The hyperthermophilic archaeon Pyrococcus furiosus (Pf) grows optimally at 100°C by fermenting peptides and sugars. It also reduces elemental sulfur to hydrogen sulfide. From Pf we are purifying and characterizing a range of metal-containing, oxidoreductase-type enzymes and redox proteins that are involved in unusual catabolic pathways. In addition, all ORFs in the Pf genome (1.9 Mb) are being cloned and expressed in an NIH-funded structural genomics initiative with the goal of obtaining 3D structures on all Pf proteins. The function of all Pf ORFs are being assessed using DNA microarrays and proteomic approaches in conjunction with metabolic and physiological analyses.

I do site-directed mutagenesis on yeast enolase, changing residues the X-ray structure indicates are important in the enzyme mechanism or involved in the interaction between the two subunits. Also literary interests: See www.historomance.com.

Heme is a key and essential compound for the vast majority of living organisms. Heme, as a cofactor in a variety of proteins, is widely acknowledged to be essential for gas transport, respiration, xenobiotic detoxification, peroxide production and destruction, fatty acid desaturation, and a variety of one electron transfer reactions. Over the past decade the number of roles identified for heme has grown substantially. It has become clear that heme is also an important intracellular regulatory ligand. Among the list of biological processes for which higher eucaryotic heme-binding proteins have now been implicated is regulation of circadian rhythm, adipogenesis, glucose homeostasis, microRNA processing, gas sensing, control of ion channels, and intra- and intercellular signal transduction. The list of bacterial heme-binding sensors seems to grow with each new journal publication, and the role of the heme-containing DevS-DevR proteins of Mycobacterium tuberculosis as regulators of passage into the dormant stage has attracted considerable attention for obvious biomedical reasons. Dietary heme also serves as a significant source of iron for many organisms including pathogenic bacteria. A search of PubMed for “heme” yields over 2500 listed publications in the past year. The Dailey lab’s research focuses on the enzymes responsible for heme biosynthesis. Current studies involve structure/function investigations of the terminal enzymes of heme biosynthesis and their relationship to the human genetic diseases known as porphyrias, biochemical characterization of the enzymes from both eukaryotic and prokaryotic organisms, identification and characterization of novel and previously unidentified genes involved in heme synthesis and transport, protein-protein interactions among heme synthesis enzymes, and regulation of expression and translocation of heme synthetic enzymes.

Glucose inhibition in cancer cells

We study how protein glycosylation affects cellular communication as well as protein folding using biochemical, cell biological and animal studies. The forms of glycosylation we study affect development, birth defects, and cancer.
 

Heme is a cofactor found in essentially all aerobic organisms and a majority of anaerobes and facultative organisms. Most organisms that possess heme synthesize it themselves. With the exception of Caenorhabditis elegans and related helminthes, heme acquired via dietary sources is generally degraded to release free iron and is not utilized as a source of cellular heme. While the traditional textbook roles for heme as a cofactor include hemo- and myoglobins, cytochromes and a handful of enzymes, considerable evidence has emerged that demonstrates a central role for heme in regulation of gene transcription, as a gas sensor, in the regulation of circadian rhythm, during development and in RNAi processing. Disordered heme metabolism can have profound developmental and health consequences. Our laboratory is exploring these mechanisms in atomic detail in order to provide a better foundation for future drug and therapy development.

Understanding intracellular and intercellular heme transport

Pectin is a family of complex polysaccharides present in all plant primary cell walls. Pectin plays multiple roles in plant growth, development, and defense responses; in part through contributing to cell wall strength, wall ion exchange and sieving properties, cell-cell adhesion, and cell-cell communication. Pectin is a food fiber and a commercial gelling agent that has beneficial effects on human health. Our long term goal is to decipher how the 53 distinct enzyme activities required for pectin synthesis interact to synthesize pectin and to modify pectin synthesis in order to study pectin function. Towards this goal we are purifying, cloning, and characterizing the biosynthetic enzymes; many of which are Golgi localized and membrane bound enzymes. Current emphasis is on the galacturonosyltransferase and the methyltransferase that synthesize the pectic polysaccharide homogalacturonan.

Research in the Moremen lab focuses on the structure, enzymology, regulation, and localization of enzymes involved in the biosynthesis, recognition, and catabolism of mammalian glycoproteins. Carbohydrate structures on glycoproteins contribute to many biological recognition events between molecules and between cells in an organism. Alterations in the synthesis and degradation of these structures can also occur in human genetic disease. Work in the Moremen lab is focused on (1) the characterization of enzymes involved in mammalian glycoprotein biosynthesis and catabolism and the functionally defective forms of these enzymes involved in human genetic disease and (2) the identification and characterization of carbohydrate-binding proteins and their roles in vertebrate development and physiology.

Our research focuses on the function of glycoconjugates in the regulation of cell adhesion. 1) investigation of the mechanism how glycosyltransferases and oligosaccharide expression regulate cell adhesion, migration, and invasiveness; 2) structure and function of the glycosyltransferase GlcNAc-T V to develop an inhibitor as a cancer therapeutic; 3) identification of glycoprotein glycoforms diagnostic for carcinomas; 4) function of a novel endothelial cell lectin, most likely in pathogen surveillance; 5) structural determination of a new family of animal and fungal lectins, the X-type lectins; 6) functions of lectins in animal development and as ligands for BT toxins.

Research in our lab is mainly focused on proteases:

  • The CaaX proteases: Rce1 and Ste24 mediate a proteolytic cleavage event associated with the maturation of proteins that contain a covalently attached isoprenyl lipid at their C-terminus (e.g. Ras, nuclear lamins, fungal pheromones). We are investigating the mechanism and specificity of these proteases in an effort to better understand their role in human disease (e.g. Ras and cancer; lamins and progeria).
  • The M16A proteases: This evolutionarily conserved metalloprotease family includes the human insulin-degrading enzyme, which mediates degradation of amyloidogenic peptides such as the Abeta peptide associated with Alzheimer’s disease. We are investigating the mechanism and specificity of these enzymes to better understand their physiological role in the cell.

Our research program is multi-disciplinary, allowing for exposure to the disciplines of biochemistry, cell biology, chemistry, genetics, and microbiology.

Biophysical analysis of metallobiochemical systems using X-ray absorption spectroscopy; systems biology approaches to discovery of transcriptional regulation of microbiological hydrogen production as part of an alternative energy project.

Our laboratory utilizes multiple model systems including zebrafish to study the developmental consequences of impaired lysosomal catabolism of glycoproteins. We are focused on understanding how the mislocalization and inappropriate activity of specific enzymes impacts the normal development and function of several tissues including cartilage.

RNA-guided invader defense in prokaryotes: Archaea and bacteria (both pathogenic and beneficial) are constantly attacked and destroyed by viruses and other genome invaders. We are working to delineate a series of newly-identified RNA-mediated immune systems that protect prokaryotes from viruses and other invaders - the CRISPR-Cas systems. This exciting research is leading to new ways to strengthen beneficial microorganisms that produce food, pharmaceuticals and biofuels, to combat disease-causing bacteria, and to prevent the spread of antibiotic resistance.

Our laboratory is interested in how post-translational modifications of proteins increase functional diversity. Primarily, we are interested in glycosylation, with a focus regarding: 1. O-GlcNAc in Type II diabetes and stem cell biology 2. O-Mannosylation in Congenital Muscular Dystrophy and viral entry into host cells 3. Glycoproteins as biomarkers in human disease, specifically pancreatic cancer and metabolic syndrome 4. Development of technology-based approaches, primarily mass-spectrometry, for quantitive proteomics/ glycomics/ glycoproteomics.

Glycosylation regulates the activities of proteins via multiple intrinsic and extrinsic mechanisms. A remarkable example, in our laboratory’s view, is the glycosylation of a subunit of the E3(SCF)ubiquitin ligases, Skp1. Skp1 glycosylation, which depends on its prior oxygen-dependent prolyl hydroxylation, promotes assembly and presumably the activities of this enzyme family toward the degradation of a whole host of cell regulatory factors. This mechanism underlies oxygen-sensing by a variety of unicellular organisms, including the social amoeba Dictyostelium and the human pathogen Toxoplasma gondii. Our multidisciplinary approaches to understand this mechanism, and exploit our understanding to control parasite virulence, embrace structural biology, metabolomics, bioinformatics, molecular genetics, enzymology, cell and developmental biology, and parasitology.

X-ray crystallographic and biochemical studies of nucleotide sugar metabolism