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From the Department of Pathology,*
Karl-Franzens
University, Graz, Austria; the Division of Cell
Biology,
German Cancer Research Center,
Heidelberg, Germany; Protein Analysis
Facility,
German Cancer Research Center,
Heidelberg, Germany; and the Institute of
Neuropathology,
University Hospital of
Zürich, Zürich, Switzerland
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Abstract |
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1-antitrypsin
aggregates. The presence of p62 along with other stress proteins and
ubiquitin in cytoplasmic inclusions indicates deposition as aggregates
as a third line of defense against misfolded proteins in addition to
refolding and degradation.
Exploration of the conditions under which cytoplasmic protein
aggregates accumulate as well as elucidation of their biological
significance is all the more interesting since cytoplasmic inclusions
consisting of abnormal proteins are hallmark lesions of a considerable
number of human diseases, also designated as protein aggregation
diseases, such as neurofibrillary tangles in neurons of patients with
Alzheimer’s disease, Lewy bodies in neurons of patients with
Parkinson’s disease, Lewy body-like or skein-like inclusions in
amyotrophic lateral sclerosis, inclusions in skeletal muscle fibers in
patients with inclusion body myopathies, aggregates of mutated
1-anti-trypsin in hepatocytes of patients with 1-antitrypsin
deficiency, Mallory bodies (MBs) in hepatocytes of patients with
alcoholic steatohepatitis, and a variety of non-alcoholic chronic toxic
and degenerative liver disorders as well as in hepatocellular neoplasms
.7-9
To obtain new insight into the pathogenesis of such inclusions we
analyzed the protein components of MBs and studied their role in MB
formation. MBs are irregularly shaped cytoplasmic inclusions typically
present in hepatocytes. They consist of abnormally phosphorylated,
ubiquitinated, and cross-linked cytokeratins (CKs) and non-CK
components.9-15
MBs can be reproduced in mice by chronic
intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC).16
Using mass spectrometry, we identified p62 as a
novel and major component of MBs isolated from DDC intoxicated mouse
livers. p62 (also designated STAP, A170, and ZIP) was originally
identified as a ligand for the SH2 domain of
p56lck17-20
and was shown to
contain several functional domains, such as a zinc finger motif,
proline rich regions, and a PEST sequence. It was shown that p62
interacts with PKC-
and is involved in the activation of NF-
B and
p38 MAPK.17,19,21-23
Besides a role in signal
transduction p62 might be involved in the cellular stress response
since, on the one hand, its expression is markedly increased on a
variety of stress stimuli, such as exposure to oxidative stress, sodium
arsenite, cadmium, and ionophore and, on the other hand, p62 binds to
polyubiquitinated proteins.18,24-26
We found that p62
binds specifically to aggregates of misfolded and ubiquitinated
proteins and that it is present in cytoplasmic inclusions in diverse
human diseases suggesting a general role of p62 in the cellular
response to abnormal proteins in protein aggregation diseases.
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Materials and Methods |
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For induction of MBs, Swiss Albino mice (strain Him OF1 SPF; Institute of Laboratory Animal Research, Himberg, Austria) were fed a diet containing 0.1% DDC (Aldrich, Steinheim, Germany). After 2 months of DDC feeding, mice were killed and liver tissue was immediately processed for isolation of MBs. For immunofluorescence analysis aliquots were snap-frozen in methylbutane precooled with liquid nitrogen.
MBs were isolated by sucrose gradient centrifugation and high salt extraction as described previously.11 For 2D gel electrophoresis, MB material was dissolved in TISO buffer (2 mol/L thiourea, 8 mol/L urea, 4% CHAPS, 20 mmol/L Trizma base, and 30 mmol/L DTT; all reagents from Sigma-Aldrich, Deisenhofen, Germany) and separated by isoelectric focusing using non-linear immobilized pH gradient strips ranging from pH 3.5 to 10 (Pharmacia, Uppsala, Sweden) followed by separation on sodium dodecyl sulfate-polyacrylamide gradient gels (4 to 16%). Gels were either stained with Coomassie blue or transferred onto nylon membranes for immunoblotting as described.27 Reactivity of the antibodies (see below) was detected using enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
Analysis of Mallory Body Components by Mass Spectrometry
To obtain specific peptide fragments MB protein spots were excised
from the gel, digested with trypsin in 40 mmol/L ammonium bicarbonate
at 37°C overnight, and the reaction was stopped by freezing. For
matrix-assisted laser desorption ionization–time of flight mass
spectrometry (MALDI-TOF) the samples were prepared on thin film spots
according to Jensen et al. 28
Briefly, 0.3 µl of
nitrocellulose containing saturated solution of
-cyno-4-hydroxycinnamic acid (Sigma-Aldrich) in acetone were
deposited onto individual spots on the target. Subsequently, 0.8 µl
of 10% formic acid and 0.4 µl of digested sample were loaded on top
of the thin film spots and dried at ambient temperature. To remove
salts the spots were washed with 10% formic acid and
H2O. MALDI mass spectra were recorded in the
positive ion mode with delayed extraction on a Reflex II time-of-flight
instrument (Bruker-Daltonic, Bremen, Germany) equipped with a SCOUT
multiprobe inlet and a 337 nm nitrogen laser. Ion acceleration voltage
was set to 20.0 kV, the reflector voltage was set to 21.5 kV, and the
first extraction plate was set 15.4 kV. Mass spectra were obtained by
averaging 50 to 200 individual laser shots. Calibration of spectra was
performed internally by two-point linear fit using the autolysis
products of trypsin at m/z 842.50 and
m/z 2211.10.
Sample preparation for postsource decay (PSD) analysis was achieved by
cocrystallization of matrix with ZipTip C18 (Millipore, Bedford, MA)
concentrated samples. In brief, the peptides in the supernatant of
in-gel digestion were absorbed to a prewashed (50% acetonitrile/water)
and equilibrated (0.1% trifluoroacetic acid/water) ZipTip C18 by
repeated applications. Following washing of the ZipTip C18 by
equilibration buffer the peptides were eluted from the ZipTip C18 with
1 µl of matrix (-cyano-4-hydroxycinnamic acid saturated in 50%
acetonitrile/water, Sigma-Aldrich). PSD analysis was performed in the
reflection mode with delayed extraction by setting an ion gate width of
40 Da around the ion of interest. Data were acquired in 14
segments by decreasing the reflector voltage in a stepwise fashion. For
each segment 100 to 200 individual laser shots were accumulated. The
fragment ion spectrum was obtained by pasting together all segments to
a single spectrum using FAST software provided by Brucker. Fragment ion
calibration was performed externally with fragment masses of the
adrenocorticotropic hormone (ACTH) 18–19 clip.
Singly charged monoisotopic peptide masses were used as inputs for database searching. Searches were performed for best hits against the NCBInr database using the ProFound algorithm developed at the Rockefeller University, New York (see W. Zhang & B.T. Chait, http://prowl.rockefeller.edu/cgi-bin/ProFound) and the Protein Prospector software developed at the University of California, San Francisco (see P.R. Baker and K.R. Clauser, http://prospector.ucsf.edu). Isoelectric points were allowed to range from 0 to 14, and the oxidation of methionine was included as a side reaction. Up to one missed tryptic cleavage was considered, and a mass tolerance for monoisotopic peptide masses was set to ± 0.1 Da. Searches for fragment masses from PSD experiments were performed against NCBIncr database using the MS-Tag search algorithm provided by the Protein Prospector software package. Parent mass tolerance was set to ±50 ppm and fragment ion tolerance was set to ±1500 ppm.
Antibodies, Immunofluorescence Microscopy, and Immunohistochemistry
Antibodies to p62 were raised in guinea pigs against the two peptides (peptide p62NT, MASLTVKAYLLGKEDAAREIC, and p62CT, CNYDIGAALDTIQYSKHPPPL) conjugated to keyhole limpet hemocyanin as described.29 Additional antibodies used were: 1) monoclonal antibodies: Ks 8.7 and Ks 18.04 (Progen, Heidelberg, Germany) to CK 8 and CK 18, respectively, SMI 31 that recognized phosphorylated epitopes present on tau and neurofilament proteins (Sternberger Monoclonal Inc., Baltimore, MD), anti-HSP/C 70 (Novocastra Laboratories, Newcastle-on-Tyne, UK), and anti-his tag (Roche Diagnostics, Mannheim, Germany); 2) polyclonal antibodies: anti-ubiquitin (StressGene, Victoria, BC, Canada), anti-HSP 25/27 (StressGene); anti CK 8 and CK 18 (produced in our laboratory); 3) secondary antibodies: FITC-conjugated goat anti-mouse Ig (Zymed, San Francisco, CA); TRITC-conjugated swine anti-rabbit Ig (Dako, Glostrup, Denmark); goat anti-guinea pig (Jackson Immuno Research Laboratory, West Grove, PA); horseradish peroxidase-conjugated rabbit anti-mouse Ig (Dako); and horseradish peroxidase-conjugated rabbit anti-guinea pig Ig (Dako). For negative control, primary antibodies were replaced by serum, isotype-matched immunoglobulins, and antibody binding was inhibited by addition of the corresponding peptides (160 µg/ml), respectively. Immunofluorescence specimens were analyzed with a MRC600 (BioRad, Richmond, CA) laser-scanning confocal device attached to a Zeiss Axiophot. Immunohistochemistry on paraffin-embedded human tissues using avidin-biotin complex detection method was performed as described.27
Cloning of the p62 cDNA and Transfection Experiments
Full-length human p62 cDNA was isolated from human liver tissue by reverse transcription-PCR using the primers corresponding to positions 1425–1402 and 53–70 of the published p62 sequence.19 PCR products were cloned into the pCRII vector using the TA cloning kit (Invitrogen, Groningen, The Netherlands). For transfection the p62 cDNA was subcloned into NSI I site of a multiple cloning site which has been introduced in the expression vector pHß April-1 harboring the human ß-actin promoter.30 Furthermore the following cDNA expression constructs were used for transfection: LK440-H8 (human CK 8),31 LK440-H18 (human CK 18),31 and pRBG4-ubiquitin-his-myc (kindly provided by R. Kopito). HepG2 cells and CHO-K1 cells were seeded 18 hours before transfection onto glass coverslips. Transfection was performed using the adenovirus-augmented transferrin receptor-mediated gene delivery system essentially as described by Wagner et al.32 Immunofluorescence analysis was performed 17 to 24 hours after transfection.
Human Tissues
Formaldehyde-fixed and paraffin-embedded human tissue samples were obtained from the Institutes of Pathology of the Universities at Graz and Zurich, respectively. Diagnoses and number of cases studied are listed in below.
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Results |
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. Besides CKs there was another major
MB component with an apparent molecular mass corresponding to 65 to 68
kd and an isoelectric pH value in the range of pH 4.5. This component
had been shown to be recognized by the antibody SMI 31, which binds to
phosphoepitopes present on tau protein as well as on neurofilaments in
neurofibrillary tangles and Lewy bodies.14,33
|
. According to
the NCBInr database the best hit for peptide masses obtained from
tryptic digest of the SMI-31-reactive protein (Figure 1a
, spot 4) was
the oxidative stress protein A 170 from mouse, which is a homologue to
the human p62 ubiquitin-binding protein. An additional finding of
MALDI-TOF analysis was that CK 8 was covalently modified by
polyubiquitin (Figure 1)
. Using the NCBInr database and searching with
the masses of tryptic peptides obtained from spot 5 in the "single
protein only" mode, by far the best hit was CK 8 from mouse; CK 8
from other species ranked next with lower probabilities and scores.
Database search with the residual masses of spot 5 indicated the
presence of an additional protein with ubiquitin as the best hit. CK 8
and ubiquitin were also specifically detected in spot 5 by PSD fragment
ion analysis. Therefore the higher molecular weight of CK 8 in spots 5
and 6, compared to the authentic molecular weight position of CK 8 in
spot 7, is due to ubiquitination (see also hits for spot 1 in Figure 1b
). As estimated on the basis of Coomassie blue staining intensity, p62 was as abundantly present in MBs as CKs and, therefore, CKs and p62 have to be considered as the major components of MBs. In addition to p62, HSP 70 and HSP 25 were also identified as MB components in the 2D gels. However, the concentrations of the latter two components were much lower than that of p62 and CK.
To show that the proteins identified by mass spectrometry were indeed
integral MB components, polyclonal antibodies to N- and C-terminal
amino acid sequences of p62 were raised in guinea pigs. Antibodies to
the other MB components were commercially available. 2D immunoblots
showed that the antibodies to p62 were specific and recognized the same
protein as the SMI 31 antibody (Figure 2)
.
|
. Since
p62 was already detectable in the smallest MBs, which, at least in
part, reflect newly formed MBs, p62 is suggested to have a central role
in MB formation. HSP 25 and HSP 70 were also present in MBs (Figure 3, c and d)
. These two proteins, however, were detected in most but not
all MBs. The same was noted for ubiquitin in accordance with previous
reports (Figure 3b)
.7,12
|
. This means that p62 by itself is not
able to induce MB formation. Based on the fact that in MBs CKs 8 and 18
are not present in an equimolar ratio and are not assembled as regular
intermediate filaments,9
we tested whether p62 is able to
bind to improperly assembled CK. For this purpose, we transfected
CHO-K1 cells, which do not endogenously express CK, with human CK 8.
When overexpressed in the absence of its partner CK18, CK 8 was unable
to form intermediate filaments and accumulated as cytoplasmic
aggregates (for review on intermediate filament assembly see reference
34). With double-label immunofluorescence microscopy using antibodies
to CK 8 and p62, we observed that endogenous p62 was associated with
some CK aggregates (not shown). This indicates that the occurrence of
improperly assembled CKs provides a stimulus for the cell to induce
endogenous p62. Furthermore, p62 apparently selectively associates with
misfolded but not with regularly assembled CKs. Since p62 is known to
bind polyubiquitin in a non-covalent manner and ubiquitination of CKs
has been reported previously,35
we analyzed whether the
association of p62 with CK 8 is mediated via ubiquitin by
cotransfecting CK 8 with a tagged ubiquitin expression construct
(Figure 4, b and e)
. Double-label immunofluorescence microscopy using
either antibodies to p62 and CK 8 or antibodies to ubiquitin and CK 8
demonstrated that endogenous p62 associated with CK 8 as described
above, indicating that transfection of ubiquitin in addition to CK 8
neither influenced the distribution of CK 8 nor had any effect on the
association of the endogenous p62 with CK. Furthermore, the
distribution of ubiquitin matched that of CK 8, which demonstrates that
improperly assembled CK becomes heavily ubiquitinated and binding of
p62 to CK is most likely mediated by ubiquitin (Figure 4e)
. The next
question to be answered was whether the amount of endogenous p62 was a
limiting factor for its association with the overexpressed CK. This was
addressed in a triple transfection experiment where CK 8, tagged
ubiquitin, and p62 were cotransfected into CHO-K1 cells. In this
situation in which both CK 8 and p62 were overexpressed all three
components colocalized and the amounts of accumulated CK 8 and p62 were
comparable. The resulting cytoplasmic inclusions, therefore, closely
resembled MBs (Figure 4, c and d)
. To further demonstrate that the
association of p62 with CK was indeed a consequence of the accumulation
of abnormally folded proteins, we cotransfected CK 8 and its partner,
CK 18, together with the tagged ubiquitin.35
This
experiment led to formation of CK intermediate filament bundles, which
were negative both for ubiquitin and p62 (Figure 4f)
.
|
, p62
is a common denominator of cytoplasmic inclusions in a variety of
chronic degenerative diseases, some genetic diseases, and tumors of the
liver and brain. All 10 human liver biopsies with alcoholic hepatitis
investigated showed strong reactivity of all MBs with the polyclonal
p62 antibody (Figure 5a)
. Furthermore, in five cases of
1-antitrypsin deficiency, in which accumulation of misfolded
proteins is caused by mutation of the 1-antitrypsin gene, we
observed p62 reactivity of some larger irregularly shaped
1-antitrypsin deposits (Figure 5c)
. In these liver specimens,
however, most of the 1-antitrypsin deposits were negative for p62.
This can be explained by the fact that 1-antitrypsin is a secretory
protein and mutated 1-antitrypsin accumulates in the endoplasmic
reticulum.36
Under certain circumstances, 1-antitrypsin
deposits leak from the endoplasmic reticulum into the cytoplasm, where
they are exposed to p62. Hepatocellular carcinoma is another liver
disease which may feature different types of cytoplasmic
inclusions.27
Figure 5e
shows a hepatocellular carcinoma
with intracytoplasmic hyaline bodies, which are strongly stained with
the p62 antibodies. These findings of occurrence of p62 in cytoplasmic
inclusions in several liver diseases are also pertinent to a variety of
diseases of the brain. In all 10 cases of Alzheimer’s disease,
neurofibrillary tangles stained for p62 (Figure 5g)
. Some tangles,
however, were not detected by the antibodies. In all five cases of
Parkinson’s disease, Lewy bodies constantly reacted with the p62
antibodies (Figure 5i)
. Furthermore, in all five cases of pilocytic
astrocytoma most of the Rosenthal fibers, which are cytoplasmic
inclusions consisting of glial fibrillary acidic protein and ubiquitin,
reacted with the p62 antibodies (Figure 5k)
.
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Discussion |
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All stress proteins detected in MBs are known to be involved in the cellular response to misfolded proteins. HSP 70 mediates refolding whereas HSP 25 prevents formation of misfolded protein aggregates by temporarily binding misfolded proteins which are then delivered either to HSP 70 for refolding or to the proteasome complex for degradation.40 In acute stress situations these mechanisms are apparently able to prevent deposition of misfolded proteins as aggregates. In chronic stress, as it is the case in alcoholic liver disease, age-associated degenerative diseases as well as in certain genetic disorders, however, the capacity of these rescue mechanisms might become limiting, leading to accumulation of aggregated proteins.5 It has been reported that chronically stressed cells have reduced capacities to induce HSP expression.41 Furthermore, chronic stress may result in impairment of the proteasome degradation machinery.42 With regard to MB formation it is noteworthy that chronic oxidative stress seems to be involved in all human diseases associated with MB occurrence, such as alcoholic and non-alcoholic steatohepatitis as well as drug-induced liver injury.42,43 Moreover, chronic oxidative stress might also be the common pathogenetic principle in neurodegenerative diseases featuring cytoplasmic inclusions.44-46 It is interesting in this context, particularly with respect to chronic neurodegenerative diseases associated with ubiquitinated neuronal protein inclusions and apoptotic cell death, that p62 is up-regulated in cultured neuronal cells during initiation of apoptosis and proteasomal inhibition.47 This suggests a protective role of p62 in pathological conditions affecting the central nervous system, eg, oxidative stress, which favor the appearance of ubiquitinated abnormal proteins and protein aggregation.
This study is one of the first to demonstrate p62 as a common component of cytoplasmic inclusions in various chronic toxic and degenerative diseases. p62 is induced in cells in response to misfolded CKs and is associated with protein aggregates. p62 per se does not lead to aggregate formation but binds to aggregated misfolded proteins and the binding is dependent on prior ubiquitination. The role of p62 in these aggregates, however, is still elusive. One possible function of p62 could be modulation of the stability and biological behavior of cytoplasmic protein aggregates. Aggregated misfolded proteins expose hydrophobic residues at their surface. These hydrophobic residues could act as a trap for other misfolded cellular proteins which are expected to occur in repeated or chronic stress situations. As mentioned above, MBs consist mostly of CKs and stress proteins. It is surprising that CKs preferentially accumulate in MBs while other cellular proteins, which would also be expected to be misfolded in stress situations, do not. An explanation for this situation could be that, on the one hand, CKs become a preferred substrate to modification by oxidative stress, and, on the other hand, the accumulation of HSPs and particularly the association of p62 prevent further binding of other misfolded proteins.
Another consequence of the accumulation of p62 in cytoplasmic
aggregates could be that the redistribution of p62 within the cell has
some influence on the other cellular functions of p62. p62 not only
acts as a ubiquitin-binding protein but also is involved in different
signaling pathways. Its role in the activation of NF-B could be
particularly important in the context of oxidative stress since the
level of NF-B is known to be a major modulator of the cellular
effects of stress-induced injury.48,49
In conclusion, we identified p62 as a novel and common component of cytoplasmic inclusions in various protein aggregation diseases. The presence of p62 along with other stress proteins, such as HSP 70, HSP 25, and ubiquitin in protein aggregates indicates that, particularly in chronic stress situations, a third line of defense against misfolded proteins, namely deposition as aggregates, gains importance in addition to refolding and degradation. The nature of the misfolded protein as well as of proteins that associate with aggregate could determine whether the resulting protein aggregate is harmful to the cell and thus aggravates tissue injury or whether it lacks deleterious effects, as it is the apparently case with MBs.38,39
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Acknowledgements |
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Footnotes |
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Supported by grant S7401-MOB from the Austrian Science Fund to K.Z.
Accepted for publication October 9, 2001.
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