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Free radical damage to proteins and its role in the immune response
Molec. Aspects IVied. Vol. 12, pp. 121-128, 1991 Printed in Great Britain. All rights reserved.
0098-2997/91 $0.00 + .50 ~) 1991 Pergamon Press pie.
FREE RADICAL DAMAGE TO PROTEINS AND ITS ROLE IN THE IMMUNE RESPONSE Roger Dean and Jeremy Simpson Heart Research Institute, 145 Missenden Road, Camperdown 2050, N.S.W., Australia
Introduction Free radicals are generated in many contexts within the immune response. In some cases they are part of the mode of action of immune effector cells. In other cases their generation is more a consequence of the progression of an immune response. In this brief review I will concentrate on the role of free radical damage to proteins in the immune response. While it has been traditional to study free radical damage to lipids, and the process of lipid peroxidation in pathological circumstances, the role of protein damage is probably more central to the action of some of the immune effector cells. In addition, protein damage is an inevitable part of degradative and remodeling processes in the immune response. There has been considerable advance in the study of free radical damage to proteins in the last five years, particularly through the work of Schuessler (e.g. Schuessler and Schilling, 1984) Stadtman and Levine (e.g. Rivett and Levine, 1987), Davies (e.g. reviewed Davies, 1986), and their colleagues, and through that of our own group. Other talks in this meeting will describe specific studies on immunoglobulins. Therefore I will survey the kinds of free radical damage which can be inflicted upon proteins in the first part of the paper. I will then discuss the consequences of such damage and its relation to the immune response.
The Nature of Free Radical Damage to Proteins in the Immune Response Systems for Investigation of Free Radical Damage to Protein Table 1 summarises the main kinds of experimental system used in such studies. While experiments using Fenton chemistry and enzymatic radical generation are widely available, they are complex and imprecise (Sutton and Winterbourn, 1989). However, this of course reflects the complexity of real living systems. On the other hand much useful and precise information can be obtained by using defined radical-generating systems such as cobalt gamma-radiolysis. While this is an unphysiological system, it does not allow control of generation of the physiologically relevant radicals. An intermediate level of control is offered by the thermolabile azo-compounds and these
121
122
R. Dean and J. Simpson
can be supplied to whole organisms (Terao and Niki 1988). They generate primary peroxyl radicals but secondary radicals may result from radical-radical reactions, so that the interpretation of the action of these compounds is not quite as simple as that of the gamma radiolytically generated defined radicals.
Table 1. Systems for investigation of radical damage to proteins Radical generating system
Radical available
Degree of control of radical generation
"/-Radiolysis
Most (aerobic or anaerobic)
Qualitative and quantitive control possible
Thermolabile azo compounds
Mainly peroxy
Rate can be kept constant and fairly accurately known, however phase effects influence the rate and there are secondary radicals generated in lower yield
Fenton chemistry
Superoxide, hydroxyl radicals; higher states of metals
Imprecise and complex
Enzymatic reactions
e.g. Xanthine oxidase and hypoxanthine: superoxide radical is the primary product. If metals are available complex reactions ensue
In a system with no available metal these reactions can be well controlled
Influences of Transition Metals Other papers in this meeting have discussed in some detail the relevance of iron and copper amongst transition elements to radical reactions in vivo. Here it probably suffices to point out that iron and copper may be bound to proteins and influence radical reactions thereon (reviewed Chevion, 1988). Histidine is the amino acid most able to chelate copper but specialised binding sites are known on some proteins for both copper and iron. In general the effect of such binding is to reduce the participation of the metals in bulk-phase redox cycling reactions. Sometimes this may localise damage to the protein, but in other cases (Table 2) radicals can be released into the bulk phase even when their interconversion involves protein-bound metals. We have recently studied this in some detail (Simpson and Dean, in press) and find that for many chelators of metals there is a complex dose response of radical-generation in the bulk phase. Thus for example, low concentrations of histidine (Simpson et al., 1988) or protein may gradually increase the availability of a fixed concentration of metal for radical reactions. However, as the concentration of chelator is increased a maximum is reached and subsequently there is depression of radical generation in the bulk phase. There are of course scavenging effects of such high concentrations of agents such as protein and histidine whereby radicals are still generated but rather than reacting with the detection system, react with the protein or amino acid (Dean et al., 1989).
Free Radicals and Proteins
123
In the immune response, as also mentioned elsewhere in this volume, metals may be displaced from their normal protein binding sites as a result of damaging reactions upon the surface of the protein effected by incident radicals. Thus the concept of misplaced transition metals is important in inflammatory disease and there are instances of haemorrhage of metals into inflammatory sites and into other chronic pathological sites such as the atherosclerotic plaque. Such misplaced metal may have drastic influence on radical reactions. The metal zinc competes with copper for binding sites on biological molecules and thereby defends against such damaging reactions of misplaced metals (reviewed Willson, 1989; Lovering and Dean, unpublished).
Free Radical Generation in the Immune Response Table 2 summarises the wide range of sources of free radicals which may be relevant in the immune response. It can be seen that radicals are both controlled products of effector cells within the immune system and also the consequence of poorly controlled progression of pathological aspects of the response. Thus, effector cells such as granulocytes, mononuclear phagocytes and lymphocytes, for their several different purposes can generate free radicals in controlled ways. On the other hand, the increased availability of transition metals or the development of peroxides upon already damaged biological macromolecules, may give rise to a source of further free radical reactions which can be damaging in the immune response. Normally the immune response involves inflicting damage on foreign entities such as invading organisms or on damaged tissue, so that the damaging reactions of radicals are not necessarily deleterious. However, in chronic immunological responses, as in some chronic inflammatory diseases, this damaging capacity of free radical reactions may become seriously deleterious.
Table 2. Somesources of free radical in the immune response Site
Reaction sources
Extracellular
Auto-oxidationreactions
Triggered fluxes from cells released extra-cellularly and into phagasomes
The "respiratoryburst" oxidase. This electron transport system present on granulocytesand mononuclear phagocytesgives rise to superoxide radicals and is activated by def'med triggers
Other radical fluxes which may be released from cells
Most cells, possibly in association with triggeringof cell division (e.g. proliferating lymphocytes). Untriggered macrophagesetc. (e.g. oxidising extracellular low density lipoprotein)
Intracellular fluxes
Auto-oxidationreaction Enzymatic reaction. Lipid and protein peroxidation
124
R. Dean and J. Simpson
Kinds of Damage Inflicted upon Proteins by Free Radicals Table 3 summarises some of the biochemical and chemical information as to the nature of damage inflicted upon proteins by free radicals. Much of the data summarised there has been obtained by experiments using defined in vitro conditions. What is less clear as yet is the extent to which such protein damage occurs in vivo. Table 3. Protein damage inflicted by free radicals Radical
Kinds of damage
02
Sulphydryl oxidation, but few other changes
HO2"
Limited fragmentation and crosslinking of soluble proteins. More extensive damage to membrane proteins
OH" and probably higher oxidation states of transition metals
Fragmentation, amino acid modification. Cross-linking (particularly when oxygen absent or limiting)
Radicals generated by local Fenton reactions with metal bound to the protein
Amino acid oxidation and fragmentationof polypeptide. Note that the reactive intermediates may also be available in the bulk fluid phase (Simpson and Dean, in press)
The information given above is supported by the following selected references from our own work: Dean et al., 1988a,b; Dean and Cheesman, 1987; Wolff and Dean, 1986, 1987; Dean et al., 1986; and other work is reviewed in Davies, 1986; Rivett and Levine, 1987. It is clear that polypeptides displaying radical damage can be detected in vivo. For example, in the atherosclerotic plaque one can detect the apo-protein of low density lipoprotein in a fragmented form and in which the lysines residues (and presumably other amino acids) have been modified in a manner consistent with free radical damage (Shaikh et al., 1988). This particular protein has been studied extensively and it probably reveals the kind of damage which takes place on many proteins during immune responses (Jessup etal., 1989a,b) and of course atherosclerosis involves inflammatory and immune components as we will discuss in the next section. Some of the proteins of the body which are longer lived also can be detected with modifications consistent with free radical damage: e.g. modified amino-acid residues in collagen (Dean et al., 1989). The fact that the demonstration in vivo of such modified protein is an infrequent event in the literature, is partly due to the fact that protein studies have been undertaken only recently, but also to the fact that of the consequences of protein damage which we shall discuss in the next section, involve the removal of the damaged protein. Demonstration that free radical damage to proteins produces modified entities which then may be removed by degradation, requires many more in vivo
Free Radicals and Proteins
125
measurements of such modified products and probably precursor product relationships will have to be established by means of radioactive tracer experiments.
Consequences of Free Radical Damage to Proteins in the Immune Response Inactivation of Protein Function In many circumstances, the first consequence of even very limited free radical damage to proteins is inactivation of their function. For example, enzymes can be inactivated by slight fluxes of radicals. This is true of proteinases, but also of proteinase inhibitors (Dean et al., 1989b; Reddy et al., 1989). In the immune system, the most important application of this is probably in the process of cytolysis. Some of the cells which are effectors of cell mediated cytolysis, for example the mononuclear phagocytes, are avid free radical generators and free radical fluxes are often triggered by contact with target cells. However, many other cytolytic components are produced by such professional killer cells, and in contrast there are other killer cells (e.g. NK cells) which lack the respiratory burst oxidase present on mononuclear phagocytes. As a consequence, the quantitative importance of free radical generation for cytolysis in these circumstances is somewhat unclear. What is clear however is that free radical damage inflicted from the exterior of cells can be lytic. There is more about this in some of the other manuscripts in this meeting, but it is worth emphasising here that protein damage may be a key component in such cytolysis. Thus in an experimental system in which macrophage cell lines were exposed to exogenously generated radical fluxes we found that lipid peroxidation and protein damage were normally concomitant and preceded cell death. One of the earliest events along this pathway we could detect was membrane depolarisation, which occurred within a few minutes of exposure of the cells to the radical fluxes. By means of lipophilic antioxidants such as butylated hydroxytoluene we were able to show that lipid peroxidation can be completely suppressed in these circumstances and yet membrane depolarisation, protein damage and subsequent lysis are all virtually unaffected. Thus protein damage may well be an important component in some of these cytolytic mechanisms and in our particular case, the inactivation of certain membrane transporters seems to be critical (Richards et al., 1988).
Degradation of Damaged Protein Most denatured or inactive proteins, however generated, are relatively rapidly removed by the cellular proteolytic machinery (reviewed Wolff etal., 1986). Thus for example intracellular abnormal proteins are degraded selectively and rapidly. Extracellular damaged proteins may also be endocytosed and degraded fairly efficiently and in some cases this is simply due to the release of the protein from a fixed extra-cellular matrix in which it was previously inaccessible (Wolff et al., 1986). Thus, in the immune response, extracelluar protein damage is often an important step in removal of tissue in a wounded site, or of organisms in an inflammatory site, prior to reconstruction of the area. In the case of neutrophil-mediated damage to such extracellular tissue, a clear role for free radicals in contributing to the process has been established by the work of Weiss and colleagues (e.g. Reddy et al., 1989). In the case of macrophages, such clear cut evidence has been very difficult to obtain (e.g. Dean and Schnebli, 1989), although the generation of modified forms of low density lipoprotein by macrophages has been well documented and does lead to enhanced endocytosis and degradation by the same cell (Jessup et al., 1989; Jessup et al., 1990). This may be an example of many processes in which macrophages use free radical damage as a part of the degradative mechanism, though it is one of the few for which such clear cut evidence exists.
126
R. Dean and J. Simpson
Persistent Modified Proteins The case just mentioned of low density lipoprotein is a case where protein modified by free radical damage can be detected in vivo in quite sizable pools. Thus damaged low density lipoprotein including damaged molecules of its polypeptide Apo-B can be detected in atherosclerotic vessel walls (Shaikh et al., 1988). No doubt this lipoprotein is undergoing accelerated endocytosis and is being degraded intracellularly but nevertheless its rate of generation suffices to maintain a detectable pool. This maybe somewhat unusual. However, a well known example has been in the literature for much longer: the aging pigment lipofuschin. This accumulates in lysosomes of cells in a variety of tissues of aging organisms. It is a complex incompletely characterised entity but it does include damaged polypeptides. It seems that the polypeptides may be so altered and sequestered by the lipid with which they are bound that in spite of their co-existence within lysosomes (with a huge array of degradative hydrolases), their turnover seems to be quite slow (Wolff et al., 1988). The reason for this slow degradation has not been elucidated and would be very interesting. A very interesting possibility which is addressed in another paper is that limited modifications to proteins may produce autoantigens which are not sufficiently perturbed in conformation to be immediately fully degraded, but which can nevertheless be recognised by the immune system as a trigger for generation of auto-antibodies. One could readily envisage that a modified protein in the extracellular phase could be generated during radical reactions in the immune response and then be endocytosed because of its increased exposure of hyrophobic moieties due to unfolding. Such hydrophobicity in itself is sufficient to enhance the endocytosis of many damaged proteins. However a further consequence might be that the intracellular transport of the modified protein might be distinct from that of the normal protein or of the normal catabolic pathway and hence permit the antigen to be processed and presented in different sites within the cell. Again the example of low density lipoprotein is interesting, since as mentioned already, modified low density lipoprotein can be detected in man, and in addition, auto-antibodies to low density lipoprotein are quite common and may have a bearing on pathology (e.g. Palinski et al., 1988).
Reactive Moieties on Free Radical Damaged Protein In our recent work, together with Jan Gebicki, Macquarie University, Sydney, we have characterised the generation of hydroperoxides upon protein (Thomas etal., 1989). While hydroperoxides have been studied extensively on lipids, and are known to be reactive entities (e.g. Hunt et al., 1989b) their production on protein has been neglected for many years. It is not clear precisely how hydroperoxides on proteins decay, and whether reactive entities are produced during this process, but it is clear that these hydroperoxides are rather unstable and that after a few days at 4°C most hydroperoxides have been destroyed. We have also demonstrated another reactive moiety upon radical damaged protein, which as yet remains undefined chemically (Simpson and Dean, 1989). We find that after radical damage several proteins become able to reduce the metal centre in Cytochrome C, and free iron and free copper from their higher valency to lower valency states. While the function of reducing free metals is possessed by some native proteins, and largely depends on their free cysteine residues, the generation of a vastly increased quantity of reducing capacity is a novel finding and may be important in view of the participation of transition metals in Fenton chemistry (Sutton and Winterborn, 1989). In some circumstances such reactions can be limited by failure to reduce metal which has become oxidised during the reactions and a reducing moiety present on protein might ensure the completion of Fenton chemistry in its vicinity and thus be an important influence upon damage at this site. This area of study is under intense investigation in our lab at present since it may have substantial bearing on the immune and inflammatory responses.
Free Radicals and Proteins
127
References Chevion, M. (1988) Free Radicals Biol. Meal.5, 27-37. Davies, KJA, Goldberg AL (1987) J. Biol Chem 262, 8220-8226 Dean RT, Thomas SM, Gamer AC (1986). Biochem J 240, 489-494. Dean RT, Wolff SP, McElligott MA (1989a) Free Radical Res. Communication 7 97-103. Dean RT, Nick HP, Schnebli HJP (1989b) Biochem Biophys Res Commun 159, 821-827. Dean RT, Cheeseman KH (1987) Biochem Biophys Res Commun 148,1277-1282. Dean RT, Schnebli HP (1989) Biochim Biophys Acta 992, 174-180. Hunt JV, Dean RT, Wolff SP (1988) Biochem J 256, 205-212. Hunt JV, Simpson JA, Dean RT (1988) Biochem J 250, 87-93. Jessup W, Bedwell S, Dean RT (1989a) Biochem J 262, 707-712. Jessup W, Bedwell S, Kwok K, Dean RT (1989b) Agents and Actions, Supplement 26, 214-6 Jessup W, Rankin SM, de Whalley CV, Hoult JRS, Scott J, Leake DS (1990) Biochem J. 269, 399-400. Palinski W, Gunner, GC, Butler SW, Parasarathy S, Steinberg D, Witztum J (1988) Circulation Suppl. II-14. Reddy UY, Pizzo SV, Weiss SJ. (1989) J. Biol Chem 264, 13801-9. Richards DCH, Jessup W, Dean RT (1988) Biochim Biophys Acta 946, 281-288. Rivett AJ, Levine RL (1987) Biochem. Soc. Trans. 15, 816-818. Schuessler H, Schilling K (1984) Int. J. Radiat. Biol. 45, 267-287. Shaikh M, Martini S, Quniney JR, Baskerville P, La Ville E, Browse NL, Duffied R, Turner PR and Lewis B (1988) Atherosclerosis, 69, 165-172. Simpson JA, Cheeseman KH, Smith SE, Dean RT (1988), Biochem J 254, 519-523. Simpson JA, and Dean RT (1989) Proc. AUst. Biochem. Soc., p. 42. Sutton HC, Winterbourn C. (1989) Free Radicals in Biol. Med. 6, 53-60 Terao K, Niki E (1986) J. Free Rad Biol Med 2, 193-201. Thomas SM, Jessup W, Dean RT, Gebicki J (1989) Analyt. Biochem. 176, 353-359. Wilson RL (1989) in "Zinc in Human Biology" (Mills CF, ed.) pp. 147-172, Springer, Berlin. Wolff SP, Dean RT (1986) Biochem J 234, 399-403.
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Wolff SP, Gamer A, Dean RT (1986) Trends in Biochemical Science 11, 27-31. Wolff SP, Dean RT, (1987) Biochem J 245,243-250.
0098-2997/91 $0.00 + .50 ~) 1991 Pergamon Press pie.
FREE RADICAL DAMAGE TO PROTEINS AND ITS ROLE IN THE IMMUNE RESPONSE Roger Dean and Jeremy Simpson Heart Research Institute, 145 Missenden Road, Camperdown 2050, N.S.W., Australia
Introduction Free radicals are generated in many contexts within the immune response. In some cases they are part of the mode of action of immune effector cells. In other cases their generation is more a consequence of the progression of an immune response. In this brief review I will concentrate on the role of free radical damage to proteins in the immune response. While it has been traditional to study free radical damage to lipids, and the process of lipid peroxidation in pathological circumstances, the role of protein damage is probably more central to the action of some of the immune effector cells. In addition, protein damage is an inevitable part of degradative and remodeling processes in the immune response. There has been considerable advance in the study of free radical damage to proteins in the last five years, particularly through the work of Schuessler (e.g. Schuessler and Schilling, 1984) Stadtman and Levine (e.g. Rivett and Levine, 1987), Davies (e.g. reviewed Davies, 1986), and their colleagues, and through that of our own group. Other talks in this meeting will describe specific studies on immunoglobulins. Therefore I will survey the kinds of free radical damage which can be inflicted upon proteins in the first part of the paper. I will then discuss the consequences of such damage and its relation to the immune response.
The Nature of Free Radical Damage to Proteins in the Immune Response Systems for Investigation of Free Radical Damage to Protein Table 1 summarises the main kinds of experimental system used in such studies. While experiments using Fenton chemistry and enzymatic radical generation are widely available, they are complex and imprecise (Sutton and Winterbourn, 1989). However, this of course reflects the complexity of real living systems. On the other hand much useful and precise information can be obtained by using defined radical-generating systems such as cobalt gamma-radiolysis. While this is an unphysiological system, it does not allow control of generation of the physiologically relevant radicals. An intermediate level of control is offered by the thermolabile azo-compounds and these
121
122
R. Dean and J. Simpson
can be supplied to whole organisms (Terao and Niki 1988). They generate primary peroxyl radicals but secondary radicals may result from radical-radical reactions, so that the interpretation of the action of these compounds is not quite as simple as that of the gamma radiolytically generated defined radicals.
Table 1. Systems for investigation of radical damage to proteins Radical generating system
Radical available
Degree of control of radical generation
"/-Radiolysis
Most (aerobic or anaerobic)
Qualitative and quantitive control possible
Thermolabile azo compounds
Mainly peroxy
Rate can be kept constant and fairly accurately known, however phase effects influence the rate and there are secondary radicals generated in lower yield
Fenton chemistry
Superoxide, hydroxyl radicals; higher states of metals
Imprecise and complex
Enzymatic reactions
e.g. Xanthine oxidase and hypoxanthine: superoxide radical is the primary product. If metals are available complex reactions ensue
In a system with no available metal these reactions can be well controlled
Influences of Transition Metals Other papers in this meeting have discussed in some detail the relevance of iron and copper amongst transition elements to radical reactions in vivo. Here it probably suffices to point out that iron and copper may be bound to proteins and influence radical reactions thereon (reviewed Chevion, 1988). Histidine is the amino acid most able to chelate copper but specialised binding sites are known on some proteins for both copper and iron. In general the effect of such binding is to reduce the participation of the metals in bulk-phase redox cycling reactions. Sometimes this may localise damage to the protein, but in other cases (Table 2) radicals can be released into the bulk phase even when their interconversion involves protein-bound metals. We have recently studied this in some detail (Simpson and Dean, in press) and find that for many chelators of metals there is a complex dose response of radical-generation in the bulk phase. Thus for example, low concentrations of histidine (Simpson et al., 1988) or protein may gradually increase the availability of a fixed concentration of metal for radical reactions. However, as the concentration of chelator is increased a maximum is reached and subsequently there is depression of radical generation in the bulk phase. There are of course scavenging effects of such high concentrations of agents such as protein and histidine whereby radicals are still generated but rather than reacting with the detection system, react with the protein or amino acid (Dean et al., 1989).
Free Radicals and Proteins
123
In the immune response, as also mentioned elsewhere in this volume, metals may be displaced from their normal protein binding sites as a result of damaging reactions upon the surface of the protein effected by incident radicals. Thus the concept of misplaced transition metals is important in inflammatory disease and there are instances of haemorrhage of metals into inflammatory sites and into other chronic pathological sites such as the atherosclerotic plaque. Such misplaced metal may have drastic influence on radical reactions. The metal zinc competes with copper for binding sites on biological molecules and thereby defends against such damaging reactions of misplaced metals (reviewed Willson, 1989; Lovering and Dean, unpublished).
Free Radical Generation in the Immune Response Table 2 summarises the wide range of sources of free radicals which may be relevant in the immune response. It can be seen that radicals are both controlled products of effector cells within the immune system and also the consequence of poorly controlled progression of pathological aspects of the response. Thus, effector cells such as granulocytes, mononuclear phagocytes and lymphocytes, for their several different purposes can generate free radicals in controlled ways. On the other hand, the increased availability of transition metals or the development of peroxides upon already damaged biological macromolecules, may give rise to a source of further free radical reactions which can be damaging in the immune response. Normally the immune response involves inflicting damage on foreign entities such as invading organisms or on damaged tissue, so that the damaging reactions of radicals are not necessarily deleterious. However, in chronic immunological responses, as in some chronic inflammatory diseases, this damaging capacity of free radical reactions may become seriously deleterious.
Table 2. Somesources of free radical in the immune response Site
Reaction sources
Extracellular
Auto-oxidationreactions
Triggered fluxes from cells released extra-cellularly and into phagasomes
The "respiratoryburst" oxidase. This electron transport system present on granulocytesand mononuclear phagocytesgives rise to superoxide radicals and is activated by def'med triggers
Other radical fluxes which may be released from cells
Most cells, possibly in association with triggeringof cell division (e.g. proliferating lymphocytes). Untriggered macrophagesetc. (e.g. oxidising extracellular low density lipoprotein)
Intracellular fluxes
Auto-oxidationreaction Enzymatic reaction. Lipid and protein peroxidation
124
R. Dean and J. Simpson
Kinds of Damage Inflicted upon Proteins by Free Radicals Table 3 summarises some of the biochemical and chemical information as to the nature of damage inflicted upon proteins by free radicals. Much of the data summarised there has been obtained by experiments using defined in vitro conditions. What is less clear as yet is the extent to which such protein damage occurs in vivo. Table 3. Protein damage inflicted by free radicals Radical
Kinds of damage
02
Sulphydryl oxidation, but few other changes
HO2"
Limited fragmentation and crosslinking of soluble proteins. More extensive damage to membrane proteins
OH" and probably higher oxidation states of transition metals
Fragmentation, amino acid modification. Cross-linking (particularly when oxygen absent or limiting)
Radicals generated by local Fenton reactions with metal bound to the protein
Amino acid oxidation and fragmentationof polypeptide. Note that the reactive intermediates may also be available in the bulk fluid phase (Simpson and Dean, in press)
The information given above is supported by the following selected references from our own work: Dean et al., 1988a,b; Dean and Cheesman, 1987; Wolff and Dean, 1986, 1987; Dean et al., 1986; and other work is reviewed in Davies, 1986; Rivett and Levine, 1987. It is clear that polypeptides displaying radical damage can be detected in vivo. For example, in the atherosclerotic plaque one can detect the apo-protein of low density lipoprotein in a fragmented form and in which the lysines residues (and presumably other amino acids) have been modified in a manner consistent with free radical damage (Shaikh et al., 1988). This particular protein has been studied extensively and it probably reveals the kind of damage which takes place on many proteins during immune responses (Jessup etal., 1989a,b) and of course atherosclerosis involves inflammatory and immune components as we will discuss in the next section. Some of the proteins of the body which are longer lived also can be detected with modifications consistent with free radical damage: e.g. modified amino-acid residues in collagen (Dean et al., 1989). The fact that the demonstration in vivo of such modified protein is an infrequent event in the literature, is partly due to the fact that protein studies have been undertaken only recently, but also to the fact that of the consequences of protein damage which we shall discuss in the next section, involve the removal of the damaged protein. Demonstration that free radical damage to proteins produces modified entities which then may be removed by degradation, requires many more in vivo
Free Radicals and Proteins
125
measurements of such modified products and probably precursor product relationships will have to be established by means of radioactive tracer experiments.
Consequences of Free Radical Damage to Proteins in the Immune Response Inactivation of Protein Function In many circumstances, the first consequence of even very limited free radical damage to proteins is inactivation of their function. For example, enzymes can be inactivated by slight fluxes of radicals. This is true of proteinases, but also of proteinase inhibitors (Dean et al., 1989b; Reddy et al., 1989). In the immune system, the most important application of this is probably in the process of cytolysis. Some of the cells which are effectors of cell mediated cytolysis, for example the mononuclear phagocytes, are avid free radical generators and free radical fluxes are often triggered by contact with target cells. However, many other cytolytic components are produced by such professional killer cells, and in contrast there are other killer cells (e.g. NK cells) which lack the respiratory burst oxidase present on mononuclear phagocytes. As a consequence, the quantitative importance of free radical generation for cytolysis in these circumstances is somewhat unclear. What is clear however is that free radical damage inflicted from the exterior of cells can be lytic. There is more about this in some of the other manuscripts in this meeting, but it is worth emphasising here that protein damage may be a key component in such cytolysis. Thus in an experimental system in which macrophage cell lines were exposed to exogenously generated radical fluxes we found that lipid peroxidation and protein damage were normally concomitant and preceded cell death. One of the earliest events along this pathway we could detect was membrane depolarisation, which occurred within a few minutes of exposure of the cells to the radical fluxes. By means of lipophilic antioxidants such as butylated hydroxytoluene we were able to show that lipid peroxidation can be completely suppressed in these circumstances and yet membrane depolarisation, protein damage and subsequent lysis are all virtually unaffected. Thus protein damage may well be an important component in some of these cytolytic mechanisms and in our particular case, the inactivation of certain membrane transporters seems to be critical (Richards et al., 1988).
Degradation of Damaged Protein Most denatured or inactive proteins, however generated, are relatively rapidly removed by the cellular proteolytic machinery (reviewed Wolff etal., 1986). Thus for example intracellular abnormal proteins are degraded selectively and rapidly. Extracellular damaged proteins may also be endocytosed and degraded fairly efficiently and in some cases this is simply due to the release of the protein from a fixed extra-cellular matrix in which it was previously inaccessible (Wolff et al., 1986). Thus, in the immune response, extracelluar protein damage is often an important step in removal of tissue in a wounded site, or of organisms in an inflammatory site, prior to reconstruction of the area. In the case of neutrophil-mediated damage to such extracellular tissue, a clear role for free radicals in contributing to the process has been established by the work of Weiss and colleagues (e.g. Reddy et al., 1989). In the case of macrophages, such clear cut evidence has been very difficult to obtain (e.g. Dean and Schnebli, 1989), although the generation of modified forms of low density lipoprotein by macrophages has been well documented and does lead to enhanced endocytosis and degradation by the same cell (Jessup et al., 1989; Jessup et al., 1990). This may be an example of many processes in which macrophages use free radical damage as a part of the degradative mechanism, though it is one of the few for which such clear cut evidence exists.
126
R. Dean and J. Simpson
Persistent Modified Proteins The case just mentioned of low density lipoprotein is a case where protein modified by free radical damage can be detected in vivo in quite sizable pools. Thus damaged low density lipoprotein including damaged molecules of its polypeptide Apo-B can be detected in atherosclerotic vessel walls (Shaikh et al., 1988). No doubt this lipoprotein is undergoing accelerated endocytosis and is being degraded intracellularly but nevertheless its rate of generation suffices to maintain a detectable pool. This maybe somewhat unusual. However, a well known example has been in the literature for much longer: the aging pigment lipofuschin. This accumulates in lysosomes of cells in a variety of tissues of aging organisms. It is a complex incompletely characterised entity but it does include damaged polypeptides. It seems that the polypeptides may be so altered and sequestered by the lipid with which they are bound that in spite of their co-existence within lysosomes (with a huge array of degradative hydrolases), their turnover seems to be quite slow (Wolff et al., 1988). The reason for this slow degradation has not been elucidated and would be very interesting. A very interesting possibility which is addressed in another paper is that limited modifications to proteins may produce autoantigens which are not sufficiently perturbed in conformation to be immediately fully degraded, but which can nevertheless be recognised by the immune system as a trigger for generation of auto-antibodies. One could readily envisage that a modified protein in the extracellular phase could be generated during radical reactions in the immune response and then be endocytosed because of its increased exposure of hyrophobic moieties due to unfolding. Such hydrophobicity in itself is sufficient to enhance the endocytosis of many damaged proteins. However a further consequence might be that the intracellular transport of the modified protein might be distinct from that of the normal protein or of the normal catabolic pathway and hence permit the antigen to be processed and presented in different sites within the cell. Again the example of low density lipoprotein is interesting, since as mentioned already, modified low density lipoprotein can be detected in man, and in addition, auto-antibodies to low density lipoprotein are quite common and may have a bearing on pathology (e.g. Palinski et al., 1988).
Reactive Moieties on Free Radical Damaged Protein In our recent work, together with Jan Gebicki, Macquarie University, Sydney, we have characterised the generation of hydroperoxides upon protein (Thomas etal., 1989). While hydroperoxides have been studied extensively on lipids, and are known to be reactive entities (e.g. Hunt et al., 1989b) their production on protein has been neglected for many years. It is not clear precisely how hydroperoxides on proteins decay, and whether reactive entities are produced during this process, but it is clear that these hydroperoxides are rather unstable and that after a few days at 4°C most hydroperoxides have been destroyed. We have also demonstrated another reactive moiety upon radical damaged protein, which as yet remains undefined chemically (Simpson and Dean, 1989). We find that after radical damage several proteins become able to reduce the metal centre in Cytochrome C, and free iron and free copper from their higher valency to lower valency states. While the function of reducing free metals is possessed by some native proteins, and largely depends on their free cysteine residues, the generation of a vastly increased quantity of reducing capacity is a novel finding and may be important in view of the participation of transition metals in Fenton chemistry (Sutton and Winterborn, 1989). In some circumstances such reactions can be limited by failure to reduce metal which has become oxidised during the reactions and a reducing moiety present on protein might ensure the completion of Fenton chemistry in its vicinity and thus be an important influence upon damage at this site. This area of study is under intense investigation in our lab at present since it may have substantial bearing on the immune and inflammatory responses.
Free Radicals and Proteins
127
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