Cone photoreceptor phosphodiesterase PDE6H inhibition regulates cancer cell growth and metabolism, replicating the dark retina response.

BACKGROUND: PDE6H encodes PDE6γ', the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth. METHODS: From an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model. RESULTS: PDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ'-deficient tumours. CONCLUSIONS: Our results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ' depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

Bdellovibrio bacteriovorus uses chimeric fibre proteins to recognize and invade a broad range of bacterial hosts.

Predatory bacteria, like the model endoperiplasmic bacterium Bdellovibrio bacteriovorus, show several adaptations relevant to their requirements for locating, entering and killing other bacteria. The mechanisms underlying prey recognition and handling remain obscure. Here we use complementary genetic, microscopic and structural methods to address this deficit. During invasion, the B. bacteriovorus protein CpoB concentrates into a vesicular compartment that is deposited into the prey periplasm. Proteomic and structural analyses of vesicle contents reveal several fibre-like proteins, which we name the mosaic adhesive trimer (MAT) superfamily, and show localization on the predator surface before prey encounter. These dynamic proteins indicate a variety of binding capabilities, and we confirm that one MAT member shows specificity for surface glycans from a particular prey. Our study shows that the B. bacteriovorus MAT protein repertoire enables a broad means for the recognition and handling of diverse prey epitopes encountered during bacterial predation and invasion.

N-Acyloxymethyl-phthalimides deliver genotoxic formaldehyde to human cells.

Formaldehyde is a pollutant and human metabolite that is toxic at high concentrations. Biological studies on formaldehyde are hindered by its high reactivity and volatility, which make it challenging to deliver quantitatively to cells. Here, we describe the development and validation of a set of N-acyloxymethyl-phthalimides as cell-relevant formaldehyde delivery agents. These esterase-sensitive compounds were similarly or less inhibitory to human cancer cell growth than free formaldehyde but the lead compound increased intracellular formaldehyde concentrations, increased cellular levels of thymidine derivatives (implying increased formaldehyde-mediated carbon metabolism), induced formation of cellular DNA-protein cross-links and induced cell death in pancreatic cancer cells. Overall, our N-acyloxymethyl-phthalimides and control compounds provide an accessible and broadly applicable chemical toolkit for formaldehyde biological research and have potential as cancer therapeutics.

Formaldehyde reacts with N-terminal proline residues to give bicyclic aminals.

Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

Species identification of silks by protein mass spectrometry reveals evidence of wild silk use in antiquity.

Silk has been a luxurious commodity throughout modern human history and sericulture has played an important role in ancient global trade as well as technological and cultural developments. Archaeological findings suggest that prior to domestication of the mulberry silkworm (Bombyx mori) silks were obtained from a range of silk-producing moth species with regional specificity. However, investigating the origins of sericulture is difficult as classification of silks by species-type has proved technically challenging. We therefore investigated a range of methods for solubilising modern and archaeological silks and developed a mass spectrometry-based proteomics method that was able to successfully differentiate modern Bombyx, Antheraea, and Samia-produced silks down to the species level. We subsequently analysed archaeological silk materials excavated from the ancient city of Palmyra. Solubilisation behaviour and proteomic analysis provided evidence that the Palmyra silks were constructed from wild silk derived from Antheraea mylitta, the Indian Tasar silkworm. We believe this is the first species-level biochemical evidence that supports archaeological theories about the production and trade of Indian wild silks in antiquity.

An Integrated Taxonomy for Monogenic Inflammatory Bowel Disease.

BACKGROUND & AIMS: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. METHODS: To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). RESULTS: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. CONCLUSION: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

Clerodane Diterpenoids from an Edible Plant Justicia insularis: Discovery, Cytotoxicity, and Apoptosis Induction in Human Ovarian Cancer Cells.

OBJECTIVES: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. METHODS: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/ß)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. CONCLUSIONS: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

Human Blood and Bird Egg Proteins Identified in Red Paint Covering a 1000-Year-Old Gold Mask from Peru.

We analyzed a red paint sample from the surface of a gold mask excavated from a Middle Sicán elite tomb in Peru. The mask covered the face of the principal male and dates from ca. 1000 AD, a period when many painted precious metal objects were produced. The paint's inorganic pigment was identified more than 30 years ago as cinnabar (a mercuric sulfide scarlet-red to brown-red mineral), but the identity of the effective organic binder remained a mystery. Fourier transform infrared (FTIR) analysis of the sample indicated a proteinaceous composition, and no lipids were recovered from an N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatized extract of the sample analyzed by gas chromatography-mass spectrometry (GC-MS). Proteomics analysis by nanoLC-MS/MS identified unique peptides in the sample, which were matched to human blood and bird egg proteins via Uniprot database searches. These included immunoglobulin heavy chain, immunoglobulin G, serum albumin, and ovomucoid. Cinnabar-based paints were typically used in the context of social elites and ritually important items. The presence of human blood would support previous ideas that red cinnabar paint may represent "life force" intended to support "rebirth". As the red paint sample came from the first scientifically excavated Sicán gold mask, the results suggest a method to authenticate similar unprovenanced masks now in private and museum collections. Proteomics data set identifier https://doi.org/10.5287/bodleian:1ajYbBgQP.

Differentiation of human induced pluripotent stem cells to authentic macrophages using a defined, serum-free, open-source medium.

Human induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built.

Terminalia bentzoë, a Mascarene Endemic Plant, Inhibits Human Hepatocellular Carcinoma Cells Growth In Vitro via G0/G1 Phase Cell Cycle Arrest.

Tropical forests constitute a prolific sanctuary of unique floral diversity and potential medicinal sources, however, many of them remain unexplored. The scarcity of rigorous scientific data on the surviving Mascarene endemic taxa renders bioprospecting of this untapped resource of utmost importance. Thus, in view of valorizing the native resource, this study has as its objective to investigate the bioactivities of endemic leaf extracts. Herein, seven Mascarene endemic plants leaves were extracted and evaluated for their in vitro antioxidant properties and antiproliferative effects on a panel of cancer cell lines, using methyl thiazolyl diphenyl-tetrazolium bromide (MTT) and clonogenic cell survival assays. Flow cytometry and comet assay were used to investigate the cell cycle and DNA damaging effects, respectively. Bioassay guided-fractionation coupled with liquid chromatography mass spectrometry (MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopic analysis were used to identify the bioactive compounds. Among the seven plants tested, Terminaliabentzoë was comparatively the most potent antioxidant extract, with significantly (p < 0.05) higher cytotoxic activities. T. bentzoë extract further selectively suppressed the growth of human hepatocellular carcinoma cells and significantly halted the cell cycle progression in the G0/G1 phase, decreased the cells' replicative potential and induced significant DNA damage. In total, 10 phenolic compounds, including punicalagin and ellagic acid, were identified and likely contributed to the extract's potent antioxidant and cytotoxic activities. These results established a promising basis for further in-depth investigations into the potential use of T. bentzoë as a supportive therapy in cancer management.

Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity.

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii.

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.

Systematic characterization of chromatin modifying enzymes identifies KDM3B as a critical regulator in castration resistant prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for prostate cancer (PCa) patients who fail surgery or radiotherapy. While initially effective, the cancer almost always recurs as a more aggressive castration resistant prostate cancer (CRPC). Previous studies have demonstrated that chromatin modifying enzymes can play a critical role in the conversion to CRPC. However, only a handful of these potential pharmacological targets have been tested. Therefore, in this study, we conducted a focused shRNA screen of chromatin modifying enzymes previously shown to be involved in cellular differentiation. We found that altering the balance between histone methylation and demethylation impacted growth and proliferation. Of all genes tested, KDM3B, a histone H3K9 demethylase, was found to have the most antiproliferative effect. These results were phenocopied with a KDM3B CRISPR/Cas9 knockout. When tested in several PCa cell lines, the decrease in proliferation was remarkably specific to androgen-independent cells. Genetic rescue experiments showed that only the enzymatically active KDM3B could recover the phenotype. Surprisingly, despite the decreased proliferation of androgen-independent cell no alterations in the cell cycle distribution were observed following KDM3B knockdown. Whole transcriptome analyses revealed changes in the gene expression profile following loss of KDM3B, including downregulation of metabolic enzymes such as ARG2 and RDH11. Metabolomic analysis of KDM3B knockout showed a decrease in several critical amino acids. Overall, our work reveals, for the first time, the specificity and the dependence of KDM3B in CRPC proliferation.

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase.

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ±â€¯0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.