Biology subjects

Giner, J.-L.

Publications and source records attributed to Giner, J.-L..

6 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↗

bioRxiv2026

A sterol reductase responsible for the unusual 8(14)-unsaturation in bacterial sterol production and degradation

Sterols are a class of lipids that play a crucial role in human health through their essential physiological roles and as a point of interaction between commensal and pathogenic bacteria. The biosynthesis and modification of these lipids is a well-characterized process in many eukaryotes and increasingly in bacteria. However, the proteins responsible for formation of the unusual 8(14)-unsaturation found in the sterols produced by aerobic methanotrophs, dinoflagellates, nematodes, and marine sponges, remains unknown. Here, we utilize a heterologous expression system to identify a bacterial 8,14-sterol reductase (8,14-Bsr) responsible for generating the 8(14)-unsaturation in the aerobic methanotroph Methylococcus capsulatus. This enzyme modifies the direct product of C-14 demethylation, reducing one double bond in the nuclear core structure and isomerizing the other to produce an 8(14)-sterol. We subsequently tested the requirement of putative active site residues for catalysis through site directed mutagenesis, identifying residues likely involved in interacting with the sterol substrate and directly catalyzing this reaction. Bioinformatic analysis of the distribution of 8,14-Bsr reveals it is unique to the bacterial domain, found primarily in the Methylococcaceae family, the Mycobacteriales order, and yet uncultured members of the Myxococcota phylum. Further phylogenetic analysis of 8,14-Bsr suggests it shares an evolutionary history with the C-14 demethylase in these organisms and that these two enzymes were likely inherited together. These results provide insight into novel sterol biochemistry, further delimiting sterol biosynthesis in the bacterial domain from eukaryotes and illustrating the importance of molecular characterization to identify bacterial proteins that interact with sterols. ImportanceMany of the eukaryotic proteins required for the biosynthesis of sterols, such as cholesterol, have been characterized. However, the pathways governing analogous processes in the bacterial domain are less characterized. Here, we identify an 8,14-sterol reductase in aerobic methanotrophs. This enzyme carries out a unique biochemical reaction, saturating and isomerizing double bonds in the nuclear core structure to produce an 8(14)-sterol. This reductase is restricted to the bacterial domain, further separating the evolution of bacterial sterol production from eukaryotes. Additionally, we find this reductase is prevalent in members of the sterol degrading order Mycobacteriales, highlighting a potential role for this protein in the remodeling of host sterol production by these pathogens.

microbiology↗

bioRxiv2024

The evolutionary history and modern diversity of triterpenoid cyclases

Cyclic terpenoids are a class of lipid compounds containing immense structural and functional diversity, with many cyclic triterpenoids acting as regulators of the physical properties and spatial organization of lipid membranes. Cyclic terpenoids are also readily preserved as terpane fossils, such as steranes and hopanes, forming a rich record of the evolution of life on Earth. Formation of the multiple ring structure of all cyclic terpenoids is catalyzed by terpenoid cyclase enzymes, among which are whole clades of proteins - many from environmental metagenomes and uncultured organisms - whose substrates and products are completely unknown. We investigate the function of these divergent cyclases through biochemical assays, and the evolutionary processes that produced them by testing and applying a variety of evolutionary models. We find deep divergence between the diterpenoid cyclases and triterpenoid cyclases, with other clades branching between the two, rooting the triterpenoid cyclase subtree, between squalene-hopene cyclases and sterol cyclases. Through a simple test of evolutionary rate shifts, we find an elevated evolutionary rate in the enzyme active site on the squalene-hopene cyclase stem, potentially indicative of positive selection. Finally, by testing the activity of divergent cyclases for a variety of substrates, we find a group of early-branching sterol cyclases from bacteria that synthesize arborinols, two of which produce the molecular precursor to a Permian "orphan biomarker." Together, our data present an evolutionary framework for triterpenoid cyclases that can inform both the biochemical potential of these proteins and their products occurrence in the geological record.

evolutionary biology↗

bioRxiv2022

Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursors enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2- carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/497760v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@63c641org.highwire.dtl.DTLVardef@6cee38org.highwire.dtl.DTLVardef@18bd310org.highwire.dtl.DTLVardef@cdfdc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Sterol side chain alkylation by sponge and bacterial methyltransferases

Sterol lipids are required by most eukaryotes and are readily preserved as sterane molecular fossils. These geologic steranes are broadly interpreted as biomarkers for ancient eukaryotes1,2 although diverse bacteria also produce sterols3. Steranes with side-chain methylations can act as more specific biomarkers4 if their sterol precursors are limited to particular extant eukaryotes and are absent in bacteria. An abundance of one such sterane, 24-isopropylcholestane, in late Neoproterozoic rocks has been attributed to marine demosponges and potentially represents the earliest evidence for animals on Earth5. However, debates over this interpretation6-14 continue given the potential for alternative sources of 24-isopropylcholestane and the lack of experimental evidence demonstrating the function of enzymes that methylate sterols to give the 24-isopropyl side-chain. Here we show that sterol methyltransferases from both sponges and bacteria are functional and identify three bacterial methyltransferases each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We identified two of these propylating enzymes in a demosponge metagenome suggesting bacterial symbionts contribute to 24-isopropyl sterol biosynthesis in demosponges. Our results demonstrate yet-uncultured bacteria have the genomic capacity to synthesize side-chain alkylated sterols and should therefore be considered when interpreting side-chain alkylated sterane biomarkers in the rock record.

microbiology↗

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↗