Proteins

Cell metabolism is a tightly controlled process that uses numerous feedback and feed-forward mechanisms to provide the necessary requirements to sustain growth. Many of these regulatory mechanisms are mediated through the post-translational modification of enzymes that serve to modulate activity and function. My laboratory studies the link between cell metabolism, protein post-translational modifications, and gene expression. We utilize mass spectrometry to investigate both novel and established metabolic feedback mechanisms and how these go awry in disease. Current work centers on histone modifications derived from cell metabolism and how these modifications are disrupted in diabetes and cancer.

The Horton lab uses a variety of biochemical and biophysical methods to investigate DNA binding proteins. Recent projects include the discovery of a novel mechanism of regulation of enzyme activity using filamentation. Filamentation, or self-association into polymers of varied lengths, by enzymes has only recently been appreciated as a widespread phenomenon, although the purpose of filamentation is not known in most cases. We discovered this phenomenon in 2010 in a sequence specific endonuclease, SgrAI, and have now determined its high resolution structure via cryo-electron microscopy. We have also performed a full kinetic analysis showing that filamentation greatly expedites the activation of the enzyme, and also allows for the sequestration of enzyme activity onto only a subset of available substrates. The other major project in the lab concerns the triggering of autoimmune diseases in genetically susceptible individuals. We study proteins from the human parvovirus B19, a virus which often precedes the development of autoimmune diseases like rheumatoid arthritis, autoimmune hepatitis, and lupus. We study how these proteins interact with cellular components to modulate the immune system into loss of self-tolerance.

Mechanistic studies of the Nrf2/Keap1 signaling pathway Oxidative stress, an imbalance between production and removal of reactive oxygen species, can damage biological macromolecules including DNA, proteins and lipids ( Oxidative damage to biological macromolecules can have profound effects on cellular functions and has been implicated in cancer, inflammation, neurodegenerative diseases, cardiovascular diseases and aging. Eukaryotic cells have evolved anti-oxidant defense mechanisms to neutralize reactive oxygen species (ROS) and maintain cellular redox homeostasis. One of the most important cellular defense mechanisms against ROS and electrophilic intermediates is mediated through the ARE (antioxidant responsive element, or electrophile responsive element) sequence in the promoter regions of phase II and antioxidant genes. The ARE-dependent cellular defense system is controlled by the transcription factor Nrf2. Recent advances in the mechanistic studies of this pathway have provided the following models for Nrf2 regulation: Keap1, a key player in the activation of this pathway, has been identified to function as a molecular switch to turn on and off the Nrf2-mediated antioxidant response. Under basal condition, Keap1 is in the off position and functions as an E3 ubiquitin ligase, constantly targeting Nrf2 for ubiquitination and degradation. As a consequence, the constitutive levels of Nrf2 are very low. The switch is turned on when oxidative stress or chemopreventive compounds inhibit the activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase, resulting in increased levels of Nrf2 and activation of its downstream target genes. The switch is turned off again upon recovery of cellular redox homeostasis; Keap1 travels into the nucleus to remove Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the nuclear export signal (NES) in Keap1. In the cytosol, the Nrf2-Keap1 complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in degradation of Nrf2. We are currently working on the detailed steps of the Nrf2-Keap1-ARE pathway in response to oxidative stress and to chemopreventive compounds The protective role of Nrf2 in arsenic-induced toxicity and carcinogenicity Another direction of our research is to understand the molecular mechanisms of toxicity/carcinogenicity of environmental pollutants and the endogenous cellular defense systems to cope with pollutants. Drinking water contaminated with arsenic is a worldwide public health issue. Arsenic has been classified as a human carcinogen that induces tumors in the skin, lung, and bladder. Arsenic damages biological systems through multiple mechanisms, one of them being reactive oxygen species. The ARE-Nrf2-Keap1 signaling pathway, activated by compounds possessing anti-cancer properties, has been clearly demonstrated to have profound effects on tumorigenesis. More significantly, Nrf2 knockout mice display increased sensitivity to chemical toxicants and carcinogens and are refractory to the protective actions of chemopreventive compounds. Therefore, we hypothesize that activation of the ARE-Nrf2-Keap1 pathway acts as an endogenous protective system against arsenic-induced toxicity and carcinogenicity. The following Specific Aims are intended to further elucidate the mechanism of Nrf2-activation in protection from arsenic-induced toxicity/tumorigenicity. We will (1) determine the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and cell transformation using a model cell line UROtsa, (2) define the molecular mechanisms of activation of the ARE-Nrf2-Keap1 pathway by arsenic, sulforaphane, and tBHQ, and (3) define the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and tumorigenicity using Nrf2 knockout mouse as a model. So far, we have demonstrated a protective role of Nrf2 against arsenic-induced toxicity using cell culture and Nrf2-/- mouse model. We have provided evidence demonstrating that Nrf2 protects against liver and bladder injury in response to six weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. Recently, we submitted another manuscript to Toxicological and Applied Pharmacology, reporting our long-term study of the effect of Nrf2 on arsenic-mediated cell transformation and tumor formation. In this study, we provide evidence demonstrating the importance of Nrf2 activation in preventing the carcinogenetic effects induced by long-term exposure to low-dose arsenic both in vitro and in vivo. The UROtsa cell line was used to show that daily exposure to the Nrf2 inducer, tBHQ, alleviated arsenic-induced hypomethylation and cell transformation. Moreover, tBHQ treatment reduced tumorigenicity of arsenic-transformed cells in SCID mice. Chronic treatment with arsenic also compromised the Nrf2-dependent defense response in the bladder epithelium in Nrf2+/+ implicating the important role of Nrf2 in protecting against arsenic-induced carcinogenicity. This study supports the advantages of using dietary supplements specifically targeting Nrf2 as a chemopreventive strategy to protect humans from various environmental insults that may occur on a daily basis. Identification and development of Nrf2 activators into dietary supplements/ for disease preventio Identification and development of Nrf2 inhibitors into therapeutic drugs to enhance the efficacy of cancer treatment High-throughput screening of Nrf2 activators or inhibitors: we are screening a chemical library and a natural product library to identify compounds that are able to activate or inhibit ARE-luciferase activity using a stable cell line established in our laboratory, MDA-MB-231-ARE-Luc. Based on the critical role of Nrf2 in disease prevention, using Nrf2 activators to boost our antioxidant response represents an innovative strategy to enhance resistance to environmental insults. Once Nrf2 activators are identified and the specificity of these compounds in activating Nrf2 is validated, the compounds will then be tested for the mechanism by which they confer cellular protection and the feasibility of using these compounds for disease prevention using various disease models. On the other hand, recent findings point to the “dark side” of Nrf2, as studies have shown that Nrf2 promotes cancer formation and contributes to chemoresistance. Using a genetic approach, we have provided evidence that the level of Nrf2 correlates well with cancer cell resistance to several therapeutic drugs, demonstrating that Nrf2 is likely responsible for chemoresistance. More recently, we have reported a study on Nrf2 expression in endometrial cancer patients (117 cases). We found no detectable Nrf2 expression in complex hyperplasia, 28% Nrf2 positive cases in endometrial endometrioid carcinoma (type I), and 89% Nrf2 positive cases in endometrial serous carcinoma (type II). Please note that type II endometrial cancer is the most malignant and recurrent carcinoma among various female genital malignancies. Furthermore, inhibition of Nrf2 by overexpressing Keap1 sensitized SPEC-2 cells, which are derived from type II endometrial cancer, or SPEC-2 xenografts to cisplatin using both cultured cells and SCID mouse models. These studies demonstrate that Nrf2 contributes to chemoresistance in many cancers originating from different organs and illustrate the urgent need for identification of Nrf2 inhibitors and for the development of Nrf2 inhibitors into druggable compounds to enhance the efficacy of cancer treatment. We have identified the very first Nrf2 inhibitor and characterized its use to sensitize cancer cells to chemotherapy. We just submitted a manuscript to Science reporting our discovery. The following is the abstract of the manuscript: “The major obstacle in cancer treatment is the resistance of cancer cells to chemotherapy. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive to cancer cell survival. We have identified brusatol as a selective Nrf2 inhibitor that is able to sensitize cancer cells and xenografts to chemotherapeutic drugs by enhancing the degradation of Nrf2 and inhibiting the Nrf2-dependent antioxidant response. These results suggest that brusatol can be developed into an adjuvant drug to enhance the efficacy of cancer treatments”. The importance of this project: (i) the use of an Nrf2 inhibitor to enhance the efficacy of cancer therapeutics represents a novel approach to caner treatment. Nrf2 inhibitors may be used in a broad spectrum across many types of cancers and chemotherapeutic drugs to increase the effectiveness of cancer treatment. Development of brusatol into an adjuvant for clinical use to sensitizer many cancer types to treatment will have an enormous impact on human health worldwide. (ii) Brusatol will be extremely useful for the mechanistic investigation of Nrf2 regulation by complex cellular networks. Cross talk between the Nrf2 signaling pathway and others During the last couple of years, crosstalk between the Nrf2 pathway and other important pathways has emerged. Our group has identified two separate branches that converge with the Nrf2 pathway, the p53-p21(Cip1/WAF1) pathway and the autophagy pathway. Crosstalk is mediated by the direct interaction between p21 and Nrf2, and Keap1 with p62, respectively. In the p53-p21 study, we provide molecular and genetic evidence suggesting that the previously suggested antioxidant function of p53 or p21 is mediated through activation of the Nrf2 pathway. Mechanistically, p21 is able to stabilize Nrf2 by competing away Keap1, thus, activating the Nrf2-mediated antioxidant response. Therefore, the interaction between Nrf2 and p21 represents a fine-tuning mechanism between life and death according to the level of stress. In the study with p62 and Nrf2, we reported a novel mechanism of Nrf2 activation by autophagy deficiency through a direct interaction between Keap1 and p62. In response to stress, cells can utilize several cellular processes, such as autophagy, a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1-dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a non-canonical cysteine-independent mechanism. These work was published in Molecular Cell and Molecular and Cellular Biology, respectively, both are high profile journals. Furthermore, both articles were highlighted in Molecular Cell and Science Signaling, respectively, indicating the importance and high impact of the projects.

Catharine Smith, PhD, focuses on epigenetic mechanisms of gene expression, particularly their regulation through signaling pathways and their modulation by anti-cancer drugs. Epigenetic mechanisms play a very important role in transcriptional regulation of genes but the specifics of these mechanisms require ongoing study. A very exciting new area of research focuses on how these mechanisms are disrupted during tumorigenesis but may also be harnessed to treat cancer. Signaling pathways control the expression of key genes in non-cancerous cells but are often misregulated during the process of oncogenesis. Chromatin proteins and transcription factors that interact with chromatin are often targets of these pathways. Two projects in the lab are directed at the interface of signaling pathways and chromatin. First, Dr. Smith is interested in the mechanism by which the female reproductive steroid, progesterone, regulates target genes in the physiological context of chromatin. Chronic progestin exposure has been linked to increased incidence of breast cancer in post-menopausal women on hormone-replacement therapy. However, the function of the progesterone receptor in mammary tissue and its role in oncogenesis are not well understood. Current studies in this area are directed at the role of chaperone proteins in determining how the progesterone receptor functions at target genes in chromatin and how it is impacted by other signaling pathways.Second, her lab has discovered a novel cAMP signaling pathway that regulates cell cycle progression and are focused on identifying specific components and targets of this pathway.Third, histone deacetylases (HDACs) are key transcriptional regulatory proteins. Inhibitors that target these enzymes have shown great promise as anti-cancer drugs and are currently in clinical trials. However, a lack of knowledge of HDAC biology has made it difficult to predict which tumors will respond to these drugs. HDACs are known to participate in gene repression, but recent work indicates that they are also transcriptional coactivators. Further studies on the mechanism of gene repression through HDAC inhibitors will provide insight into the role of these enzymes as coactivators.

William Montfort, PhD, determines the atomic structures of proteins and seeks to understand how protein structure gives rise to protein function – both in vitro and in living cells. At their heart, the problems have a fundamental structure-function question, but also address questions of importance to human health. Approaches include X-ray crystallography, rapid kinetic measurements, spectroscopy, theory, protein expression, drug discovery, molecular genetics and related techniques.Dr. Montfort is particularly interested in nitric oxide signaling mechanisms. Nitric oxide (NO) is a small reactive molecule produced by all higher organisms for the regulation of an immensely varied physiology, including blood pressure regulation, memory formation, tissue development and programmed cell death. He is interested in two NO signaling mechanisms: binding of NO to heme and the nitrosylation (nitrosation) of cysteines. NO, produced by NO synthase, binds to soluble guanylate cyclase (sGC) at a ferrous heme center, either in the same cell or in nearby cells. Binding leads to conformational changes in heme and protein, and to induction of the protein’s catalytic function and the production cGMP. NO can also react with cysteine residues in proteins, giving rise to S-nitroso (SNO) groups that can alter protein function. He continues to study the mechanistic details surrounding cGMP and SNO production, and the signaling consequences of their formation.For reversible Fe-NO chemistry, Dr. Montfort is studying soluble guanylate cyclase and the nitrophorins, a family of NO transport proteins from blood-sucking insects. Our crystal structures of nitrophorin 4 extend to resolutions beyond 0.9 angstroms, allowing us to view hydrogens, multiple residue conformations and subtle changes in heme deformation. For reversible SNO chemistry, he is studying thioredoxin, glutathione S-nitroso reductase (GSNOR) and also sGC. For regulation in the cell, Dr. Montfort and his group have constructed a model cell system based on a human fibrosarcoma called HT-1080, where sGC, NO synthase, thioredoxin and GSNOR can be manipulated in a functional cellular environment. With these tools, they are exploring the molecular details of NO signaling and whole-cell physiology, and undertaking a program of drug discovery for NO-dependent diseases. Keywords: Structural Biology, Drug Discovery, Cancer, Cardiovascular Disease

Xianchun Li, PhD, is interested in understanding the physiological, biochemical, molecular and evolutionary bases of fundamental processes in the life history of insects such as embryonic polarity, metamorphosis, developmental commitment, host usage and environmental adaptation. One focus of his research is to elucidate the reciprocal signaling interactions between plants and insects, and the resulted on-going defense (in the case of plants) / counterdefense (in the case of herbivorous insects) phenotypic arm race over ecological time scale, with emphasis on the genetic machinery that percepts and transduces the reciprocal cues into genome and regulate defense / counterdefense phenotypes. Working systems include Helicoverpa zea, the corn earworm, a polyphagous noctuide of economic importance, and Drosophila melanogaster, the fruit fly, a model organism. State of arts and traditional techniques are combining to identify the cues and to uncover the signaling transduction cascade that links environmental cues, gene expression and the resulted defense/counterdefense phenotypes. This research may lead to characterization of genes for designing new insecticides and/or genetically modifying crops. The second focus of Dr. Li’s research is to define, globally, the regulatory triangle between nuclear receptors (NRs), their ligands, and cytochrome P450s (P450s) in Drosophila melanogaster. Nuclear receptors (NRs) constitute a transcription factor superfamily that has evolved to sense and bind endogenous (e.g., hormones) and/or exogenous (e.g., naturally-occurring or synthetic xenobiotics) signal compounds, resulting in differential expression of particular target genes, which underlies a range of fundamental biological processes, including growth, development, reproduction, behavior, host usage, and environmental adaptation. Many of those cue chemicals, namely NR ligands, are synthesized and/or metabolized by members of the P450s gene superfamily, whose expression may be regulated by certain NRs. Bioinformatics analyses as well as systematic functional genomic techniques such as microarray, X-ChIP, mutation and ectopic expression will be combined to define the genome-wide regulatory interaction loops between NRs and P450s as well as to assign, at least partially, functions of individual NRs and P450s in the life history of fruit fly. Given the evolutionary conservations of homologous NRs and P450s between vertebrates and invertebrates, the results obtained in this project are expected to provide insights into the reciprocal regulatory interactions between NRs and P450s in other animals including humans as well as to provide great insights into new avenue for human NR ligand identification and NR-related drug design. The third focus of his research is to investigate the molecular mechanisms of Bt and conventional insecticide resistance, which is a major threat in current IPM system. In collaboration with Dr. Bruce Tabashnik, Timothy Dennehy, and Yves Carriere in our Department, Dr. Li is going to compare Bt toxin binding affinity and other defects of natural (s, r1, r2, r3) and artificial mutant PBW (Pink Bollworm) cadherin proteins and thus define the key functional domains of PBW cadherin.

Carol Gregorio, PhD, performs research in her lab that is focused on identifying the components and molecular mechanisms regulating actin architecture in cardiac and skeletal muscle during normal development and disease. Control of actin filament lengths and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends of the filaments. Striated muscle is an ideal model system to test for the functional properties of various actin regulatory proteins due to the precise organization and polarity of cytoskeletal components within repeating sarcomeric units (for example, the ~1 mm long actin filaments are easily resolved by light microscopy). Using this system, she can combine advanced cell biological and biochemical approaches with direct tests of physiological function in live beating muscle cells.The research objectives of the laboratory can be broadly summarized as follows: 1) understanding the cellular mechanisms involved in the assembly, regulation and maintenance of contractile proteins in cardiac muscle in health and disease; 2) deciphering the mechanisms critical for precisely specifying and maintaining the lengths of actin filaments; 3) discovery of novel models of de novo cardiac muscle assembly, with special emphasis on differentiating murine embryonic stem (ES) cells to study all stages of heart muscle development. Actin is an indispensable structural element of cells and is the major component of heart muscle. Changes in actin, caused by genetic mutations, which have been identified in humans, are a frequent cause of several forms of cardiomyopathy. Her lab is determining how genetic defects in this protein affect muscle force generation and muscle contraction, leading to sudden cardiac death.

Hendrikus Granzier, PhD, studies the mechanisms whereby the giant filamentous protein titin (the largest protein known) influence muscle structure and function. His lab has shown that titin functions as a molecular spring that mediates acute responses to changing pathophysiological states of the heart. They also study the role of titin in cardiac disease, using mouse models with specific modifications in the titin gene, including deciphering the mechanisms that are responsible for gender differences in diastolic dysfunction. An additional focus of Dr. Granzier’s lab is on nebulin, a major muscle protein that causes a severe skeletal muscle disease in humans. Based on previous work, they hypothesize that nebulin is a determinant of calcium sensitivity of contractile force. To test this and other concepts, he uses a nebulin knockout approach in the mouse. Research is multi-faceted and uses cutting-edge techniques at levels ranging across the single molecule, single cell, muscle, and the intact heart. His research group is diverse and has brought together individuals from several continents with expertise ranging from physics and chemistry to cell biology and physiology.

Joan Curry, PhD, stands in the field of research related to interfacial chemistry, which is a focus within physical chemistry. Within interfacial chemistry, she focuses on chemistry of molecules at the interfaces where solids and liquids come together. The term solid here includes mineral and bacterial surfaces found in soils and sediments, metal and oxide machine surfaces and cell surfaces found in the human body. Molecules can be water and ions that bathe soil surfaces, organics that lubricate machine parts and large biomacromolecules, such as proteins and lipopolysaccharides, attached to cells that mediate cell adhesion. Her specific interests are: 1) determining the effect of confinement on liquids in general and lubricants in particular and 2) characterizing the adhesive properties of cell surface biomacromolecules. The primary goal of this work is to understand how biomacromolecules that cover cell surfaces influence the interaction and adhesion of cells with other cells and with solid surfaces. Cells can be either bacteria or human cells. It is important to understand bacterial adhesion because it is the first step in biofilm formation, which has numerous undesirable consequences ranging from heat exchanger fouling to medical implant infections. Currently, very little is known about how bacterial surface biomacromolecules mediate adhesion and therefore it is still not possible to control or manipulate the process. Human cell adhesion is also mediated by biomacromolecules, in particular proteins that bind to one another through specific lock and key mechanisms. The structure of many cell adhesion proteins is well known but their function is still poorly understood. In collaboration with Ronald Heimark (Surgery), Dr. Curry is working to understand how heavy metals such as cadmium affect the binding of cell adhesion proteins called cadherins. This work will help scientists understand better how heavy metals may lead to birth defects and in adults could accelerate cardiovascular disease. This work is experimental and involves direct force measurements between biomembrane covered mica surfaces with the Surface Forces Apparatus (SFA). With the SFA it is possible to measure the magnitude and distance dependence of molecular forces acting between two flat surfaces with angstrom and nanonewton resolution.