Research Interests
The primary focus of the Systems Biology laboratory is identifying and understanding the genes and signaling pathways that, when mutated, contribute to the pathophysiology of cancer and neurodegeneration. We take advantage of RNA interference (RNAi) and novel proteomic approaches to identify the enzymes that control cell growth, proliferation, and survival. For example, after screening the human genome for more than 600 kinases and 200 phosphatases—called the “kinome” and “phosphatome”, respectively—that act with chemotherapeutic agents in controlling apoptosis, we identified several essential kinases and phosphatases whose roles in cell survival were previously unrecognized. We are asking several questions. How are these survival enzymes regulated at the molecular level? What signaling pathway(s) do they regulate? Does changing the number of enzyme molecules present inhibit waves of compensatory changes at the cellular level (system-level changes)? What are the system-level changes after reduction or loss of each gene?
Key regulators of mitochondrial signaling and apoptosis
Mitochondria are dynamic organelles best known for producing more than 90% of cellular ATP and for releasing cytochrome c during apoptosis. They also modulate ion homeostasis, oxidize carbohydrates and fatty acids, and participate in numerous other molecular signaling pathways. Disruption of mitochondrial function contributes to at least 50 diseases, including cancer and neurodegenerative disorders, underscoring the importance of identifying the molecular components that regulate the normal and pathological functions of these organelles. Similar to the discovery of the Bcl-2 family members that play key roles in mitochondrial apoptosis, the discovery of phosphatases and kinases that regulate mitochondrial function will provide critical insights into the physiology of this organelle and how that physiology is disrupted in disease.
We are particularly interested in kinases and phosphatases that mediate the pro-apoptotic and cell survival functions of mitochondria. Various intrinsic cell-death stimuli compromise the integrity of mitochondria and cause the release of cytochrome c into the cytoplasm. That release represents a key event in the apoptotic cascade, leading to apoptosome (Apaf1/cytochrome c) formation and initiator caspase (caspase 9) activation in the cytosol. We have active mitochondrial systems biology projects studying, for example, whether a cellular energy deficit is required to permit entry into apoptosis, and whether we can identify druggable enzymes (kinases and phosphatases) that regulate mitochondrial phosphorylation status and intrinsic apoptosis.
Key regulators of lipid signaling and autophagy
Macroautophagy is a dynamic process whereby portions of the cytosol are encapsulated in double-membrane vesicles and delivered to a lysosome for degradation. The lipid phosphatidylinositol-3-phosphate, or PI(3)P, is concentrated on autophagic vesicles and recruits effector proteins critical for this process. We are particularly interested in kinases and phosphatases that regulate lipid signaling and autophagy. With this in mind, production of PI(3)P by the class III phosphatidylinositol 3-kinase (PI3K) Vps34 has been well established; however, phosphatases that dephosphorylate this lipid during autophagy are unknown. To identify such enzymes, we screened human phosphatase genes by RNA interference (RNAi) and found that loss of a single phosphatase, a dual-domain protein tyrosine phosphatase (PTP), increases cellular PI(3)P and hyperactivates autophagy. Further, we discovered that the catalytic domains localize to PI(3)P-positive vesicles and their presence reduces cellular PI(3)P. Intriguingly, structural docking revealed the active site accommodates PI(3)P with proper orientation for catalysis, and we have data to suggest that this enzyme functions as a direct PI(3)P phosphatase. Our findings suggest a novel role for PTPs and provide insight into the regulation of autophagy. We also have drug discovery projects investigating the role of autophagic flux in chemoresistance and chemosensitivity. Mechanistic knowledge of autophagy is critical for understanding and targeting therapies for several human diseases, including cancer, Alzheimer disease, and Parkinson disease, in which abnormal autophagy may be pathological.
The most prominent group of enzymes that modify membrane lipids is the family of PI3K. An often underappreciated aspect of PI3K is that there are eight different PI3K isoforms, each with distinct characteristics. They are organized by substrate specificity and structure into three different PI3K classes, Class I, Class II, and Class III. By far the most well studied of the PI3Ks are the Class I isoforms. The Class I alpha isoform of PI3K is arguably the most commonly mutated kinase in the human genome. These mutations result in an increased level of kinase activity and a subsequent cellular growth advantage through downstream activation of the AKT/TSC2/mTOR pathway. Understanding the contribution and role of all eight PI3K isoforms, in disease and in normal biology, is an important component to unlocking the potent mechanisms controlling lipid signaling. We have ongoing projects investigating the relative contribution of each isoform to cellular growth, autophagy, and chemosensitivity as a gateway to investigating each isoform’s relative function in human disease.
Parkinson disease–associated genes in cancer and neurodegeneration
Mitochondrial homeostasis is a crucial feature of the cell, and it is therefore proposed that altered mitochondrial bioenergetics, particularly the disruption of mitochondrial fission and fusion, may play an important role in cancer and in neurodegeneration. We have ongoing projects investigating the role of PTEN-induced kinase 1 (PINK1 or PARK6) and its role in mitochondrial dynamics. The objective of this project is to determine the cellular consequences of deregulated mitochondrial fission and fusion and whether or not this dynamic process provides unique and novel molecular target.
The receptor tyrosine kinases are frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence or presence of growth factor stimulation. We sought to identify mechanisms that promote ligand-dependent or ligand-independent receptor tyrosine kinase activation, using informatics to searching for kinases that are coordinately dysregulated with receptor tyrosine kinases in human tumors. Our bioinformatic analysis has identified Parkinson disease–associated kinases that are amplified and overexpressed in human cancers. Down-regulation of these kinases compromises receptor tyrosine kinase activation and reduces signaling flux through the PI3K/mTOR pathway. The resulting metabolic crisis induces cell death, indicating that these kinases are required for efficient tumor cell growth and survival in the context of receptor tyrosine kinase amplification.
Mouse models of Parkinson and Alzheimer disease
Parkinson disease (PD) is a progressive neurodegenerative condition primarily characterized by severe loss of locomotor function, which results from the specific depletion of regulatory dopaminergic (DA) neurons in the midbrain substantia nigra. The vast majority of PD cases occur in sporadic fashion and are identified primarily after the sixth decade of life. While several explanations have been suggested to account for this observation, it is currently unclear why the single most important risk factor for developing PD is increased age. We have ongoing genetic mouse modeling projects to specifically delete expression of genes from dopaminergic neurons in the mouse brain using conditional knockout technology. We are using this mouse model to determine whether the loss of specific genes in DA neurons recapitulates key features of PD in whole-animal models or in cultured primary cells.
We are also utilizing transgenic mice that express the mutant human tau protein under the control of an endogenous mouse promoter. The expression of the mutant human tau is higher than the expression of the endogenous mouse tau protein. This hyperphosphorylated, insoluble mutant human tau protein accumulates in the brain with age. We have projects testing these transgenic mice with regards to life span, neuron degeneration, and the onset of neurofibrillary tangle formation. These Alzheimer disease lesions are predicted to be similar to those found in brain lesions of human Alzheimer patients.
