An NMR‐Based Biosensor to Measure Stereospecific Methionine Sulfoxide Reductase Activities in Vitro and in Vivo**

Abstract

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases.I nh ealthy cells, MetOxi sr educed to methionineb yt wo families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the So rR-diastereoisomerso fm ethionine-sulfoxides, respectively.T od irectly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups.W e demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby,weestablish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOxa s ah ighly sensitive tool for studying therapeutic targets of oxidative stress-related human diseasesa nd redox regulated signaling pathways. Oxidationo fm ethionine side chains is ah allmark of cellular ageing and oxidative stress. [1] Methionine oxidationp roduces methionine-sulfoxides (MetOx), with ac hiral centera tt he sulfur atom giving rise to two diastereoisomers, designated Rand S-MetOx (Figure S1 ai nt he Supporting Information). [2] Under physiological conditions, methionine sulfoxides are re

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…Purified protein/nucleic acid can be microinjected in millimeter wide single-cell oocytes like those of Xenopus laevis or Danio rerio . The latter ones have been utilized only very recently. , We focus on the better established X. laevis oocytes below. These are quite appealing systems for in-cell NMR studies: (i) only ∼170 of them are necessary to fill a 5 mm NMR tube; (ii) they can be manipulated and microinjected cell by cell in about 1 h; (iii) they do not require a specific wet-lab, even though a source of oocytes has to be found (commercial companies sell them at ∼$1–2 a piece); (iv) they can stand the injection of about 20–50 nL of high-concentration material, which generates ∼20–50-fold intracellular dilutions; (v) they can survive and maintain their integrity about 18 h in the NMR tube without any medium replenishment; and (vi) they are well-known model systems in biology .…”

“…Fluorine is often present in drug compounds (∼25% of approved drugs) . A number of 19 F-NMR reporters have been designed for a broad variety of enzymatic activities, and often tested in mice for MRI purposes. , 15 N-labeled reporters of kinases and of methionine sulfoxide reductases have also been used in cells. , …”

Unsaturated fatty acids profiling in liveC. elegansusing real-time NMR spectroscopy

“…Moreover, a continuous NMR flow system was used on 13 C-enriched water fleas to assess the effects of growth conditions or stressors on organismal metabolism in real time 27,28 . Other applications of high resolution NMR studies in live animals include the monitoring of enzymatic reductions of exogenously delivered, 15 N isotopically enriched, oxidized methionine derivatives in zebrafish embryos (Danio rerio), a widely used vertebrate model system for biological studies 29 . Finally, Park and co-workers recently applied NMR spectroscopy on live, 13 C-isotopically enriched C. elegans, to characterize time-dependent metabolic changes of N2 and mutant worms with impaired AMPactivated kinase (AAK) activity, a central regulator of cellular metabolism 30 .…”

Danio rerio Oocytes for Eukaryotic In-Cell NMR

“…33,34 Sańchez-Loṕez et al used transparent early stage Danio rerio (zebrafish) embryos to assess enzyme activity in vivo. 35 To exploit the transparency of zebrafish, while maintaining the stable cellular environment, we employ unfertilized zebrafish oocytes to assess their utility for in-cell NMR. Here, we validate the detectability and cytoplasmic location of a test protein (Figure 1), the 7 kDa N-terminal src homology 3 domain (SH3) domain of the Drosophila melanogaster signal transduction protein Drk, and then quantify its stability and dynamics.…”

“…Selenko et al bypassed these limitations by microinjecting the model B1 domain of the streptococcal immunoglobulin G-binding protein (GB1) into Xenopus laevis oocytes. Oocytes are useful for studying the effects of the cytoplasmic milieu on proteins because they are fixed in development, which minimizes changes due to the cell cycle and growth. , Sánchez-López et al used transparent early stage Danio rerio (zebrafish) embryos to assess enzyme activity in vivo . To exploit the transparency of zebrafish, while maintaining the stable cellular environment, we employ unfertilized zebrafish oocytes to assess their utility for in-cell NMR.…”