Bryan C. Dickinson, Ph.D. - US grants

Project Summary Thousands of proteins in the human proteome are subjected to modification by reversible lysine acetylation, which is now recognized as a key regulator of diverse processes such as epigenetics and metabolism. The careful balance between the expression and activity of lysine acetyl transferases (KATs) and lysine deacetylases (KDACs) maintains the acetylome of a cell. Our long-term goal is to develop a mechanistic understanding of how lysine acetylation is controlled and how the mark effects cell state. Our current focus is to develop a new class of small molecule fluorescent chemical tools that report on lysine deacetylation activities in living cells with spatial resolution. We will deploy this new family of chemical tools to test the hypothesis that KDAC signaling is in part mediated by subcellular distribution, which controls access to substrates and local cofactors. This hypothesis could help explain some of the ambiguous results associated with lysine acetylation that have been observed, as the biological consequence of modulation of a specific KDAC isoform may be masked by other isoforms in a cell-type or disease specific manner. We postulate that a key to understanding how lysine acetylation is regulated is to monitor the overall amounts of KAT and KDAC activities with spatial-temporal resolution. Our primary biological interests right now deal with roles of KDACs outside of the nucleus, in particular in the mitochondria and the cytoplasm, while pursing mechanistic studies in the context of metabolism and breast cancer.

PROJECT SUMMARY The long-term goal of this project is to develop a rapid, cost-effective, efficacious evolution platform for the creation of selective inhibitors of target protein-protein interactions (PPIs). The emergence of cancer systems biology approaches has revealed a plethora of PPI hubs that are critical for cancer acquisition, maintenance, and immune evasion. However, disrupting these PPIs with pharmacophores, either therapeutically or for basic science discovery, has been challenging, in large part due to a lack of methods to select for functional activity and inadequate molecular libraries. Our new proposed system, ?rapid evolution of PPI inhibitors? (rePPI-i), will synergize two recently developed technologies: 1) phage-assisted continuous evolution (PACE), and 2) activity-responsive RNA polymerases (ARs), to solve this long-standing problem. In the rePPI-i system, bacteriophage will carry an evolving population of genetically-encoded therapeutic leads, and the phage life cycle will depend on those leads disrupting a target PPI selectively over an off-target PPI. Therefore, in a matter of days, billions of targets can be screened through hundreds of rounds of rapid evolution. Due to the versatility of the AR PPI detection system, rePPI-i will be capable of moving from target identification to a library of PPI inhibitors in a matter of weeks, dramatically accelerating the drug discovery process. Moreover, PPIs are often considered ?undruggable? targets with traditional pharmacological approaches due to the difficulty in: 1) disrupting the often extensive macromolecular interfaces; and 2) the limits of molecular libraries and screening approaches currently utilized in cancer target campaigns. This paradigm will be broken by rePPI-i due to the power of rapid, continuous evolution and the resultant ability to generate highly optimized peptide-based inhibitor molecules. To develop and validate this novel approach, we will evolve both linear and cyclic peptides, as well as small structured proteins, to disrupt the c-Myc/Max interaction and the BCL-2 family protein Mcl-1, both validated oncogenic targets in need of therapeutics. Our technology will result in a new paradigm for pharmacological development of PPI inhibitors in addition to discovering therapeutic leads for these two important cancer targets. rePPI-i has the potential to not only rapidly accelerate PPI inhibitor drug discovery and open up previously ?undruggable? targets to pharmacological intervention, but also lowers the ?activation barrier? to targeting interactions, allowing researchers to explore more interventions. Once optimized, rePPI-i will be both simple and inexpensive to employ for new targets of interest, democratizing the drug discovery process and allowing cancer researchers to more readily develop therapeutic leads for novel targets.

PROJECT SUMMARY Deciphering the complex underpinning of brain structure and function requires a complete understanding of how molecular contacts between cells in the brain are regulated. Currently, there are a lack of tools to measure these intercellular protein-protein interactions (PPIs) with requisite sensitivity, precluding a complete understanding of endogenous regulatory mechanisms. Moreover, as intercellular contacts are guided by thousands of diverse PPIs, it is critical to be able to study multiple interactions simultaneously, which is presently not possible. Here, we propose to develop a completely new category of split protein-based biosensors using RNA polymerases (RNAPs). The concept is that an RNAP-based intercellular detection strategy should offer substantial improvements in sensitivity, due to the signal amplification made possible by nucleic acid amplification technologies, and multidimensionality, due to the capacity of orthogonal RNAPs to drive distinguishable RNA output signals. In preliminary data, we show an evolved split RNAP that can detect target PPIs in vivo with more than 1,200-fold dynamic range, and also show in proof-of-concept experiments that two interactions can be monitored simultaneously. We propose to develop the split RNAP platform into a new set of tools, reagents, and protocols for the study of intercellular PPIs, through the completion of three aims. First, we will transition the binary PPI analysis platform to mammalian cells for intercellular PPI detection, using the well-studied neurexin (NRX) and neuroligin (NLG) interaction as a model system. Second, we will expand the scope of detection by evolving a panel of orthogonal split RNAPs for the detection of at least four simultaneous PPIs. In addition, we will develop new imaging techniques to quantify the interactions using super-resolution imaging platforms, both for binary and multidimensional PPI detection. Finally, we will test and showcase the capabilities of the system by interrogating the ?NRX interactome? using coculture experiments with primary neurons and engineered reporter cells. During the course of completing all of these steps, we will compare our technologies to the most successful split protein reporters, with metrics including sensitivity, dynamic range, and resolution as the primary attributes. Together, completion of this work will lay the foundation for a completely new approach for interrogating the intercellular interactions that guide synapse formation and brain function. We will make our new tools and protocols widely available to the broader community, accelerating and improving the work done within the BRAIN initiative. Through collaborations with other BRAIN initiative researchers, we will ultimately deploy our new technologies to uncover the complex code that guides neural coding in intact brain circuits.

PROJECT SUMMARY Both neurodevelopment and the synaptic plasticity events that underlie learning and memory rely heavily on tightly regulated gene expression programs and rapid, finely-tuned translation of messenger RNA (mRNA) transcripts. The degradation, stability, and translation of mRNA has, in recent years, been found to be regulated by adenosine methylation, which alters both transcript structure and the recruitment of RNA-binding proteins that inform these activities. Recent identification of and experimentation with the methyltransferases (?writers?), demethylases (?erasers?), and specific methyladenosine binding proteins (?readers?) have established that these epitranscriptomic mRNA regulatory processes are both dynamic and tightly regulated. Although the most well-studied of these modifications is N6-methyladenosine (m6A), N1- methyladenosine (m1A) has also recently emerged as a prevalent epitranscriptomic mark. Current methods used to explore these modifications require large sample sizes and are inherently low-resolution. These limitations preclude them from mapping and quantifying the epitranscriptome in specific brain regions, or in clinical biospecimens. Here, we describe preliminary development of innovative technologies to precisely sequence and probe the function of specific m6A and m1A modifications. We propose to leverage these foundations in the service of the following specific aims: 1) Evolve and establish high-resolution, antibody-free m6A and m1A mapping platforms for brain analysis, 2) Design and validate a molecular toolkit to manipulate transcript-specific m6A and m1A modifications in vivo, and 3) Catalog m6A and m1A modifications in the brain across development, neuron populations, activity state, and in synapses, and determine their function in relation to learning and memory. Our findings will illuminate how the epitranscriptomic landscape and specific mRNA transcripts in discrete neuronal populations regulates gene expression to inform complex neuronal processes, such as development, learning and memory, and how perturbations thereof result in abnormal brain function such as learning impairment. Importantly, this translational, functional validation of our new tools, which will be made available to the research community, provides a strong foundation for their usage to specifically interrogate how mRNA modifications are perturbed in other pathological contexts.

Project Summary Protein lipidation is a dynamic post-translational modification (PTM) that affects subcellular trafficking, co-factor binding affinity, and membrane localization of proteins, which in turn influence downstream signaling cascades. In particular, cyclic S-acylation and -deacylation of proteins at specific cysteine residues is emerging as a key link between circulating lipid levels and the regulation of essential biological processes, including those involved in cellular growth, metabolism, and neurological health. In-depth study of this PTM, however, has proven technically difficult, in large part due to the paucity of selective, effective chemical inhibitors for the enzymes that catalyze its installation and removal. The proposed research program is designed to generate novel chemical technologies, namely small molecule probes and inhibitors, in the service of illuminating the involvement of regulated protein S-acylation in both normal and pathophysiological contexts. These goals will be realized through two complementary chemical and cellular biology research areas. One area will involve measuring, manipulating, and determining the targets of the ?writers? of S-acylation, DHHCs. To do so, pan-active DHHC inhibitors will be identified using newly developed and optimized screening and selectivity profiling assays in combination with rationally and computationally designed molecules, as well as a library of putative inhibitors. Validated inhibitors and proteomics-based methods will then be used to identify the specific protein targets of DHHCs in live cells to more precisely describe their involvement in various disease states. The second research area will utilize our recently validated chemical tools to study the biological function of the ?erasers? of S-acylation, APTs, with particular emphasis on the involvement of these enzymes in cellular redox homeostasis and metabolic disease. The expected outcome of this multidisciplinary research program is two-fold: generating a collection of chemical tools and assays for the study of this important PTM, and describing its biological function and influence in normal and disease states.