Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes.

Activity-Based Protein Profiling to Probe Relationships between Cytochrome P450 Enzymes and Early-Age Metabolism of Two Polycyclic Aromatic Hydrocarbons (PAHs): Phenanthrene and Retene.

A growing body of literature has linked early-life exposures to polycyclic aromatic hydrocarbons (PAH) with adverse neurodevelopmental effects. Once in the body, metabolism serves as a powerful mediator of PAH toxicity by bioactivating and detoxifying PAH metabolites. Since enzyme expression and activity vary considerably throughout human development, we evaluated infant metabolism of PAHs as a potential contributing factor to PAH susceptibility.

Glutathione Transferase Photoaffinity Labeling Displays GST Induction by Safeners and Pathogen Infection.

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry.

Affinity- and activity-based probes synthesized from structurally diverse hops-derived xanthohumol flavonoids reveal highly varied protein profiling in .

Xanthohumol, the principle prenylflavonoid found in hops () and a reported anti-inflammatory agent, has great potential for pharmaceutical interventions related to inflammatory disorders in the gut. A suite of probes was prepared from xanthohumol and its structural isomer isoxanthohumol to enable profiling of both protein affinity binding and catalytic enzyme reactivity. The regiochemistry of the reactive group on the probes was altered to reveal how probe structure dictates protein labeling, and which probes best emulate the natural flavonoids.

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing.

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies.

The global anaerobic metabolism regulator is necessary for the degradation of food dyes and drugs by .

This work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of to degrade azo dyes due to their impacts on bacterial transcription and metabolism.

Snekmer: a scalable pipeline for protein sequence fingerprinting based on amino acid recoding.

Motivation: The vast expansion of sequence data generated from single organisms and microbiomes has precipitated the need for faster and more sensitive methods to assess evolutionary and functional relationships between proteins. Representing proteins as sets of short peptide sequences (kmers) has been used for rapid, accurate classification of proteins into functional categories; however, this approach employs an exact-match methodology and thus may be limited in terms of sensitivity and coverage. We have previously used similarity groupings, based on the chemical properties of amino acids, to form reduced character sets and recode proteins.

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber.

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome's functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin.

An activity-based probe targeting the streptococcal virulence factor C5a peptidase.

Development of profiling strategies to provide high resolution understanding of enzymes involved in bacterial infections remains an important need. These strategies help resolve enzyme mechanisms of actions and can guide therapeutic development. We have developed a selective new activity-based probe (ABP) targeting a highly conserved surface bound enzyme, C5a peptidase, present in several pathogenic .

Profiling How the Gut Microbiome Modulates Host Xenobiotic Metabolism in Response to Benzo[]pyrene and 1-Nitropyrene Exposure.

The gut microbiome is a key contributor to xenobiotic metabolism. Polycyclic aromatic hydrocarbons (PAHs) are an abundant class of environmental contaminants that have varying levels of carcinogenicity depending on their individual structures. Little is known about how the gut microbiome affects the rates of PAH metabolism.

Bile salt hydrolase profiling by fluorogenic probes in the human gut microbiome.

Bile is a digestive fluid produced in the liver and stored in the gallbladder. It participates in absorption of fatty nutrients and vitamins, and aids in elimination of metabolic waste and toxins. The major chemical components of bile are bile salts that, apart from their function in digestion, are also known to participate in cell signaling by binding host farnesoid X (FXR), vitamin D (VDR), and G-protein coupled bile acid (TGR5) receptors.

A transcriptional relationship with a natural product disrupts mitochondrial biogenesis.

A limonoid natural product disrupts mitochondrial biogenesis and overcomes resistance. In this issue of Cell Chemical Biology, Cho et al. describe an anti-melanoma strategy in which a transcriptional target gene enables the anti-proliferative activity of the limonoid, harrpernoid D.

Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 μmol/kg/d) of benzo[]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site.

Nutritional markers and proteome in patients undergoing treatment for pulmonary tuberculosis differ by geographic region.

Introduction: Contemporary phase 2 TB disease treatment clinical trials have found that microbiologic treatment responses differ between African versus non-African regions, the reasons for which remain unclear. Understanding host and disease phenotypes that may vary by region is important for optimizing curative treatments.

Methods: We characterized clinical features and the serum proteome of phase 2 TB clinical trial participants undergoing treatment for smear positive, culture-confirmed TB, comparing host serum protein expression in clinical trial participants enrolled in African and Non-African regions.

Anaerobic gut fungi are an untapped reservoir of natural products.

Anaerobic fungi (class Neocallimastigomycetes) thrive as low-abundance members of the herbivore digestive tract. The genomes of anaerobic gut fungi are poorly characterized and have not been extensively mined for the biosynthetic enzymes of natural products such as antibiotics. Here, we investigate the potential of anaerobic gut fungi to synthesize natural products that could regulate membership within the gut microbiome.

Ligand- and Structure-Based Analysis of Deep Learning-Generated Potential α2a Adrenoceptor Agonists.

The α2a adrenoceptor is a medically relevant subtype of the G protein-coupled receptor family. Unfortunately, high-throughput techniques aimed at producing novel drug leads for this receptor have been largely unsuccessful because of the complex pharmacology of adrenergic receptors. As such, cutting-edge ligand- and structure-based assessment and deep learning methods are well positioned to provide new insights into protein-ligand interactions and potential active compounds.

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health.

Activity-Based Protein Profiling of Chitin Catabolism.

The microbial catabolism of chitin, an abundant and ubiquitous environmental organic polymer, is a fundamental cog in terrestrial and aquatic carbon and nitrogen cycles. Despite the importance of this critical bio-geochemical function, there is a limited understanding of the synergy between the various hydrolytic and accessory enzymes involved in chitin catabolism. To address this deficit, we synthesized activity-based probes (ABPs) designed to target active chitinolytic enzymes by modifying the chitin subunits N-acetyl glucosamine and chitotriose.

Detecting differential protein abundance by combining peptide level P-values.

The majority of methods for detecting differentially abundant proteins between samples in label-free LC-MS bottom-up proteomics experiments rely on statistically testing inferred protein abundances derived from peptide ionization intensities or averaging peptide level statistics. Here, we statistically test peptide ionization intensities directly and combine the resulting dependent P-values using the Empirical Brown's Method (EBM), avoiding error introduced through the estimation of protein abundances or summarizing test statistics. We show that on a spike-in proteomics dataset, a peptide level approach using EBM outperforms differential abundance detection using a protein level approach and several analysis workflows, including MSstats.

Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates.

Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities.

Probe-enabled approaches for function-dependent cell sorting and characterization of microbiome subpopulations.

Understanding the roles that individual species or communities play within a microbiome is a significant challenge. The complexity and heterogeneity of microbiomes presents a challenge to researchers looking to unravel the function that microbiomes serve within larger environments. While identification of the species and proteins present in a microbiome can be accomplished through genomics approaches, strategies that report on enzyme activity are limited.

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity.

Selection, Succession, and Stabilization of Soil Microbial Consortia.

Soil microorganisms play fundamental roles in cycling of soil carbon, nitrogen, and other nutrients, yet we have a poor understanding of how soil microbiomes are shaped by their nutritional and physical environment. In this study, we investigated the successional dynamics of a soil microbiome during 21 weeks of enrichment on chitin and its monomer, -acetylglucosamine. We examined succession of the soil communities in a physically heterogeneous soil matrix as well as a homogeneous liquid medium.

Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection, responsible for millions of infections each year. Despite this high prevalence, the elucidation of the molecular mechanisms of Chlamydia pathogenesis has been difficult due to limitations in genetic tools and its intracellular developmental cycle. Within a host epithelial cell, chlamydiae replicate within a vacuole called the inclusion.

Benzo[ a]pyrene Induction of Glutathione S-Transferases: An Activity-Based Protein Profiling Investigation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules.