Specialty/Research Focus:
Genomics and proteomics; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Protein Function and Structure; Proteins and metalloenzymes
Research Summary:
Research in my laboratory is directed at understanding basic questions concerning structure-function relationships in ion channels. We are especially interested in using a variety of naturally-occurring toxins as probes of both ion transport and gating processes in these transmembrane macromolecules. To better understand the nature of these toxin binding sites, we have cloned and expressed synthetic genes which encode polypeptide modulators of voltage-sensitive sodium and calcium channels and also of a peptidic blocker of the gastric-chloride channel. By creating designed mutant forms of these toxins, and characterizing their functional properties and 3-dimensional structures, we hope to construct a picture of the channel region involved in binding. Complementary mutagenesis of channel products helps to define the structure of the channel itself, and this information is not accessible via other experimental approaches. Definition of toxin regions essential for this interaction will also allow us to design useful pharmacological agents directed at the target channel.
Specialty/Research Focus:
Bioinformatics; Genomics and proteomics; Molecular and Cellular Biology; Molecular genetics; Gene Expression; Transcription and Translation
Research Summary:
Our research group is interested in how regulatory proteins are targeted to the correct DNA binding sites at the correct time. Transcription factors are directed to their genomic targets by DNA sequence, local chromatin structure, and protein-protein interactions. These modulators of transcription factor binding are not independent but function both cooperatively and competitively to regulate where transcription factors bind. Understanding how these modulators affect transcription factor binding in vivo remains a major unsolved biological problem.
We use the model organism Saccharomyces cerevisiae to address the disconnect between the presence of the correct DNA binding sequence and true regulatory protein binding, integrating both experimental and computational approaches to: i) investigate transcription factor binding in response to environmental stress, ii) identify and characterize the mechanisms directing transcription factor target selection, and iii) and develop bioinformatics tools to analyze and interpret ChIP-seq experiments and chromatin structural patterns.
Research Summary:
Protein Methylation in Growth and Differentiation and Copper in Brain Development
A role for protein methylation in the signal transduction pathways for growth and differentiation is beginning to be elucidated. PC12 cells elaborate neurites when exposed to nerve growth factor (NGF). We recently found that NGF regulates the methylation of several proteins during differentiation and that the methylation of these proteins is essential for outgrowth of neurites. Among the proteins methylated are RNA-binding proteins that are hypothesized to regulate gene expression at the RNA level. Our goal is to identify the methylated proteins involved in nerve cell development and to determine the mechanisms for how they affect nerve cell differentiation.
Copper deficiency is known to affect brain development, and Menkes disease is fatal due to impaired brain development from low brain copper. A reduction in S-adenosylhomocysteine hydrolase (SAHH) levels, as occurs in copper deficiency, may affect brain development by inhibiting protein methylation. This is because when SAH, the substrate for SAHH accumulates, all methyltransferases and methylation reactions are inhibited. We are examining whether copper deficiency affects protein methylation in copper deficient neonatal mouse brain and PC12 cells. Yet another approach that we are using is to examine a SAHH-disruption mutant in yeast. These cells exhibit markedly defective growth and some abnormal properties related to copper. We are testing the hypotheses that protein methylation is required for growth and that SAHH plays a role in copper trafficking in yeast.
Specialty/Research Focus:
Neurology; Cytoskeleton and cell motility; Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Signal Transduction; Inherited Metabolic Disorders; Transgenic organisms
Research Summary:
My laboratory seeks to understand the molecular basis of myelination and myelin diseases. Myelin is a multi-lamellar sheath that invests large axons and permits rapid conduction of nerve signals. Failure in myelin synthesis and myelin breakdown cause several important neurological diseases, including multiple sclerosis, leukodystrophies and peripheral dysmyelinating neuropathies.
In some of these diseases, genetic mutations cause defects in cytoskeletal, adhesion and signaling molecules. I work with a team of undergraduate and graduate students, postdoctoral fellows, technicians, senior scientists and many international collaborators to discover how these molecules normally coordinate cell-cell and cell-extracellular matrix interactions to generate the cytoarchitecture of myelinated axons. We use a variety of approaches, including generation of mice carrying genetic abnormalities, cultures of myelinating glia and neurons, imaging, biochemistry and morphology to understand the role of these molecules in normal and pathological development. By comparing normal myelination to the abnormalities occurring in human diseases, we aim to identify molecular mechanisms that pharmacological intervention might correct.
For example, we described how the protein dystroglycan associates with different proteins, some of which impact human neuropathies, depending on a proteolitic cleavage that can be regulated to improve the disease. Similarly, we found that molecules such as integrins and RhoGTPAses are required for glia to extend large processes that will become myelin around axons. In certain neuromuscular disorders, defective signaling pathways that converge on these molecules cause failure to produce or mantain an healthy myelin Finally, in collaborations with scientists and clinicians in the Hunter J. Kelly Research Institute, we are generating transgenic forms of GalC, an enzyme deficient in Krabbe leukodystrophy, to investigate which cells requires the enzyme. Investigating how GalC is handled may help find a cure for this devastating disease.
Specialty/Research Focus:
Autoimmunity; Cell growth, differentiation and development; Gene Expression; Immunology; Molecular and Cellular Biology; Molecular genetics; Signal Transduction; Transcription and Translation; Transgenic organisms
Specialty/Research Focus:
Bioinformatics; Genomics and proteomics; Immunology; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Neurobiology; Gene Expression
Research Summary:
Molecular Basis of Erythroid Differentiation
Our research has four long-term themes: 1.) Analysis of the molecular basis of differential gene expression among tissues and during development with hemoglobin synthesis and red blood cell development as models; 2.) Application of molecular and genetic advances to inherited diseases; 3.) Iron metabolism; 4.) Study of gene variation in populations and divergence of gene loci during evolution.
Some examples of our unfinished and/or planned projects in each category are as follows:
1a. and 3.) The Belgrade rat and hemoglobin deficit mouse, mutant strains that are genetically iron deficient, are each undergoing analysis to determine the defective step in iron metabolism. The Belgrade rat has a mutation that inactivates DMT1 (divalent metal transporter 1). Our data and those of others indicate that DMT1 plays a critical role in GI uptake of iron and in systemic iron trafficking. Endosomal acidification also appears defective in Belgrade rat reticulocytes. 1b.) We are determining the role of intracellular heme concentration in erythroid differentiation by comparing these strains to normal animals. 2a.) A strain of mice has a disease which is a very good model for human beta-thalassemia; we are using these mice to investigate the pathology and treatment of iron-overload - the main cause of death in human patients. 2b.) It should be feasible to design objective, automatable chemical techniques to supplant or at least supplement the reticulocyte count in hematology. 4.) Every adult rat has at least seven distinct hemoglobin chains. We are trying to analyze the genetic basis for this and to learn why rats have so many (and other mammals apparently so few). The initial approach involves analyzing in RNA sequences (cDNAs) from rat reticulocyte cDNA libraries and characterizing globin gene DNA from genomic sequences.
Specialty/Research Focus:
Bioinformatics; Cell growth, differentiation and development; Gene Expression; Genomics and proteomics; Molecular Basis of Disease; Molecular and Cellular Biology; Molecular genetics; Neurobiology; Stem Cells; Transgenic organisms
Research Summary:
We are working on the Nuclear Factor I family of transcription factors. We are investigating their roles in development and analysis of their "regulomes", that is, all the genes regulated by the specific transcription factor. Our work focuses on loss-of-function mutations of the NFI genes in the mouse and C. elegans.
Specialty/Research Focus:
Bioinformatics; Cell growth, differentiation and development; Gene Expression; Genomics and proteomics; Molecular genetics; Signal Transduction
Research Summary:
The long term goal of the research conducted in my lab is to learn about the general principles that organisms use to acquire and metabolize the essential nutrient iron. Since in eukaryotes, iron metabolism depends on the activity of copper-containing enzymes called ferroxidases, we examine the trafficking copper in cells as well. The first challenge for a cell is to scavenge these two metals from the environment. This is true for a yeast cell in culture, or for an epithelial cell in your intestine. The second challenge is to efficiently and correctly partition these metals in the cell for subsequent utilization and storage. Ultimately the cell or organism will have to regulate the accumulation of these metals and to ensure that they are not allowed to roam "free" since both are toxic.
Iron and copper are essential micronutrients. They are required in fundamental cellular processes such as cellular respiration in all organisms, and for vital physiologic functions such as oxygen transport in blood and muscle, and the synthesis of the "elastic" material in blood vessels and and "connective" material in joint ligaments. However, these metals are also intrinsically toxic. This toxicity results from their strong tendency to generate oxygen radicals which in turn destroy key cellular components. The essentiality of these metals and their toxicity are illustrated by the diverse genetic disorders in copper and iron metabolism that result in a variety of human disease.
Specialty/Research Focus:
Cardiology
Research Summary:
Research in my laboratory is focused on stem cell biology, engineering, and therapeutic applications with an emphasis on cardiovascular repair.
We have explored the immunomodulatory property of bone marrow mesenchymal stem cells (MSCs) in our cell transplantation studies, and found that large quantities of human and porcine MSCs can be implanted in immunocompetent pigs, mice, and hamsters without inducing inflammatory immune responses in the host. Our research shows that MSCs improve cardiac function in the porcine myocardial ischemia and hamster heart failure models. Implanted MSCs promote tissue regeneration by recruiting bone marrow progenitor cells and activating local host stem cell niches. These processes are mediated by inter-tissue cross-talk mechanisms involving signaling molecules such as JAK/STAT3, integrins, VEGF receptors, and Wnt/b-catenin. Our long-term goal is to generate clinically relevant stem cell information that may be used to achieve robust therapeutic effects for a broad spectrum of human diseases and lower the cost of future stem cell therapy.
Specialty/Research Focus:
Bioinformatics; Genomics and proteomics; Molecular genetics
Specialty/Research Focus:
Microbial Pathogenesis; Molecular and Cellular Biology; Gene Expression; Regulation of metabolism
Research Summary:
One research goal is to investigate the structure-function relationships and regulation of the human pyruvate dehydrogenase complex (PDC). We investigate the catalytic mechanism of the pyruvate dehydrogenase (PDH) component and its interactions with the dihydrolipoamide acetyltransferase (E2) component of PDC. We also determine the loci of interactions between PDH kinases (four PDK isoenzymes) and the lipoyl domains of E2.
Using a PDC-knockout mouse line we investigate the importance of glucose metabolism as a source of energy for fetal development as well as the role of PDC in glucose-stimulated insulin secretion by pancreatic beta cells.
Another research goal is to investigate diet-induced metabolic programming during early life. We investigate (i) the effects of an altered nutrition during the immediate postnatal life on development of adult-onset obesity and (ii) the effects of maternal obesity on fetal programming. Current research focuses on the role of the hypothalamic signaling pathways in rodents with diet-induced obesity and also in the progeny of obese mothers.
Specialty/Research Focus:
Molecular and Cellular Biology; Molecular Basis of Disease; Molecular genetics; Proteins and metalloenzymes; Gene Expression; Transcription and Translation
Research Summary:
I. Molecular mechanisms of eukaryotic RNA polymerase II transcription initiation and elongation.
Our laboratory utilizes combined genetic, biochemical and molecular biological approaches to investigate the molecular mechanisms involved in transcription initiation and elongation by RNA polymerase II (RNAPII). Studies in both the budding yeast Saccharomyces cerevisiae and human cells have resulted in the identification and biochemical characterization of mutants of RNAPII and the general transcription factors TFIIB and TFIIF that coordinately affect transcription start site utilization and transcript elongation. The focus of our present work is to utilize a variety of biochemical approaches to test the hypothesis that yeast and human TFIIF induce global conformational changes in RNAPII that result in structural and functional changes in the polymerase active center.
II. Molecular mechanisms of transcription and replication by RNA polymerases of non-segmented negative strand (NNS) RNA viruses.
NNS RNA viruses are the causative agents for a multitude of modest to severe human diseases. Due to their high potential for human pathogenicity, this family of viruses, which among others include the Filoviridae (Ebola, Marburg), the Rhabdoviridae (rabies, vesicular stomatitus) and the Paramyxoviridae (measle, mumps, parainfluenza, and respiratory syncytial virus), is currently a high priority area for investigation. Despite years of research, surprisingly little is known regarding the molecular mechanisms by which the RNA-dependent RNA polymerase of these viruses is modulated to carry out the fundamentally critical and sequential functions of messenger RNA synthesis (transcription) versus full-length genome replication. We have expanded the scope of our research program by utilizing our conceptual and methodological expertise in the study of RNAPII to investigate the molecular mechanisms governing the activities of the RNA polymerase from vesicular stomatitis virus (VSV), a prototype and well-established experimental negative strand RNA virus that primarily infects horses, cattle, swine and insects. The initial objective of these studies is to define the biochemical composition and functions of the VSV RNA polymerase complexes during the transcriptional and replicative phases of RNA synthesis. The determination of the compositions of these complexes, initially in VSV and subsequently in other NNS RNA viruses, will enable subsequent structural determinations of the transcriptional and replicative complexes, with the ultimate goal of designing and generating specific inhibitors.
Specialty/Research Focus:
Neurodegenerative disorders; Apoptosis and cell death; Ion channel kinetics and structure; Membrane Transport (Ion Transport); Molecular Basis of Disease; Neurobiology; Neuropharmacology; Protein Folding; Protein Function and Structure; Proteins and metalloenzymes; Signal Transduction; Toxicology and Xenobiotics
Specialty/Research Focus:
Genomics and proteomics; Molecular and Cellular Biology; Gene Expression
Research Summary:
My laboratory is interested in understanding the transcriptional control mechanisms that dictate epithelial cell development and differentiation. Specifically, we seek to understand the functional role of a p53-family member, p63 and Ets family of proteins in epithelial cells such as those of the skin and mammary glands. Towards this end, we have developed and characterized transgenic mice in which the normal expression pattern of these crucial factors is altered by both gain-of-function (Tet-inducible transgenic system) and loss-of-function (knockout) experiments. Our broad objectives are to elucidate the molecular mechanism by which transcription factors such as p63 and Ets proteins regulate their target genes and how such regulation of specific pathways dictate cell fate, development and differentiation. We utilize broad biochemical and genetic approaches, cell culture systems and state of the art genome-wide interrogation techniques to answer questions about differentiation of progenitor/stem populations and to examine molecular consequences of altered expression of transcription factors. These studies will not only help better understand the normal physiological processes but also lead to novel mechanistic insights into the pathophysiology of wide range of disease including cancer.
Specialty/Research Focus:
DNA Replication, Recombination and Repair; Genome Integrity; Molecular and Cellular Biology; Molecular genetics; Protein Function and Structure
Specialty/Research Focus:
Microbiology; Gene Expression; Molecular and Cellular Biology; Signal Transduction
Research Summary:
We are interested in developing an integrated mechanistic view of how organisms coordinate the actions of their replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. We utilize a combination of biochemical, biophysical, and genetic approaches to investigate the molecular mechanisms of DNA replication and DNA repair in Escherichia coli. Current efforts are focused on understanding the mechanisms by which the actions of high fidelity and error-prone lesion bypass DNA polymerases are coordinated with each other, as well as other proteins involved in DNA metabolism.
We are also interested in understanding the mechanisms that contribute to DNA mutagenesis in the opportunistic human pathogen, P. aeruginosa. P. aeruginosa is a particular problem for individuals afflicted with cystic fibrosis. We are particularly interested in determining the contribution of mutagenesis and DNA repair to clonal expansion and pathoadaptation of P. aeruginosa during colonization of cystic fibrosis airways.
Specialty/Research Focus:
Cell growth, differentiation and development; Membrane Transport (Ion Transport); Molecular and Cellular Biology; Signal Transduction; Inherited Metabolic Disorders; RNA
Research Summary:
Regulation of Kidney Epithelial Cell Growth, Transport and Differentiation
Our laboratory is investigating the molecular mechanisms by which hormones, growth factors and extracellular matrix proteins regulate kidney tubule epithelial cell growth and functional differentiation in vitro. An established canine kidney epithelial cell line, MDCK, and isolated "mutants" are currently being utilized to examine the actions of growth regulatory on the expression of several proteins including the Na+, K+-ATPase and laminin, a glycoprotein in the extracellular matrix. The effects of novel growth regulatory factors on the expression of proteins involved in gluconeogenesis, membrane transport, renal disease and growth control in primary renal cell cultures are being examined. Primary kidney epithelial cells differentiate into nephrons in a reconstituted extracellular matrix proteins is the subject of study.
Research Summary:
Dr. Willsky’s research focuses on the role of oxovanadium compounds in cellular metabolism. V is a trace metal believed to be required for growth. Oral administration of oxovanadium compounds alleviates the symptoms of Diabetes in animal models and humans. The techniques of genetics, microbiology, molecular biology, biochemistry, pharmacology, magnetic resonance spectroscopy, and cell physiology are used. The diabetes-altered gene expression of genes involved in lipid metabolism, oxidative stress and signal transduction is returned to normal by V treatment of rats with STZ-induced diabetes, as demonstrated using DNA microarrays. Inhibition of tyrosine protein phosphatases is believed to be a major cause of the insulin-like effects of V. Our results implicate the interaction of V with cellular oxidation-reduction reactions as being important in the anti-diabetic mechanism of V complexes. A new project in the lab studies the mode of action of medicinal plant mixtures used by the native healers of Peru.