Lipophilic Substrates Research Articles

Microglia drive diurnal variation in susceptibility to inflammatory blood-brain barrier breakdown.

The blood-brain barrier (BBB) is critical for maintaining brain homeostasis but is susceptible to inflammatory dysfunction. While transporter-dependent efflux of some lipophilic substrates across the BBB shows circadian variation due to rhythmic transporter expression, basal transporter-independent permeability and leakage is nonrhythmic. Whether daily timing influences BBB permeability in response to inflammation is unknown. Here, we induced systemic inflammation through repeated LPS injections either in the morning (ZT1) or evening (ZT13) under standard lighting conditions; we then examined BBB permeability to a polar molecule that is not a transporter substrate, sodium fluorescein. We observed clear diurnal variation in inflammatory BBB permeability, with a striking increase in paracellular leak across the BBB specifically following evening LPS injection. Evening LPS led to persisting glia activation as well as inflammation in the brain that was not observed in the periphery. The exaggerated evening neuroinflammation and BBB disruption were suppressed by microglial depletion or through keeping mice in constant darkness. Our data show that diurnal rhythms in microglial inflammatory responses to LPS drive daily variability in BBB breakdown and reveal time of day as a key regulator of inflammatory BBB disruption.

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.

The Catabolic Network of Aromatoleum aromaticum EbN1T.

The denitrifying betaproteobacterium Aromatoleum aromaticum EbN1T is a facultative anaerobic degradation specialist and belongs to the environmental bacteria studied best on the proteogenomic level. This review summarizes the current state of knowledge about the anaerobic and aerobic degradation (to CO2) of 47 organic growth substrates (23 aromatic, 21 aliphatic, and 3 amino acids) as well as the modes of respiratory energy conservation (denitrification vs. O2-respiration). The constructed catabolic network is comprised of 256 genes, which occupy ∼7.5% of the coding regions of the genome. In total, 219 encoded proteins have been identified by differential proteomics, yielding a proteome coverage of ∼74% of the network. Its degradation section is composed of 31 peripheral and 4 central pathways, with several peripheral modules (e.g., for 4-ethylphenol, 2-phenylethylamine, indoleacetate, and phenylpropanoids) discovered only after the complete genome [Arch Microbiol. 2005 Jan;183(1):27-36] and a first proteomic survey [Proteomics. 2007 Jun;7(13):2222-39] of A. aromaticum EbN1T were reported. The activation of recalcitrant aromatic compounds involves a suite of biochemically intriguing reactions ranging from C-H-bond activation (e.g., ethylbenzene dehydrogenase) via carboxylation (e.g., acetophenone carboxylase) to oxidative deamination (e.g., benzylamine), reductive dearomatization (benzoyl-CoA), and epoxide-forming oxygenases (e.g., phenylacetyl-CoA). The peripheral reaction sequences are substrate-specifically induced, mediated by specific transcriptional regulators with in vivo response thresholds in the nanomolar range. While lipophilic substrates (e.g., phenolics) enter the cells via passive diffusion, polar ones require active uptake that is driven by specific transporters. Next to the protein repertoire for canonical complexes I-III, denitrification, and O2-respiration (low- and high-affinity oxidases), the genome encodes an Ndh-II, a tetrathionate reductase, two ETF:quinone oxidoreductases, and two Rnf-type complexes, broadening the electron transfer flexibility of the strain. Taken together, the detailed catabolic network presented here forms a solid basis for future systems biology-level studies with A. aromaticum EbN1T.

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids.

Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP-independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T-component, or TatT, is predicted to be a periplasmic-facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long-chain fatty acids. Our bioinformatics, quantitative analyses of protein-ligand interactions, and high-resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.

A Membrane-Embedded Macromolecular Catalyst with Substrate Selectivity in Live Cells.

Substrate selectivity is one of the most attractive features of natural enzymes from their "bind-to-catalyze" working flow and is thus a goal for the development of synthetic enzyme mimics that mediate abiotic transformations. However, despite the recent success in the preparation of substrate-selective enzyme mimics based on single-chain nanoparticles, examples extending such selectivity into living systems have been absent. In this article, we report the in cellulo substrate selectivity of an enzyme-mimicking macromolecular catalyst based on a cationic dense-shell nanoparticle (DSNP) scaffold. With a systematic study on DSNP's structure-activity relationship, we demonstrate that the DSNP has excellent membrane affinity that is governed by several contributing factors, namely, charge density, type of charge, and particle size, and the best-performing phosphonium-rich DSNP can be used as a membrane-embedded catalyst (MEC) for efficient on-membrane synthesis. Importantly, the DSNP catalyst retains its selectivity toward lipophilic and anionic substrates when working as an MEC for on-membrane ligation. The usefulness of such substrate selectivity and on-membrane catalysis strategy was exemplified with several molecules of interest with low cell permeability and anionic nature, which were successfully transported into eukaryotic cells by after their formation directly on the cell membrane.

SEC14-GOLD protein PATELLIN2 binds IRON-REGULATED TRANSPORTER1 linking root iron uptake to vitamin E.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. Here, we describe that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

Strategies for Transferring Photobiocatalysis to Continuous Flow Exemplified by Photodecarboxylation of Fatty Acids.

The challenges of light-dependent biocatalytic transformations of lipophilic substrates in aqueous media are manifold. For instance, photolability of the catalyst as well as insufficient light penetration into the reaction vessel may be further exacerbated by a heterogeneously dispersed substrate. Light penetration may be addressed by performing the reaction in continuous flow, which allows two modes of applying the catalyst: (i) heterogeneously, immobilized on a carrier, which requires light-permeable supports, or (ii) homogeneously, dissolved in the reaction mixture. Taking the light-dependent photodecarboxylation of palmitic acid catalyzed by fatty-acid photodecarboxylase from Chlorella variabilis (CvFAP) as a showcase, strategies for the transfer of a photoenzyme-catalyzed reaction into continuous flow were identified. A range of different supports were evaluated for the immobilization of CvFAP, whereby Eupergit C250 L was the carrier of choice. As the photostability of the catalyst was a limiting factor, a homogeneous system was preferred instead of employing the heterogenized enzyme. This implied that photolabile enzymes may preferably be applied in solution if repair mechanisms cannot be provided. Furthermore, when comparing different wavelengths and light intensities, extinction coefficients may be considered to ensure comparable absorption at each wavelength. Employing homogeneous conditions in the CvFAP-catalyzed photodecarboxylation of palmitic acid afforded a space-time yield unsurpassed by any reported batch process (5.7 g·L-1·h-1, 26.9 mmol·L-1·h-1) for this reaction, demonstrating the advantage of continuous flow in attaining higher productivity of photobiocatalytic processes.

A combined “AIE + ESIPT” fluorescent probe for detection of lipase activity

Lipases are acyl hydrolases that play a key role in fat digestion and monitoring of acute pancreatitis by cleaving long-chain triglycerides into polar lipids. Due to an opposite polarity between the hydrophilic enzyme and their lipophilic substrates, lipase reaction occurs at the interface between the aqueous and the oil phases. It is quite challenging to develop a specific probe to detect lipase activity, especially in homogeneous systems. Herein we designed a blue fluorescent probe HBT-LDC for specific detection of lipase activity based on both aggregation induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) effect. The probe shows significant advantages, such as high selectivity and excellent sensitivity, no self-quenching at high concentration, large Stokes shift, low cytotoxicity, and good biocompatibility. The linear range for in vitro quantification of lipase is 0.2–1.3 mg/mL with a detection limit of 0.01 mg/mL. The probe has been successfully applied to the evaluation of commercial lipase and the in vivo bioimaging sensing exogenous lipase activity in HeLa cells. The results revealed that the probe could serve as a potential tool in lipase related drug discovery and disease diagnostics.

Testing enabling techniques for olefin metathesis reactions of lipophilic substrates in water as a diluent

SummaryOlefin metathesis reactions of diverse polyfunctional substrates were conducted in water emulsions using two hydrophobic ruthenium catalysts in the presence of air. Instead of using surfactants to increase the efficiency of the metathesis reaction in water, ultrasound and microwave techniques were tested on a small-scale reaction, whereas conventional heating and mechanical stirring were effective enough to provide high conversion and selectivity on a larger scale. The developed conditions extend known protocols for the aqueous metathesis methodology, utilizing relatively low catalyst loadings and allowing for simple product isolation and purification. The established synthetic protocol was successfully adopted in the large-scale synthesis of a pharmaceutically related product – sildenafil (Viagra) derivative.

Enhanced sophorolipid production and effective conversion of waste frying oil using dual lipophilic substrates

A highly efficient sophorolipid (SL) fermentation process using dual lipophilic substrates (DLS) was developed. Using DLS consisting of palm oil and oleic acid in the flask fermentation with SL-producing yeast Starmerella bombicola, the productivity and yield of SL improved 2.4 and 1.7 times, in comparison to when using palm olein alone. In the jar fermentation process with DLS, the SL productivity and yield of 127 g/L and 62.4% were achieved in 7 days, with the complete consumption of residual lipophilic substrates in the culture medium. This process was successfully applied to the effective conversion of waste frying oil to SL, maintaining a high SL productivity and yield of 142.8 g/L and 72% in 9 days. It is concluded that the newly developed fermentation process with DLS can be useful for the industrial production of SL from renewable biological sources, especially waste frying oils.

Myristic Acid Derived Sophorolipid: Efficient Synthesis and Enhanced Antibacterial Activity.

Microbial glycolipids are one of the most interesting alternatives to chemical-based surfactants as they exhibit improved biodegradability and less toxicity. However, their potential has been limited because of specificity of the yeast toward fatty acids having a carbon 16 or carbon 18 chain. This study focuses on sophorolipid (SL) production by the yeast Starmerella bombicola using myristic acid, a medium-chain carbon-14 fatty acid that has not been used as a substrate for SL production. The production was optimized for inoculum size and lipophilic substrate concentration. Furthermore, we also studied the effect of medium-chain fatty acid on yeast cell growth and optimized the process for excellent yield. The myristic acid SL (MASL) so synthesized consisted of mono- and diacetylated forms with preferential glycosylation at the methyl end group, as determined by high-resolution mass spectrometry. Individual congeners of the crude mixture were separated using dry column chromatography and then structurally characterized by mass spectrometry. The synthesized MASL was also shown to have promising surface tension, lowering abilities with a low CMC of 14 mg/L. The SL derived from myristic acid exhibited superior antibacterial activity as compared to SL derived from oleic acid. MASL was also found to be more potent against Gram-positive organisms as compared to Gram-negative organisms. This work, therefore, demonstrates successful synthesis of myristic acid-derived SL and its superior antibacterial activity, establishing a promising future for this biosurfactant.

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure\u2013function properties of PATELLIN2

Key messageSEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity.SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

Hydrolytic Nanozymes

In 2004 we first reported catalytic nanoparticles, that are able to cleave phosphate diesters with very high efficiency (Angew. Chem. Int Ed, 2004, 43, 6165–6169) and dubbed them “nanozymes” for the similarity of their behavior with natural enzymes, both in terms of efficiency and mechanism of action. Since then the field has impressively expanded and a search on the web of science at the time of submitting this contribution returned almost 1,000 entries. This minireview highlights what has been done in the field focusing specifically on hydrolytic nanozymes, the focal point of the research in our group since its very beginning. Special emphasis is given to the advantage of bringing catalytic units in the confined space of a nanosystem in terms of inducing the cooperation between them, favoring the interaction with substrates, and altering the local environment. We will try to answer to questions like: why can a lipophilic substrate be transformed by these catalysts even in an aqueous environment? Why may the pH in the catalytic loci of the nanosystem be different from that of the bulk solution even in the presence of buffers? Why are most of these nanosystems better than monovalent ones?

One-pot Synthesis of Betulin Triterpenoid Quaternized Pyridine Derivatives and their Antimicrobial Activity

Background: A wide range of biological activity, relatively low toxicity and multiple pharmacological effects of triterpenoids are major advantages of these compounds in the prevention and treatment of various diseases. They include the lupane- type triterpenoids that proved to be a promising platform for the synthesis of analogs with a wide range of biological activities, including anti-inflammatory, antitumor, antiparasitic and antiviral properties. The main disadvantage complicating the use of all known derivatives of lupane acids in medical practice is low bioavailability associated with poor solubility in biologic fluids, limiting their effective interaction with the biological targets. Objective: The objective of this study is the synthesis of new amphiphilic betulin derivatives on the base of pyridinium salts with antifungal and antibacterial activity. Methods: In this study we have developed an effective one-pot method for the preparation of new quaternized pyridine derivatives 4-6 of the betulinic series based on the reaction of the initial triterpenes 1-3 with the Tempo+Br3 - reagent in the pyridine. The synthesized and initial compounds were tested for their antimicrobial and antifungal activity. Results: The data presented in this document indicate that all synthesized compounds 4-6 exhibited high activity against both gram-positive Staphylococcus aureus bacteria and gram-negative Pseudomonas aeruginosa strains, as well as Candida albicans and Cryptococcus neoformans fungi with the >90% coverage of the inhibition zone. The best result in a series of compounds 4-6 was found for the derivative 6 at the minimum inhibitory concentration of 1 µg/ml against S. aureus bacteria, C. albicans and C. neoformans fungi at the concentration of 8 µg/ml. Conclusion: Thus, we have demonstrated the first example of the pyridine quaternization using the betulin triterpenoids as the lipophilic substrates and Tempo+Br3 - cation. The obtained quaternized pyridine analogs of betulin triterpenes showed high antibacterial and antifungal activity in comparison with the initial compounds.

Role of Steroids on the Membrane Binding Ability of Fatty Acid Amide Hydrolase.

Background: Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that gets in contact with a lipophilic substrate in the lipid bilayer, and then cleaves it into water soluble products. FAAH plays a critical role in modulating in vivo content and biological activity of endocannabinoids (eCBs), and its function is affected by membrane lipids. Increasing evidence suggests that also steroids can modulate endocannabinoid signaling, both in the central nervous system and at the periphery.Methods: In this study, we interrogated the effect of six steroids with relevant biological activity (testosterone, hydrocortisone, estradiol, pregnenolone, progesterone, and cortisone) on the membrane binding ability of rat FAAH. The experimental data analysis obtained by Fluorescence Resonance Energy Transfer Spectroscopy was paralleled by computational docking analysis.Results: Our data revealed distinct effects of the different steroids on the interaction of rat FAAH with model membranes. Among them, pregnenolone was found to be the most effective in raising rat FAAH affinity for model membranes. A possible binding pocket for steroid molecules was identified by docking analysis in the membrane-embedded region of the enzyme; such a pocket could account for the observed increase of the membrane affinity in the presence of the tested molecules.Conclusions: Overall, the results point to steroids as new regulators of FAAH interaction with membranes, which may impact the biological activity of eCBs.

UDP-glucuronosyltransferases: Structure, Function and Drug Design Studies.

UDP-glucuronosyltransferases (UGTs) are important phase II metabolic enzymes responsible for approximately 40-70% of endo and xenobiotic reactions. It catalyzes the transfer of glucuronic acid to lipophilic substrates, converting them into hydrophilic compounds that are excreted. There are 22 active human UGTs that belong to 4 families. This review focuses on human UGTs, highlighting the most current issues in order to connect all information available and allowing a discussion on the challenges already solved and those in which we need to move forward. Although, several UGTs studies have been conducted, the most recent ones addressing drug-drug interactions and polymorphism issues, there are still bottlenecks to overcome. Tridimensional structure is difficult to obtain due to overexpression, purification, and crystallization problems as well as the action mechanism - since overlapping of substrate specificities renders impasses on the identification of which isoform is responsible for a particular drug metabolic pathway. For this reason, bioinformatic tools are gaining more space, since it is a faster and less expensive reliable methodology that complements in vitro and in vivo researches. Combinations of quantum and molecular methods have become increasingly common, leading to the incorporation of enzyme features comprising their structure, dynamics and chemical reactions. Breakthroughs related to the enzyme, not only enable the discovery of new drugs essential for the treatment of various diseases, but also provide an improved action of the existing drugs.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl β-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Mass Spectrometry of Bacterial Outer Membrane Enzymes within the Native Lipid Environment

Lipid complexity of bacterial membranes and the dynamic interaction of lipophilic substrates with membrane‐embedded enzymes reflect a demand for a robust detection approach that unravels the local lipids and hydrophobic substrates/products either bound or within the milieu of membrane enzymes. In this study, we used styrene maleic acid lipid particle (SMALP) nanodiscs and electrospray ionization‐ion mobility spectrometry–mass spectrometry (ESI‐IMS) mass spectrometry to address the stability of lipid‐bound states of two bacterial β‐barrel enzymes.We overexpressed and purified different oligomeric states of two model β‐barrel enzymes with their co‐associated lipids (i.e. enzyme substrates/products) from the outer membrane of E.coli using various polymer formulations. We further examined the stability of the intact lipid‐protein nanodiscs in solution as well as in the gas phase. Our results, thus far, has demonstrated the higher efficacy of the native nanodiscs over conventional detergents in isolating lipid populations bound to the enzymes, also an improved thermal stability of the resulting β‐barrel complexes in solution. We have, interestingly, observed that some polymers are more compatible with the required conditions in mass spectrometry hence overcoming limitations (e.g. low pH which is known as destabilizing factor for the original SMALP nanodiscs) in the structural analysis of lipid‐bound states of membrane proteins in the gas phase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Working at the membrane interface: Ligand-induced changes in dynamic conformation and oligomeric structure in human aromatase.

Aromatase catalyzes the biosynthesis of estrogens from androgens. Owing to the physiological importance of this conversion of lipophilic substrates, the interaction with the lipid bilayer for this cytochrome P450 is crucial for its dynamics that must allow an easy access to substrates and inhibitors. Here, the aromatase-anastrozole interaction is studied by combining computational methods to identify possible access/egress routes with the protein inserted in the membrane and experimental tools aimed at the investigation of the effect of the inhibitor on the protein conformation. By means of molecular dynamics simulations of the protein inserted in the membrane, two channels, not detected in the starting crystal structure, are found after a 20-nSec simulation. Trypsin digestion on the recombinant protein shows that the enzyme is strongly protected by the presence of the substrate and even more by the inhibitor. DSC experiments show an increase in the melting temperature of the protein in complex with the substrate (49.3 °C) and the inhibitor (58.7 °C) compared to the ligand-free enzyme (45.9 °C), consistent with a decrease of flexibility of the protein. The inhibitor anastrozole enters the active site of the protein through a channel different from that used from the substrate and promotes a conformational change that stiffens the protein conformation and decreases the protein-protein interaction between different aromatase molecules.

Recognition of Histone H3K14 Acylation by MORF

The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states invitro and invivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.