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INTRODUCTION |
Critical to the ability of a cell to reestablish cellular
homeostasis after a range of different environmental stresses is the
removal of oxidatively modified proteins by the proteasome (1, 2). The
proteasome represents approximately 1% of the total cellular protein
and is present in the cytosol and nuclei of all mammalian cells in two
major forms (i.e. the 20 S and 26 S proteasomes) (3). The 20 S proteasome is a 700-kDa complex with a 10-20-Å-diameter opening
into an internal cavity that provides a sequestered environment for
proteolysis. The 26 S proteasome is a 2000-kDa complex containing two
19 S regulatory complexes bound to the 20 S multicatalytic core. The 26 S proteasome is responsible for the degradation of the majority of
cellular proteins through an ATP-dependent and
ubiquitin-mediated pathway (4-6). In contrast, the 20 S proteasome
core selectively degrades a range of different oxidized proteins in an
ATP-independent manner and has been suggested to represent the primary
mechanism in the rapid removal of oxidized proteins after oxidative
stress (7-11). The signal for recognition and degradation of oxidized
proteins by the 20 S proteasome is unknown but has been suggested to
involve (i) exposure of hydrophobic surfaces after oxidative
modification, (ii) recognition of molecular "markers" associated
with the oxidative modification of specific amino acid side chains, and
(iii) increases in the conformational flexibility of oxidized proteins
(12-16).
To distinguish which signals are involved in targeting an oxidized
protein for degradation by the 20 S proteasome, we have investigated
the mechanisms involving the recognition and cleavage of oxidized
calmodulin (CaM).1 CaM was
chosen because of its key role in intracellular signaling and its
functional sensitivity to conditions of oxidative stress (17, 18).
After oxidative modification of multiple methionines to their
corresponding sulfoxides, CaM is unable to activate a range of
different target proteins involved in intracellular signaling (19, 20).
These results suggest that CaM oxidation has the potential to result in
large changes in cellular function as a result of changes in both
calcium signaling and energy metabolism (21). Consistent with this
suggestion, CaM isolated from senescent brain contains multiple
methionine sulfoxides that may be partially responsible for observed
alterations in calcium signaling associated with a range of age-related
diseases (17). Although intracellular enzymes exist that can reduce
(i.e. repair) oxidized methionines and partially restore CaM
function (22, 23), it is essential that rapid turnover mechanisms exist
to clear oxidatively modified CaM (CaMox) so as to maintain
normal calcium signaling. In this respect normal CaM turnover is slow
(t1/2
18 h) in comparison with many
transcription factors and other cellular regulatory molecules (24, 25).
However, it has been shown that the proteasome selectively degrades
post-translationally modified CaM after deamidation of selected
asparagines through a ubiquitin-independent cellular degradative
mechanism (16). It is therefore of interest to determine whether the
oxidation of methionines in CaM facilitates recognition and degradation by the 20 S proteasome and, if so, identify the molecular determinants that are responsible for the degradation of CaM after methionine oxidation.
To identify the mechanisms underlying the degradation of oxidized CaM
by the 20 S proteasome, in vitro conditions were used to
selectively oxidize variable numbers of methionines in CaM (18, 19).
Under these conditions no amino acids other than methionine are
oxidized, and the pattern of methionine oxidation is similar to that
found in CaM isolated from senescent brain (17, 18, 21). We assessed
the ability of the 20 S proteasome to degrade CaMox and
identified recognition elements within CaMox that promote
degradation. Our results demonstrate that the 20 S proteasome
selectively degrades oxidized CaM in preference to native (unoxidized)
CaM. Although neither hydrophobicity nor the presence of methionine
sulfoxide are signals for the degradation of oxidized CaM, the rate of
degradation correlates with the extent of secondary structural loss.
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EXPERIMENTAL PROCEDURES |
Materials--
Fluorescamine, fluorogenic peptides
(Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-AMC),
Leu-Leu-Glu-
-naphthylamine (LLE-Na), N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin
(LSTR-AMC)), 7-amino-4-methylcoumarin,
-naphthylamine, and
5-bromo-4-chloro-3-indolyl phosphate were obtained from Sigma.
1-Anilinonaphthalene-8-sulfonate (ANS) was purchased from Molecular
Probes (Eugene, OR). Polyclonal antibodies to the
and
subunits
of the 20 S proteasome were purchased from Calbiochem. Alkaline
phosphatase-conjugated secondary antibodies, goat anti-rabbit IgG, and
anti-mouse IgM were purchased from Zymed Laboratories
Inc. (South San Francisco, CA). Immobilon-P polyvinylidene
difluoride membrane (0.45 µm) for Western immunoblotting was from
Millipore (Bedford, MA). All reagents for sodium dodecyl sulfate
polyacrylamide gel electrophoresis (PAGE) were supplied by Bio-Rad.
Benchmark prestained protein ladder molecular weight markers were from
Life Technologies, Inc.
Purification of 20 S Proteasome--
The 20 S proteasome was
purified from frozen livers of 4-6-month-old Fischer 344 rats
essentially as described previously (26). The final purified proteasome
was made to a concentration of approximately 1 mg/ml in 50 mM potassium phosphate (pH 7.0), 0.1 M KCl and
stored at
70 °C. The purity of the proteasome, assessed by
SDS-PAGE and Western immunoblotting, was similar to that previously
reported (26). The maximal catalytic activity of the chymotrypsin-like,
trypsin-like, and peptidylglutamyl peptide hydrolase activities of the
proteasome were, respectively, 0.3, 0.1, and 0.1 µmol/min/mg as
assayed using the fluorogenic peptides Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-AMC),
Leu-Leu-Glu-
-naphthylamine (LLE-Na), and
N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin (LSTR-AMC).
These catalytic activities were independent of the free calcium
concentration (measured in reaction buffers containing 0.1 mM EGTA or after additions of either 100 and 500 µM calcium) and were comparable with values previously
reported (27). Protein concentrations were determined using the
bicinchoninic acid (BCA) protein assay reagents obtained from Pierce,
using bovine serum albumin as the standard.
Expression and Purification of Methionine Sulfoxide
Reductase--
A clone encoding the bovine liver isoform of
methionine sulfoxide reductase, kindly provided by Drs. Brot and
Moskovitz, was expressed in Escherichia coli as a fusion
protein with glutathione S-transferase and purified as described
previously using a glutathione-Sepharose 4B affinity column (28).
Expression and Purification of CaM--
A single isoform of CaM
corresponding to the cDNA encoding vertebrate CaM provided by
Professor Sam George (Duke University) was subcloned into the
expression vector pALTER-Ex1, overexpressed in E. coli
strain JM109, and purified essentially as described previously using
phenyl-Sepharose CL-4B and weak anion exchange HPLC (20, 29). Protein
concentration was determined using a micro-BCA protein assay reagent
kit (Pierce) using desalted CaM as the protein standard. The
concentration of CaM standard was determined using the published molar
extinction coefficient (
277 nm = 3029 M
1 cm
1) for calcium-saturated CaM (29, 30).
CaM Oxidation--
Methionines in CaM were oxidized essentially
as described previously (18) by incubating 60 µM CaM (1 mg/ml) in 50 mM HOMOPIPES (pH 5.0), 0.1 M KCl, 1 mM MgCl2, and 0.1 mM CaCl2 with 50 mM
H2O2 at 25 °C for times ranging from 1 h to 24 h. Hydrogen peroxide concentration was determined by using
the published extinction coefficient,
240 = 39.4 ± 0.2 M
1 cm
1 (31). The reaction was stopped by dialyzing
the sample overnight at 4 °C against multiple changes of distilled
water (5 × 1 liter) buffered with ammonium bicarbonate (pH
7.7).
CaM Proteolysis by the 20 S Proteasome--
Rates of proteolysis
using oxidized CaM as a substrate for the proteasome were determined
using two different assays that involved 1) monitoring the
disappearance of the integrated intensity of CaM bands on
SDS-polyacrylamide gels and 2) measurement of the initial release of
peptides generated by proteasome cleavage using fluorescamine, which
forms a fluorescent adduct with the amino termini of peptides (15). The
rate of CaM degradation was linear with respect to proteasome
concentration. Identical experimental conditions were used for both
assays, which involved the incubation of CaM (600 nM) with
the proteasome (5 nM) at 37 °C in 50 mM
HEPES (pH 7.5), 10 mM MgCl2, 100 mM
KCl, and 0.1 mM CaCl2.
Mass Spectrometric Analysis--
Electrospray ionization mass
spectrometry (ESI-MS) was used to 1) quantify the distribution of CaM
oxiforms and determine the average methionine sulfoxide concentration
for each sample and 2) identify masses of CaM peptides resulting from
proteolytic cleavage, essentially as described previously (17-19, 32).
Identification of released peptides was assisted by the software GPMAW
(Lighthouse Data, Aalokken 14, DK-5250; Odense SV, Denmark), which
permitted a unique identification for approximately one-half of the
released peptides. The calculated lengths of the remaining peptides
were determined within one amino acid based on identification of two or
three peptides whose theoretical masses were consistent with the
experimentally measured masses of the released peptides. Tentative identification of these peptides relied on predicted cleavage motifs
and permitted the calculation of the average size of the released
peptide (33).
Circular Dichroism Spectroscopy of CaM--
Circular dichroism
(CD) spectra were measured using a Jasco J-710 spectropolarimeter and a
temperature-jacketed spectral cell with a path length of 1.0 cm.
Spectra were recorded of CaM (0.6 nM) in 25 mM
Tris (pH 7.5), 0.1 M KCl, 10 mM
MgCl2, and 0.1 mM CaCl2 at 1-nm
intervals between 202 and 240 nm at 37 °C. The apparent helical
content was determined by the method of ridge regression using the
computer program Contin (34).
Evaluation of Hydrophobicity of Oxidized CaM--
The surface
hydrophobicity of CaM was evaluated from the change in fluorescence
spectra of ANS, which has previously been shown to accurately measure
the surface hydrophobicity of a range of different model proteins (35).
After incubation of CaM (0.3 µM) for 30 min in the
presence of a 10-fold molar excess of ANS, the fluorescence emission
was scanned from 450 to 600 nm (
ex = 372 nm). The
maximal fluorescence intensity and associated emission wavelength were
determined after subtraction of the emission spectrum of ANS in the
absence of protein.
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RESULTS |
Resolution of CaM Oxiforms--
To address the selectivity and
structural requirements of the proteasome in the degradation of
oxidized CaM (CaMox), we first used ESI-MS to identify the
extent of oxidation and distribution of oxidized CaM molecules that
will be used as potential substrates. Before oxidative modification,
CaM exhibits a single major ESI-MS peak corresponding to a mass of
16,707 ± 3 Da (Fig. 1A),
in close agreement with the theoretical average mass of vertebrate CaM expressed in E. coli (16, 705.4). In vitro
oxidative modification of CaM (see "Experimental Procedures") for
increasing periods of time resulted in the appearance of additional
peaks that differ in mass by 16 atomic mass units, which correspond to
the nine possible CaM oxiforms. After correction for the charge-induced dissociation product generated in the mass spectrometer (32), the area
of each peak in the ESI-MS spectra provided an estimate of the relative
abundance of each CaM species. In this manner the average number of
oxygens incorporated into each CaM sample after in vitro
oxidation was calculated (Fig. 1).

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Fig. 1.
Electrospray ionization mass specta of native
and oxidized CaM after deconvolution of multiply charged ions.
Spectra corresponding to native CaM (A) or subsequent to
exposure with 50 mM H2O2 for
increasing amounts of time results in average methionine sulfoxide
concentrations of 3.6 ± 0.2 (B), 6.1 ± 0.4 (C), or 7.7 ± 0.5 (D) mol/mol of CaM. For
mass spectrometry analysis, 30 µg of CaM in 5 mM ammonium
bicarbonate (pH 7.1) and 0.1 mM EGTA were trapped,
desalted, and then directly infused (on line) into an Autospec EQ mass
spectrometer as described previously (32). Masses of native and
oxidized CaM were resolved to within 3 atomic mass units, where the
respective masses are 16,705 Da (CaM), 16,721 [CaM(O)1],
16,737 [CaM(O)2], 16,753 [CaM(O)3], 16,769 [CaM(O)4], 16,785 [CaM(O)5], 16,801 [CaM(O)6], 16,817[CaM(O)7], 16,833 [CaM(O)8], and 16,849 [CaM(O)9]. The
relative fractions (fi) of the different CaM
oxiforms [CaM(O)i] were determined by spectral deconvolution
after correction for charge-induced dissociation (32). The average
number of oxygens (N) incorporated into each sample is
indicted in the figure and was obtained using the following
equation.
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(Eq. 1)
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An alternative resolution of CaM oxiforms was obtained by
electrophoretic separation on SDS-polyacrylamide gels, taking advantage of differences in the electrophoretic mobilities of CaM with varying degrees of methionine oxidation (17). Native (unoxidized) CaM migrates
as a single band with the greatest electrophoretic mobility (Fig.
2A). After oxidative
modification, multiple protein bands were resolved whose relative
mobilities decreased after increasing amounts of oxidative
modification. It should be noted that the large shifts in the relative
mobility of CaM oxiforms are the result of methionine oxidation, as no
other protein modifications are present under these experimental
conditions. By comparison with the mobility of different CaM oxiforms
separated and identified by weak anion exchange HPLC and ESI-MS,
respectively, we have shown that each protein band on SDS-PAGE
corresponds to CaM oxiforms having different integral numbers of
methionine sulfoxide from 0 to 9 and that the relative mobility of the
CaM oxiforms is inversely related to the number of oxidized
methionines. Thus, the most extensively oxidized CaM, migrating with an
apparent molecular mass near 18 kDa, corresponds to CaMox,
containing an average of approximately 9 methionine sulfoxides in each
CaM molecule.

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Fig. 2.
Resolution of CaM oxiforms using
SDS-PAGE. A, native CaM (lane 1) and CaM
after oxidative modification of an increasing number of methionines
(lanes 2-5) (corresponding to the following average number
of methionine sulfoxides: 1.1 ± 0.1 (lane 2), 2.6 ± 0.2 (lane 3), 6.6 ± 0.4 (lane 4), and 8.2 ± 0.6 (lane 5)) demonstrate electrophoretic resolution of CaM
oxiforms. In addition, oxidized CaM is shown before (lane 6)
and after enzymatic repair by methionine sulfoxide reductase
(lane 7), which, respectively, contain 7.7 ± 0.5 and 4.2 ± 0.3 methionine sulfoxide/CaM. B, the time course is shown
for the degradation of CaMox (2.6 methionine
sulfoxides/CaM) by 20 S proteasome after incubation at 37 °C in 50 mM HEPES (pH 7.5), 0.1 M KCl, 10 mM
MgCl2, and 0.1 mM CaCl2 for 0, 3, and 6 h, where the respective concentrations of CaM and the 20 S
proteasome were 12.5 µM and 95 nM. The
average extent of CaM oxidation was determined using ESI-MS, as
described in the legend to Fig. 1. Each lane represents 5 µg (A) or 3 µg (B) of protein applied to a
15% (m/v) polyacrylamide SDS gel (61). Mobilities of 15- and 20-kDa
molecular mass markers are indicated on the left side of each
panel. MCP, 20 S proteasome.
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Selective Degradation of Oxidized CaM--
We have investigated
whether native or oxidized CaM was a substrate for the proteasome using
SDS-PAGE to resolve different oxiforms of CaM both before and after
various times of exposure to proteasome. For the oxidized CaM sample
before exposure to the proteasome, three major bands appear
corresponding to native CaM and CaM containing as many as nine
methionine sulfoxides; several intermediate oxiforms are present in
very low abundance that have a reduced mobility (Fig. 2B).
In the absence of the proteasome, neither native nor oxidized CaM are
degraded over the 6-h time course of the experiment. In contrast, the
single band corresponding to extensively oxidized CaM is completely
degraded with 3-h exposure to the proteasome. Under these conditions
there is limited degradation of minor bands corresponding to CaM
oxiforms, with an intermediate mobility over the subsequent 3 h.
These results demonstrate that extensively oxidized CaM is
preferentially degraded by the 20 S proteasome.
Non-processive Degradation of CaMox--
To
investigate the recognition signals and mechanism of degradation of
extensively oxidized CaM (8.2 ± 0.6 methionine sulfoxides/CaM) by
the 20 S proteasome, we used mass spectrometry to measure the masses of
the peptides released from the proteasome. After exposure of
CaMox to the proteasome, CaMox and released
peptides were separated from the proteasome using a 100-kDa molecular
weight cutoff filter. Their respective masses were identified by
ESI-MS. After 1 h of degradation, approximately 10% of
CaMox was degraded by the proteasome, and the released
peptides were in large molar excess relative to the proteasome.
Thus, the majority of these peptides represent products released from
the proteasome before separation. At this time point, masses for 26 peptides were identified whose size distribution varied between 1188 and 4977 Da, which corresponds to peptide lengths between 10 and 39 amino acids. Of the resolved masses, 15 peptides were uniquely
identified that varied in mass between 1187.6 and 3589.9 Da (Table
I; Fig.
3A). From these data, it is
apparent that cleavage occurs preferentially on the carboxyl-terminal side of Glu, Leu, Asp, Phe, Lys, and Ala (Fig.
4). The preferential cleavage of these
sites in CaMox agrees with earlier results using other
protein substrates, suggesting that specific amino acid side chains are
preferentially recognized for cleavage (33, 36). In contrast, after
24 h of CaM exposure to proteasome, 25 peptides were identified in
the mass spectra. The mass distribution for these peptides varied
between 672 and 1430 Da, which corresponds to peptide lengths between 6 and 12 amino acids. Of these peptides, eight were uniquely identified
(Table I). Although these peptides also involved the preferential
cleavage of the peptide bond on the carboxyl-terminal side of side
chains previously implicated to enhance the proteolytic susceptibility
of protein substrates, it is apparent that the majority of these
peptides involve new cleavage sites. In conjunction with the large
decrease in the average size of the released peptides after incubation
of CaMox with the proteasome for long times, these results
suggest that CaMox is initially cleaved into large pieces
that are subsequently further degraded by the proteasome.
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Table I
Peptides released after incubation of CaMox with the 20 S
proteasome
There were no ambiguities in the mass assignments of the peptide ions,
and all peptides were detected with an accuracy of 0.05% of the
theoretical mass of the peptide of interest.
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Fig. 3.
Masses of released peptides after incubation
of 20 S proteasome with CaMox. Distribution of peptide
masses obtained from oxidized CaM (8.2 ± 0.6 methionine
sulfoxides/CaM) after incubation with 20 S proteasome for 1 h
(A) or 24 h (B), corresponding to partial and
complete degradation. Experimental conditions are as described in the
legend to Fig. 2. Peptide masses were identified using ESI-MS.
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Fig. 4.
Map of peptide intermediates observed after a
1-h incubation of CaMox with 20 S Proteasome. Peptides
identified by ESI-MS are indicated above the sequence of CaM.
Methionine residues are represented by bold letters.
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Rate of CaMox Degradation Correlates with Decreases in
Secondary Structure--
Methionine oxidation has previously been
shown to result in a large reduction in the
-helical content of CaM,
indicating the methionines function to stabilize the native
conformation of CaM (22). To determine whether these structural changes
may predispose CaMox to degradation by the proteasome, we
investigated whether changes in the secondary structure of CaM
resulting from methionine oxidation correlate with the sensitivity of
degradation by the proteasome. CD spectroscopy was used to estimate
changes in the
-helical content of CaM resulting from methionine
oxidation. From the CD spectra, it is apparent that a progressive
increase in the molar ellipticity ([
]) is observed, indicating a
loss of secondary structure that correlates with the extent of
oxidation (Fig. 5). The
-helical
content was calculated using a non-linear least squares-fitting
algorithm (34). After the oxidation of all nine methionines,
approximately one-half of the native
-helical content of CaM is
lost. These results confirm that methionine oxidation disrupts the
secondary structure of CaM and that the decrease in
-helical content
depends on the extent of methionine oxidation. Furthermore, observed
changes in the molar ellipticity correlate with the rates of
degradation by the proteasome, irrespective of whether the initial
release of peptides is measured using the fluorescamine assay or if
slower rate of disappearance of protein bands is detected using
SDS-PAGE (Fig. 6).

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Fig. 5.
Secondary structure of CaM.
A, CD spectra for native (unoxidized) CaM ( ) and after
oxidation of an average of 3.0 ( ), 4.9 ( ), and 8.2 ( )
methionines to their corresponding methionine sulfoxides.
Lines represent fits to the data using the method of ridge
regression to estimate the -helical content (34), which was
approximately 66% ( ), 52% ( ), 43% ( ), and 35% ( ).
B, ellipticity and -helical content are plotted for CaM
oxidized to varying extents. The line represents the least
squares fit (r = 0.97, p = 0.0001). Experimental
conditions involved 0.6 µM CaM in 25 mM Tris
(pH 7.5), 0.1 M KCl, 10 mM MgCl2,
and 0.1 mM CaCl2 at 37 °C.
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Fig. 6.
Correlation between rates of CaM degradation
and secondary structure. CaM degradation was measured by the
disappearance of the integrated intensity of all CaM bands on SDS-PAGE
( , ) or the reaction of free amines generated by peptide bond
cleavage with fluorescamine ( , ) after 1) variable extents of
in vitro oxidation ( , ), 2) calcium binding to
oxidized CaM containing 8.2 ± 0.5 methionine sulfoxides/CaM ( ), or
3) enzymatic reduction of CaMox by methionine sulfoxide
reductase ( ), as described in Fig. 2. Experimental conditions are as
described in the legend to Fig. 2.
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Decreased Degradation of CaMox after Partial Repair by
Methionine Sulfoxide Reductase--
Methionine sulfoxide reductase has
previously been shown to reduce (i.e. repair) methionine
sulfoxides in CaMox to induce partial refolding and to
restore the function of oxidized CaM (22). After repair, an average of
4.2 ± 0.3 methionine sulfoxides remain in each CaM, and the
distribution of oxidized methionines is very different from that
initially obtained after in vitro oxidation by
H2O2 (22). There are corresponding differences in the relative mobility of CaMox after repair compared
with that observed after in vitro oxidation (Fig.
2A). Therefore, since this physiological repair system has
the potential to modulate the rate of degradation of CaMox
by the proteasome and because of differences in the pattern of
methionine oxidation, it is of interest to investigate the sensitivity
of repaired CaM to degradation by the proteasome. After the repair of
fully oxidized CaM by methionine sulfoxide reductase there is a 30%
reduction in the rate of degradation by the proteasome accompanied by a
corresponding decrease in the molar ellipticity, which indicates an
increase in
-helical content (Fig. 6).
Calcium-dependent Changes in Secondary Structure of CaM
Correlate with Degradation Rate--
To distinguish whether the
increased degradation rate of CaMox by the proteasome
reflects a preferential recognition of methionine sulfoxide or, rather,
to the resulting loss of CaM secondary structure, we took advantage of
the fact that the
-helical content of extensively oxidized CaM
increases dramatically upon calcium binding, so that calcium-saturated
CaMox assumes a native-like structure (22, 32).
Accordingly, we have compared the calcium dependence of the degradation
rate of extensively oxidized CaM containing 8.2 ± 0.5 methionine
sulfoxides with changes in the secondary structure, as measured by the
molar ellipticity at 222 nm. Upon increasing the calcium concentration,
the rate of degradation by the proteasome decreases by approximately
75% with corresponding decreases in the molar ellipticity (Fig.
7). Since the range of calcium
concentrations used in this experiment did not alter the catalytic
efficiency of the proteasome, as assayed independently with fluorogenic
peptides (data not shown), these calcium-dependent
differences in the rate of degradation of CaMox reflect
calcium-induced conformational changes. Furthermore, since calcium
binding results in an increased solvent exposure of methionine side
chains in CaM, the rate of CaM degradation depends on the loss of
native structure rather than solvent exposure of methionine sulfoxide
(Fig. 6).

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Fig. 7.
Calcium-dependent changes in
degradation rate and secondary structure of CaMox. The
relationship between apparent secondary structural changes measured as
the ellipticity at 222 nm in the CD spectra and rates of degradation of
CaMox (8.2 methionine sulfoxides/CaM) by 20 S proteasome
after incubation at 37 °C in 10 mM Tris (pH 7.5), 0.1 M KCl, 10 mM MgCl2, and 0.1 mM EGTA or after the addition of either 0.1 mM
CaCl2 or 0.5 mM CaCl2. Rates of
CaMox degradation were measured by the appearance of free
amines using fluorescamine, as described under "Experimental
Procedures." MCP, 20 S proteasome.
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Hydrophobicity and CaMox Degradation--
The
oxidant-induced exposure of hydrophobic regions within proteins has
been suggested to uncover a universal recognition motif that targets
proteins for degradation by the 20 S proteasome (13-15). However,
since calcium activation results in the exposure of methionine-rich
(hydrophobic) binding pockets in CaM, the previous results showing that
calcium binding diminishes the rate of proteasome-mediated degradation
argues against this suggestion. Therefore, it is of interest to
directly determine the relationship between hydrophobicity and the
degradation of CaMox by the proteasome. These measurements have taken advantage of the large increase in the fluorescence intensity and changes in the emission maximum of ANS associated with
binding to surface-exposed hydrophobic sequences (35). In comparison to
apo-CaM, ANS binding to calcium-activated CaM results in a large
fluorescence increase and a blue-shift in the emission maximum of ANS
(data not shown), consistent with the calcium-dependent
exposure of hydrophobic pockets within each of the opposing globular
domains of CaM (37-39). The initial differences in ANS fluorescence
associated with calcium binding to native CaM are diminished as a
result of methionine oxidation, so that upon oxidation of the majority
of the nine methionines in CaM essentially no differences in the ANS
fluorescence to either apo- or calcium-activated CaM are retained.
Under these conditions there is a 4-fold difference in the rate of
degradation of CaMox by the proteasome (Fig. 7). Thus,
these results suggest that the exposure of hydrophobic residues have
little or no effect with respect to the recognition and degradation of
CaMox by the proteasome.
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DISCUSSION |
Protein Degradation by the Proteasome--
Post-translational
modifications decrease the half-life of a range of different proteins
by enhancing their rates of degradation, suggesting that they mark
proteins for degradation (12). It is generally believed that the
degradative mechanisms that enhance protein turnover are important to
the maintenance of cellular function. However, although a range of
different protein modifications have been identified that correlate
with enhanced rates of degradation by cellular proteases, relatively
little is known regarding how site-specific protein modifications
enhance their rates of degradation by the proteasome. In this respect,
the oxidative modification of a number of proteins has previously been
shown to correlate with the exposure of hydrophobic surfaces, resulting
in enhanced rates of proteolysis by the 20 S proteasome (7, 9, 10, 13,
15, 40-42). Results from these studies have been interpreted to
suggest that a universal recognition signal for target protein degradation by the 20 S proteasome involves partial protein unfolding and the exposure of hydrophobic surfaces. However, a simple
interpretation of this previous data has been complicated by the
oligomeric structure of the proteins studied, and their tendency to
undergo self-association after oxidative modification. In addition, the
significance of the in vitro oxidative modifications to
cellular physiology is unclear, as neither the products of their
in vitro oxidation nor evidence that these proteins are
oxidized in cells under conditions of physiological significance have
been presented. It is, therefore, important to identify the degradative
mechanisms of oxidatively modified proteins that (i) have been shown to
be present in cells, (ii) have the potential to modulate cellular
metabolism, and (iii) are normally degraded by the proteasome. With
respect to the first two criteria, previous measurements have
demonstrated that multiple methionines are oxidatively modified to
their corresponding sulfoxides in the calcium-signaling protein CaM,
isolated from senescent brain, resulting in the inability to fully
activate a range of different target proteins (17, 19, 20, 43).
Furthermore, post-translational modifications in CaM involving the
deamidation of asparagines induced by in vitro storage
result in its selective degradation by the proteasome relative to
other proteins (16, 44, 45).
Ubiquityl-calmodulin derivatized at Lys12 has been
identified in cells; a corresponding ubiquitin-calmodulin ligase is
likely to mediate this derivatization (46, 47). However,
ubiquitinylation does not result in the degradation of ubiquitinated
CaM by the 26 S proteasome (16, 46). Rather, ubiquitinylation of CaM appears to regulate the binding and activation of CaM to target proteins (e.g. glycogen phosphorylase kinase) and is not
coupled to the mechanism of protein degradation by the proteasome.
These results are consistent with the suggestion that ubiquitin
conjugation can have consequences other than direct targeting to the
proteasome (48) and suggest the physiological relevance of measuring
the mechanisms underlying the degradation of CaM by the 20 S
proteasome. CaM is a relatively long-lived signaling protein
(t1/2
18 h) in comparison to the short
half-lives of many transcription factors and other cellular regulatory
molecules (24, 25). Thus, cellular regulatory mechanisms that
stringently maintain constant levels of functional CaM, such as the
degradation of excess or nonfunctional CaM, are important in the
maintenance of cellular homeostasis (49).
Mechanism of Degradation--
The observation of the nonprocessive
degradation of CaMox by the proteasome is consistent with
earlier observations in which the degradation of many other protein
substrates has been observed to involve the initial cleavage into large
protein fragments at early times of digestion that can dissociate and
then rebind and be further digested by the proteasome (50-54). The
dissociation of large protein fragments from the proteasome suggests
the opportunity for other cellular protease systems to also be involved
in the degradation of cellular proteins (12, 54). However, in a number of cases the proteasome has been shown to degrade proteins in a
processive manner, involving the release of relatively homogeneous population of small peptides (55-57). Although the underlying reasons for observed differences in the mechanisms of protein degradation are
unclear, it is significant that the majority of these earlier studies
have involved chemically modified and unfolded proteins that are not
likely to be present in the cellular milieu. In contrast, it is likely
that oxidized CaM containing multiple methionine sulfoxides is a
physiological substrate of the proteasome (17).
The lack of a functional correlation between changes in the substrate
hydrophobicity and CaMox degradation by the proteasome suggests that hydrophobic sequences do not function in the recognition of CaMox by the 20 S proteasome. Rather, the best
correlation is observed between protein degradation rates and decreased
-helical content induced by either CaM oxidation or calcium binding.
The nonprocessive digestion of CaMox into a limited number
of large fragments that are released and further digested suggests that partial protein unfolding is sufficient to initiate recognition and
cleavage by the 20 S proteasome, without the need for global protein
unfolding to allow access of substrate into the internal cavity of the proteasome.
Conclusions and Future Directions--
The oxidative modification
of methionines in CaM results in a loss of secondary structure and the
preferential degradation by the 20 S proteasome into multiple large
fragments that are subsequently degraded into small peptides with an
average length of about eight amino acids. The preferential degradation
of oxidized CaM is consistent with the observation that methionine
oxidation destabilizes the
-helical structural elements (58-60) and
suggests that conformationally disordered structures may serve as
recognition elements for the proteasome. Future experiments aimed at
identifying the mechanism of degradation will require direct structural
measurements of protein intermediates bound to the proteasome, which
will permit the identification of specific structural motifs that
facilitate binding and degradation.