Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP₆-Binding Proteins

Abstract

Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems.

Using Biotinylated myo-Inositol Hexakisphosphate to Investigate Inositol Pyrophosphate–Protein Interactions with Surface-Based Biosensors

“…47 A biotinylated version of InsP 6 had been reported in the past, but this reagent was not suitable for the current application, due to its relatively short and hydrophobic linker. 48 We therefore decided to pursue two synthetic strategies to obtain InsP 6 conjugates in which the biotin is attached via a hydrophilic polyethylene glycol (PEG) linker. The first approach sought to introduce the biotin-containing linker early in the synthesis, facilitating purification and isolation steps of protected intermediates along the synthetic route (Scheme S1), but this strategy proved to be inefficient.…”

“…Several examples exist in which biotinylated, negatively charged small molecules have been applied in binding measurements of surface-based biosensors . A biotinylated version of InsP 6 had been reported in the past, but this reagent was not suitable for the current application, due to its relatively short and hydrophobic linker . We therefore decided to pursue two synthetic strategies to obtain InsP 6 conjugates in which the biotin is attached via a hydrophilic polyethylene glycol (PEG) linker.…”

The impact of “super-dosing” phytase in pig diets on growth performance during the nursery and grow-out periods1

“…Phosphoinositides are stored in the plasma membrane and are a source of lower derivative inositols, which are used as secondary messengers in insulin signaling and intracellular Ca signaling ( Croze and Soulage, 2013 ). Inositol derivatives are synthesized within the animal de novo ( Jiao et al, 2015 ). Thus, by increasing dietary inositol, there may be less need for inositol synthesis by the animal, or perhaps there is an increase in inositol usage, which is contributing to the observed improvements in growth performance.…”

Identity and functions of inorganic and inositol polyphosphates in plants

“…This analogue was then linked to an affinity resin, allowing for protein pull-down, much like a co-immunoprecipitation, followed by mass spectrometry analysis to identify novel PP-InsP-interacting proteins in yeast . Similar approaches, using amine-tethered or biotinylated InsPs, were employed to identify putative PP-InsP binding proteins (Jiao et al, 2015;Gregory et al, 2016). These tagged InsP versions provide the added benefit of enabling attachment to a chip for SPR detection methods (Gregory et al, 2016), allowing for precise quantification of InsP-protein interactions that is critical to accurately distinguish PP-InsP isomer-binding preferences.…”