Distinct RNAN-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates

Abstract The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems. Significance Statement Non-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.

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Distinct RNA N-Demethylation Pathways Catalyzed by Non-Heme Iron ALKBH5 and FTO Enzymes Enable Regulation of Formaldehyde Release Rates

10.26434/chemrxiv.12816224.v1 ◽

2020 ◽

Author(s):

Joel D.W. Toh ◽

Steven W. M. Crossley ◽

Kevin Bruemmer ◽

Eva J. Ge ◽

Dan He ◽

...

Keyword(s):

Cellular Localization ◽

Slow Release ◽

Direct Detection ◽

Heme Iron ◽

Rna Regulation ◽

Substrate Preferences ◽

Non Heme Iron ◽

Rapid Release ◽

Demethylation Pathway ◽

Covalent Intermediate

Abstract The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems. Significance Statement Non-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.

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Measuring nucleus mechanics within a living multicellular organism: Physical decoupling and attenuated recovery rate are physiological protective mechanisms of the cell nucleus under high mechanical load

Molecular Biology of the Cell ◽

10.1091/mbc.e20-01-0085 ◽

2020 ◽

Vol 31 (17) ◽

pp. 1943-1950

Author(s):

Noam Zuela-Sopilniak ◽

Daniel Bar-Sela ◽

Chayki Charar ◽

Oren Wintner ◽

Yosef Gruenbaum ◽

...

Keyword(s):

Mechanical Characterization ◽

Slow Release ◽

Mechanical Load ◽

Mechanical Energy ◽

High Load ◽

Cell Nuclei ◽

Release Rates ◽

Protective Mechanisms ◽

Deformation Recovery

A mechanical characterization of cell nuclei within living Caenorhabditis elegans nematodes reveals two protective mechanisms: (1) cytoskeletal decoupling of the nucleus at high load and (2) slow release rates of stored mechanical energy. Lamin knockdown softened the nucleus and attenuated deformation recovery, whereas aging increased nucleus stiffness.

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Sources of nitrogen and methods of application for flooded rice: I. Comparison of two methods of applying slow release and standard fertilizer materials

The Journal of Agricultural Science ◽

10.1017/s002185960001858x ◽

1967 ◽

Vol 69 (2) ◽

pp. 189-196 ◽

Cited By ~ 3

Author(s):

G. V. Simsiman ◽

S. K. De Datta ◽

J. C. Moomaw

Keyword(s):

Medium Release ◽

Grain Yield ◽

Field Experiments ◽

Slow Release ◽

Fertilizer Treatment ◽

Plant Growth And Development ◽

Release Rates ◽

Stages Of Growth ◽

Rough Rice ◽

Methods Of Application

Field experiments with rice were conducted on a flooded soil comparing two standard fertilizers with N fertilizers with various release rates. For each of these fertilizer materials two methods of application were compared: broadcast and incorporated, and placement at 15 cm depth. The .slow release materials resulted in a lower grain yield than did fast or medium release rate or standard fertilizer materials. They released N too slowly during the early growth of rice to permit optimum plant growth and development.Placement of 80 kg/ha N at 15 cm depth increased the plant N content at all stages of growth and significantly increased the grain yield compared with the broadcast-and-incorporated fertilizer treatment. The highest grain yield in the experiment (7701 kg/ha rough rice) was obtained from a medium release material, which was 88 % more efficient in increasing grain yield per unit of N when placed at 15 cm than when broadcast-and-incorporated.The recovery of fertilizer N was 68% when deeply placed and 38 % when broadcast-andincorporated.

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579 In situ and in vitro evaluation of a slow release form of nitrate for ruminants: Nitrate release rates, rumen nitrate metabolism and production of methane, hydrogen, and nitrous oxide

Journal of Animal Science ◽

10.2527/asasann.2017.579 ◽

2017 ◽

Vol 95 (suppl_4) ◽

pp. 283-284

Author(s):

C. Lee ◽

R. C. Araujo ◽

K. M. Koenig ◽

K. A. Beauchemin

Keyword(s):

Nitrous Oxide ◽

Slow Release ◽

Release Rates ◽

In Vitro Evaluation ◽

Nitrate Metabolism ◽

Release Form

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Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-296120180054 ◽

2018 ◽

Author(s):

Jaires Gomes de Oliveira Filho ◽

Lorena Lopes Ferreira ◽

Fernanda de Oliveira Silva ◽

Karolina Martins Ferreira Menezes ◽

Elen Regozino Muniz ◽

...

Keyword(s):

Slow Release ◽

Marked Change ◽

Rhipicephalus Sanguineus ◽

Repellent Activity ◽

Control Group ◽

Release Rates ◽

Slow Release Formulations ◽

Rhipicephalus Sanguineus Sensu Lato ◽

Higher Doses ◽

Preferred Hosts

Abstract Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due to the production of the allomones benzaldehyde and 2-hexanone. Our previous published work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increase repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates, were tested on artificially-infested dogs. Ten dogs were randomly assigned to two groups with five dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted once a day for 40 days. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three-fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. We also hypothesize that the avoidance of less-preferred hosts by ticks relies on olfactory-mediated perception of specific blends of volatile cues from less preferred hosts.

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Slow-Release Fertilizer for Rehabilitating Oligotrophic Streams: a Physical Characterization

Water Quality Research Journal ◽

10.2166/wqrj.2000.005 ◽

2000 ◽

Vol 35 (1) ◽

pp. 73-94 ◽

Cited By ~ 11

Author(s):

Megan S. Sterling ◽

Kenneth I. Ashley ◽

Abigail B. Bautista

Keyword(s):

Slow Release ◽

Specific Growth ◽

Habitat Degradation ◽

Inorganic Nitrogen ◽

Dissolved Inorganic Nitrogen ◽

Release Rates ◽

Pellet Size ◽

Slow Release Fertilizer ◽

Periphyton Biomass ◽

Dominance Pattern

Abstract A combination of habitat degradation and overharvesting of anadromous salmonids in many of B.C.’s oligotrophic watersheds has prompted the addition of inorganic nutrients to streams, which increases autotrophic production and aids in the restoration of salmonid production. A new slow-release fertilizer (7-40-0, N-P2O5-K2O, percent by weight) was examined to determine its phosphate (PO43-) release rates using laboratory and field trough experiments. A series of indoor trough experiments indicated that the fertilizer pellet dissolution rate (0.393 g ? days-0.401) was independent of the experimental range of water velocity (0.03-0.30 m . s-1), fertilizer pellet size (2–9 g) and water temperature (8–14.5°C). Resulting phosphate additions (0.5-5 µg P . L-1) in outdoor trough experiments increased periphyton biomass and altered the dominance pattern of periphytic diatoms. An optimal phosphate concentration for periphyton biomass was achieved with calculated 3.0 µg P . L-1 phosphate additions from May to June. In June to July, periphyton biomass increased proportionately to fertilizer additions. Saturation of the relative specific growth rate of the diatom community occurred with 1.0 µg P . L-1 phosphate additions. Nitrogen analysis was not conducted since inorganic nitrogen is typically available in non-limiting concentrations (i.e., >50 µg . L-1 dissolved inorganic nitrogen) in the majority of B.C.’s oligotrophic salmonid streams. These studies indicated that slow-release fertilizer may be effective in stimulating autotrophic production and restoring salmonid production in nutrient deficient streams.

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Influence of vegetable oil on the synthesis of bioactive nanocarriers with broad spectrum photoprotection

Open Chemistry ◽

10.2478/s11532-014-0503-9 ◽

2014 ◽

Vol 12 (8) ◽

pp. 837-850 ◽

Cited By ~ 16

Author(s):

Gabriela Niculae ◽

Ioana Lacatusu ◽

Nicoleta Badea ◽

Aurelia Meghea ◽

Raluca Stan

Keyword(s):

Broad Spectrum ◽

Seed Oil ◽

Slow Release ◽

Physical Stability ◽

Entrapment Efficiency ◽

Uv Filters ◽

Release Rates ◽

Cosmetic Formulations ◽

Lipid Nanocarriers ◽

Pomegranate Seed

AbstractDue to their unique features, most nanostructured lipid carriers (NLCs) in association with vegetable oils that exhibit UV filtering properties and bioactivity could be used in many cosmetic formulations. Therefore, in this work, a new application of pomegranate seed oil (PSO) in the cosmetic sector was developed, based on the synthesis of bioactive lipid nanocarriers loaded with various UV filters by the hot high pressure homogenization technique. To get broad spectrum photoprotection, different UVA and UVB filters have been used (Avobenzone — AVO, Octocrylen-OCT, Bemotrizinol — BEMT). The influence of the solid lipids combined with PSO on the particle size, physical stability and entrapment efficiency was investigated using 8 nanocarrier systems. An improved physical stability and an appropriate size were obtained for NLCs prepared with Emulgade, carnauba wax and PSO (e.g. −30.9÷-36.9 mV and 160÷185 nm). NLCs showed an entrapment efficiency above 90% and assured slow release rates of UV filters, especially for BEMT (5%). The developed nanocarriers have been formulated into safe and effective sunscreens containing low amounts of synthetic UV filters coupled with a high percent of natural ingredients. The highest SPF of 34.3 was obtained for a cream comprising of 11% PSO and 3.7% BEMT

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Granule Structure and Rate of Release with Starch-Encapsulated Thiocarbamates

Weed Science ◽

10.1017/s004317450006152x ◽

1980 ◽

Vol 28 (6) ◽

pp. 685-690 ◽

Cited By ~ 14

Author(s):

M. M. Schreiber ◽

M. D. White

Keyword(s):

Internal Structure ◽

Slow Release ◽

Control Technique ◽

Scanning Electron Micrographs ◽

Release Rates ◽

Slow Release Formulations ◽

Granule Structure ◽

Quality Control Technique ◽

Fast Release ◽

Scanning Electron

Scanning electron micrographs that depict the surface and internal structure of several starch-encapsulated granular formulations of EPTC (S-ethyl dipropylthiocarbamate) and butylate (S-ethyl diisobutylthiocarbamate) relate well with herbicidal release rates previously reported. Fast-release formulations appear to have a porous surface with little distinctive internal structure. Diffusion would be expected to be more rapid as fewer diffusion barriers are present. Slow-release formulations have a smooth, hard surface with irregular surface pores and a honeycombed internal structure. The slower release characteristics are associated with stronger oxidants used for crosslinking xanthate in the entrapment of herbicides. The scanning electron microscope would be an excellent quality control technique in the preparation of starch-encapsulated herbicides.

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Influence of pressure release rate and protein concentration on the formation of pressure-induced casein structures

Journal of Dairy Research ◽

10.1017/s002202990700252x ◽

2007 ◽

Vol 74 (3) ◽

pp. 283-289 ◽

Cited By ~ 19

Author(s):

Eva Merel-Rausch ◽

Ulrich Kulozik ◽

Jörg Hinrichs

Keyword(s):

Release Rate ◽

Protein Concentration ◽

Slow Release ◽

Hydrodynamic Diameter ◽

Pressure Treatment ◽

Release Rates ◽

Casein Micelles ◽

High Pressure Treatment ◽

Pressure Release ◽

Casein Solution

The formation of pressure-induced casein structures (600 MPa for 30 min at 30°C) was investigated for different pressure release rates (20 to 600 MPa min−1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min−1, while lower release rates resulted in less firm gels (200 MPa min−1) or liquid structures (20 MPa min−1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min−1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment.

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