Accumulation of polyubiquitinated proteins: A consequence of early inactivation of the 26S proteasome.

The proteasomal degradation system is one of the most important protein degradation systems in the cytosol and nucleus. This system is present in two major forms: the ATP-stimulated 26S/30 S proteasome or the ATP-independent 20S core proteasome. While the first recognize ubiquitin-tagged target proteins and degrade them, the 20S proteasome works also independent from ATP, but requires partially unfolded substrates. While the role of the proteasome in the selective removal of oxidized proteins is undoubted, the debate about a selective ubiquitination of oxidized proteins is still ongoing. Here we demonstrate, that under some conditions of oxidative stress an accumulation of oxidized and of K48-ubiquitinated proteins occurs. However, the removal of oxidized proteins seems not to be linked to ubiquitination. In further experiments, we could show that the accumulation of ubiquitinated proteins under certain oxidative stress conditions is rather a result of a different sensitivity of the 26S proteasome and the ubiquitination machinery towards oxidants.

Non-enzymatic cleavage of Hsp90 by oxidative stress leads to actin aggregate formation: A novel gain-of-function mechanism.

Aging is accompanied by the accumulation of oxidized proteins. To remove them, cells employ the proteasomal and autophagy-lysosomal systems; however, if the clearance rate is inferior to its formation, protein aggregates form as a hallmark of proteostasis loss. In cells, during stress conditions, actin aggregates accumulate leading to impaired proliferation and reduced proteasomal activity, as observed in cellular senescence. The heat shock protein 90 (Hsp90) is a molecular chaperone that binds and protects the proteasome from oxidative inactivation. We hypothesized that in oxidative stress conditions a malfunction of Hsp90 occurs resulting in the aforementioned protein aggregates. Here, we demonstrate that upon oxidative stress Hsp90 loses its function in a highly specific non-enzymatic iron-catalyzed oxidation event and its breakdown product, a cleaved form of Hsp90 (Hsp90cl), acquires a new function in mediating the accumulation of actin aggregates. Moreover, the prevention of Hsp90 cleavage reduces oxidized actin accumulation, whereas transfection of the cleaved form of Hsp90 leads to an enhanced accumulation of oxidized actin. This indicates a clear role of the Hsp90cl in the aggregation of oxidized proteins.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress.

Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed.

The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome.

One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome.

The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium.

Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2.

Protein Oxidation in Aging: Does It Play a Role in Aging Progression?

SIGNIFICANCE: A constant accumulation of oxidized proteins takes place during aging. Oxidation of proteins leads to a partial unfolding and, therefore, to aggregation. Protein aggregates impair the activity of cellular proteolytic systems (proteasomes, lysosomes), resulting in further accumulation of oxidized proteins. In addition, the accumulation of highly crosslinked protein aggregates leads to further oxidant formation, damage to macromolecules, and, finally, to apoptotic cell death. Furthermore, protein oxidation seems to play a role in the development of various age-related diseases, for example, neurodegenerative diseases. RECENT ADVANCES: The highly oxidized lipofuscin accumulates during aging. Lipofuscin formation might cause impaired lysosomal and proteasomal degradation, metal ion accumulation, increased reactive oxygen species formation, and apoptosis. CRITICAL ISSUES: It is still unclear to which extent protein oxidation is involved in the progression of aging and in the development of some age-related diseases. FUTURE DIRECTIONS: An extensive knowledge of the effects of protein oxidation on the aging process and its contribution to the development of age-related diseases could enable further strategies to reduce age-related impairments. Strategies aimed at lowering aggregate formation might be a straightforward intervention to reduce age-related malfunctions of organs.

HSP90 cleavage associates with oxidized proteins accumulation after oxidative stress.

Oxidative stress, OS, has been associated to a variety of phenomena as cancer progression, neurodegeneration and ageing itself. At a molecular level, OS leads to protein carbonylation, a non-enzymatic irreversible event and common feature of aged cells. Carbonylated proteins are dysfunctional and can accumulate, in the form of protein aggregates that alter cellular functionality. To cope with carbonylated proteins, cells employ the proteasome, the main non-lysosomal structure for carbonylated proteins turnover. However, if the degrading rate is inferior to carbonylated proteins formation rate, protein aggregates form. In a previous study, in oxidative stress challenged Jurkat cells we could verify that cytoplasmatic actin becomes heavily carbonylated and forms oxidized actin aggregates, which lead to proliferation impairment and proteasome activity diminishment, similar to senescence like states. Because under these oxidative conditions there is a proteostasis disturbance, such as oxidized proteins (especially actin) accumulation and proteasome activity impairment, Hsp90 involvement was studied. Hsp90, a molecular chaperone, assists oxidized proteins degradation and also protects the 20S proteasome from oxidative inactivation. We reasoned that the mechanism by which protein aggregates can form, mainly happens due to Hsp90 lesser functionality, which we attribute to cleavage. In our study, we were able to verify that cleaved Hsp90 is present in protein aggregates and that it occurs before actin insolubilization, verified after fractioning soluble and insoluble cellular extracts. We are convinced, supported by our data, that cleaved Hsp90 is an important intervenient for oxidized protein accumulation and proteasome inactivation. However, further studies should follow to confirm our hypothesis.

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress.

After oxidative stress, proteins that are oxidatively modified are degraded by the 20S proteasome. However, several studies have documented an enhanced ubiquitination of yet unknown proteins. Because ubiquitination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner this raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However, we were able to confirm an increase in ubiquitinated proteins 16 h after oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide-treated cells, as well as from control cells and cells treated with lactacystin, an irreversible proteasome inhibitor, and identified some of these proteins by MALDI tandem mass spectrometry. As a result we obtained 24 different proteins that can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified, ubiquitinated proteins confirms the thesis that ubiquitination upon oxidative stress is not a random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins.