SYNOPSIS Phosphatidylinositol-5-phosphate (PtdIns5P) was discovered in 1997 (Rameh, L.E.; Tolias, K.F.; Duckworth, B.C. and Cantley, L.C. 1997, Nature 390, 192–196) but very little is known about its regulation and function. Hitherto, studies of PtdIns5P regulation have been hindered by the inability to measure cellular PtdIns5P using conventional HPLC, owing to poor separation from PtdIns4P. Here we present a new HPLC method for resolving PtdIns5P from PtdIns4P, which makes possible accurate measurements of basal and inducible levels of cellular PtdIns5P in the context of other phosphoinositides. Using this new method, we found that PtdIns5P is constitutively present in all cells examined (epithelial cells, fibroblasts and myoblasts, among others) at levels typically 1% to 2 % of PtdIns4P levels. In the β-pancreatic cell line BTC6, which is specialized in insulin secretion, PtdIns5P levels were higher than in most cells (2.5% to 4% of PtdIns4P). Using subcellular fractionation, we found that the majority of the basal PtdIns5P is present in the plasma membrane, but it is also enriched in intracellular membrane compartments, especially in smooth endoplasmic reticulum (SER) and/or Golgi, where high levels of PtdIns3P were also detected. Unlike PtdIns3P, PtdIns5P was also found in fractions containing very low-density vesicles. Knockdown of PtdIns5P 4-kinase (PIP4k) leads to accumulation of PtdIns5P in light fractions and fractions enriched in SER/Golgi, while treatment with Brefeldin A results in a subtle, but reproducible, change in PtdIns5P distribution. These results indicate that basal PtdIns5P and the PtdIns5P pathway for PtdIns(4,5)P2 synthesis may play a role in Golgi-mediated vesicle trafficking.

We describe a general synthetic strategy for developing high affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify a best binder. We describe epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

Phosphatidylinositol-5-phosphate 4-kinases (PIP4ks) are a family of lipid kinases that specifically use phosphatidylinositol 5-phosphate (PI-5-P) as a substrate to synthesize phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2). Suppression of PIP4k function in Drosophila results in smaller cells and reduced target of rapamycin complex 1 (TORC1) signaling. Here we showed that the γ isoform of PIP4k stimulated signaling through mammalian TORC1 (mTORC1). Knockdown of PIP4kγ reduced cell mass in cells in which mTORC1 is constitutively activated by Tsc2 deficiency. In Tsc2 null cells mTORC1 activation was partially independent of amino acids or glucose and glutamine. PIP4kγ knockdown inhibited the nutrient-independent activation of mTORC1 in Tsc2 knockdown cells and reduced basal mTORC1 signaling in wild-type cells. PIP4kγ was phosphorylated by mTORC1 and associated with the complex. Phosphorylated PIP4kγ was enriched in light microsomal vesicles, whereas the unphosphorylated form was enriched in heavy microsomal vesicles associated with the Golgi. Furthermore, basal mTORC1 signaling was enhanced by overexpression of unphosphorylated wild-type PIP4kγ or a phosphorylation-defective mutant and decreased by overexpression of a phosphorylation mimetic mutant. Together these results demonstrate that PIP4kγ and mTORC1 interact in a self-regulated feedback loop to maintain low and tightly regulated mTORC1 activation during starvation.

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We report on peptide-based ligands matured through the protein catalyzed capture (PCC) agent method to tailor molecular binders for in vitro sensing/diagnostics and in vivo pharmacokinetics parameters. A vascular endothelial growth factor (VEGF) binding peptide and a peptide against the protective antigen (PA) protein of Bacillus anthracis discovered through phage and bacterial display panning technologies, respectively, were modified with click handles and subjected to iterative in situ click chemistry screens using synthetic peptide libraries. Each azide-alkyne cycloaddition iteration, promoted by the respective target proteins, yielded improvements in metrics for the application of interest. The anti-VEGF PCC was explored as a stable in vivo imaging probe. It exhibited excellent stability against proteases and a mean elimination in vivo half-life (T 1/2 ) of 36 min. Intraperitoneal injection of the reagent results in slow clearance from the peritoneal cavity and kidney retention at extended times, while intravenous injection translates to rapid renal clearance. The ligand competed with the commercial antibody for binding to VEGF in vivo. The anti-PA ligand was developed for detection assays that perform in demanding physical environments. The matured anti-PA PCC exhibited no solution aggregation, no fragmentation when heated to 1008C, and > 81% binding activity for PA after heating at 908C for 1 h. We discuss the potential of the PCC agent screening process for the discovery and enrichment of next generation antibody alternatives.

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In this study, we investigated the preparation of living bacteria-nanoparticle hybrids mediated by surface-displayed peptides. The assembly of metallic nanoparticles on living bacteria has been achieved under mild conditions utilizing metal-peptide interactions, whereas the viability of the bacterial cells was greatly preserved. Escherichia coli was engineered with inducible gene circuits to control the display of peptides with desired sequences. Several designed peptide sequences as well as known gold-binding peptides were expressed on the cell surface using enhanced circularly permuted outer membrane protein X (eCPX) scaffolds. Driven by metal-peptide affinity, "biofriendly" citrate-stabilized gold nanoparticles were self-assembled onto the surface of bacteria with displayed peptides, which required overcoming the repulsive force between negatively charged nanoparticles and negatively charged cells. The bacteria/Au nanoparticle hybrids were highly viable and maintained the ability to grow and divide, which is a crucial step toward the creation of living material systems. Further activity and preservation of the bacterial hybrid assembly was demonstrated. The method described herein enables the conjugation of bacterial surfaces with diverse metal-rich nanoparticles in an inducible, and therefore easily controlled, manner. The expressed peptide sequences can be easily modified to alter the binding affinity and specificity for a wide variety of materials to form on-demand, high-density living biohybrids.

Peptide capture agents have become increasingly useful tools for a variety of sensing applications due to their ease of discovery, stability, and robustness. Despite the ability to rapidly discover candidates through biopanning bacterial display libraries and easily mature them to Protein Catalyzed Capture (PCC) agents with even higher affinity and selectivity, an ongoing challenge and critical selection criteria is that the peptide candidates and final reagent be selective enough to replace antibodies, the gold-standard across immunoassay platforms. Here, we have discovered peptide affinity reagents against abrax, a derivative of abrin with reduced toxicity. Using on-cell Fluorescence Activated Cell Sorting (FACS) assays, we show that the peptides are highly selective for abrax over RiVax, a similar derivative of ricin originally designed as a vaccine, with significant structural homology to abrax. We rank the newly discovered peptides for strongest affinity and analyze three observed consensus sequences with varying affinity and specificity. The strongest (Tier 1) consensus was FWDTWF, which is highly aromatic and hydrophobic. To better understand the observed selectivity, we use the XPairIt peptide-protein docking protocol to analyze binding location predictions of the individual Tier 1 peptides and consensus on abrax and RiVax. The binding location profiles on the two proteins are quite distinct, which we determine is due to differences in pocket size, pocket environment (including hydrophobicity and electronegativity), and steric hindrance. This study provides a model system to show that peptide capture candidates can be quite selective for a structurally similar protein system, even without further maturation, and offers an in silico method of analysis for understanding binding and down-selecting candidates.