Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Schematic for genetically encoding 22 amino acids. (A) Genetically encoding acK and Sec: acKRS ligates acK to tRNA Pyl , which reassigns all UAG stop codons to acK. This allows insertion of acK at a specific UAG codon in TrxR1. For Sec insertion, tRNA Sec is aminoacylated with serine (Ser) by seryl-tRNA synthetase (SerRS), followed by conversion of Ser-tRNA to Sec-tRNA by selenocysteine synthase (SelA). The elongation factor SelB binds Sec-tRNA Sec as well as the SecIS, which we designed into the 3′ UTR of the TrxR1 mRNA. The SecIS localizes Sec-tRNA Sec at the UGA codon, allowing for Sec insertion at a single UGA stop codon. (B) The two-plasmid system required for production of acetylated TrxR1: the pTech vector expresses AcKRS and tRNA Pyl , required for reassignment of UAG codons to acK. The pET vector expresses TrxR1 with a TAG at the desired acetylation sites (141, 200, or 307), as well as SecIS in the 3′ UTR (see the “ ” section). In this system, UAG genetically encodes acK, while SecIS following downstream of the UGA codon is required for Sec insertion. The Sec synthesis and insertion system is endogenous to Escherichia coli , encoded in the E. coli genome (SelA, SelB, SelC, SelD, SerRS). 3′ UTR, 3′ untranslated region; acK, N ɛ -acetyl-lysine; acKRS, mutant pyrrolysyl-tRNA synthetase ( N ɛ -acetyl-lysine-tRNA synthetase); Sec, selenocysteine; SecIS, selenocysteine insertion sequence; TrxR1, thioredoxin reductase 1. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Plasmid Preparation, Mutagenesis, Sequencing

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Thioredoxin Reductase 1 and acetylated Thioredoxin Reductase 1 Protein Yields and Activity

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Activity Assay, Variant Assay, DTNB Assay

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Physical identification of acK and Sec in acTrxR1 K200 . Mass spectrometry data for other TrxR1 variants are shown in – . Liquid chromatography tandem mass spectrometry of polypeptides from trypsin digested acTrxR1 K200 , demonstrating (A) acK and (B) Sec incorporation at the desired locations. acTrxR1, acetylated TrxR1. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Mass Spectrometry, Liquid Chromatography

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Activity of TrxR1 and acTrxR1 variants. In vitro activity assay of WT and acTrxR1 variants using a colorimetric (A) DTNB reduction assay measuring TNB production (412 nm) with 50 n M of each TrxR1 variant, (B) a 9,10-phenanthrenequinone reduction assay with 100 n M of each TrxR1 variant monitoring NADPH depletion (340 nm), or (C) a Trx1-insulin linked assay using 200 n M of TrxR1 and 6 μ M Trx1 monitoring NADPH depletion (340 nm). In the Trx1 insulin linked assay, TrxR1 reduces Trx1, which then reduces insulin before being reduced again by TrxR1. (D) The relative activity of the TrxR1 variants for each substrate, where the velocity (TNB/s or NADPH consumed/s) of WT TrxR1 is set to 1. These velocities were calculated from the data shown (A – C) . (* p < 0.05, ** p < 0.01, *** p < 0.005). Error bars represent one standard deviation based on triplicate measurements. A no enzyme control has been subtracted from all enzyme assays shown. DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NADPH, β-nicotinamide adenine dinucleotide 2′-phosphate; Trx1, thioredoxin 1; WT, wild type. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Activity Assay, In Vitro, Variant Assay, Standard Deviation

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: CD spectra of TrxR1 variants. CD spectra demonstrates properly folded WT TrxR1 (A) and acTrxR1 (B–D) variants, indicating no changes in protein folding caused by cotranslational incorporation of acK. Data are collected in units of mean residue ellipticity (in deg cm 2 /dmol). CD, circular dichroism. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques:

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Effect of HDAC on TrxR1 variants. The in vitro activity of TrxR1 variants, using 3.75 n M TrxR1 incubated with or without HDAC3, was measured by monitoring TNB production (412 nm). The relative activity for each variant incubated with ( filled bars ) or without ( open bars ) HDAC3, where the initial velocity of unacetylated TrxR1 incubated without HDAC3 is set to 1. Error bars represent one standard deviation (** p < 0.01, *** p < 0.005, ns, not significant). All error bars represent one standard deviation of triplicate measurements. HDAC3, histone deacetylase 3.

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: In Vitro, Activity Assay, Incubation, Variant Assay, Standard Deviation, Histone Deacetylase Assay

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Mechanistic basis for acetylation-dependent enhanced TrxR1 activity. (A) Acetylation of K141 ( blue ), K200 ( purple ), or K307 ( red ) on the surface of TrxR1 dimers interferes with salt bridges and hydrogen bonds (B , C) in the TrxR1 tetramer interface. Close-up view of the interactions at the tetramer interface with (B) K141, (C) K200, and K307 [PBD: 4KPR and 3EAN ]. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Activity Assay

Journal: Antioxidants & Redox Signaling

Article Title: Acetylation Regulates Thioredoxin Reductase Oligomerization and Activity

doi: 10.1089/ars.2017.7082

Figure Lengend Snippet: Characterization of WT TrxR1 and acTrxR1 oligomerization. (A) Different oligomeric states of the TrxR1 variants were separated in native conditions on a size exclusion column. His 6 -TrxR1 present in the fractions was visualized by Western blotting (anti-His). Arrows indicate TrxR1 subunits (**) or covalently linked and nonproductive TrxR1 dimers (*) formed during tetramerization. M indicates a molecular weight marker. (B) DTNB activity assays (100 n M TrxR1) demonstrated that high-activity dimers were successfully separated from low-activity tetramers and monomers. Relative activity is based on initial velocity (TNB/s), where the unacetylated dimer is set to 1. Error bars represent one standard deviation based on triplicate experiments. (C) The percent of TrxR1 existing as dimers, tetramers, monomers, or covalently linked inactive dimers was plotted for each TrxR1 variant. Percentages are calculated based on densitometry of the Western blot (A) . To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: In an attempt to enhance UAG translation with acK, we expressed TrxR1 variants in an E. coli RF1 deletion strain and in E. coli BL21 (DE3) (C2527H; New England Biolabs, Ipswich, MA).

Techniques: Western Blot, Molecular Weight, Marker, Activity Assay, Standard Deviation, Variant Assay