Farren Isaacs - US grants

? DESCRIPTION (provided by applicant): Protein phosphorylation is one of the most common and critical post-translational modifications governing signaling cascades in humans. Phosphorylation of protein kinases governs their activity and regulation. The importance of regulation by phosphorylation is further emphasized by the fact that protein kinases comprise nearly 2% of the human proteome and numerous kinases have been implicated in processes that control cell proliferation, motility, and apoptosis in healthy and diseased human cells. While identification of phosphorylation sites within the human proteome has dramatically progressed in recent years, our understanding of phosphorylation cascades is limited due to a distinct lack of knowledge of which kinases are responsible for each phosphorylation event and the specific arrangement of phosphorylation sites leading to an active kinase that phosphorylates its target substrate. Establishing direct connections of all human kinases to the phosphoproteome and revealing a systems-level diagram of human signaling networks also remain defining challenges. Since phosphorylation plays a central role in protein-protein interactions through phospho-binding domains, new approaches that can address these questions in a comprehensive and unbiased fashion are needed. Studying protein phosphorylation has been limited by the inability to generate phosphoproteins with the specificity of natural systems. Genetically encoded non-standard amino acids (NSAAs) have recently enabled site-specific incorporation of phosphoserine into proteins. We showed that a genomically recoded organism (GRO), in which all TAG stop codons were converted to TAA and the deletion of RF-1, converted TAG to an open sense codon dedicated for incorporating phosphoamino acids. Importantly, this technological breakthrough enables site-specific expression of human phosphoproteins in an engineered bacterial system (i.e., GRO containing phosphoserine orthogonal translation system, OTS). Furthermore, it provides a platform technology to address questions probing the connectivity of the human kinome and the functional landscape of phospho-binding domains. Here, we aim to further develop and apply this technology to generate optimized platforms to address functional questions surrounding the phosphoserine component of the human phosphoproteome (Aim 1). These new, enhanced platforms will enable studies to identify STE20 kinase substrates that will directly inform future research into multiple human disease pathways as well as define a general strategy to elucidate human kinase substrates (Aim 2). Finally, we aim to identify phosphorylation sites that are drivers of protein-protein interactions in general, followed by, a systematic screen of the STE20 substrates in a coordinated effort to assign biological function to a portion of the human phosphoproteome (Aim 3).

Developing synthetic routes to polymers consisting of a defined sequence of monomers is a current challenge in chemistry. Proteins are natural polymers, and are specific sequences of amino acids put together easily and efficiently by the living cell. The NSF Center for Genomically Encoded Materials (C-GEM) takes a bio-inspired approach making a sequence of synthetic polymers by re-engineering the cell's systems. In this case, the cell becomes a new translational machine that synthesizes new chemical polymers of specific sequence and length. NSF C-GEM establishes a new, transformative field of chemistry and fosters innovation at the chemistry-biology-materials frontier. The applications of the new materials with novel properties range from information storage to anti-counterfeiting and drug delivery, from environmental remediation to drug discovery. NSF C-GEM engages scientists and non-scientists in a variety of research and educational activities, including improved communication to the public. These education and participation programs integrate research with training to establish a diverse chemical workforce. NSF C-GEM presents a new online platform and data management system, GEM-Net, to promote data sharing within and outside the research team. NSF C-GEM is also developing an online video game that allows citizen scientists to participate in the research process and track results.

The NSF Center for Genomically Encoded Materials, NSF C-GEM, is focused on the challenge of preparing polymers whose monomers and dispersity are defined with protein-like control. Sequence-defined polymers possess extraordinary potential for information storage, anti-counterfeiting, drug delivery, environmental remediation, and even drug discovery, but strategies to prepare them are barely in their infancy. NSF C-GEM's approach synthesizes sequence-defined chemical polymers by repurposing the E. coli translational apparatus. The translation machinery, which normally promotes bond formation between alpha-amino acids, now promotes bond-forming reactions between monomers that are the building blocks for aramids, polyolefins, polyurethanes, and other polymers. The approach demands orthogonal enzymes that acylate orthogonal tRNAs with each monomer, efficiently and in vivo; orthogonal ribosomes that accept these tRNAs as substrates and elongate the products; genomically-recoded organisms with multiple open codons to enable mRNA-templated synthesis of sequence-defined polymers; and high-resolution structural data to deepen understanding and inform design. The project develops the multi-disciplinary tools and technologies that demonstrate the proof of concept of the approach and establish practical methodologies to this aim. NSF C-GEM fosters innovation at the chemistry-biology-materials frontier, and engages scientists and non-scientists in research and educational activities. A diverse set of education and participation programs integrates research with training, establishes a diverse chemical workforce, and improves communication with the public.

The translation apparatus is the cell's factory for protein biosynthesis, stitching together amino acid substrates into sequence-defined polymers (proteins) from a defined genetic template. The extraordinary synthetic capability of the protein biosynthesis system has driven extensive efforts to harness it for societal needs in areas as diverse as energy, materials, and medicine. For example, recombinant protein production has transformed the lives of millions of people through the synthesis of biopharmaceuticals and industrial enzymes. In nature, however, only limited sets of protein monomers are utilized, thereby resulting in limited sets of biopolymers (i.e., proteins). Expanding nature's repertoire of ribosomal monomers could yield new classes of enzymes, therapeutics, materials, and chemicals with diverse chemistry. In the short term, this will expand the genetic code in a unique and transformative way. In the long-term, knowledge gained will allow researchers to diversify, evolve and repurpose the ribosome and the entire protein synthesis system to generate non-natural polymers as new classes of sequence-defined, evolvable matter. This proposal will also promote interdisciplinary education, including the specific expansion of STEM education and career opportunities for underrepresented minorities and women. Students will be trained to integrate principles from genome engineering, systems biology, and synthetic biology. As a form of outreach, the investigators will create experiential learning modules that bring synthetic biology research to K-12 and undergraduate classrooms and connect students to the science being done at our institutions. This new outreach program will ensure that advances made in this project benefit a broader community and will contribute to motivating and training young scientists and engineers.

In this project, the investigators seek to repurpose the translation apparatus for making new proteins containing multiple mirror-image D-alpha-amino acids. By seamlessly melding the integration of bottom-up engineering design, genome engineering, and full-scale systems optimization, the project will create a new framework for studying and engineering translation and the genetic code. This framework will be instrumental in sustaining the much-anticipated transformation in expanding the range of genetically encoded chemistry in living systems, with the potential for significant breakthroughs. For example, understanding the structural and substrate flexibility of the protein synthesis machinery may provide insights into life's origin and also lead to general rules for engineering protein synthesis to meet societal needs. From an engineering perspective, the research could enable scalable mirror image polypeptide synthesis, opening the door to cheaper and more effective peptidomimetic enzymes, materials, and drugs, and expanding the scope of synthetic and chemical biology. In sum, it is expected that this project will provide new directions for academic and industrial enterprises related to engineering translation, while simultaneously training the next generation of scientists and engineers to be full participants in the work force.

Project Summary  This proposed work seeks to develop a suite of enabling technologies capable of producing synthetic  phosphoproteins with the goal of transforming the field of human protein signaling from one that is purely  observation?based into one that biosynthesizes designer proteins to achieve a comprehensive understanding of  complex signaling networks. The importance of phosphorylation is emphasized by the fact that  phosphorylated proteins control most aspects of normal cellular homeostasis.  Aberrations in protein  phosphorylation can drive cancer, hypertension, diabetes, and neurodegenerative disorders.  Thus,  understanding differential patterns of protein phosphorylation in disease states is of extreme physiological  and clinical interest.  Analysis of phosphorylated amino acid residues has been limited by our inability to  control these chemical modifications due to a lack of phosphomimetics that fully recapitulate the chemistry of  phosphorylated residues. Current progress toward the elucidation of phospho?signaling networks is  hampered by the lack of methods to produce proteins containing specific combinations of phosphorylated  amino acids. In particular, synthetic chemistry is inadequate for total phosphoprotein synthesis, and  conventional biological methods do not control phosphorylation levels. We have recently developed a new  technology, albeit limited to phosphoserine (pSer), that enables the synthesis of recombinant phosphoproteins.   This technology directs phosphorylated amino acids into their physiologically relevant positions within  proteins yet our functional understanding of protein phosphorylation will remain incomplete without access  to phosphotyrosine (pTyr) and phosphothreonine (pThr) containing proteins. Specific Aims: In Aim 1, we will  utilize mutagenesis and laboratory evolution to engineer an optimized tyrosyl aminoacyl?tRNA synthetase for  phosphotyrosine.  In Aim 2, we will provide a solution to this problem by engineering an aminoacyl?tRNA  synthetase that can charge a phosphothreonine onto a special tRNA that reads a dedicated open codon.  Unique to our approach, we will also employ our genomically recoded E. coli cells in which open stop codons  can be converted into new sense codons that encode pThr and pTyr into precise locations in recombinant  proteins. Significance: The overall outcome of our studies will be an enabling technology for the expression of  pTyr and pThr containing proteins that will broadly enable research into disease mechanisms and can be used  directly to develop new therapies for human disease. This will be the first technology able to re?create human  disease networks that are ?difficult? or ?impossible? to infiltrate, and will establish the paradigm for  addressing other post?translational modifications. More broadly, the proposed work will enable the re?design  of programmable signaling networks comprising proteins with natural and synthetic nonstandard amino acids  capable of expanding networks beyond their natural functions and of producing novel synthetic polymers  with diverse chemistries. 

A hallmark of life on Earth is homochirality, or the fact that many of the key biological molecules - proteins, nucleic acids, sugars, and lipids - possess the same chirality. The term chirality refers to the property of an object to be distinguishable from its mirror image. We often refer to this property colloquially as handedness, as our left and right hands are not superimposable yet are mirror images of one another. These properties motivate the exploration of constructing and studying mirror biomolecules. In this project, the researchers seek to take the first steps toward building a mirror synthetic cell, providing a unique lens through which we will attain a fundamental understanding of chirality in biological molecules, systems, and processes. From an applied perspective, the work could enable production of entirely new classes of materials and mirror drugs endowed with improved stability and activity. Creating substances that were previously impossible to create will lead to the next-generation of renewable biotechnology and medical products. This proposal will also promote interdisciplinary education, including the specific expansion of STEM education and career opportunities for underrepresented minorities and women. To educate the public, the research team will engage the artistic community to illustrate the science of chirality through art, culminating in a 'Mirror World' exhibit that will be displayed at local museums. By doing so, the research team aim to communicate the importance of molecular handedness to the public, ensuring that advances made in this project benefit a broader community and contribute to inspiring and training young scientists and engineers.

In this project, the researchers seek to design, construct, and safely deploy synthetic mirror cells in which all of the key molecules - nucleic acids, proteins, carbohydrates, and lipids - exist in chiral states opposite to their natural forms. Toward this goal, the team will develop the capabilities to synthesize mirror DNA, RNA, and proteins; predict the physiology of cells with mirror components; and assess the risks and rewards of mirror life. If successful, this project will transform basic science, bioengineering, and open up new applications in biotechnology. Synthetic mirror cells will offer a unique lens to help decipher the role of chirality across multiple scales of life and elucidate why natural life has focused on one chirality. The researchers will develop a foundation for mirror cells via five coupled research, education, and outreach activities: (1) developing schemes for chemically synthesizing mirror biomolecules; (2) repurposing the natural biological machinery to synthesize mirror nucleic acids and proteins; (3) developing a computational framework for predicting the physiological impact of alternative chirality; (4) identifying gaps in the current ethical, legal, and environmental framework for synthetic cells and proposing new metrics for assessing the risks and rewards of mirror life; and (5) inspiring and educating the public about the potential of mirror life by working with artists to develop a museum exhibit titled 'The Mirror World.' Looking forward, this work will be a foundation for the know-how and capabilities to design, produce, evaluate, and safely deploy synthetic mirror cells with transformative potential in biotechnology, medicine, and industry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The breadth of genomic diversity endows organisms with rich biosynthetic capabilities and allows them to adapt to diverse environments. This species diversity in biological systems has tremendous potential to solve global challenges, such as the remediation of hazardous waste, producing new drugs and designer cells to alleviate human diseases, and the synthesis of novel chemicals and materials to ensure environmental sustainability. These challenges motivate the need to develop entirely new functional genomic tools, and enabling technologies to modify genomes on a large scale. Specifically, methods are needed for parallel and continuous directed evolution of gene networks or genomes to enhance understanding and expedite the design and evolution of organisms with prescribed functions. In this project, the investigators address these challenges through the development of multiplex genome engineering technologies in non-conventional yeast species. This project also promotes interdisciplinary education, including the specific expansion of STEM education and career opportunities for underrepresented minorities and women. Further, the investigators create experiential learning modules that bring genome engineering research to K-12 and undergraduate classrooms and connect students to the science in the laboratory. This new outreach program ensures that advances made in this project benefit a broader community and contribute to motivating and training young scientists and engineers.

In this project, we seek to develop cross-species multiplex genome engineering technologies to enable gene function discovery in non-conventional yeast (NCY) species. By integrating state-of-the-art molecular biology, laboratory evolution, and synthetic biology technologies, this project creates a new framework for studying and generating genetic variation. This framework is focused on establishing cross-species genome editing capabilities in nonconventional yeast with improved capabilities of delivering synthetic DNA in cells. The ability to introduce many targeted genome modifications at base-pair level precision establishes a general strategy for multiplex combinatorial genome engineering in NCYs and more broadly for eukaryotes. These advances enable the construction of targeted sets of genetic variants that can be functionally studied to elucidate causal links between genotype and phenotype and reprogram cellular behavior. In the long-term, the present work will transform the way scientists perform large-scale genetic modifications in non-conventional yeast species and usher in an era wherein globally reprogramming cellular behavior will become simple, inexpensive, and empower researchers to uncover new biological phenomena, elucidate causal links between genotype and phenotype, and design and reprogram organisms. In sum, this project provides new directions for academic and industrial efforts related to genome engineering, while simultaneously training the next generation of scientists and engineers to be full participants in the work force.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY Healthy and diseased physiological states are governed by a complex web of interacting proteins that confer the collective behavior observed in cells. The precise placement and chemical composition of post-translational modifications (PTMs) decorated across proteins determines their structure, function, and impart specificity for cellular signaling. Current progress toward the elucidation of PTM-mediated signaling and function is hampered by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific combinations of modified amino acids. Recent advances in synthetic and chemical biology have successfully demonstrated the ability to encode diverse nonstandard amino acids (nsAAs), including physiologically relevant PTMs, into proteins. In particular, recent advances in the development of genomically recoded organism (GROs) ? recoded strains of E. coli with open coding channels ? and engineered translation systems that encode PTMs (e.g., phosphoserine) have allowed activation of human phosphoproteins. These capabilities have precisely defined active protein states, map substrate networks, and implicate new function for disease-relevant mutations. However, two important challenges have emerged that preclude a comprehensive understanding of these protein networks and limit the translation of such insights into targeted clinical solutions. First, the precise arrangement and contributions of distinct PTMs that lead to active protein states is often unknown and hard to decipher. Second, the development of small molecules that target PTMs at molecular precision to modulate protein activity is a defining challenge for the development of new drugs. Specific Aims: In this proposal, we seek to leverage a strong foundation of genomic, biomolecular and proteomic technologies, expertise in systems and synthetic biology, and preliminary data to construct a genomically recoded organism (GRO) with three open codons in E. coli (Aim 1), engineer translational machinery that reassigns sense and stop codons for site-specific incorporation of multiple nonstandard amino acids that encode post-translational modifications into proteins (Aim 2), and utilize these technologies to develop a synthetic biology platform that synthetically activates disease-relevant protein networks targeted for isolation of new drug candidates (Aim 3). Significance: This work will be significant because it will enable the synthetic activation of physiologically relevant protein networks at the molecular level in GROs. These activated protein systems can elucidate complex biomolecular interactions that underlie disease and recapitulate human protein networks that are difficult to study and manipulate in their native contexts. Challenging these activated protein networks to small molecule libraries establishes a rapid and facile new approach to probe biomarkers at molecular specificity and sets the stage for a new synthetic-biology based drug discovery platform.