University Of Illinois At Urbana-Champaign

NIH Research Projects · FY 2025 · 2000-07

This is a competing continuation application to renew the Research Training Program in Toxicology and Environmental Health at the University of Illinois at Urbana-Champaign. The Program, established in 2000, educates predoctoral and postdoctoral trainees in reproductive/endocrine toxicology, neurotoxicology, nutritional toxicology, nanotoxicology, and cancer toxicology. The Program unites several long-standing areas of research excellence on the University of Illinois campus—environmental toxicology, reproductive biology, neuroscience, nutritional sciences, cancer biology, chemistry, and bioengineering. A total of 21 faculty members from 7 departments in 5 colleges will serve as preceptors for the Training Program, making this a truly interdisciplinary Program. Of these preceptors, 16 including the Program Director and Associate Director, have been affiliated with the Program for many years, providing long-term stability and continuity to the program. New preceptors were added to the Program to expand training opportunities in our 5 focus areas. These preceptors brought additional expertise in environmental epidemiology/heath, reproductive/endocrine toxicology, nutritional toxicology, and cancer toxicology. The preceptors are well funded, collaborate extensively, and have a wealth of experience mentoring graduate students and postdoctoral trainees. Together, the preceptors currently have 59 federally funded research grants and 20 grants from other sources totaling over $15.5 million dollars/year in direct costs. Collaborations among labs working at the molecular, cellular, whole animal, and human health levels provide trainees with the unique opportunity to directly participate in translational research. Selection of the 4 predoctoral and 3 postdoctoral trainees supported by this Program is based on academic success, strength of the proposed research, relevance of the research to Program goals, and commitment to toxicology and environmental health. Trainees are appointed for 2 years. The Program offers a broad range of graduate level courses in toxicology. In addition to fulfilling departmental requirements, all predoctoral trainees take basic toxicology, systems toxicology, and at least one other advanced toxicology course. Postdoctoral trainees conduct independent research. All pre- and postdoctoral trainees attend weekly toxicology research seminars, a monthly toxicology journal club, and a course on research ethics in toxicology, coordinated by the Director and team taught by the preceptors. Trainees also attend career development workshops and take a grant writing class. They are required to present their research in the toxicology seminar series and strongly encouraged to attend national or international meetings to present their work. Trainees are expected to pursue toxicology- related research careers in academia, government, or industry.

NIH Research Projects · FY 2025 · 2000-07

Project Summary Hydrogenase enzymes are pervasive, being found in bacteria, archaea, and some higher organisms. These enzymes are hosted by some pathogens, often in anaerobic environments including the human gut. The hydrogenases mediate the most fundamental chemical reaction: the interconversion of H2 with protons and reducing equivalents. The enzymes are structurally exceptional with an array of distinctive cofactors, especially the site of H2 binding and release. Interest in such enzymes stems from three angles: the possibility that some pathogens could be controlled rationally, the excitement about their unusual structures, and the commercial implications of hydrogen production/oxidation in the context of fuel cells. Two major classes of hydrogenases exist, [NiFe]- and [FeFe]-hydrogenases. This project is almost exclusively focused on the latter. More specifically, this project aims to elucidate the biosynthesis of the active site of [FeFe] enzymes, the faster hydrogenase and the one most amenable to development for other applications. This project is timely because we have just defined the sequence by which the three maturase enzymes build the active site. In parallel with their unusual structures, the construction (biosynthesis) of the active site proceeds unusually. The first subproject aims to make the first Fe-containing intermediate, "Compound B". The next two projects tackle how B is converted to an inorganic Fe-S-CN-CO monomer. The fourth project examines the coupling of this monomer to give an inorganic dimer. The final and fifth project examines the retrofitting of this Fe2 entity with an organic cofactor. In this program collateral projects address allied themes of still broader interest. One involves expanding our knowledge of iron complexes of amino acids. Another contributes to the biosynthesis of [NiFe]-hydrogenases. One spin-off project critically examines the premises of the Iron-Sulfur Theory of the origin of life by examination of the first Fe-S-CN-CO complexes.

NIH Research Projects · FY 2025 · 1999-02

Transcriptional regulation is fundamental to most basic molecular and cell biological processes. Our long-term goals are to determine how 10 and 30 nm chromatin fibers fold into large-scale chromatin domains, how these chromatin domains are moved and positioned within nuclei relative to specific nuclear bodies and compartments, and what this means for DNA functions such as transcription and replication. Our first Aim is to dissect both transcription independent and transcription dependent mechanisms for genome positioning relative to nuclear speckles. Our rationale is that gene positioning relative to nuclear speckles modulates levels of gene expression possibly for thousands of genes, and therefore dissection of these mechanisms will reveal novel aspects of gene regulation. This Aim builds on strong preliminary work. During the last grant cycle, we discovered gene expression amplification after nuclear speckle contact. We also discovered a surprising conservation of genome distance to nuclear speckles, with small distance shifts relative to speckles highly correlated with large changes in gene expression and with many inducible genes “pre-positioned” near nuclear speckles even before transcriptional activation. Our second Aim is to map, visualize, reconstitute, and then dissect cis and trans determinants for large- scale chromatin domains with distinct levels of compaction; these domains can be visualized by live-cell microscopy and electron microscopy but are not yet measured by current genomic methods such as Hi-C and are not well-preserved by conventional FISH methods. Our rationale is that this level of large-scale compaction modulates both transcriptional initiation and elongation rates and therefore our analysis of chromatin domains with different levels of large-scale chromatin compaction will reveal novel aspects of gene regulation. This Aim also builds on strong preliminary work. During the last grant cycle, we used high slopes of TSA-seq scores to identify unusually decondensed large-scale chromatin domains (DLCDs). DLCDs mapped predominately to Hi-C compartment, subcompartment, and TAD boundaries separating active and repressed chromatin domains. Acidic activators and chromatin factors recruited by acidic activators showed the highest enrichment over DLCDS among hundreds of chromatin-modifying factors. Strikingly, this observed enrichment connects with results from the early years of this grant showing that among four classes of transcriptional activators, only acidic transcriptional activator domains showed the common activity of inducing large-scale decondensation of an engineered heterochromatic chromosome region. During this last grant cycle, we also made technological advances in developing TSA-MS to identify proteins localizing to immunostained nuclear bodies and in the manipulation and transgenesis of large DNA constructs.

NIH Research Projects · FY 2025 · 1999-02

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a major group of natural products (NPs) produced in all domains of life. The number of RiPP families continues to expand rapidly based on genome sequencing. RiPPs a·re well suited for genome mining for new natural products by heterologous expression of biosynthetic gene clusters (BGCs) and for library generation of cyclic peptides to recognize diverse targets. Furthermore, RiPPs appear to be widely encoded in the human microbiome and have recently been implicated in causation and prevention of human disease. For the RiPP field to advance, a better understanding of the post-translational modification processes is important. Similarly, tools need to be developed to take advantage of the substrate tolerance of the biosynthetic enzymes. In the current application we describe our progress towards these goals and our proposed studies for continued advances. This application focuses mostly on the lanthipeptides, a group of polycyclic peptides with macrocyclic thioether crosslinks. Individual lanthipeptides have a variety of biological activities including antimicrobial, antiviral, antifungal and anti-allodynic. At least four different routes to lanthipeptides have evolved and lanthipeptide BGCs are ubiquitous amongst the RiPP family in sequenced genomes. For one of these four pathways, substrate recognition is reasonably understood but for the other three this information is lacking and will be a focus of continued research . Another poorly understood aspect is the factors that control the ring topology of the multiple thioether rings that are formed and this question will be a focus of investigation. Furthermore, previous work suggests that lanthipeptide biosynthesis takes place in enzyme complexes that may require its substrate. A method for catalytically competent covalent attachment of the substrate to the key enzyme will be used to try and structurally characterize such enzyme complexes. In addition, the mechanisms of a family of enzymes that make amino acid-derived NPs in a novel pathway will be investigated and the pathways in which they operate will be determined. Improved engineering technology to disrupt protein-protein interactions with cyclic peptides will be developed. RELEVANCE (See instructions): Based on historical precedent, the most likely group of compounds to deliver new antibiotics to fight antibiotic resistance are natural products. Genome sequences offer unprecedented access to new natural products that could be future antibiotics but for this information to be used, biosynthetic enzymes that make the natural products need to be better understood. This application aims to obtain this information.

NIH Research Projects · FY 2024 · 1994-05

We have learned that most oxidative toxicity arises when oxygen species attack enzymic iron centers and that cellular defenses work by blocking, reversing, or by-passing the resultant injuries. Yet key observations remain unexplained. In Aim 1 we will investigate why superoxide stress precludes the use of sulfate as a sulfur source, and we will examine why thioredoxins and glutaredoxins are strongly induced as part of the cellular reaction to hydrogen peroxide. Extensive work has led us to the proposal that intracellular cysteine and redoxins help to repair damaged metalloenzyme centers. This model would identify a key connection between sulfur redox state and ROS. Two enzymes dedicated to anaerobic metabolism—pyruvate:formate lyase activating enzyme and alcohol dehydrogenase—have been suggested to be inactivated by iron-centered oxidation events when cells are aerated. This would comprise a clever exploitation of reaction types that are usually harmful. The goal of Aim 2 is to test this striking idea. This hypothesis leads to notions of how the cell might seamlessly restore anaerobic metabolism when anoxia is restored. Protein carbonylation (Aim 3) has long been used as a convenient marker of oxidative stress—but the underlying events and physiological impact are unclear. Our data indicate that carbonylation is focused upon relatively few proteins rather than the full proteome, and we suspect that these proteins are mononuclear Fe(II) enzymes. Global mass spectrometry will identify them by name. We will also test the idea that methionine sulfoxide is a disproportionate Fenton product that reductases can repair. The novelty is that methionine may be oxidized by a secondary electron-hopping event, rather than by direct attack. Finally, in Aim 4 we will take a transcriptomic approach to fully define the OxyR peroxide response. We hope to explain our discovery that OxyR activation per se compromises cells fitness, to the point of prohibiting growth on acetate. It is not surprising that a stress response should exert a price, but we do not yet recognize why any OxyR-driven adaptation would have such a profound effect. The emergent theme of oxidative stress is the tendency of oxygen species to react with iron centers, and of cells to respond with layers of defensive tactics. Our four Aims will build upon this knowledge by tackling persistent questions, with the overall goal of assembling a picture of oxidative stress that is detailed, quantitative, and unified.

Other NSERC · FY 2024

specialized metabolites, plant biochemistry, plant lipids, evolution, natural diversity, substrate specificity, fatty acid synthesis

Other NSERC · FY 2024

Biochemistry, Catalysis, Physical Organic Chemistry, Chemical Biology, Enzymology

Other NSERC · FY 2024

Computer Science Theory, Computational Geometry, Algorithms, Data Structures, Approximation algorithms, Fine grained algorithms, Upper bounds, Lower bounds

Other NSERC · FY 2024

machine learning, catalysis, linear regression, organic synthesis, methodology, improving organic chemistry, artificial intelligence, AI, organic chemistry, synthesis

Other NSERC · FY 2024

combinatorics, graph theory, Hamiltonicity, induced subgraphs