The proteasome, a large protease complex in cells, is the major machinery for protein degradation. It was previously considered a humble garbage collector, performing housekeeping duties to remove misfolded or spent proteins. Until recently, the interests of immunologists in proteasomes were focused largely on its role in antigen processing. Its real importance in cell biology has only been revealed contemporarily due to the availability of relatively specific inhibitors. It has now become increasingly clear that many aspects of immune responses highly depend on proper proteasome activity. Recently, a proteasome inhibitor has been successfully used to prevent acute as well as ongoing heart allograft rejection in mice. Such inhibitors are also efficacious in treating several autoimmune diseases, such as arthritis, psoriasis, and probably type I diabetes, in animal models. Phase II and III clinical trials of proteasome inhibitors in treating various tumors have shown promising results, and the side-effects of these drugs are tolerable. Therefore, proteasome inhibition represents a new and promising frontier in immunosuppressant development.
The proteasome is the major machinery in cells for protein degradation. In this article, we will review recent developments in proteasome research, and discuss prospects of using proteasome inhibitors as a new category of immunosuppressants in transplantation. Sections I to VI cover structure and function of the proteasome, and its roles in cell biology, with an emphasis on events relevant to immune responses. Scientists engaging in basic research might find useful information there. Sections VII and VIII review translational research on proteasome inhibitors in transplantation, autoimmune diseases and cancers, and discuss future perspectives of using proteasome inhibitors to prevent graft rejection. Clinician scientists might benefit from these sections.
I.Structure and Mechanisms of Action of Proteasomes
The core structure of the proteasome is referred to as the 20S proteasome, according to its sedimentation rate [see (1,2) for more detailed review]. It is cylinder shaped and composed of multiple subunits. The 20S proteasome exists in all three domains of life: archaea, bacteria and eukarya (1). Its protease activities are located in the inner cavity of the cylinder and are thus insulated by the cylinder wall. Such compartmentalization of proteases prevents indiscriminate digestion of proteins that are not to be eliminated. The cylinder is built of four stacked rings with seven subunits in each ring [Figure 1 and (1)]. In eukarya, the outer two rings are made of seven different α subunits, while the two inner rings are composed of seven different β subunits (3). Only the β1, β2 and β5 subunits contain protease activities.
There are five better-characterized protease activities in the proteasome: chymotrypsin-like, tyrosine-like, peptidylglutamyl-peptide hydrolyzing (also called caspase-like), activity which cleaves after branched chain residues, and activity which cleaves between neutral amino acids (4). Chymotrypsin-like activity is rate limiting (5,6).
It is noteworthy that through a poorly understood mechanism, degraded products of the proteasome fall in a narrow size-range of 7–9 residues (1). This size-range must have profound influence on the evolution of the MHC class I antigen presentation system, which appeared much later during evolution and adopted the peptide size range established by the proteasome. The immune system in turn influenced the evolution of the proteasome. When high eukaryotic cells are stimulated by inflammatory cytokines, such as IFN-γ or TNF-α, three new enzymatic active subunits, β1i, β2i and β5i, are transcriptionally induced and take the place of their constitutive homologs during proteasome neosynthesis (Figure 2), (7–9). The proteasomes with these new subunits are termed immunoproteasomes, and are abundant in the spleen but almost absent in the brain. The exchange of β1 with β1i leads to down-regulation of caspase-like activity and increased chymotrypsin-like activity (10,11). Such a change in specificity conforms to the requirement of class I peptides, which normally have hydrophobic C-terminals in the mouse, and hydrophobic or basic C-terminals in the humans. The alteration of enzymatic specificity by β2i and β5i replacement is not consistently found (11–13).
The 20S proteasome can only degrade unfolded proteins or short peptides in an ATP-independent fashion (1). The majority of proteasomes in eukaryotic cells exist in a 26S form, which is composed of the 20S core with a 19S regulatory complex attached to each of its ends. The 26S proteasome digests intact but ubiquitylated proteins in an ATP-dependent fashion. It presents in cytoplasm as well as in nuclei, and degrades about 70–90% of cellular proteins (5). The 19S complex contains ATPase activity and is responsible for recognizing ubiquitylated protein substrates.
Protein ubiquitylation is a mechanism that enables the 26S proteasome to specifically and timely degrade proteins that are destined to be destroyed (14). It involves at least three essential steps. First, ubiquitin is ATP-dependently activated by ubiquitin-activating enzyme E1. Then, one of several ubiquitin-conjugating enzymes (E2s) transfers ubiquitin from E1 to one of E3s, which are ubiquitin ligases belonging to a large family with a growing number of members. Recently, a novel ubiquitylation factor E4 has been reported to enhance the efficiency of E3 (15). It is to be noted that ubiquitylation is not solely for protein degradation via the proteasome, but may have proteasome-independent regulatory functions in cell biology. For example, the association of a centromere protein E with kinetochores during mitosis, and activation of IkB kinase depend on ubiquitylation, but do not involve the proteasome (16,17). Recent studies have also revealed important roles of ubiquitylation in regulating the RNA polymerase II machinery without involving proteolysis [see (18) for more detailed review] Interestingly, ubiquitylation is also essential in budding of retroviruses, including HIV-1 and HIV-1 (19–21).
The 20S proteasome can also bind to 11S regulators (Figure 1), which only exist in higher eukaryotes. The 11S regulator does not enable the 20S proteasome to degrade intact proteins or ubiquitylated proteins. Rather, it enhances the peptide degradation by the 20S proteasome, or enlarges the peptide repertoire (22,23) in an ATP- and ubiquitin-independent fashion. Moreover, the 11S regulator is drastically induced by IFN-γ. It is thus speculated that the 11S regulator is evolved to meet the need of antigen processing. This is supported by the finding that HIV-1 virus escapes from immune surveillance by preventing the formation of 20S/11S complexes (24). As free peptides are short-lived and rare in vivo, from where the 20S/11S complex gets its substrates is an intriguing question. Possibly, the 19S and 11S regulators attach to the 20S proteasome on each end, and such a structure might allow the proteasome to degrade proteins like the 26S, and generate short peptides in an 11S-regulated way. The physical existence of such a 19S/20S/11S entity (Figure 2), now termed ‘the hybrid proteasome’, has recently been proved (25), and it accounts for one fourth of the proteasomes of the HeLa cells. However, the hybrid proteasome does not behave exactly as postulated. It degrades a model protein in an ATP-dependent but ubiquitylation-independent fashion, which is halfway the behavior of a 26 proteasome; on the other hand, its activity is indeed enhanced by IFN-γ, as expected. Whether it degrades other proteins in this fashion is yet to be assessed.
II. Role of the Proteasome in T- and B-Cell Activation
In any immune responses, including graft rejection, T- and B cells first need to be activated. The activation process requires a large number of positive and negative regulatory cellular proteins, whose presence, levels or activation in cells have to be tightly controlled. The proteasome is indispensable in irreversibly eliminating these regulatory proteins as well as in their synthesis and activation in certain cases.
A well-studied case is NF-κB. It was initially identified as a nuclear transcription factor binding to the B site of the intronic promoter of the Ig k chain (26). It was later found that this factor positively regulates many important genes involved in immune responses [see (27) for more detailed review], such as the TCR β chain, IL-2, IL-6, IL-8, G-CSF, GM-CSF, TNF-α, IFN-β, CD25, CD54, CD62E, CD62L, iNOs, MHC class I α chain and β2 microglobulin, certain MHC class II α and β chains and their invariable chain Ii, etc. (27–29). NF-κB comprises two subunits, which can be p50 or p65 homodimers, or p50/p65 heterodimers. A recent study showed that, rather than being derived from a p105 precursor through proteasome-dependent degradation (30), the p50 subunit is generated by novel cotranslational biogenesis requiring the 26S proteasome (31). NF-κB is normally associated with its inhibitory factors IκBα and IκBβ, and is located in the cytoplasm in an inactive status. Upon receiving activation signals from a variety of stimuli, IκBα and IκBβ are degraded by the 26S proteasome (32,33), and NF-κB is then translocated into nuclei for its transcriptional regulation. Therefore, the proteasome is essential for both the synthesis and the activation of this important transcription factor.
Another transcription factor AP-1 exists in many types of cells. Several immunologically important genes, such as IL-2 (34), IFN-γ (34), IL-5 (35), and CD95L (36), have AP-1 binding sites in their promoter regions. The best-characterized AP-1 comprises two subunits, Jun and Fos, both of which rely on the ubiquitin-proteasome pathway for their elimination (37,38). Similarly, rapid elimination of a number of other transcription factors, such as c-Myc (39), c-Myb (40), OBF-1 (41), STAT3 (42), STAT4 (43), STAT5b (44), HIF-1α (45), Smad1 and Smad2 (46,47), and IRF-1 (48), all depend on the proteasome. To name a few target genes and the function of these transcription factors, c-Myc along with its partners modulates the gene expression of FasL (49) and CXCR4 (50); c-Myb is a transcription factor for the enhancer of the pre-T-cell receptor α chain gene (51), and plays both positive and negative roles in CD4 gene expression (52); OBF-1 is essential in the formation of the germinal center of secondary lymphoid organs (41); STATs respond to many cytokine stimuli and activate cytokine-responsive genes; HIF-α induces target genes during hypoxia, which often leads to inflammation, or apoptosis (45); Smad1 and 2 activate TGF-β-responsive genes (46,47); IRF-1 is an activator of IFN genes and IFN-inducible genes (48).
The proteasome is also responsible for the removal of several nonreceptor kinases, which are pivotal in T- and B-cell signaling. Included in the list of such kinases are Lyn (53), Src and Fyn (54), Syk and Zap-70 (55), ERK3 (56), Raf-1 (57), and the p21-activated protein kinase (PAK) family member γ-PAK (58). A couple of other non-kinase signaling molecules, such as suppressors of cytokine signaling SOCS (59) and Ras guanine nucleotide exchange factor Ras-GRF2 (60), depend on the proteasome for degradation as well.
Most of the above-described transcription factors and signaling molecules play positive regulatory roles in activation of T- and B cells, as well as monocytes/macrophages and dendritic cells. Proteasome inhibition leads to accumulation of these positive regulators at the end of their normal function. It is conceivable, although not proven, that the abnormal elevation of these molecules at an inappropriate time will cause aberrant cellular events, which prevent these cells from being fully activated.
III. Role of the Proteasome in Cell-Cycle Control
During immune responses, T- and B cells need to undergo rounds of proliferation for clonal expansion. Such proliferation is also required for their differentiation. Cell cycling is a highly regulated and programmed event. The protein levels of many pivotal cell-cycle regulators oscillate during different phases of the cycle as a way of modulating their functions [see (61) for more detailed review]. The proteasome is responsible for timely degradation of many of these proteins. These proteins can be classified into three groups.
In the first group, the detrimental effect on cell-cycle progression due to failed degradation of these regulators has been proven. For instance, cyclin E is degraded in the late S phase via the proteasome pathway (62–64), and failure to do so impedes progression through the S phase (65). Degradation of cyclin A via the proteasome is essential for exit from mitosis (66). Cdc25 A phosphatase is rapidly degraded by the proteasome, and its overexpression disturbs G1-S phase progression (67).
In the second group, the regulators are degraded through the proteasome during a particular phase of the cycle, although further studies are needed to prove that their degradation is essential for cell-cycle progression. Included in this group are cyclin Ds (68,69), Cdc6 (70), and Skp-2, the component of the Skp1p-cullin-F-box protein (SCF) complex (71), which is a ubiquitin ligase involved in cell-cycle control. The last-mentioned shows that even a component of the ubiquitin–proteasome machinery regulating cell-cycle progression is under the control of the proteasome in certain cases.
The third group is composed of several inhibitor proteins repressing CDK activity. For this group, it is easy to understand that proteasome inhibition will result in accumulation of CDK inhibitors, and, hence, inhibition of cell cycle progression. Cip/Kip family inhibitors comprise p27kip1, p57kip2 (not expressed in immune cells), and p21cip1. P27kip1 is expressed at a high level in the G0 and G1 phases. Before the onset of the S phase, it needs to be degraded via the proteasome pathway (72), and failure to do so halts the cells in the G1 phase (73). P21cip1, although a pan-CDK inhibition, is also required in the early G1 phase (62). Both its induction and degradation are proteasome dependent (62,74), but it has yet to be determined whether such modulation is obligatory for immune cell-cycle progression. The other CDK inhibitor family, the INK4 family, comprises p16ink4a, p15ink4b, p18ink4c and p19ink4d. Only the p19ink4d level fluctuates during the cell cycle, and this protein is degraded through the proteasome (75). However, p19ink4d expression in T cells is very low (62), and its relevance in T- and B-cell cycling needs to be established.
IV. Role of the Proteasome in Cell Adhesion and Migration
Proper cell–cell interaction during immune responses is often required. For example, T cells need to interact with antigen-presenting cells for T cell receptor (TCR) activation as well as for costimulating signals [see (76) for more detailed review]; leukocyte–endothelial cell interaction is necessary for leukocytes to migrate from the blood stream to extravascular sites in response to inflammatory stimulation, for their homing to lymphoid organs, and for them to reach their targets after differentiation [reviewed in more detail in (77)]. Such cell–cell interaction depends on the expression of adhesion molecules on immune as well as endothelial cells. The proteasome pathway is required for expression of a number of adhesion molecules, such as CD54, CD11a, CD62E, and VCAM-1 (78,79). Mechanistically, proteasome inhibitors mostly suppress the transcription of these adhesion molecules, but in some cases, they decrease the expression or affinity of these molecules via undefined post-transcriptional mechanisms (78,80). Chemotaxis is also important for immune cell migration, and T-cell chemotactic activity induced by IL-16 and RANTES is proteasome dependent (81). Thus, proteasome inhibitors can repress the antigen presentation, costimulation, chemotaxis, homing and cytotoxic activities of lymphocytes by suppressing cell–cell interaction and cell migration.
V.Role of the Proteasome in Apoptosis of Cells of T-, B- and Monocytes Lineage
Disturbances in cell activation, cell cycling or cell–cell interaction often lead to apoptosis [see (82–84) for more detailed review]. As the proteasome is essential in these processes, its inhibition should have a major impact on cell survival. Indeed, it has been shown that activated T cells, Jurkat cells (62), and monoblast U937 cells (85) undergo apoptosis after proteasome inhibition. Mechanistically, such an apoptosis-promoting effect of proteasome inhibition is probably due to the accumulation of pro-apoptotic factors, which are short-lived and normally channeled to the proteasome for degradation. For example, a pro-apoptotic Bcl-2 family member, Bik, rapidly increases its protein level due to decreased degradation in lymphocytes after proteasome inhibition, and such an increase is sufficient to cause T- and B-cell apoptosis (86). In other cell types, the degradation of pro-apoptotic factors, such as Bid (87), Bax (88), Nix (89), Id1, Id2 and Id3 (90), occurs through the proteasome pathway. However, whether these factors play critical roles in apoptosis of T cells, B cells, monocytes/macrophages, or dendritic cells has not been established. Obviously, the proteasome does not act on a one-way street. It also degrades anti-apoptotic factors, such as Bcl-2 (91), TC3 (92), IAP and XIAP (93), in various cell types. IAP and XIAP accumulation after proteasome inhibition is reportedly a mechanism for the anti-apoptotic effect of proteasome inhibitors on thymocytes (93). Thus, proteasome inhibition can be pro- or anti-apoptotic for particular cell types at a particular stage, depending on the overall balance of these pro- and anti-apoptotic forces. For mature and activated lymphocytes, proteasome inhibition normally leads to their apoptosis.
VI. Proteasome and Antigen Presentation
The proteasome is critical in MHC class I-restricted antigen presentation, and this is probably the best-known proteasome function to most immunologists. The role of the proteasome in antigen presentation is not the focus of this article, as several comprehensive reviews are readily available (94,95). Conceivably, near-complete inhibition will prevent short peptide generation, and result in empty class I molecules, which cannot be efficiently transported from the ER to the cell surface. In the case of transplantation, this might mean reduced alloantigenicity, although further study is needed to prove such a speculation.
VII. Novel Proteasome Inhibitors and Their Application in Transplantation and Autoimmune Diseases
The proteolytic sites in the 20S proteasome initially attack the peptide bonds by the hydroxyl group on an N-terminal threonine of the β subunits. This is a mechanism which is distinct from that of other proteases (96), and is the theoretical base for some inhibitors to be more specific to the proteasome than to non-proteasome proteases. Our knowledge of proteasome functions has been greatly enhanced since the discovery of the first relatively specific proteasome inhibitor lactacystin (97), which was initially extracted from Streptomyces (98). Although lactacystin inhibits all three major proteasome activities (chymotrypsin-like, trypsin-like, and caspase-like), and inhibition of the first two is irreversible when tested with purified proteasomes, it rapidly loses its activity in biological aqueous solution (62,99). Its analog, PS519, seems to have better stability, because it can effectively treat psoriasis in vivo in a scid-hu mouse model (100) and reduce ischemia-induced infarction as well as leukocyte infiltration in a rat model of focal cerebral ischemia (101,102). Toxicology data on PS519 are not yet publicly available.
Realization of the proteasome's importance in various aspects of cell biology has prompted increased efforts to search for new and existing compounds capable of inhibiting its activity. A classical strategy to design protease inhibitors is to generate short peptides based on the preferences of proteases, and then add various chemical groups to protect the peptide bonds. Along this line of strategy, peptide aldehydes (103) and, recently, peptide phthalimides (104), peptide boronic acids (105,106) and peptide vinyl sulfonates (107) have been developed as competitive protease inhibitors. Based on empirical selection, some inhibitors of this category, such as dipeptide boronic acid (DPBA, also named PS341, MLN341), are more specific to proteasome activity than to non-proteasome proteases.
DPBA/PS341 has been successfully used to prevent acute as well as ongoing mouse heart allograft rejection (108). At therapeutic dose levels, this compound has no apparent acute toxicity in the liver, kidney, pancreas and heart, according to blood biochemistry in mice. This is the first study that intentionally and successfully used a proteasome inhibitor as an immunosuppressant to treat graft rejection. DPBA/PS341 has also been effective in treating streptococcal cell wall-induced polyarthritis in a rat model (105) and human multiple myeloma (106,109,110). Presently, more than 30 separate phase II and III clinical trials are under way to study the efficacy of this proteasome inhibitor in treating breast, colon, lung and prostate cancers (107). The reported side-effects in patients include low fever and/or fatigue, and non-dose-limiting thrombocytopenia (106). Low-grade diarrhea and peripheral neuropathy are observed in some patients. However, it seems the compound could be tolerated overall. These clinical trials have relieved a major concern that since every cell has proteasomes, proteasome inhibitors might be too toxic. This should not be too surprising, because most somatic cells are in a quiescent state, and need much less proteasome activity to regulate their activities, compared to tumor cells or active immune cells, which are intended targets. As long as proteasome activity is not completely inhibited for a prolonged period, the target cells would receive most of the blow.
A new group of proteasome inhibitors, TMC-95 A, and its diasteromers TMC-95B, C and D, were isolated in 2000 from fermentation broth of Apiospora montagnei (111,112). TMC-95 A has a chemical structure totally different from that of known proteasome inhibitors. It binds to the proteasome noncovalently and competitively, and specifically inhibits proteasome activity. In vitro and in vivo data for this novel inhibitor on the immune system are not yet available.
There are several old natural compounds with newly discovered proteasome-inhibiting activity. Epoxomicin was isolated from Actinomycetes in 1992 as an antitumor agent (113), and was found in 1999 to inhibit proteasome activity (114). Epoxomicin covalently binds to the proteasome and primarily inhibits its chymotrypsin-like activity with higher potency than lactacystin. In vivo, epoxomicin inhibits delayed-type hypersensitivity in a mouse model. No other in vivo data are currently available.
Vinblastine, an alkaloid anticancer drug developed in the early 1960s (115), is capable of disrupting microtubules (116). A recent report has shown that it can reversibly inhibit major protease activities of the proteasome (117). It is not clear whether its effect on microtubules is derived from its proteasome-inhibiting action. Tea polyphenols are another interesting category of compounds, which have been shown to inhibit chymotrypsin-like activity of the proteasome in vitro and in vivo at concentrations found in the serum of green tea drinkers (118). The possible application of vinblastine and polyphenols as immunosuppressants for transplantation and autoimmune diseases has yet to be explored.
Gliotoxin, a metabolite of Aspergillus fumigatus Fresenius and other fungi, was isolated in the 1960s (119). It has a profound immunosuppressive effect and plays important roles in the pathogenesis of aspergillosis. Gliotoxin was used successfully to prevent human fetal pancreas graft rejection in 1988 (120), mouse skin allograft rejection in 1990 (121), and mouse thyroid allograft rejection in 1995 (122). Chronic treatment of diabetes-prone BB rats with gliotoxin significantly reduced diabetes incidence in this model (123). Its mechanism was not clearly elucidated. In 1999, gliotoxin was found to be a noncompetitive inhibitor of the chymotrypsin-like activity of the proteasome. If the proteasome is the only target of gliotoxin, then a proteasome inhibitor was used successfully but unknowingly as early as in 1988 in organ transplantation. Its effect as a proteasome inhibitor in organ transplantation and autoimmune diseases is worth further investigation.
VIII. Conclusions and Future Perspectives
The proteasome plays pivotal roles in many aspects of cell biology, and the overall effect of proteasome inhibition is to down-regulate cell activation, proliferation as well as cell–cell interaction, and to promote apoptosis. Therefore, the proteasome is a legitimate and very promising target of immunosuppressants and anticancer drugs. Most of the current immunosuppressants are based on mechanisms inhibiting cellular anabolic processes, such as macromolecule synthesis and modification. Proteasome inhibition represents a novel and previously unexplored direction to achieve immunosuppression by blocking a cellular catabolic process, i.e. protein degradation. Proteasome inhibition-based immunosuppressants could be very useful in preventing graft rejection as a component of drug combination, because they work with a totally different mechanism from the current front-line immunosuppressants. Since proteasome inhibitors can effectively suppress activated lymphocytes and induce their apoptosis, they could also be very promising in treating rejection episodes, which are normally diagnosed after lymphocytes are already activated and front-line drugs are not effective. If proteasome inhibitors are given periodically for a long duratioin to transplant patients in addition to regular immunosuppressants, they might induce apoptosis of chronically activated alloantigen-specific T- and B cells and lead to their clonal reduction. One would hope that chronic rejection might thus be ameliorated. Currently, increasing efforts are being made to find proteasome inhibitors from novel molecules as well as from old compounds. We hope soon to have more options of various inhibitors suppressing different protease activities of the proteasome, including those activities nested only in subunits induced during immune responses. Probably, not all protease activities in the proteasome are equally important in controlling all aspects of cell biology. Selective inhibition of certain protease activities of the proteasome will be a strategy to differentially repress immune responses while affecting other cellular functions as little as possible. Needless to say, proteasome inhibition will have much broader applications in various pathological conditions in addition to controlling graft rejection.
This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MT-15673, PPP-57321), the CIHR/Canadian Blood Service Partnership Program, the Kidney Foundation of Canada, the Heart and Stroke Foundation of Quebec, the Roche Organ Transplantation Research Foundation, Switzerland (ROTRF #474950960), the Juvenile Diabetes Research Foundation, U.S.A. (#5-2001-540), and the J-Louis Levesque Foundation to J.W. J.W. is a National Researcher of the Quebec Health Research Foundation. The author acknowledges the editorial assistance of Mr Ovid Da Silva, Redacteur, Research Support Office, Research Centre, CHUM-Hotel-Dieu.