The proteasome is the main protease for degrading oxidized proteins. We asked whether altered proteasome function contributes to the accumulation of oxidized muscle proteins with aging. Proteasome structure, function, and oxidation state were compared in young and aged F344BN rat fast-twitch skeletal muscle. In proteasome-enriched homogenates from aged muscle, we observed a two- to threefold increase in content of the 20S proteasome that was due to a corresponding increase in immunoproteasome. Content of the regulatory proteins, PA700 and PA28, relative to the 20S were reduced 75% with aging. Upon addition of exogenous PA700, there was a twofold increase in peptide hydrolysis in aged muscle, suggesting the endogenous content of PA700 is inadequate for complete activation of the 20S. Measures of catalytic activity showed a 50% reduction in specific activity for proteasome-enriched homogenates with aging. With purification of the 20S, proteasome specific activity was equivalent between ages, indicating that endogenous regulators inhibit proteasome in aged muscle. Significantly less degradation of oxidized calmodulin by the 20S from aged muscle was observed. Partial rescue of activity for aged 20S by DTT implies oxidation of functionally significant cysteines. These results demonstrate significant age-related changes in proteasome structure, function, and oxidation state that could inhibit removal of oxidized proteins.

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We describe a strategy for the identification of carbonylated proteins from complex protein mixtures that combines biotin hydrazide labeling of protein carbonyl groups, avidin affinity chromatography, multiplexed iTRAQ reagent stable isotope labeling, and analysis using pulsed Q dissociation (PQD) operation on an LTQ linear ion trap mass spectrometer. This strategy provided the ability to distinguish biotin hydrazide labeled, avidin purified, carbonylated proteins from non-carbonylated background proteins with affinity for the avidin column, derived from a control sample. Applying this strategy to the identification of crudely enriched rat skeletal muscle mitochondrial protein isolates, we generated a catalogue of over 200 carbonylated proteins by virtue of their quantitative enrichment compared to the control sample. The catalogue contains many mitochondrial localized proteins shown to be susceptible to carbonyl modification for the first time, including numerous transmembrane proteins involved in oxidative phosphorylation. Other oxidative modifications (e.g. nitrosylation, hydroxylation) were also identified on many of the carbonylated proteins, providing further evidence of the susceptibility of these proteins to oxidative damage. The results also demonstrate the utility of PQD operation on the LTQ instrument for quantitative analysis of iTRAQ reagent-labeled peptide mixtures, as well as the quantitative reproducibility of the avidin-affinity enrichment method.

One of the remarkable features of skeletal muscle is its adaptability. Skeletal muscle adaptations are characterized by modifications of morphological, biochemical, and molecular variables that alter the functional attributes of specific skeletal muscle fiber types. Skeletal muscle adaptation is diverse and the magnitude of change is dependent on many factors, such as activity pattern, age, and muscle fiber type composition. The adaptation of skeletal muscle in the adult population is well described. In contrast, the adaptation of skeletal muscle in the older population is less documented, especially in the area of inactivity-induced alterations. Age-related changes in skeletal muscle may play a significant role in the magnitude of change with inactivity and influence the rehabilitation process for the older adult.A consistent feature of age and inactivity is limb muscle atrophy and the loss of peak force and power. Differences exist in the rate and mechanisms of muscle wasting and in the susceptibility of a given fiber type to atrophy. Most likely, the rapid muscle wasting might be in part due to a decrease in protein synthesis coupled with an increased degradation. Besides the quantitative change in muscle mass, age and inactivity induce important qualitative changes in the structure of key skeletal muscle proteins that are manifested in alterations in contractile properties.Therefore, the purpose of this clinical commentary is to identify the major effects of age and inactivity on skeletal muscle structure and function, and discuss potential therapeutic interventions. Special emphasis will be placed on how alterations in muscle structure affect function and on the cellular and molecular mechanisms of the age-related and inactivity-induced muscle changes.

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