Enzyme fragment complementation driven by nucleic acid hybridization sans self-labeling protein.

A modified enzyme fragment complementation assay has been designed and validated as a turn-on biosensor for nucleic acid detection in dilute aqueous solution. The assay is target sequence-agonistic and uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified enzymatically at their C-termini to steramers, sterol-linked oligonucleotides. The Drosophila hedgehog autoprocessing domain, DHhC, serves as the self-cleaving enzyme for the NanoBiT-steramer bioconjugations.

Enzyme Fragment Complementation Driven by Nucleic Acid Hybridization.

A modified protein fragment complementation assay has been designed and validated as a gain-of-signal biosensor for nucleic acid:nucleic acid interactions. The assay uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified at their C-termini to steramers, sterol-modified oligodeoxynucleotides. The hedgehog autoprocessing domain, DHhC, served as a self-cleaving catalyst for these bioconjugations.

Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules.

Paracatalytic inducers are antagonists that shift the specificity of biological catalysts, resulting in non-native transformations. In this Chapter we describe methods to discover paracatalytic inducers of Hedgehog (Hh) protein autoprocessing. Native autoprocessing uses cholesterol as a substrate nucleophile to assist in cleaving an internal peptide bond within a precursor form of Hh.

There's more to enzyme antagonism than inhibition.

A native enzyme's usual assurance in recognizing their physiological substrate(s) at the ground state and on going to the transition state can be undermined by interactions with selected small molecule antagonists, leading to the generation of abnormal products. We classify this mode of enzyme antagonism resulting in the gain-of-nonnative-function as paracatalytic induction. Enzymes bound by paracatalytic inducers exhibit new or enhanced activity toward transformations that appear aberrant or erroneous.

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators.

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value.

Cholesterol Chip for the Study of Cholesterol-Protein Interactions Using SPR.

Cholesterol, an important lipid in animal membranes, binds to hydrophobic pockets within many soluble proteins, transport proteins and membrane bound proteins. The study of cholesterol-protein interactions in aqueous solutions is complicated by cholesterol's low solubility and often requires organic co-solvents or surfactant additives. We report the synthesis of a biotinylated cholesterol and immobilization of this derivative on a streptavidin chip.

Nanomolar, Noncovalent Antagonism of Hedgehog Cholesterolysis: Exception to the "Irreversibility Rule" for Protein Autoprocessing Inhibition.

Hedgehog (Hh) signaling ligands undergo carboxy terminal sterylation through specialized autoprocessing, called cholesterolysis. Sterylation is brought about intramolecularly in a single turnover by an adjacent enzymatic domain, called HhC, which is found in precursor Hh proteins only. Previous attempts to identify antagonists of the intramolecular activity of HhC have yielded inhibitors that bind HhC irreversibly through covalent mechanisms, as is common for protein autoprocessing inhibitors.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid.

Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection.

Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous "hedgehog" protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis.

Specificity Distorted: Chemical Induction of Biological Paracatalysis.

We define paracatalysis as the acceleration of a reaction that appears abnormal or nonphysiological. With the high specificity of enzymes, side reactivity of this kind is typically negligible. However, enzyme paracatalysis can be amplified to levels that are biologically significant through interactions with a special class of small molecule "antagonist", here termed a paracatalytic inducer.

Insights on developing a field hospital formulary and medication distribution process in preparation for a second surge of COVID-19 cases.

Purpose: The coronavirus disease 2019 (COVID-19) pandemic has caused health systems across the country to plan for field hospitals to care for patients outside of traditional healthcare settings in the event of a second surge. Here we describe key considerations for the implementation of pharmacy operations and a field hospital formulary at an offsite location within a 2-week time frame.

Summary: Development of an offsite field hospital formulary is first dependent on the location and patient population defined for the field hospital.

Subverting Hedgehog Protein Autoprocessing by Chemical Induction of Paracatalysis.

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs through the action of the precursor's C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction.

General Base Swap Preserves Activity and Expands Substrate Tolerance in Hedgehog Autoprocessing.

Hedgehog (Hh) autoprocessing converts Hh precursor protein to cholesterylated Hh ligand for downstream signaling. A conserved active-site aspartate residue, D46, plays a key catalytic role in Hh autoprocessing by serving as a general base to activate substrate cholesterol. Here we report that a charge-altering Asp-to-His mutant (D46H) expands native cholesterylation activity and retains active-site conformation.

Protein-Nucleic Acid Conjugation with Sterol Linkers Using Hedgehog Autoprocessing.

Hedgehog (Hh) precursor proteins contain an autoprocessing domain called HhC whose native function is protein cleavage and C-terminal glycine sterylation. The transformation catalyzed by HhC occurs in from a precursor protein and exhibits wide tolerance toward both sterol and protein substrates. Here, we repurpose HhC as a 1:1 protein-nucleic acid ligase, with the sterol serving as a molecular linker.

Sterol A-ring plasticity in hedgehog protein cholesterolysis supports a primitive substrate selectivity mechanism.

Cholesterolysis of Hedgehog family proteins couples endoproteolysis to protein C-terminal sterylation. The transformation is self-catalyzed by HhC, a partially characterized enzymatic domain found in precursor forms of Hedgehog. Here we explore spatial ambiguity in sterol recognition by HhC, using a trio of derivatives where the sterol A-ring is contracted, fused, or distorted.

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate.

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >10-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol -OH group is replaced by the hyper nucleophilic -OOH group.

Illuminating cytochrome P450 binding: Ru(ii)-caged inhibitors of CYP17A1.

New Ru(ii)-caged abiraterone complexes were synthesized that exhibit strong absorption in the visible region and release the steroidal CYP17A1 inhibitor abiraterone upon exposure to low energy visible light in buffer and prostate cancer cells. Photoinduced release results in abiraterone binding to its CYP17A1 target in an inhibitory mode.

A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.

Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps.

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off-target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis.

Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling.

Discussions of therapeutic suppression of hedgehog (Hh) signaling almost exclusively focus on receptor antagonism; however, hedgehog's biosynthesis represents a unique and potentially targetable aspect of this oncogenic signaling pathway. Here, we review a key biosynthetic step called cholesterolysis from the perspectives of structure/function and small molecule inhibition. Cholesterolysis, also called cholesteroylation, generates cholesterol-modified Hh ligand via autoprocessing of a hedgehog precursor protein.

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor.

Hedgehog (Hh) proteins function in cell/cell signaling processes linked to human embryo development and the progression of several types of cancer. Here, we describe an optical assay of hedgehog cholesterolysis, a unique autoprocessing event critical for Hh function. The assay uses a recombinant Förster resonance energy transfer (FRET)-active Hh precursor whose cholesterolysis can be monitored continuously in multi-well plates (dynamic range=4, Z'=0.

Zinc inhibits Hedgehog autoprocessing: linking zinc deficiency with Hedgehog activation.

Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand.

Active site targeting of hedgehog precursor protein with phenylarsine oxide.

Hedgehog proteins, signaling molecules implicated in human embryo development and cancer, can be inhibited at the stage of autoprocessing by the trivalent arsenical phenyl arsine oxide (PhAs(III) ). The interaction (apparent Ki , 4 × 10(-7) M) is characterized by an optical binding assay and by NMR spectroscopy. PhAs(III) appears to be the first validated inhibitor of hedgehog autoprocessing, which is unique to hedgehog proteins and essential for biological activity.