CHAPTER 32
Ubiquitin-Proteasome Pathway
in the Pathogenesis of Liver Disease
Samuel W. French, Fawzia Bardag-Gorce
32.1
Introduction
The discovery of the ubiquitin-proteasome pathway
as a regulated system of protein digestion within all
cells [32] has led to an appreciation of the impor-
tance of protein turnover control. This control is a
mechanism for regulation of cellular processes and
quality control of intracellular proteins. Many liver
cell functions are regulated by this mechanism of
protein degradation. These include cell cycle check
points and activation of transcription factors such
as nuclear factor-κB (NF-κB) (Chapter 29), and hy-
poxia inducible factor-1α (HIF-1α) (Chapter 26)
[28, 67, 71]. The loss of proteasomes or the inhibi-
tion of the ubiquitin-proteasome pathway can lead
to hepatocellular injury including proliferation and
apoptosis [74, 107], and hepatic inclusions of ag-
gregated cytokeratins [24]. Liver cell gene expres-
sions, dependent on transcription factor activation
by the proteasome, could impede the inflammatory
response of the liver and the response to hypoxic in-
jury. The importance of the ubiquitin-proteasome
pathway to the homeostatic mechanisms involved
in liver injury is the focus of this review.
32.2
Ubiquitin-Proteasome Pathway
The enzymes that catalyze ubiquitin activation,
conjugation and ligation are depicted as E1, E2, and
E3, respectively in Fig. 32.1. Ubiquitin is indicated
as a shaded circle. Note that the polyubiquitinated
protein indicated by Cn+1 docks at the 19S protea-
some. Alpha (α) units of the 20S proteasome par-
ticle are shown in gray, while the beta (β) subunits
are shown in white. Both types of subunits make up
the 20S catalytic core of the proteasome. The arrows
shown at the 19S proteasome indicate that the ubiq-
uitinated protein is deubiquitinated by proteasomal
deubiquitinase before the protein unfolds and enters
32
the chamber of the proteasome to undergo digestion.
Ubiquitin (shown as Ub) exists as a pool of free mol-
ecules (upper left of Fig. 32.1), which can be attached
either singly or in polyubiquitin chains to the sub-
strate protein (bottom center). The polyubiquitinat-
ed protein substrate is then degraded by the 26S pro-
teasome to smaller peptides, which can then either
be degraded to amino acids by other proteinases or
be presented on the surface of the cell membrane in
the immune cells. The beta subunits in the middle
of the 20S proteasome include chymotrypsin-like
and trypsin-like catalytic enzymes, which digest
the proteins that enter the chamber. The entrance of
the proteins into the digestion chamber is gated by
alpha subunits. In this way the digestion of proteins
is selectively regulated. Proteins that are digested by
the 26S proteasomes must first be targeted to pro-
teasomes by at least a 4 polyubiquitin chain where
the C-terminal glycine is covalently bound to lysine
residues in the protein destined to be digested by the
proteasome.
32.3
Ubiquitination of Proteins Destined
for Digestion by the Proteasome
Initially, in the conjugation phase of ubiquitin to the
protein destined for proteolysis by the proteasome,
ubiquitin is first activated through the ATP-depend-
ent formation of thiol ester with a cysteine residue of
the ubiquitination-activating enzyme (E1) (Fig. 32.1).
The carboxyl group of the last amino acid of ubiqui-
tin, a glycine-glycine dipeptide, is first activated by
adenylation. A thiol group in the activating enzyme
(E1), which initiates the ubiquitination enzyme cas-
cade, attaches the ubiquitin-carboxyl-adenosine 5’-
monophosphate to form a E1-ubiquitin thiol ester.
Ubiquitin is then transferred to a cysteine residue
on a ubiquitin-conjugating enzyme (E2) (Fig. 32.1).
Ubiquitin is then transferred to a lysine residue of
the protein substrate in a reaction catalyzed by a
ubiquitin-protein ligase (E3) (Fig. 32.1) [38, 40]. All
378 PART II: Specific Signaling Pathways
ATP
known E3s utilize one of two catalytic domains,
HECT domains or a RING finger [81].
The ubiquitin-proteasome system of regulated
protein digestion is highly selective where signal-
dependent proteolysis directs the time and location
of protein turnover in the cell [38, 40]. The selection
of proteins to be degraded by the proteasome oc-
curs at the point of conjugation, though the E2s and
E3s have different substrate specificities. Each E3,
in conjunction with its cognate E2, binds substrates
that share a particular ubiquitination signal, usual-
ly a small primary sequence motif [53, 62, 80]. Rec-
ognition of the primary signal targets the substrate
to be labeled with a second signal, i.e. the polyu-
biquitin chain formation. Only the polyubiquitin
chain signal is recognized by the proteasome. The
phenomenon of polyubiquitinated secondary signal
formation provides a large number of substrates for
the proteasome, which are predestined for selective
digestion.
The recognition of the secondary signal by the
proteasome is true only for the proteasome and
differs in this way from other ATP-dependent pro-
teases in the cell [100]. The ubiquitin molecules are
linked covalently to one another through K48-G76
isopeptide bonds. The first ubiquitin binds a lysine
in the substrate. This supports residues in the sub-
strate to generate an isopeptide bond [16]. The af-
finity of binding of the polyubiquitin chain to the
proteasome increases 100-fold as the chain length
increases from two to four ubiquitins, but increases
Fig. 32.1. Scheme
of the ubiquitin-proteasome
pathway
Deubiquitination
only tenfold when eight more ubiquitin molecules
are added [100]. Therefore, the length of the polyu-
biquitin chain determines the rate of substrate deg-
radation by the proteasome, after the chain binds to
the 19S proteasome, possibly because of the nature
of the deubiquitinating enzyme chain reaction. If
the chain is too short before degradation is initiat-
ed, the substrate will be deubiquitinated, but not de-
graded. A longer ubiquitin chain would allow more
time for unfolding of the substrate and opening the
gate to allow entrance of the substrate into the cata-
lytic chamber of the 20S proteasome for digestion.
The ubiquitin-proteasome system is regulated
at the level of ubiquitination or at the level of the
proteasome activity. The targeting motif can be the
N-terminal residue, a sequence, a domain, or a post-
translational change such as phosphorylation that
results from externally generated signal transduc-
tion. Phosphorylation of either the substrate or the
ligase can activate binding of the ligase. Binding can
be enhanced by ubiquitin-like proteins (UBL) by in-
creasing the affinity of the ligases to the E2 compo-
nent of the conjugating apparatus, especially in the
case of phosphorylated substrates. UBL proteins in-
teract with the polyubiquitinin domain (UBA). The
UBL domain probably acts to facilitate the interac-
tion with UB-containing proteins, targeting them to
the proteasome [17]. Linking elements on proteins
such as UBA and UBL may increase the efficiency of
the proteasome process.
 CHAPTER 32: Ubiquitin-Proteasome Pathway in the Pathogenesis of Liver Disease
32.4
Ubiquitin-Proteasome Mechanics
The processing of short-lived regulatory and struc-
tural proteins within the cell for degradation by
the ubiquitin-proteasome system is extraordinarily
complex and has recently been reviewed elsewhere
in detail [32]. The process involves the folding of
intracellular proteins by heat shock proteins and
chaperones [35, 37]; polyubiquitination by three
enzymes in sequence (ubiquitin-activating enzyme,
E1; ubiquitin-conjugating enzyme, E2s; and ubiqui-
tin-protein ligase, E3s); assembly of the 20S protea-
some containing the catalytic subunits; assembly
of the 19S proteasome including the base and lid;
assembly of the 26S subunit; ATP, and deubiquiti-
nating enzyme. There are thousands of proteasomes
in hepatocytes that are mobile in the cytosol and
localized to the nuclear pore, endoplasmic reticu-
lum, and the nucleus [33]. In pathologic conditions,
where proteins accumulate to form an aggresome,
the proteasomes localize in the aggresomes [42].
When isolated from the liver they are found as 20S,
20S with one 19S, and 20S with two 19S attachments
(26S) [28]. The 20S proteasome does not require
ubiquitination of the proteins or ATP to digest pro-
teins. This includes oxidized proteins [91, 97]. Thus,
the digestion of proteins by the 26S proteasome is
regulated by the polyubiquitinated protein and
ATP-dependent mechanisms.
Other proteins involved in delivering the ubiq-
uitinated proteins to the proteasome for digestion
include CHIP, Rad 23, AG-1, and P62 (ZIP). These
proteins dimerize and bind to polyubiquitin chains
and the proteasome to facilitate proteolysis through
a hypothetical UBA/UBL [18, 30, 35–37, 83, 102]. The
protein substrates for proteasome digestion are nu-
merous and the list is still growing [24, 101]. These
include p53, Cdc 25, Cdc 18, IKBα, β-catenin, CD4,
HIF-α, EGF-R, PDGF-R, Mdm2, and MATα2, which
are involved in liver diseases. Each has a different
ubiquitin ligase.
32.5
Experimental Alcoholic Liver Disease
and Proteasomal Catalytic Activity
Donohue [22] has recently reviewed the literature
summarizing the evidence implicating the ubiq-
uitin-proteasome system in ethanol-induced liver
disease. He first introduced the concept [21] but, to
date, it has largely been ignored in recent reviews
of the mechanisms of alcohol-induced liver injury
379
[69]. The concept of liver injury through this mech-
anism applies to both alcoholic and non-alcoholic
liver disease in man [98, 99], as well as experimental
ethanol-induced liver disease in rats and mice [4, 6,
21, 23, 28].
32.6
Functional Consequences
of Ethanol Inhibition
of the Proteasome in the Liver
Donohue et al. [21] first showed that the activities
of three proteolytic enzymes located in the 20S pro-
teasome, i.e., chymotrypsin-like, trypsin-like, and
peptidyl-glutamyl-peptide hydrolase, were inhibited
in rats fed ethanol intragastrically for 2 months but
not in rats fed ethanol ad lib. The inhibition of these
proteasome enzyme activities correlated with an in-
crease in liver weight, compared to pair-fed controls.
The liver total proteins also increased significantly
compared to pair-fed controls, suggesting that the
increase in liver weight and protein content was a
result of the inhibition of protein proteolysis asso-
ciated with ethanol intake. The relative amounts of
proteasome subunits did not change with ethanol,
suggesting that the inhibition of catalytic activity
was not due to reduced enzyme protein levels. The
proteasome inhibition was shown in the standard
activities, i.e. in the SDS-stimulated 20S proteasome
activities, and in the ATP-stimulated 26S protea-
some activities, indicating that the defect involved
the 26S proteasome as well as the 20S proteasome.
The inhibition of proteasome activity corre-
lated with an increase in malondialdehyde in liver
homogenates, suggesting that malondialdehyde ad-
ducts resulting from increased lipid peroxidation
caused by alcohol could be involved in proteasome
inhibition. None of these changes was noted in the
rats fed ethanol ad lib, suggesting that a dose-re-
sponse relationship is responsible for proteasome
inhibition [21]. The studies of Fataccioli et al. [23]
corroborated those of Donohue et al. [21]. In addi-
tion, when CYP2E1 inhibitors were fed with ethanol,
inhibition of the proteasome chymotrypsin-like ac-
tivity was significantly reduced, suggesting that free
radical damage may be the mechanism of inhibition
of the proteasome caused by ethanol feeding. Li-
pid peroxidation correlated inversely with chymot-
rypsin-like activity. Again, ad lib feeding of ethanol
did not cause these associations with proteasome
inhibition, nor was there an increase in formation of
malondialdehyde, protein thiols, or protein carbon-
yls. Also, ad lib feeding of ethanol did not increase
the total liver protein levels.
380 PART II: Specific Signaling Pathways
Rouach et al. [88] found that rats fed ethanol
intragastrically had increased levels of malondial-
dehyde, protein thiols, and protein carbonyls in the
liver. The increase in oxidized proteins correlated
positively with the severity of the liver pathology.
Thus, the inhibition of proteasome activity and the
increase in protein oxidation may be related because
the proteasome eliminates oxidized proteins [91].
32.7
Other Functional Consequences
of Proteasome Inhibition
Ethanol stabilizes proteins normally degraded by
the proteasome. Hypothetically, this would include
CYP2E1, NF-κB, and HIF-1α. In the case of CYP2E1,
it has been shown that ethanol withdrawal leads to an
overnight reduction in the ethanol-induced CYP2E1
and that this rapid loss in CYP2E1 in the liver can be
prevented by injecting a potent proteasome inhibi-
tor, PS-341 [7]. This change in CYP2E1 levels cor-
related with a return of the proteasome, inhibited
by ethanol, to normal levels of activity 24 h after
ethanol withdrawal. This is compelling evidence
that CYP2E1 was stabilized by ethanol inhibition of
the proteasome. Proteasome inhibitor treatment of
HepG2 cells in vitro increases the level of CYP2E1
threefold by preventing degradation by the protea-
some (stabilizes CYP2E1) [79]. In the case of NF-κB
stabilization, it was shown that ethanol fed intragas-
trically for 2 months was associated with the lack of
NF-κB activation [34], which could be the result of
inhibition of proteasome digestion of IκBα that pre-
vents NF-κB activation. NF-κB would be expected to
be activated by oxidative stress caused by ethanol in
this model. In the case of HIF-1α, the nuclear activa-
tion of this transcription factor is increased at high
blood alcohol levels and expression of genes regu-
lated by HIF-1α is also increased, including eryth-
ropoietin, iNOS, and VEGF gene expression [7, 70],
suggesting that the normal elimination of HIF-1α by
the proteasome is inhibited by ethanol feeding.
32.8
The Mechanism of Ethanol-Induced Inhibition
of Proteasome Activity in the Liver
Ethanol feeding does not inhibit proteasome cata-
lytic activity directly [10]. The inhibition does not
occur after 2 weeks of ethanol feeding. Inhibition of
the proteasome was not found until 1 month of feed-
ing rats ethanol. This suggested that an oxidized
product of CYP2E1 generated by ethanol CYP2E1
would be a likely mechanism of inhibition. Indeed,
transgenic mice, in which the gene for CYP2E1 had
been knocked out, failed to develop proteasome
activity inhibition even after 1 month of ethanol
feeding [4]. One candidate product is 4-hydrox-
ynonenal (4-HNE), an end product of lipid peroxi-
dation, which results from the hydroxyethyl radical
generated by CYP2E1 during ethanol oxidation [2].
Indeed, 4-HNE is increased in the liver of rats fed
ethanol intragastrically [26]. 4-HNE progressively
increased in the liver during 1 month of ethanol
feeding, peaking when the proteasome catalytic ac-
tivity had become inhibited [10]. 4-HNE adducts are
formed in the liver in alcoholic liver disease. Their
presence correlated with the morphologic severity
of liver disease [89].
The possibility of adduct forming between a pro-
teasome subunit and 4-HNE accounting for the loss
of proteolytic activity was investigated. When the
20S proteasome was isolated from the livers of rats
fed ethanol and controls, no loss of catalytic activity
was found. Also, no 4-HNE adduct was found when
the 20S proteasome was studied by two-dimension-
al (2D)-electrophoresis and Western blot. However,
when the 26S proteasome was isolated, and studied
by 2D-electrophoresis and Western blot, there was
loss of proteasome activity, and a 4-HNE adduct to
a 44-kDa subunit was found in the alcohol-fed rats.
The 2D membrane, stripped of the 4-HNE antibody
and stained with an antibody to Rpt4, an ATPase
subunit of the regulatory complex 19S part of the
26S proteasome, showed that 4-HNE was adducted
to this subunit in the ethanol-fed rat liver. Thus,
proteasomal catalytic activity could be lost because
the subunit that controls the opening of the catalytic
channel was altered by 4-HNE adduction [10].
Another possible mechanism that could explain
the loss of proteasome catalytic activity after ethanol
feeding is phosphorylation of a regulatory subunit of
the proteasome. Previous in vitro studies had shown
that ethanol stimulated phosphorylation of proteins
in liver cells through a protein kinase C (PKC) route
[49]. Okadaic acid, which inhibits phosphatase ac-
tivity and increases phosphorylated proteins in the
liver, caused profound inhibition of proteasome
activity in vivo [8]. Proteasomes isolated from the
liver of rats fed ethanol and controls were studied
for subunit phosphorylation using an antibody to
phosphothreonine. Using 2D-electrophoresis and
Western blotting, a hyperphosphorylated subunit,
alpha 7, was found in the proteasomes isolated from
the liver. Inhibition of proteolytic activity of the
20S proteasome could result from phosphorylation
of this subunit because the α subunits are also in-
volved in opening the mouth of the catalytic cham-
ber of the 26S proteasome.
 CHAPTER 32: Ubiquitin-Proteasome Pathway in the Pathogenesis of Liver Disease
32.9
Some Consequences of Inhibition
of the Proteasome: Apoptosis
Apoptosis in the liver is increased in alcoholic liver
disease [27]. Apoptosis is also increased in the liver
from viral hepatitis and drug hepatitis. Inhibition
of the proteasome is one mechanism involved in
apoptosis. When rats were injected with the potent
proteasome inhibitor PS-341 (IP 0.5 mg/kg), apop-
tosis increased compared to controls [28] (Fig. 32.2).
The livers were examined for apoptosis 4 h post in-
jection using TUNEL staining. Positive-stained
nuclei were increased, as were cells that stained
positive for activated caspase-3 (hepatocytes and
sinusoidal cells). Alanine aminotransferase (ALT)
was increased. Proteasome chymotrypsin-like ac-
tivity was decreased in liver homogenates (~ 85%
decrease). The way in which proteasome inhibition
leads to apoptosis is complex. Many pro- and anti-
apoptosis proteins are regulated by the proteasome
[28]. These include Bax, Bcl-2, p53, mdm 2, IKBα,
caspase 3, caspase 8 c-1AP1, XiAP, NF-κB precursor,
and Bid [74]. Pro-apoptosis proteins are p53, Bax,
and Bid. Anti-apoptosis proteins are c-IAPI, XIAP,
381
Fig. 32.2a–f. Comparison of ethanol feeding
and proteasome inhibition effects on liver cells.
a–d Hematoxylin and eosin-stained sections
from the liver of rats fed ethanol for 1 month
(a), or fed ethanol for 1 month and given PS-
341 (b) or rats given PS-341 only (c, d). Note
that both ethanol and PS-341 caused apoptosis
(a, c). e, f Hepatocytes stained by TUNEL from
the liver of a rat fed ethanol for 1 month (e) or
a rat fed ethanol for 1 month and given PS-
341 (f). PS-341 treatment caused apoptosis (f)
mainly when combined with ethanol (e)
Bd-2, and NF-κB. Thus, the ubiquitin-proteasome
system is an essential player in the regulation of
proteins involved in programmed cell death in the
liver and other organs.
32.10
Role of the Ubiquitin-Proteasome Pathway
in Liver Cell Aggresome (Mallory Body)
Formation
Mallory bodies are formed by damaged liver cells
in a great variety of different chronic liver diseases
[41]. Thus, they are a sign of more severe liver injury
[3, 90]. In alcoholic liver disease, ubiquitin conju-
gates (polyubiquitinated proteins) are increased in
the blood [99] and ubiquitin in the liver correlates
with the presence of Mallory bodies in non-alco-
holic steatohepatitis [3]. Thus, there is a general in-
crease in polyubiquitinated proteins in an environ-
ment in which Mallory bodies form. The association
of Mallory bodies with ubiquitinated cytokeratins
in liver cells has been known since 1988 [64, 75].
Only recently has the role of the ubiquitin-protea-
some pathway in the pathogenesis of Mallory body
382 PART II: Specific Signaling Pathways
Fig. 32.3a, b. Theoretical model of the role of heat shock pro-
teins (hsp) in normal protein degradation and MB formation. (a)
Normal cytokeratin degradation. (b) Cytokeratin aggregation
and formation of MBs in the presence of stress and/or UBB+1. Ub
formation been appreciated. The new concept is
based on the aggresome phenomenon, which biolo-
gists have described in a variety of tissues where
protein aggregates accumulate around the centro-
some [42, 104, 106]. The idea was first formulated as
a consequence of events in the degradation pathway
of liver cell cytokeratins. It started with a conforma-
tional change in the cytokeratin proteins caused by
phosphorylation, where α helices decreased and β
sheets increased, causing the cytokeratins to stick
to each other [109]. According to the concept of ag-
gresome formation, β-sheet conformational change
is an important mechanism for protein aggregation
[54]. Conformational changes in Mallory bodies
have been documented by infrared spectroscopy
[44]. Hyperphosphorylation of cytokeratin 8 by
hepatocytes from mouse livers forming Mallory
bodies was documented by 32P incorporation into
cytokeratins of hepatocytes in tissue culture using
2D-electrophoresis [15]. Mallory bodies containing
hyperphosphorylated proteins have been found in
a model of Mallory body formation in hepatocytes.
Phosphothreonine antibody was used in Western
blots of Mallory body protein [72]. Hyperphosphor-
ylation of cytokeratins in Mallory-body-forming liv-
ers has been characterized further by finding phos-
phorylation of multiple sites on amino acid residues
in the cytokeratin proteins [94]. The turnover of cy-
tokeratins is signaled by phosphorylation, followed
ubiquitin, hsp 70 heat shock protein 70, hsp 90 heat shock protein
90, MB Mallory body, UBB+1 mutant ubiquitin, cytokeratin. (Re-
printed from [86], with permission from Elsevier)
by ubiquitination. Proteolysis of the cytokeratins by
the proteasome then occurs [56].
To test the hypothesis that hyperphosphorylation
induces Mallory body formation, drug-primed mice
were given an acute dose of okadaic acid and the liv-
ers were monitored for the appearance of Mallory
bodies. Fifteen minutes after okadaic acid injection,
phosphorylated protein aggregates formed in liver
cells. The aggregates stained positive with cytokera-
tin antibody [109]. True Mallory bodies formed by
day 2 and progressed with daily injections of oka-
daic acid. The Mallory bodies stained positive for
phosphothreonine in tissue slices and by Western
blot in the Mallory body band. Human liver cell
Mallory bodies also stained positive with the phos-
phothreonine antibody. Similar cytokeratin aggre-
gates formed in hepatocytes in tissue culture when
exposed to okadaic acid [13, 92].
32.11
Role of Heat Shock Proteins
in Mallory Body Formation
Heat shock proteins act as chaperones for altered
cytoplasmic proteins that have been targeted for
digestion by the proteasome after polyubiquitina-
tion (Fig. 32.3) [105]. Heat shock treatment of drug-
 CHAPTER 32: Ubiquitin-Proteasome Pathway in the Pathogenesis of Liver Disease
Fig. 32.4a–c. Human liver cells with numerous Mallory bodies
due to alcohol abuse. The liver section was double stained with
antibodies to cytokeratin 18 (a) and to p62 (b) and viewed with
primed mice induces Mallory bodies after upregula-
tion of heat shock protein expression [108]. When
human or mice livers containing Mallory bodies
were stained with antibodies to heat shock proteins
70 and 90, they stained positive, indicating that both
heat shock proteins were pathogenetically linked to
Mallory body formation [86, 95]. The expression of
heat shock protein 70 and 90 as determined by West-
ern blot correlated with the number of Mallory bod-
ies expressed [9].
32.12
Role of p62
in Mallory Body Formation
P62 (Z1P) links polyubiquitinated proteins to the
proteasome. P62 is a major component of hyaline
bodies in hepatocellular carcinomas [93], Mallory
bodies [96], as well as Alzheimer’s disease neurofi-
brillary tangles and synucleipathies [59, 60] are in-
volved in p62-ubiquitin binding in aggresome for-
mation. ZIP/p62 is a scaffolding protein that binds
to polyubiquitin at its UBA domain [30, 82]. Human
Mallory bodies stain strongly with p62 antibody
(Fig. 32.4).
32.13
Role of Microtubules
in Mallory Body Formation
Aggresomes require microtubules to be present,
in order for them to form. Colchicine and nocoda-
383
a confocal microscope. a, and b images were merged in c. Note
the merged image shows the Mallory bodies, indicating colo-
calization of p62 and cytokeratin 18 (×2,500)
zole prevent aggresome formation [42, 104, 106].
Miniaggregates are transported on microtubules
towards the microtubule organizing center at the
centrosome, driven by the minus-end motor protein
dynein [29, 43]. Using a new tissue culture model
of Mallory body formation, microtubule inhibitors
totally blocked Mallory body formation [87]. Only
small cytokeratin-ubiquitin positive aggregates
formed at the cell periphery, whereas the control liv-
er cultures formed numerous Mallory bodies in the
absence of the microtubular inhibitors (Fig. 32.5).
Tubulin and Tau isoforms located in microtubules
localized in the Mallory body [51, 84].
32.14
Role of Transglutaminase
in Mallory Body Formation
Transglutaminase was found in the Mallory bodies
and in Western blots and was induced when Mallory
bodies were induced [9]. Northern blots showed that
transglutaminase gene expression was upregulated
when Mallory bodies were formed at the same time
that cytokeratin 8 was overexpressed. Increased
transglutaminase activity and cross-linking of cy-
tokeratins were reported earlier [19, 110, 111]. Cross-
linking of cytokeratin by transglutaminase in hepa-
tocytes may account for the insolubility of Mallory
bodies in the SDS cocktail used in Western blots
[78].
384 PART II: Specific Signaling Pathways
Fig. 32.5a–c. Primary culture of mouse liver cells forming Mal-
lory bodies. The liver cells were double stained for cytokeratin
18 (green, a) and for ubiquitin (red, b) and viewed with a confocal
microscope. c a merged picture from a and b. Note the merged
32.15
Role of Proteasome Inhibition
in the Formation of Mallory Bodies
Proteasome inhibitors induced aggresome forma-
tion in vitro, presumably because the inhibition of
proteolysis causes an accumulation of proteins in
the cell [42, 106]. This means that inhibition of the
proteasome is central to Mallory body formation
[109]. In fact, injection of a potent proteasome in-
hibitor in drug-primed mice induces Mallory body
formation after 2 days [24]. The proteasome inhibi-
tor PS-341 is selective in its action on the protea-
some and is an effective anticancer drug because
proteasome inhibition causes apoptosis of cancer
cells [1]. PS-341 also induces aggresome formation
in hepatocytes in vitro after going through a stage of
collapse of cytokeratin from the plasma membrane
followed by retraction of the cytokeratin around
the nucleus [85] at 5 h. Later (24 h), aggresomes are
formed in place of the contracted cytokeratin. When
Mallory bodies form in mouse liver, the cytokeratin
disappears in the cytoplasm (ghost cell formation).
Proteasomes are localized at the edge of the Mallory
body and the proteasomes in the cytoplasm are di-
minished [5].
32.16
Role of the Frameshift Mutant
of Ubiquitin in Mallory Body Formation
Recently, a frameshift mutant of ubiquitin, UBB+1,
was shown to be present in Mallory bodies in hu-
image shows the Mallory bodies in yellow, indicating colocaliza-
tion of ubiquitin and cytokeratin 18. Confocal fluorescent micro-
scopy (×2,500)
man and mouse livers [24, 68]. The UBB+1 mutant
was first reported to be present in neuronal cell ag-
gresomes in Alzheimer’s disease [103]. This mutant
is a result of molecular misreading of the ubiquitin
B gene during transcription (a dinucleotide dele-
tion), which results in a mutant ubiquitin that has
lost the binding site in the C prime end of ubiquitin.
It therefore cannot function to bind misfolded pro-
teins destined for proteolysis by the ubiquitin-pro-
teasome pathway. This is not a functional ubiquitin.
Worse, UBB+1 is polyubiquitinated by wild-type
ubiquitin, but the resultant polyubiquitin chains
are refractory to disassembly by deubiquitinating
enzymes (Fig. 32.6). This potentially inhibits the
degradation of a polyubiquitinated substrate by pu-
rified 26S proteasomes [61]. In vitro studies using
primary liver cultures showed that UBB+1 transfec-
tion induced cytokeratin-ubiquitin aggresomes in
hepatocytes within 4 h of incubation [11]. In vitro
studies used a ubiquitin-proteasome pathway mix-
ture to form a reconstituted ubiquitin-proteasome,
which includes a cytokeratin immunoprecipitate.
This mixture contained Mallory bodies, added
ubiquitin, a ubiquitination system with the enzymes
E1, E2, and E3, purified proteasomes, and an enzyme
mixture for generating ATP. The mixture was in-
cubated for 4 and 24 h [9]. The Mallory body band
(insoluble protein at the top of the gel located in the
loading gel) increased when the mixture was added.
Likewise, the ubiquitinated protein fraction was in-
creased. Adding UBB+1 to the mixture increased the
Mallory body band further [11]. Adding PS-341 also
increased the Mallory body band. The combination
of UBB+1 and PS-341 did not increase the Mallory
body band further, suggesting that the two treat-
ments were acting through the same mechanism.
 CHAPTER 32: Ubiquitin-Proteasome Pathway in the Pathogenesis of Liver Disease
Ubiquitination of UBB+1 occurred. The proteasomes
localized within the Mallory body, indicating that,
as occurs in aggresomes in vivo, the proteasomes
were bound to the aggresome. This might explain
how UBB+1 interferes with the ubiquitin-proteasome
pathway of proteolysis.
The question arises: does UBB+1 expression lead
to cell death? In the case of neurons, UBB+1 expres-
sion does lead to cell death [20]. Aggresome forma-
tion under many differing circumstances may cause
death of the cell [12, 63].
32.17
Role of Cytokeratin 8 and 18 Overexpression
in Mallory Body Formation
During primary tissue culture of liver cells from
Mallory-body-forming livers, there was an increase
in synthesis of cytokeratin 8 and cytokeratin 18 as
measured by Western blot [15]. Cytokeratin 8 gene
expression was also markedly upregulated in Mal-
lory-body-forming livers [72]. The increase of cy-
tokeratins in hepatocytes may overload the ubiqui-
tin-proteasome pathway of cytokeratin proteolysis,
which then may cause cytokeratin accumulation to
form the Mallory body. Several studies, where tissue
culture cells were transfected with plasmids con-
taining cDNA coded for cytokeratin 8 or cytokera-
385
Fig. 32.6. Molecular misreading and Mallory
body formation. Representation of how target
proteins can be tagged efficiently for protea-
somal destruction (right side) and how this oc-
curs less efficiently (left side) when the mutant
ubiquitin UBB+1 is involved in the ubiquitina-
tion. UBB+1 has lost its ability to ubiquitinate
because it does not have a terminal Gly resi-
due. It is refractory to deubiquitination by the
deubiquitinase (isopeptidase) enzyme. In fact,
the mutant ubiquitin becomes ubiquitinated
itself, and inhibits the proteasome in a domi-
nant-negative way. (Reprinted from [68], with
permission from Elsevier)
tin 18 to cause overexpression, led to the formation
of cytokeratin aggresomes. But these aggresomes
failed to form the characteristic filaments seen in
Mallory bodies [14, 39, 73].
Mallory bodies incorporate newly synthesized
cytokeratin directly in tissue culture as shown by
[35S]methionine pulse labeling using Western blots
and electron microscopy autoradiography of deter-
gent-extracted hepatocytes in culture where only the
cytoskeleton remained [45]. In Western blots, the
radiolabel was found in the Mallory band as well as
in normal cytokeratin 8 and cytokeratin 18 bands.
Similarly, the radiolabel was incorporated into the
Mallory filaments as well as the cytokeratin fila-
ments. Cytokeratin 18 cDNA was used to transfect
Mallory-body-forming cells in mouse liver tissue
culture using a green fluorescent protein tag. The
cytokeratin localized in both the normally distrib-
uted cytokeratin but was also mainly concentrated
in the Mallory body. This suggested that the nascent
human cytokeratin went both to the normal fila-
ments as well as directly to the Mallory body after
synthesis [84, 85].
386 PART II: Specific Signaling Pathways
32.18
Functional Consequence
of the Loss of Cytokeratin Organization
Due to Aggresome (Mallory Body) Formation
The function of liver cell cytokeratins and the vul-
nerability of liver cells exposed to toxins and viral
damage have been reviewed elsewhere [52, 57, 76,
77]. Mutations in cytokeratin 8 and cytokeratin 18
were found in patients with cryptogenic cirrhosis,
hepatitis C, and other liver diseases. The question
is: do changes in the cytokeratin organization due
to mutations or other etiologies interfere with liver
function and increase vulnerability of the liver to in-
jury? For instance, hyperphosphorylation of the liv-
er cell cytokeratins changes their organization, but
does this lead to loss of function [55]? Cytokeratin
reorganizes extensively during mitoses, apoptosis
and polarization of the cell [66]. In in vitro studies
using transfection of cells with mutant cytokeratins,
the cells were more vulnerable to injury. Cytokerat-
ins have viscoelastic properties, which make cells
resistant to physical stress. Lack of cytokeratins in
K18/8-deficient mice hepatocytes disturbs the pro-
gression of the cell cycle. These cells are more likely
to develop apoptosis in response to FAS [31, 58, 65].
The cytokeratins of the liver anchor and support the
bile canaliculus, forming the pericanalicular sheath
supporting bile flow [47, 48]. Likewise the cytokera-
tin filaments of the liver cell are involved in transhe-
patic transport of vesicles [50].
The cytokeratin cytoskeleton of liver cells that
form Mallory bodies is profoundly altered so that
only the Mallory body stains for keratin, leaving the
rest of the cell unstained, the so-called “empty cell”
or “ghost cell” [46]. Mallory body cells lose their bile
canaliculi and cytokeratin in the cytoplasm. This is
associated with cell enlargement and rounding. The
cytokeratin network supports the nucleus, the nu-
clear pores and the centrioles. The Mallory-body-
containing cells in primary tissue culture were un-
able to form bile canaliculi or secrete fluorescent
diacetate, compared with controls [25].
32.19
Concluding Remarks
It can be concluded that the inhibition of the ubiq-
uitin-proteasome pathway of protein degradation
has profound effects on the structure and function
of liver cells, including loss of cell shape and inter-
nal organization of organelles, polarity, and the bile
secretory apparatus. Vulnerability of the liver cell
to FAS-induced apoptosis is increased. Cytokeratin
aggresome formation (Mallory bodies) may further
reduce proteolysis by the proteasome.
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