To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with 35 S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to deglutathionylate itself. These data suggest that the intracellular glutathione͞glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions. T hioredoxin (Trx; ref. 1), a ubiquitous redox protein, is an essential cofactor electron donor for ribonucleotide reductase, but also has many other cellular functions, including regulation of transcription factors, apoptosis, and antioxidant activity and can act exogenously as a redox active growth factor (1, 2). The catalytic activity of Trx resides in its active site where the two redox active Cyss (Cys-31 and Cys-34 in human Trx) undergo reversible oxidation͞reduction. In addition to the conserved Cys residues in the active site, three additional structural Cys residues (Cys-61, Cys-68, and Cys-72) are present in the structure of human Trx.The present paper describes the experiments that led us to the conclusion that Trx can undergo glutathionylation in T cells exposed to oxidative stress. Protein glutathionylation, the formation of a disulfide between a Cys in a protein and the Cys in the tripeptide glutathione (GSH), is a modification that can be induced in cells by oxidative stress. Glutathionylation can occur by direct oxidation of a protein and GSH, by a thiol-disulfide exchange between a protein Cys and oxidized glutathione (GSSG), and also with the intermediacy of S-nitrosoglutathione (GSNO; refs. 3 and 4). Glutathionylation of proteins is reversible, as those proteins can be reduced by glutaredoxins (5, 6), and the process serves to regulate protein functions by the redox state of the cell (i.e., by the GSSG͞GSH ratio; refs. 7-9).To demonstrate the glutathionylation of Trx, we first labeled the intracellular GSH pool of the T-cell blasts with [ 35 S]Cys and analyzed the cell lysate by two dimensional electrophoresis under nonreducing conditions. A spot was found in the autoradiographic protein map with IP͞M r corresponding to those of Trx. In the second part of the study, we incubated recombinant human Trx in the presence or ...

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. ␣-and ␥-enolase, ATP synthase ␤ chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, ␣-enolase nitration at Tyr 43 , target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.

Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin ␣-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and ␤-actin. NAC pretreatment augmented integrin ␣-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease, and protein aggregation has been proposed as a possible pathogenetic mechanism. However, the aggregate protein constituents are poorly characterized so knowledge on the role of aggregation in pathogenesis is limited.Methodology/Principal FindingsWe carried out a proteomic analysis of the protein composition of the insoluble fraction, as a model of protein aggregates, from familial ALS (fALS) mouse model at different disease stages. We identified several proteins enriched in the detergent-insoluble fraction already at a preclinical stage, including intermediate filaments, chaperones and mitochondrial proteins. Aconitase, HSC70 and cyclophilin A were also significantly enriched in the insoluble fraction of spinal cords of ALS patients. Moreover, we found that the majority of proteins in mice and HSP90 in patients were tyrosine-nitrated. We therefore investigated the role of nitrative stress in aggregate formation in fALS-like murine motor neuron-neuroblastoma (NSC-34) cell lines. By inhibiting nitric oxide synthesis the amount of insoluble proteins, particularly aconitase, HSC70, cyclophilin A and SOD1 can be substantially reduced.Conclusion/SignificanceAnalysis of the insoluble fractions from cellular/mouse models and human tissues revealed novel aggregation-prone proteins and suggests that nitrative stress contribute to protein aggregate formation in ALS.

There is evidence that alterations in the normal physiological activity of PrP C contribute to prion-induced neurotoxicity. This mechanism has been difficult to investigate, however, because the normal function of PrP C has remained obscure, and there are no assays available to measure it. We recently reported that cells expressing PrP deleted for residues 105-125 exhibit spontaneous ionic currents and hypersensitivity to certain classes of cationic drugs. Here, we utilize cell culture assays based on these two phenomena to test how changes in PrP sequence and/or cellular localization affect the functional activity of the protein.We report that the toxic activity of ⌬105-125 PrP requires localization to the plasma membrane and depends on the presence of a polybasic amino acid segment at the N terminus of PrP. Several different deletions spanning the central region as well as three disease-associated point mutations also confer toxic activity on PrP. The sequence domains identified in our study are also critical for PrP Sc formation, suggesting that common structural features may govern both the functional activity of PrP C and its conversion to PrP Sc .Prion diseases or transmissible spongiform encephalopathies comprise a group of fatal neurodegenerative disorders in humans and animals that can be sporadic, infectious, or genetic in origin (1, 2). The prion protein (PrP C ) is a membrane-anchored glycoprotein with no widely agreed-upon physiological function, although its ability to convert into a self-propagating isoform (PrP Sc ) is associated with development of prion diseases. PrP C , thus, plays a crucial role in prion pathogenesis as a substrate for generation of PrP Sc , a conclusion that has been demonstrated by the resistance of PrP-null mice to prion infection (3). In addition, however, there is evidence that PrP C is required for delivery of a toxic signal during prion propagation. This is demonstrated by the fact that brain tissue from PrP-null mice grafted into wild-type animals remains healthy despite the presence of copious amounts of PrP Sc from the surrounding host brain (4). Moreover, conditional genetic ablation of neuronal PrP C allows pathological and clinical recovery of prioninfected mice (5). Therefore, prion neurotoxicity is likely due to subversion of normal PrP C function rather than loss of PrP C or gain of PrP Sc activity (6). However, progress in investigating this mechanism has been hampered by a lack of understanding of the physiological role of PrP C and the absence of assays to measure the functional activity of the protein. To address this issue, our laboratory has recently developed two assays that measure toxicity of PrP mutants expressed in cultured cells. The first assay is a drugbased cellular assay (DBCA) 3 that measures cell death resulting from increased accumulation of two classes of drugs that are normally used to select transfected cell lines (aminoglycosides and bleomycin analogues) (7, 8). The second assay utilizes whole-cell patch clamping to measure the la...

In prion diseases, the infectious isoform of the prion protein (PrP Sc ) may subvert a normal, physiological activity of the cellular isoform (PrP C ). A deletion mutant of the prion protein (⌬105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP C and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing ⌬105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of ⌬105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing ⌬105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP Sc) binds to the cellular form (PrP C), generating additional molecules of PrP Sc. While several regions of the PrP C molecule have been suggested to play a role in PrP Sc formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23–31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP Sc. These results, in combination with biochemical data, demonstrate that residues 23–31 represent a critical site on PrP C that binds to PrP Sc and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to β-sheet-rich oligomers that bind to PrP C.

Peptidylprolyl isomerase A (PPIA), also known as cyclophilin A, is a multifunctional protein with peptidyl-prolyl cis-trans isomerase activity. PPIA is also a translational biomarker for amyotrophic lateral sclerosis, and is enriched in aggregates isolated from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. Its normal function in the central nervous system is unknown. Here we show that PPIA is a functional interacting partner of TARDBP (also known as TDP-43). PPIA regulates expression of known TARDBP RNA targets and is necessary for the assembly of TARDBP in heterogeneous nuclear ribonucleoprotein complexes. Our data suggest that perturbation of PPIA/TARDBP interaction causes 'TDP-43' pathology. Consistent with this model, we show that the PPIA/TARDBP interaction is impaired in several pathological conditions. Moreover, PPIA depletion induces TARDBP aggregation, downregulates HDAC6, ATG7 and VCP, and accelerates disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Targeting the PPIA/TARDBP interaction may represent a novel therapeutic avenue for conditions involving TARDBP/TDP-43 pathology, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration.