Download Chemical Genomic Profiling for Identifying

TOXICOLOGICAL SCIENCES 95(1), 182–187 (2007)
doi:10.1093/toxsci/kfl131
Advance Access publication October 16, 2006
Chemical Genomic Profiling for Identifying Intracellular Targets of
Toxicants Producing Parkinson’s Disease
Julie Doostzadeh,*,1 Ronald W. Davis,† Guri N. Giaever,†,‡ Corey Nislow,†,‡ and James W. Langston*
*The Parkinson’s Institute, Sunnyvale, California 94087; †Stanford Genome Technology Center, California 940304; and
‡University of Toronto Donnelley Centre for Cellular and Biomolecular Research, Toronto, Ontario M5S 3E1, Canada
Received June 19, 2006; accepted October 5, 2006
The yeast deletion collection includes ~4700 strains deleted for
both copies of every nonessential gene. This collection is a powerful resource for identifying the cellular pathways that functionally
interact with drugs. In the present study, the complete pool of
~4700 barcoded homozygous deletion strains of Saccharomyces
cerevisiae were surveyed to identify genes/pathways interacting
with 1-methyl-4-phenylpyridinium (MPP+) and N,N-dimethyl4-4-bipiridinium (paraquat), neurotoxicants that can produce
Parkinson’s disease. Each yeast mutant is molecularly ‘‘barcoded’’
the collections can be grown competitively and ranked for sensitivity by microarray hybridization. Analysis data from these
screens allowed us to determine that the multivesicular body
pathway is an important element of toxicity induced by both
MPP+ and paraquat. When yeast genes that when deleted showed
sensitivity to MPP+ and paraquat toxicity were analyzed for their
homology to human genes, 80% were found to have highly
conserved human homologs (with e < 108). Future work will
address if these human genes may also functionally interact with
MPP+ and paraquat toxicity.
Key Words: chemogenomics; neurotoxicants; Parkinson’s disease.
The search for a link between environmental factors and the
risk of developing Parkinson’s disease (PD) has been investigated for over 20 years. One interesting observation is that
the structurally simple molecule, MPTP (1-methyl-4-phenyl1,2,3,6-tetrahydropyridine) could induce many of the motor
features of PD in humans (Langston, 1985; Langston et al.,
1984). In the intervening years, many studies have highlighted
links between PD and exposure to insecticides and herbicides
(Hertzmann et al., 1990; Seidler et al., 1996). However, to date
no causative agent has been unequivocally identified. One of the
few compounds linked to PD is paraquat (N,N-dimethyl-4-4bipiridinium) (Broussolle and Thobois, 2002; Di Monte, 2003;
Hertzmann et al., 1990; Liou et al., 1997) a widely used herbicide that is structurally similar to MPTP.
It has been suggested that paraquat exerts its toxicity by
several potential mechanisms: (1) generation of the superoxide
anion and the formation of more toxic reactive oxygen species;
(2) oxidation of cellular nicotinamide adenine dinucleotide phosphate (reduced), a major source of reducing equivalents for the
intracellular reduction of paraquat; and (3) lipid peroxidation,
which results in the oxidative degeneration of cellular polyunsaturated fatty acids (Suntres, 2002). Of particular relevance to
PD are several recent studies indicating that paraquat damages
nigrostratial dopaminergic neurons when administrated to mice
either alone or in combination with other toxicants (Brooks et al.,
1999; McCormack et al., 2002; Thiruchelvam et al., 2002).
The molecular mechanism by which MPTP exerts its toxicity
is well characterized. Because of its lipophilicity, MPTP readily
crosses the blood-brain barrier, and once in the brain, it is
biotransformed to 1-methyl-4-phenylpyridinium (MPPþ) a
reaction catalyzed by monoamine oxidase type B (MAO-B)
(Chiba et al., 1985). This reaction takes place in glia and MPPþ
accumulates in mitochondria, where it disrupts cellular respiration (Gluck et al., 1994) and causes neuronal cell death.
Because both of toxins are selective for the system known to be
vulnerable in PD (the dopaminergic nigrostriatal system)
elucidating the molecular basis of cell death triggered by MPPþ
and paraquat could provide valuable insight into the mechanisms underlying their toxicity.
The goal of this study was to identify cellular targets or target
pathways that underlie the mechanisms of paraquat and MPPþ
toxicity, by taking advantage of a genome-wide assay known as
‘‘chemogenomic profiling.’’ This technology allows for profiling
of the relative sensitivity to drugs or toxins of the gene products
of the entire genome in yeast (Brown et al., 2006; Giaever et al.,
2002, 2004; Lum et al., 2004; Steinmetz et al., 2002; Winzeler
et al., 1999) and targets or target pathways can be identified. Our
results provide a comprehensive in vivo snapshot of the genomewide cellular response to MPPþ and paraquat perturbation.
METHODS
1
To whom correspondence should be addressed at The Parkinson’s
Institute, 1170 Morse Ave., Sunnyvale, CA 94087. Fax: (408) 734-8522.
E-mail: [email protected].
Strains and media. Yeast strains were grown in yeast peptone dextrose
(YPD) media at 30C. The homozygous deletion pool was constructed as
The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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GENES IMPLICATED IN MPPþ AND PARAQUAT TOXICITY
183
described (Giaever et al., 2004; Pierce et al., 2006) and stored in 20-ll aliquots at
80C. Aliquots of the pool were thawed and diluted in YPD to an optical density
at 600 nm (OD600) of 0.0625 and a final volume of 700 ll. Compound was then
added and the pool was grown for five generations in a Tecan GENios microplate
reader (Tecan, Durham, NC). Cells were maintained in logarithmic phase by
robotically diluting cultures every five doublings using a Packard Multiprobe II fourprobe liquid-handling system (PerkinElmer, Wellesley, CA) (Giaever et al., 2002).
Experimental design. Our experimental design is detailed in Giaever et al.
(2002). Briefly, 0.062 OD600 units of homozygous deletion pool is diluted into
700 ll of YPD containing compound. This volume of media permits 300 individual cells of each deletion strain to be sufficiently represented at the beginning of the experiment. Cells are grown for five generations until the OD600
reaches 2.0 and cells are harvested.
Harvested cells are subjected to genomic DNA extraction according to the
Zymo Research (YeaStar DNA) kit. Fifteen microliters of genomic DNA is
added to a 100 ll of PCR reaction, one with ‘‘UPTAG’’ primers and another
with ‘‘DOWNTAG’’ primers.
Thirty microliters of each TAG PCR products are mized and hybridized to
TAG3 microarrays (Giaever et al., 2002), stained, and scanned as described in
(Fig. 1).
Data analysis. Fitness-defect scores were calculated for each strain in the
pool for each experiment. These scores are based on a tag-specific algorithm
that takes into account the intensities of each tag on the experimental array and
the corresponding intensities on a set of control arrays performed on the pool
without compound (the control set) (Giaever et al., 2004).
Tag intensities are log transformed, mean normalized, and the intensities are
averaged into a single value. A mean and standard deviation are calculated for
the uptag and downtag intensities for each strain across the set of control arrays.
A z-score for upstream and downstream tags for each strain is then calculated
by taking the difference of the average intensities between the control and
treatment and dividing by the standard deviation of the control-set intensities.
The result is two z-score values for the upstream and downstream tags; these are
then averaged into a single fitness-defect score for the strain.
RESULTS AND DISCUSSION
Paraquat and MPPþ
MPTP is rapidly converted into MPPþ, a potent neurotoxin,
by MAO-B. Another prototypic neurotoxicant that shares structural homology with MPPþ is paraquat, a common herbicide
(Fig. 2). Because the molecular mechanisms involved in MPPþ
and paraquat are not completely understood, and because there
is sufficient homology between yeast and human biochemical
pathways we tested the hypothesis that the chemogenomic
assay could reveal new insights into mechanism of action of
MPPþ and paraquat.
Genes Displaying Sensitivity to MPPþ and Paraquat Toxicity
The sensitivity of 4700 homozygous deletion yeast strains
was tested in the presence of sublethal doses of MPPþ (250lM)
and paraquat (2000lM). These doses were derived from a serial
dose response curve to define conditions in which wild-type
yeast cells manifest 10% reduction in growth rate. Sensitivity for
each treated and untreated strain was obtained by calculating the
fitness values for homozygous collections in rich media (YPD)
by monitoring the abundance of the molecular barcodes over
time as described (Giaever et al., 2002; Winzeler et al., 1999).
FIG. 1. KanMX gene replacement cassette. A PCR-generated (Baudin,
1993; Wach, 1994) deletion strategy was used to systematically replace each
yeast open reading frame from its start-to-stop codon with a KanMX module and
two unique 20-mer molecular bar codes. The presence of the tags can be detected
via hybridization to a high-density oligonucleotide array, enabling growth
phenotypes of individual strains to be analyzed in parallel. At the beginning of
the experiment, all 4700 homozygous strains are mixed together at equal abundance and allowed to grow in the presence of compound. Most strains will be
unaffected by drug and be present at equal abundance over time. Those strains
that are drug sensitive, will rapidly become underrepresented in this pool and
eventually be depleted altogether. If we had a snapshot of the relative abundance
of the strains as a function of time we could identify which strain grows the
slowest and therefore identify the drug target. DNA array intensity reflects tag
abundance ¼ strain abundance ¼ strain growth or drug sensitivity.
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DOOSTZADEH ET AL.
FIG. 2. Two-dimensional molecular structures of paraquat (left) and
MPPþ (right).
Analysis of the data allowed us to identify 96 genes for
MPPþ and 141 genes for paraquat with a growth defect in the
homozygous deletion collection. The list of genes showing a
growth defect in the presence of MPPþ and paraquat are pro-
vided in Figure 3 and supplementary data. None of these genes,
when deleted in homozygous diploids, showed a growth defect
in the presence of dimethyl sulfoxide (vehicle control). Those
genes showing sensitivity to MPPþ and paraquat (96 genes for
MPPþ and 141 genes for paraquat) are dispensable for viability
and, in the absence of compound, and are not required for
normal growth rate as a homozygote, suggesting that the slowgrowth phenotype of these strains is a direct result of MPPþ
and/or paraquat toxicity. The description, biological process,
molecular function, cellular component, and analyzed array
information for all screened genes are provided as supplementary material. The genes enriched for sensitivity to both
compounds were specific, that is to say, they were not generally
enriched in the majority of other conditions that this collection
has been subjected to (see Brown et al., 2006; Giaever et al.,
2002, 2004; Steinmetz et al., 2002).
A broad functional distribution of enriched genes, genes that
are showing sensitivity in the presence of MPPþ and paraquat,
when deleted in the homozygote, is illustrated in supplementary
figures (saccharomyces genome database [SGD] Gene Ontology
[GO] Term Mapper; Dwight et al., 2002). The frequency of those
categories across the genome is compared to the frequency of
those genes in each category that are sensitive to MPPþ and
paraquat. The functional distribution analysis (Fig. 4) revealed
that those genes that confer sensitivity to paraquat, are categorized in processes such as cellular localization, establishment of
localization, vesicle-mediated transport, endosome transport,
FIG. 3. Scatter plot visualization of results from homozygous profiling of Paraquat (a) and MPPþ (b). The X axis represents the sensitivity of each strain when
treated for five generations of growth in YPD in 5mM paraquat and 250lg/ml MPPþ. All strains with a sensitivity score of 1.5 or greater are highlighted. This value
comprises all strains that are at least three standard deviations more sensitive than the mean of all strains.
GENES IMPLICATED IN MPPþ AND PARAQUAT TOXICITY
185
FIG. 4. Broad functional classification of the yeast genome that manifests sensitivity to paraquat (a) or MPPþ (b) from the GO Term Mapping function
available on the SGD website (www.yeastgenome.org). The classifications represent general GO parental terms (GO Slim) for the process ontology. The frequency
of those categories across the genome is shown in magenta and is compared to the frequency of those genes in each category that are sensitive to paraquat (a) and
MPPþ (b).
vacuolar transport, late endosome transport, and ubiquitindependent protein catabolism. The broad functional distribution
of enriched genes for MPPþ were overrepresented for the negative
regulation of biological processes (e.g., negative regulation of
metabolism and transcription as well as for ubiquitin-dependent
protein catabolism). These results indicate that both enriched
genes sets for MPPþ and paraquat are highly enriched for similar
cellular but distinct from the genome as a whole.
Ubiquitin-dependent Protein Catabolism via the
Multivesicular Body Pathway is Engaged in Toxicity of
Both MPPþ and Paraquat
A more precise functional map was determined for enriched
genes for both MPPþ and paraquat using (SGD GO Term
Mapper; Dwight et al., 2002). This functional map of the genes
sensitive to MPPþ and paraquat revealed processes related to
ubiquitin-dependent protein catabolism via the multivesicular
body pathway with a p value < 108 and a p value < 1010 in
the presence of MPPþ, and paraquat, respectively. Genes in
ubiquitin-dependent protein catabolism via the multivesicular
body pathway showing very similar sensitivity to MPPþ and to
paraquat include STP22, VPS25, DID4, VPS24, SNF7, SRN2,
VPS36, VPS20, SNF8, and VPS 28. The yeast vacuolar protein
sorting (VPS) proteins have human homologs, and are known
components of the endosomal sorting complex required for
transport (ESCRT) complexes I, II, and III, each of which is
required for sorting proteins to the lumenal membranes of
multivesicular bodies (Bowers et al., 2004).
The functional map for enriched genes showing slow growth
in the presence of paraquat revealed that other processes are
engaged in upstream of ubiquitin-dependent protein catabolism
via the multivesicular body pathway (with a p value < 108)—
these processes include (1) late endosome to vacuole transport
(NHX1, VPS27, VPS8, VPS60, DID4, VPS24, DID2, SNF7,
VPS38, VPS20, VPS4); (2) protein targeting to vacuole (VPS27,
CCZ1, STP22, ATG18, MON1, VPS25, DID2, ARP6, SRN2,
VPS36, VPS9, SNF8, VPS28, VPS30); (3) intracellular protein
transport (LST7, LST4), vacuolar transport (CCZ1 and BRO1);
and (4) endosome transport (NHX1, VPS27). For all yeast
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genes (n ¼ 26) involved in processes with p values < 108 the human homologs were analyzed using the SGD data base (www.
yeastgenome.org)—the description, e-values, and percentage
of human homologs for these yeast genes were also determined
(SGD GO and PSIBLAST [www.ncbi.nlm.nih.gov/BLAST/
Blast]). The alignment of enriched yeast genes compared to
their respective human homologs revealed a high sequence
homology (> 80%) for 76% of those genes. These data suggest
that chemogenomic assays such has those presented here may be
a valuable for filtering and prioritizing both toxins and genes
involved in Parkinson’s syndromes.
Defining the functional interactions between specific gene
products and toxicants is fundamental for determining the
molecular mechanisms that underlie their toxicity. Chemogenomic profiling of various compounds demonstrated that this
genome-wide assay specifically allows the identification of gene
products or in this study—genetic pathways that functionally
interact with compounds or toxins (Giaever et al., 2004; Lum
et al., 2004). In the our study, we used yeast as a model organism
to interrogate the complete genome set of homozygous yeast
deletion strains to enrich genes that show sensitivity to MPPþ
and paraquat. We identified 95 genes and 140 genes that exhibit
slow growth, as homozygotes, when exposed to MPPþ and paraquat, respectively. Several of these genes include TSA1, ALD1,
TCH1, FET3, FTR1, and CCC2 encode yeast homologs of
human proteins involved in pathways important for paraquat
toxicity.
It will be of great interest to investigate the role of these
homologs in PD model. Because MPPþ cytotoxicity is known
to involve respiratory chain coplex I alterations, which
Saccharomyces cerevisiae lacks, it is important to assay the
contribution of this complex to the toxicity we observe. However, our results do implicate the ubiquitin-dependent catabolism in both MPPþ and paraquat toxicity. A functional map of
enriched genes revealed that ubiquitin-dependent protein catabolism via the multivesicular body pathway is most likely engaged in the processes underlying MPPþ and paraquat toxicity
( p value < 108). Functional maps of enriched genes that show
slow growth in the presence of paraquat revealed that processes
such as late endosome to vacuole transport, protein targeting to
vacuole, intracellular protein transport, vacuolar transport, and
endosome transport are affected by paraquat. Current models
suggest that the ESCRT complexes recognize cargo via the
ubiquitin tag, and mediate sorting into the lumenal multivesicular
bodies (MVB) membranes. The Vps27p-Hse1p complex binds
phosphatidylinositol-3 phosphate. Vps27 recruits ESCRT-I
(VPS23, VPS28, VPS 37) from the cytoplasm to the endosome,
where ESCRT-I interacts with monoubiquitinated cargo. Consequently, ESCRT-I activates ESCRT-II (VPS22, VPS25, VPS
36), which in turn initiates the oligomerization of a group of at
least four small coiled-coil proteins (Vps2, Vps24, Vps20,
Snf7), resulting in the formation of ESCRT-III. The ESCRT-III
(VPS20, VPS32) complex concentrates the MVB cargo and
recruits additional factors such as Bro1 and the AAA-type
adenosine triphosphatase Vps4. Bro1 functions in the recruitment
of the deubiquitinating enzyme Doa4 that removes the ubiquitin
tag from cargo proteins.
Mounting pathological, genetic, and experimental evidence
suggests that dysfunction of the ubiquitin-dependent protein
catabolism, either at the level of the proteasome itself or at the
level of ubiquitination, plays a role in the pathogenesis of PD
(McNaught and Jenner, 2001). Our results clearly indicate that
toxicity mediated by MPPþ and paraquat is regulated by nonoverlapping sets of conserved genes and engages ubiquitindependent protein catabolism. Respectively, 80 and 76% of
enriched yeast genes showing sensitivity to MPPþ and paraquat
toxicity (with e < 108) have highly conserved homologs in the
human genome. The utilization of mammalian animal models
in which single genes along the ubiquitin-proteasome pathway
are manipulated can be used as powerful tools to expand the
results of this study to mammals.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
ACKNOWLEDGMENTS
This study was supported by a grant from Centers for Parkinson’s Disease
Environmental Research—National Institute of Environmental Health Sciences/National Institutes of Health. We thank Anjani Attili, Carla Camos Alves,
Sheri Mohandessi, and Rama Yerramilli for bioinformatic assistance.
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