Eukaryotes, including humans, are 'petri dishes', hosting an abundant and a rich prokaryotic 'microbiome'. The Garg Lab aims to understand the molecular interactions between a eukaryotic host and its microbiome, and how these molecular interactions dictate human health and disease. Using a concoction of innovative tools including bioinformatics, clinical microbiology, mass spectrometry, DNA sequencing, and mass spectrometry-based 2D and 3D spatial imaging, we aim to delineate specific molecules that modulate the dynamics of microbial involvement in our response to genetic and environmental triggers of disease. We characterize the biosynthesis of these small molecule natural products to innovate developement of new therapeutics.
Electron- and Photon-stimulated Interface and Surface Processes. Dr. Orlando's group utilizes state-of-the-art ultra-high vacuum (UHV) surface science systems equipped with UV-laser sources and low-energy electron beams to stimulate reactions (such as the production of hydrogen) on a variety of substrates and interfaces. Sensitive laser detection schemes (resonance-enhanced multiphoton ionization) are used to probe the reaction dynamics. Approaches based on quantum mechanical interference to control desorption and patterning of surfaces at the nano- and meso-scale are also being developed.
Environmental Chemistry and Planetary Surface Science. Water is ubiquitous in terrestrial and planetary atmospheres and environments. Dr. Orlando's group studies "wet" interfaces using nanoscale films of ice grown in UHV. Radical and ion-molecule reactions are then initiated using electron- and photon-beam sources. These experiments are relevant to understanding the photochemistry of stratospheric cloud particles and magnetospheric processing of icy satellite surfaces in the Jovian system.
Biophysical Chemistry. The mechanisms of DNA damage and repair have been studied extensively, though the role intrinsic waters of hydration play in initiating damage have not been elucidated. Dr. Orlando's group will carry out electron- and photon-irradiation studies of DNA:water interfaces to examine the importance of direct vs. indirect damage.
A majority of antibiotics and drugs that we use in the clinic are derived or inspired from small organic molecules called Natural Products that are produced by living organisms such as bacteria and plants. Natural Products are at the forefront of fighting the global epidemic of antibiotic resistant pathogens, and keeping the inventory of clinically applicable pharmaceuticals stocked up. Some Natural Products are also potent human toxins and pollutants, and we need to understand how these toxins are produced to minimize our and the environmental exposure to them.
We as biochemists ask some simple questions- how and why are Natural Products produced in Nature, what we can learn from Natural Product biosynthetic processes, and how we can exploit Nature's synthetic capabilities for interesting applications?
Broadly, we are interested in questions involving (meta)genomics, biochemistry, structural and mechanistic enzymology, mass spectrometry, analytical chemistry, and how natural product chemistry dictates biology.
Nature has provided us with an arsenal of agents that have proven clinically useful in the treatment of many human diseases, and this is particularly apparent for infectious diseases and cancer. Resistance to current anticancer and antimicrobial chemotherapies will always necessitate the discovery and development of additional therapeutic compounds, both by screening of natural products and by synthetic design. Biosynthetic engineering is a promising tool that could be coupled with these proven techniques to generate novel bioactive metabolites. Dr. Kelly's group examines natural products biosynthesis and its applications from chemical and microbiological perspectives.
Dr. Kelly's group is interested in the biosynthesis of polyketide and nonribosomal peptide antibiotics in addition to the biosynthesis of post-translationally modified peptide antibiotics. We aim to understand the assembly of central scaffolds that appear in families of metabolites that vary in their biological activity according to unique peripheral modifications. This requires a detailed understanding of the enzymes responsible for construction of these molecules, including their catalytic mechanism and substrate specificity. Strategies and techniques from organic chemistry, biochemistry, molecular biology, and microbiology will be infused together to accomplish this task. Ultimately, we will apply the information gleaned from these studies to direct the biosynthesis of designer metabolites possessing antimicrobial or anticancer activities.
We study folding and structure of RNA and DNA as modulated by sequence, covalent damage, anti-cancer drugs, proteins, other nucleic acid molecules. The oldest assembly in biology is the ribosome, which is a primary focus of our efforts. Ancient ribosomal structure and function, from beyond the root of the tree of life, can be inferred from extant structure/function combined with phylogeny, evolutionary theory, biophysical chemistry, bioinformatics and molecular biology. We use all of these approaches to construct models of ancient ribosomes, which we then study by biochemical methods. Three-dimensional structure, being more conserved over evolutionary time than sequence, offers some of the most important guideposts in our journeys down the base of the tree of life.
Structural biology, protein misfolding, amyloid, glaucoma, crystallography, and molecular biophysics
The Lieberman research group focuses on biophysical and structural characterization of proteins involved in misfolding disorders. One major research project in the lab has been investigations of the glaucoma-associated myocilin protein. The lab has made major strides toward detailed molecular understanding of myocilin structure, function, and disease pathogenesis. Our research has clearly demonstrated similarities between myocilin glaucoma and other protein misfolding disorders, particularly amyloid diseases. The work has led to new efforts aimed at ameliorating the misfolding phenotype using chemical biology approaches. Our second project involves the study of membrane-spanning proteolytic enzymes, which have been implicated disorders such as Alzheimer disease. Our group is tackling questions surrounding discrimination among and presentation of transmembrane substrates as well as the enzymatic details of peptide hydrolysis. In addition to the biochemical characterization of intramembrane aspartyl proteases, our group is developing new crystallographic tools to improve the likelihood of determining structures of similarly challenging membrane proteins more generally.
Bioorganic Chemistry, Biochemistry and Drug Design. The overarching research objective of our laboratory is to delineate the chemical basis of the molecular recognition events employed by biomolecules to drive important biological processes and how perturbation of these events, by natural and synthetic ligands, can be used to understand the molecular basis of various human disease conditions, especially cancer, viral- and bacterial-infections; and to use the information gleaned from such perturbation studies to arrive at potentially new therapeutic solutions for these conditions. Individual research project involves a unique blend of the tools of synthetic organic chemistry with biochemistry and molecular biology. Enumerated below are specific interrelated research projects that are currently underway:
RNA-Small Molecule Interaction. RNAs adopt intricate and structurally diversed motifs susceptible to direct interactions by small molecules in similar manner to proteins. An atomic level understanding of RNA-small molecule interactions will aid identification of a myriad of biologically useful molecules including novel RNA structural probes; and new anti-viral, anti-tumor and antibacterial agents. One class of nucleic acid targeting drugs are the anthracyclines. Literature evidence suggests that anthracyclines partly derived their anti-tumor activities through DNA-intercalation mediated "poisoning' of the eukaryotes topoisomerase II. Despite their chemical similarity to DNA and direct role in gene expression, little is known about the extent to which RNA interactions with anthracyclines contribute to anthracyclines' biological activities. The primary objective of this research is to elucidate the molecular features essential for the interaction of anthracyclines with the iron responsive elements (IREs), the hairpin loops located in the untranslated regions of mRNAs encoding key proteins involved in iron metabolism; and the effects of such interactions on the formation of the crucial RNA-protein complexes that regulate intracellular iron homeostatis.
Targeted Histone Deacetylase (HDAC) Inhibition. One nucleic acid associated protein that is of current interest to us is the Histone Deacetylase (HDAC). HDACs and histone acetyltransferases (HATs) are two functionally opposing enzymes, which tightly regulate the chromatin structure and function via sustenance of equilibrium between the acetylated- and deacetylated-states of nucleosomal histones. Aberrations in intracellular histone acetylation-deacetylation equilibrium have been linked to the repression of a subset of genes resulting in excessive proliferation and are implicated in a number of malignant diseases. HDACs function as part of multiprotein complexes that catalyze the removal of acetyl groups from the -amino groups of specific lysine residues located near the N-termini of nucleosomal core histones. Inhibition of HDACs activity results in the weakening of the bond between histones and DNA, thus increasing DNA accessibility and gene transcription. This has recently been clinically validated as a new therapeutic strategy for cancer treatment with the FDA approval of SAHA for the treatment of cutaneous T cell lymphoma. To date, several other structurally distinct small molecule HDAC inhibitors have been reported, however, most of these agents non-selectively inhibit the deacetylase activity of class I/II HDAC enzymes. Toward improving the therapeutic index of current HDAC inhibitors, we are developing new HDAC inhibitors for targeted cancer therapy applications. Our goals here are two-fold: (1) to develop HDAC inhibitors that sensitize cancer cells to nucleic acid interacting anticancer drugs and (2) to develop methodology for cell type-selective delivery of HDAC inhibitors.
Design and Synthesis of Novel Bioconjugates for Molecular Delivery Applications. We are also investigating new molecular delivery systems for nanoparticles and small molecule drugs. In collaboration with Professor El-Sayed's group, we are developing new approaches for targeting gold nanorods into the nucleus of live cells. Additionally, we are exploring the periplasmic protein secretion pathway to deliver cell wall damaging agents to the bacterial periplasm. This effort could result in novel diagnostic and therapeutic strategies.
Engineering Nuclear Hormone Receptors. Nuclear hormone receptors control the expression of genes in response to small molecule hormones. In performing this activity, the receptors must specifically recognize small molecules, DNA, and other proteins. The amino acids that recognize each of these substrates are varied using genetic engineering techniques until a receptor with novel recognition is created. The original and new receptors are studied using a variety of biophysical techniques to elucidate the principles behind the new activity. This exercise provides both new knowledge for future protein engineering and real materials for research and medical applications.
We engineer new receptors to turn on gene expression in response to specifically chosen small molecules. The small molecule may be chosen for its properties in the specific application. For applications in gene therapy and use as a tailored gene switch, the small molecule may be chosen due to its long or short pharmacokinetic half-life, because it crosses the blood-brain barrier, or other tissue specificity. For applications in synthetic biology gene circuits, the small molecule may be chosen for its interactions with other circuits in a network. Another application is in assembling the biosynthetic components (enzymes) to synthesize the small molecule via genetic selection.