Research

Strategies for Elucidating the Ecto-Interactome

Jan. 14, 2018, 11:02 p.m.

Ecto
Ecto

Defining the entire ensemble of molecular interactions and networks present in an organism (i.e., the Interactome) is essential for understanding the function, dynamics and logic underlying complex cellular systems. Large-scale efforts, involving both yeast-two-hybrid approaches and affinity-mass spectrometry, have begun to define large portions of the eukaryotic Interactome. While these approaches have proven effective for evaluating cytoplasmic interactions, they are inadequate for defining the interactions involving the 1/3 of the human proteome represented by secreted proteins and the ectodomains of integral membrane proteins. These proteins and their interactions are vital to cellular and multi-cellular processes as they detect the developmental, morphogenetic and environmental cues that are central to normal physiology and pathology. These receptor:ligand complexes also offer enormous promise as therapeutic targets and for the development of biologics to treat autoimmune diseases, infectious diseases and malignancies. At present there exists no platform to support the discovery of these extracellular interactions. To address this challenge, we have recently established strong proof-of-concept for two distinct high-throughput platforms for mapping of the “Ecto-Interactome”, the entire set of interactions formed by secreted and cell surface proteins. These efforts exploit a multi-disciplinary team, composed of protein chemists, automation specialists and biologists, which is merging multiple protein expression/presentation strategies with cutting-edge cell microarray and flow cytometry technologies. The optimization and implementation of these platforms promises to have transformative impact by revealing extracellular interactions and networks that yield novel insights into normal physiology, disease and therapeutic strategies. We are positioned to make significant progress in assessing the feasibility of defining the Ecto-Interactome. Together with the considerable body of cytoplasmic interactions that is accruing, these studies will provide important insights into the full range of molecular circuitry that integrates multiple disparate signals into cellular and multi-cellular function.

Novel Strategies for Precision T-Cell Therapies

Jan. 14, 2018, 11:02 p.m.

TCell
TCell

Immunotherapies, including biologics and cell-based therapies, are emerging as highly promising and effective strategies for the treatment of cancer. Accompanying the great potential of these approaches are continuing challenges, including 1) untargeted global immune modulation associated with biologics, resulting in serious side effects; 2) challenges in scalability of cell-based therapies (e.g., adoptive/CAR-T therapies) and bispecifics, 3) lack of differentiation, as most efforts are focused on relatively few therapeutic targets and mechanisms; and 4) the lack of flexible platforms to rapidly and efficiently target new indications and mechanisms. To address these challenges, we describe a novel class of soluble precision biologics for the treatment of cancer. Our approach manipulates antigen-specific (i.e., clonal) lymphocyte populations by covalently linking single chain peptide-MHC (sc-pMHC) and costimulatory molecules in a manner that recapitulates the proximity, orientation and overall organization experienced at the immunological synapse. The sc-pMHC unit serves to selectively target distinct T cell clones for the delivery of a modulatory domain that can represent any potential costimulatory function. These constructs are generated as Fc-fusion proteins (i.e., IgG) for enhanced avidity and stability. This combined targeting:modulation construct is referred to as synTac (artificial immunological Synapse for T-cell Activation). Using this strategy we have already demonstrated clonal-specific T cell proliferation and activation in vitro, and clonal T cell expansion in vivo. The extreme specificity associated with these reagents eliminates the extensive side effects associated with currently used immunotherapeutics and the highly modular design supports a wide range of indications and therapeutic mechanisms via substitution of the disease relevant peptide epitope and comodulatory modules, respectively.

Function and Mechanism of Viperin, A radical antiviral SAM protein

Jan. 14, 2018, 11:02 p.m.

Viperin
Viperin

Viral infections of all kinds continue to represent major public health challenges and demand new therapeutic strategies. Viperin (virus-inhibitory protein, endoplasmic reticulum associated, interferon (IFN) inducible), a member of the radical S-adenosylmethionine (RS) superfamily of enzymes, is an interferon inducible protein that inhibits the replication of a remarkable range of viruses, including Chikungunya virus, Bunyamwera virus, Tick-born encephalitis virus, influenza A virus, human cytomegalovirus, West Nile virus, hepatitis C virus, sindbis virus, Japanese encephalitis virus, HIV and numerous other DNA and RNA viruses. Viperin has been suggested to elicit these far-reaching antiviral activities through interaction or co-localization with a large number of functionally unrelated host and viral proteins. All of these interactions are based on indirect methods (e.g., yeast-two-hybrid and immunoprecipitation), and none have been validated by direct biochemical approaches. The mechanisms underlying viperin’s sweeping antiviral activity remain enigmatic and it is unclear how a single protein (i.e., viperin) can participate in such a broad playlist of interactions to inhibit this wide array of viruses. Instead, we favor a more general mechanistic explanation for these antiviral activities; one that involves a viperin-mediated enzymatic transformation that modulates specific cellular processes common to all of these viruses. We demonstrate that, contrary to all previous work, viperin converts cytidine triphosphate (CTP) to a novel CTP-related triphosphate via an S-adenosylmethionine (SAM)-dependent radical mechanism analogous to other members of the RS superfamily. The in vivo function of this new molecules remains to be defined; but may include 1) selective “poisoning” of viral RNA and DNA polymerases, 2) modulation/inhibition of cytidylyl transferases, which use CTP as a substrate, and are required for lipid biosynthesis (e.g., phosphatidylethanolamine, phosphatidylcholine) and 3) a role as a novel signaling molecule. All of these possibilities would provide a unified mechanism for viperin antiviral function, as each proposed mechanism relies on the radical-based enzymatic properties of viperin to modulate fundamental processes (replication, membrane dynamics and signaling) critical to all viral species.

Nucleic Acid Programmable Protein Arrays

Feb. 12, 2018, 7:18 p.m.

nappa_cap
nappa_cap

Protein microarrays are ideal high-throughput methods for investigating protein:protein interactions, including biomarker discovery (screening serum against antigen proteins), on a single array. Conventional arrays are generated by expressing, purifying, and spotting thousands proteins on a microscope slide; however there are technical and cost challenges associated with the purification of thousands of different proteins. Nucleic Acid Programmable Protein Array (NAPPA) is an alternative method that overcomes these challenges by translating the proteins in situ on the array surface. Arrays are generated by printing protein-encoding plasmid DNA at high density on a chemically modified slide. DNA is transcribed and translated into functional proteins in situ using coupled in vitro transcription and translation (IVTT) in human cell lysates. Using human cell lysate provides the cellular translational machinery required for proper protein folding and post-translational modifications. Expressed proteins are immobilized in situ, enabling screening of the array shortly after protein production; by reducing the experimental time, issues with protein stability are reduced and reproducibility increased. NAPPA slides programmed with potential antigens are challenged with patient serum to detect specific antibody response, allowing biomarker discovery and analysis of antigen drift in cancer, infectious diseases, and autoimmune diseases.

We are in the process of developing NAPAA in house, utilizing an Arrayjet piezoelectric microarray printer which is capable of printing rapidly and reproducibly on glass slides and high density nanowell arrays.

The Ligand Discover Project

March 13, 2018, 1:33 p.m.

The development of strategies, tools and infrastructure for the discovery of novel microbial metabolism represents a major challenge to the post-genomic biological community. The functional properties of solute binding proteins (SBPs) make them particularly amenable to large-scale functional annotation, as the first step in a catabolic pathway is frequently the passage of a metabolite across the cellular membrane by SBP- dependent transport machinery. The ability to identify the initial reactant (or a closely related molecule) for a catabolic pathway provides an immediate toe-hold by placing significant constraints on the regions of chemical space which need to be considered and, in conjunction with knowledge of colocalized and coregulated genes, begins to define details of the in vivo biochemical transformations operating within the metabolic pathway. Accordingly, a central goal of this project is to evaluate the wide-spread utility of targeting SBPs and related proteins for initial functional insight. We have implemented a high-throughput differential scanning fluorimetry (DSF) assay for the interrogation of ligands/metabolite libraries carefully constituted for specific subfamilies of SBPs. In parallel, high resolution structural characterization of SBP-ligand complexes will reveal the determinants responsible for ligand recognition, and delineate “specificity boundaries” required for confidently defining the limits of annotation transfer. The Ligand Discovery Project will specifically examine SBPs involved in carbohydrate and amino acid metabolism and expand this strategy to include the ligand-responsive transcriptional regulators which combine an SBP-like domain with a DNA-binding module. Finally, this strategy will be applied to the microbes that compose the human gut microbiome. Combining experimentally defined SBP ligands and high resolution structural information with genome neighborhood networks (Metabolism Project) and additional insights gleaned from computational approaches (Modeling Project) results in a powerful multidisciplinary strategy for the discovery of new metabolism. When systematically applied to the human gut microbiome, these approaches will result in the discovery of new metabolic pathways important for communication between members of the gut community, communication between the gut microbes and the human host, and in particular will expand our understanding of the impact of microbial metabolism on human health and disease. Most importantly, the strategies and tools described in this application will enable the discovery of novel metabolism by the entire community.