Rensselaer Polytechnic Institute

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY This research program will investigate the general hypothesis that understanding the conformational diversity of proteins will provide new insights into their biology, and enable medical research. It is directed to two classes of systems: Integral Membrane Proteins (IMPs) and viral-host interactions. IMPs play critical roles as gate keepers, receptors, transporters, homeostasis regulators, and drug targets. These functions are mediated by the conformational plasticity of the IMP in the membrane environment. IMPs are challenging to prepare, and even more challenging to reconstitute in appropriate membrane mimicking environments. Cost-effective technologies for isotope-enrichment in condensed volumes, hybrid approaches combining NMR with evolutionary co-variation (ECs), novel methods of contact prediction, and innovative modeling methods from the protein structure prediction community, will be applied to structure-function studies of IMPs. These IMPs, chosen from important human pathogens, including E. coli, K. pneumoniae, and P. aeruginosa, are potential targets for antibiotic discovery. ECs will also be combined with NMR data to determine structures of multiple “native states” of proteins. The second component of our program is directed to viral – host biomolecular complexes, and antiviral drug discovery. We will utilize innovative paramagnetic NMR methods, together with small angle X-ray scattering (SAXS), electron-electron double resonance spectroscopy (DEER), and Förster resonance energy transfer (FRET), to rigorously define dynamic interdomain structural distributions conferred by the partially-ordered linkers of the murine Moloney Leukemia Virus (MLV) integrase (IN). These data will be interpreted in the context of maximum occupancy probabilities (MaxOcc), and used to probe the role(s) of this flexibility in the gene integration mechanisms of g-retroviruses. Interdomain linkers also function to provide flexibility needed for binding partner promiscuity. We will also determine how the interdomain linker sequences of influenza Non-Structural Protein 1 (NS1) confer appropriate plasticity to define its specificity and affinity for host proteins and RNAs. This structural and functional promiscuity underlies NS1’s mechanisms for suppressing the cellular innate immune response to influenza infection, and rigorous characterization of its dynamic structural basis will provide fundamental information for live-attenuated virus vaccine development. We will also apply our platform to investigate drugs that inhibit SARS-CoV2 virus by binding its main protease (Mpro). We have identified three drugs, already approved for use in humans, originally designed to inhibit the NSP3/4A protease of hepatitis C virus, that also inhibit SARS-CoV2 in viral replication assays at low micromolar concentrations. Our computational docking studies have also identified several other FDA- approved drugs that may inhibit Mpro. Enzyme kinetic, biophysical chemistry, and X-ray crystallography studies will be used to characterize complexes formed between these protease inhibitor drugs and Mpro, and to develop their potential as COVID-19 therapeutics, or as lead compounds for new therapeutic development.

NIH Research Projects · FY 2025 · 2021-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Biomolecular Science and Engineering Training Program at Rensselaer Polytechnic Institute (RPI) is dedicated to the education of predoctoral students at the interface of biology, chemistry and engineering, focusing on the quantitative linkages that define this interface and prepare trainees for careers in biotechnology. The Program’s overarching objective is to provide the trainees with a keen understanding of the interdisciplinary nature of research, how it depends on fundamental underpinnings in both science and engineering, how it leads to innovative new scientific advances and technologies, and how basic science and engineering research can lead to new discoveries and commercial products that benefit society. The Training Program has been designed with key Program objectives to provide each trainee with a strong foundation in rigorous experimental design emphasizing rigor and reproducibility: to conduct ethical, responsible and productive predoctoral research with integrity and appropriate time-to-degree; to communicate and work effectively in teams; and to gain knowledge, professional skills and experiences for careers in biomedical research. Our Training Program at RPI increases interactions among students from four departments: Biological Sciences, Biomedical Engineering, Chemical and Biological Engineering, and Chemistry and Chemical Biology, through courses, structured activities, research mentoring and training, and industrial experiences. Key activities of the Training Program include the following 12 Action Items: [A1] a core course entitled “Perspectives in Biomolecular Science and Engineering” that is taken by all trainees; [A2] a set of four courses that maximize didactic training among the key disciplines; [A3] a daylong technology commercialization boot camp to learn about fundamentals of intellectual property and participate in an entrepreneurial program; [A4] a high-profile seminar series that provides exposure to contemporary research in academia and industry; [A5] a student-run seminar program, in which trainees present their research to their peers and Program faculty; [A6] exposure to enrichment activities about human health, physiology, and disease, thereby promoting a healthcare innovation and entrepreneurial ecosystem; [A7] an annual retreat to network externally and hold discussions on contemporary research areas and careers in biotechnology; [A8] dual advising and multidisciplinary thesis committee membership for Ph.D. thesis work; [A9] an industrial internship; [A10] Exposure/interaction with other NIH funded programs; [A11] training in responsible conduct of research, scientific rigor and reproducibility; and [A12] participation in activities for professional development. Program committees and a trained evaluator conduct evaluations of the training process and outcomes to ensures that the trainees satisfy these requirements and that the overall Program goal and objectives are met through continuous process improvement.

NIH Research Projects · FY 2024 · 2020-09

Neurofibrillary tangles (NFT) is a pathological hallmark of Alzheimer’s disease (AD). NFT is composed of aggregated tau protein and is correlated to cognitive decline in dementia progression. NFT spreads in the brain in a prion-like manner, mediated by the interaction between tau and neuronal surface glycans. The long-term goal is to study the structural details of tau-glycan interaction and to discover drugs to disrupt this interaction for disease-modifying treatment of AD. In this proposal, we will study the tau-heparan sulfate interaction from at the molecular (aim 1), the cellular (aim 2), and organismal level and carry out drug discovery (aim 3).

NIH Research Projects · FY 2024 · 2020-09

Summary Membrane organization in eukaryotic cells is controlled by ADP ribosylation factors (Arfs), small GTPases that function as molecular switches to activate signaling cascades. Arfs regulate vesicular transport of lipids and proteins between the ER and the Golgi (Class I-Arf1) and endosome-plasma membrane trafficking (Class III-Arf6), implicating Arf function in cytokinesis, cell shape, organelle transport, mitochondrial and lipid droplet function and pH-dependent regulation of cell size. Mutations in Arfs or their partners have been linked to genetic neurological diseases causing severe malformation of the cerebral cortex or mental retardation. Moreover, many pathogenic bacteria and viruses commandeer Arfs as they invade cells, thereby promoting infection. Our overall goal is to understand the nucleotide exchange transitions of Arf GTPases, the mechanisms of which cannot be deduced from their static structures. We hypothesize that the Arf conformations specifically recognized by their cognate exchange factors correspond to significantly disrupted excited states that are populated at very low levels under standard conditions. Specifically, we aim to map the GDP/GTP switches of Arf1 and Arf6 (Aims 1 and 2), and using mutational analysis, establish the underlying molecular mechanisms of their functional specificity (Aim 3). Our approach combines experimental biophysical tools (multi- dimensional NMR, SAXS and fluorescence) with pressure perturbation and coarse-grained molecular dynamics simulations constrained by our data, to provide structural ensembles and pseudo-free energy landscapes that will reveal functionally relevant excited states implicated in Arf function and specificity. These excited state structures will provide novel target sites for inhibiting Arf signaling pathways, offering new avenues for developing approaches to mitigate the invasive capacity of bacteria and viruses. More generally, the pressure-based mapping approach proposed here represents a powerful means to characterize elusive states of proteins implicated in their functions.

NIH Research Projects · FY 2025 · 2018-08

Project Summary/Abstract: Circadian rhythms are highly conserved, 24-hour, oscillations that tune human physiology to the day/night cycle, enhancing fitness by ensuring that appropriate activities occur at biologically advantageous times. Disruption of proper circadian timing negatively impacts the human long-term medical outlook, making understanding the mechanism underlying circadian regulation over cellular physiology critical to human health. Circadian rhythms are controlled via a transcription-translation based negative feedback loop, or clock. The current paradigm for circadian regulation over physiology, termed the clocks “output”, is that transcriptional programing generated by the clock drives temporally-specific waves of gene expression. However, our research has revealed that transcriptional programing cannot wholly account for clock output, as we discovered weak correlation between mRNAs and proteins that oscillate with a circadian periodicity, particularly in the circadian regulation of immunometabolism. The mechanisms that control this post-transcriptional regulation are unknown, but we have shown that intrinsic protein disorder in the repressive complex of the clock may control the formation of macromolecular complexes to time clock output post-transcriptionally. Our immediate research goal is to identify specific pathways by which the clock imparts post- transcriptional control over the immune response at the biophysical, molecular, and physiological levels. We hypothesize that circadian post-transcriptional metabolic regulation can tune immune-tissue and sex-specific rhythms via the formation of time-of-day defined macromolecular protein complexes that are centered around the repressive complex of the circadian clock. To test this hypothesis, we will create a Conformational/Temporal Interactome (CiTI) map of circadian repressive complex proteins. We will also investigate the contribution of sex-specific metabolic post-transcriptional regulation to immune cell functions to demonstrate the effects of metabolic oscillations on the basal immune response. As a mechanism for keeping time, circadian feedback loops are highly conserved and much of what is understood about the molecular clock comes from the investigation of clock model systems. We will therefore exploit the simplicity and reproducibility of fungal and mammalian model systems to cost-effectively address our hypotheses. Due to the conservation of clock architecture, our findings will have the potential to define several novel and unrecognized paradigms in clock regulation over cellular physiology, including the sources and effects of circadian post-transcriptional regulation. These newly defined paradigms will further our long-term goal of elucidating the fundamental principles of circadian timing by identifying the mechanistic underpinnings of circadian control over cellular physiology.