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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3050-3056
RAPID COMMUNICATION
By
From the Departments of Molecular and Human Genetics, Pharmacology
and Medicine, Baylor College of Medicine, Houston, TX 77030; and the
Hematology Branch, National Heart, Lung and Blood Institute, Bethesda,
MD.
The FAC protein encoded by the Fanconi anemia (FA) complementation
group C gene is thought to function in the cytoplasm at a step before
DNA repair. Because FA cells are susceptible to mitomycin C, we
considered the possibility that FAC might interact with enzymes
involved in the bioreductive activation of this drug. Here we report
that FAC binds to NADPH cytochrome-P450 reductase (RED), a microsomal
membrane protein involved in electron transfer, in both transfected
COS-1 and normal murine liver cells. FAC-RED interaction requires the
amino-terminal region of FAC and the cytosolic, membrane-proximal
domain of the reductase. The latter contains a known binding site for
flavin mononucleotide (FMN). Addition of FMN to cytosolic lysates
disrupts FAC-reductase complexes, while flavin dinucleotide, which
binds to a distinct carboxy-terminal domain, fails to
alter FAC-RED complexes at concentrations similar to FMN. FAC is also
functionally coupled to this enzyme as its expression in COS-1 cells
suppresses the ability of RED to reduce cytochrome c in the presence of
NADPH. We propose that FAC plays a fundamental role in vivo by
attenuating the activity of RED, thereby regulating a major
detoxification pathway in mammalian cells.
© 1998 by The American Society of Hematology.
THE AUTOSOMAL RECESSIVE disease Fanconi
anemia (FA) can lead to birth defects, bone marrow failure, and myeloid
leukemia.1,2 Although the disorder is genetically
heterogeneous, there are several shared features that include
chromosome breakage, enhanced sensitivity to mitomycin C (MMC) and to
related bifunctional alkylating agents (also called crosslinkers),
delays in the G2 phase of the cell cycle, and predisposition to
apoptosis.2 The hypersensitivity to crosslinking agents has
served as the basis for assigning FA cells to at least eight different
complementation groups3-5 and for cloning the disease genes
in two groups.6,7 The genes for FA groups A
(FAA),8 C (FAC),3
and D (FAD)9 have been mapped to different
chromosomal loci, suggesting that mutations in several distinct genes
can give rise to a similar disease phenotype.4 The genes
for complementation groups A7,10 and C6 are
present in single copies and encode unique proteins, which are
expressed at low levels in most tissues. The The distinctive sensitivity of FA cells to crosslinkers has led us to
consider the possibility that FAC modulates the toxicity of these
agents directly or indirectly. MMC and diepoxybutane (DEB) are among
the most popular agents in this group. MMC is an antineoplastic drug
that requires metabolic activation to unmask its cytotoxic
function.21 The reduction of MMC by cellular enzymes generates highly reactive species that can generate interstrand crosslinks in double-stranded DNA. In turn, reactive oxygen metabolites can degrade DNA and contribute to the cytotoxicity of MMC. The relative
contribution of these pathways to the pathogenesis of FA is not clear.
However, it is noteworthy that the chromosomal instability can be
attenuated by low oxygen tension and exacerbated by normal or high
concentrations of oxygen.22-24
One approach to deciphering the function of FAC may be through the
identification of its binding partners, which include at least three
ubiquitous cytoplasmic proteins.17,25 Because FA cells are
highly sensitive to MMC, we investigated whether FAC interacts with
enzymes involved in the bioreductive activation of this
drug.26 A key enzyme in this pathway is NADPH:cytochrome c
(P-450) reductase (RED; EC 1.6.2.4), a 77-kD integral microsomal enzyme
that can transfer electrons from NADPH to an isozyme of the cytochrome
P450 family26-33 as well as to cytochrome c. Tethered by a
short hydrophobic sequence to the microsomal membrane, RED extends into
the cytosol and contains binding sites for several prosthetic groups,
including flavin mononucleotide (FMN), flavin dinucleotide (FAD), and
NADPH. Electrons donated by NADPH are initially transferred internally
from FAD to FMN, then externally to one of the cytochromes P450 in
microsomes. An outcome of this chain of events is the oxidative
metabolism of various drugs, xenobiotics, and endogenous substrates,
such as steroids and fatty acids.
A potential interaction between FAC and RED seemed attractive for
several reasons. First, during attempts to identify FAC-associated proteins, cytoplasmic proteins in the 69- to 90-kD range were found to
bind to glutathione-S-transferase (GST)-FAC, but not to
GST.25 Second, similar to the phenotype of FA cells, RED overexpression in a non-FA cell line was shown to induce MMC
hypersensitivity,33 and acquired resistance to MMC
correlated with reduced activity of RED.34 Here we show
that FAC binds to the cytosolic domain of RED, which can be inhibited
in vitro by FMN. In vivo, FAC suppresses the catalytic function of RED.
These data suggest a model in which an important component of the
defect in FA group C cells involves the uncoupling of FAC-RED
interaction. Without appropriate attenuation of RED activity by FAC,
reactive species (eg, of MMC or oxygen metabolites) could accumulate
and affect cell viability.
Expression plasmids.
Full-length human FAC and RED cDNAs as well as cDNAs encoding human
cytochrome P4501A1, NADPH:Quinone Oxidoreductase1 (NQO1), NADPH:Quinone
Oxidoreductase2 (NQO2), BclXL, p34cdc2 kinase,
and cyclin B were cloned into either pMT2 (gift of Dr R. Wise, Brigham
and Women's Hospital, Boston, MA) or pcDNA3 (Invitrogen, Carlsbad,
CA). Wild-type FAC and a panel of deletion mutants generated by
polymerase chain reaction were also subcloned as fusion cDNAs upstream
of the human IgG1 heavy-chain cDNA, as
before.17 Recombinant GST-FAC expressed in Escherichia
coli was prepared as described previously.25
Preparation and analysis of liver cellular extracts.
Livers from three C57BL/6 mice were homogenized in ice-cold
homogenization buffer (50 mmol/L Tris-HCl [pH 7.4], 0.25 mol/L sucrose]. Nuclei and unbroken cells were pelleted by centrifugation at
3,000g for 10 minutes, and mitochondria were pelleted by a further centrifugation at 9,000g for 20 minutes. The clarified supernatant was then centrifuged at 100,000g for 60 minutes to yield cytosol (supernatant) and microsome (pellet). The latter fraction
was resuspended in homogenization buffer before protein interaction
studies. Each fraction was immunoprecipitated with affinity-purified
anti-FAC antibodies raised against the GST-FAC recombinant protein, as
described,35 or with a control antibody against
MxA36 prepared by the same affinity-purification procedure. After incubation of lysates with each antibody in phosphate-buffered saline (PBS) containing 0.1% NP-40 for 1 hour, immune complexes were
precipitated with protein A-agarose, washed, and analyzed by
immunoblotting. Protein concentrations were determined by the Bradford
assay (Bio-Rad, Richmond, CA) corrected for detergent effects.
Transfection and immunoprecipitation (IP).
COS-1 cells were transfected by the diethyl aminoethyl
(DEAE)-dextran method. For metabolic labeling, cells were
preincubated for 1 hour in Dulbecco's Modified Essential Medium
(DME) lacking cysteine and methionine, followed by
incubation in the same medium containing
Expre35S35S label (0.2 mCi/mL; DuPont,
Wilmington, DE) for 1 hour at 37°C. Monolayers were then washed in
PBS and lysed in 0.4 mL lysis buffer (20 mmol/L Tris-HCl [pH 8.0], 50 mmol/L NaCl, 0.1% NP-40, 2 mmol/L EDTA, and protease inhibitors).
Supernatants were incubated for 1 hour with either pre-immune serum or
affinity-purified anti-FAC antibody in the presence or absence of the
indicated competitors or in higher concentrations of NP-40. Immune
complexes were then precipitated with protein A-agarose beads
(Bio-Rad). After washing in lysis buffer, beads were boiled in Laemmli
sample buffer containing reducing agents and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
autoradiography.
Western analysis of immune complexes.
Lysates of transfected COS-1 cells or mouse liver extracts were
analyzed either directly by immunoblotting or after IP with anti-FAC or
anti-RED antibodies. Immune complexes precipitated with protein
A-agarose beads were resolved by SDS-PAGE and transferred to
polyvinyldifluoride membranes (DuPont) by electroblotting. After
blocking with 10% nonfat milk and 1% bovine serum albumin (BSA),
membranes were reacted sequentially with a primary antibody (either
anti-FAC or anti-RED antiserum [Novus Molecular Inc, San Diego, CA])
and horseradish peroxidase-conjugated goat anti-rabbit IgG (GIBCO-BRL,
Grand Island, NY), and bands were visualized by chemiluminescence
(DuPont).
Yeast two-hybrid analysis.
RED deletion mutants generated by PCR were cloned into the vector
pAD-GAL4 (Stratagene, La Jolla, CA) downstream of the GAL4 transcriptional activation domain,37 and full-length human
FAC cDNA was cloned into the vector pBD-GAL4 Cam. Both inserts were under the control of the ADH1 promoter. After cotransformation of the
yeast host strain YRG-2, a filter color assay was used to assess the
transcriptional activation of lacZ ( Enzyme assays.
Ten- to 50-µL aliquots of COS-1 cell lysates were incubated with 20 µmol/L NADPH, 0.6 µmol/L cytochrome c, and 50 mmol/L potassium phosphate (pH 7.6) in a final volume of 1 mL, as before.38
An increase in absorbance at 550 nm due to the NADPH-dependent
reduction of cytochrome c was taken as an index of RED activity. Enzyme activity was calculated using the extinction coefficient of cytochrome c (18.5 cm FAC binds to RED in transfected COS-1 cells.
We used several strategies to test the hypothesis that an interaction
between FAC and RED takes place in vivo. First, COS-1 cells were
transiently transfected with a combination of mammalian expression
vectors encoding FAC and RED and analyzed by metabolic 35S
labeling and IP. Cytosolic lysates from cells expressing FAC alone
showed the expected 63-kD protein when immunoprecipitated with anti-FAC
antibodies, while lysates from cotransfected cells showed an additional
band of
Fac-RED complexes in normal liver cells.
Based on our earlier observation that anti-human FAC antibodies can
cross-react with the murine orthologue of FAC, fac,40 and
the assumption that FAC-RED interactions may be conserved in other
mammals, we attempted to detect fac-RED protein complexes in extracts
of non-FA mouse livers. Because RED is primarily microsomal, we
prepared cytosolic and microsomal extracts and attempted to detect
fac-RED protein complexes by sequential IP and immunoblotting experiments. As expected, the microsomal fraction contained
significantly greater amounts of RED than the cytosolic fraction
(Fig 2). When each fraction was
immunoprecipitated with either anti-FAC antibody or anti-MxA and
immunoblots probed with anti-RED antibodies, fac-RED complexes were
found in both cytosolic and microsomal extracts. Consistent with the
known location of RED in microsomes, fac-RED complexes were
significantly more abundant in the microsomal extracts (Fig 2).
Conversely, IP with anti-RED antibody and probing of immunoblots with
anti-FAC antibody also showed fac-RED complexes. These results
demonstrate that fac-RED complexes can be detected in a normal tissue
extract, and that the distribution of the complex correlates with the
known subcellular location of RED.
Binding domain localization on FAC.
To determine the region of FAC that is necessary for this interaction,
we generated a series of carboxy-terminal truncated mutants fused to
the constant region of the human IgG1 heavy-chain cDNA, as
described previously.17 After coexpression of these constructs with full-length RED in COS-1 cells, single-step IP with
protein A-agarose beads showed that residues within the region 8-149 of FAC are necessary for binding to RED
(Fig 3). Thus, the amino-terminal domain of
FAC is required for interaction with RED.
Binding domain of FAC on RED.
Considerably more is known about the functional organization of RED
than of the FAC protein.29-32 The amino-terminal region of
RED is homologous to FMN-containing bacterial flavodoxins, and the
carboxy-terminus is homologous to FAD-containing ferrodoxin NADP+ reductases. Furthermore, the FMN- and
FAD/NADPH-binding domains can be dissected into distinct structural and
functional units, which bind to their respective
cofactors.26,27 To delineate the FAC-binding domain of RED
and, if more than one domain is involved, discern quantitative
differences, we performed reciprocal mapping experiments using the
yeast two-hybrid system.37 Deletion mutants of RED were
fused to the transcriptional activation domain of the GAL4 protein
(AD-RED), while FAC was fused to the DNA-binding domain of GAL4
(BD-FAC; Fig 4A). Transformation with
AD-RED or BD-FAC alone did not result in transcriptional activation
(data not shown). However, transformants expressing either full-length RED or deletion mutants encoding either residues 1-274 (membrane anchor
and FMN-binding domain) or 61-274 (containing the FMN-binding domain,
but lacking the membrane anchor) turned blue in the presence of BD-FAC
in a filter color assay. There was no interaction between BD-FAC and
AD-RED constructs lacking the FMN-binding domain. Thus, the cytosolic,
membrane-proximal region of RED that is known to bind to FMN also binds
FAC. The proximity of FAC to the microsomal membrane is compatible with
our previous observation that approximately one third of the total
intracellular pool of FAC associates with internal
membranes.16,17
Effect of cofactors on FAC-RED interaction.
To assess whether known RED cofactors affect the interaction of
FAC with RED, we cotransfected COS-1 cells with expression constructs
encoding these cDNAs and immunoprecipitated FAC-RED complexes in the
presence or absence of known RED cofactors. The intensity of bands
corresponding to RED and FAC on a representative autoradiogram (Fig 4B)
were quantified by densitometry (data not shown), and the degree of
protein-protein interaction was expressed as the ratio of RED to FAC in
control relative to the experimental samples. The inclusion of 0.1 mmol/L FMN in lysates caused a greater than 95% reduction in FAC-RED
complex formation. Similar concentrations of FAD did not appear to have
any effect. Cytochrome c also partially inhibited this interaction,
albeit at a 10-fold higher concentration. Finally, we were unable to
show that FMN in the range 0 to 1.0 mmol/L binds directly to
recombinant GST-FAC immobilized to glutathione-agarose beads (data not
shown). Taken together, these results show that FMN can compete with
FAC for interaction with RED.
Suppression of RED activity by FAC.
We also determined whether the expression of FAC could affect the
catalytic activity of RED in vivo. COS-1 cells transfected with RED
expressed dose-dependent levels of reductase activity (Fig 5A). However, cotransfection of COS-1
cells with RED and FAC, but not RED and the empty expression vector,
suppressed the activity of RED by 3.2- to 3.6-fold. Interestingly, the
extent of suppression was independent of the amount of transfected FAC plasmid DNA over a 10-fold range, and FAC was not able to abolish RED
activity completely. By contrast, the catalytic activity of NQO1 was
not affected by coexpression of NQO1 with FAC (Fig 5B). These results
demonstrate that (1) the catalytic activity of RED can be attenuated by
FAC; (2) the final determinant of reductase activity in this cell
culture model is the intracellular level of RED, not FAC; and (3) a
fraction of RED activity is not subject to regulation by FAC.
A critical component of the cytochrome P450 monooxygenase system is the
membrane-embedded microsomal enzyme RED, which is essential for the
activation of cytochrome P450 enzymes that are involved in the
oxidation of many xenobiotics and endogenous compounds. Abnormal
metabolism of one or several of these compounds could contribute to the
pathogenesis of FA. Here we show that FAC binds to the cytosolic domain
of RED (Fig 3) and attenuates its ability to transfer electrons (Fig
5). This observation provides the first insight into the molecular
function of FAC in the regulation of an important cellular
detoxification pathway. Our earlier studies had suggested that FAC
interacts with at least three cytoplasmic proteins17,25;
RED is one such binding protein.
Submitted July 2, 1998;
accepted August 18, 1998.
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Abstract
Introduction
Abstract
163-kD
protein encoded by the FAA gene contains a nuclear localization
signal (NLS), but otherwise it is devoid of any sequence motifs that may suggest a biological function,
-galactosidase activity)
as an indicator of a physical interaction between AD-RED and BD-FAC,
and the intensity of the color reaction was scored in a
semi-quantitative manner by visual inspection.
1mmol/L
which may be expected to be detergent-sensitive
-deoxyguanosine, a marker of
oxidative damage, in the genomic DNA of FA lymphoblasts treated with
hydrogen peroxide
)-diepoxybutane by cDNA-expressed human cytochrome P450s and by mouse, rat, and human liver microsomes: Evidence for preferential hydration of meso-diepoxybutane in rat and human liver microsomes.
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