A Small Molecule Inhibitor of Redox-Regulated Protein Translocation into Mitochondria

Article (PDF Available)inDevelopmental Cell 25(1):81-92 · April 2013with40 Reads
DOI: 10.1016/j.devcel.2013.03.006 · Source: PubMed
The mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis.
Developmental Cell
A Small Molecule Inhibitor of Redox-Regulated
Protein Translocation into Mitochondria
Deepa V. Dabir,
Samuel A. Hasson,
Kiyoko Setoguchi,
Meghan E. Johnson,
Piriya Wongkongkathep,
Colin J. Douglas,
Johannes Zimmerman,
Robert Damoiseaux,
Michael A. Teitell,
and Carla M. Koehler
Department of Chemistry and Biochemistry
Department of Pathology and Laboratory Medicine
Molecular Screening Shared Resource
Molecular Biology Institute
Jonsson Comprehensive Cancer Center and Broad Stem Cell Research Center
UCLA, Los Angeles, CA 90095, USA
Present address: National Institute of Neurological Disorders and Stroke, Building 35, Room 2C1014, 35 Convent Drive, Bethesda,
MD 20815, USA
Present address: Charite
Medical School, 10117 Berlin, Germany
*Correspondence: koehler@chem.ucla.edu
The mitochondrial disulfide relay system of Mia40
and Erv1/ALR facilitates import of the small translo-
case of the inner membrane (Tim) proteins and
cysteine-rich proteins. A chemical screen identified
small molecules that inhibit Erv1 oxidase activity,
thereby facilitating dissection of the disulfide relay
system in yeast and vertebrate mitochondria. One
molecule, mitochondrial protein import blockers
from the Carla Koehler laboratory (MitoBloCK-6),
attenuated the import of Erv1 substrates into yeast
mitochondria and inhibited oxidation of Tim13 and
Cmc1 in in vitro reconstitution assays. In addition,
MitoBloCK-6 revealed an unexpected role for Erv1
in the carrier import pathway, namely transferring
substrates from the translocase of the outer mem-
brane complex onto the small Tim complexes.
Cardiac development was impaired in MitoBloCK-
6-exposed zebrafish embryos. Finally, MitoBloCK-6
induced apoptosis via cytochrome c release in
human embryonic stem cells (hESCs) but not in
differentiated cells, suggesting an important role for
ALR in hESC homeostasis.
The mitochondrion has translocons of the outer membrane
(TOM) and inner membrane (TIM) to import proteins from the
cytosol. Proteins with a typical N-terminal targeting sequence
are imported via the TIM23 pathway, whereas polytopic
inner membrane proteins use the TIM22 import pathway
(Chacinska et al., 2009; Mokranjac and Neupert, 2009). In
contrast, most of the proteins imported into the intermembrane
space (IMS) lack a mitochondrial targeting sequence and
employ diverse routes for mitochondrial import (Herrmann
and Hell, 2005).
A recently identified pathway in the IMS mediates oxidation
of imported proteins that require disulfide bonds to acquire
their native conformation (Deponte and Hell, 2009; Koehler and
Tienson, 2009; Riemer et al., 2011; Sideris and Tokatlidis,
2010), such as the small Tim proteins and proteins with a twin
C motif (Cavallaro, 2010). In the small Tim proteins, the prox-
imal N-terminal cysteine residues serve as internal targeting
sequences that are recognized by the IMS oxidoreductase
Mia40 (Milenkovic et al., 2009; Sideris et al., 2009), which func-
tions as a receptor to mediate translocation across the outer
membrane (Chacinska et al., 2004). Mia40 contains a redox-
active cysteine pair that is maintained in an oxidized state by
the sulfhydryl oxidase Erv1 (Tienson et al., 2009). As the imported
protein substrate is oxidized, electrons are passed from Mia40
to Erv1, followed by transfer to molecular oxygen or cytochrome
c (cyt c )(Bien et al., 2010; Dabir et al., 2007). Subsequently, cyt c
can be reoxidized by cyt c oxidase of the respiratory chain (Bien
et al., 2010) or by cyt c peroxidase (Dabir et al., 2007). Thus,
Mia40 and Erv1 constitute a mitochondrial disulfide relay system
that is also evolutionarily conserved.
Erv1 belongs to the Erv/ALR sulfhydryl oxidase family, and
homologous proteins are found in the endoplasmic reticulum
(Erv2) of yeast, in the extracellular environment (Quiescin sulfhy-
dryl oxidase), and in the poxvirus family (E10R) (Gerber et al.,
2001; Senkevich et al., 2002; Thorpe et al., 2002). In addition
to protein translocation, the role of Erv1 in various cellular path-
ways is exemplified by a number of defects observed in cells that
lack functional Erv1 protein. For example, Erv1 is required for the
maturation of cytosolic iron-sulfur cluster-containing proteins
(Lange et al., 2001). In erv1 mutant yeast, heme maturation is
impaired (Dabir et al., 2007). Also, mutations in mammalian
Erv1 homolog, ALR, result in an autosomal-recessive myopathy
(Di Fonzo et al., 2009), and ALR has an essential prosurvival role
in the maintenance of murine embryonic stem cells (Todd et al.,
2010b) and in the regeneration of Drosophila imaginal discs
(McClure et al., 2008).
Erv1 has several key functions in the IMS, necessitating the
characterization of its homolog, ALR, to uncover basic mecha-
nisms in mitochondrial assembly in vertebrate systems. Because
Erv1 donates electrons to cyt c, Erv1/ALR may have a central
Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc. 81
role in apoptotic pathways that lead to cyt c release (Dabir et al.,
2007). Classically, mitochondrial protein import has been stud-
ied using yeast genetics and biochemical assays. However,
new approaches are needed to elucidate disease mechanisms
and dissect essential functions in mammalian cells. Here, we
report a small molecule screening approach to identify Erv1 in-
hibitors, with the goal of developing a set of probes that can
modulate the pathway quickly and recapitulate disease pheno-
types. We have taken advantage of the previously developed
in vitro Amplex Red assay for monitoring Erv1 activity to identify
inhibitors (Dabir et al., 2007). Our results indicate that the small
drug-like inhibitor characterized here is specific for Erv1/ALR
and can be used to reveal normal functions and disease mecha-
nisms in mammalian mitochondria.
A Chemical Screen to Identify Inhibitors of Erv1 Oxidase
We previously developed an assay to test the sulfhydryl oxidase
activity of recombinant Erv1 protein based on the oxidation of a
nonphysiologic substrate, dithiothreitol (DTT), which produces
hydrogen peroxide (H
)(Dabir et al., 2007). H
was measured using a standard fluorometric assay with Amplex
Red and horseradish peroxidase (HRP). The assay was adapted
in high throughput format, and a chemical screen was conduct-
ed on an integrated robotic system with plate scheduling (Fig-
ure S1A available online). Briefly, diversity-oriented commercial
libraries of 50,000 drug-like compounds from Chembridge
(Lumsden et al., 2007; Webb, 2005), Kwon (Castellano et al.,
2007), and Asinex (Lumsden et al., 2007)at10mM concentration
were screened for inhibition of Erv1 activity. Erv1 (10 mM) was
aliquoted into 384-well plates followed by compound addition
with robotic pinning into the assay wells. DMSO (1%, vehicle)
was included in several plate columns as a carrier control with
the pinned compounds. As a negative control, 10 mM catalyti-
cally inactive Erv1 (Erv1C133S) was also aliquoted into several
plate columns. Incubation of the pinned compounds with Erv1
for 1 hr at 25
C was followed by addition of Amplex Red-HRP
and then DTT (20 mM) to initiate the oxidase assay. After
12 min, the reaction was in the kinetic linear range and a high
signal-to-noise ratio was achieved. Fluorescence intensity was
measured, and reactions that were inhibited by more than
50% were picked as potential Erv1 inhibitors and selected for
secondary analysis. In total, 184 primary candidate inhibitors
were identified (Figure S1B). Forty plates were processed
with a Z
greater than 0.8 across the screen, indicating that the
screen was consistent and robust.
To eliminate false positives, a counter screen was used to test
whether the small molecule compounds directly inhibited the
Amplex Red-HRP assay. H
(800 nM) was reacted with
Amplex Red-HRP in the presence of the small molecules; this
is the approximate amount of H
that was produced by Erv1
during the assay. Those compounds that did not inhibit the
Amplex Red assay directly and showed >50% inhibition of
Erv1 activity (29 compounds) were selected for additional
characterization and designated as mitochondrial protein import
blockers from the Carla Koehler lab (MitoBloCK) compounds
based on their potential to inhibit Erv1 activity. Of these potential
‘lead’ inhibitors, MitoBloCK-6 was chosen for additional
analysis. Figure S1C verifies that MitoBloCK-6 does not directly
hinder the Amplex Red-HRP reaction.
MitoBloCK-6 Inhibits Erv1 Activity In Vitro
MitoBloCK-6 is 2,4-dichloro-6-(((((phenylamino)phenyl)imino)
methyl)phenol) from the Chembridge library (Figure 1A), consist-
ing of a 3,5-dichlorosalicylaldehyde derivative. Upon reordering,
MitoBloCK-6 showed the same Erv1 inhibitory activity as the
original aliquot from the Chembridge library. The inhibitory con-
centration at which Erv1 protein activity is reduced by 50% (IC
for MitoBloCK-6 in the in vitro Amplex Red-HRP assay was
900 nM (Figure 1B). We also tested MitoBloCK-6 as an inhibitor
of ALR (Farrell and Thorpe, 2005) and the yeast paralog in the
endoplasmic reticulum, Erv2 (Gross et al., 2002), using the
in vitro Amplex Red-HRP assay. The IC
for MitoBloCK-6-inhib-
iting ALR and Erv2 was 700 nM and 1.4 mM, respectively (D.V.D.
and C.M.K., unpublished data).
To determine whether MitoBloCK-6 generally impaired redox
active enzymes, we investigated the oxidative folding properties
of protein disulfide isomerase (PDI). MitoBloCK-6 did not inhibit
the ability of PDI to reduce insulin (Figure S1D). Because
MitoBloCK-6 may potentially hinder flavin adenine dinucleotide
(FAD)-containing enzymes, succinate dehydrogenase activity
of the mitochondrial respiratory chain was measured in the
presence of MitoBloCK-6 (Figure S1E). Isolated mitochondria
were incubated in a Clarke-type oxygen electrode, and oxygen
consumption was measured with succinate addition. The oxy-
gen consumption rate was indicative of well-coupled mitochon-
dria, and subsequent addition of DMSO vehicle or MitoBloCK-6
did not alter the oxygen consumption rate. As controls, succi-
nate dehydrogenase activity was disrupted with the inhibitor
malonate, and carbonyl cyanide m-chlorophenylhydrazone
(CCCP) addition indicated that respiring mitochondria could be
uncoupled. Because a 3,5-dichlorosalicylaldehyde is a potential
degradation product of MitoBloCK-6 and the 3,5-dichlorosalicy-
laldehyde moiety may instead inhibit Erv1 (Doorn and Petersen,
2003), commercially available 3,5-dichlorosalicylaldehyde re-
placed MitoBloCK-6 in the in vitro Amplex Red-HRP assay (Fig-
ure 1C). The addition of 100 mM 3, 5-dichlorosalicylaldehyde did
not inhibit Erv1 activity. We assessed MitoBloCK-6 stability in
our screening conditions at pH 6.5 and 7.4 using liquid chroma-
tography-mass spectrometry (LC-MS) analysis (Figure S2A).
Analysis at pH 3.4 was also included, because an acidic pH
favors hydrolysis of the imine linkage to release the 3,5-dichlor-
osalicylaldehyde (Kirdant et al., 2011). MitoBloCK-6 was stable
over this pH range, as supported by a similar retention time
(3.03 min) and a constant area under the curve in the LC-MS
analysis (Figure S2A).
Because aldehydes covalently modify lysine residues in pro-
teins by forming a Schiff base (Volkmann et al., 2011; Yamagata
et al., 1993), we tested whether a potential aldehyde derived
from MitoBloCK-6 covalently modified Erv1 using mass spec-
trometry (Figure S2B). The addition of 75 mM 3, 5-dichlorosalicy-
laldehyde to 25 mM Erv1 yielded a spectrum with a single, minor
peak at 175 Da, which corresponds to a small amount (<5%) of
the 3, 5-dichlorosalicylaldehyde bonding to one position in Erv1;
in contrast, most of the Erv1 migrated as the unmodified protein,
indicating that the lysine residues in Erv1 are not highly reactive.
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MitoBloCK-6 Inhibits Erv1 in Mitochondria
82 Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc.
Using lysozyme as a control protein, a small fraction of 3,
5-dichlorosalicylaldehyde again attached covalently, but most
of the lysozyme was unmodified (Figure S2C). MitoBloCK-6
(75 mM) addition to Erv1 (25 mM) generated a spectrum in which
a small fraction (<3%) of MitoBloCK-6 likely degraded to 3,
5-dichlorosalicylaldehyde that covalently modified Erv1 (Fig-
ure S2B); MitoBloCK-6 was specific for Erv1, because the lyso-
zyme spectrum lacked a similar peak that was shifted by 175 Da
and additional peaks were not detected (Figure S2C). As
a control for the assay, formaldehyde treatment to lysozyme
yielded a spectrum in which unmodified lysozyme was replaced
with lysozyme bonded with at least 7–9 formaldehyde groups
(Figure S2C). Thus, MitoBloCK-6 is a stable compound that
does not markedly bond to Erv1.
MitoBloCK-6 Inhibits Erv1-Dependent Import
in Mitochondria
The import of Erv1 substrates was tested with an in organello
import assay. Substrates included twin CX
C proteins (Mia40,
Figure 1. MitoBloCK-6 Inhibits Erv1 Activity
(A) The structure of MitoBloCK-6, Erv1 SAR com-
pound-1 (ES-1) and compound-2 (ES-2), and 3,5-
(B) IC
analysis of MitoBloCK-6 in the in vitro Erv1
activity assay. Ten micromolar Erv1 was incu-
bated with varying concentrations of MitoBloCK-
6, as described for the chemical screen.
(C) As in (B), IC
analysis with 3,5-di-
chlorosalicylaldehyde and Erv1.
(D) As in (B), IC
analysis with ES-2 and Erv1
(average ± SD, n = 3).
See also Figures S1 and S2.
Cmc1, Cox19, and Cox17), twin CX
protein Tim8, and Erv1 (Figures 2 and
S3; Hofmann et al., 2005; Horn et al.,
2008; Riemer et al., 2011; Terziyska
et al., 2007). Energized mitochondria
were preincubated with 20–50 mM Mito-
BloCK-6 or 1% DMSO for 15 min, fol-
lowed by the addition of the radiolabeled
substrate. A time course assay was per-
formed, and aliquots were removed and
treated with protease to remove nonim-
ported precursors. Import of the twin
C proteins and Erv1 was strongly
decreased, whereas the import of Tim8
was impaired by 40% upon treatment
with MitoBloCK-6 compared to import in
presence of 1% DMSO. We also investi-
gated the import of additional substrates,
Tim23 and ADP/ATP carrier (AAC) of the
TIM22 import pathway, and Su9-dihydro-
folate reductase (DHFR), cyt b
and Hsp60 of the TIM23 import pathway
(Figures 3 and S3). At 20 mM, the import
of Tim23 and AAC was decreased by
approximately 50% (Figures 3A and 3B),
whereas the import of TIM23 substrates was not impaired,
even with 50 mM MitoBloCK-6 (Figures 3C, S3A, and S3B).
Given that Erv1 played a role in the import of TIM22 substrates,
we investigated the import of AAC using blue-native (BN) gel
analysis (Figure 3D). Previous studies have defined the steps of
AAC translocation from the cytosol to the inner membrane using
mutants and biochemical manipulations (Curran et al., 2002;
Ryan et al., 1999; Truscott et al., 2002). Specifically, AAC accu-
mulates with the TOM machinery in a 500 kDa complex in the
tim10-2 mutant or in the absence of ATP, and then is passed
to the Tim9-Tim10 complex; the mature form of AAC subse-
quently assembles as a dimer in a 90 kDa complex in the inner
membrane. After importing AAC in the presence of MitoBloCK-
6 or control DMSO, the mitochondria were solubilized in 1%
digitonin and separated on BN gels followed by autoradiog-
raphy. In the presence of DMSO, AAC accumulated in the
90 kDa complex, which is indicative of an assembled AAC dimer
). Moreover, the AAC dimer was protected from exoge-
nous protease, verifying that AAC translocated to the inner
Developmental Cell
MitoBloCK-6 Inhibits Erv1 in Mitochondria
Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc. 83
membrane. In contrast, the addition of MitoBloCK-6 resulted in
AAC accumulation in a 500 kDa complex with the TOM com-
plex (Figure 3D), and this AAC intermediate was sensitive to
protease, confirming localization at the outer membrane. Anal-
ysis with MitoBloCK-6 supports a role for Erv1 in transferring
AAC from the TOM complex to the Tim9-Tim10 complex in
the intermembrane space. Therefore, in addition to the
cysteine-rich substrates, Erv1 plays a key role in the TIM22
import pathway.
To confirm specificity of MitoBloCK-6, we purchased two
additional compounds, termed Erv1-structure-activity relation-
ship (SAR) (ES)-1 and ES-2 for an abbreviated SAR study (Fig-
ure 1A). ES-2 but not ES-1 (D.V.D. and C.M.K., unpublished
data) inhibited Erv1 function in the in vitro assay with an IC
2.2 mM(Figure 1D). When included in the import assays, ES-2
mirrored MitoBloCK-6 in its ability to impair import, but ES-1
had no effect (Figures 2, S3C, and S3D). Thus, ES-2 and
MitoBloCK-6 seem to specifically inhibit Erv1 function, but
ES-1, like 3,5-dichlorosalicylaldehyde, did not abrogate Erv1
To verify that mitochondrial Erv1 is the target of MitoBloCK-6,
an increased abundance of Erv1 should require an increased
MitoBloCK-6 concentration to inhibit protein import. Previously,
we designed a yeast strain, in which Erv1 with a C-terminal
hexahistidine tag (designated [Erv1) was expressed from a
high-copy plasmid (Dabir et al., 2007).This strain contained an
approximate 5-fold increase in Erv1 with no aberrant phenotypes
detected. The import of Mia40, Cmc1, and AAC proteins was
tested in isolated wild-type (WT) and [Erv1 mitochondria. For
Mia40 and Cmc1, the concentration of MitoBloCK-6 that was
required to inhibit import increased from 10 mMto50mM(Figures
4A and 4B). A similar trend was detected for AAC import with
a concentration increase from 15 mMto30mM(Figure 4C).
Combined, the data strongly support that Erv1 is the target of
To evaluate the cell-based activity of MitoBloCK-6, we also
determined the minimum inhibitory concentration required to
inhibit the growth of yeast by 50% (MIC
) with the Dpdr5Dsnq2
yeast strain, in which the genes for the multidrug resistance
pumps PDR5 and SNQ2 were disrupted in the wild-type strain
(Duncan et al., 2007; Hasson et al., 2010). Deletion of these
pumps increases the steady state intracellular concentration of
Figure 2. MitoBloCK-6 Inhibits the Import of Substrates of the
Mia40/Erv1 Pathway
Radiolabeled precursors were imported into WT mitochondria in the presence
of 25 or 50 mM MitoBloCK-6, 50 mM SAR compounds, or the control 1%
DMSO. Nonimported precursor was removed by protease treatment. Pre-
cursors included (A) Mia40, (B) Cmc1, (C) Cox19, and (D) Tim8. A 10% stan-
dard (Std) from the translation reaction is included. Import reactions were
quantitated using a BioRad FX Molecular Imager and the affiliated Quantity 1
software; 100% was set as the amount of precursor imported into WT mito-
chondria at the endpoint in the time course.
See also Figure S3 and Table S1.
Figure 3. MitoBloCK-6 Inhibits the Import of Substrates of the TIM22
Import Pathway but Not the TIM23 Import Pathway
(A–C) As in Figure 2, import assays were performed. Precursors included
TIM22 import substrates (A) Tim23 and (B) AAC and (C) TIM23 substrate Su9-
DHFR. Aliquots were removed at the indicated time points, and samples were
treated with carbonate extraction to confirm that Tim23 and AAC were inserted
into the inner membrane.
(D) AAC was imported in the presence of DMSO or 25 mM MitoBloCK-6, ali-
quots were removed at indicated time points, and samples were subjected to
Blue-Native PAGE analysis followed by autoradiography (left panel) or im-
munoblotted with antibodies against Tom40 (right panel). AAC
marks the AAC
dimer, and AAC/TOM marks AAC that accumulates in the TOM complex.
Percent import calculated as in Figure 2.
See also Table S1.
Developmental Cell
MitoBloCK-6 Inhibits Erv1 in Mitochondria
84 Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc.
drugs in yeast. The MIC
was 15.2 mM(Figure 4D), which is
similar to the IC
concentration that inhibited protein import.
As in the import assays (Figures 4A–4C), we measured the
with the Dpdr5Dsnq2 strain overexpressing Erv1 from a
high-copy plasmid (Dabir et al., 2007). The MIC
increased to
28.3 mM when Erv1 was overexpressed (Figure 4E).
Mitochondria Are Not Damaged by MitoBloCK-6
A potential mechanism by which MitoBloCK-6 could alter protein
translocation is to nonspecifically permeabilize membranes,
resulting in the release of mitochondrial proteins, particularly
from the IMS. We have previously shown that MitoBloCK-2, an
inhibitor of the TIM22 import pathway, nonspecifically permea-
bilizes mitochondrial membranes (Hasson et al., 2010). We incu-
bated energized mitochondria with 1% DMSO or MitoBloCK-6
followed by centrifugation. Released proteins were recovered
in the supernatant fraction and analyzed by Coomassie staining
for the collective release of proteins (Figure S4A) and by immu-
noblot assay for key proteins (Figure S4B). The results from
Coomassie staining indicated that MitoBlock-6 did not alter
mitochondrial membrane integrity, because proteins were not
released into the supernatant fraction (Figure S4A). Similarly,
immunoblot analysis showed that marker proteins aconitase
(matrix), AAC and Tim54 (inner membrane), and IMS proteins
Mia40, Ccp1, and cyt c were not released with MitoBloCK-6 or
DMSO treatment (Figure S4B).
Another potential mechanism by which MitoBloCK-6 may
disrupt protein translocation is indirect, by dissipation of the
membrane potential (Dc) or disruption of oxidative phosphoryla-
tion, both of which can be measured with a Clark-type oxygen-
sensing electrode (Figure S4C; Claypool et al., 2008). Isolated
mitochondria were incubated in a 0.5 ml chamber at 25
C with
an oxygen electrode, and respiration was initiated with reduced
nicotinamide adenine dinucleotide. The measured oxygen con-
sumption rate was indicative of well-coupled mitochondria.
The subsequent addition of DMSO vehicle or MitoBloCK-6 did
not alter the oxygen consumption rate. As a control, mitochon-
dria were treated with the protonophore CCCP, and respiration
increased drastically, indicative of uncoupled mitochondria
(Figure S4C). Taken together, MitoBloCK-6 does not alter
mitochondrial function or disrupt mitochondrial integrity and
functions biochemically as a specific inhibitor of Erv1.
MitoBloCK-6 Impairs Substrate Oxidation
To determine how MitoBloCK-6 inhibited Erv1 function, we
investigated whether MitoBloCK-6 altered Erv1 interactions
with partner proteins in isolated mitochondria (Figure 5A). Mito-
BloCK-6 was preincubated with mitochondria isolated from the
Erv1-His strain followed by solubilization in 1.0% digitonin, and
Erv1-His was purified with Ni
agarose. In DMSO-treated cells,
a small fraction of the Mia40 and cyt c copurified with Erv1, as
reported previously (Tienson et al., 2009). However, in the pres-
ence of MitoBloCK-6, binding of Mia40 and cyt c to Erv1 was
decreased by 75% and 95%, respectively (Figure 5A).
If MitoBloCK-6 interferes with Mia40-Erv1 binding, then the
oxidation of substrates may be inhibited in vitro. We therefore
evaluated Tim13 oxidation and subsequent production of H
in vitro (Figure 5B; Tienson et al., 2009 ). Erv1 was preincubated
with DMSO or MitoBloCK-6 for 1 hr at 25
C. Then, the oxidation
of Tim13 was reconstituted by incubating reduced Tim13 with
catalytic amounts of Erv1 and Mia40 in an aerobic environment.
Oxidation was monitored over a time course by the addition of
4-acetamido-4-maleimidylstilbene-2, 2-disulfonic acid (AMS)
followed by nonreducing SDS-PAGE and immunoblot analysis
with antibodies against Tim13. AMS addition causes an increase
in molecular mass of 0.5 kDa per addition to a cysteine residue.
In the presence of DMSO, reconstitution proceeded normally
and approximately 80% was oxidized after 3 hr. By contrast,
only 15% of Tim13 was oxidized in the presence of Mito-
BloCK-6 (Figure 5B). As Tim13 was oxidized, H
was monitored using the Amplex Red-HRP assay (Figure 5C;
Tienson et al., 2009). The addition of MitoBloCK-6 caused a sig-
nificant decrease in H
production compared to the control
reactions. We also tested the oxidation of Cmc1 (Bourens
et al., 2012), a substrate of Mia40/Erv1 pathway, with Erv1
Figure 4. Inhibition of Import by MitoBloCK-6 Is Dependent on the
Concentration of Erv1 in Mitochondria
(A–C) Import assays of precursors (A) Mia40, (B) Cmc1, and (C) AAC were
performed as described in Figure 2 into mitochondria derived from wild-type
(WT) yeast or yeast overexpressing Erv1 with a hexahistidine tag ([Erv1) (Dabir
et al., 2007). The concentration of MitoBloCK-6 was varied from 5 to 50 mMas
indicated. A 10% standard (Std) from the translation reaction was included.
analysis of the WT yeast strain lacking the drug pumps (Dpdr5
Dsnq2) with varying concentrations of MitoBloCK-6 (average ± SD, n = 6).
(E) As in (D), MIC
analysis of the Dpdr5 Dsnq2 yeast strain that overexpresses
Erv1-His from a high-copy plasmid ([Erv1) (average ± SD, n = 6).
See also Figure S4 and Table S1.
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MitoBloCK-6 Inhibits Erv1 in Mitochondria
Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc. 85
(Figure 5D) and ALR (Figure 5E). An increase in MitoBloCK-6
concentration correlated with a dose-dependent decrease in
production. Thus, MitoBloCK-6 specifically blocks the
oxidation of Tim13 and Cmc1 in vitro for both Erv1 and ALR.
As an additional test for MitoBloCK-6 inhibition of Erv1 oxi-
dase activity, we measured the oxygen consumption rate by
Erv1 with an oxygen electrode in the presence of excess DTT
(Dabir et al., 2007). When Erv1 was added alone or with
DMSO, the oxygen consumption rate was similar (Figure S4D).
By contrast, the addition of MitoBloCK-6 resulted in a concentra-
tion-dependent decrease in the oxygen consumption rate.
Results from these analyses show that MitoBloCK-6 selectively
inhibits Erv1 and ALR oxidase activity in vitro.
MitoBloCK-6 Inhibits ALR Function in Vertebrate
The long-term goal in developing the MitoBloCK compounds is
to adapt them for studies in vertebrate mitochondria, such as
recapitulating biochemical phenotypes similar to those in cells
derived from patients with mutations in ALR (Di Fonzo et al.,
2009). In addition, MitoBloCK-6 may be useful for studies of
apoptosis, iron sulfur cluster and heme export (Dabir et al.,
2007), and cell differentiation (Todd et al., 2010b), because
ALR has been implicated in these pathways. Since MitoBloCK-
6 inhibits ALR oxidase activity in vitro, we asked whether Mito-
BloCK-6 disrupts mitochondrial function in mammalian cells by
investigating mitochondrial morphology, a general readout for
mitochondrial defects. HeLa cells were transiently transfected
with mitochondrial matrix-targeted Su9-EGFP and colabeled
with Mitotracker-Red (Figure S5A). Cells were treated with
50 mM MitoBloCK-6 for 12–16 hr, and mitochondrial morphology
and integrity was visualized by microscopy. In cells treated with
DMSO, Su9-EGFP colocalized with Mitotracker staining and the
mitochondrial network was distributed as in the untreated cells.
However, the addition of CCCP caused the mitochondrial
network to collapse around the nucleus. MitoBloCK-6 addition
did not disrupt the mitochondrial network (Figure S5A), even at
concentrations up to 100 mM MitoBloCK-6 (unpublished data).
We also examined cell viability with a 1-(4, 5-dimethylthiazol)-
3, 5-diphenylformazan (MTT) assay (Figure S5B). MitoBloCK-6
(100 mM) did not significantly reduce cell viability. In addition,
treatment of HEK293 cells with MitoBloCK-6 showed similar re-
sults (D.V.D. and C.M.K., unpublished data). Because Erv1
passes electrons to cyt c, ALR may play a role in apoptosis in
mammalian cells. Therefore, we queried specifically whether
cyt c was released in cells exposed to MitoBloCK-6 (Figure S5C).
Cells incubated with a positive control, staurosporine, showed
cyt c release and detection in the cytoplasmic fraction as an indi-
cation of apoptosis. However, 50 mM MitoBloCK-6 treatment for
12–16 hr failed to initiate cyt c release (Figure S5C). Whereas Mi-
toBloCK-6 inhibits ALR function in vitro, this inhibitory activity is
surprisingly lacking in HeLa and HEK293 cells.
ALR was identified in a set of common genes that are enriched
in embryonic, neuronal, and hematopoie tic stem cells (Ivanova
et al., 2002; Ramalho-Santos et al., 2002), and ALR has a prosur-
vival role in maintaining human embryonic stem cells (hESCs)
(Todd et al., 2010a). Thus, ALR may have a specific and different
role in hESCs and induced pluripotent stem cells than in differen-
tiated cells, such as HeLa and HEK293 cells. Therefore, we
determined whether MitoBloCK-6 affected hESC survival.
HSF1 hESCs and normal human dermal fibroblasts, which repre-
sent a differentiated cell type, were exposed with 20 mM Mito-
BloCK-6 or 0.1% DMSO and visualized using bright field micro-
scopy (Figure S6A), including staining with Coomassie brilliant
blue to visualize colony morphologies (Figure S6B; Mochizuki
and Furukawa, 1987
). MitoBloCK-6 exposure resulted in marked
cell death, whereas DMSO exposure did not cause cell
death or alter overall colony morphology. MitoBlock-6 may
trigger stem cell apoptosis. Release of cyt c was examined in
Figure 5. MitoBloCK-6 Impairs Substrate Oxidation In Vitro and Dis-
rupts Erv1 Binding
(A) Mitochondria from a strain expressing C-terminal histidine-tagged Erv1
were incubated with 50 mM MitoBloCK-6 or 1% DMSO for 30 min at 25
followed by solubilization in 1% digitonin buffer. As a control, 100 mg of extract
was withdrawn (T), and 500 mg lysate was incubated with Ni
-agarose beads.
The beads were washed and bound proteins (B) were eluted with SDS-PAGE
sample buffer. To test effectiveness of binding, 100 mg of the unbound protein
fraction (S) was also included. Proteins were analyzed by immunoblotting with
polyclonal antibodies against Mia40, Erv1, and cyt c.
(B) Recombinant Erv1 was preincubated with MitoBloCK-6 or 1% DMSO for
C, and then Erv1 (1 mM) was incubated with reduced Tim13 (15 mM)
and Mia40 (1 mM) in a time-course assay (Tienson et al., 2009 ). Aliquots were
removed at the indicated times, and free thiols on Tim13 were modified with
AMS addition. Oxidized and reduced Tim13 were detected by nonreducing
SDS-PAGE and immunoblotting with antibodies against Tim13.
(C–E) The same reconstitution assay was performed as in (B) with reduced
Tim13 (C), reduced Cmc1 (D and E) or mammalian ALR (E), and H
duction was monitored over a 30 min time period with the indicator Amplex
Red and displayed as pmol H
(one-way ANOVA, n = 3).
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MitoBloCK-6 Inhibits Erv1 in Mitochondria
86 Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc.
HSF1 cells exposed to MitoBloCK-6 (Figure 6A) using antibodies
against cyt c and visualized by fluorescence microscopy (Water-
house et al., 2001). MitoBloCK-6 addition resulted in a shift in cyt
c localization from mitochondria (marked with Tomm20) into the
cytosol (shown as diffuse staining that did not overlap with
Tomm20 staining). SAR compound ES-2, but not ES-1, also
caused cyt c release from mitochondria. Quantification indicated
that the number of cells in which cyt c was released was similar
with addition of MitoBloCK-6, ES-2, or actinomycin D, a known
apoptosis inducer (Figure 6B). In contrast, treatment with the
broad caspase inhibitor N-(2-Quinolyl)valyl-aspartyl-(2,6-difluor-
ophenoxy)methyl ketone (Q-VD-OPH) and the vehicle 0.1%
DMSO did not alter mitochondrial morphology or cause cyt c
release. In addition, downstream events in apoptosis, poly-
ADP-ribose polymerase (PARP) and caspase-3 cleavage, were
also detected with MitoBloCK-6 exposure (Figure 6C).
To support that MitoBloCK-6 specifically inhibited the survival
of hESCs and not of differentiated cells, HSF1 cells were induced
to differentiate with 10 mM retinoic acid followed by MitoBloCK-6
Figure 6. MitoBloCK-6 Induces Apoptosis
in hESCs
(A) HSF1 cells were treated with 20 mM Mito-
BloCK-6, ES-1, or ES-2 for 8 hr. As a positive
control, apoptosis was induced in cells by treat-
ment with 20 mM actinomycin D (ActD) for 8 hr.
Downstream caspases were inhibited by simulta-
neous addition of 25 mM Q-VD-OPH (caspase in-
hibitor) for 8 hr. Cells were fixed and analyzed by
immunofluorescence microscopy using anti-
bodies against cyt c (gree n) and Tomm20 (red).
Merged images are also depicted in panels with
Hoescht staining (blue) to mark nuclei. Scale bar,
20 mm.
(B) Quantification of data obtained in (A) and rep-
resented as percentage of cells that lost the
mitochondrial cyt c staining at 5 hr (white bars) or
8 hr (solid black bars) but retained Tomm20
staining. Data were collected from three inde-
pendent experiments. Error bars represent stan-
dard deviation (average percent ± SD; n = 4).
(C) As in (A), HSF1 cells were treated with 20 mM
MitoBloCK-6 or 20 mM ActD for the indicated time.
Whole cell extracts were analyzed by SDS-PAGE
and immunoblotted with antibodies for caspase-3
fragment and PARP. Tomm40 was included as a
loading control.
(D) As in (A), HSF1 cells were treated with 20 mM
MitoBloCK-6 for the indicated times, followed by
staining for alkaline phosphatase activity. Scale
bar, 500 mm.
(E) Analysis of alkaline phosphatase activity in
HSF1 cells after treatment with 0.1% DMSO,
20 mM MitoBloCK-6, 20 mM ES-1, or 20 mM ES-2
for 16 hr. Scale bar, 500 mm.
See also Figures S5 and S6.
exposure (Figure S6). Again, the images
show that colony morphology remained
intact when HSF1 cells were differenti-
ated with retinoic acid treatment and cells
did not die. To assess the earliest time
point at which MitoBloCK-6 perturbed
hESC viability, a time course assay was performed and hESCs
were stained for alkaline phosphatase activity (Shamblott et al.,
1998). hESC viability started to decline after 5 hr posttreatment
(Figure 6D). Twenty micromolar SAR compounds ES-1 and ES-
2 were applied to hESCs and stained for alkaline phosphatase
activity. Whereas ES-1 had no effect on cell growth, ES-2
inhibited cell growth similar to MitoBloCK-6 (Figure 6E). Taken
together, MitoBloCK-6 does not inhibit mitochondrial func-
tion in differentiated cells, but hESCs were susceptible to
MitoBloCK-6 and apoptosis was induced. The data suggest
a key role for ALR in hESC maintenance and show that
MitoBloCK-6 is a small molecule reagent that identifies this
Having characterized the effects of MitoBloCK-6 in vitro and
in primary cell culture systems, we applied MitoBloCK-6 to
developing zebrafish embryos, which is a useful in vivo verte-
brate model. The effect of MitoBlock-6 on mitochondrial func-
tion and zebrafish development was tested using previously
established parameters (Mendelsohn et al., 2006; Murphey
Developmental Cell
MitoBloCK-6 Inhibits Erv1 in Mitochondria
Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc. 87
and Zon, 2006). Zebrafish embryos were placed in either 1%
DMSO or 2.5 mM MitoBloCK-6 at 3 hr postfertilization (hpf)
and allowed to develop until 72 hpf. Higher concentrations of
MitoBloCK-6 were toxic to the fish. MitoBloCK-6- but not
DMSO-incubated embryos displayed ventral curvature of the
body and cardiac edema (Figures 7A and 7B). Furthermore,
we also treated fish with MitoBloCK-6 from 3–24 hpf, followed
by removal of MitoBloCK-6, and the zebrafish embryos were
identical to those exposed to DMSO at 72 hpf, indicating
that the effects of MitoBloCK-6 are reversible (M.E.J. and
C.M.K., unpublished data). Because ALR may play a role in
FeS cluster assembly and export (Lange et al., 2001), erythro-
poiesis may be defective (Shaw et al., 2006). Therefore, em-
bryos were stained with o-dianisidine, which binds to heme
(Lumsden et al., 2007), as a method to visualize hematopoietic
development. Whereas embryos exposed to 1% DMSO or
MitoBloCK-6 showed normal hematopoiesis, embryos treated
with MitoBloCK-6 showed erythrocyte pooling along the yolk
sac prior to entering the lower chamber of the heart and an
absence of red blood cells in the tail (Figures 7D and 7E).
To assess if the observed phenotypes were caused by ALR
inhibition via MitoBloCK-6, one-cell embryos were also injected
with 4 ng of a translation initiation codon (ATG) morpholino tar-
geted to ALR ( Figures 7C and 7F). This morpholino prevents
ALR translation in embryos. The phenotypes observed from
the morpholino-injected embryos were identical to that of
MitoBlock-6 exposure, suggesting that ALR is targeted.
Cardiac development was also investigated in a transgenic
zebrafish line, in which DsRed is targeted to mitochondria
under control of the heart-specific cardiac myosin light chain
promoter cmlc2 (Figures 7G–7I; Shu et al., 2007). Cardiac
development at day 3 in embryos exposed to DMSO was
similar to that of wild-type fish in that the heart is looped and
the mitochondria are also very bright (Figures 7G–7I). In
contrast, MitoBloCK-6 exposure retarded cardiac development
in that the hearts failed to loop by day 2, instead becoming
stringy and extended. In addition, the mitochondria were less
fluorescent (Figure 7H), which is likely indicative of dysfunc-
Figure 7. MitoBloCK-6 Treatment Impairs
Cardiac Development in Zebrafish
Embryos (3 hpf) were treated with 2.5 mM Mito-
BloCK-6 (B, E, and H) or 1% DMSO (A, D, and G)
or embryos were injected with an ATG morpholino
against ALR (C and F). Development was visual-
ized by microscopy at 72 hpf (A–C). Erythrocytes
were visualized by o-dianisidine staining at 72 hpf
(D–F); arrows indicate regions of red blood cell
accumulation in wild-type fish. Fluorescence
microscopy of zebrafish hearts (72 hpf) that con-
tained a mitochondrial-targeted DsRed included
embryos treated with 1% DMSO (G), 2.5 mM
MitoBloCK-6 (H), and buffer only (I).
See also Figure S7.
tional mitochondria. This developmental
defect is supported by a decreased
heart rate of 50% and 25% in embryos
treated with MitoBloCK-6 and the ALR
morpholino, respectively.
To confirm that the target of MitoBloCK-6 in vivo is indeed
ALR, embryos were treated with suboptimal concentrations of
MitoBloCK-6 (1 or 2 mM) and the ATG morpholino (1 or 2 ng)
in different combinations (Figure S7). Lower concentrations
of either MitoBloCK-6 or ATG morpholino did not impair
development; cardiac tissue was relatively normal and Ds-Red
fluorescence marking mitochondria was similar to the fish
treated with 1% DMSO. As zebrafish were treated with 2 mM
MitoBloCK-6 and 1 to 2 ng of ATG morpholino, defects in
development were additive. Specifically, the embryos displayed
cardiac edema and decreased fluorescence in cardiac tissue;
this phenotype was similar to that in embryos treated with either
2.5 mM MitoBloCK-6 or 4 ng ATG morpholino (Figure 7). Taken
together, the data strongly suggest that MitoBloCK-6 specifically
targets and blocks ALR function in zebrafish, which is marked
by impaired cardiac development.
We identify MitoBloCK-6 as a selective inhibitor of the Mia40/
Erv1 redox-mediated import pathway. Based on the assay in
which oxidation of substrate DTT by Erv1 was inhibited, the
mechanism by which MitoBloCK-6 may attenuate Erv1 activity
is to potentially interfere with binding or electron transfer be-
tween Mia40, cyt c, and/or oxygen. MitoBloCK-6 is a stable
compound. The hydroxyl group at the ortho position likely
stabilizes the compound (Crugeiras et al., 2009), and a similar
class of molecules has been identified in a small molecule screen
for inhibitors of Type III secretion (Nordfelth et al., 2005).
Import of CX
C proteins was reduced more than CX
C pro-
tein Tim8. Pfanner and colleagues have shown that a ternary
complex is formed by the substrate, Mia40, and Erv1 (Stojanov-
ski et al., 2008); MitoBloCK-6 may potentially interfere with the
formation of this ternary complex in a substrate-specific manner.
Strong inhibition of Mia40 import by MitoBloCK-6 was also
unexpected, because full-length Mia40 in yeast uses the
TIM23 pathway (as in Figure 2A), but a truncated version, similar
to human Mia40, that contains the core cysteine residues uses
Developmental Cell
MitoBloCK-6 Inhibits Erv1 in Mitochondria
88 Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc.
the Mia40/Erv1 pathway (Chacinska et al., 2008). That Mito-
BloCK-6 blocks Mia40 import suggests that the Erv1 pathway
may be important for coordinating disulfide assembly in the im-
ported Mia40, because mia40 mutants with cysteine mutations
that prevent correct disulfide bond formation are not viable (Ter-
ziyska et al., 2009). Surprisingly, import of substrates of the
TIM22 pathway (AAC and Tim23) was also reduced, which sug-
gests a broader role for the Mia40/Erv1 pathway in protein trans-
location. The potential role of Erv1 in the TIM22 pathway may be
difficult to dissect with yeast mutants; a subset of yeast mutants
did not show a defect in the carrier import pathway (Mesecke
et al., 2005; Rissler et al., 2005), whereas our import studies
with erv1 mutants resulted in general pleiotropic defects in
import (D.V.D. and C.M.K., unpublished data). Redox regulation
seems to be important in the TIM22 pathway, because the small
Tim proteins may undergo redox regulation and the cysteine-rich
protein Hot13 may also participate (Curran et al., 2004). Alterna-
tively, MitoBloCK-6 inhibition of Erv1 may change the redox
potential of the IMS, which may alter import ability of the small
Tim proteins. Additional experiments will be required to deter-
mine how MitoBloCK-6 specifically alters the TIM22 pathway.
ALR Has a Key Function in hESC Maintenance
and Zebrafish Development
Our strategy of screening with the yeast protein Erv1 was also
constructive, because MitoBloCK-6 inhibited the human homo-
log ALR with an improved IC
of 700 nM. High-resolution
crystallography and nuclear magnetic resonance studies of
four Erv1 family proteins, Arabidopsis thaliana Erv1 (Vitu et al.,
2006), rat ALR (Wu et al., 2003), human ALR (Banci et al.,
2011), and yeast Erv2 (Gross et al., 2002), reveal that the struc-
ture is highly conserved. Thus, our screen has produced small
molecules that work across species. This also has been shown
in an in vivo screen, in which we determined that MitoBloCK-1
of yeast Tim10 also inhibited Tim10 in mammalian mitochondria
(Hasson et al., 2010). Furthermore, Nunnari and colleagues
identified mdivi-1 as an inhibitor of the yeast fission component
Drp1 (Cassidy-Stone et al., 2008). mdivi-1 also abrogates
mammalian Drp1 and retards apoptosis by preventing mito-
chondrial outer membrane permeabilization.
Whereas MitoBloCK-6 inhibits activity of the Erv1 family
in vitro, a surprising finding was that MitoBloCK-6 did not inhi-
bit growth or function of differentiated cells in vivo. An initial
reason may be that a factor in the media inhibited MitoBloCK-
6 action. However, several types of media were tested, including
the permissive hESC media with differentiated cells, and
MitoBloCK-6 remained inactive. In contrast, MitoBloCK-6 spe-
cifically induced apoptosis in hESCs, suggesting ALR may
have a distinct role in pluripotent stem cell maintenance.
Published studies support a role for ALR in stem cells, because
ALR expression is enriched in embryonic, neuronal, and hema-
topoietic stem cells (Ivanova et al., 2002; Ramalho-Santos
et al., 2002). ALR has been reported to have a prosurvival role
in maintaining mouse pluripotent embryonic stem cells by
interacting with Drp1 (Todd et al., 2010a). However, Drp1 is a
cytosolic protein mediating mitochondrial fission, and it is not
apparent how IMS-localized ALR associates with Drp1; our
data support the model that ALR inactivation by MitoBlock-6
results in cyt c release, and the mitochondrial network collapses
as a consequence of apoptosis (Parone et al., 2006). We and
others have shown that Erv1 and ALR shuttle electrons to cyt c
(Bihlmaier et al., 2007; Dabir et al., 2007; Farrell and Thorpe,
2005). In differentiated cells, approximately 85% of the cyt c
population is distributed in the cristae in association with the
respiratory complexes, and 15% is located in the IMS in the
region between the inner and outer membrane (Bernardi and
Azzone, 1981); this 85% population of cyt c is released from
the cristae during apoptosis in differentiated cells (Scorrano
et al., 2002). However, hESC mitochondria lack numerous
cristae and display decreased respiration compared to differen-
tiated cells (Zhang et al., 2011), so the population of cyt c that
associates with ALR may be the critical pool that is released dur-
ing apoptosis. As a result of our preliminary finding, MitoBloCK-6
is an excellent tool to understand the contribution of mitochon-
drial to pluripotent stem cell function and differentiation. In
addition, MitoBloCK-6 may be important in translational strate-
gies to remove pluripotent hESCs that ‘fail-to-differentiate’ in
hESC transplantation studies. Removal of hESCs prior to trans-
plantation in patients is important because hESCs induce tera-
tomas in the wrong environment (Tang et al., 2011). Additional
studies are ongoing to understand how MitoBloCK-6 induces
apoptosis in hESCs.
In contrast to differentiated culture cells, zebrafish provide a
powerful model system for characterizing ALR function, because
cells are not transformed and are in their normal physiologic
setting of cell-cell and cell-extracellular matrix interactions (Mur-
phey and Zon, 2006). The embryos are also in simple buffered
water, so MitoBloCK-6 uptake may be enhanced. Defects
in mitochondrial biogenesis in zebrafish display varied pheno-
types. Mutations in the Tomm22 import component result in
defects in liver development (Curado et al., 2010), and mutations
in Fe-S cluster biogenesis typically impact erythropoiesis (Shaw
al., 2006; Wingert et al., 2005). Indeed, MitoBloCK-6 also
elicited gross morphologic and cardiac defects in zebrafish
that were akin to ALR downregulation. Overall, characterization
of MitoBloCK-6 supports that the chemical approach is valid
for developing probes to study protein translocation and under-
stand the role of protein import in development.
High-Throughput Screen for Erv1 Modulators
The primary chemical screen used fresh recombinant Erv1 (in buffer 30 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 100 mM NaCl,
1 mM EDTA) at a concentration of 10 mM, which was expressed as described
previously. A Titertek multidrop (Beckman Coulter) was used to dispense 25 ml
Erv1 or 25 ml of catalytically inactive enzyme Erv1C133S into wells of a clear
bottom 384-well plate (Greiner Bio One). A Biomek FX (Beckman Coulter)
was used to pin transfer 0.5 ml of compound from 1 mM stock or DMSO to
respective wells. Approximate screening concentration was 12.5 mM. After
completed compound transfer, all plates were incubated at 25
C in a humid-
ified incubator for 1 hr. A Titertek multidrop was used to dispense 15 mlof
Amplex Red-HRP (Sigma) mix into all wells of the 384-well plate. The final con-
centration of Amplex Red and HRP were 46 mM and 0.092 U/ml, respectively.
The Amplex Red-HRP solution was shielded from light during the entire
experiment. The plates were incubated for an additional 10 min, and then
15 ml of the substrate DTT (20 mM) was added to initiate the reduction of O
to H
. The plates were incubated for 12 min to achieve a maximal signal-
to-noise ratio in the kinetic liner range. Plates were then read at an endpoint
using an excitation wavelength of 545 nm and an emission wavelength of
590 nm. All operations were performed by an automated plate scheduler to
Developmental Cell
MitoBloCK-6 Inhibits Erv1 in Mitochondria
Developmental Cell 25, 81–92, April 15, 2013 ª2013 Elsevier Inc. 89
ensure consistency across the screening run. We chose compounds that
inhibited Erv1 activity by greater than 50%. Using a similar screening method-
ology as above, hit compounds were reconfirmed. Compounds that were
available were ordered from Asinex and Chembridge and assayed for IC
using a similar automated technique in 384-well plates, as previously
described. Serial dilutions of purchased compounds were performed with
robotic automation in 100% DMSO. Subsequently, compounds were pinned
into assay plate wells containing 10 mM Erv1, Erv2, or ALR.
Assays in hESCs
The hESC line HSF1 (NIH-UC01; UCLA Embryonic Stem Cell Research
Oversight committee-approved) was cultured in Stem Pro SFM (GIBCO)
supplemented with 10 ng/ml basic fibroblast growth factor on Matrigel (BD
Biosciences)-coated plates under 5% CO
, 95% air. Differentiation involved
culturing cells in Stem Pro SFM with 10 mM retinoic acid (Acros Organics) for
4 days. Cells were treated with the indicated concentration of the MitoBloCK
compounds or 0.1% DMSO as a control. For cytochrome c release analysis
using microscopy, cells were exposed to 20 mM actinomycin D (Sigma),
MB-6, ES-1, or ES-2 with 25 mM broad caspase inhibitor Q-VD-OPH (EMD
Millipore) (Caserta et al., 2003). Treatment with Q-VD-OPH and 0.1% DMSO
alone or in combination did not affect cell morphology. Following treatment,
cells were fixed with 3.7% formaldehyde for indirect immunofluores cence
study or lysed with Triton buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1%
Triton X-100, 1mM EDTA) for analysi s by SDS-PAGE. Bright field images
were acquired with Exi Blue (QImaging). Immunofluorescent images were
acquired with a 63X oil immersion objective on an LSM 5 PASCAL laser
scanning microscope (Carl Zeiss). Antibodies against cyt c (BD PharMingen),
Tomm20 (Santa Cruz), cleaved caspase-3 (Cell Signaling), and poly-ADP-
ribose polymerase (Cell Signaling) were purchased from the indicated ven-
dors. Nuclei were visualized by Hoechst (0.12 mg/ml) staining after straining.
Alkaline phosphatase activity staining was performed with the leukocyte
alkaline phosphatase kit (Sigma) as per manufacturer’s protocol. Coomassie
brilliant blue staining was performed by staining cells with Coomassie
brilliant blue solution (0.25% Coomassie brilliant blue R250, 45% methanol,
10% acetic acid) for 1 hr at room temperature. Cells were washed with phos-
phate-buffered saline followed by visualization, as described above.
Statistical Analysis
Quantitative analysis was performed in GraphPad Prism 5 software unless
otherwise stated. Statistical tests for significant deviation between samples
were performed using one-way ANOVA followed by Bonferroni’s posttest.
The alpha threshold for significance was <0.05 for all tests.
MitoBloCK-6 was analyzed using a battery of established in vitro, yeast,
mammalian cell-based, and zebrafish assays. These are described in detail
in the Supplemental Information. Yeast strains are listed in Table S1.
Supplemental Information includes seven figures, one table, and Supple-
mental Experimental Procedures and can be found with this article online at
We thank Dr. A. Barrientos (University of Miami) for the Cmc1 antibody,
J. Wijaya, J. Steffen, T. Hioe, S. Irving, and J. Hotter for excellent technical
assistance, and Dr. M. Jung (UCLA) for discussions about small molecule
chemistry. We acknowledge the use of the Chemical Database Service at
Daresbury. This work is supported by CIRM grants RS1-00313 and
RB1-01397 (to M.A.T. and C.M.K.) and NIH grants GM073981 (to C.M.K.
and M.A.T.), GM61721 (to C.M.K.), MH085683 (to C.M.K.), PNEY018228
(to M.A.T.), P01GM081621 (to M.A.T.), CA156674 (to M.A.T.), CA90571 (to
M.A.T.), and S10RR025631 (to P.W.). S.A.H. is a recipient of a USPHS
NRSA (GM084 96), and M.E.J. and C.J.D. are recipients of a USPHS
NRSA (GM07185) from NIH. D.V.D. is the recipient of a postdoctoral
fellowship from the United Mitochondrial Disease Foundation and NIH
(1F32GM084568). K.S. is a recipient of a CIRM training grant (TG2-01169).
P.W. is supported by the Development and Promotion of Science and Tech-
nology Talents Project from the Royal Thai Government. D.V.D., S.A.H.,
M.E.J., C.J.D., K.S., M.A.T., R.D., and C.M.K. designed research; D.V.D.,
S.A.H., K.S., M.E.J., P.W., and C.J.D. performed research. D.V.D., S.A.H.,
J.Z., R.D., and C.M.K. contributed to reagents and analytical tools. D.V.D.,
M.E.J., C.J.D., K.S., M.A.T., and C.M.K. wrote the paper.
Received: May 9, 2011
Revised: January 29, 2013
Accepted: March 6, 2013
Published: April 15, 2013
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    • "When all four cysteine residues were converted to serines (C–S) no interaction was seen with Mia40 and no localization to mitochondria was seen (Fig. 4C). MitoBloCK-6 has been shown to be a selective inhibitor of the Mia40/Erv1 pathway (Dabir et al., 2013). To evaluate whether MNRR1 is imported via this pathway, 293 cells were co-transfected with a Flag-tagged expression plasmid for MNRR1 and an HA-tagged expression plasmid for Akt. "
    [Show abstract] [Hide abstract] ABSTRACT: Our understanding of stress-associated regulatory mechanisms for mitochondria remains incomplete. We now report a new regulator of mitochondrial metabolism, the coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) which, based on the functionality described here, is renamed MNRR1 (Mitochondria Nuclear Retrograde Regulator 1). It functions in a novel way by acting in two cellular compartments, mitochondria and nucleus. In normally growing cells most MNRR1 is located in mitochondria; during stress most MNRR1 is now located in the nucleus. MNRR1 is imported to the mitochondrial intermembrane space by a Mia40-mediated pathway, where it binds to cytochrome c oxidase (COX). This association is required for full COX activity. Decreased MNRR1 levels produce widespread dysfunction including reduced COX activity, membrane potential, and growth rate, and increased reactive oxygen species and mitochondrial fragmentation. In the nucleus, MNRR1 acts as a transcription factor, one of whose targets is the COX subunit 4 isoform, COX4I2, which is transcriptionally stimulated by hypoxia. This MNRR1-mediated stress response may provide an important survival mechanism for cells under conditions of oxidative or hypoxic stress, both in the acute phase by altering mitochondrial oxygen utilization and in the chronic phase by promoting COX remodeling.
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    • "Human patients with a homozygous mutation in Erv1/ALR exhibit respiratory-chain deficiency, myopathy, congenital cataract, sensorineural hearing loss, and delayed development [59, 91] (Figure 4). In zebrafish the formation of heart and liver is impaired upon chemical inhibition or silencing of Erv1/ALR [78, 79]. In addition, chemical inhibition of Erv1/ALR induces apoptosis in human embryonic stem cells [79]. "
    [Show abstract] [Hide abstract] ABSTRACT: Disulfide bond formation drives protein import of most proteins of the mitochondrial intermembrane space (IMS). The main components of this disulfide relay machinery are the oxidoreductase Mia40 and the sulfhydryl oxidase Erv1/ALR. Their precise functions have been elucidated in molecular detail for the yeast and human enzymes in vitro and in intact cells. However, we still lack knowledge on how Mia40 and Erv1/ALR impact cellular and organism physiology and whether they have functions beyond their role in disulfide bond formation. Here we summarize the principles of oxidation-dependent protein import mediated by the mitochondrial disulfide relay. We proceed by discussing recently described functions of Mia40 in the hypoxia response and of ALR in influencing mitochondrial morphology and its importance for tissue development and embryogenesis. We also include a discussion of the still mysterious function of Erv1/ALR in liver regeneration.
    Full-text · Article · Nov 2013
  • [Show abstract] [Hide abstract] ABSTRACT: The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs, how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny, and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d, the in vitro assessment requires another day, and the in vivo assessment requires up to 12 additional weeks.
    Full-text · Article · Mar 2014
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