The rockefeller university

Event Details

Type

Other Seminars

Speaker(s)

4:00 p.m.: Yael David, Ph.D., associate member, Chemical Biology Program, Memorial Sloan Kettering Cancer Center, Leveraging Chemical Biology to Uncover Epigenetic Mechanisms in Disease

4:30 p.m.: Yukti Dhingra, Ph.D., postdoctoral fellow, The Rockefeller University, RNA Polymerase Inhibitors Reveal Active Site Motions Essential for the Nucleotide Addition Cycle

4:45 p.m.: Oliver Swart, Ph.D., postdoctoral fellow, New York University, A Recognition Code for Duplex RNA

5:00 p.m.: Charles Warren, graduate student, Tri-Institutional Program in Chemical Biology, Weill Cornell Medicine, Global Protein-Ligand Binding Affinity Profiling

5:15 p.m.: Tom Muir, Ph.D., Van Zandt Williams Jr. Class of 1965 Professor of Chemistry, Princeton University, Harnessing Inteins in Synthetic Biology From Triggers to Logic Gates

Speaker bio(s)

Yael David: Leveraging Chemical Biology to Uncover Epigenetic Mechanisms in Disease

Abstract:

The epigenetic regulation of DNA-templated processes is central to cellular identity and function, and its dysregulation underlies diverse pathological states, including cancer and chronic viral infection. These epigenetic pathways are particularly susceptible to disruption in contexts of genomic instability, which often give rise to non-canonical chromatin structures such as those found in micronuclei, extrachromosomal DNA, and viral minichromosomes. We recently revealed how these atypical chromatin structures are subject to—and drivers of—unique epigenetic programs. Using chemical biology tools and epigenomic profiling, we defined the chromatin landscape of micronuclei and showed that their rupture exposes transcriptionally active chromatin to cytoplasmic sensors, triggering inflammatory cascades. We further characterized the formation and modification of extravesicular chromatin fragments, which propagate oncogenic signaling beyond the nucleus. In the context of infection, we investigated the covalently closed circular DNA (cccDNA) of Hepatitis B virus, a viral minichromosome maintained in host cells, and identified novel epigenetic regulatory features. Finally, we uncovered metabolism-epigenetic axis that drives T cell dysfunction in tumors and describe how chemical epigenetic reprogramming is being harnessed to engineer next-generation CAR T cells with enhanced efficacy. Together, these studies illustrate the power of chemical biology to illuminate fundamental chromatin biology and open new avenues for therapeutic development targeting epigenetic vulnerabilities.

Yukti Dhingra: RNA Polymerase Inhibitors Reveal Active Site Motions Essential for the Nucleotide Addition Cycle

Abstract:

The nucleotide-addition cycle (NAC) of bacterial RNA polymerases (RNAPs) involves conformational changes in key structural elements near the active site, such as the Rim -Helices/F-loop (RH-FL), trigger loop (TL), and bridge helix (BH). Most RNAP structures have an open active site, where the RH-FL and TL are in open conformations. RNAP bound to the DNA template with the incoming NTP substrate sometimes has a closed active site, where the TL closes on the substrate and the RH-FL closes onto the TL. CBR, an E. coli (Eco) RNAP inhibitor, binds in a pocket formed by the N-terminal end of the BH and the RH-FL, likely interfering with RNAP conformational dynamics during the NAC. However, the precise mechanism by which CBR inhibits RNAP is yet to be defined. To investigate the CBR inhibition mechanism, we determined cryo-EM structures of Eco RNAP on a nucleic-acid scaffold known to give rise to multiple RH-FL and TL conformational states. Structures were determined with and without CBR. Without CBR, we observed two major conformational states: State 1 (Eco) with an open active site (69% of particles), and State 2 (Eco) with a closed active site (31% of particles). When CBR was present, only State 1 (Eco) (open active site) was observed. Although CBR is ineffective against M. tuberculosis (Mtb) RNAP, AAP binds to the same pocket in Mtb RNAP and inhibits its NAC. Using an elongation scaffold with an RNA transcript lacking a 3'-OH group, in the absence of AAP we determined two states of Mtb RNAP: State 1 (Mtb) with an open active site (30% of particles), and State 2 (Mtb), a pre-incorporation state with the incoming NTP substrate and a fully closed active site (70% of particles). In the presence of AAP, we only observed an open active site. From these results we draw two conclusions: 1) Since CBR and AAP interact directly with the RH-FL but not the TL, we conclude that CBR and AAP inhibit the RNAP NAC by preventing closure of RH-FL. 2). The closure of RH-FL stabilizes the closed state of the TL and is required for an efficient RNAP NAC. This structural mechanism is likely universal across all domains of life, given the conservation of these structural elements in all multisubunit RNAPs.

Oliver Swart: A Recognition Code for Duplex RNA

Abstract:

The scientific and therapeutic potential for rationally designable ligands which can specifically target RNA is massive; however, their development remains a challenging feat due to the large variety and dynamic nature of RNA structures. Our work narrows its focus to the simplest and most common RNA structure, the A-form double helix. While many ligands have been known to bind this structure there is an insufficiency of ligands which can do so in a sequence-specific manner. Drawing inspiration from a unique RNA binding protein, tomato aspermy virus 2b (TAV2b), We have developed a peptidomimetic ligand which prioritizes sequence-specific base readout in RNA major grooves. This ligand displayed selective binding to helical RNAs with repeated ‘GC’ sequence motifs, including the CUG-repeat implicated in myotonic dystrophy type 1 and the CAG-repeat sequence of Huntington’s disease. With the peptidomimetic as a foundation, current work has focused on developing cell-stable analogues to establish therapeutic effects of repeat-associated ligands. Additionally further development of this ligand class has focused on enhancing sequence readout ability, which has returned stronger binding affinities and an improved ability to target non-repeat sequences of RNA. This work demonstrates both a novel bulge-independent targeting strategy for repeat expansion diseases and more generally provides a much-needed expansion of the RNA-targeting toolbox.

Charles Warren: Global Protein-Ligand Binding Affinity Profiling

Abstract:

Protein-ligand binding, selectivity, and affinity dictate the effects of drugs and endogenous molecules in cells. Currently, potential protein-ligand interactions are identified by qualitative interpretation of proteomic, transcriptomic, or genomic data, then binding affinities of hits are measured using purified proteins or engineered reporter systems to validate and quantify the strength of individual interactions. Few methods enable simultaneous target identification and biophysical affinity measurement, and these either apply to specific enzyme classes or proteins with ligand-dependent shifts in stability. Here we describe a general platform, termed Affinity Map, which leverages competitive binding analysis and high throughput proteomics for global quantitative binding affinity profiling. We show that this method is applicable to major classes of ligands, including small molecules, linear peptides, cyclic peptides, and proteins, and can measure affinities for proteins in cell lysates, organ extracts, and live cell surfaces.

Tom Muir: Harnessing Inteins in Synthetic Biology From Triggers to Logic Gates

Abstract:

Inteins are auto-processing domains found in organisms from all domains of life. These proteins are consummate molecular escape artists that spontaneously excise themselves, in a traceless manner, from proteins in which they are embedded. Chemical biologists have long exploited various facets of intein reactivity to modify proteins in myriad ways for both basic biological research as well as translational applications. Here I discuss our recent efforts to engineer inteins for protein engineering applications in the test tube and in cells. I will also describe the development of an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. 

Open to

Tri-Institutional