The proteasomal system and HNE-modified proteins
Introduction
Protein oxidation is a continuous process that occurs at low rates during the normal metabolism of aerobic cells and at increased rates in several pathological situations. The degree of protein oxidation caused by oxidants is dependent on several factors, including the nature, relative location, and flux rate of the oxidant, and the presence (or absence) of antioxidants. Both the oxidation of free amino acids and the oxidation of peptides and proteins have been studied by many laboratories. Numerous amino acids are known to be susceptible to oxidation. Besides the oxidation of amino acid side chains in proteins several changes can occur in the protein backbone, such as fragmentation of polypeptide chains, and both intra- and intermolecular cross-linking (Davies, 1987; Davies and Delsignore, 1987; Davies et al., 1987a, Davies et al., 1987b; Stadtman, 1993; Vogt, 1995).
Proteins may also be oxidatively modified via secondary mechanisms resulting from reactions of free radicals with other cellular constituents, such as lipids, carbohydrates, and nucleic acids. The process of lipid peroxidation has been intensively studied and it is known today that numerous products, some of them very reactive, are formed during this fascinating chain reaction. Reactive aldehydes, like malondialdehyde and 4-hydroxynonenal, are of special importance due to their formation rate, their frequent measurement by many laboratories, and their high reactivity with proteins. Of particular interest is the fact that both aldehydes are bi-functional compounds, which are able to form protein cross-links (Grune et al., 1997; Stadtman, 1993).
4-Hydroxynonenal is extensively metabolized, possibly in order to prevent the reaction of this aldehyde with proteins (Grune et al., 1994; Siems et al., 1997). Therefore, only a small share of the aldehyde formed actually reacts with proteins. In experimental models this amount is between 0.5% and 10% of the aldehyde present (Grune et al., 1994; Siems et al., 1997). This rate is actually probably higher than in the real in vivo situation due to the high 4-hydroxynonenal concentrations used in these experiments. Various chemical reactions of 4-hydroxynonenal with proteins have been carefully studied (Esterbauer et al., 1991; Grune et al., 1994; Siems et al., 1997; Uchida et al., 1994, Uchida et al., 1993). It is quite clear that 4-hydroxynonenal (HNE) can inhibit enzymes (Esterbauer et al., 1991) by chemical modification, which raises interesting questions about the fate of these 4-hydroxynonenal modified proteins. Since it is known that oxidized proteins are selectively degraded in mammalian cells, one may reasonably postulate that oxidatively (or 4-hydroxynonenal) modified proteins might be degraded by the same proteolytic machinery, the proteasomal system.
Section snippets
The proteasome
Mammalian cells appear to possess several major pathways for general protein degradation: lysosomal proteases, calcium-dependent proteases, and the proteasomal system. Proteins that enter cells from the outside as well as several intracellular proteins, especially long-lived ones or proteins from various organelles, are degraded within lysosomes. True intracellular proteins are degraded by the intracellular proteasomal system (Rock et al., 1994). The proteasomal system consists of the so called
Degradation of oxidized proteins
Oxidation can induce many changes in proteins, including amino acid modification, fragmentation or aggregation (Berlett and Stadtman, 1997; Davies, 1986; Davies, 1987; Davies and Delsignore, 1987; Davies et al., 1987a, Davies et al., 1987b; Dean et al., 1997; Grune et al., 1997; Huang et al., 1995; Shang et al., 1994; Stadtman, 1993). Some of these oxidative modifications cause an increased susceptibility of oxidized proteins towards proteolysis, possibly due to an increase in surface
HNE-modified proteins and the proteasomal degradation
Unfortunately the literature about the degradation of HNE-modified proteins is very limited. We performed some studies ourselves, demonstrating, that HNE-modified histones are degraded preferentially by the proteasomal system (Table 1). Obviously the recognition and the degradation of HNE-modified isolated histones is concentration dependent. In our experiments we were able to find a maximal degradation of histones (1 mg/ml) treated with 5 μM HNE. Such a bell-shaped proteolytic response by the
Protein cross-linking by HNE
As already mentioned, a fairly broad spectrum of protein aggregate formation initially occurs not due to covalent cross-links, but because of new hydrophobic and electrostatic interactions (Davies, 1987; Davies and Delsignore, 1987; Davies et al., 1987a, Davies et al., 1987b; Grune et al., 1997; Sommerburg et al., 1998). This aggregated material can then be chemically modified by a great variety of cellular metabolites, including aldehydic lipid peroxidation products (Grune et al., 1997;
Proteasome inhibition by HNE-cross-linked proteins
Interestingly HNE-modified proteins actually become poor substrates for the proteasome, after cross-linking reactions. This was revealed by studies from our group (Table 1), and others. We have reported that heavily oxidized and cross-linked protein aggregates accumulate in cells because they inhibit the proteasome (Sitte et al., 2000a, Sitte et al., 2000b, Sitte et al., 2000c). HNE-cross-linked proteins are also able to inhbit the turnover of other proteins by inhibiting the proteasome (
Conclusions
As discussed in this mini-review, HNE is a highly reactive lipid peroxidation product able to modify proteins and form intermolecular cross-linked protein aggregates. Whereas moderately HNE-modified proteins are degraded by the proteasomal system, extensively modified proteins form extensive cross-links and are poor substrates for proteasomal degradation. Moreover, such cross-linked proteins are able to inhibit the proteasome, and further impair cellular protein turnover. Despite our present
Acknowledgments
T.G. was supported by the DFG. K.J.A.D. was partially supported by NIH/NIEHS grant number ES03598.
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