hsp90 could be associated with an increase in the levels of free 20S proteasome. Although there was more free 20S proteasome, the total amount remained unchanged. Finally, they also noticed that the loss of the functional 26S proteasome in the mutant cells occurred faster than cell death, showing that the
disassembly process was not due to cell death.
Overall, the studies done by Imai et al. showed the connection between the inactivation of the Hsp90 chaperone and the disassembly and functional loss of the 26S proteasome. Based on this study, it is clear that the functional integrity of the 26S proteasome is dependent on the Hsp90 chaperone, but further research has been conducted to explore more cellular complexes that either affect or are affected by the 26S proteasome. In recent years, there have been data showing that the 26S proteasome, aside from protein degradation, also plays a role in DNA damage response (DDR). In particular, a 2014 study done by Narayanaswamy et al. examined the effect of the urokinase-type plasminogen activator receptor (uPAR) on the assembly of the 26S proteasome and consequently, the effect on the DDR system after DNA damage.
To study the effect uPAR has on the 26S proteasome, Narayanaswamy et al. (2104) worked with vascular smooth muscle cells (VSMC), which are not terminally differentiated. They divided the cells into three groups: a control group of untreated cells, a group treated with an anti-cancer drug Doxorubicin (Dox) only, and group treated with both Dox and uPAR silencers. Like many anti-cancer drugs, Dox induces DNA damage, particularly double-stranded breaks, and DDR signaling. The use of the Dox was to insure that there would be sufficient proteasome activity related to DDR to be observed. After treatment, the cells were examined using mass spectrometry to look at both the ATPase and non-ATPase 19S subunits. Although there was no significant change in the recruitment of the ATPase subunits, there was a considerable decrease in three non-ATPase subunits in the uPAR-silenced cells. The decrease of the recruitment of PSMD6, PSMD7, and PSMD13 is shown in Table 1.
Narayanaswamy et al. (2014) also found data to suggest that uPAR specifically regulates the translocation of PSMD6 to the nucleus for assembly of the 26S proteasome. For this part of the study, they were using MMS, a different chemical agent that commonly produces single-stranded, rather than double, breaks in DNA. By treating the cells with MMS, Narayanaswamy et al. (2014) could observe the proteasome activity better since it would be upregulated. Once again, they also treated cells to be uPAR-silenced to observe this effect on the proteasome activity. To analyze the location of the PSMD6 and the proteasome activity, they used immunochemistry and cell fractionation assessments shown in Figure 4. Figure 4A compares the proteasome activity in the MMS treated cells with both active and silenced uPAR. In the resting (control) cells, PSMD6 was localized mainly in the cytoplasm. Figure 4B showed that it was only after treatment with MMS that the cells began to localize the PSMD6 in the nucleus, suggesting that there was increased assembly of the 26S proteasome in the nucleus to combat the DNA damage done by the MMS. However, in the cells in which the uPAR was silenced, the nuclear translocation of PSMD6 stopped as well, as shown in Figure 4C.
Finally, Narayanaswamy et al. (2014) determined the role of uPAR in single-stranded DNA break signaling and repair. For this experiment, they treated the VSMC with MMS and observed the results of this treatment on ATR and Chk1 kinases in the cells using western blotting (Figure 5A). Their findings showed that after the MMS treatment, the activation of the Chk-1 kinase was significantly impaired due to phosphorylation only in the cells that were uPAR-deficient. To go even further, they treated VSMC cells from mice with various amounts of the MMS and observed the survival rates of those cells after 24 hours. Figure 5B shows that with the higher amounts of MMS, and therefore DNA damage, the survival rates of the cells was significantly decreased when the uPAR was impaired. Their data provide a great deal of evidence that cells need uPAR to successfully conduct DNA single-stranded break signaling and DNA repair.
Due to the genomic instability that can be caused by initial damage to the DNA, small mistakes in the DNA can quickly develop into many serious diseases including cancer, cardiovascular and neurodegenerative disorders, immune deficiencies and more. In a 2013 study, done by Zhu et al., researchers explored a less-commonly studied condition called renal interstitial fibrosis. For their research, they used renal interstitial fibroblasts, also known as NRK-49F cells, which they induced using a transforming growth factor, TGF-β1. When a cell becomes damaged, its ability to go into apoptosis is vital to the prevention of the cell from multiplying and causing a more serious condition.
In their research, Zhu et al.(2013) first treated the NRK-49F cells with or without the TGF-β1. They then also treated those cells with or without MG-132, a proteasome inhibitor to observe the effect of the inactivation of the proteasome in the fibroblasts. They used gel electrophoresis to analyze their results after 24 hours and found DNA ladders indicating DNA damage when cells were treated with MG-132 (Figure 6).
Finally, Zhu et al.(2014) looked at the effect of MG-132 on the expression of the p53, p21, and p27 proteins in NRK-49F cells. To do so, they treated the NRK-49F cells with specific concentrations of MG-132, and then observed the levels of each protein in the cells. They also treated some of the cells with both the MG-132 and TGF-β1. Based on the data shown in Figure 7, they found that there was a significant increase in the levels of p53 and p21 proteins in the cells that were stimulated with both the MG-132 and TGF-β1 compared to the cells that were treated only with the MG-132. However, the levels of p27 protein did not vary significantly. These finding are important because they showed that proteasome inhibition in the fibroblasts could lead to the upregulation of p53, which signals cell death, and p21, a cyclin-dependent inhibitor that induces cell growth arrest. Overall, these data showed that the addition of MG-132 could promote apoptosis and inhibit cell growth through the upregulation of p53 and p21 proteins, opening new ideas for treatment of renal interstitial fibrosis and other similar diseases.
As many recent studies have demonstrated, regulation of the 26S proteasome can simultaneously lead to the regulation of many other cellular complexes. There is a great deal of evidence showing the functions of the 26S proteasome in the cell including both protein degradation and DNA damage repair. However, there are many times when cells become damaged beyond repair capabilities, and they are viable to fall into a tumorous or other disease state. When these cells proliferate, serious conditions can occur in the organism overall. This is why regulation of the 26S proteasome in various ways has become such an interesting area of research. Many of these studies have been aimed at inhibiting the 26S proteasome because of its vital functions in properly working cells. Normally, the 26S proteasome works to remove non-native proteins or signal for DNA repair, but in the disease state, the proteasomesystem is overwhelmed by the amount of damage. In this paper, impairing the Hsp90 chaperone, inhibiting the uPAR system, and shutting down the 26S proteasome itself were all observed at possible methods for disease treatments. Hopefully further studies will be done in the future to determine which of these methods of 26S proteasome inhibition is the most effective for disease treatment.
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