Expression and distribution of the class III ubiquitin-conjugating enzymes in the retina

Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Molecular vision (Impact Factor: 1.99). 11/2010; 16(260-62):2425-37.
Source: PubMed
ABSTRACT
Mounting evidence implicates chronic oxidative stress as a significant pathogenic factor in the development and progression of retinopathies, including age-related macular degeneration (AMD). The age-dependent toxic accumulation of oxidatively damaged proteins, lipids, and DNA in susceptible cells of the retina arises, at least in part, from a decreased capacity to eliminate these damaged biomolecules. The goal of this study was to determine the expression patterns and function of class III ubiquitin-conjugating enzymes (UbcM3, UBE2E2, and UbcM2) in the retina. These enzymes have been implicated in the ubiquitin-dependent degradation of oxidatively damaged and misfolded proteins.
Complementary western blotting and immunohistochemistry was performed with specific antibodies to determine the retinal cell expression pattern of each enzyme. Additional analyses using antibodies raised against UbcM2 were performed to determine the relative levels of the enzyme in lysates derived from various mouse organs as compared to the retina. An established light-damage model of oxidative stress-induced retinal degeneration was used to determine alterations in the susceptibility of mice harboring a single intact allele of UbcM2. Ubiquitin charging and auto-ubiquitylation assays were done to assess the catalytic state of UbcM2 following photo-oxidative stress.
Expression of the class III ubiquitin-conjugating enzymes in the retina, from highest to lowest, is UbcM2>UbcM3>UBE2E2. In addition to being the most robustly expressed, UbcM2 is further distinguished by its expression in photoreceptors and retinal pigment epithelial cells. UbcM2 is expressed in most mouse tissues analyzed and is most abundant in the retina. Studies using a bright-light-damage model of acute oxidative stress in mice harboring a single disrupted allele of UbcM2 revealed that a 58% reduction in enzyme levels did not increase the susceptibility of photoreceptors to acute photo-oxidative toxicity. This result may be explained by the observation that UbcM2 retained an intact and functional active site following exposure to acute bright light.
The class III ubiquitin-conjugating enzymes, and in particular UbcM2, are expressed in the retina and may function to counter the accumulation of oxidatively damaged and misfolded proteins. A 58% reduction in UbcM2 does not increase the susceptibility of photoreceptors to an acute photo-oxidative stress, suggesting the existence of compensating enzymes and/or that the remaining UbcM2 activity is sufficient to target oxidatively damaged proteins for destruction.

Full-text

Available from: Kendra Plafker
Expression and distribution of the class III ubiquitin-conjugating
enzymes in the retina
Saima Mirza,
1
Kendra S. Plafker,
1
Christopher Aston,
2
Scott M. Plafker
1
1
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK;
2
General Clinical Research
Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK
Purpose: Mounting evidence implicates chronic oxidative stress as a significant pathogenic factor in the development
and progression of retinopathies, including age-related macular degeneration (AMD). The age-dependent toxic
accumulation of oxidatively damaged proteins, lipids, and DNA in susceptible cells of the retina arises, at least in part,
from a decreased capacity to eliminate these damaged biomolecules. The goal of this study was to determine the expression
patterns and function of class III ubiquitin-conjugating enzymes (UbcM3, UBE2E2, and UbcM2) in the retina. These
enzymes have been implicated in the ubiquitin-dependent degradation of oxidatively damaged and misfolded proteins.
Methods: Complementary western blotting and immunohistochemistry was performed with specific antibodies to
determine the retinal cell expression pattern of each enzyme. Additional analyses using antibodies raised against UbcM2
were performed to determine the relative levels of the enzyme in lysates derived from various mouse organs as compared
to the retina. An established light-damage model of oxidative stress-induced retinal degeneration was used to determine
alterations in the susceptibility of mice harboring a single intact allele of UbcM2. Ubiquitin charging and auto-
ubiquitylation assays were done to assess the catalytic state of UbcM2 following photo-oxidative stress.
Results: Expression of the class III ubiquitin-conjugating enzymes in the retina, from highest to lowest, is
UbcM2>UbcM3>UBE2E2. In addition to being the most robustly expressed, UbcM2 is further distinguished by its
expression in photoreceptors and retinal pigment epithelial cells. UbcM2 is expressed in most mouse tissues analyzed and
is most abundant in the retina. Studies using a bright-light-damage model of acute oxidative stress in mice harboring a
single disrupted allele of UbcM2 revealed that a 58% reduction in enzyme levels did not increase the susceptibility of
photoreceptors to acute photo-oxidative toxicity. This result may be explained by the observation that UbcM2 retained
an intact and functional active site following exposure to acute bright light.
Conclusions: The class III ubiquitin-conjugating enzymes, and in particular UbcM2, are expressed in the retina and may
function to counter the accumulation of oxidatively damaged and misfolded proteins. A 58% reduction in UbcM2 does
not increase the susceptibility of photoreceptors to an acute photo-oxidative stress, suggesting the existence of
compensating enzymes and/or that the remaining UbcM2 activity is sufficient to target oxidatively damaged proteins for
destruction.
The retina is highly susceptible to oxidative stress and
damage due to its robust oxygen consumption, exceptionally
high content of polyunsaturated fatty acids, and exposure to
bright light. Together, these factors create a chronic oxidative
burden that can result in damage to retinal proteins, DNA, and
lipids [1]. Elimination of these oxidatively damaged
biomolecules is required to prevent the toxicity that can result
from their accumulation [2]. The accumulation of these
damaged biomolecules is a hallmark of numerous
neurodegenerative disorders, including age-related macular
degeneration (AMD) [3]. The ubiquitin (Ub) proteolytic
system (UPS) plays an integral role in destroying misfolded
and oxidatively damaged proteins [4,5], and multiple lines of
evidence implicate a critical function for this system in
countering oxidative stress in the retina and lens. Evidence in
Correspondence to: Scott M. Plafker, Department of Cell Biology,
940 Stanton L. Young Blvd, BMSB 538, University of Oklahoma,
Oklahoma City, OK, 73104; Phone: (405) 271-8001 (ext. 45512);
FAX: (405) 271-3548; email: scott-plafker@ouhsc.edu
support of this comes from studies showing that inhibition of
the UPS in the retina, by either pharmacological means or with
mutant Ub, leads to the deleterious accumulation of oxidized
proteins [6,7].
The central player of the UPS is Ub, a highly conserved
76-amino acid polypeptide that is post-translationally
attached to target proteins. Protein ubiquitylation is performed
by an enzyme cascade consisting of a Ub-activating enzyme
(E1), a Ub-conjugating enzyme (E2), and a Ub protein ligase
(E3) [8]. In humans, there are two different E1s, at least 38
E2s, and 600–1,000 E3s [9]. Substrate selection and
specificity are conferred primarily through the pairing of
particular E2–E3 combinations. Ub is conjugated to an
internal lysine of a target protein, and in the case of
polyubiquitylation, subsequent Ubs are attached sequentially
to a lysine of the previously added Ub. The best-studied fate
of polyubiquitylation is that the modified protein gets targeted
to the 26S proteasome for degradation. However, particular
configurations of polyUb chains can result in non-proteolytic
outcomes for the target protein. In addition, substrates can be
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Received 26 April 2010 | Accepted 13 November 2010 | Published 18 November 2010
© 2010 Molecular Vision
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regulated in non-proteolytic ways by the addition of a single
Ub, a process referred to as monoubiquitylation. Analogous
to the removal of phosphorylation by protein phosphatases,
balance in the UPS is achieved by a set of Ub C-terminal
hydrolases/deubiquitylating isopeptidases that cleave Ub
from substrates (all reviewed in [10]).
Analysis of the retina for the expression and distribution
of UPS components has demonstrated the presence of
numerous enzymes in select cell types. For example, four
different Ub-conjugating enzymes (E2
14K
, E2
20K
, E2
25K
, and
E2
35K
) have been identified in bovine rod outer segments
[11]. PGP 9.5, a Ub C-terminal hydrolase, is only present in
retinal ganglion and horizontal cells [12], whereas the Ub
hydrolase UCH-L3 is enriched in photoreceptor inner
segments [13]. The E3 ligases Nedd4 and Siah1 have recently
been identified in retinal ganglion cells and Müller cells,
respectively [14,15]. Kelch-like 7 (KLHL7), a substrate
adaptor for the cullin 3-based E3 ligase, is expressed in rod
photoreceptors, and the identification of three different
missense mutations has linked this protein to hereditary
retinitis pigmentosa [16].
It is likely that these various enzymes represent only a
small subset of the UPS components present in the retina. We
undertook the present study to analyze the expression and
distribution of the class III E2s in the retina. The human class
III E2s are called UBE2E1, UBE2E2, and UBE2E3. The
mouse versions are referred to as UbcM3, UBE2E2, and
UbcM2, respectively, and each is identical to its human
counterpart [17] (see Table 1). These enzymes share a host of
properties, including: 1) a steady-state nuclear distribution
[18,19], 2) entering the nucleus via the importin-11 transport
receptor [18,19], 3) binding common E3 ligase partners (e.g.,
[20,21]), and 4) interacting with the N-terminal domain of
various cullin proteins [21]. An additional distinguishing
feature among these enzymes is that each has a unique N-
terminal domain of 40–60 residues [17]. The rationale for
analyzing the retinal expression patterns of these enzymes is
threefold. First, these enzymes are functional homologs of a
pair of S. cerevisiae E2s, Ubc4, and Ubc5, that mediate the
degradation of misfolded and oxidatively damaged proteins
[17,22-24]. Second, we recently reported that these enzymes
can directly bind the master antioxidant transcription factor
Nrf2 [25]. Moreover, we demonstrated that UbcM2 can
stabilize and transcriptionally activate Nrf2 and that these
functions are largely mediated by a unique cysteine residue
present in the class III E2s [25]. In the context of the retina,
Nrf2 has been shown to play an important role in conferring
protection from photo-oxidative and electrophilic stress
[26-34]. Third, we have shown in cultured retinal pigment
epithelial cells that UbcM2 is required for proliferation [35].
Depletion of the enzyme by small interfering RNA (siRNA)
caused a robust increase in the cell-cycle inhibitor p27
Kip1
.
However, the mechanism underlying this cell-cycle effect is
unknown.
Using antibodies specific for each enzyme, we have
discovered that these three enzymes display differential
distributions in the mouse retina. Interestingly, only UbcM2
was detectable in the nuclei of retinal pigment epithelial cells
and in photoreceptors. Furthermore, we demonstrate that
UbcM2 is expressed in most mouse tissues analyzed but is
most abundant in the retina. Studies using a bright-light
damage model of acute oxidative stress in mice harboring a
single disrupted allele of UbcM2 revealed that a 58%
reduction in enzyme levels did not increase the susceptibility
of photoreceptors to photo-oxidative toxicity. These light-
damage studies further revealed that UbcM2 retains its
catalytic capacity following exposure to acute bright light.
Together, these studies are the first to describe the retinal
expression pattern of the class III E2s and to test the concept
that a full complement of one of these enzymes, UbcM2, is
necessary to protect photoreceptors from an acute oxidative
insult.
METHODS
Antibodies: Anti -UbcM3, anti -UBE2E2, and anti -UbcM2
were raised in rabbits against recombinant His
6
-S-tagged
polypeptides corresponding to the unique N-terminal
extension of each enzyme. The His
6
-S tag consists of a hexa-
histidine stretch followed by the S-peptide from RNase A, and
the combination is encoded in the series of pET30 vectors
from Novagen EMD Biosciences (Darmstadt, Germany).
Purified glutathione S-transferase (GST) fusions of each
enzyme were used for affinity purification. anti-glutathione
S-transferase (anti-GST) was purchased from Bethyl
Laboratories (Montgomery, TX) and anti-UbcH6 from
Boston Biochem (Cambridge, MA).
Western and dot blotting: GST- and His
6
-S-tagged
recombinant fusion proteins were expressed from pGEX-2T
and pET30a, respectively, and purified from BL21(star) E.
coli, as described previously [21]. For the dot blot assays, five
TABLE 1. CLASS II E2 NOMENCLATURE.
Mouse
Human
UbcM3 UBE2E1, UbcH6
UBE2E2 UBE2E2, UbcH8
UbcM2 UBE2E3, UbcH9, E2–23K, UBCE4
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sheets of nitrocellulose were spotted with 10, 1, or 0.1 ng of
either GST, GST-UbcM3, GST-UBE2E2, or GST-UbcM2
and then air dried. Blots were blocked for 60 min in 5% nonfat
dried milk in TBST (20 mM Tris-HCl [pH 7.4], 150 mM NaCl,
0.1% Tween-20) and incubated with primary antibodies
diluted as follows: anti-GST (1:1,000), anti-UbcM3 (1:100),
anti-UBE2E2 (1:100), anti-UbcM2 (1:1,000), and anti-
UbcH6 (1:1,000). Primary antibodies were detected with a
horseradish peroxidase-conjugated goat antirabbit secondary
antibody diluted at 1:2,500 followed by enhanced
chemiluminescence.
Small interfering RNA transfections: Synthetic pools of
UbcM2-specific or UbcM3-specific siRNAs as well as control
siCON siRNA (all from Dharmacon, Inc.) were combined
with oligofectamine (Invitrogen, Carlsbad, CA) for 20 min
before being added to 5×10
5
HeLa cells in 12-well dishes. A
final concentration of 60 nM siRNA was added to the cultured
cells. Four hours following the addition of siRNA, 1 ml of
DMEM (cat. # 10–013-CV; Mediatech, Inc.) supplemented
with 10% fetal calf serum, 100 units/ml penicillin, and 0.1 mg/
ml streptomycin sulfate was added to each well. Samples were
harvested 72 h post transfection and processed for western
blotting.
Light-damage experiments: All animal care procedures
complied with the ARVO Resolution on the Use of Animals
in Research and with the rules and regulations of the OUHSC
Institutional Animal Care and Use Committee. A modification
of an established bright-light damage protocol was followed
[36,37]. Six-week old, albino SvEv mice (wild type [WT] and
UbcM2
+/−
) [35] (Of note, this albino strain is a mixed SvEv/
Bl6 line) that were born and raised in dim cyclic light (5 lux,
12 h:12 h on–off cycle) were dark adapted overnight before
being exposed to 3,000 lux of white, cool, diffuse fluorescent
light for 6 h in a light box with reflective surfaces and a wire
top. Each cage housed a single animal, and animals had
unrestricted access to food and water. Following the light
stress, animals were returned to the dim cyclic light for 1 week
before being sacrificed by asphyxiation with carbon dioxide.
Negative control animals were treated identically except they
were not exposed to the bright light. For the auto-
ubiquitylation studies, animals were exposed to 3,000 lux for
6 h and then immediately sacrificed and retinas harvested for
lysates. For immunohistochemical studies, enucleated eyes
were fixed overnight at room temperature in Perfix (20%
isopropanol, 2% trichloroacetic acid, 4% paraformaldehyde,
and 2% zinc chloride) followed by embedding in paraffin.
Five-μm-thick sections were cut along the vertical meridian
and stained with hematoxylin and eosin. Starting at the optic
nerve head (ONH) and extending to the superior and inferior
ora serrata, outer nuclei layer (ONL) counts were collected at
225-μm intervals. Spider graphs were compiled from pooled
data with error bars representing standard deviations.
Immunohistochemistry: Heat-induced epitope retrieval
(HIER) was performed on paraffin-embedded sections, using
a pressurized Decloaking Chamber (Biocare Medical;
Concord, CA) in citrate buffer (pH 6.0) at 99 °C for 18 min.
Slides were incubated in 3% hydrogen peroxide, followed by
normal serum and BSA at room temperature for 20 min each.
After incubation with primary antibodies (anti-UbcM3 diluted
1:250; anti -UBE2E2 diluted 1:250; anti -UbcM2 diluted
1:2,000), the slides were incubated in polymer-horseradish
peroxidase-conjugated secondary antibody (DAKO,
Glostrup, Denmark) and developed with diaminobenzidine
(Sigma, St. Louis, MO). Counterstaining was accomplished
with analine blue (Sigma). Slides were examined with a Nikon
80i microscope and DXM1200C camera, and images captured
using NIS-Elements software (Nikon; Tokyo, Japan). Image
processing was done with Adobe Photoshop (version 8.0). Of
note, the dilutions of antibodies used were based on pilot
studies done to optimize the signal to noise ratio. Further,
because anti-UbcM2 was more sensitive than anti-UbcM3 and
anti-UBE2E2 (based on the GST-fusion dot blots [Figure
1A]), we diluted the anti-UbcM2 antibody 1:2,000 but the
other two antibodies 1:250 to normalize for this difference.
Mouse tissue analysis: The generation of SvEv/129 UbcM2
heterozygotes (UbcM2
+/−
) has been described previously
[35]. Extracts were made by homogenizing tissues in Tissue
Protein Extraction Reagent (ThermoScientific, Waltham,
MA) followed by a 30-min incubation in a 4 °C thermomixer
shaking at 1,000 rpms. Lysates were then clarified by
centrifugation at 16,000× g in a 4 °C tabletop centrifuge.
Protein concentrations of clarified lysates were determined by
bicinchoninic acid assay (ThermoScientific). Retinas were
isolated by the “Winkling” procedure [38]. Briefly,
immediately following sacrifice, forceps were placed around
the optic nerve and the eye was lifted slightly. A razor blade
was used to cut the globe along the equator, thus permitting
removal of the cornea and lens. Further raising of the eye with
the forceps caused detachment and subsequent extrusion of
the retina from the globe. Equal micrograms of retinal lysates
were resolved by sodium dodecyl sulfate PAGE (SDS–
PAGE) and processed for western blot analysis. Comparable
loading of samples was monitored by amido black staining of
the nitrocellulose membranes.
Ubiquitin-charging and in vitro auto-ubiquitylation assays:
Ub-charging assay—Mouse retinal lysates were isolated
and protein concentrations determined as described above.
Equal amounts of protein were then solubilized with
nonreducing SDS–PAGE buffer (50 mM Tris-HCl [pH6.8], 4
M urea, 2% SDS, 10% glycerol, and 0.001% bromophenol
blue) [39]. Subsequently, samples were heated for 15 min in
a 30 °C water bath, and then one-half of each sample was
transferred to a new tube and combined with 1 μl β-
mercaptoethanol (14.3 M) to reduce the thiolester bond
between the active site cysteine of UbcM2 and Ub. Thirty
micrograms of each nonreduced and reduced sample in
parallel was then resolved by SDS–PAGE in a 4 °C cold room
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at 100 V constant, electrotransferred to nitrocellulose at room
temperature, and analyzed by anti-UbcM2 western blotting.
In vitro ubiquitylation assay—For each
sample, 50 μg
of clarified retinal lysate was combined with anti-UbcM2 and
protein A Sepharose for 2 h at 4 °C. Immunoprecipitations
were washed with 20 column volumes of ice-cold wash buffer
(10 mM HEPES-KOH [pH 7.4], 55 mM potassium acetate,
1 mM magnesium acetate, 0.1 mM EGTA, 0.25% Tween-20,
150 mM NaCl) before being combined with a ubiquitylation
cocktail containing recombinant human E1 (0.14 μg/reaction;
Boston Biochem), Ub (16 μg/reaction), and an energy-
regenerating system (100 mM Tris-HCl [pH 7.4], 0.4 mM
MgATP (from 100 mM stock of MgATP using 100 mM
magnesium acetate, 20 mM HEPES [pH 7.5], ATP and water.
This stock was then diluted to a final concentration of
0.4 mM), 1 mM MgCl
2
, 0.2 mM DTT, 2mM phosphocreatine,
0.2% Tween-20, and 0.5 mg creatine phosphokinase) for 90
min at 37 °C. Reactions were terminated by the addition of
concentrated (4×) Laemmli solubilizing buffer, resolved by
SDS–PAGE, and analyzed by anti-UbcM2 western blotting.
Statistical analyses for light-damage studies: Results were
expressed as mean±standard deviation. The outcome of
interest for the primary analysis was the amount of light
damage measured for each animal in the light-damaged group
as the ONL count at a particular position (relative distance
from the optic nerve head) minus the average of the negative
control animals of the same sex and genotype at that same
position. The primary analysis determined whether there were
significant differences in the amount of damage between the
two genotypes: WT and UbcM2 heterozygotes (UbcM2
+/−
).
Figure 1. Characterization of the
sensitivity and
specificity of class III
ubiquitin conjugating enzyme (E2)
antibodies. A: Dot blot assay using
recombinant glutathione S-transferase
(GST) or GST-E2 fusion proteins. The
indicated recombinant proteins (10, 1,
or 0.1 ng) were spotted on pieces of
nitrocellulose paper in quintuplicate.
The blots were blocked in 5% milk/
TBST and then incubated with anti-
GST, anti-UbcM3, anti-UBE2E2, anti-
UbcM2, or a commercial antibody
against human UbcM3 (anti-UbcH6).
To the right of the blots is a diagram of
the class III E2s highlighting the relative
location of the conserved catalytic core
domain (UBC), the number of residues
in each protein, and the residue
corresponding to the end of the unique
N-terminal extension. B: Mouse retinal
lysate (10 or 30 μg) was resolved by
sodium dodecyl sulfate PAGE (SDS–
PAGE) in quadruplicate, transferred to
nitrocellulose, and probed with the
indicated antibodies. The migration of
UbcM2 and UbcM3 is indicated to the
right of the anti-UbcH6 blot. Two
distinct isoforms of UbcM3 are detected
(arrows). The migration of molecular
weight markers is indicated on the left.
C: siRNA experiments in HeLa cells to
demonstrate that targeted knockdown of
UbcM2 results in loss of the band
denoted as UbcM2 but does not affect
UbcM3 expression (left blot), and
targeted knockdown of UbcM3 results
in loss of detection of both isoforms of
the enzyme (right blot). The migration
of molecular weight markers is
indicated to the left of the blots.
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Secondary analyses used the absolute ONL counts to
determine sex and genotype differences between negative
control animals. Analyses used generalized linear models with
genotype and sex effects nested within position to allow for
determination of significance of the overall sex or genotype
effect, while accounting for differences between positions.
These were followed by post hoc pair-wise tests within each
site, with a Scheffe correction for multiple comparisons.
Descriptive statistics were calculated in Microsoft Excel
(Microsoft Corporation), while all statistical analyses used
SAS (version 9.1, SAS Institute Inc., Cary, NC).
RESULTS
The human genome encodes at least 38 different E2s [9], but
the tissue distributions, E3 ligase partners, substrates, and
functions of many of these enzymes remain unknown. Of this
large group of enzymes, the class III E2s (UBE2E1, UBE2E2,
and UBE2E3) are of particular interest due to their high
conservation among metazoans. For example, the respective
mouse and human counterparts of all three enzymes are 100%
identical [17]. In this study all of the work is done with the
mouse enzymes, and we therefore refer to the enzymes and
their respective antibodies by the mouse nomenclature (Table
1). UBE2E1 is referred to as UbcM3, UBE2E2 is UBE2E2,
and UBE2E3 is UbcM2.
The high level of conservation (>95%) among the class
III E2s suggests that these three enzymes have functional
overlap and therefore might be distinguished by their
respective tissue and/or cellular distributions. To address this,
we developed rabbit polyclonal antibodies against the three
enzymes, taking advantage of the fact that each possesses a
unique N-terminal domain (Figure 1A). Antibodies were
raised against recombinant His
6
-S fusions of each N-terminal
domain and affinity purified using GST fusions of each. Dot
blot assays using the GST-fusion proteins established the
specificity and sensitivity of each antibody. Anti-UbcM3 and
anti-UBE2E2 readily detected 1 ng of their respective GST
fusions, whereas anti-UbcM2 detected as little as 0.1 ng of
GST-UbcM2 (Figure 1A). None of the antibodies appreciably
cross-reacted with GST fusions of the other two enzymes. An
anti-GST control dot blot was done to show that equivalent
amounts of the various fusion proteins were spotted. A
commercial antibody, referred to hereafter as anti-UbcH6,
raised against the human form of UbcM3 (also known as
UbcH6) was also tested. This antibody detected all of the class
III E2s, which precluded its use for subsequent
immunohistochemical (IHC) studies. Of note, anti-UbcH6 has
a higher sensitivity for GST-UbcM3 compared to anti-
UbcM3.
As the retina is subjected to chronic oxidative stress [1]
and the class III E2s are functional homologs of the yeast
enzymes that degrade oxidatively damaged proteins [17,
22-24], we next examined which of the enzymes is expressed
in the mouse retina (Figure 1B). The three enzymes migrate
at 20–25 kDa in SDS–PAGE. We found that UbcM2 was
readily detectable but UBE2E2 was not. Using our anti-
UbcM3 antibody, we did not detect UbcM3. In contrast the
commercial anti-UbcH6 antibody detected UbcM3 as well as
UbcM2 and a third faster migrating band that represented a
second isoform of UbcM3. The identification of this faster
migrating band as a second isoform of UbcM3 is based on
siRNA experiments targeting the expression of either UbcM2
or UbcM3. Expression of the band identified as UbcM2 was
reduced by UbcM2-specific siRNA (Figure 1C, left panel),
whereas expression of the two UbcM3 bands was reduced by
UbcM3-specific siRNA (Figure 1C, right panel). A control
siRNA (siCON) did not affect the expression of either
enzyme. The different results regarding UbcM3 expression in
the retina using anti-UbcM3 and anti-UbcH6 are consistent
with the higher sensitivity of anti-UbcH6 relative to anti-
UbcM3 (Figure 1A). Further, the nearly comparable detection
of both UbcM2 and UbcM3 by commercial anti-UbcH6 but
the approximately tenfold higher sensitivity of the antibody
for UbcM3 (Figure 1A) indicates that UbcM2 is expressed in
the retina at higher levels than UbcM3. These data show that
expression of the class III E2s in the retina, from highest to
lowest, is UbcM2>UbcM3>UBE2E2.
The high sensitivity of anti-UbcM2 prompted us to
determine the tissue distribution of the enzyme and to compare
expression levels to those detected in the retina. Various
organs were harvested from a 7-month-old mouse, and 30 μg
of each lysate was resolved by SDS–PAGE for western
blotting with anti-UbcM2. Amido black staining of the blot
was performed to demonstrate comparable loading. These
experiments demonstrated that UbcM2 is most prominently
expressed in the retina (Figure 2, lane 9). The enzyme is
second-most prominently expressed in the brain and spleen
(Figure 2, lanes 1 and 8, respectively), with lower amounts
detectable in the heart, kidney, liver, stomach, pancreas, and
lung (Figure 2, lanes 2, 3, 4, 6, 7, and 10, respectively).
Strikingly, skeletal muscle lysates contained a prominent band
migrating at ~75 kDa but had no band at the predicted
migration for UbcM2 (Figure 2, lane 5). The identification of
this 75-kDa band is unknown. These data reveal that UbcM2
is enriched in the retina and ubiquitously expressed at lower
levels in most tissues and organs analyzed.
To corroborate and extend the findings from the retinal
lysate western blots, we determined the temporal retinal cell
expression pattern of UbcM3, UBE2E2, and UbcM2. To
accomplish this, we performed IHC staining of paraffin-
embedded sections of retinas from 6-, 11-, and 31-week-old
mice. As we reported previously [19], all of the class III E2s
have a steady-state nuclear distribution, and therefore we
analyzed the sections for nuclear immunohistochemical
staining. In addition we used anti-UbcM3 and anti-UBE2E2
at dilutions of 1:250 but anti-UbcM2 at a dilution of 1:2,000
as a means of normalizing the antibodies for their relative
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sensitivities (Figure 1A). These experiments demonstrated
minimal nuclear staining for UbcM3 at all time points (Figure
3A,D,G), although we could detect faint cytoplasmic
speckling in the inner segments of the photoreceptors and in
the ganglion cell layer. Because UbcM3 is a nuclear enzyme
[19], this labeling is likely to be nonspecific. Faint nuclear
UBE2E2 was detected in some nuclei of the inner nuclear
layer (i.e., second order neurons) and in ganglion cells (Figure
3B,E,H). A range of nuclear UbcM2 expression was detected
in the inner nuclear layer, and robust staining was apparent in
ganglion cells, whereas moderate UbcM2 expression was
evident in the ONL (i.e., photoreceptor nuclei; Figure
3C,F,I). Closer examination of the retinal pigment epithelial
(RPE) layer revealed nuclear staining for UbcM2 (Figure
3L,O,R), but not UbcM3 or UBE2E2, at all three time points
(Figure 3J,K,M,N,P,Q). The relatively higher IHC detection
of UbcM2 versus the other two enzymes is consistent with the
western blotting results (Figure 1B). Together, these data
show that, within the detection limits of the antibodies used,
UbcM2 is enriched in the retina, especially in RPE cells,
compared to UbcM3 and UBE2E2.
To test for a potential antioxidant/protective function of
UbcM2 in the retina, we used an established bright-light
damage model of acute oxidative stress and retinal
degeneration [37,40,41]. In this assay, albino mice are reared
in the dark and then exposed to 3,000 lux of cool white light
for 6 h. Exposure to the light induces an oxidative stress that
is lethal to photoreceptors. Thus, by counting the rows of
nuclei in the ONL a week after light exposure (to allow
clearance of the dead photoreceptors), the susceptibility of the
retina to photo-oxidative insult can be quantified and
evaluated. To determine if a full complement of UbcM2 is
required for countering oxidative stress in the retina, we
compared the toxicity of the light challenge in mice
heterozygous for UbcM2 versus wild-type littermates.
Disruption of a single UbcM2 allele reduces expression of the
enzyme in the retina by 58±3% (Figure 4A, graph). We were
unable to perform these experiments in UbcM2 null mice as
disruption of both alleles is embryonic lethal (unpublished
results). Control animals were maintained in dim light for the
entire experiment. Isolated retinas were paraffin embedded,
sectioned, stained with hematoxylin and eosin (Figure 4B),
and then analyzed for the number of layers comprising the
ONL (Figure 4C). From these experiments, we found that
3,000 lux was sufficient to induce the loss of three to four
layers of nuclei flanking the ONH in both UbcM2
Figure 2. UbcM2 is enriched in the
retina and expressed in most organs.
Thirty micrograms of lysate from the
brain, heart, kidney, liver, skeletal
muscle, stomach, pancreas, spleen,
retina, and lung of a 7-month-old mouse
were subjected to anti-UbcM2 western
blotting (top blot). The same blot was
stained with Amido Black to
demonstrate comparable protein levels
in each lysate (bottom blot). The
migration of molecular weight markers
is shown to the left of the blots.
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Figure 3. Differential expression of class III ubiquitin conjugating enzymes (E2s) in cells of the mouse retina. A-I: Shown here are
representative photomicrographs of paraffin-embedded sections labeled with the indicated antibodies. Mouse age in weeks is shown in the
bottom right corner of each panel. Abbreviations are as follows: RPE represents retinal pigment epithelium; OS represents outer segments;
IS represents inner segments; ONL represents outer nuclear layer; OPL represents outer plexiform layer; INL represents inner nuclear layer;
IPL represents inner plexiform layer; GCL represents ganglion cell layer. The magnification bar represents 50 μm, and all photomicrographs
were taken at the same magnification. J-R: Enlarged sections are shown of images from (J) highlighting the presence or absence of RPE
labeling with the various antibodies. Black arrowheads mark unlabeled RPE nuclei and white arrows indicate nuclei labeled with anti-UbcM2.
The magnification bar represents 25 μm.
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heterozygotes and their wild-type littermates (Figure 4B-D).
Interestingly, we observed that UbcM2 heterozygote females
displayed an apparently higher sensitivity to the bright-light
challenge, although this difference was not statistically
significant (data not shown). These data reveal that a 58%
reduction in UbcM2 levels does not increase the susceptibility
of the retina to acute photo-oxidative toxicity, as measured by
bright-light-induced photoreceptor death.
We also analyzed the catalytic state of UbcM2 in
response to bright-light stress. The catalytic state of an E2 is
reflected by the relative amount of enzyme that is charged with
Ub on its active site cysteine. For these experiments, mice
were either maintained in dim light or exposed to 3,000 lux
for 6 h and sacrificed. Retinal lysates were generated under
nonreducing conditions to maintain the thiolester linkage
between the active site cysteine of UbcM2 and the COOH-
terminal glycine of Ub. The lysates were subsequently split in
half and resolved in parallel by nonreducing and reducing
SDS–PAGE followed by anti-UbcM2 western blotting. The
rationale for this methodology is that the attachment of Ub to
the active site cysteine results in a slower migrating, ~30-kDa
band in nonreducing SDS–PAGE. The addition of β-
mercaptoethanol reduces the thiolester bond, thereby
removing Ub from the active site and collapsing the 30-kDa
band to the faster migrating 23-kDa band (Figure 5A, compare
top and bottom blots) [19]. These experiments revealed that
in both WT and UbcM2
+/−
retinas, the level of Ub-charged
UbcM2 was maintained in the bright-light challenged retinas
as compared to the dim-light control samples (Figure 5A,
compare lanes 2,6,7 to lanes 1,3,4,5). This result is somewhat
surprising considering that previous studies show particular
E2s lose Ub from their active sites in response to oxidative
stress (e.g., [39]).
We next performed auto-ubiquitylation assays using
UbcM2
+/−
retinal lysates to determine if the capacity of the
enzyme to transfer Ub was affected by an acute oxidative
stress. Of note, heterozygote mice were chosen for these
experiments because the data in Figure 4 implied that
expression of the enzyme from a single intact allele provided
sufficient catalytic activity to protect photoreceptors from an
acute photo-oxidative stress. For this assay, UbcM2 was
immunoprecipitated from retinal lysates derived from six
control animals and six light-challenged animals (Figure 5B,
left panel) and combined with recombinant E1, Ub, and an
ATP-regenerating system for 90 min at 37 °C. Reaction
products were resolved by SDS–PAGE and analyzed by anti-
UbcM2 western blotting. Auto-ubiquitylation of UbcM2 is
detected by the generation of a slower migrating band(s) in
this assay. These experiments showed that UbcM2 auto-
ubiquitylation was comparable between the control and light-
challenged samples (Figure 5B, lanes 15–20 versus 21–26).
Notably, by far the most prominent product of the auto-
ubiquitylation reaction was mono-ubiquitylated UbcM2, in
agreement with previously reported results for this enzyme
[42]. These data show that UbcM2 retains an intact active site
cysteine (i.e., not oxidized) following an acute photo-
oxidative insult and can therefore function in the in vitro assay
to get charged with Ub and undergo auto-ubiquitylation.
Identical results were obtained with UbcM2 isolated from wt
retinas (not shown). Together, the thiolester and
ubiquitylation data reveal that in response to acute bright-light
challenge, the steady-state levels of Ub-charged UbcM2
remain constant and the enzyme retains its capacity to transfer
Ub, indicating maintenance of an intact and functional active
site.
DISCUSSION
In this report we describe the expression and distribution of
the class III Ub-conjugating enzymes in the retina. These three
enzymes share 95% identity in their 150 amino acid catalytic
core domains and are thus principally distinguished at the
amino acid level by their unique N-terminal domains [17]. We
have determined that these enzymes are further distinguished
by their apparent cellular distribution in the retina.
Specifically, UbcM2 appears to be the most highly expressed
of the three, followed by UbcM3 and then UBE2E2 (Figure
1B and Figure 3C,F,I). These expression patterns and levels
appear to be
established in young mice and sustained as the
mice age (Figure 3A-I). These conclusions are derived from
data generated with rigorously characterized affinity-purified
antibodies specific for each enzyme (Figure 1A). Of the three
enzymes, UbcM2 is also the only one we detected in the nuclei
of RPE cells and in photoreceptors (Figure 3B,C,F,I). This
relative enrichment of UbcM2 in the retina was further
demonstrated by an analysis of the enzyme’s distribution in
different mouse tissues and organs (Figure 2).
Despite the discovery of numerous E3 ligases that can
interact with UbcM2 [20,21,43], to date no substrates have
been identified; thus the function(s) of the enzyme remains an
open question. Previous work in the yeast S. cerevisiae
established that a pair of nearly identical E2s, referred to as
Ubc4/5, mediates the degradation of oxidatively damaged and
misfolded proteins [17,22-24]. Furthermore, exogenous
UbcM2 expression partially rescued the cold-sensitive growth
phenotype of a yeast strain lacking Ubc4/5 [17]. An
implication of these findings is that UbcM2 functions in
mammalian cells to ubiquitylate oxidatively damaged
and misfolded proteins and thereby targets these
potentially toxic species for destruction by the 26S
proteasome. We attempted to test this idea using a bright-light
stress model of photo-oxidative stress [37,40,41]. These
studies were done by comparing the toxicity of acute bright
light to photoreceptors
in mice harboring a single intact
UbcM2 allele versus their wild-type littermates. We were
unable to generate homozygous UbcM2-knockout mice for
these studies because complete loss of the enzyme is
embryonic lethal (unpublished data). Although inconvenient
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Figure 4. Mice harboring a single intact allele of UbcM2 are not more susceptible to light-induced retinal degeneration. A: Inactivation of a
single UbcM2 allele reduces expression of the enzyme by 58%. Equal amounts (10 μg) of retinal lysates derived from a UbcM2
+/−
mouse and
a wild-type (WT) littermate were subjected to denaturing and reducing sodium dodecyl sulfate PAGE (SDS–PAGE) followed by anti-UbcM2
western blotting. The migration of molecular weight markers is shown on the left. The asterisk denotes a nonspecific band serving as a loading
control. The graph depicts the relative level of expression of UbcM2 in WT versus heterozygous littermates (n=3 of each genotype) as
determined using a desktop scanner and Image J software. B: Representative hematoxylin and eosin (H&E) stained paraffin-embedded sections
are shown from mice (UbcM2
+/−
and WT littermates) 7 days after acute bright-light challenge. Control denotes animals that were maintained
in dim light for the entire experiment. Abbreviations of retinal cell layers are as described in the legend for Figure 3. The magnification bar
in panel A represents 50 μm. C: A Spider graph representing data compiled from the indicated number of animals for each experimental
condition. The error bars represent the standard deviation. Outer nuclear layer (ONL) rows are plotted along the y-axis with inferior and
superior distances in mm from the optic nerve head (ONH) depicted along the x-axis. There was no statistically significant difference in ONLs
between WT and UbcM2
+/−
control animals or between WT and UbcM2
+/−
light-damaged animals.
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Figure 5. Acute bright-light stress does not reduce the steady-state level of Ub-charged UbcM2 or the catalytic activity of the enzyme. A:
Lysates derived from the retinas of wt mice (lanes 1 and 2) and UbcM2
+/−
mice (lanes 3–7) maintained in dim light (lanes 1, 3, 4, 5) or exposed
to 3,000 lux for 6 h (lanes 2, 6, 7) were resolved by nonreducing (top blot) or reducing (bottom blot) sodium dodecyl sulfate PAGE (SDS–
PAGE) followed by anti-UbcM2 western blotting. Ub-charged enzyme is evident as a slower migrating band in nonreducing SDS–PAGE (top
blot, indicated by “UbcM2~Ub”). This band collapses to uncharged UbcM2 in samples exposed to reducing agent (bottom blot). Each lane
represents lysate from an individual mouse. B: Left blot— Shown in this blot is the enzyme used in the auto-ubiquitylation assay. The enzyme
was immunoprecipitated from UbcM2+/− retinal lysates. Each lane corresponds to lysate from an individual mouse. Lane 1 contains antibody
(Ab) only to distinguish bands derived from the Ab versus those IPed by the Ab. Right blot—IPed UbcM2 was combined with recombinant
E1, Ub, and energy and