Metabolic Underpinnings of Cancer and Stem Cells

These studies have led to the initiation of a significant effort in the lab to understand the fundamental metabolic underpinnings of cancer and stem cells. We are employing in vitro and in vivo systems in conjunction with biochemical, genetic, metabolomic and cell biological approaches.

In summary, our goal to functionally annotate the mitochondrial proteome has enabled discovery of biochemical functions important for mitochondria, elucidation of the genetic basis of two human diseases and catalysis of future studies with a direct impact on common human diseases. In addition to the six projects described above, we are actively pursuing the functions of a number of additional protein families.

Integrated Control of Metabolism by PASK-dependent Signaling

We have a growing but incomplete understanding of the mechanisms whereby the body senses its nutrient status and responds to adapt cellular and organismal behavior accordingly. The resulting energetic efficiencies are of obvious evolutionary importance as organisms faced a variety of challenging environmental situations, including prolonged exertion, episodic food shortage and competition for resources. In modern human societies, however, these adaptations often have negative health consequences. One clear example is the propensity of most mammals to store excess ingested calories, primarily as fat, in anticipation of an ensuing period of food scarcity. That period rarely comes in our society today and the result is obesity, with all of its attendant comorbidities.

We are interested in the functions of PASK, an evolutionarily conserved serine/threonine kinase, in coupling nutrient status with metabolic state, energy storage and growth. PASK-/- mice were resistant to high-fat diet induced obesity, hepatic steatosis and insulin resistance. This phenotype appears to be due to hypermetabolism in PASK-/- mice in vivo as measured by indirect calorimetry and in isolated skeletal muscle. These findings suggest an important physiological role of PASK in regulating metabolism and controlling energy balance in mammals (Hao et al. 2007).

A major focus of our lab is to understand the mechanisms by which PASK controls both cellular and organismal energy metabolism. Specifically, we are working to understand how PASK regulates mitochondrial metabolism in skeletal muscle (and probably in many cell types). We are also working to understand how PASK controls hepatic lipid metabolism (as described in Hao, et al. 2007). We have strong in vivo data, using genetics and pharmacology, that PASK plays a key role in mediating the effects of a Western diet to promote dyslipidemia and disease (Wu et al. 2014).


My laboratory is currently exploring three areas.  While distinct, these programs are all centered upon cellular metabolic homeostasis—the concept that cells must constantly monitor their nutrient, metabolic and hormonal environments and adjust their behavior accordingly.  First, as much of metabolic control is enacted at the level of mitochondria—in ways that are mostly not understood—we initiated a project to functionally annotate the eukaryotic mitochondrial proteome.  To date, this effort has elucidated the genetic basis of two human diseases and several fundamental mitochondrial functions.  Second, we have developed and validated an experimental platform for the identification of low affinity protein-metabolite interactions.  It has enabled discovery of novel metabolite-based allostery, which is the primary ancestral and acute modality for metabolic control—not discussed in detail (Orsak et al. 2012). Finally, the discovery of conserved metabolic effectors and allosteric regulation culminate in our efforts on an ancient and allosterically regulated control circuit centered on the nutrient sensing PASK protein kinase, which plays an evolutionarily conserved role in partitioning available carbon between its various fates. Using both broad and deep approaches, our goal is to define the molecules and their interactions that enable robust and responsive cellular metabolic control.  The projects described next are examples of the questions we ask and the approaches we take to ask them, but we are always looking for new ways to understand metabolism and how it integrates with cell behavior.

Functional Annotation of the Mitochondrial Proteome

Mitochondria are small but complex organelles with a disproportionately large impact on human health. Changes in mitochondrial enzyme activities, respiratory capacity, genome sequence and superoxide generation play important roles in the pathogenesis of heart failure, cancer, neurodegenerative disorders such as Parkinson's, Alzheimer's and Huntington's disease and in aging and longevity. The best current inventory of mammalian mitochondrial resident proteins consists of 1098 proteins (Pagliarini et al. 2008).  Surprisingly, nearly 300 of these proteins are uncharacterized (Pagliarini et al. 2008).  This includes many that are highly conserved throughout eukarya, a strong indication that they perform a fundamentally important function.  The genes that encode the mitochondrial proteome are heavily represented amongst known human disease genes, with about 20% of predicted human mitochondrial proteins implicated in one or more hereditary diseases (Andreoli et al. 2004, Elstner et al. 2008).  Presumably, the quarter of the mitochondrial proteome that is uncharacterized contains many others that await discovery.  Making this connection would be greatly facilitated by an understanding of the genetic connections, biochemical properties and physiological functions of these proteins.  Therefore, elucidating the functions of these uncharacterized, conserved mitochondrial proteins will not only explain important aspects of mitochondrial biology, but will also provide a framework for identifying new human disease genes. 

As a first step toward this goal, we bioinformatically identified many mitochondrial protein families that are pan-eukaryotic and unstudied. Using yeast mutants generated in our lab, we have analyzed the loss-of-function growth phenotypes for each. We have also determined the subcellular and sub-mitochondrial localization for each protein. Such higher throughput, standardized studies have then been used to guide specific hypotheses for a subset of proteins.  These projects, all focused on proteins for which no function had ever been described, have progressed to various levels of understanding; six are published.

  1. Hao, et al (2009) Science (Hao et al. 2009),(Hao and Rutter 2009). We found that Sdh5 is necessary for the assembly of the succinate dehydrogenase complex (Complex II) of the electron transport chain, where it catalyzes the insertion of the obligate flavin-adenine dinucleotide cofactor into the catalytic subunit. As a result of these mechanistic functional studies, we were able to discover that familial mutations in human SDH5 (SDHAF2) cause a paraganglioma tumor syndrome.

  2. Heo, et al (2010) Mol Cell (Heo et al. 2010),(Taylor and Rutter 2011). Our understanding of mitochondrial protein quality control has lagged behind that of the cytosol and other organelles. We found that Vms1 is required for the stress-responsive mitochondrial recruitment of Cdc48/VCP/p97 and Npl4, which play a role in protein extraction and degradation. Further studies led to the conclusion that Vms1 is a critical component of a previously unknown system for mitochondrial protein quality control, eliminating damaged or misfolded proteins that promote progressive mitochondrial dysfunction. We are in the process of characterizing Vms1-/- mice generated in our lab.

  3. Chen, et al (2012) Cell Metabolism (Chen et al. 2012). Mitochondria actively control the production and removal of reactive oxygen species. One key mechanism is the regulated organization of the electron transport chain complexes into large macromolecular assemblies (called supercomplexes), which are believed to facilitate efficient electron flow thereby limited reactive oxygen species production. The components and regulation of the supercomplex assembly system are almost completely unknown. We discovered that Rcf1 is required for the normal assembly of respiratory supercomplexes in yeast and mammals. Deletion of the RCF1 gene caused impaired respiration and elevated mitochondrial oxidative stress and damage. The identification of the function of this conserved protein family enables us, for the first time, to genetically probe the importance of respiratory supercomplexes in mitochondrial function and integrity.

  4. Chen, et al (2014) EMBO J. (Chen et al. 2014). Msp1 is a AAA-ATPase protein that we found to be integral to the mitochondrial outer membrane, with the catalytic domain facing the cytosol. Through a combination of genetics and biochemistry, we showed that it extracts proteins that mis-localize to the mitochondrial outer membrane, which we observed as a surprisingly common phenomenon. We demonstrated this function for the protein family in yeast, human cells and knockout mice, with profound impact on mitochondrial function and metabolic phenotype (Chen et al. 2014).

  5. Van Vranken, et al (2014) Cell Metabolism (Van Vranken et al. 2014). The key observation that enabled discovery of the function of the Sdh8 protein was its stoichiometric co-purification with Sdh1. We went on to show that Sdh8 acts downstream of Sdh5 to facilitate the incorporation of FAD-conjugated Sdh1 into the intact SDH complex (Figure 1). In its absence, yeast, flies and mammalian cells exhibit impaired SDH assembly and activity and impaired respiration. Our data strongly suggest that the sole function of the Sdh8 protein family is to chaperone assembly of the SDH complex, which is likely necessary because, as we found, in the absence of Sdh8, Sdh1 conjugated with the redox-active FAD cofactor catalyzes the production of toxic reactive oxygen species. We are currently exploring the disease implications of this recent discovery.

  6. Bricker, et al (2012) Science (Bricker et al. 2012). The fate of pyruvate is one of the most important metabolic decisions made by eukaryotic cells. Most normal, differentiated mammalian cells partition pyruvate primarily toward transport into mitochondria where it is oxidized for efficient ATP production. The partitioning of pyruvate in stem cells, cancer cells and failing hearts, however, is different—away from mitochondrial oxidation. Our ability to understand the molecular basis for these metabolic distinctions has been hampered by the surprising fact that the mitochondrial pyruvate transporter had not been identified until now. We discovered a protein complex consisting of Mpc1 and Mpc2 that constitutes the major mitochondrial pyruvate transporter in yeast, Drosophila, and humans. Empowered by this discovery, we found that three families with children suffering from lactic acidosis and hyperpyruvatemia had causal mutations in MPC1.