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.
Many of the protein signaling networks that are critical for the emergence and progression of diseases are difficult to target due to their complex interactions and incomplete biochemical characterization of the underlying regulatory mechanisms. Among the numerous knowledge gaps is the role of post-translational modification (PTM) correlated to disease states, and barriers to their biochemical synthesis is a defining challenge. This work seeks to develop new genomic and synthetic biology technologies capable of encoding multiple, site-specific combinations of physiologically relevant PTMs into proteins to synthetically activate `undruggable' biomarkers to isolate small molecules that modulate the activity of protein targets and establish a new drug discovery platform.