Biology subjects

Callahan, B. P.

Publications and source records attributed to Callahan, B. P..

4 recordsLinked to original sources

bioRxiv2026

In vitro reconstitution of vertebrate Sonic Hedgehog protein cholesterolysis

Extracellular secretion of the oncogenic sonic hedgehog signaling ligand is contingent on its release from a precursor protein through peptide bond cholesterolysis, mediated by the hedgehog C-terminal domain, SHhC. In this work, we describe the in vitro reconstitution of cholesterolysis activity for SHhC domains from vertebrate model organisms, Xenopus laevis (Xla) and Danio rerio (Dre). Cholesterolysis is assayed continuously in multi-well plates by monitoring changes in fluorescence resonance energy transfer (FRET) from an engineered precursor construct, expressed in E. coli and purified in soluble form. Using this FRET assay, we found that Xla and Dre SHhC exhibit high substrate stereospecificity, accepting cholesterol, (KM, 1-2 {micro}M, cholesterolysis t1/2 of [~]11 min) while rejecting the 3-alpha epimer, epi-cholesterol (KM > 100 {micro}M, t1/2 > 10 hr). By screening a 96-member detergent/surfactant library for compatibility with SHhC activity, we identify cationic detergents that inhibit cholesterolysis and find a shared preference for the zwitterionic n-dodecyl-phosphocholine (DPC, Fos-choline-12), which supported the fastest reaction kinetics. Lastly, we report that alanine point mutation at a conserved aspartate residue (D46A) in Xla SHhC and Dre SHhC blocks cholesterolysis; however, activity could be chemically rescued with rationally designed hyper-nucleophilic sterols. Of those sterols, 2-beta carboxy cholestanol was active as a substrate with D46A variants only; the remaining sterols were accepted by both D46A and wild-type SHhC. In summary, we have established the first in vitro kinetic assay to continuously monitor enzymatic activity of wild-type and mutant vertebrate SHhC domains in multi-well plates, a key step toward pharmacological manipulation of Sonic hedgehog protein biosynthesis in vivo.

biochemistry↗

bioRxiv2023

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 Drosophila hedgehog autoprocessing domain, DHhC, served as a self-cleaving catalyst for these bioconjugations. In the presence of ssDNA or RNA with segments complementary to the steramers and adjacent to one another, the two NanoBiT fragments productively associate, reconstituting NanoBiT enzyme activity. NanoBiT luminescence in samples containing nM ssDNA or RNA template exceeded background by 30-fold and as high as 120-fold depending on assay conditions. A unique feature of this detection system is the absence of a self-labeling domain in the NanoBiT bioconjugates. Eliminating that extraneous bulk broadens the detection range from short oligos to full-length mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/572427v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@f0706dorg.highwire.dtl.DTLVardef@1653d01org.highwire.dtl.DTLVardef@18843bcorg.highwire.dtl.DTLVardef@1170e47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

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 turn-over by an enzymatic domain, called HhC. HhC is found in precursor Hh proteins only. Through cholesterolysis, HhC is cleaved from the precursor. Attempts to identify molecules that inhibit intramolecular cleavage/sterylation activity of HhC have resulted in antagonists that bind HhC irreversibly through covalent mechanisms, as is commonplace for protein autoprocessing inhibitors. Here we report an exception to the "irreversibility rule" for protein autoprocessing inhibition. Using a FRET-based activity assay for HhC, we screened a focused library of sterol-like analogs for HhC cholesterolysis inhibitors. We identified and validated four structurally related noncovalent inhibitors, which were then used for SAR studies. The most effective derivative, tBT-HBT, binds HhC reversibly with an IC50 of 300 nM. An allosteric binding site for tBT-HBT, encompassing interactions from the two subdomains of HhC, is suggested by kinetic analysis, mutagenesis studies, and photoaffinity labeling. A striking resemblance is found between the inhibitors described here and a family of noncovalent, allosteric activators of HhC, which we described previously. The inhibitor/activator duality appears to be mediated by the same allosteric site, which displays sensitivity to subtle differences in the structure of a heterocycle substituent on the effector molecule.

biochemistry↗

bioRxiv2021

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. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 x 10-12 M) reported wild-type SARS-CoV-2 nucleic acid at [≥]1 x 10-9 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents reported to date.

biochemistry↗