Downhill exercise alters immunoproteasome content in mouse skeletal muscle

Content of the immunoproteasome, the inducible form of the standard proteasome, increases in atrophic muscle suggesting it may be associated with skeletal muscle remodeling. However, it remains unknown if the immunoproteasome responds to stressful situations that do not promote large perturbations in skeletal muscle proteolysis. The purpose of this study was to determine how an acute bout of muscular stress influences immunoproteasome content. To accomplish this, wildtype (WT) and immunoproteasome knockout lmp7-/-/mecl1-/-(L7M1) mice were run downhill on a motorized treadmill. Soleus muscles were excised 1 and 3 days post-exercise and compared to unexercised muscle(control). Ex vivophysiology, histology and biochemical analyses were used to assess the effects of immunoproteasome knockout and unaccustomed exercise. Besides L7M1 muscle being LMP7/MECL1deficient, no other major biochemical, histological or functional differences were observed between the control muscles. In both strains, the downhill run shifted the force-frequency curve to the right and reduced twitch force, however did not alter tetanic force or inflammatory markers. In the days post-exercise, several of the proteasome 's catalytic subunits were upregulated. Specifically, WT muscle increased LMP7 while L7M1 muscle instead increased ≤ 5. These findings indicate that running mice downhill results in subtle contractile characteristics that correspond to skeletal muscle injury, yet does not appear to induce a significant inflammatory response. Interestingly, this minor stress activated the production of specific immunoproteasome subunits; that if knocked out, were replaced by components of the standard proteasome. These data suggest that the immunoproteasome may be involved in maintaining cellular homeostasis.This study was supported by the Elaine and Robert Larson Endowed Vision Research Chair (to DAF), the National Institutes of Health/National Institute of Aging (T32-AG29796 to CWB), an anonymous benefactor for Macular Degeneration Research, the Lindsay Family Foundation and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology and Visual Neurosciences. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. (Elaine and Robert Larson Endowed Vision Research Chair; T32-AG29796 - National Institutes of Health/National Institute of Aging; Lindsay Family Foundation; Research to Prevent Blindness)Accepted manuscrip

Denervation-induced activation of the standard proteasome and immunoproteasome

The standard 26S proteasome is responsible for the majority of myofibrillar protein degradation leading to muscle atrophy. The immunoproteasome is an inducible form of the proteasome. While its function has been linked to conditions of atrophy, its contribution to muscle proteolysis remains unclear. Therefore, the purpose of this study was to determine if the immunoproteasome plays a role in skeletal muscle atrophy induced by denervation. Adult male C57BL/6 wild type (WT) and immunoproteasome knockout lmp7-/-/mecl-1-/- (L7M1) mice underwent tibial nerve transection on the left hindlimb for either 7 or 14 days, while control mice did not undergo surgery. Proteasome activity (caspase-, chymotrypsin-, and trypsin- like), protein content of standard proteasome (β1, β5 and β2) and immunoproteasome (LMP2, LMP7 and MECL-1) catalytic subunits were determined in the gastrocnemius muscle. Denervation induced significant atrophy and was accompanied by increased activities and protein content of the catalytic subunits in both WT and L7M1 mice. Although denervation resulted in a similar degree of muscle atrophy between strains, the mice lacking two immunoproteasome subunits showed a differential response in the extent and duration of proteasome features, including activities and content of the β1, β5 and LMP2 catalytic subunits. The results indicate that immunoproteasome deficiency alters the proteasome's composition and activities. However, the immunoproteasome does not appear to be essential for muscle atrophy induced by denervation.T32 AG029796 - NIA NIH HH

Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase

AbstractMethionine sulfoxide (MetSO) in calmodulin (CaM) was previously shown to be a substrate for bovine liver peptide methionine sulfoxide reductase (pMSR, EC 1.8.4.6), which can partially recover protein structure and function of oxidized CaM in vitro. Here, we report for the first time that pMSR selectively reduces the D-sulfoxide diastereomer of CaM-bound L-MetSO (L-Met-D-SO). After exhaustive reduction by pMSR, the ratio of L-Met-D-SO to L-Met-L-SO decreased to about 1:25 for hydrogen peroxide-oxidized CaM, and to about 1:10 for free MetSO. The accumulation of MetSO upon oxidative stress and aging in vivo may be related to incomplete, diastereoselective, repair by pMSR

Identification of a novel phosphorylation site in ataxin-1

AbstractSpinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein–protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1

In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals

AbstractSarcoplasmic reticulum (SR) Ca-ATPase of young adult (5 months) and aged (28 months) Fischer 344 male rat skeletal muscle was analyzed for posttranslational modifications as a result of biological aging and their potential functional consequences. The significant differences in the amino acid composition were a 6.8% lower content of sulfhydryl groups and a ca. 4% lower content of Arg residues of the Ca-ATPase from old as compared to young rats. Based on a total of 24 Cys residues the difference in protein thiols corresponds to a loss of 1.5 mol Cys/mol Ca-ATPase as a result of in vivo aging. The loss of Cys residues was not accompanied by a loss of enzyme activity though the `aged' Ca-ATPase was more sensitive to heat inactivation, aggregation, and tryptic digestion. A comparison of the total sulfhydryl content of all SR proteins present revealed a 13% lower amount for SR vesicles isolated from aged rats. Compared to the alterations of Cys and Arg, there was only a slight and probably physiologically insignificant increase of protein carbonyls with aging, i.e. from 0.32 to 0.46 mol carbonyl groups per mol of Ca-ATPase. When SR vesicles from young rats were exposed to AAPH-derived peroxyl radicals, there was a loss of ca. 1.38×10−4 M total SR sulfhydryl groups per 4 mg SR protein/ml (corresponding to ca. 25%) and a loss of 9.6×10−5 M Ca-ATPase sulfhydryl groups (corresponding to ca. 31%) per 1.6×10−5 M initiating peroxyl radicals, indicating that the stoichiometry of sulfhydryl oxidation was ≥6 oxidized thiols per initiating AAPH-derived peroxyl radical. Besides Cys, the exposure to AAPH-derived radicals caused a slight loss of Ca-ATPase Arg, Met, and Ser residues. Most importantly, the SR Ca-ATPase exposed to this low concentration of peroxyl radicals displayed physical and functional properties quantitatively comparable to those of SR Ca-ATPase isolated from aged rats, i.e. no immediate loss of activity, increased susceptibility to heat inactivation, aggregation, and tryptic digestion. Moreover, a comparison of kinetically early tryptic fragments by HPLC-electrospray MS and N-terminal sequencing revealed that similar peptide fragments were produced from `aged' and AAPH-oxidized Ca-ATPase which were not (or kinetically significantly later) generated from the `young' Ca-ATPase, suggesting some conformational changes of the Ca-ATPase as a result of aging and AAPH-exposure. All except one of these peptides originated from locations remote from the nucleotide-binding and calcium-binding sites. The latter results suggest that aging and AAPH-exposure may target similar Cys residues, mainly at locations remote from the nucleotide-binding and calcium-binding sites, rationalizing the fact that Cys oxidation did not immediately cause inactivation of the Ca-ATPase. Our results provide a quantitative estimate of a net concentration of reactive oxygen species, here peroxyl radicals, which induces physical and chemical alterations of the SR Ca-ATPase quantitatively comparable to those induced by in vivo aging

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1 alpha in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1 alpha dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1 alpha dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.Peer reviewe

Glutathione Metabolism and the Novel Role of Mitochondrial GSH in Retinal Degeneration

Glutathione (GSH) is present ubiquitously, and its role as a crucial cellular antioxidant in tissues, including the retina, is well established. GSH’s antioxidant function arises from its ability to scavenge reactive oxygen species or to serve as an essential cofactor for GSH S-transferases and peroxidases. This review summarizes the general functions, retinal distribution, disorders linked to GSH deficiency, and the emerging role for mitochondrial GSH (mGSH) in retinal function. Though synthesized only in the cytosol, the presence of GSH in multiple cell organelles suggests the requirement for its active transport across organellar membranes. The localization and distribution of 2-oxoglutarate carrier (OGC) and dicarboxylate carrier (DIC), two recently characterized mitochondrial carrier proteins in RPE and retina, show that these transporters are highly expressed in human retinal pigment epithelium (RPE) cells and retinal layers, and their expression increases with RPE polarity in cultured cells. Depletion of mGSH levels via inhibition of the two transporters resulted in reduced mitochondrial bioenergetic parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) and increased RPE cell death. These results begin to reveal a critical role for mGSH in maintaining RPE bioenergetics and cell health. Thus, augmentation of mGSH pool under GSH-deficient conditions may be a valuable tool in treating retinal disorders, such as age-related macular degeneration and optic neuropathies, whose pathologies have been associated with mitochondrial dysfunction

Carbonic anhydrase III and four-and-a-half LIM protein 1 are preferentially oxidized with muscle unloading

The identities of proteins that show disuse-related changes in the content of oxidative modification are unknown. Furthermore, it is unknown whether the global accumulation of oxidized proteins is greater in aged animals with muscle disuse. The purposes of this study are 1) to identify the exact proteins that show disuse-related changes in oxidation levels and 2) to test the hypothesis that the global accumulation of oxidized proteins with muscle disuse would be greater in aged animals. Adult and old rats were randomized into four groups: weight bearing and 3, 7, or 14 days of hindlimb unloading. Soleus muscles were harvested to investigate the protein oxidation with unloading. Slot blot, SDS-PAGE, and Western blot analyses were used to detect the accumulation of 4-hydroxy-2-nonenol (HNE)- and nitrotyrosine (NT)-modified proteins. Matrix-assisted laser desorption ionization-time of flight and tandem mass spectroscopy were used to identify modified proteins. We found that global HNE- and NT-modified proteins accumulated significantly with aging but not with muscle unloading. Two HNE and NT target proteins, four-and-a-half LIM protein 1 (FHL1) and carbonic anhydrase III (CAIII), showed changes in the oxidation levels with muscle unloading. The changes in the oxidation levels happened to adult rats but not old rats. However, old rats had higher baseline levels of HNE-modified FHL1. In summary, the data suggest that the muscle unloading-related changes of protein oxidation are more significant in specific proteins and that the changes are age related