The present study demonstrated, for the first time to our knowledge in intact animals, that forced PA28αOE is sufficient to upregulate 11S proteasomes in cardiomyocytes in which overexpressed PA28α binds and stabilizes its partner, PA28β. By monitoring degradation of a surrogate misfolded protein substrate of the UPS, we found that PA28αOE enhanced proteasome proteolytic function in cardiomyocytes in intact mice without causing a concomitant pathology. We thus established what we believe to be a novel inducible Tg mouse model of benign cardiac proteasome functional enhancement. Given the prevalence of proteasome dysfunction in heart disease (3
), this mouse model should provide a valuable tool for exploring the therapeutic potential of manipulating cardiac proteasomal activity and for determining the contribution of proteasome dysfunction in cardiac pathogenesis. Using this mouse, we demonstrated that improving proteasome function inhibited aberrant protein aggregation, attenuated cardiac hypertrophy, and delayed premature death in a well-established DRC mouse model, demonstrating that PFI plays a critical role in the genesis of DRC, a bona fide cardiac proteinopathy. We were also able to demonstrate, for the first time to our knowledge, that PA28αOE protected against myocardial I/R injury in intact animals.
An 11S subcomplex can be either a homoheptamer of PA28γ or a heteroheptamer of PA28α and PA28β (26
). In the present study, we found that upregulation of the 11S could be achieved by forced CR-PA28αOE in intact mice. We hypothesize that concurrent upregulation between PA28α and PA28β is mediated by mutual stabilization at the protein level. Transcript levels of PA28β were not significantly changed during forced PA28αOE. As shown previously and in the present study, PA28α and PA28β proteins interact with each other. The degradation rate of PA28β was also significantly decreased by PA28αOE in cultured cardiomyocytes (23
), which is consistent with the proposed hypothesis. PA28α and PA28β interaction may prevent both from being recognized by the degradation machinery and thus result in mutual stabilization. This is also consistent with a previous report that PA28α protein cannot be detected in PA28β knockout mice (29
Damaged or misfolded proteins usually undergo conformational changes with exposure of their hydrophobic sequences and subsequent ubiquitination (12
). This process is mimicked by the degron CL1 fused GFP (GFPdgn or GFPu). By manipulating PA28αOE in mouse hearts, we demonstrated here that GFPdgn levels were inversely correlated to protein levels of PA28α. The decreases in GFPdgn protein levels were caused by a posttranslational mechanism, because neither the steady-state level nor the polysomal association of GFPdgn mRNA in the heart was significantly altered by CR-PA28aOE. Indeed, in an ancillary study using cultured cardiomyocytes, we found that PA28αOE destabilizes a similarly modified GFP and reduces the steady level of oxidized proteins during oxidative stress (23
). These findings suggest that upregulation of the 11S, at least those formed by PA28α and PA28β, can enhance degradation of misfolded proteins in cardiomyocytes. This is further supported by our findings from cultured cardiomyocytes that PA28αOE enhanced the protein degradation of CryABR120G
, a bona fide misfolded protein (36
Consistent with our in vitro findings that the abundance of the bona fide substrate normal proteins of the UPS was not affected by PA28αOE-induced proteasome functional enhancement (23
), no significant changes in cardiac gene expression, cardiac growth, cardiac histology, or heart function were observed in the hearts of mice with PA28αOE, which indicates that the intracellular homeostasis of normal proteins is not markedly perturbed by PA28αOE. We conclude that the increase of proteasome proteolytic function by upregulation of the 11S enhances the removal of abnormal proteins, but has little effect on the turnover of normal proteins.
Upregulation of 11S proteasomes via CR-PA28αOE protected against the pathogenesis, and thereby improved the outcome, of a well-documented mouse model of proteinopathy. Expression of CryABR120G
, a bona fide misfolded protein, triggers aberrant aggregation and thereby damages the cell via a number of potential mechanisms (14
). One of the suspects is PFI, which leads to accumulation of ubiquitinated proteins and further facilitates aberrant protein aggregation, forming a vicious cycle (1
). Conceivably, severe PFI can have adverse effects on cell function. It was recently shown that PFI can activate the calcineurin/NFAT pathway and facilitate adverse remodeling of a pressure-overloaded heart in mice (39
). We have also recently demonstrated that disruption of the COP9 signalosome induced cardiomyocyte-restricted UPS impairment, which subsequently led to cardiomyocyte death, dilated cardiomyopathy, and premature death in mice (40
). Here, PFI attenuation in DRC hearts via CR-PA28αOE broke the vicious cycle between PFI and aberrant protein aggregation; therefore, the steady levels of the ubiquitinated proteins and detergent-resistant oligomers (Figure ) as well as the abundance of microscopic protein aggregates (Figure ) were all substantially reduced. Remarkably, enhancement of proteasome function via CR-PA28αOE substantially slowed the progression of CryABR120G
-based DRC, as evidenced by decreased cardiac hypertrophy and prolonged survival (Figure ). These findings revealed a role for PFI in DRC development and the compelling potential of modulating the 11S proteasome as a therapeutic strategy to treat proteinopathies.
Previous studies showed PFI in I/R hearts (20
). Here, we found that CR-PA28αOE prevented and/or attenuated I/R injury–induced cardiac malfunction and significantly reduced infarct size, demonstrating that enhancing proteasome proteolytic function in cardiomyocytes protects against acute I/R injury. Increased oxidative stress is a major pathogenic factor in I/R injury (35
). We have recently shown that PA28αOE protects against oxidative stress in cultured cardiomyocytes, likely through enhancing the removal of oxidized proteins (23
). On one hand, both oxidative stress and oxidized proteins impair proteasome function (21
). On the other hand, proteasome impairment slows down the removal of the toxic oxidized proteins, thereby forming a vicious cycle in I/R hearts. Enhancing proteasome function and subsequently protecting against I/R injury interrupted and attenuated the pathogenic cycle. Alternatively, the observed protection of CR-PA28αOE against acute I/R injury may be interpreted as a preconditioning-like effect. This interpretation implies 2 possibilities: first, CR-PA28αOE represents a mild insult to the heart and triggers mechanisms that protect the heart from I/R injury; and second, CR-PA28αOE–induced proteasome functional enhancement acts like a mediator and/or executor of the preconditioning process to counter the pathogenic factors of I/R injury. The data in the present study and reported by others overwhelmingly favor the latter possibility, because (a) preserving proteasome function was recently shown as an important mechanism underlying ischemic preconditioning (20
); (b) our comprehensive baseline characterization of mice with CR-PA28αOE during their first year did not detect any adverse effects; and (c) we demonstrated here that the same enhancement of cardiac proteasome function remarkably rescued CryABR120G
-based DRC. Hence, this study provides compelling evidence that PFI plays an important role in acute myocardial I/R injury.
The mechanism by which PA28αOE enhances UPS-mediated degradation of abnormal proteins is not clear at this time, but our data support the notion that the 11S particle formed by PA28α and PA28β functions as an alternative activator for 20S and increases proteasome proteolytic activity directed at the abnormal or denatured/misfolded proteins. The 20S core can be capped by 11S at one end and 19S at the other to form a hybrid proteasome, or by 11S at both ends (25
). Association of the 11S with the 20S subcomplex stimulates the 20S peptidase activity (25
). Upregulation of 11S proteasomes in cardiomyocytes did not cause detectable changes in the protein levels of representative 19S or 20S subunits. Our gel filtration experiments using native myocardial protein extracts revealed that CR-PA28αOE increased the hybrid proteasome and the subpopulation of 20S proteasomes with both ends capped with the 11S. This was further demonstrated by our 20S IP experiments. More PA28α, but less Rpt6 (a bona fide 19S subunit), was co-immunoprecipitated with the α4 subunit of 20S from mouse hearts with CR-PA28αOE compared with that from littermate control hearts without PA28αOE. Our previous in vitro study showed that this increased association of the 11S with the 20S increases ATP-dependent and -independent proteasome peptidase activities in cultured cardiomyocytes (23
At the molecular level, an important shared pathological change between CryABR120G
-based DRC and I/R injury was increased production of misfolded/damaged proteins, which places increased demands on UPS-mediated protein degradation. Like normal proteins, misfolded proteins (with some exceptions) are generally ubiquitinated first and then degraded by the proteasome, in which the 19S is required for uptake of the ubiquitinated proteins. We hypothesize that PA28αOE increases the subpopulation of hybrid proteasomes (i.e., 19S-20S-11S), thereby allowing the UPS to respond to the increased demand. However, the functional significance of the hybrid proteasome has not been formally determined. We speculate that the hybrid proteasome is better equipped to degrade misfolded proteins than is the conventional 26S proteasome (19S-20S-19S or 19S-20S). It was previously shown that the association of 11S increases the peptidase activities of the 20S (42
); meanwhile, the associated 19S allows the hybrid proteasome to uptake ubiquitinated protein substrates. The rate-limiting step for UPS-mediated degradation of a native protein is the ubiquitination step, which often requires exposure or posttranslational maturation of its ubiquitination signal. For a misfolded protein, its ubiquitination signals, such as surface exposure of a patch of hydrophobic residues or cryptic ubiquitination signals that are normally buried in properly folded proteins, are born with the misfolding; hence, proteasome is conceivably the rate-limiting step (4
). This may explain why the homeostasis of normal proteins is not perturbed in PA28αOE cells and hearts, but remains a hypothesis to be further tested.
Notably, 11S proteasomes can be upregulated by IFN-γ (41
), a cytokine that has been clinically used to treat disease (44
). IFN-γ also increases expression of the inducible proteasome peptidase subunits (β1i, β2i, and β5i), which leads to the replacement of corresponding conventional peptidase subunits (β1, β2, and β5) and thereby the formation of immunoproteasomes (43
). Interestingly, a recent report compellingly demonstrated that upregulation of the immunoproteasome by IFNs not only plays a previously recognized role in helping antigen presentation, but also facilitates the removal of damaged proteins generated by IFN-induced oxidative stress (45
). Hence, it will be interesting to test whether upregulation of 11S proteasomes by pharmacological means mitigates the progression of heart disease in a model that displays PFI.