Annika Höhn

Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a very complex and multifactorial metabolic disease characterized by insulin resistance and β cell failure leading to elevated blood glucose levels. Hyperglycemia is suggested to be the main cause of diabetic complications, which not only decrease life quality and expectancy, but are also becoming a problem regarding the financial burden for health care systems. Therefore, and to counteract the continually increasing prevalence of diabetes, understanding the pathogenesis, the main risk factors, and the underlying molecular mechanisms may establish a basis for prevention and therapy. In this regard, research was performed revealing further evidence that oxidative stress has an important role in hyperglycemia-induced tissue injury as well as in early events relevant for the development of T2DM. The formation of advanced glycation end products (AGEs), a group of modified proteins and/or lipids with damaging potential, is one contributing factor. On the one hand it has been reported that AGEs increase reactive oxygen species formation and impair antioxidant systems, on the other hand the formation of some AGEs is induced per se under oxidative conditions. Thus, AGEs contribute at least partly to chronic stress conditions in diabetes. As AGEs are not only formed endogenously, but also derive from exogenous sources, i.e., food, they have been assumed as risk factors for T2DM. However, the role of AGEs in the pathogenesis of T2DM and diabetic complications—if they are causal or simply an effect—is only partly understood. This review will highlight the involvement of AGEs in the development and progression of T2DM and their role in diabetic complications.

Protein oxidation in aging and the removal of oxidized proteins

Reactive oxygen species (ROS) are generated constantly within cells at low concentrations even under physiological conditions. During aging the levels of ROS can increase due to a limited capacity of antioxidant systems and repair mechanisms. Proteins are among the main targets for oxidants due to their high rate constants for several reactions with ROS and their abundance in biological systems. Protein damage has an important influence on cellular viability since most protein damage is non-repairable, and has deleterious consequences on protein structure and function. In addition, damaged and modified proteins can form cross-links and provide a basis for many senescence-associated alterations and may contribute to a range of human pathologies. Two proteolytic systems are responsible to ensure the maintenance of cellular functions: the proteasomal (UPS) and the lysosomal system. Those degrading systems provide a last line of antioxidative protection, removing irreversible damaged proteins and recycling amino acids for the continuous protein synthesis. But during aging, both systems are affected and their proteolytic activity declines significantly. Here we highlight the recent advantages in the understanding of protein oxidation and the fate of these damaged proteins during aging. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Lipofuscin: formation, effects and role of macroautophagy.

Oxidative stress plays a crucial role in the development of the aging process and age dependent diseases. Both are closely connected to disturbances of proteostasis by protein oxidation and an impairment of the proteasomal system. The final consequence is the accumulation of highly cross-linked undegradable aggregates such as lipofuscin. These aggregates of damaged proteins are detrimental to normal cell functions. Here we provide an overview about effect of these aggregates on the proteasomal system, followed by transcription factor activation and loss of cell viability. Furthermore, findings on the mechanism of radical genesis, proteasomal inhibition and the required components of lipofuscin formation were resumed.

Lipofuscin-bound iron is a major intracellular source of oxidants: role in senescent cells.

Aging is accompanied by an intracellular accumulation of lipofuscin, a hydrophobic yellow-brownish material that accumulates especially in the lysosomal compartment, where it can be neither degraded nor exocytosed from the cell. The intracellular effects of accumulating lipofuscin are still a subject of speculation. In addition to the demonstrated inhibition of the proteasome, it was proposed that lipofuscin is cytotoxic because of its ability to incorporate transition metals such as copper and iron, resulting in a redox-active surface, able to catalyze the Fenton reaction. This characteristic of lipofuscin may contribute to an increased level of radical formation and oxidatively modified cellular components such as proteins, lipids, and RNA/DNA, which has been shown to be extensive in aging cells. In this study for the first time the lipofuscin-mediated formation of oxidants and the role of iron in this process are directly shown in a model of senescent fibroblasts, as well as in vitro with artificial lipofuscin. We demonstrate that this oxidant production is independent of mitochondria and has cytotoxic effects. The ability of lipofuscin to produce oxidants is dependent on the amount of transition metals incorporated. Although the amount of oxidants formed by cellular lipofuscin turned out to be moderate, it is chronic and thus lipofuscin is able to catalyze its own formation.

Lipofuscin inhibits the proteasome by binding to surface motifs.

Lipofuscin, a highly oxidized aggregate, consists of covalently cross-linked proteins, lipids, and sugar residues and is one of the major life-span-limiting factors in postmitotic aging cells. An artificial model of this material, showing characteristics and effects comparable to those of the natural form, has turned out to be very useful for in vitro studies. Artificial lipofuscin was used to investigate its effects on the viability of human fibroblasts, its rate of uptake, and its ability to inhibit the proteasomal system. The inhibition of the proteasomal system is one of the major aspects of the cytotoxic effects of lipofuscin. We present here that this proteasomal inhibition is due to proteasomal binding to the lipofuscin surface motifs, degradable by protease K. Furthermore, removal of the surface peptide structures by protease K strongly reduces the cytotoxic effects of lipofuscin and binding of cellular proteins and proteasomes to intracellular protein aggregates.

The proteasome and the degradation of oxidized proteins: Part II – protein oxidation and proteasomal degradation

Here, we review the role of oxidative protein modification as a signal for recognition and degradation of proteins. It was clearly demonstrated that the ATP- and ubiquitin-independent 20S proteasome is playing a key role in the selective removal of oxidized proteins. Furthermore, the current knowledge of the substrate susceptibility on the degradation of oxidized proteins and the role of the immunoproteasome will be highlighted.

Pathophysiological importance of aggregated damaged proteins

Reactive oxygen species (ROS) are formed continuously in the organism even under physiological conditions. If the level of ROS in cells exceeds the cellular defense capacity, components such as RNA/DNA, lipids, and proteins are damaged and modified, thus affecting the functionality of organelles as well. Proteins are especially prominent targets of various modifications such as oxidation, glycation, or conjugation with products of lipid peroxidation, leading to the alteration of their biological function, nonspecific interactions, and the production of high-molecular-weight protein aggregates. To ensure the maintenance of cellular functions, two proteolytic systems are responsible for the removal of oxidized and modified proteins, especially the proteasome and organelles, mainly the autophagy-lysosomal systems. Furthermore, increased protein oxidation and oxidation-dependent impairment of proteolytic systems lead to an accumulation of oxidized proteins and finally to the formation of nondegradable protein aggregates. Accordingly, the cellular homeostasis cannot be maintained and the cellular metabolism is negatively affected. Here we address the current knowledge of protein aggregation during oxidative stress, aging, and disease.

The Proteasome Is an Integral Part of Solar Ultraviolet A Radiation-induced Gene Expression

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

Advanced-glycation-end-product-induced formation of immunoproteasomes: involvement of RAGE and Jak2/STAT1

AGEs (advanced glycation-end products) accumulate during aging and several pathologies such as Alzheimers disease and diabetes. These protein products are known to inhibit proteolytic pathways. Moreover, AGEs are known to be involved in the activation of immune responses. In the present study we demonstrate that AGEs induce the expression of immunoproteasomal subunits. To elucidate a molecular basis underlying the observed effects we were able to demonstrate an activation of the Jak2 (Janus kinase 2)/STAT1 (signal transducer and activator of transcription 1) pathway. Inhibition of Jak2 by AG-490 and STAT1 by specific siRNA (small interfering RNA) abolished AGE-induced expression of immunoproteasomal subunits. Furthermore, silencing of RAGE (receptor for AGEs) revealed that AGE-induced up-regulation of the immunoproteasome is mediated by a RAGE signalling process. Thus we have described for the first time that the signalling pathway of Jak2 and STAT1 activated by AGEs via RAGE is involved in the induction of the immunoproteasome.

Accumulation of modified proteins and aggregate formation in aging.

Increasing cellular damage during the aging process is considered to be one factor limiting the lifespan of organisms. Besides the DNA and lipids, proteins are frequent targets of non-enzymatic modifications by reactive substances including oxidants and glycating agents. Non-enzymatic protein modifications may alter the protein structure often leading to impaired functionality. Although proteolytic systems ensure the removal of modified proteins, the activity of these proteases was shown to decline during the aging process. The additional age-related increase of reactive compounds as a result of impaired antioxidant systems leads to the accumulation of damaged proteins and the formation of protein aggregates. Both, non-enzymatic modified proteins and protein aggregates impair cellular functions and tissue properties by a variety of mechanisms. This is increasingly important in aging and age-related diseases. In this review, we will give an overview on oxidation and glycation of proteins and the function of modified proteins in aggregate formation. Furthermore, their effects as well as their role in aging and age-related diseases will be highlighted.