Selective degradation of oxidatively modified protein substrates

by the proteasome

Tilman Grune,

a

Katrin Merker,

a

Grit Sandig,

a

and Kelvin J.A. Davies

b,*

a

Neuroscience Research Center, Medical Faculty (Charit

ee) Humboldt University Berlin, Schumannstr. 20/21, 10117 Berlin, Germany

b

Ethel Percy Andrus Gerontology Center, and Division of Molecular and Computational Biology, University of Southern California,

Los Angeles, CA 90089-0191, USA

Received 9 March 2003

Abstract

Oxidative stress in mammalian cells is an inevitable consequence of their aerobic metabolism. Oxidants produce modifications to

proteins leading to loss of function (or gain of undesirable function) and very often to an enhanced degradation of the oxidized
proteins. For several years it has been known that the proteasome is involved in the degradation of oxidized proteins. This review
summarizes our knowledge about the recognition of oxidized protein substrates by the proteasome in in vitro systems and its
applicability to living cells. The majority of studies in the field agree that the degradation of mildly oxidized proteins is an important
function of the proteasomal system. The major recognition motif of the substrates seems to be hydrophobic surface patches that are
recognized by the 20S ÔcoreÕ proteasome. Such hydrophobic surface patches are formed by partial unfolding and exposure of hy-
drophobic amino acid residues during oxidation. Oxidized proteins appear to be relatively poor substrates for ubiquitination, and
the ubiquitination system does not seem to be involved in the recognition or targeting of oxidized proteins. Heavily oxidized proteins
appear to first aggregate (new hydrophobic and ionic bonds) and then to form covalent cross-links that make them highly resistant
to proteolysis. The inability to degrade extensively oxidized proteins may contribute to the accumulation of protein aggregates
during diseases and the aging process.
Ó 2003 Elsevier Science (USA). All rights reserved.

Keywords: Protein oxidation; Proteolysis; Proteasome; Lysosomes; Calpains; Free radicals

Life in an oxygen environment inevitably involves the

production of free radicals and other oxidants. One of
the consequences of this process is the continuous for-
mation of oxidatively modified proteins, both within
cells and in extracellular fluids. Since modified proteins
often experience significant loss of function (or gain of
undesirable function) they have to be replaced by the
cellular protein synthesis machinery. To avoid excessive
accumulation of damaged proteins, such non-functional
oxidized proteins have to be removed by proteolytic
systems. Substantial evidence, accumulated over the
past 20years, strongly suggests that the proteasomal
system is responsible for degrading oxidized proteins in
the cytoplasm, nucleus, and endoplasmic reticulum of
eucaryotic cells [1–31]. These findings have been ob-

tained with purified components of the proteasomal
system, with cell lysates, and with intact cells of various
lineages [1–31].

It has been concluded that mild oxidation of globu-

lar, soluble proteins enhances their proteolytic suscep-
tibility and makes them targets of the proteasomal
system [23,32,33]. An increasing body of literature in-
dicates that mildly oxidized proteins are readily de-
graded, whereas severe oxidation stabilizes proteins due
to aggregation, cross-linking, and/or decreased solubil-
ity, thus increasing their half-lives [21–24,27–31]. The
inability to degrade extensively oxidized proteins may
contribute to certain disease states, including various
neurodegenerative diseases such as AlzheimerÕs disease
[34], or ParkinsonÕs disease [35], and could also play a
significant role in the aging process [36–38].

The aim of this review is to summarize the current

knowledge about the protein substrates that have been

Biochemical and Biophysical Research Communications 305 (2003) 709–718

www.elsevier.com/locate/ybbrc

BBRC

*

Corresponding author. Fax: [phone].

E-mail address:

[e-mail]

(K.J.A. Davies).

0006-291X/03/$ - see front matter

Ó 2003 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(03)00809-X

used in studies of protein oxidation and proteolysis. In
particular, we have focused our attention on those
substrates that have been used to test the selectivity of
the proteasome for the oxidized forms of intracellular
proteins.

Oxidative protein modification

The degree of protein oxidation caused by a given

oxidant depends on many factors, including the nature,
relative location, and flux rate of the oxidant, and the
presence (or absence) of antioxidants. Both the oxida-
tion of free amino acids and the oxidation of peptides
and proteins have been studied by many laboratories,
and numerous amino acids are known to be susceptible
to oxidation [4–6,11,16,18,39,40]. Although chemical
reactions occur during amino acid side chain oxidation,
the products of these processes differ only minimally in
molecular weight from those of the original amino acid
residues. On the other hand fragmentation of polypep-
tide backbones can occur, leading to the formation of
protein fragments which are not true peptides (these are
formed during proteolytic degradation of polypeptides),
but protein fragments with derivatized terminal amino
acids [4–6,11,41]. Depending on the conditions of the
oxidation and the oxidant itself, these fragments will
vary in length and in rate of formation.

Protein aggregates can also form during free radical

reactions [5,6,23,38,41]. It is suggested that the formation
of protein aggregates occurs initially on a non-covalent
basis, largely involving such forces as hydrophobic
bonds and electrostatic interactions. Subsequently, these
aggregates tend to form covalent cross-links due to re-
actions between carbon-, oxygen-, and nitrogen-centered
radicals of amino acid side chains. One of the most

thoroughly investigated cross-links is the formation of a
2,2

0

-biphenyl bond between two tyrosyl radicals, to form

dityrosine or bityrosine [16]. During the process of co-
valent cross-linking of protein aggregates, non-protein
components such as carbohydrates and oxidized lipids
can also react and add to the growing oxidized mass of
material [29,42,43]. It is now clear that these protein
aggregates are poor substrates for proteases, which re-
sults in their accumulation within cells [15–17,23,29,32,
38,41].

Accumulation of cross-linked proteins

Several diseases, and aging processes, are accompa-

nied by the accumulation of cross-linked proteins. This
accumulation of oxidized protein aggregates can occur
both extracellularly, and within various cellular com-
partments. Differences in the effects of protein aggre-
gates on various cellular or organismal functions may be
expected, depending on the rate of formation and the
exact location of such aggregates. In several cases ag-
gregated/cross-linked material will be autophagocyto-
sed, resulting in a major accumulation of the material in
lysosomes [44,45].

The accumulation of oxidized proteins can result

from several kinds of malfunctions of cellular metabo-
lism. As demonstrated in Fig. 1 the accumulation of a
protein can be the result of a genetically determined
lower proteolytic susceptibility, or an innate proneness
to aggregation (Fig. 1, pathway 1). The oxidation of
protein aggregates in this case is most likely the result of
the prolonged half-life of such proteins. A prominent
example of such a mutation is the Huntington disease,
where the mutated Huntigtin protein has a prolonged
poly-Glu-strech that causes aggregation. Under other

Fig. 1. Possible mechanisms for the accumulation of oxidized proteins. If a given protein accumulates, the corresponding gene might be mutated
(pathway 1), the protein might undergo excessive or artificial posttranslational modification (pathway 2) or the balance between synthesis and
degradation might be disturbed due to poor metabolic regulation (pathway 3). The accumulating (non-degraded) protein might in all cases undergo
secondary oxidation.

710

T. Grune et al. / Biochemical and Biophysical Research Communications 305 (2003) 709–718

conditions, an increase in posttranslational modification
can be the reason for the formation of protein aggre-
gates (Fig. 1, pathway 2). Since posttranslational mod-
ifications

change

the

proteolytic

susceptibility

of

substrates, some proteins will experience decreased
proteolysis. Alternatively, a high formation rate of
posttranslationally modified proteins might simply
overwhelm the capacity of the proteolytic systems. Ex-
amples for posttranslational modifications changing the
proteolytic susceptibility of substrates include oxidative
modifications [1–31] and phosphorylation of the tau
protein [46]. Furthermore an imbalance between protein
synthesis and proteolytic capacity might be the reason
for an accumulation of cross-linked proteins (Fig. 1,
pathway 3). Examples are the ceroid lipofuscinoses with
their decline in proteolytic capacity [47,48].

With time such aggregated proteins are transferred

into a highly cross-linked and reactive material, referred
to as lipofuscin, ceroid or AGE-pigment-like fluoro-
phores by several authors [49,50]. The involvement of
free radicals as one of the initial steps in the formation
of fluorescent oxidized/cross-linked aggregates has been
postulated [51,52].

Degradation of oxidized proteins

Following exposure to oxidants one can detect

changes in the proteolytic susceptibility of a number of
protein substrates (Fig. 2). This change in proteolytic
susceptibility has a biphasic response. At moderate
oxidant concentrations proteolytic susceptibility in-
creases, whereas at higher oxidant concentrations a de-
crease (sometimes even below the Ôbasal degradationÕ
level) in proteolytic susceptibility occurs (see Fig. 2).
Between these extremes, the oxidant reaches an Ôoptimal
concentrationÕ characterized by a Ômaximal degradationÕ
rate for the given conditions. The increase in degrada-
tion or the Ôproteolytic stimulationÕ is the ratio of the
Ô

maximal degradationÕ to the Ôbasal degradation.Õ

As one can see in Table 1, a large number of varied

proteins have been used as substrates to test for oxidant-
induced proteolytic stimulation. The phenomenon seems
to be a common feature of all globular, soluble proteins
with defined secondary and tertiary structures. The or-
igin of the protein, however, whether extracellular, cy-
tosolic, nuclear, native or recombinant, seems not to
have any importance. Essentially ÔstructurelessÕ proteins,
such as casein, are inherently good substrates for pro-
teolysis and their susceptibility is not increased by mild
oxidation; although it can be decreased by heavy oxi-
dation.

In addition to numerous proteolytic substrates, vari-

ous oxidizing systems, employing either bolus treat-
ments or fluxes of oxidants, have been used to oxidize
many proteins (Tables 1 and 2). Although the concen-

tration or dosage of the oxidants reported in Tables 1
and 2 varies widely, due to the different reactivity and
stability of each oxidizing agent, an increase in proteo-
lytic susceptibility could always be achieved at an Ôop-
timal oxidant exposure.Õ Naturally, the increase in
proteolytic susceptibility depends on the oxidizing
agent, the protein substrate, and the exact experimental
conditions (Table 2). However, the increase in proteo-
lytic stimulation differs also as a function of the nature
and the source of the proteolytic enzyme(s) employed
(Tables 3 and 4). Furthermore, several reports have
demonstrated that increased proteolytic susceptibility is
limited to a certain oxidant concentration range, which
is often very tight [53], implicating the possibility of
missing this Ôoptimal concentrationÕ range in any ex-
perimental setup. This reveals one of the potential dif-
ficulties in measuring the in vitro effects of oxidants on
the proteolytic susceptibility of a given protein sub-
strate.

On the other hand a number of different sources for

the proteasomal system have been employed by various

Fig. 2. Proteolytic susceptibility of oxidized proteins. Due to increasing
oxidation, the proteolytic susceptibility of the substrate increases to a
certain point (Ôoptimal oxidant concentrationÕ) but at higher oxidant
concentrations begins to decline. Initially, oxidation causes charge
rearrangements within the protein substrate, with random refolding
and partial exposure of internal hydrophobic amino acid Ôpatches.Õ
These hydrophobic patches bind to the core 20S proteasome and en-
able the protein substrate to be fully degraded. With further oxidation,
however, the surface hydrophobicity of the substrate protein continues
to increase, due to further oxidation-induced unfolding, and these
hydrophobic patches attract each other (or are repelled by the aqueous
environment) so quickly that proteasome has difficulty competing, and
aggregation becomes a significant problem. Over time, new covalent
bonds and ionic bonds form between oxidized amino acid residues in
the aggregates, making them highly resistant to any form of proteo-
lysis. For further information see text.

T. Grune et al. / Biochemical and Biophysical Research Communications 305 (2003) 709–718

711

Table 1
Overview of the most common oxidized protein substrates used for in vitro degradation assays

Protein

Oxidant

Proteasome source

Reference

Hemoglobin

H

2

O

2

, phenylhydrazine

Rabbit erythrocytes and reticulocytes, human erythrocytes, isolated

proteasome (20S and 26S), and C9 and K562 cells

[5,13,15–18,21,22,71–74]

Superoxide dismutase

O


2

, H

2

O

2

,



OH, SIN-1

Rabbit reticulocytes and erythrocytes, human erythrocytes and

reticulocytes, bovine erythrocytes, isolated proteasome,
and C9 and RAW264 cells

[5,9,13,21,24,71,75]

Laminin

H

2

O

2

Rabbit erythrocytes and reticulocytes, human erythrocytes, and

primary microglia

[76]

Bovine serum albumin



OH, phenylhydrazine O


2

, H

2

O

2

Rabbit reticulocytes and erythrocytes, human erythrocytes, isolated

proteasome, and mice kidney cells

[3,5,13,14,74,77–79]

a

-Casein



OH, AGE, CML, H

2

O

2

Rabbit erythrocytes and reticulocytes, human erythrocytes, isolated

proteasome, and K562 cells

[3,5,13,80]

Aconitase

H

2

O

2

, SNAP, ONOO



, SIN-1

Isolated proteasome

[24]

Myosin, ovalbumin, collagenase,

and carbonic anhydrase

SIN-1

Isolated proteasome

[24]

Myoglobin

SIN-1, H

2

O

2

Isolated 20S proteasome

[24,56]

Ferritin

SIN-1, ONOO



, NDPO

2

, rose

bengal + light, DEA-NO, X/XO,
H

2

O

2

Rabbit erythrocytes and reticulocytes, human erythrocytes, isolated

proteasome, K562, CH E36, and ts20E1 mutant cells

[24,53,65,81–83]

Catalase

H

2

O

2

, SIN-1

Isolated proteasome

[5,24]

Histones 1, 2A, 2B, 3, and 4

H

2

O

2

K562, C9 cells, isolated proteasome

[27,28]

Protein disulfide isomerase

H

2

O

2

C9 cells

[84]

Ezrin

H

2

O

2

Primary mice liver cells

[85]

Lysozyme

FeCl

3

/Ascorbate H

2

O

2

CH E36 and ts20E1 mutant cells, isolated proteasome

[65,86]

Glutamine synthetase

O


2

, FeCl

3

/ascorbate, HNE,

FeSO

4

/citrate

Rat liver

[1,2,42,43,58,87,88]

G6PDH

HNE, FeSO

4

/Citrat, AGE, CML,

H

2

O

2

Isolated 20S proteasome

[42,43,53,89,90]

Calmodulin

H

2

O

2

HeLa cells, isolated 26S proteasome

[90,91]

a

, b

L

, b

H

, c-Crystallin

60

Co-irradiation,



OH, H

2

O

2

, UV

Isolated proteasome, bovine lens epithelial cells

¼ BLEC,

rabbit erythrocytes, and mice macrophages

[20,92–95]

Apolipoprotein B (human)

CuCl

2

B lymphoblastoid cell line

¼ LCL

[96]

Tetanus toxin



OH

RAW264 cells

[97]

Myelin basic protein

¼ MBP

H

2

O

2

Primary microglia

[76]

712

T.
Grune

et
al.
/

Biochemic

al
and

Biophysical

Research

Communicati

ons

305

(2003)

709–718

investigators, using diverse sources for crude cell lysates,
purified cell lysates or isolated proteasomes (see Tables
1–4). Since in a large number of these experiments the
species of the substrate protein in question and the
source of the proteasome do not match, one can assume
a general, species overlapping mechanism for the rec-
ognition of oxidized proteins. Therefore, it can be con-
cluded that the removal of ‘‘minimally’’ oxidized
proteins is an essential function for maintaining cellular
homeostasis by preventing the accumulation of highly
oxidized and cross-linked proteins, which are no longer
degradable and which may threaten cellular/organismal
viability.

Recognition of the oxidized protein substrates by the
proteasome

It has been shown that erythrocytes and reticulocytes

from rabbits, cows, and human beings, as well as rat
muscles in vitro, rat hepatocytes, fibroblasts, macro-

phages, tumor cells, and Escherichia coli cells [1,2,9,14–
16,21,22,24,30,31,54–57] are able to selectively degrade
oxidatively modified proteins. What forms the recogni-
tion motif of oxidized proteins for the proteasome is one
of the key research questions surrounding the fate of
oxidized proteins. The selective oxidation of several
amino acids, and their resulting products, has to be
taken into account as possible recognition markers.
Levine et al. [58], for example, could clearly demon-
strate an increase in the proteolysis of glutamine syn-
thetase after oxidizing a threshold level of methionine
residues. Lasch et al. [59] found a clear correlation be-
tween tyrosine oxidation and proteasomal degradation
of RNase A. Numerous other examples of single amino
acid changes that correlate with altered proteolytic
susceptibility can be found in the literature. Although
such findings are important, one has to be aware that
many protein oxidation processes correlate with the
oxidation of several amino acids and with changes in
the secondary, tertiary, and even quaternary structures
of substrate proteins. Furthermore, as one can see in

Table 2
Increased proteolytic susceptibility of ferritin after treatment with various oxidants

Oxidant

Optimal oxidant concentration

Proteolytic stimulation

References

SIN-1

2 mM

12.6-fold

[24]

SIN-1

5 lmol

 mg protein

1

4-fold

[82]

ONOO



100 lM

1.9-fold

[81]

NDPO

2

50 lM

2.9-fold

[81]

DEA-NO

1 lmol

 mg protein

1

2.5-fold

[82]

X/XO

0.75 nmol

 mg protein

1

4.5-fold

[81]

H

2

O

2

10 lmol

 mg protein

1

5.5-fold

[83]

H

2

O

2

15 mM

3.7-fold

[65]

H

2

O

2

10 lmol

 mg protein

1

3-fold

[81]

Table 4
Increase in proteolytic susceptibility of superoxide dismutase after treatment with hydrogen peroxide under various experimental conditions

Proteasome source

Optimal H

2

O

2

concentration (mM)

Proteolytic stimulation

References

Isolated proteasome

1028-fold

[71]

C9 cells

15

5.4-fold

[22]

Rabbit erythrocytes

304-fold

[75]

Bovine erythrocytes

302.5-fold

[75]

Bovine erythrocytes

303.4-fold

[9]

Rabbit erythrocytes

3013-fold

[13]

Rabbit reticulocytes

3012-fold

[13]

Table 3
Increased proteolytic susceptibility of hemoglobin after treatment with the hydroxyl radical under various experimental conditions

Proteasome source

Optimal



OH concentration

Proteolytic stimulation

References

Rabbit erythrocytes

20nmol



OH/nmol protein

9-fold

[13]

Rabbit reticulocytes

20nmol



OH/nmol protein

10-fold

[13]

Isolated proteasome

20nmol



OH/nmol protein

5.5-fold

[15]

Human erythrocytes

25 nmol



OH/nmol protein

7.5-fold

[15]

Isolated proteasome

20nmol



OH/nmol protein

18-fold

[71]

Rabbit erythrocytes

150nmol



OH/0.33 mg protein

2.6-fold

[5]

Rabbit reticulocytes

150nmol



OH/0.33 mg protein

12.9-fold

[5]

T. Grune et al. / Biochemical and Biophysical Research Communications 305 (2003) 709–718

713

Table 5, the substrate proteins that have been studied
differ widely in amino acid content, and therefore also in
the oxidizable amino acid side chains, yet all show an
oxidation-induced increase in proteolytic susceptibility.

Several years ago we proposed that oxidized proteins

are partially unfolded due to the loss of regular sec-
ondary and tertiary structures within the domain of the
oxidative impact [15,17,23]. Lasch et al. [59] were able to
demonstrate an up to 50% unfolding of RNase A under
conditions allowing an increase in proteolytic suscepti-
bility. Such unfolding is clearly accompanied by an ex-
posure of hydrophobic patches from the interior of the
protein globule to the outside. This is a possible reason
for the aggregation of oxidized proteins through hy-
drophobic interactions. On the other hand, it was also
proposed that these sites might serve as recognition
motifs for proteolysis. The strong correlation between
the increase in proteolytic susceptibility and the increase
in hydrophobicity of the protein substrate was demon-
strated by separation of substrates, according to their
hydrophobicity, by hydrophobic interaction chroma-
tography [15,17] or by using fluorescence labels detect-
ing the surface hydrophobicity of proteins [58,60]. Since
it was shown that the proteasome has a preference to
bind hydrophobic and aromatic amino acids [61] the
recognition of these hydrophobic ‘‘unfolded’’ patches by
the proteasome seems likely. However, although it is
assumed that this is the main mechanism for recognition

of oxidized proteins in vitro it is not yet clear whether
this is also the main mechanism responsible for the re-
moval of oxidized proteins from living cells.

Role of further components of the proteasomal system in
the recognition of oxidized proteins

Numerous studies have been performed using the

isolated 20S ÔcoreÕ proteasome to degrade oxidized
proteins. However, since our knowledge about the
proteasomal system, its components, and its coordi-
nated action with various ubiquitination systems is quite
extensive, the question arises as to which form of the
proteasome is involved in the degradation of oxidized
proteins. Today it is accepted that the proteasome is just
the core proteolytic particle of a whole system of regu-
latory factors, many of which interact with the ubiqui-
tination system and several heat shock and chaperone
proteins [62–64]. Therefore, the question was raised
whether any of these factors is involved in the recogni-
tion of oxidized proteins. Clearly in studies using the
isolated 20S ÔcoreÕ proteasome it was demonstrated that
this protease is able to recognize oxidized proteins [1–
31]. However, whether this is a process with physiolog-
ical relevance in living cells was, at first, unclear.
Therefore, more complex systems such as cell lysates
were used to test the degradation of oxidized proteins.

A number of early studies demonstrated that ATP

has no stimulating effect on the degradation of oxidized
proteins in cell lysates, thus denying involvement of the
19S/PA700 proteasome activator [22]. In fact ATP ac-
tually inhibits the degradation of all oxidized proteins
investigated so far in cell free lysates, by some 10–20%.
Furthermore, no evidence has been reported that oxi-
dized proteins are specifically recognized by the ubiq-
uitination system. In contrast, we were recently able to
demonstrate that there is no ubiquitination of oxidized
and highly degradable ferritin and lysozyme in an in
vitro system, whereas the heat denatured forms of these
proteins were ubiquitinated [65]. The lack in ubiquiti-
nation of oxidized proteins may be due to oxidative side
chain modification of lysine residues, which are the
binding site for ubiquitin. The accumulation of ubiqui-
tinated proteins and ubiquitinated oxidized proteins due
to oxidative stress which was reported by Shang and
Taylor [20] and Shang et al. [66] is probably, therefore, a
non-specific effect.

Recognition of oxidized proteins in cells

Although it is generally accepted that oxidized, un-

folded proteins can be degraded by the isolated 20S
Ô

coreÕ proteasome in vitro, it has been rather less clear if

this same form of the proteasome actually has physio-

Table 5
Comparison of the amino acid composition of commonly used sub-
strates in oxidation-degradation assays and their maximal proteolytic
stimulation

Amino acid

Hemoglobin

Ferritin

L

L

-chain

SOD1

Total number

147

136

154

Ala

10.2

13.2

6.5

Cys

1.4

0.7

2.6

Asp

4.8

2.9

7.1

Glu

5.4

5.1

6.5

Phe

5.4

2.9

2.6

Gly

8.8

5.9

16.2

His

6.1

1.5

5.2

Ile

05.9

5.8

Lys

7.5

9.6

7.1

Leu

11.6

8.8

5.8

Met

1.4

1.5

0.6

Asn

4.1

0.7

4.5

Pro

4.8

4.4

3.2

Gln

2.7

5.9

1.9

Arg

2.013.2

2.6

Ser

3.4

4.4

6.5

Thr

4.8

7.4

5.2

Val

12.2

3.7

9.1

Trp

1.4

00

.6

Tyr

2.02.2

0

Maximal proteolytic

stimulation
[Reference]

28-fold [71]

12.6-fold [24]

18-fold [71]

714

T. Grune et al. / Biochemical and Biophysical Research Communications 305 (2003) 709–718

logical relevance in living cells. Rivett [1,2] demonstrated
the selective degradation of oxidatively modified gluta-
mine synthetase in a non-lysosomal pathway by a cy-
tosolic protease. Subsequently, numerous studies have
demonstrated that this key enzyme is the proteasome,
although the literature is confusing because at least 20
different names have been used for the same proteinase.
Since the proteasome is responsible for the degradation
of most soluble intracellular proteins it appeared quite
reasonable that proteasome would also be responsible
for the degradation of oxidized intracellular proteins.
Perhaps the clearest answer about the degradation of
oxidized proteins by the proteasomal system was pro-
vided by means of anti-sense oligodeoxynucleotides di-
rected against the (essential) C2 proteasomal subunit.
Anti-sense treated cells are essentially depleted of the
proteasome and are no longer able to increase protein
turnover after oxidative stress, to degrade oxidatively
modified proteins [21,22,24]. Additionally it was shown
that the proteasome-specific or selective inhibitors such
as lactacystin, b-lactone, NLVS, and epoxomicin all
prevent the removal of oxidized groups from the protein
pool [55,71,84].

One particular difficulty with deciding which form of

the proteasome is important in degrading oxidized
proteins in vivo was an assumption, by several in the
field, that 20S core proteasome would not exist without
bound regulatory proteins in living cells. Thus, many
researchers felt that proteasome would always be found
with bound 19S or 11S regulators, to form the 26S
proteasome or the Ôimmunoproteasome,Õ respectively.
Recently, the actual distribution of all the proteasome
forms has been carefully assessed, and the presence of
free 20S ÔcoreÕ proteasome particles in living cells has
been demonstrated [67,68]. In fact, these important
studies have actually revealed that the concentration of
free 20S ÔcoreÕ proteasome exceeds all other proteasome
forms in living cells by 2- to 3-fold.

Many, perhaps most, intracellular proteins are de-

graded by the 26S proteasome system (the 20S core
proteasome, with a 19S regulatory subunit at each end)
in a process that requires initial ubiquitination of the
protein substrate. The primary and secondary structures
of proteins make their lysine residues either more or less
prone to ubiquitination by a series of ubiquitin activat-
ing, ubiquitin conjugating, and ubiquitin ligating en-
zymes. The result is that proteins eventually obtain a
long polyubiquitin Ôtail.Õ This hydrophobic polyubiquitin
chain is recognized and bound by the 19S regulators of
the 26S proteasome. Energy from ATP hydrolysis is then
used by the 19S regulators to remove the polyubiquitin
chain and to unfold the substrate protein. The unfolded,
de-ubiquitinated substrate protein is then ÔfedÕ into the
cylinder of the 20S ÔcoreÕ proteasome, where it is pro-
teolytically degraded in a process that requires no ATP.
Thus, ubiquitin serves only to ÔtagÕ a protein for prote-

olysis, and proteins whose structure allows rapid ubiq-
uitination will undergo rapid turnover in vivo. Similarly,
energy from ATP hydrolysis is only required to remove
polyubiquitin chains from substrate proteins and to un-
fold globular proteins. Our studies of protein oxidation
and proteolysis have consistently shown a lack of ATP
involvement in the degradation of oxidized proteins in
mammalian cells and cell extracts [5–7,9,13–18,21–24,
27–29,71,77,78,83–85]. The possible involvement of the
ubiquitination system in the recognition of oxidized
cellular proteins was recently overruled by the recent
work of Shringarpure et al. [65]. In this work we were
able to show that cells deficient in the ubiquitination
system, and therefore unable to ubiquitinate proteins, do
not lose the ability to degrade oxidized proteins. There-
fore, it seems clear that the proteasome is responsible for
the degradation of oxidized non-ubiquitinated proteins.

We have proposed that partial denaturation, partial

unfolding, and exposure of internal Ôhydrophobic pat-
chesÕ of amino acids are the key to selective recognition
and degradation of oxidized proteins by the 20S pro-
teasome

[4–6,13,15–17,21–24,28,29,56,57,65,71,77,78].

In our model, the hydrophobic patches of oxidized
proteins can bind to the a-subunits at the entrance to the
20S proteasome core cylinder. This step would be im-
portant because the entrance to the 20S core proteasome
cylinder is normally closed. Binding of such partially
unfolded, oxidized substrates is then proposed to acti-
vate further substrate unfolding and complete proteol-
ysis. Recent work by other groups [69,70] has provided
independent evidence to support the idea that substrate
binding can result in proteasome opening and activa-
tion, without ATP or ubiquitin.

In closing, it now seems clear that the 20S core pro-

teasome plays a major role in the degradation of oxi-
dized

proteins

in

the

cytoplasm,

nucleus,

and

endoplasmic reticulum of eucaryotic cells. Diminished
proteasome activity and consequent diminished clear-
ance of oxidized proteins in aging [41,56,57,71,89] and
various diseases [29,43,71,98–100] underlies the impor-
tance of this physiological function. The possibility that
either, or both, the 26S proteasome and the immuno-
proteasome may also degrade oxidized proteins by
mechanisms that do not require ATP or ubiquitin still
exists, however [70]. Furthermore, it is also still possible
that other proteasome regulators and/or chaperone
proteins may be involved.

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