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Current
Projects
Timing is
important for all life processes. A life form without timing control would
deteriorate into a chaotic and futile mess, and various timing mechanisms
have to be coordinated in order to achieve the optimal health for the host
organism (see Figure, copied from Science, 309, 1197, 2005). We are interested
in studying how life is controlled by various timing mechanisms and how
they interact to coordinate life processes, using both budding yeast and
mice as model systems. In yeast, work in our lab and others have shown a
robust metabolic cycle where the culture alternates between oxidative and
reductive metabolism. We are currently investigating how this robust metabolic
cycle interacts with yeast cell cycle to maintain culture homeostasis and
genome integrity. In mice, we are interested in studying the potential regulatory
role of metabolism in circadian rhythm. Specifically, our lab had cloned
a transcription factor, named NPAS2, which is related to the master regulator
of circadian rhythm, Clock. NPAS2 mutant mice showed abnormal sleeping behavior
and did not adapt well to restricted feeding (RF). Interestingly, NPAS2
binds heme, and its DNA binding activity is modulated by CO and cellular
redox state. One experiment we are undertaking is to examine the molecular
basis for the RF defect in these mutant mice. In summary, the ultimate goal
of my research is to demonstrate that metabolic oscillation is crucial for
distinct timing mechanisms in both yeast and mammals.
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One of our
projects focuses on the investigation of novel molecular pathways implicated
in psychiatric disorders in hopes of advancing the understanding and treatment
of mental illness. Currently, we are investigating the role of neuronal
PAS domain protein 3 (NPAS3) in a putative animal model of schizophrenia.
NPAS3 is a brain-enriched basic helix-loop-helix PAS domain transcription
factor that is disrupted by translocation in affected members of a family
with schizophrenia. We have shown that mice harboring compound disruptions
in the genes for NPAS3 and the paralogous brain-specific transcription factor
neuronal PAS domain protein 1 (NPAS1) manifest behavioral and neuroanatomical
abnormalities reminiscent of schizophrenia. The Drosophila ortholog of NPAS3
and NPAS1, trachealess (Trh), controls stem cell proliferation by regulating
expression of breathless, a Drosophila fibroblast growth factor (FGF) receptor.
We have learned that npas3-/- mice are specifically deficient in FGF-mediated
hippocampal neurogenesis, as illustrated by the figure below showing virtual
abolishment of basal hippocampal neural precursor cell proliferation in
mice deficient in NPAS3 (lower left panel) relative to wild type mice (upper
left panel), as measured through immunohistochemical visualization of incorporation
of the thymidine analog bromodeoxyuridine.
We have further shown that this deficiency in hippocampal proliferation
results in selectively decreased thickness of the dentate gyrus granule
cell layer in mice deficient in NPAS3. We thus propose that aberrant neurogenesis
is implicated in the pathophysiology of schizophrenia through affecting
the normal functioning of critical brain regions, such as the hippocampus.
Our current work centers around behavioral, neuroanatomical, and neurochemical
studies of mice deficient in NPAS3 and/or NPAS1 to study the possible
role of neurogenesis in the adult CNS, specifically as it pertains to
mental illness.
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Budding yeast
grown under continuous, nutrient-limited conditions exhibit robust, highly
periodic cycles in the form of respiratory bursts. Comprehensive microarray
studies reveal that over half of the yeast genome is expressed periodically
during these metabolic cycles. Genes encoding proteins having a common function
exhibit similar temporal expression patterns, and genes specifying functions
associated with energy and metabolism tend to be expressed with exceptionally
robust periodicity. Essential cellular and metabolic events are coordinated
by the metabolic cycle, demonstrating that key processes
in a simple eukaryotic cell are compartmentalized in time. Thus, we have
described an ultradian metabolic cycle that is fundamentally important to
the life of a cell and surprisingly similar to the circadian cycle. In future
work, we plan to use a combination of approaches to identify the metabolites
and proteins that control the yeast metabolic cycle and determine how they
establish the cycle. This system offers the unique opportunity to reveal
how a cell optimizes its metabolism and coordinates fundamental processes
with cellular metabolic or redox state over time. Ultimately, understanding
the logic behind the yeast metabolic cycle will lead to fundamental insights
into biological temporal compartmentalization and the circadian cycle of
higher eukaryotes.
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Sprouty proteins were
initially discovered as the product of the Sprouty gene of D. melanogaster.
Flies bearing an inactivating mutation in the Sprouty gene display enhanced
branching morphogenesis in the larval tracheal system. Mutations in the
Branchless and Breathless genes attenuate branching morphogenesis. Branchless
and Breathless encode, respectively, the fibroblast growth factor (FGF)
and FGF receptor. Since Sprouty is an intracellular protein, it has been
hypothesized that this protein acts as a specific inhibitor of receptor
tyrosine kinase signaling. Whereas considerable evidence has accumulated
in support of this prediction, the precise functional mechanism used by
Sprouty to inhibit tyrosine kinase signaling remains unclear. In order
to initiate biochemical experiments pertinent to Sprouty function we expressed
and purified the mammalian Sprouty2 protein. Most fundamental to our observations
is the discovery that the conserved cysteine-rich "Sprouty" domain coordinates
an iron:sulfur complex. These and other features of the large oligomeric
assembly provide evidence that Sprouty proteins contain a dedicated sensing
capacity responsive to nitric oxide, redox state or both.
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A biochemical
screen was employed to identify PASK dependent phosphorylation targets.
This brute force technique encompassed a complex fractionation scheme utilizing
various chromatographic separation techniques to generate partially purified
pools of proteins that maintained native multiprotein interactions. In short,
a soluble S-100 extract was generated from 150 liters of HeLa cells. This
was then fractionated into approximately 1000 partially purified pools of
protein via ammonium sulfate precipitation, phenyl sepharose, Ni-NTA agarose,
Cibacron blue sepharose, anion exchanger, cation exchanger, and gel filtration
columns. Fractions were then individually assayed for PASK-dependent protein
phosphorylation with the addition of ?-32P ATP and -/+ recombinant PASK.
Bands specifically phosphorylated in a PASK-dependent manner were excised
and polypeptides identified by mass spectrometry. Phosphorylation sites
of identified substrates were then mapped via in-gel trypsinolysis, HPLC
separation of peptides and MALDI-TOF. Seven distinct proteins were identified
as bona fide in vitro substrates of PASK. Two of them, Alanyl-tRNA Sythetase
and Ribosomal protein S3A are involved in translation. Two others, GAPDH
and Pyruvate Kinase are enzymes in glycolysis. The other four substrates
play roles in intermediary metabolism. Mapping of the PASK-dependent phosphorylation
sites has revealed a substrate consensus motif, RXXS/T, and is shedding
light on the functional mechanisms of these phosphorylation events. Consistent
with yeast PASK, these studies demonstrate a critical evolutionarily conserved
role of PASK in the regulation of protein synthesis and sugar metabolism
that may participate in the in the etiology of cancer and diabetes.
Yeast, when grown in continuous cultures, demonstrate synchronized respiratory
and metabolic oscillations characteristic of distinct oxidative and reductive
phases. Glycogen is one of the many parameters, including dissolved oxygen
concentration, ATP, NADH, respiratory rates and genome wide transcription
levels, that cyclically change during these metabolic oscillations. We
have previously demonstrated glycogen and sugar flux regulation by PAS
Kinase (PASK) in S. cerevisiae through phosphorylation and inhibition
of UDP-glucose pyrophosphorylase and glycogen synthase. We hypothesized
that the cyclic fluctuation in glycogen levels during these metabolic
oscillations would be regulated by PASK dependent phosphorylation. Yeast
PASK knock-out (KO) and PASK-TAP tagged strains were generated. Aliquots
of yeast culture from TAP tagged and KO strains were harvested every 30
minutes across the 4-hour respiratory cycle. Immunoprecipitated TAP-tagged
PASK was incubated in vitro with an exogenous substrate to determine its
kinase activity. Glycogen levels were also determined spectroscopically,
in both wild-type and KO strains, at each time point. Phospho-specific
antibodies to glycogen synthase (Gsy2p) were used to investigate PASK
dependent phosphorylation of Gsy2p during the cycle. Consistently, PASK
activity and glycogen levels cycled - peaking in the reductive phase.
Unexpectedly, glycogen levels continued to cycle in the PASK KO strains
even though phosphorylation of glycogen synthase, using phospho-specific
antibodies, was absent in the KO. However, glycogen levels were markedly
elevated in the PASK null strain as compared to wild-type. Cyclic glycogen
levels may be alternatively explained by known allosteric regulation of
glycogen synthase activity by glucose 6-phosphate levels. Thus PASK dependent
phosphorylation of glycogen synthase is not responsible for the cyclic
fluctuation of glycogen, but appears to play a regulatory role in glycogen
metabolism in the transition between different phases of the yeast metabolic
cycle.
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