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SOD1 deficiency decreases proteasomal function, leading to the accumulation of ubiquitinated proteins in erythrocytes
Accepted Manuscript SOD1 deficiency decreases proteasomal function, leading to the accumulation of ubiquitinated proteins in erythrocytes. Takujiro Homma, Toshihiro Kurahashi, Jaeyong Lee, Eun Sil Kang, Junichi Fujii PII:
S0003-9861(15)30023-0
DOI:
10.1016/j.abb.2015.07.023
Reference:
YABBI 7038
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 25 May 2015 Revised Date:
16 July 2015
Accepted Date: 27 July 2015
Please cite this article as: T. Homma, T. Kurahashi, J. Lee, E.S. Kang, J. Fujii, SOD1 deficiency decreases proteasomal function, leading to the accumulation of ubiquitinated proteins in erythrocytes., Archives of Biochemistry and Biophysics (2015), doi: 10.1016/j.abb.2015.07.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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SOD1 deficiency decreases proteasomal function, leading to the accumulation of
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ubiquitinated proteins in erythrocytes.
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3 Takujiro Homma, Toshihiro Kurahashi, Jaeyong Lee, Eun Sil Kang and Junichi Fujii*
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Department of Biochemistry and Molecular Biology, Graduate School of Medical
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Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan
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Running title: Elevated protein aggregation in SOD1-deficient red blood cells
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*Correspondence author: Junichi Fujii
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e-mail: [email protected]
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Tel. +81-23-628-5229; Fax +81-23-628-5230
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Abstract
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We previously demonstrated that elevated levels of ROS in red blood cells (RBCs) are
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responsible for anemia in SOD1-deficient mice, suggesting that the oxidative
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stress-induced massive destruction of RBCs is an underlying mechanism for
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autoimmune hemolytic anemia. In the current study, we examined the issue of how
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elevated ROS are involved in the destruction of RBCs and the onset of anemia from the
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view point of the proteolytic removal of oxidatively-damaged proteins. We found that
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poly-ubiquitinated proteins had accumulated and had undergone aggregation in RBCs
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from SOD1-deficient mice and from phenylhydrazine-induced anemic mice. Although
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the protein levels of the three catalytic components of the proteasome, β1, β2, and β5,
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were not significantly altered, their proteolytic activities were decreased in the
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SOD1-deficient RBCs. These data suggest that oxidative-stress triggers the dysfunction
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of the proteasomal system, which results in the accumulation of the aggregation of
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poly-ubiquitinated proteins. We conclude that an oxidative stress-induced malfunction
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in the scavenging activity of proteasomes accelerates the accumulation of damaged
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proteins, leading to a shortened life span of RBCs and, hence, anemia.
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Key words
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SOD1, oxidative stress, proteasome, anemia, protein degradation
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Introduction Reactive oxygen species (ROS) cause cellular damage by oxidizing lipids, proteins,
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and nucleic acids and are believed to be major contributors to aging and various
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diseases [1]. Disulfide bond and carbonyl adduct formation are typically observed in
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oxidized proteins [2]. The accumulation of oxidized proteins represents a hallmark of
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cellular aging and has been proposed to cause a disturbance in cellular homeostasis
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[3,4].
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The accumulation of oxidatively-damaged proteins dramatically accelerate the
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development of senescence in anucleate cells such as red blood cells (RBCs) and
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eventually leads to cell death. Indeed, elevated levels of ROS in a peroxiredoxin 2
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(Prx2) or SOD1 deficiency cause the lifespan of RBCs to be shortened, resulting in the
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development of anemia [5–7]. It appears that the continuous oxidative destruction of
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RBCs then leads to an aberrant immune response to RBCs, which results in
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autoimmune hemolytic anemia (AIHA) [8]. The hypothetical role of oxidative stress in
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the onset of AIHA are supported by findings that ROS levels are elevated in RBCs from
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AIHA-prone NZB mice and coincide with AIHA onset [9]. The overexpression of
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SOD1 in erythroid cells improved the phenotypes not only in SOD1-deficient NZB
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congenic mice but also in the mice possessing intrinsic SOD1 [10]. While a causal
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connection between oxidative stress and anemia is becoming evident, the issue of
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specifically how elevated ROS leads to the destruction of RBCs remains unclear.
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During periods of oxidative stress, protein oxidation is accelerated and becomes a
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threat to cell survival. Proteasomes play a key role in maintaining cellular homeostasis
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by the proteolytic removal of damaged proteins and abnormal proteins [11].
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Proteasomes are present in two forms, 20S and 26S, in cells. The 20S core proteasome
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contains three main catalytic activities: caspase-like, trypsin-like, and chymotrypsin-like
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activity, catalyzed by β1, β2, and β5 subunits, respectively, which cleave the peptide
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bond at the carboxyl end of acidic residues, basic residues, and hydrophobic residues,
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respectively [12]. The 26S proteasome, which is capped at one or both ends of the 20S
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proteasome by a 19S regulatory component composed of multiple ATPases which are
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necessary for unfolding the protein substrates, degrades ubiquitinated proteins in an
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ATP-dependent manner [13]. The 20S proteasome alone degrades non-ubiquitinated
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proteins without consuming ATP.
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Proteasomes are also involved in the removal of the oxidized proteins unless they
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are in heavily aggregated or cross-linked forms [14]. After proteolytic degradation,
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nucleated cells can replace the lost proteins with newly synthesized ones by gene
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expression. However sustained oxidative stress accelerates the production of damaged
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proteins, which eventually exceeds proteasomal removal capacity, leading to their
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accumulation in the cells [15,16]. Independent of oxidative stress, the age-related
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impairment of proteasome function has also been reported in several cell types or
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organs [17–19]. Since the proteasome is essential for removing abnormal proteins, a
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functional impairment can cause serious damage to long-lived cells with low
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regenerating ability, such as neurons. Notably, dysfunction of the ubiquitin-proteasome
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system has been implicated in the development of neurodegenerative disorders such as
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Parkinson's disease [20], Alzheimer's disease [21], and prion diseases [22,23]. Their
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build-up promotes a vicious cycle in which the proteasome becomes further impaired.
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In the current study, we report on attempts to elucidate the mechanism
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responsible for how oxidative stress causes the destruction of RBCs from the view point
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of the elimination of damaged proteins. The findings reported herein suggest that
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proteasome function becomes impaired as the result of oxidative stress. This, in turn,
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results in the accumulation of protein aggregates in RBC, thus leading to their early
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destruction.
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85 Materials and methods
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Antibodies
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Anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-β-actin (Santa Cruz
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Biotechnology, sc-69879), anti-Hemoglobin alpha (Proteintech, 14537-1-AP), anti-β1
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(Enzo Life Sciences, BML-PW8140), anti-β2 (Enzo Life Sciences, BML-PW9300),
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anti-β5 (Enzo Life Sciences, BML-PW8895), and anti-19S (Enzo Life Sciences,
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BML-PW8830)
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peroxidase-conjugated anti-mouse (sc-2005) and anti-rabbit (sc-2004) IgG antibodies
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were purchased from Santa Cruz Biotechnology. The anti-SOD1 antibody has been
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described elsewhere [6].
purchased
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from
the
indicated
vendors.
Horseradish
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were
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Mice
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C57BL/6 SOD1 hetero-knockout mice, originally established by Matzuk [24], were
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purchased from Jackson Laboratories (Bar Harbor, ME) and backcrossed more than 10
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times with C57BL/6 males. Genotypic analysis of the mice was performed using PCR
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with specific primers, and SOD1-competent (WT) and SOD1-deficient mice were used
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in this study. The animal room was maintained under specific pathogen-free conditions
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at a constant temperature of 20-22 ºC with a 12 h alternating light-dark cycle. Animal
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experiments were performed in accordance with the Declaration of Helsinki under the
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protocol approved by the Animal Research Committee at Yamagata University.
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Immunoblotting
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RBCs collected from WT and SOD1-deficient mice were washed three times with
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phosphate buffered saline (PBS), and lysed in 20 mM Tris-HCl (pH 7.5). The lysate was
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centrifuged at 15,000 rpm for 10 min in a microcentrifuge. After centrifugation, the
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supernatant (soluble fraction) was collected and protein concentrations were determined
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using Pierce® BCA™ Protein Assay Kit (Pierce). The pellet was washed twice with
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PBS and re-suspended in SDS-sample buffer (insoluble fraction). The proteins were
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separated on 12% or 15% SDS–polyacrylamide gels and blotted onto polyvinylidene
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difluoride (PVDF) membranes (GE Healthcare). The blots were blocked with 5% skim
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milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and were then
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incubated overnight with the primary antibodies diluted in TBST containing 1% skim
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milk. After three washings with TBST, the blots were incubated with horseradish
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peroxidase-conjugated second antibody. After washing, the bands were detected using
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Immobilon western chemiluminescent HRP substrate (Millipore) on an image analyzer
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(ImageQuant LAS500, GE Healthcare).
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122 Culture of RBCs
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RBCs were collected from WT and SOD1-deficient mice through the tail vein, washed
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three times with PBS, and suspended in DMEM (Wako, 044-29765) supplemented with
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110 mg/l sodium pyruvate, 0.1% BSA, 100 U/ml penicillin, and 100 mg/ml
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streptomycin, and dispensed into 1.5 ml tubes at a density of 1.0 × 107 cells/50 µl; they
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were maintained in a CO2 incubator (5% CO2, 95% air, 37 °C). For superoxide
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generation, RBCs were treated with various doses (5, 10, 25, 50, 100 µM) of menadione
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sodium bisulfite (Sigma, M-5750) for 1 h in the CO2 incubator.
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Proteasomal activity assays
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The proteasomal activities in RBCs were determined by fluorescence assays as
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described previously [25]. Briefly, proteins were extracted from the RBCs with 20 mM
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Tris-HCl (pH 7.5). The lysates were then centrifuged at 15,000 rpm for 10 min, the
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protein concentration in the supernatants were measured. From each RBC sample, 50
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µg of total protein was incubated with 200 µM of proteasome substrate
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z-Leu-Leu-Glu-MCA
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trypsin-like activity), or Suc-Leu-Leu-Val-Tyr-MCA (for chymotrypsin-like activity) in
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100 µl assay buffer (20 mM Tris–HCl, pH 7.5) at 37°C. All substrates were purchased
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from Peptide Institute, Inc. (Osaka, Japan). After incubation, the fluorescence intensity
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was measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific)
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with an excitation filter of 365 nm and an emission filter of 460 nm.
caspase-like
activity),
Boc-Leu-Arg-Arg-MCA
(for
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(for
Phenylhydrazine-induced hemolytic anemia
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Hemolytic anemia was induced by an administration of phenylhydrazine (PHZ). At day
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0, WT mice (12 weeks old) were randomly divided into the two groups, the PBS group
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and the PHZ group (n=3 in each group). A solution of PHZ in PBS was injected
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intraperitoneally at a dose of 60 mg/kg on day 0 and day 1. Blood was collected from
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the tail vein prior to the administration and on day 2. Hematological analyses were
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performed using an automated hematology analyzer (Celltac-α Nihon Kohden, Tokyo,
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Japan).
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153 Statistical analysis
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Statistical analysis was performed using GraphPad Prism 4 software.
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Results
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Elevated ubiquitinated proteins and accelerated aggregation in SOD1-deficient
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RBCs
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To investigate the degradation of ubiquitinated proteins by proteasomes, we
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isolated RBCs from WT and SOD1 KO mice and subjected the lysates to detection of
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ubiquitinated proteins. We analyzed the soluble and insoluble fractions of the RBC
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lysate separately. The levels of ubiquitinated proteins were elevated in both the soluble
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and insoluble fractions from SOD1-deficient RBCs compared to WT RBCs (Fig. 1).
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The unibiquitinated proteins in the insoluble fraction (Fig. 1B) were more abundant in
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the high molecular weight range compared to those in the soluble fraction (Fig. 1A),
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which is consistent with the fact that poly-ubiquitinated proteins are sparingly soluble.
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We next estimated the change in the ubiquitinated proteins in RBCs under
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cultural conditions. We harvested RBCs at 0, 24, and 48 h after incubation and
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examined the levels of ubiquitinated protein in the soluble and insoluble fractions by
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immunoblotting. During the incubation periods, the level of ubiquitinated proteins in
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the soluble fraction gradually decreased (Fig. 2A), but those in the insoluble fraction
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concomitantly increased, and this was more pronounced for proteins in the high
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molecular weight range (Fig. 2B). The transition of the ubiquitinated proteins from the
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soluble to the insoluble fraction was more prominent in SOD1-deficient RBCs than WT
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RBCs. Thus, an SOD1 deficiency enhanced the ubiquitination of proteins, which
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resulted in the accelerated formation of protein aggregates in RBCs.
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The proteasomal activities decreased in RBCs under SOD1 deficiency
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To elucidate the reason for the elevated ubiquitination in the SOD1-deficient
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RBCs, we analyzed the protein levels of major subunits in proteasomes by
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immunoblotting. Unexpectedly, however, no differences were observed in the three
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catalytic subunits β1, β2, and β5 for the 20S component and a subunit for the 19S
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component of proteasomes between the WT and SOD1-deficient RBCs (Fig. 3).
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Since oxidation can affect protein function without changing protein levels via the
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oxidative modification of catalytic amino acid side chains, we assessed the caspase-like
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(β1), trypsin-like (β2), and chymotrypsin-like (β5) activities of three main catalytic
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subunits of the proteasome in the RBC lysates. As a result, all three proteolytic activities 8
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were markedly decreased in SOD1-deficient RBCs compared to WT RBCs (Table 1). To confirm that oxidative stress actually affected proteasomal function in vitro,
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we incubated WT RBCs with various doses of superoxide-producing pro-oxidant
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menadione and then subjected the RBC proteins to immunoblotting and the proteolytic
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activity assay. Immunoblotting revealed that the levels of ubiquitinated proteins in the
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soluble fraction increased at lower doses of menadione but rather decreased at higher
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doses (Fig. 4A). In case of the insoluble fraction, the levels of ubiquitinated proteins
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increased in the menadione-dose dependent manner (Fig. 4B), suggesting the transition
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of the polyubiquitinated proteins from the soluble fraction to the insoluble fraction. All
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three proteolytic activities of the proteasomal components were significantly decreased
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as the result of the menadione treatment (Supplementary Fig. 1).
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Robust accumulation of ubiquitinated proteins and α-globin cross-linked proteins
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in RBCs of experimental hemolytic anemia
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To evaluate involvement of the accumulation of ubiquitinated proteins in hemolytic
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anemia, we next challenged WT mice with phenylhydrazine (PHZ) to induce hemolytic
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anemia. The PHZ treatment resulted in a significant decrease in the RBC counts
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(549±51 vs 1000±28 104/µL, p<0.001), HCT (24.6±2.2 vs 49.3±1.2 %, p<0.001), MCV
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(44.8±0.2 vs 49.3±0.1 fL, p<0.01) and increased the WBC counts (831±31 vs 76±2
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102/µL, p<0.001) and MCH (25.3±1.2 vs 14.5±0.1 pg, p<0.001). The PBS treatment had
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no effect on hematological parameters (data not shown).
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We then examined the levels of ubiquitinated proteins in both the soluble and
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insoluble RBC fractions by immunoblotting. While the administration of PHZ resulted
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in a decreased level of ubiquitinated proteins in the soluble fraction, the levels of
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ubiquitinated proteins in the insoluble fraction were markedly elevated compared with
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the PBS treatment (Fig. 5). PHZ causes oxidative stress within RBCs and results in the
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oxidation of hemoglobin, leading to the formation of inclusions called Heinz bodies
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[26]. When we applied an α-globin antibody to the immunoblotting, an accumulation of
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proteins that were cross-linked with α-globin were found only in the insoluble fraction
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of RBCs from the PHZ-administered mice. In addition, all three proteolytic proteasome
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activities were markedly decreased in RBCs from the PHZ-administered mice compared
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to control RBCs (Table 2).
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221 Discussion
In the current study, we show that ubiquitinated proteins had accumulated in
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RBCs from SOD1-deficient mice (Figs. 2 and 3), which is consistent with the
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observations that oxidative stress induces the oxidative modification and ubiquitination
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of proteins [27,28]. The findings also indicate that ROS inhibits these proteasomal
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activities, without affecting the overall protein levels (Fig. 4 and Table 1). Since the
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dysfunction of the proteasome system would further enhance the accumulation of
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damaged proteins, this oxidative damage in the proteasome system would be expected
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to trigger a vicious cycle and consequently induce the destruction of RBCs, which leads
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to anemia in SOD1-deficient mice.
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Among the proteasome components, the 26S proteasome, which is involved in the
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ATP-dependent degradation of ubiqutinated proteins, is much more susceptible to
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oxidative stress than the 20S proteasome [14,29,30]. The 26S proteasome is
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disassembled into a 20S core proteasome and a 19S regulatory component by mild
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oxidative stress. Txnl1/TRP32, a member of the thioredoxin family of proteins, binds to
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a subunit Rpn11 of the 19S proteasome and may be involved in the redox-sensitive
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conversion of proteasomes [31]. In terms of substrate specificity, the 20S proteasome
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selectively recognizes and degrades oxidized proteins, while the 26S proteasome, even in
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the presence of ATP, is not very effective in eliminating oxidized proteins [14]. No
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changes were detected in a subunit for the 19S proteasome component between WT and
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SOD1-deficient RBCs (Fig. 4), which suggests that the oxidative stress-induced
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conversion from the 26S to the 20S proteasome is not likely the mechanism responsible
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for the accumulation of the ubiquitinated proteins. The RBC contains about 10 times
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more 20S proteasomes than 26S proteasomes although the cellular concentrations of
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proteasomes in RBCs is much lower compared to nucleated cells [32]. Continuous
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oxidative stress, which is caused by the release of ROS from hemoglobin autoxidation,
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appears to be involved in the function of 26S and 20S proteasomes in RBCs.
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We observed that the size and amounts of several proteasomal components were
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also not changed in SOD1-deficient RBCs (Fig. 4). In cultured cells, the nuclear
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factor-erythroid 2-related factor-2 (Nrf2) is involved in proteasomal gene expression in
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response to oxidative stress [33–35]. It has also been shown that immunoproteasomes,
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which contain all three IFN-γ–inducible catalytic β subunits in the same 20S complex
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and are thought to be more resistant to oxidants, are up-regulated upon oxidative stress
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and contribute to the removal of oxidized proteins [36–38]. Thus proteasomal activity
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can be elevated under conditions of moderate oxidative stress and protein homeostasis
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can be maintained by responding to the oxidative insult. Contrary to nucleated cells,
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RBCs lack nuclei and hence are incapable of replacing damaged proteins, thereby
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oxidative stress easily entraps RBCs into a vicious cycle which faces severe oxygen
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toxicity compared with other tissues.
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The age-related decrease in proteasomal activity appears to be due to
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post-translational modifications, since the amount of the proteases remained unchanged
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while their activity was significantly decreased [39,40]. We have also demonstrated a
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decreased proteasomal function in SOD1-deficient RBCs (Table 1). A significant
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decline in proteasome activity upon severe oxidative stress has been reported in cultured
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cells [29,30]. Moreover, the treatment of mice with a superoxide generator, paraquat,
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reportedly induces the accumulation of ubiquitinated proteins [41]. Among the
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proteolytic activities in proteasomes, caspase-like activity is the most profoundly
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affected during aging [17–19]. Caspase-like proteasomal activities in livers from old
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male rats are approximately 50% lower than those from young male rats [18]. Although
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the trypsin-like activity of the proteasome is also sensitive to oxidation, the heat shock
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protein 90 appears to protect the activity by direct interactions in aged rats [18].
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While severe oxidative stress causes extensive protein oxidation, which includes
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direct fragmentation, cross-linking, and the aggregation of proteins, massively
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aggregated proteins become progressively resistant to proteasomal degradation,
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ultimately causing dysfunction. PHZ is a hemolytic agent that interacts with hemoglobin
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to generate superoxide [42,43] and, in hemoglobin, would be expected to cause oxidative
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damage, such as fragmentation [44], leading to the formation of Heinz bodies. We
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challenged mice with PHZ to induce hemolytic anemia, and found that highly
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ubiquitinated proteins and cross-linked hemoglobin accumulated in RBCs (Fig. 5).
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These heavily oxidized and cross-linked proteins, especially large aggregates, actually
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bind irreversibly to proteasomes, resulting in inhibition [45–47], leading to a vicious
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cycle with progressive defects in protein degradation. Thus, it is likely that oxidative
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insults to RBCs trigger the accumulation of ubiquitinated proteins, which consequently
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disturbs proteasome activity, thus leading to the destruction of RBC. Since SOD1 plays
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an essential role in eliminating superoxide, the deficiency would enhance oxygen
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toxicity and reduce the lifespan of RBCs to 60% of the WT RBCs [6].
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In conclusion, the catalytic activities of proteasomes are susceptible to oxidative
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inactivation, which results in the accumulation of ubiquitinated proteins in
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SOD1-deficient RBCs. Since these oxidatively-damaged proteins tend to form large
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aggregates, which in turn leads to the dysfunction of proteasomal function. Thus, the
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fatal vicious cycle is triggered by an SOD1 deficiency and disturbs protein homeostasis,
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which would render RBCs to be more susceptible to oxidative modification, shorten
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their lifespan, ultimately resulting in the development of anemia.
295 Acknowledgments
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J.L. is a scholarship recipient from the Otsuka Toshimi Scholarship Foundation.
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E.S.K. is supported by the Tokyo Biochemical Research Foundation Postdoctoral
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Fellowship for Asian Researchers in Japan. This work was supported by the Strategic
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Young Researcher Overseas Visits Program for Accelerating Brain Circulation from the
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Japan Society for the Promotion of Sciences (S2402).
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liver multicatalytic proteinase activity and protection from oxidative inactivation by heat-shock protein 90. Arch. Biochem. Biophys. 331: 232–240; 1996. Conconi, M.; Friguet, B. Proteasome inactivation upon aging and on oxidation-effect of HSP 90. Mol. Biol. Rep. 24: 45–50; 1997.
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Kapeta, S.; Chondrogianni, N.; Gonos, E. S. Nuclear erythroid factor 2-mediated proteasome activation delays senescence in human fibroblasts. J. Biol. Chem. 285: 8171–8184; 2010. Takabe, W.; Matsukawa, N.; Kodama, T.; Tanaka, K.; Noguchi, N. Chemical structure-dependent gene expression of proteasome subunits via regulation of the antioxidant response element. Free
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Pickering, A. M.; Koop, A. L.; Teoh, C. Y.; Ermak, G.; Grune, T.; Davies, K. J. a. The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem. J. 432: 585–594; 2010.
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Ding, Q.; Reinacker, K.; Dimayuga, E.; Nukala, V.; Drake, J.; Butterfield, D. A.; Dunn, J. C.; Martin, S.; Bruce-Keller, A. J.; Keller, J. N. Role of the proteasome in protein oxidation and
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neural viability following low-level oxidative stress. FEBS Lett. 546: 228–232; 2003. Hussong, S. a.; Kapphahn, R. J.; Phillips, S. L.; Maldonado, M.; Ferrington, D. a.
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Shibatani, T.; Nazir, M.; Ward, W. F. Alteration of rat liver 20S proteasome activities by age and
food restriction. J. Gerontol. A. Biol. Sci. Med. Sci. 51: B316–B322; 1996. Hayashi, T.; Goto, S. Age-related changes in the 20S and 26S proteasome activities in the liver of male F344 rats. Mech. Ageing Dev. 102: 55–66; 1998.
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Damage Princ. Pract. Appl. 2010. Goldberg, B.; Stern, A. The generation of O2 - by the interaction of the hemolytic agent,
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phenylhydrazine, with human hemoglobin. J. Biol. Chem. 1975. p. 2401–2403. Goldberg, B.; Stern, a.; Peisach, J. The mechanism of superoxide anion generation by the interaction of phenylhydrazine with hemoglobin. J. Biol. Chem. 251: 3045–3051; 1976.
Di Cola, D.; Battista, P.; Santarone, S.; Sacchetta, P. Fragmentation of human hemoglobin by
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oxidative stress produced by phenylhydrazine. Free Radic. Res. Commun. 6: 379–386; 1989. Friguet, B.; Stadtman, E. R.; Szweda, L. I. Modification of glucose-6-phosphate dehydrogenase
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by 4-hydroxy-2-nonenal. Formation of cross-linked protein that inhibits the multicatalytic protease. J. Biol. Chem. 269: 21639–21643; 1994.
Sitte, N.; Huber, M.; Grune, T.; Ladhoff, a; Doecke, W. D.; Von Zglinicki, T.; Davies, K. J. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts. FASEB J. 14: 1490–1498; 2000.
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Shringarpure, R.; Grune, T.; Sitte, N.; Davies, K. J. 4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer’s disease. Cell. Mol. Life Sci. 57: 1802–1809; 2000.
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Oxidative Damage: Principles and Practical Applications. Biomarkers Antioxid. Def. Oxidative
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Figure legends
437 Fig. 1. Levels of ubiquitinated proteins in WT and SOD1-deficient RBCs.
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The representative immunoblots of WT and SOD1-deficient (KO) RBCs probed with
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antibodies against ubiquitin (Ub), β-actin, and SOD1 in soluble (A) or insoluble (B)
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fraction are shown (n=3 for each group).
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Fig. 2. Levels of ubiquitinated proteins in WT and SOD1-deficient RBCs under culture
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conditions.
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Proteins were extracted from WT and SOD1-deficient (KO) RBCs at 0, 24, and 48 h
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after isolation and subjected to immunoblotting. The representative immunoblots
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probed with antibodies against ubiquitin (Ub), β-actin, and SOD1 in soluble (A) or
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insoluble (B) fraction are shown (n=3 for each group).
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Fig. 3. Levels of proteasome subunits in WT and SOD1-deficient RBCs.
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The representative immunoblots of WT and SOD1-deficient (KO) RBCs probed with
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antibodies against the β1 (A), β2 (B), β5 (C), and 19S (D) subunits of the proteasome
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are shown. Bottom panels depict the quantification of each proteins normalized to the
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corresponding β-actin. Values are the mean ± SD of mice (n=3 for each group). ns, not
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significant.
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Fig. 4. Levels of ubiquitinated proteins in menadione-treated RBCs.
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WT RBCs were treated with the indicated doses of menadione (Men). The
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representative immunoblots probed with antibodies against ubiquitin (Ub) and β-actin in
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the soluble (A) or insoluble (B) fraction are shown.
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Fig. 5. Levels of ubiquitinated proteins in WT RBCs during hemolytic anemia.
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RBCs from WT mice treated with PBS or phenylhydrazine (PHZ) were collected and
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subjected to immunoblotting. The representative immunoblots probed with antibodies
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against ubiquitin (Ub), β-actin, and α-globin (Hb) in soluble (A) or insoluble (B)
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fraction are shown (n=3 for each group).
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468 Table 1. The proteasomal activities in WT and SOD1-deficient RBCs.
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WT and SOD1-deficient (KO) RBCs were collected, and caspase-like, trypsin-like, and
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chymotrypsin-like activities were monitored. Values represent the mean ± SD of three
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mice per group. Statistical analysis was performed by Student t-test. The asterisks
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denote a significant difference between WT and KO RBCs. *, P < 0.05; ***, P< 0.001.
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FU: Fluorescence unit.
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Caspase-like activity Trypsin-like activity
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Chymotrypsin-like activity
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WT
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3.37 ± 0.17
2.04 ± 0.21***
5.77 ± 0.40
4.85 ± 0.30*
3.92 ± 0.40
3.28 ± 0.46* (FU/h/µg protein)
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477 Table 2. The proteasomal activities in PHZ-administered and control RBCs.
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RBCs from WT mice treated with PBS or phenylhydrazine (PHZ) were collected, and
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caspase-like, trypsin-like, and chymotrypsin-like activities were monitored. Values
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represent the mean ± SD of three mice per group. Statistical analysis was performed by
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Student t-test. The asterisks denote a significant difference between PBS and PHZ. **,
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P< 0.01. FU: Fluorescence unit.
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Caspase-like activity Trypsin-like activity
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Chymotrypsin-like activity
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PBS
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PHZ
2.38 ± 0.27
0.35 ± 0.40**
2.69 ± 0.20
0.60 ± 0.62**
3.40 ± 0.54
0.59 ± 0.49** (FU/h/µg protein)
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Highlights
Poly-ubiquitinated proteins accumulated in RBCs from SOD1-deficient mice 5
Poly-ubiquitinated proteins accumulated also in RBCs of phenylhydrazine-treated mice.
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Proteolytic activities of the proteasome were decreased in the SOD1-deficient RBCs. An oxidative stress-induced malfunction of proteasomes may be a cause for anemia.
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Supplementary Fig. 1
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**
** **
** **
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Men (μM)
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Relative activity (%)
Caspase-like activity
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Relative activity (%)
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Relative activity (%)
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Chymotrypsin-like activity
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** **
Men (μM)
Supplementary Fig. 1. The proteasomal activities in menadione-treated RBCs. RBCs were treated with the indicated doses of menadione (Men) and subjected to proteolytic activity assays. Data are expressed as the mean ± SD (n=3 for each group). Statistical analysis was performed by one-way ANOVA with Dunnett's post hoc tests. **, P < 0.01.
S0003-9861(15)30023-0
DOI:
10.1016/j.abb.2015.07.023
Reference:
YABBI 7038
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 25 May 2015 Revised Date:
16 July 2015
Accepted Date: 27 July 2015
Please cite this article as: T. Homma, T. Kurahashi, J. Lee, E.S. Kang, J. Fujii, SOD1 deficiency decreases proteasomal function, leading to the accumulation of ubiquitinated proteins in erythrocytes., Archives of Biochemistry and Biophysics (2015), doi: 10.1016/j.abb.2015.07.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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SOD1 deficiency decreases proteasomal function, leading to the accumulation of
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ubiquitinated proteins in erythrocytes.
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3 Takujiro Homma, Toshihiro Kurahashi, Jaeyong Lee, Eun Sil Kang and Junichi Fujii*
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Department of Biochemistry and Molecular Biology, Graduate School of Medical
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Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan
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Running title: Elevated protein aggregation in SOD1-deficient red blood cells
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*Correspondence author: Junichi Fujii
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e-mail: [email protected]
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Tel. +81-23-628-5229; Fax +81-23-628-5230
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Abstract
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We previously demonstrated that elevated levels of ROS in red blood cells (RBCs) are
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responsible for anemia in SOD1-deficient mice, suggesting that the oxidative
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stress-induced massive destruction of RBCs is an underlying mechanism for
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autoimmune hemolytic anemia. In the current study, we examined the issue of how
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elevated ROS are involved in the destruction of RBCs and the onset of anemia from the
20
view point of the proteolytic removal of oxidatively-damaged proteins. We found that
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poly-ubiquitinated proteins had accumulated and had undergone aggregation in RBCs
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from SOD1-deficient mice and from phenylhydrazine-induced anemic mice. Although
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the protein levels of the three catalytic components of the proteasome, β1, β2, and β5,
24
were not significantly altered, their proteolytic activities were decreased in the
25
SOD1-deficient RBCs. These data suggest that oxidative-stress triggers the dysfunction
26
of the proteasomal system, which results in the accumulation of the aggregation of
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poly-ubiquitinated proteins. We conclude that an oxidative stress-induced malfunction
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in the scavenging activity of proteasomes accelerates the accumulation of damaged
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proteins, leading to a shortened life span of RBCs and, hence, anemia.
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Key words
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SOD1, oxidative stress, proteasome, anemia, protein degradation
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Introduction Reactive oxygen species (ROS) cause cellular damage by oxidizing lipids, proteins,
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and nucleic acids and are believed to be major contributors to aging and various
36
diseases [1]. Disulfide bond and carbonyl adduct formation are typically observed in
37
oxidized proteins [2]. The accumulation of oxidized proteins represents a hallmark of
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cellular aging and has been proposed to cause a disturbance in cellular homeostasis
39
[3,4].
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The accumulation of oxidatively-damaged proteins dramatically accelerate the
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development of senescence in anucleate cells such as red blood cells (RBCs) and
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eventually leads to cell death. Indeed, elevated levels of ROS in a peroxiredoxin 2
43
(Prx2) or SOD1 deficiency cause the lifespan of RBCs to be shortened, resulting in the
44
development of anemia [5–7]. It appears that the continuous oxidative destruction of
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RBCs then leads to an aberrant immune response to RBCs, which results in
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autoimmune hemolytic anemia (AIHA) [8]. The hypothetical role of oxidative stress in
47
the onset of AIHA are supported by findings that ROS levels are elevated in RBCs from
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AIHA-prone NZB mice and coincide with AIHA onset [9]. The overexpression of
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SOD1 in erythroid cells improved the phenotypes not only in SOD1-deficient NZB
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congenic mice but also in the mice possessing intrinsic SOD1 [10]. While a causal
51
connection between oxidative stress and anemia is becoming evident, the issue of
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specifically how elevated ROS leads to the destruction of RBCs remains unclear.
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During periods of oxidative stress, protein oxidation is accelerated and becomes a
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threat to cell survival. Proteasomes play a key role in maintaining cellular homeostasis
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by the proteolytic removal of damaged proteins and abnormal proteins [11].
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Proteasomes are present in two forms, 20S and 26S, in cells. The 20S core proteasome
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contains three main catalytic activities: caspase-like, trypsin-like, and chymotrypsin-like
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activity, catalyzed by β1, β2, and β5 subunits, respectively, which cleave the peptide
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bond at the carboxyl end of acidic residues, basic residues, and hydrophobic residues,
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respectively [12]. The 26S proteasome, which is capped at one or both ends of the 20S
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proteasome by a 19S regulatory component composed of multiple ATPases which are
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necessary for unfolding the protein substrates, degrades ubiquitinated proteins in an
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ATP-dependent manner [13]. The 20S proteasome alone degrades non-ubiquitinated
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proteins without consuming ATP.
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Proteasomes are also involved in the removal of the oxidized proteins unless they
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are in heavily aggregated or cross-linked forms [14]. After proteolytic degradation,
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nucleated cells can replace the lost proteins with newly synthesized ones by gene
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expression. However sustained oxidative stress accelerates the production of damaged
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proteins, which eventually exceeds proteasomal removal capacity, leading to their
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accumulation in the cells [15,16]. Independent of oxidative stress, the age-related
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impairment of proteasome function has also been reported in several cell types or
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organs [17–19]. Since the proteasome is essential for removing abnormal proteins, a
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functional impairment can cause serious damage to long-lived cells with low
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regenerating ability, such as neurons. Notably, dysfunction of the ubiquitin-proteasome
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system has been implicated in the development of neurodegenerative disorders such as
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Parkinson's disease [20], Alzheimer's disease [21], and prion diseases [22,23]. Their
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build-up promotes a vicious cycle in which the proteasome becomes further impaired.
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In the current study, we report on attempts to elucidate the mechanism
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responsible for how oxidative stress causes the destruction of RBCs from the view point
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of the elimination of damaged proteins. The findings reported herein suggest that
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proteasome function becomes impaired as the result of oxidative stress. This, in turn,
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results in the accumulation of protein aggregates in RBC, thus leading to their early
83
destruction.
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85 Materials and methods
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Antibodies
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Anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-β-actin (Santa Cruz
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Biotechnology, sc-69879), anti-Hemoglobin alpha (Proteintech, 14537-1-AP), anti-β1
90
(Enzo Life Sciences, BML-PW8140), anti-β2 (Enzo Life Sciences, BML-PW9300),
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anti-β5 (Enzo Life Sciences, BML-PW8895), and anti-19S (Enzo Life Sciences,
92
BML-PW8830)
93
peroxidase-conjugated anti-mouse (sc-2005) and anti-rabbit (sc-2004) IgG antibodies
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were purchased from Santa Cruz Biotechnology. The anti-SOD1 antibody has been
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described elsewhere [6].
purchased
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from
the
indicated
vendors.
Horseradish
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were
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Mice
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C57BL/6 SOD1 hetero-knockout mice, originally established by Matzuk [24], were
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purchased from Jackson Laboratories (Bar Harbor, ME) and backcrossed more than 10
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times with C57BL/6 males. Genotypic analysis of the mice was performed using PCR
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with specific primers, and SOD1-competent (WT) and SOD1-deficient mice were used
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in this study. The animal room was maintained under specific pathogen-free conditions
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at a constant temperature of 20-22 ºC with a 12 h alternating light-dark cycle. Animal
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experiments were performed in accordance with the Declaration of Helsinki under the
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protocol approved by the Animal Research Committee at Yamagata University.
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Immunoblotting
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RBCs collected from WT and SOD1-deficient mice were washed three times with
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phosphate buffered saline (PBS), and lysed in 20 mM Tris-HCl (pH 7.5). The lysate was
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centrifuged at 15,000 rpm for 10 min in a microcentrifuge. After centrifugation, the
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supernatant (soluble fraction) was collected and protein concentrations were determined
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using Pierce® BCA™ Protein Assay Kit (Pierce). The pellet was washed twice with
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PBS and re-suspended in SDS-sample buffer (insoluble fraction). The proteins were
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separated on 12% or 15% SDS–polyacrylamide gels and blotted onto polyvinylidene
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difluoride (PVDF) membranes (GE Healthcare). The blots were blocked with 5% skim
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milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and were then
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incubated overnight with the primary antibodies diluted in TBST containing 1% skim
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milk. After three washings with TBST, the blots were incubated with horseradish
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peroxidase-conjugated second antibody. After washing, the bands were detected using
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Immobilon western chemiluminescent HRP substrate (Millipore) on an image analyzer
121
(ImageQuant LAS500, GE Healthcare).
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122 Culture of RBCs
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RBCs were collected from WT and SOD1-deficient mice through the tail vein, washed
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three times with PBS, and suspended in DMEM (Wako, 044-29765) supplemented with
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110 mg/l sodium pyruvate, 0.1% BSA, 100 U/ml penicillin, and 100 mg/ml
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streptomycin, and dispensed into 1.5 ml tubes at a density of 1.0 × 107 cells/50 µl; they
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were maintained in a CO2 incubator (5% CO2, 95% air, 37 °C). For superoxide
129
generation, RBCs were treated with various doses (5, 10, 25, 50, 100 µM) of menadione
130
sodium bisulfite (Sigma, M-5750) for 1 h in the CO2 incubator.
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Proteasomal activity assays
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The proteasomal activities in RBCs were determined by fluorescence assays as
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described previously [25]. Briefly, proteins were extracted from the RBCs with 20 mM
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Tris-HCl (pH 7.5). The lysates were then centrifuged at 15,000 rpm for 10 min, the
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protein concentration in the supernatants were measured. From each RBC sample, 50
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µg of total protein was incubated with 200 µM of proteasome substrate
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z-Leu-Leu-Glu-MCA
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trypsin-like activity), or Suc-Leu-Leu-Val-Tyr-MCA (for chymotrypsin-like activity) in
140
100 µl assay buffer (20 mM Tris–HCl, pH 7.5) at 37°C. All substrates were purchased
141
from Peptide Institute, Inc. (Osaka, Japan). After incubation, the fluorescence intensity
142
was measured using a microplate reader (Varioskan Flash, Thermo Fisher Scientific)
143
with an excitation filter of 365 nm and an emission filter of 460 nm.
caspase-like
activity),
Boc-Leu-Arg-Arg-MCA
(for
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(for
Phenylhydrazine-induced hemolytic anemia
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Hemolytic anemia was induced by an administration of phenylhydrazine (PHZ). At day
147
0, WT mice (12 weeks old) were randomly divided into the two groups, the PBS group
148
and the PHZ group (n=3 in each group). A solution of PHZ in PBS was injected
149
intraperitoneally at a dose of 60 mg/kg on day 0 and day 1. Blood was collected from
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the tail vein prior to the administration and on day 2. Hematological analyses were
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performed using an automated hematology analyzer (Celltac-α Nihon Kohden, Tokyo,
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Japan).
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153 Statistical analysis
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Statistical analysis was performed using GraphPad Prism 4 software.
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157
Results
158
Elevated ubiquitinated proteins and accelerated aggregation in SOD1-deficient
159
RBCs
160
To investigate the degradation of ubiquitinated proteins by proteasomes, we
161
isolated RBCs from WT and SOD1 KO mice and subjected the lysates to detection of
162
ubiquitinated proteins. We analyzed the soluble and insoluble fractions of the RBC
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lysate separately. The levels of ubiquitinated proteins were elevated in both the soluble
164
and insoluble fractions from SOD1-deficient RBCs compared to WT RBCs (Fig. 1).
165
The unibiquitinated proteins in the insoluble fraction (Fig. 1B) were more abundant in
166
the high molecular weight range compared to those in the soluble fraction (Fig. 1A),
167
which is consistent with the fact that poly-ubiquitinated proteins are sparingly soluble.
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We next estimated the change in the ubiquitinated proteins in RBCs under
169
cultural conditions. We harvested RBCs at 0, 24, and 48 h after incubation and
170
examined the levels of ubiquitinated protein in the soluble and insoluble fractions by
171
immunoblotting. During the incubation periods, the level of ubiquitinated proteins in
172
the soluble fraction gradually decreased (Fig. 2A), but those in the insoluble fraction
173
concomitantly increased, and this was more pronounced for proteins in the high
174
molecular weight range (Fig. 2B). The transition of the ubiquitinated proteins from the
175
soluble to the insoluble fraction was more prominent in SOD1-deficient RBCs than WT
176
RBCs. Thus, an SOD1 deficiency enhanced the ubiquitination of proteins, which
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resulted in the accelerated formation of protein aggregates in RBCs.
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The proteasomal activities decreased in RBCs under SOD1 deficiency
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To elucidate the reason for the elevated ubiquitination in the SOD1-deficient
181
RBCs, we analyzed the protein levels of major subunits in proteasomes by
182
immunoblotting. Unexpectedly, however, no differences were observed in the three
183
catalytic subunits β1, β2, and β5 for the 20S component and a subunit for the 19S
184
component of proteasomes between the WT and SOD1-deficient RBCs (Fig. 3).
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180
185
Since oxidation can affect protein function without changing protein levels via the
186
oxidative modification of catalytic amino acid side chains, we assessed the caspase-like
187
(β1), trypsin-like (β2), and chymotrypsin-like (β5) activities of three main catalytic
188
subunits of the proteasome in the RBC lysates. As a result, all three proteolytic activities 8
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189
were markedly decreased in SOD1-deficient RBCs compared to WT RBCs (Table 1). To confirm that oxidative stress actually affected proteasomal function in vitro,
191
we incubated WT RBCs with various doses of superoxide-producing pro-oxidant
192
menadione and then subjected the RBC proteins to immunoblotting and the proteolytic
193
activity assay. Immunoblotting revealed that the levels of ubiquitinated proteins in the
194
soluble fraction increased at lower doses of menadione but rather decreased at higher
195
doses (Fig. 4A). In case of the insoluble fraction, the levels of ubiquitinated proteins
196
increased in the menadione-dose dependent manner (Fig. 4B), suggesting the transition
197
of the polyubiquitinated proteins from the soluble fraction to the insoluble fraction. All
198
three proteolytic activities of the proteasomal components were significantly decreased
199
as the result of the menadione treatment (Supplementary Fig. 1).
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200
Robust accumulation of ubiquitinated proteins and α-globin cross-linked proteins
202
in RBCs of experimental hemolytic anemia
203
To evaluate involvement of the accumulation of ubiquitinated proteins in hemolytic
204
anemia, we next challenged WT mice with phenylhydrazine (PHZ) to induce hemolytic
205
anemia. The PHZ treatment resulted in a significant decrease in the RBC counts
206
(549±51 vs 1000±28 104/µL, p<0.001), HCT (24.6±2.2 vs 49.3±1.2 %, p<0.001), MCV
207
(44.8±0.2 vs 49.3±0.1 fL, p<0.01) and increased the WBC counts (831±31 vs 76±2
208
102/µL, p<0.001) and MCH (25.3±1.2 vs 14.5±0.1 pg, p<0.001). The PBS treatment had
209
no effect on hematological parameters (data not shown).
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We then examined the levels of ubiquitinated proteins in both the soluble and
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insoluble RBC fractions by immunoblotting. While the administration of PHZ resulted
212
in a decreased level of ubiquitinated proteins in the soluble fraction, the levels of
213
ubiquitinated proteins in the insoluble fraction were markedly elevated compared with
214
the PBS treatment (Fig. 5). PHZ causes oxidative stress within RBCs and results in the
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oxidation of hemoglobin, leading to the formation of inclusions called Heinz bodies
216
[26]. When we applied an α-globin antibody to the immunoblotting, an accumulation of
217
proteins that were cross-linked with α-globin were found only in the insoluble fraction
218
of RBCs from the PHZ-administered mice. In addition, all three proteolytic proteasome
219
activities were markedly decreased in RBCs from the PHZ-administered mice compared
220
to control RBCs (Table 2).
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221 Discussion
In the current study, we show that ubiquitinated proteins had accumulated in
224
RBCs from SOD1-deficient mice (Figs. 2 and 3), which is consistent with the
225
observations that oxidative stress induces the oxidative modification and ubiquitination
226
of proteins [27,28]. The findings also indicate that ROS inhibits these proteasomal
227
activities, without affecting the overall protein levels (Fig. 4 and Table 1). Since the
228
dysfunction of the proteasome system would further enhance the accumulation of
229
damaged proteins, this oxidative damage in the proteasome system would be expected
230
to trigger a vicious cycle and consequently induce the destruction of RBCs, which leads
231
to anemia in SOD1-deficient mice.
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Among the proteasome components, the 26S proteasome, which is involved in the
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ATP-dependent degradation of ubiqutinated proteins, is much more susceptible to
234
oxidative stress than the 20S proteasome [14,29,30]. The 26S proteasome is
235
disassembled into a 20S core proteasome and a 19S regulatory component by mild
236
oxidative stress. Txnl1/TRP32, a member of the thioredoxin family of proteins, binds to
237
a subunit Rpn11 of the 19S proteasome and may be involved in the redox-sensitive
238
conversion of proteasomes [31]. In terms of substrate specificity, the 20S proteasome
239
selectively recognizes and degrades oxidized proteins, while the 26S proteasome, even in
240
the presence of ATP, is not very effective in eliminating oxidized proteins [14]. No
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changes were detected in a subunit for the 19S proteasome component between WT and
242
SOD1-deficient RBCs (Fig. 4), which suggests that the oxidative stress-induced
243
conversion from the 26S to the 20S proteasome is not likely the mechanism responsible
244
for the accumulation of the ubiquitinated proteins. The RBC contains about 10 times
245
more 20S proteasomes than 26S proteasomes although the cellular concentrations of
246
proteasomes in RBCs is much lower compared to nucleated cells [32]. Continuous
247
oxidative stress, which is caused by the release of ROS from hemoglobin autoxidation,
248
appears to be involved in the function of 26S and 20S proteasomes in RBCs.
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241
We observed that the size and amounts of several proteasomal components were
250
also not changed in SOD1-deficient RBCs (Fig. 4). In cultured cells, the nuclear
251
factor-erythroid 2-related factor-2 (Nrf2) is involved in proteasomal gene expression in
252
response to oxidative stress [33–35]. It has also been shown that immunoproteasomes,
253
which contain all three IFN-γ–inducible catalytic β subunits in the same 20S complex
254
and are thought to be more resistant to oxidants, are up-regulated upon oxidative stress
255
and contribute to the removal of oxidized proteins [36–38]. Thus proteasomal activity
256
can be elevated under conditions of moderate oxidative stress and protein homeostasis
257
can be maintained by responding to the oxidative insult. Contrary to nucleated cells,
258
RBCs lack nuclei and hence are incapable of replacing damaged proteins, thereby
259
oxidative stress easily entraps RBCs into a vicious cycle which faces severe oxygen
260
toxicity compared with other tissues.
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The age-related decrease in proteasomal activity appears to be due to
262
post-translational modifications, since the amount of the proteases remained unchanged
263
while their activity was significantly decreased [39,40]. We have also demonstrated a
264
decreased proteasomal function in SOD1-deficient RBCs (Table 1). A significant
265
decline in proteasome activity upon severe oxidative stress has been reported in cultured
266
cells [29,30]. Moreover, the treatment of mice with a superoxide generator, paraquat,
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reportedly induces the accumulation of ubiquitinated proteins [41]. Among the
268
proteolytic activities in proteasomes, caspase-like activity is the most profoundly
269
affected during aging [17–19]. Caspase-like proteasomal activities in livers from old
270
male rats are approximately 50% lower than those from young male rats [18]. Although
271
the trypsin-like activity of the proteasome is also sensitive to oxidation, the heat shock
272
protein 90 appears to protect the activity by direct interactions in aged rats [18].
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267
While severe oxidative stress causes extensive protein oxidation, which includes
274
direct fragmentation, cross-linking, and the aggregation of proteins, massively
275
aggregated proteins become progressively resistant to proteasomal degradation,
276
ultimately causing dysfunction. PHZ is a hemolytic agent that interacts with hemoglobin
277
to generate superoxide [42,43] and, in hemoglobin, would be expected to cause oxidative
278
damage, such as fragmentation [44], leading to the formation of Heinz bodies. We
279
challenged mice with PHZ to induce hemolytic anemia, and found that highly
280
ubiquitinated proteins and cross-linked hemoglobin accumulated in RBCs (Fig. 5).
281
These heavily oxidized and cross-linked proteins, especially large aggregates, actually
282
bind irreversibly to proteasomes, resulting in inhibition [45–47], leading to a vicious
283
cycle with progressive defects in protein degradation. Thus, it is likely that oxidative
284
insults to RBCs trigger the accumulation of ubiquitinated proteins, which consequently
285
disturbs proteasome activity, thus leading to the destruction of RBC. Since SOD1 plays
286
an essential role in eliminating superoxide, the deficiency would enhance oxygen
287
toxicity and reduce the lifespan of RBCs to 60% of the WT RBCs [6].
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273
In conclusion, the catalytic activities of proteasomes are susceptible to oxidative
289
inactivation, which results in the accumulation of ubiquitinated proteins in
290
SOD1-deficient RBCs. Since these oxidatively-damaged proteins tend to form large
291
aggregates, which in turn leads to the dysfunction of proteasomal function. Thus, the
292
fatal vicious cycle is triggered by an SOD1 deficiency and disturbs protein homeostasis,
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293
which would render RBCs to be more susceptible to oxidative modification, shorten
294
their lifespan, ultimately resulting in the development of anemia.
295 Acknowledgments
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296
J.L. is a scholarship recipient from the Otsuka Toshimi Scholarship Foundation.
298
E.S.K. is supported by the Tokyo Biochemical Research Foundation Postdoctoral
299
Fellowship for Asian Researchers in Japan. This work was supported by the Strategic
300
Young Researcher Overseas Visits Program for Accelerating Brain Circulation from the
301
Japan Society for the Promotion of Sciences (S2402).
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436
Figure legends
437 Fig. 1. Levels of ubiquitinated proteins in WT and SOD1-deficient RBCs.
439
The representative immunoblots of WT and SOD1-deficient (KO) RBCs probed with
440
antibodies against ubiquitin (Ub), β-actin, and SOD1 in soluble (A) or insoluble (B)
441
fraction are shown (n=3 for each group).
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438
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442
Fig. 2. Levels of ubiquitinated proteins in WT and SOD1-deficient RBCs under culture
444
conditions.
445
Proteins were extracted from WT and SOD1-deficient (KO) RBCs at 0, 24, and 48 h
446
after isolation and subjected to immunoblotting. The representative immunoblots
447
probed with antibodies against ubiquitin (Ub), β-actin, and SOD1 in soluble (A) or
448
insoluble (B) fraction are shown (n=3 for each group).
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443
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Fig. 3. Levels of proteasome subunits in WT and SOD1-deficient RBCs.
451
The representative immunoblots of WT and SOD1-deficient (KO) RBCs probed with
452
antibodies against the β1 (A), β2 (B), β5 (C), and 19S (D) subunits of the proteasome
453
are shown. Bottom panels depict the quantification of each proteins normalized to the
454
corresponding β-actin. Values are the mean ± SD of mice (n=3 for each group). ns, not
455
significant.
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456
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450
457
Fig. 4. Levels of ubiquitinated proteins in menadione-treated RBCs.
458
WT RBCs were treated with the indicated doses of menadione (Men). The
459
representative immunoblots probed with antibodies against ubiquitin (Ub) and β-actin in
460
the soluble (A) or insoluble (B) fraction are shown.
461
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Fig. 5. Levels of ubiquitinated proteins in WT RBCs during hemolytic anemia.
463
RBCs from WT mice treated with PBS or phenylhydrazine (PHZ) were collected and
464
subjected to immunoblotting. The representative immunoblots probed with antibodies
465
against ubiquitin (Ub), β-actin, and α-globin (Hb) in soluble (A) or insoluble (B)
466
fraction are shown (n=3 for each group).
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468 Table 1. The proteasomal activities in WT and SOD1-deficient RBCs.
470
WT and SOD1-deficient (KO) RBCs were collected, and caspase-like, trypsin-like, and
471
chymotrypsin-like activities were monitored. Values represent the mean ± SD of three
472
mice per group. Statistical analysis was performed by Student t-test. The asterisks
473
denote a significant difference between WT and KO RBCs. *, P < 0.05; ***, P< 0.001.
474
FU: Fluorescence unit.
SC
RI PT
469
Caspase-like activity Trypsin-like activity
EP
476
AC C
475
TE D
Chymotrypsin-like activity
M AN U
WT
20
KO
3.37 ± 0.17
2.04 ± 0.21***
5.77 ± 0.40
4.85 ± 0.30*
3.92 ± 0.40
3.28 ± 0.46* (FU/h/µg protein)
ACCEPTED MANUSCRIPT
477 Table 2. The proteasomal activities in PHZ-administered and control RBCs.
479
RBCs from WT mice treated with PBS or phenylhydrazine (PHZ) were collected, and
480
caspase-like, trypsin-like, and chymotrypsin-like activities were monitored. Values
481
represent the mean ± SD of three mice per group. Statistical analysis was performed by
482
Student t-test. The asterisks denote a significant difference between PBS and PHZ. **,
483
P< 0.01. FU: Fluorescence unit.
SC
RI PT
478
Caspase-like activity Trypsin-like activity
EP AC C
484
TE D
Chymotrypsin-like activity
M AN U
PBS
21
PHZ
2.38 ± 0.27
0.35 ± 0.40**
2.69 ± 0.20
0.60 ± 0.62**
3.40 ± 0.54
0.59 ± 0.49** (FU/h/µg protein)
ACCEPTED MANUSCRIPT
A
B Soluble
Insoluble
KO
WT kDa
245 180 135 100 75
245 180 135 100 75
M AN U
kDa
63
63
48
Ub
35
35
AC C
20
TE D
48
25
EP
17
17
11
48
35
25 20
KO
SC
WT
RI PT
Figure 1
β-actin
SOD1
20 17
Ub
ACCEPTED MANUSCRIPT
A
RI PT
Figure 2
B
Insoluble
Soluble
KO
0 24 48 0 24 48
WT (h) kDa 245 180 135 100
M AN U
kDa
100
63
63
48
48
Ub
35
35
EP
48
TE D
35
17
β-actin
AC C
20 17
11
(h)
75
75
20
KO
0 24 48 0 24 48
245 180 135
25
SC
WT
SOD1
25 20 17
Ub
ACCEPTED MANUSCRIPT
Figure 3
B WT
KO
WT kDa
KO
RI PT
A
kDa
35
35
β1
β2 25
25
β2 / β-actin (%)
M AN U
β1 / β-actin (%)
SC
ns
ns
D
WT
TE D
C KO
WT
KO
kDa
kDa
25 20
EP
β5
35
AC C
ns 19S / β-actin (%)
ns
β5 / β-actin (%)
48
19S
ACCEPTED MANUSCRIPT
A
RI PT
Figure 4
B Soluble 0
5
10 25
50 100
Men (µM) kDa
245 180 135
245 180 135
100 75
Ub
EP
AC C
35
48
35
TE D
35
48
50 100
63
48
20
10 25
100 75
63
25
5
M AN U
kDa
0
SC
Men (µM)
Insoluble
β-actin
25 20
Ub
ACCEPTED MANUSCRIPT
Figure 5
B
Soluble PHZ
PBS
kDa
kDa
245 180 135 100
245 180 135 100
75
75
63
63
48
48
35
35
25
25
20
20 17
48 35 25
AC C
20
EP
63
TE D
17
245 180 135 100 75
245 180 135 100 75 63 48 35
Hb
Hb 25 20
17
17
11
11
48
35
Ub
M AN U
Ub
PHZ
RI PT
PBS
Insoluble
SC
A
β-actin
ACCEPTED MANUSCRIPT
1
Highlights
Poly-ubiquitinated proteins accumulated in RBCs from SOD1-deficient mice 5
Poly-ubiquitinated proteins accumulated also in RBCs of phenylhydrazine-treated mice.
RI PT
Proteolytic activities of the proteasome were decreased in the SOD1-deficient RBCs. An oxidative stress-induced malfunction of proteasomes may be a cause for anemia.
AC C
EP
TE D
M AN U
SC
10
1
Supplementary Fig. 1
ACCEPTED MANUSCRIPT
**
** **
** **
SC
Men (μM)
RI PT
Relative activity (%)
Caspase-like activity
M AN U
**
**
**
** **
TE D
Relative activity (%)
Trypsin-like activity
AC C
Relative activity (%)
EP
Men (μM)
Chymotrypsin-like activity
**
** **
** **
Men (μM)
Supplementary Fig. 1. The proteasomal activities in menadione-treated RBCs. RBCs were treated with the indicated doses of menadione (Men) and subjected to proteolytic activity assays. Data are expressed as the mean ± SD (n=3 for each group). Statistical analysis was performed by one-way ANOVA with Dunnett's post hoc tests. **, P < 0.01.