The McKnight Lab

Current Projects

Circadian Rhythm and Yeast Metabolic cycle:

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.


Mental Illness:

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.


Ultradian Metabolic cycle of yeast:

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.


Sprouty Signaling:

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.


PAS Kinase:

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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