Methylglyoxal Alters the Function and Stability of Critical Components of the Protein Quality Control

Methylglyoxal Alters the Function and Stability of Critical Components of the Protein Quality Control Carla Figueira Bento1,2, Filipa Marques1, Rosa Fernandes1, Paulo Pereira1* 1 Center of Ophthalmology and Vision Sciences, Institute for Biomedical Research in Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal, 2 PhD Programme in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal Abstract Background: Increased production and accumulation of methylglyoxal (MGO), as well as increased modification of proteins by glycoxidation, are hallmarks of aging and diabetes. MGO was shown to modify proteins and to contribute to the accumulation of damaged proteins that can be toxic to cells. However, the effect of MGO on the cell systems responsible for repairing or degrading damaged proteins is still unclear. In this study, the effect of MGO on the function of the ubiquitin- proteasome system (UPS) and on molecular chaperones, two cooperative mechanisms associated with protein quality control, was investigated. Principal Findings: In this work it is shown that treatment of cells with MGO leads to accumulation of ubiquitin conjugates and depletion of free ubiquitin. Moreover, MGO significantly decreases the proteolytic activity of the 20S proteasome. Data further shows that MGO decreases the levels of the molecular chaperones Hsc70 and Hsp90 and leads to accumulation of CHIP-, Hsp40- and ubiquitin-containing aggregates. The formation of large aggregates containing CHIP is a consequence of its binding to misfolded proteins and to molecular chaperones. Moreover, dysfunction of the chaperones/CHIP/UPS axis is associated with accumulation of oxidized and argpyrimidine-modified proteins, which is likely to be associated with decreased cell viability. Interestingly, data further shows that MGO-induced stress induces the activation of heat shock factor-1 (Hsf-1), the main transcription factor involved in the regulation of the expression of heat shock proteins (HSPs) and cell response to stress. Conclusions: The data obtained in this work suggests that MGO impairs both the UPS and the protein quality control dependent on CHIP and molecular chaperones, leading to accumulation of toxic aggregates and increased cell death. However, these MGO-induced changes appear to elicit a response from the Hsf-1 system, which is crucial to help cells to cope with cellular stress and to re-establish homeostasis. Citation: Bento CF, Marques F, Fernandes R, Pereira P (2010) Methylglyoxal Alters the Function and Stability of Critical Components of the Protein Quality Control. PLoS ONE 5(9): e13007. doi:10.1371/journal.pone.0013007 Editor: Sue Cotterill, St Georges University of London, United Kingdom Received June 22, 2010; Accepted September 6, 2010; Published September 24, 2010 Copyright: ß 2010 Bento et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the PhD fellowship SFRH/BD/15229/2004 (Carla F. Bento) and the grants POCI/SAU-OBS/57772/2004 and PDTC/SAU-OBS/ 67498/2006 (FCT - Fundacao para a Ciencia e a Tecnologia, Portugal). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction contributes to prevent the accumulation of non-functional, potentially toxic proteins. This proteolytic system is of particular MGO is a highly reactive a-oxoaldehyde that is formed in cells importance in protecting cells against harsh conditions, such as primarily from the triose phosphate intermediates of glycolysis, heat shock, glycation or oxidative stress [9], and is an important dyhydroxyacetone phosphate and glyceraldehyde 3-phosphate [1]. player in the protein quality control system in eukaryotic cells. This by-product of glycolysis induces rapid and non-enzymatic However, when the generation of obsolete proteins exceeds the modification of free amino groups of lysine and arginine residues capacity of the cell to degrade them, there is a progressive of proteins, leading to the generation of advanced glycation end accumulation of damaged proteins, which often become insoluble products (AGEs) [2]. The posttranslational modification of over time and are potentially toxic to cells [9]. Accumulation of proteins by MGO is known to contribute to aging, as well as to damaged proteins, ultimately, occurs when the proteolytic systems a number of diseases, including cancer, diabetes and other and molecular chaperones, which normally prevent aggregation disorders [1]. Indeed, hyperglycaemia triggers enhanced produc- (for example, Hsc70 and Hsp90) [10,11], cannot keep up with the tion of MGO on diabetes [1,3], leading to vascular dysfunction rate of production of misfolded or otherwise damaged proteins. [4], while AGEs exacerbate and accelerate a number of The carboxy terminus of the Hsc70-interacting protein (CHIP) characteristics of many age-related diseases [5,6]. is a key player in protein quality control since it is a molecular link MGO is known to induce aggregation and structural modifi- between chaperones and the UPS. Indeed, CHIP has both a cations in proteins through cross-linking and formation of tetratricopeptide repeat (TPR) motif and a U-box domain [12]. chemical adducts [7,8]. In eukaryotic cells, the proteasome The TPR motif associates with chaperones, such as Hsp40, Hsc70 PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e13007 MGO in Protein Quality Control and Hsp90, while the U-box domain has ubiquitin ligase activity. [1], both characterized by decreased proteasome activity [14,16]. Thus, CHIP has a critical role in protein quality control by However, the putative effect of MGO on the proteasome activity ubiquitinating misfolded or post-translationally modified proteins and on the accumulation of modified proteins remains to be through interaction with molecular chaperones [13]. clarified. It has been suggested that the activity of the proteasome can be To evaluate the effect of MGO in the proteasome activity, we compromised upon aging [14] and diabetes [15]. For example, the used a human retinal pigment epithelium cell line (ARPE-19). This levels of the proteasome activator PA28 are decreased in muscle cell type represents an appropriate model for this study since it has extracts of diabetic rats [16], which might result in accumulation a very active and functional UPS [21,22]. Moreover, the retinal of glycated and toxic proteins, typically observed in diabetic pigment epithelium (RPE) has a high rate of glucose consumption tissues. Furthermore, formation of amyloid-aggregates, which is and its ubiquitin-proteasome system has been shown to be affected usually associated with aging and diabetes, is highly enhanced by during aging and diabetes [5,6,23]. Indeed, formation of AGEs is a MGO and other aldehydes [17] and this is known to impair the trigger for the onset of age-related phenotypes on choroid and UPS, leading to apoptosis [18]. On the other hand, it has been RPE cells [6]. suggested that UPS activity decreases with aging, which may Prior to test the proteasome activity, we observed that further account for the accumulation of modified and toxic incubation of RPE cells with 1 mM or 3 mM MGO for 3 hours proteins in cells, contributing to cell injury and death [14].There is resulted in an intracellular accumulation of MGO of 0.560.12 also increasing evidence showing that molecular chaperones, such and 0.760.14 nmol MGO/mg protein, respectively, which as a-crystallin and Hsp27 [19,20], are themselves major targets of corresponds to a 1.8 and 2.3 fold increase as compared to control MGO-induced modification and aggregation. These and other (Figure 1A). Although the high concentrations used in this work evidences indicate that age- and diabetes-dependent accumulation are not found in the plasma and tissues, we observed that they of MGO is likely to account for impairment of the protein-quality induce an intracellular accumulation of MGO that is consistent control mechanism. However, the effect of MGO in the function with the levels found in tissues of diabetic animals [24]. Moreover, of UPS and on molecular chaperones is still not clear. the obtained intracellular levels of MGO using these concentra- Herein, it is shown that increased levels of MGO lead to tions are similar to those observed in other studies using cell accumulation of ubiquitin conjugates and depletion of free culture systems [25]. ubiquitin in the cell. The accumulation of ubiquitin conjugates is To assess the 20S proteasome activity, ARPE-19 cells were presumably due to both MGO-induced impairment of UPS and treated with MGO and the cell extracts were used to measure the MGO-induced disruption of chaperones. Data presented in this three peptidase activities of the 20S proteasome using fluorogenic work further shows that MGO downregulates Hsc70 and Hsp90 substrates. Data shows that these MGO concentrations decrease and promotes the formation of large aggregates containing Hsp40, the chymotrypsin-like and caspase-like activities of the 20S CHIP and ubiquitin. Consistently, increased levels of MGO lead proteasome, by about 32% and 17%, respectively (Figure

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