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1.  Changes in 20S subunit composition are largely responsible for altered proteasomal activities in experimental autoimmune encephalomyelitis 
Journal of Neurochemistry  2012;121(3):486-494.
We recently reported that the proteasomal peptidase activities are altered in the cerebellum of mice with MOG peptide-induced experimental autoimmune encephalomyelitis (EAE). To determine whether these fluctuations are caused by proteasome activation/inactivation and/or changes in the levels of individual β subunits, we characterized the proteasome subunit composition by western blotting. The results show that the rise in proteasomal peptidase activity in acute EAE correlates with an augmented expression of inducible β subunits whereas the decline in activity in chronic EAE correlates with a reduction in the amount of standard β subunits. Using pure standard (s) and immuno (i) 20S particles for calibration, we determined that the changes in the levels of catalytic subunits account for all of the fluctuations in peptidase activities in EAE. The i-20S and s-20S proteasome were found to degrade carbonylated β-actin with similar efficiency, suggesting that the amount of protein carbonyls in EAE may be controlled by the activity of both core particles. We also found an increase in proteasome activator PA28 and a decrease in inhibitor PI31 levels in acute EAE, reflecting a response to inflammation. Elevated levels of PA700 and PA28 in chronic EAE, on the other hand, may occur in response to diminished proteasomal activity in this phase. These findings are central towards understanding the altered proteasomal physiology in inflammatory demyelinating disorders.
doi:10.1111/j.1471-4159.2012.07699.x
PMCID: PMC3323733  PMID: 22353035
20S proteasome; immunoproteasome; PA28; PA700; PI31; proteasomal dysfunction; experimental autoimmune encephalomyelitis; multiple sclerosis; oxidative stress; protein carbonylation
2.  Increased carbonylation, protein aggregation and apoptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis 
ASN NEURO  2013;5(2):e00111.
Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both MS (multiple sclerosis) and its animal model EAE (experimental autoimmune encephalomyelitis). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate whether their association can be also established in vivo. In the present study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of MOG (myelin-oligodendrocyte glycoprotein)35–55 peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3 (microtubule-associated protein light chain 3)-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Taken together, the results of the present study suggest a link between protein oxidation and neuronal/glial cell death in vivo, and also demonstrate impaired proteostasis in this widely used murine model of MS.
doi:10.1042/AN20120088
PMCID: PMC3620690  PMID: 23489322
apoptosis; autophagy; experimental autoimmune encephalomyelitis; oxidative stress; protein aggregation; protein carbonylation; proteostasis; AMC, 7-aminomethyl-4-coumarin; APC, adenomatous polyposis coli protein C-terminus; CFA, complete Freund’s adjuvant; CNS, cenral nervous system; DAPI, 4′,6-diamidino-2-phenylindole; DNP, 2,4-dinitrophenyl; DNPH, 2,4-dinitrophenylhydrazine; DPI, days post-immunization; EAE, experimental autoimmune encephalomyelitis; ECL, enhanced chemiluminescence; GFAP, glial fibrillary-associated protein; HRP, horseradish peroxidase; LC3, microtubule-associated protein light chain 3; MOG, myelin-oligodendrocyte glycoprotein; MS, multiple sclerosis; TBARS, thiobarbituric acid-reacting substances; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling
3.  Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis 
Journal of neurochemistry  2007;105(3):763-772.
Protein carbonylation, the non-enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis (MS). In this study we investigated whether protein carbonyls (PCOs) also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2-dimensional-oxyblot we identified several cytoskeletal proteins, including β-actin, GFAP and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull-down experiments, which also showed an increase in the number of carbonylated β-actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in MS and EAE.
doi:10.1111/j.1471-4159.2007.05178.x
PMCID: PMC3599778  PMID: 18088377
protein carbonylation; oxidative stress; cytoskeletal degradation; experimental autoimmune encephalomyelitis; multiple sclerosis
4.  IDENTIFICATION OF MAJOR S-NITROSYLATED PROTEINS IN MURINE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS 
Journal of neuroscience research  2009;87(13):2881-2889.
Nitrosative stress has been implicated in the pathophysiology of several CNS disorders including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG35-55 peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. Using this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, α/β-tubulin, β-actin and GAPDH is increased in EAE. Other potential substrates were either not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, γ-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.
doi:10.1002/jnr.22113
PMCID: PMC3599784  PMID: 19405105
MS; nitric oxide; protein nitrosothiol; spinal cord; EAE
5.  Protein carbonylation and aggregation precede neuronal apoptosis induced by partial glutathione depletion 
ASN NEURO  2012;4(3):e00084.
While the build-up of oxidized proteins within cells is believed to be toxic, there is currently no evidence linking protein carbonylation and cell death. In the present study, we show that incubation of nPC12 (neuron-like PC12) cells with 50 μM DEM (diethyl maleate) leads to a partial and transient depletion of glutathione (GSH). Concomitant with GSH disappearance there is increased accumulation of PCOs (protein carbonyls) and cell death (both by necrosis and apoptosis). Immunocytochemical studies also revealed a temporal/spatial relationship between carbonylation and cellular apoptosis. In addition, the extent of all three, PCO accumulation, protein aggregation and cell death, augments if oxidized proteins are not removed by proteasomal degradation. Furthermore, the effectiveness of the carbonyl scavengers hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies PCOs as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. While the study focused mostly on nPC12 cells, experiments in primary neuronal cultures yielded the same results. The findings are also not restricted to DEM-induced cell death, since a similar relationship between carbonylation and apoptosis was found in staurosporine- and buthionine sulfoximine-treated nPC12 cells. In sum, the above results show for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. To the best of our knowledge, this is the first study to place direct (oxidative) protein carbonylation within the apoptotic pathway.
doi:10.1042/AN20110064
PMCID: PMC3322383  PMID: 22376187
apoptosis; cell death; glutathione depletion; proteasome; protein carbonylation; protein aggregation; Ab, antibody; ACR, acrolein; AMC, 7-amino-4-methylcoumarin; BSO, buthionine sulfoximine; CNS, central nervous system; DTT, dithiothreitol; EPO, epoxomicin; DEM, diethyl maleate; DNP, 2,4-dinitrophenyl; DNPH, 2,4-dinitrophenylhydrazine; EAE, experimental autoimmune encephalomyelitis; GAP-43, growth-associated protein 43; 4-HNE, 4-hydroxynonenal; LDH, lactate dehydrogenase; mAb, monoclonal antibody; MDA, malondialdehyde; MPTP, mitochondrial permeability transition pore; NFH, neurofilament heavy chain; NGF, nerve growth factor; nPC12, neuron-like PC12; PCO, protein carbonyl; RCS, reactive carbonyl species; ROS, reactive oxygen species; TBARS, thiobarbituric acid-reactive substances; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling; zVAD-fmk, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone
6.  Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis 
Journal of neurochemistry  2011;117(1):143-153.
Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). While oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (β1, β2, and β5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps PA28α and PA700 are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.
doi:10.1111/j.1471-4159.2011.07182.x
PMCID: PMC3055957  PMID: 21235577
Multiple sclerosis; proteases; 20S proteasome; protein carbonylation; white matter; gray matter
7.  Reduced proteasomal activity contributes to the accumulation of carbonylated proteins in chronic experimental autoimmune encephalomyelitis 
Journal of neurochemistry  2010;115(6):1556-1567.
We have recently shown that several carbonylated proteins, including GFAP, β-actin and β-tubulin, accumulate within cerebellar astrocytes during the chronic phase of MOG35–55 peptide-induced EAE in C57BL/6 mice. Since protein carbonyls cannot be repaired and there is less oxidative stress in chronic than in acute EAE, we hypothesized that the accumulation of carbonylated proteins in these animals may be due to a defect in the degradation of the modified proteins. Alternatively, oxidized proteins in chronic EAE mice may be more resistant to proteolysis. Using LPS-stimulated astrocytes and several protease inhibitors we identified the 20S proteasome as the proteolytic system responsible for the elimination of most oxidized proteins. We also discovered that the chymotrysin-like and caspase-like activities of the 20S proteasome are impaired in chronic EAE, while the amount of proteasome was unchanged. Proteasome failure in these animals was confirmed by the build-up of ubiquitinated proteins, mostly within astrocytes. In a cell-free system, carbonylated proteins from EAE mice with acute and chronic disease seem to be equally sensitive to proteasomal degradation. Altogether, the results support the notion that diminished activity of the 20S proteasome is a major contributor to the accumulation of carbonylated proteins in astrocytes of chronic EAE mice.
doi:10.1111/j.1471-4159.2010.07062.x
PMCID: PMC2996488  PMID: 20950414
astrocyte; experimental autoimmune encephalomyelitis; oxidative stress; proteasome; protein carbonylation
8.  Accumulation of protein carbonyls within cerebellar astrocytes in murine experimental autoimmune encephalomyelitis 
Journal of neuroscience research  2010;88(15):3376-3385.
Recent work from our laboratory has implicated protein carbonylation in the pathophysiology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The present study was designed to determine the changes in protein carbonylation during the disease progression, and to identify the target cells and modified proteins in the cerebellum of EAE animals, prepared by active immunization of C57/BL6 mice with MOG35-55 peptide. In this model, protein carbonylation was maximal at the peak of the disease (acute phase) to decrease thereafter (chronic phase). Double immunofluorescence microscopy of affected cerebella showed that carbonyls accumulate in white matter astrocytes, and to a lesser extent in microglia/macrophages, both in the acute and chronic phase. Surprisingly, T cells, oligodendrocytes and neurons were barely stained. By 2D-oxyblot and mass spectrometry, β-actin, β-tubulin, GFAP and HSC-71 were identified as the major targets of carbonylation throughout disease. Using a pull-down/western blot method we found a significant increase in the proportion of carbonylated β-actin, β-tubulin and GFAP in the chronic phase but not in the acute phase. These results suggest that as disease progresses from the inflammatory to the neurodegenerative phase there may be an inappropriate removal of oxidized cytoskeletal proteins. Additionally, the extensive accumulation of carbonylated GFAP in the chronic phase of EAE may be responsible for the abnormal shape of astrocytes observed at this stage.
doi:10.1002/jnr.22488
PMCID: PMC2953568  PMID: 20857508
astrocyte; cytoskeleton; experimental autoimmune encephalomyelitis; GFAP; multiple sclerosis; oxidative stress; protein carbonylation
9.  INTRACELLULAR GLUTATHIONE MEDIATES THE DENITROSYLATION OF PROTEIN NITROSOTHIOLS IN THE RAT SPINAL CORD1 
Journal of neuroscience research  2009;87(3):701-709.
Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S-nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t1/2 ~ 2h), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH-dependent enzymes (glutathione S-transferase, glutathione peroxidase and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense-III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the CNS of patients with multiple sclerosis.
doi:10.1002/jnr.21897
PMCID: PMC3213695  PMID: 18831065
nitric oxide; protein nitrosothiol; glutathione; spinal cord; denitrosylation

Results 1-9 (9)