Abstracts

C-Peptide Inhibition of Ebola Virus
Chelsea Higgins, Albert Einstein College of Medicine

Ebola virus (EBOV) is a highly pathogenic member of the Filoveridae virus family for which there are no approved vaccines or therapeutics. EBOV infection involves host and viral membrane fusion mediated by the glycoproteins GP1 and GP2; disulfide linked dimmers that are organized into trimeric spikes on the viral surface. The current model for the mechanism of EBOV membrane fusion indicates GP2 passes through a pre-fusion extended intermediate during which its N- and C-terminal heptad repeat regions (N- and CHR) are exposed before collapsing into the highly stable post-fusion helical bundle conformation, the formation of which is thought to provide the energy required for fusion. The binding of free C-peptide to the temporarily exposed NHR could prevent the helical bundle formation and therefore inhibit viral infectivity. We have shown in collaboration with the Chandran Group that a designed Ebola C-peptide does in fact inhibit infection, but only when conjugated to an arginine-rich segment that targets the C-peptide to the endosome where membrane fusion is thought to occur. Inhibition could possibly be improved through engineered modifications to the current C-peptide design, including modifications that may impact peptide secondary structure and the endosomal delivery mechanism. As an alternative to the Tat sequence, which exhibits some cytotoxicity, we have conjugated the C-peptide to the possibly less toxic arginine-rich Penetratin sequence, or a cholesterol moiety. To improve the binding energetics between the peptide and the GP2 extended intermediate, we have engineered a covalent i to i+3 linker into the C-peptide. This constraint could force the otherwise unstructured peptide to adopt an a-helical conformation, reducing the loss of entropy upon binding.

Coauthors: Jayne Koellhoffer, Emily Miller, Kartik Chandran, and Jon Lai, Albert Einstein College of Medicine

Reiterative Recombination for Pathway Engineering: From Yeast to Stem Cells
Miguel Jimenez, Columbia University

Studying and directing the complex and dynamic biology of human embryonic stem cells (hESC) requires working at the multi-gene level. However there is a fundamental gap between methods for manipulating single genes and the complex transcriptional network of hESCs that is exacerbated by the difficulty of culturing hESCs. The next generation of tools for manipulating hESCs must work at the multi-gene level in an efficient and technically robust manner. Our lab has developed a system of in vivo Reiterative Recombination that harnesses the power of homing endonuclease-stimulated homologous recombination for assembling multi-gene pathways in yeast. The methodology consists of a pair-wise system of recycling selectable markers and endonuclease recognition sites that allow indefinite assembly of DNA in vivo. This strategy obviates the need to extract and retransform increasingly larger DNA fragments. Due to its efficiency and technical simplicity we are able to construct not only single pathways but also large libraries of >104 variants enabling the optimization of metabolic pathways. We envision the expansion of such high throughput genetic tools beyond S. cerevisiae, for example in the engineering of human stem cells differentiation.

Coauthors: Zen Liu, Nili Ostrov, Dario Sirabella, Gordana Vunjak-Novakovic and Virginia Cornish, Columbia University

Metabolic and Enzymatic Characterization of the Intracellular Growth Operon in Mycobacterium tuberculosis
Suzanne T. Thomas, Stony Brook University

 
New drugs with novel mechanisms of action are required to meet the severe threat to human health posed by the emergence of multidrug and extensively drug resistant strains of Mycobacterium tuberculosis (M. tb). The ability of M. tb to metabolize cholesterol is critical for the maintenance of the M. tb infection. The intracellular growth (igr) operon is required for in vitro growth using cholesterol as a sole carbon source, however, the function of these genes and their role in cholesterol metabolism is yet to be established. Here we describe the biosynthetic preparation of isotopically labeled 13C- [1,7,15,22,26]-cholesterol and employ it as a tool to investigate the cholesterol-derived metabolite profile of the M. tb H37Rv Δigr mutant strain by high resolution LCMS. Culture supernatants from the Δigr mutant accumulate a cholesterol-derived metabolite not observed in H37Rv wild-type or complemented strains. Multidimensional NMR and mass spectral analysis revealed the structure of this cholesterol-derived catabolite to be a late stage metabolic product: methyl 1β-(2'-propanoate)-3aa-H-4α(3'-propanoic acid)-7aβ-methylhexahydro-5-indanone. The computationally annotated functions of the six genes of the igr operon are a lipid transfer protein (ltp2/Rv3540c), two MaoC-like hydratases (Rv3541c and Rv3542c), two acyl-CoA dehydrogenases (fadE29/Rv3543c and fadE28/Rv3544c), and a cytochrome P450 (cyp125/Rv3545c). Heterologous expression in E. coli and biophysical characterization demonstrated that FadE28 forms a heteromeric complex with FadE29, and that likewise, Rv3542c forms a heteromeric complex with Rv3541c; each complex exhibits a novel α₂β₂ quaternary architecture. Using synthetic substrates analogous to the metabolite identified in M. tb H37Rv Δigr mutant strain, we verified the catalytic activity of the purified, recombinant FadE28-FadE29 and Rv3541c-Rv3542c protein complexes to be dehydrogenation and hydration of the 2'-propanoate-CoA side chain. We conclude the igr operon is required for degradation of the 2'-propanoate side-chain fragment during metabolism of cholesterol by M. tb.

Coauthors: Brian C. VanderVen, Cornell University, David G. Russell, Cornell University, Nicole S. Sampson, Stony Brook University

Chemical Genetics uncovers Cyclin-Dependent Kinase Control of the Initiation-to-Elongation switch of RNA Polymerase II in Human Cells
Stéphane Larochelle, PhD, Mount Sinai School of Medicine

Kinase inhibitors are widely used to infer specific functions of individual protein kinases in vivo. However, because of active-site conservation within the kinase family, these drugs typically display a high degree of target promiscuity in vivo. Although this propensity for multiple-kinase inhibition might be beneficial in some therapeutic contexts, it is undesirable when off-target effects cause toxicity, or when the goal is to elucidate functions of a single kinase. The chemical genetic approach, whereby a mutation within the active site renders a kinase uniquely sensitive to a designed inhibitor, allows for otherwise unachievable selectivity in vivo, while maintaining structural integrity of protein complexes that contain the targeted kinase. By this strategy we uncovered a cyclin-dependent kinase (CDK) cascade essential for promoter-proximal pausing by RNA polymerase II, which is both a gene-specific regulatory strategy and an RNA quality control mechanism. The conserved DRB-sensitivity inducing factor (DSIF) is required to impose the pause, and structural considerations suggested that eviction of transcription initiation factor TFIIE would be necessary for DSIF engagement. The activity of Cdk9 (P-TEFb) is needed to overcome pausing. We show that inhibition of Cdk7—part of TFIIH—increases TFIIE retention and prevents DSIF recruitment at transcribed genes in human cells, leading to attenuated pausing. Cdk7 activates Cdk9 in vitro, and Cdk7 inhibition prevents Cdk9-activating phosphorylation in vivo. Cdk7 thus acts through TFIIE and DSIF to establish, and through Cdk9 to relieve, a kinetic barrier to elongation: incoherent feed forward that could create a window to recruit RNA-processing machinery.

Coauthors: Ramon Amat, PhD¹, Kira Glover-Cutter, PhD², Miriam Sansó, PhD¹, Chao Zhang, PhD³, Kevan M. Shokat, PhD³, David L. Bentley, PhD2, and Robert P. Fisher, MD, PhD^1
1Dept. of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, NY
²University of Colorado School of Medicine, Aurora, CO
³Howard Hughes Medical Institute and University of California San Francisco, CA

Biomimetic Diversity-Oriented Synthesis of Benzannulated Medium Rings by Oxidative Dearomatization/Ring-Expanding Rearomatization
Todd A. Wenderski, Memorial Sloan–Kettering Cancer Center

Nature has exploited medium-sized 8- to 11-membered rings in a variety of natural products to address diverse and challenging biological targets. However, due to the limitations of conventional cyclization-based approaches to medium ring synthesis, these structures remain severely underrepresented in current probe and drug discovery efforts. To address this problem, we have developed an alternative, biomimetic ring expansion approach to the diversity-oriented synthesis of medium-ring libraries. Oxidative dearomatization of bicyclic phenols affords polycyclic cyclohexadienones that undergo efficient ring expansion to form benzannulated medium-ring scaffolds found in natural products. The ring expansion reaction can be induced using one of three complimentary reagents and is energetically driven by rearomatization to a phenol ring adjacent to the scissile bond.

Coauthors: Renato A. Bauer and Derek S. Tan, Memorial Sloan–Kettering Cancer Center, New York, NY 10065

Terebrids Can Do it Too: Discovery and Characterization of a Novel Peptide Neurotoxin Tv1 from Venomous Marine Snail Terebra variegata Active on Nicotinic Receptors
Prachi Anand, PhD, Hunter College, CUNY and The American Museum of Natural History

The Conoidea superfamily comprised of cone snails, terebrids, and turrids, produce disulfide rich peptides to subdue their prey and are an exceptionally promising group for discovering therapeutically relevant natural peptide neurotoxins. For the past 30 years, efforts to characterize venomous snail peptides has focused on cone snail peptide toxins, conopeptides, leading to the successful distribution of the first cone snail drug, ziconotide (Prialt), an analgesic. In contrast to cone snails, very little is known of terebrid peptides, teretoxins. Described here is the discovery, and structural and functional characterization of a novel terebrid neurotoxin, Tv1, from Terebra variegata. Tv1 was identified using a charge-enhanced ETD chemical derivatization strategy in combination with de novo sequencing. Based on the classification of conotoxin superfamilies, Tv1, which is 21 amino acids long with six cysteines (CC-C-C-CC), belongs to mini MIII superfamily, for which there are no reported disulfide scaffolds. To characterize the disulfide connectivity of Tv1 an inventive partial reduction and dual alkylation technique was used to label the cysteine pairs followed by LC-MS/MS analysis. , Tv1's predicted disulfide scaffold is Cys1-Cys5, Cys2-Cys6 and Cys3-Cys4. The cysteine scaffold was determined using MassHunter Bioconfirm B.05 software, and further confirmed by NMR studies. Similar to other M superfamily ψ conotoxins, preliminary results indicate Tv1 significantly blocks Nicotinic Acetylcholine receptors, specifically α6β2β3 subtype in micromolar concentrations. The α6 subunit has gained increasing attention due to its putative role in nicotine reinforcement and addiction and its selective down-regulation in Parkinson's disease. Further biological assays are in progress to confirm the biological activity of Tv1. This is the first structural and biological characterization of a teretoxin peptide.

Coauthors: Alexander Grigoryan¹, Vadi Bhat, PhD², Beatrix Ueberheide, PhD³, Alexandre Kouriatov⁴, Brian Chait, PhD⁵, Jon Lindstrom, PhD⁴, Sebastian Poget, PhD⁶ and Mandë Holford, PhD
¹Hunter College, CUNY and The American Museum of Natural History
²Agilent Technologies
³New York University-Medical Center
⁴The University of Pennsylvania
⁵The Rockefeller University
⁶College of SI, CUNY

Chromatin: An Expansive Canvas for Chemical Biology
Tom W. Muir, PhD, Princeton University

Understanding protein function is at the heart of experimental biology. Perhaps one of the grandest contemporary challenges in this area is to catalogue and then functionally characterize protein posttranslational modifications (PTMs). Modern analytical techniques reveal that most, if not all, proteins are modified at some point; it is nature's way of imposing functional diversity on a polypeptide chain. Understanding the structural and functional consequences of all these PTMs is a devilishly hard problem. While standard molecular biology methods are of limited utility in this regard, modern protein chemistry has provided powerful methods that allow the detailed interrogation of protein PTMs. In this lecture I will discuss several protein ligation technologies developed for preparing modified proteins, and will highlight these methods through specific applications to chromatin biology.