Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications.

Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.inositol pyrophosphates | affinity reagents | protein pyrophosphorylation | signal transduction | metabolism S ignal transduction pathways and metabolic circuits are essential for cell homeostasis and survival. These two types of networks have historically been viewed as separate entities, but it is becoming increasingly clear that they must be coordinately regulated. Indeed, growth factor-stimulated signaling pathways can promote the metabolic activity of the cell (1). Conversely, the activity of signaling proteins can be controlled by specific metabolites (2), either by allosteric mechanisms or via nutrient-sensitive covalent modifications, such as acetylation (3) and glycosylation (4).The highly phosphorylated inositol polyphosphates (InsPs), and in particular the inositol pyrophosphates (PP-InsPs), are primed to provide additional junctures between signaling and metabolic networks (5-7). A cascade of phosphorylation reactions converts the secondary messenger inositol trisphosphate (InsP 3 ) to the fully phosphorylated inositol hexakisphosphate (InsP 6 ) (8). Subsequent action of inositol hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks) furnishes the PP-InsP messengers, a unique class of signaling molecules containing one or two high-energy phosphoanhydride bonds (Fig. 1A) (6,7,9). A number of studies have indicated a central role for PP-InsPs in metabolic reprogramming and phosphorylation-based signaling at the cellular and organismal level. For example, the biochemical properties of the IP6Ks confer an "energy sensing" function onto these enzymes (10). Because the IP6Ks have a K m for ATP between 1.0 and 1.4 mM-concentrations that are similar to the intracellular ATP levels-t...

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, the Saccharomyces cerevisiae homolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. We identified a novel phosphatase of the recently discovered atypical dual specificity phosphatase family as a physiological inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the ␤-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP 5 or IP 7 ) in vitro. In vivo, siw14⌬ yeast mutants possess increased IP 7 levels, whereas heterologous SIW14 overexpression eliminates IP 7 from cells. IP 7 levels increased proportionately when siw14⌬ was combined with ddp1⌬ or vip1⌬, indicating independent activity by the enzymes encoded by these genes. We conclude that Siw14 is a physiological phosphatase that modulates inositol pyrophosphate metabolism by dephosphorylating the IP 7 isoform 5PP-IP 5 to IP 6 .Inositol pyrophosphates are a novel class of signaling molecules that carry energy-rich diphosphate bonds. In wild-type yeast, the most abundant isoform, diphosphoinositol pentakisphosphate (PP-IP 5 or IP 7 ), 5 consists of a fully phosphorylated six carbon myo-inositol ring further pyrophosphorylated at one of the carbons. Two physiologically relevant isomers of IP 7 include 1PP-IP 5 and 5PP-IP 5 in which the pyrophosphate group is found at the 1-position or the 5-position, respectively. Sequential phosphorylation of IP 7 results in bisdiphosphoinositol tetrakisphosphate (1,5PP-IP 4 or IP 8 ) that is pyrophosphorylated at both the 1st and 5th positions (1, 2). Since their discovery more than 20 years ago, inositol pyrophosphates have been implicated in an array of processes, including cellular energetic metabolism, telomere maintenance, oxidative stress response, and vesicle trafficking in animals and fungi (1). Recent work in Saccharomyces cerevisiae found that production of inositol pyrophosphates is essential for mounting environmental stress responses (3). Inositol pyrophosphates are thought to be metabolic regulators displaying expanded cellular roles beyond classic second messengers (1).The enzymes that regulate the pools of inositol pyrophosphates are not fully described. The inositol hexakisphosphate kinases synthesize IP 7 , the most abundant inositol pyrophosphate in the cell; they add the ␤-phosphate to IP 6 at the 5th position generating 5PP-IP 5 (2). Yeast possess a single inositol hexakisphosphate kinase gene, KCS1, whereas mammals possess three homologous genes encoding inositol hexakisphosphate kinase (Fig....