Targeted disruption of specific steps of the ubiquitin–proteasome pathway by oxidation in lens epithelial cells

Hosler, Mathew R; Wang-Su, Shuh-Tuan; Wagner, B.J.

doi:10.1016/s1357-2725(02)00397-7

Cited by 17 publications

(16 citation statements)

References 43 publications

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“…Similar to previous studies (29,57), the present experiments found no alteration in the composition of the 20 S CP or 26 S proteasome after exposure to GSH/H 2 O 2 . Additionally, oxidation of Rpn1 and Rpn2 did not affect their association with the 20 S CP.…”

Section: Discussionsupporting

confidence: 92%

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

Żmijewski

Banerjee²,

Abraham

2009

Journal of Biological Chemistry

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Although increased intracellular concentrations of hydrogen peroxide (H 2 O 2 ) are associated with inhibition of 26 S proteasomal activity, the mechanisms responsible for such effects have not been well delineated. In the present studies, we found that direct exposure of purified 26 S proteasomes to H 2 O 2 had negligible effects on their activity, whereas incubation with glutathione and H 2 O 2 produced >80% decrease in chymotrypsin-like and trypsin-like activities. Rpn1 and Rpn2, which are subunits of the 19 S regulatory particle, undergo S-glutathionylation after exposure of purified 26 S proteasomes to glutathione and H 2 O 2 , as well as in HEK 293 cells and neutrophils incubated with H 2 O 2 . Increased oxidation of Rpn1 and Rpn2 cysteine thiols was also found in lung extracts from mice in which catalase was inactivated, a condition associated with augmented intracellular concentrations of H 2 O 2 and diminished 26 S proteasomal activity. Although unoxidized Rpn2 enhanced 20 S proteolytic function in vitro, such potentiation was not found when the 20 S core particle was incubated with oxidized Rpn2. The composition of 26 S proteasomes was not altered after exposure to glutathione and H 2 O 2 , with similar amounts of Rpn1 and Rpn2 in control or oxidized 26 S proteasomal complexes. These findings identify S-glutathionylation of Rpn2 as a contributory mechanism for H 2 O 2 -induced inhibition of 26 S proteasomal function.The ubiquitin-proteasomal system is a major pathway for protein degradation and is involved in multiple cellular processes, including cell cycle control, expression of cell surface receptors, and regulation of intracellular levels of structural and regulatory proteins and transcription factors (1-6). The 26 S proteasome is composed of two main components, a proteolytic 20 S core particle (CP) 2 and an ATPase containing 19 S regulatory particle (RP) (7-10). Ubiquitinated proteins are unfolded in the 19 S RP and then translocated into the 20 S CP where they are degraded. The 20 S CP is composed of four heptameric rings of ␣ and ␤ subunits, with the two inner ␤ rings having proteolytic activity. The 19 S RP consists of a lid and base subcomplex, with the base subcomplex, including the nonenzymatic Rpn1 and Rpn2 toroids, surrounded by six AAA-type ATPases. Rpn1 and Rpn2 appear to play a major regulatory role by modulating the translocation and subsequent degradation of ubiquitinated substrates in the proteolytic core of the 20 S CP (7,(11)(12)(13)(14)(15).Decreased 26 S proteasomal activity, resulting in the accumulation of intracellular proteins, has been associated with aging (16, 17) and with pathophysiologic processes, including heart failure, neurodegenerative diseases, and alcohol induced liver injury (18 -20). A number of recent studies have shown that time-limited inhibition of the 26 S proteasome is beneficial as a therapeutic intervention for malignancies, such as multiple myeloma, and in reducing inflammation and cell injury associated with ischemia-reperfusion injury and transplanta...

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Section: Discussionsupporting

confidence: 92%

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

Żmijewski

Banerjee²,

Abraham

2009

Journal of Biological Chemistry

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“…The ability of proteasomes to degrade oxidized proteins has been previously well documented in vitro (Rivett, 1985;Davies, 2001), and more recently in vivo (Grune et al, 2002a;Hosler et al, 2003;Shringarpure et al, 2003). Most of the studies assessing oxidation-induced changes in lysosomal proteolytic behavior have focused in the analysis of the enzymatic activity of cathepsins, the lysosomal proteases.…”

Section: Discussionmentioning

confidence: 99%

“…The contribution of proteasomes to the removal of oxidized proteins in vivo has been demonstrated by analyzing the consequences of blocking its catalytic activity in oxidized protein removal (Grune et al, 2002a;Hosler et al, 2003;Shringarpure et al, 2003). This approach, however, cannot be used to evaluate the contribution of autophagy in this process.…”

Section: Discussionmentioning

confidence: 99%

“…One of the most extensively analyzed protease in this respect has been the proteasome. Although most of the studies linking the proteasome to the degradation of oxidized proteins have been carried out in vitro (Rivett, 1985;Davies, 2001), recent evidence supports the participation of the proteasome in the removal of oxidized proteins in vivo also (Hosler et al, 2003;Shringarpure et al, 2003). Thus, treatment of culture cells with proteasome inhibitors or antisense oligonucleotides against essential proteasome subunits results in accumulation of oxidized proteins inside cells and diminished rates of cell survival during oxidative stress.…”

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confidence: 99%

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Activation of Chaperone-mediated Autophagy during Oxidative Stress

Kiffin

Christian

Knecht

et al. 2004

MBoC

556

524

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Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. The activity of chaperonemediated autophagy (CMA), a selective pathway for the degradation of cytosolic proteins in lysosomes, decreases with age. We have analyzed the possible participation of CMA in the removal of oxidized proteins in rat liver and cultured mouse fibroblasts. Added to the fact that CMA substrates, when oxidized, are more efficiently internalized into lysosomes, we have found a constitutive activation of CMA during oxidative stress. Oxidation-induced activation of CMA correlates with higher levels of several components of the lysosomal translocation complex, but in particular of the lumenal chaperone, required for substrate uptake, and of the lysosomal membrane protein (lamp) type 2a, previously identified as a receptor for this pathway. In contrast with the well characterized mechanism of CMA activation during nutritional stress, which does not require de novo synthesis of the receptor, oxidation-induced activation of CMA is attained through transcriptional up-regulation of lamp2a. We conclude that CMA is activated during oxidative stress and that the higher activity of this pathway under these conditions, along with the higher susceptibility of the oxidized proteins to be taken up by lysosomes, both contribute to the efficient removal of oxidized proteins. INTRODUCTIONAccumulation of oxidized protein is a common feature of aged cells (Dunlop et al., 2002;Grune et al., 2001Grune et al., , 2002bStadtman, 2001). Many physiological and pathological processes lead to the generation of free radicals and consequent damage of intracellular components, including proteins. In most of these oxidative events, damaged proteins are removed from the cell through degradation by proteases (Dunlop et al., 2002). The activity of different intracellular proteolytic systems decreases with age (Terman, 1995;Cuervo and Dice, 1998a;Friguet et al., 2000;Carrard et al., 2002;Donati et al., 2001;Merker et al., 2001;Friguet, 2002;Keller et al., 2002;Ward, 2002), and this impaired activity is considered responsible for the deficient removal of oxidized proteins in old organisms (Grune, 2000;Merker and Grune, 2000;Dunlop et al., 2002;Szweda et al., 2002).The susceptibility of oxidized proteins to proteases changes with the duration and degree of oxidative damage (Dunlop et al., 2002;Mehlhase and Grune, 2002). Thus, mild oxidation induces partial protein unfolding and facilitates proteolytic cleavage (Grune et al., 1995. However, persistent or extensive oxidative damage usually promotes protein aggregation, due to the exposure of patches of hydrophobic amino acids. Once a protein aggregates, it becomes less susceptible to proteolytic cleavage (Hoff et al., 1993;Davies, 2001;Demasi and Davies, 2003). Kinetics of degradation of oxidized proteins in vitro have been analyzed using different types of proteases (Merker and Grune, 2000;Dunlop et al., 2002;Szweda et al., 2002). One of the most extensively analyzed protease in this respect has be...

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“…Proteasomes and ubiquitin systems in the lens are involved in the removal of oxidized proteins or peptides (35,36). Despite this, truncated proteins/short peptides might accumulate in the lens because of their excessive production or because the degradation system cannot break down the fragments (37)(38)(39)(40). Earlier studies showed that the lens contains significant quantities of crystallin fragments (6,26), and the complex formed by the interaction of crystallin peptides with proteins in vivo can be modified further to form covalent multimers (5).…”

Section: Discussionmentioning

confidence: 99%

Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation

Santhoshkumar

Udupa

Raju

et al. 2008

Journal of Biological Chemistry

110

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Analysis of aged and cataract lenses shows the presence of increased amounts of crystallin fragments in the high molecular weight aggregates of water-soluble and water-insoluble fractions. However, the significance of accumulation and interaction of low molecular weight crystallin fragments in aging and cataract development is not clearly understood. In this study, 23 low molecular mass (<3.5-kDa) peptides in the urea-soluble fractions of young, aged, and aged cataract human lenses were identified by mass spectroscopy. Two peptides, ␣B-(1-18) (MDIAIHHPWIRRPFFPFH) and ␤A3/A1-(59 -74) (SD(N)AYHIERLMSFRPIC), present in aged and cataract lens but not young lens, and a third peptide, ␥S-(167-178) (SPAVQSFRRIVE) present in all three lens groups were synthesized to study the effects of interaction of these peptides with intact ␣-, ␤-, and ␥-crystallins and alcohol dehydrogenase, a protein used in aggregation studies. Interaction of ␣B-(1-18) and ␤A3/A1-(59 -74) peptides increased the scattering of light by ␤-and ␥-crystallin and alcohol dehydrogenase. The ability of ␣-crystallin subunits to function as molecular chaperones was significantly reduced by interaction with ␣B-(1-18) and ␤A3/ A1-(59 -74) peptides, whereas ␥S peptide had no effect on chaperone-like activity of ␣-crystallin. The ␤A3/A1-(59 -74 peptide caused a 5.64-fold increase in ␣B-crystallin oligomeric mass and partial precipitation. Replacing hydrophobic residues in ␣B-(1-18) and ␤A3/A1-(59 -74) peptides abolished their ability to induce crystallin aggregation and light scattering. Our study suggests that interaction of crystallin-derived peptides with intact crystallins could be a key event in age-related protein aggregation in lens and cataractogenesis.

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Targeted disruption of specific steps of the ubiquitin–proteasome pathway by oxidation in lens epithelial cells

Cited by 17 publications

References 43 publications

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

S-Glutathionylation of the Rpn2 Regulatory Subunit Inhibits 26 S Proteasomal Function

Activation of Chaperone-mediated Autophagy during Oxidative Stress

Significance of Interactions of Low Molecular Weight Crystallin Fragments in Lens Aging and Cataract Formation

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