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Altered proteasome structure, function, and oxidation in aged muscle

Deborah A. Ferrington^*,1, Aimee D. Husom^*, and LaDora V. Thompson

Departments of
^* Ophthalmology and
Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, Minnesota, USA

¹Correspondence: Department of Ophthalmology, 380 Lions Research Bldg., 2001 6th Street SE, University of Minnesota, Minneapolis 55455, MN, USA. E-mail: ferri013{at}umn.edu

SPECIFIC AIMS

The purpose of this research was to identify a potential mechanistic explanation for accumulation of oxidized proteins in aged muscle. The decrease in protein turnover observed in aged muscle points to defects in the proteolytic pathway. We focused on the proteasome, the key protease responsible for the selective degradation of oxidized proteins. This study tests the hypothesis that proteasome function declines with aging.

PRINCIPAL FINDINGS

1. Increased immunoproteasome and reduced specific activity
The relative content of the 20S catalytic core was determined by Western immunoblot analysis using antibodies that recognize specific subunits of the proteasome. A 3-fold increase in the constitutively expressed subunits was measured, indicating that aged muscle contains a significantly greater concentration of the 20S proteasome than young muscle (P<0.05). A parallel 3-fold increase in the cytokine-induced ß subunits LMP2 and LMP7 was observed. These results indicate that the 3-fold increase in 20S proteasome is entirely due to higher expression of the immunoproteasome.

The peptidylglutamyl hydrolyzing, trypsin-like, and chymotrypsin-like activities were measured in proteasome-enriched preparations using fluorgenic peptides. Comparison of the kinetic parameters (V_max, K_m) showed no age-related difference in activity. When the relative content of proteasome was taken into account, proteasome-specific activity was 50% lower in aged muscle (P<0.05).

2. Inadequate content of proteasome activators
The content of the proteasome activator proteins PA28 and PA700 was measured by Western immunoblot analysis (Fig. 1 ). No significant difference in the concentration of either regulatory protein was observed in young and aged muscle. However, when the contents of PA28 and PA700 were compared with that of the 20S catalytic core, the ratio was 75% lower in aged muscle (P<0.05). To test the possibility that PA700 content is inadequate for maximal up-regulation of the 20S catalytic core, activity was measured in proteasome-enriched homogenates in the absence and presence of exogenously added PA700. While no change in activity was observed in young muscle, PA700 stimulated activity 2-fold in aged muscle.

View larger version (24K): [in this window] [in a new window]   Figure 1. Content of proteasome regulatory proteins, PA28 and PA700, in proteasome-enriched homogenates from young (solid) and aged (hatched) muscle. A) Content was determined from immune reactions on Western immunoblot analysis using antibodies specific to the subunit of PA28 or the S4 subunit of PA700. Relative density is the immune reaction expressed relative to young rats. n = 7–8 and 5–9 for young and aged rats, respectively. B) Ratio calculated for the immune reactions of the regulatory proteins and the reaction of the 6 subunit of the 20S proteasome. C) Hydrolysis of 50 µM LLE-AMC in proteasome-enriched homogenates in the absence (no PA) or presence of PA700 (+PA). Values represent proteasome-specific activity (mean±SE). *P= 0.03, comparing activity with and without addition of PA700 for aged muscle.

3. Activity and oxidation state of the 20S proteasome
Activity measured in the 20S proteasome purified from muscle showed no age-related difference. The disparity in these results compared with the lower specific activity in homogenates suggests aged muscle contains more endogenous inhibitors. Activity tested in the presence of DTT showed no age-related difference in peptidylglutamyl hydrolyzing activity, but there was significant activation of chymotrypsin-like activity in the 20S from aged muscle. These results are consistent with reversible cysteine modifications critically linked to chymotrypsin-like activity.

4. Decreased degradation of oxidized calmodulin and partial rescue by DTT
Degradation of unoxidized and oxidized calmodulin (CaM) by the 20S from young muscle showed oxidized CaM was degraded significantly faster than the unoxidized form (P<0.05) (Fig. 2 ). For 20S from aged muscle, rates of degradation for unoxidized and oxidized CaM were equivalent. Addition of DTT did not alter the response of 20S from young muscle. In contrast, DTT caused 2-fold increase in degradation of oxidized CaM for 20S from aged muscle. Proteolysis of oxidized CaM was still significantly slower for 20S from aged muscle compared with 20S from young muscle (P=0.04).

View larger version (39K): [in this window] [in a new window]   Figure 2. Degradation of calmodulin by the 20S proteasome purified from young and aged muscle. The calmodulin substrate was either not oxidized (solid) or maximally oxidized (hatched) by peroxide prior to digests. A) CaM degradation in the absence of DTT. B) CaM degradation in the presence of 5 mM DTT. Values are mean ± SE. Results of t test analysis comparing degradation of nonoxidized with oxidized CaM, *P= 0.05, **P= 0.02, ***0.01. n = 3 for each age group.

CONCLUSIONS AND SIGNIFICANCE

A summary of the structural and functional changes in proteasome from aged muscle and the ensuing potential outcomes is shown in Fig. 3 . Structural changes that were documented in this study include up-regulation of the immunoproteasome, increased subunit oxidation, and decreased content of the proteasome activating proteins relative to the 20S in aged muscle. The differential specific activities measured in homogenates (decreased) vs. purified 20S proteasome (equivalent) suggest the presence of more endogenous inhibitors in aged muscle. Other functional analyses of proteasome showed slower degradation of oxidized CaM and inadequate activation of the 20S by PA700 with aging. The potential outcomes from these defects may include accumulation of oxidized proteins, declines in 20S regulation by PA700 and PA28, and decreased degradation of ubiquitin-conjugated proteins.

View larger version (30K): [in this window] [in a new window]   Figure 3. Model of the proteasome in aged muscle. Solid boxes summarize structural and functional changes measured in this study. Dashed box and italics indicate potential outcomes and implications of the results.

We recently reported that proteasome from predominantly type I (slow-twitch) muscle contained a 3-fold increased content of immunoproteasome and lower proteasome specific activity as measured by the hydrolysis of fluorogenic peptides. The present study of proteasome in type II (fast-twitch) muscle confirmed the initial findings and provided potential mechanistic explanations behind the general loss in proteasome function with aging. Our rationale for examining proteasome function independently in slow- and fast-twitch muscles is based on fiber type-specific differences in aging effects: muscle atrophy and declines in muscle function are accelerated in fast-twitch muscle. Since the ubiquitin-proteasome pathway is the major proteolytic system for myofibrillar breakdown, we reasoned there could be fiber type differences in the proteasome. Our data show nearly identical kinetics for hydrolysis of peptides and relative changes in subunit content, suggesting the basic composition of proteasome is similar in fast- and slow-twitch muscles.

Our analysis of subunit composition showed a 3-fold increase in constitutive subunits that correlated with an equivalent increase in the cytokine-inducible ß subunits. Since the constitutive ß subunit expression was unchanged with aging, we interpret these results to indicate that the concentration of immunoproteasome is greater in aged muscle. We did not measure the relative content of ß2 and its inducible counterpart, MECL. However, it has been shown that incorporation of LMP2 and MECL into newly synthesized proteasome is mutually dependent; by inference, we can predict that MECL must be up-regulated in aged muscle. The mechanistic basis for the age-related change in proteasome composition and expression has not been investigated. However, cultured cells respond to the inflammatory cytokine TNF- by up-regulating expression of the inducible ß subunits. With aging, levels of circulating and muscle TNF- are significantly elevated. Hence, the increased TNF- could potentially stimulate the production of immunoproteasome in aged muscle.

Dahlman and colleagues recently used high-resolution anion chromatography to resolve six distinct subtypes of skeletal muscle 20S proteasome isolated from a young rat. Approximately 95% of the proteasome population was either composed exclusively of the constitutive subunits or contained a mixture of constitutive and inducible subunits. A minor component (5%) of the total proteasome population contained only the inducible subunits. Proteasomes containing the inducible subunits had lower chymotrypsin-like and peptidylglutamyl hydrolyzing activities. In the present study, the increased content of proteasomes containing the inducible subunits provides a partial explanation for the age-related decrease in specific activity.

The central role the immunoproteasome plays in immune function via generation of immunogenic peptides for antigen presentation has been established. However, the expression of cytokine-induced subunits in immune-privileged tissue such as the retina, lens epithelial cells, and the brain implies the immunoproteasomes may perform other undescribed nonimmune functions. In the current study we asked if the increased expression of immunoproteasome in aged muscle facilitates the degradation of oxidized proteins. Using CaM as our model protein, we found there was no appreciable degradation of oxidized CaM by 20S from aged muscle in the absence of the reducing agent DTT. While addition of DTT increased the degradation of oxidized CaM by aged muscle 20S, the rate of degradation was still slower than young muscle 20S. These results suggest that increased abundance of immunoproteasome does not provide a selective advantage for degrading oxidized proteins. These results highlight the requirement for a reducing environment to achieve maximal activity in aged proteasome. Aged organisms may be less effective in recovering from an oxidative insult due to their decreased ability to eliminate oxidized proteins.

The up-regulation of proteasome function measured by hydrolysis of the AAF peptide and degradation of oxidized CaM in the presence of DTT is consistent with the oxidation of critical cysteine residues in aged proteasome. These results support the idea that the chymotrypsin-like activity determines the rate of protein breakdown. Partial rescue of activity by DTT suggests at least a portion of cysteine residues are irreversibly oxidized. Examples of cysteine modifications not reversible by DTT included oxidation of the sulfhydryl group to sulfonic acid and covalent attachment of 4-hydroxynonenal. Oxidation of residues other than cysteine could contribute to proteasome inhibition. These results introduce an interesting paradox: the protease responsible for removing oxidized proteins is itself oxidized.

Association of PA28 and PA700 with the 20S proteasome significantly increases proteolysis of fluorogenic peptides and proteins. Binding of PA28 with the 20S core containing the inducible ß subunits aids in the generation of immunogenic peptides. Thus, the lower relative ratio of PA28 relative to the 20S catalytic core observed in aged muscle may have a negative impact on immune function. Association of PA700 with the 20S core forms the 26S proteasome, the species that recognizes and degrades ubiquitin-conjugated proteins. Selective degradation of ubiquitinylated proteins by the 26S proteasome regulates many pathways critical for cell survival, including signal transduction, apoptosis, and the cell cycle. In the current study, we demonstrated proteasome activation by exogenously added PA700 in only aged muscle. These results suggest the content of endogenous PA700 is inadequate for maximal activation of the 26S proteasome in aged muscle. While the lower ratio of PA700 relative to the 20S in aged muscle provides a likely explanation for these results, it is possible the endogenous PA700 may contain defects (i.e., oxidative modifications) that prevent its binding to and activation of the 20S. Nonetheless, it is likely that the decreased assembly of the 26S proteasome could result in delayed degradation of ubiquitin-conjugated proteins and the loss in coordinated cell signaling.

An additional explanation for the lower content of regulatory proteins relative to the 20S could be this is an adaptive response to the increased demand for degrading oxidized proteins in aged muscle. The lower levels of regulatory proteins would guarantee that a population of 20S would remain uncomplex and available to degrade oxidized proteins.

Results from this study have provided potential mechanistic explanations for the decline in proteasome function in aged muscle. The loss of proteasome function could allow oxidized proteins to accumulate in aged muscle.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2578fje; doi: 10.1096/fj.04-2578fje

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