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• W3008848398 abstract "Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp72. Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp72 of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp72 apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ± 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp72 apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp72 apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I–deficient mice, 5-OHTrp72 apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp72 impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo. Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp72. Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp72 of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered Saccharomyces cerevisiae tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp72 apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity in vitro (41.73 ± 6.57% inhibition; p < 0.01). Additionally, 5-OHTrp72 apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp72 apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; p < 0.001, p < 0.001, and p < 0.001, respectively). Following injection into apoA-I–deficient mice, 5-OHTrp72 apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; p < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp72 impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both in vitro and in vivo. Apolipoprotein A-I (apoA-I) 2The abbreviations used are: apoA-Iapolipoprotein A-ITAGamber nonsense codonAIKOapoA-I knockoutABCA1ATP-binding cassette subfamily A member 1ABCATP-binding cassetteBAECbovine aortic endothelial cell8-Br-cAMP8-bromo-cylic AMPCADcoronary artery diseaseFBSfetal bovine serumGCEgenetic code expansionHDLhigh-density lipoproteinHDL-chigh density lipoprotein-cholesterol2-OHTrp2-oxindolyl alanine5-OHTrp5-oxindolyl tryptophanHOClhypochlorous acidIPTGisopropyl β-d-1-thiogalactopyranosideLPDFlipoprotein-deficient fractionLBLuria brothMPOmyeloperoxidasencAAnon-canonical amino acidoxTrpoxidized tryptophanPON1paraoxonase 1sfGFPsuperfolder green fluorescent proteinMS/MStandem mass spectrometry (MS2)Trp-RStryptophanyl tRNA synthetaseVCAM1vascular cellular adhesion moleculeODoptical densityGnHClguanidine chlorideDTPAdiethylenetriaminepentaacetic acidDMEMDulbecco's modified Eagle's mediaANOVAanalysis of variance2,3-diOHTrp2,3-dioxindolyl alanine. is the most abundant protein component in plasma high-density lipoproteins (HDLs), which are heterogeneous assemblies of bioactive molecules including proteins and lipids (1Kontush A. 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Proteomic analyses of apoA-I recovered from human atheroma revealed that the protein is heavily oxidized, bearing the oxidative footprint of myeloperoxidase (MPO), an enzyme secreted by activated leukocytes (29DiDonato J.A. Huang Y. Aulak K.S. Even-Or O. Gerstenecker G. Gogonea V. Wu Y. Fox P.L. Tang W.H. Plow E.F. Smith J.D. Fisher E.A. Hazen S.L. Function and distribution of apolipoprotein A1 in the artery wall are markedly distinct from those in plasma.Circulation. 2013; 128 (23969698): 1644-165510.1161/CIRCULATIONAHA.113.002624Crossref PubMed Scopus (80) Google Scholar, 30Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.J. Clin. Invest. 2004; 114 (15314690): 529-54110.1172/JCI200421109Crossref PubMed Scopus (636) Google Scholar). Lesion apoA-I is dysfunctional, lacking cholesterol acceptor activity, and has disease-promoting pro-inflammatory activity (30Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.J. Clin. Invest. 2004; 114 (15314690): 529-54110.1172/JCI200421109Crossref PubMed Scopus (636) Google Scholar, 31Huang Y. DiDonato J.A. Levison B.S. Schmitt D. Li L. Wu Y. Buffa J. Kim T. Gerstenecker G.S. Gu X. Kadiyala C.S. Wang Z. Culley M.K. Hazen J.E. Didonato A.J. et al.An abundant dysfunctional apolipoprotein A1 in human atheroma.Nat. Med. 2014; 20 (24464187): 193-20310.1038/nm.3459Crossref PubMed Scopus (272) Google Scholar). Understanding the functional consequences of apoA-I modification has been the subject of intense investigation with efforts focused on recapitulating the physiological modifications in vitro using MPO-catalyzed modifications with chlorinating and nitrating oxidation systems. These studies are limited by oxidation on all susceptible residues, thereby confounding interpretation of functional studies and assignment of impaired activities to specific sites. Although natural amino acid substitution studies confirmed the relevance of oxidation at specific residues, the approach was also limited by unknown consequences of incorporating a nonoxidizable amino acid on the structure/function of the protein. Thus, the functional consequence of site-specific (single-site) oxidation of apoA-I remains poorly understood. Site-directed natural amino acid mutagenesis studies support a crucial role for apoA-I Trp72 in ABCA1-dependent efflux activity (31Huang Y. DiDonato J.A. Levison B.S. Schmitt D. Li L. Wu Y. Buffa J. Kim T. Gerstenecker G.S. Gu X. Kadiyala C.S. Wang Z. Culley M.K. Hazen J.E. Didonato A.J. et al.An abundant dysfunctional apolipoprotein A1 in human atheroma.Nat. Med. 2014; 20 (24464187): 193-20310.1038/nm.3459Crossref PubMed Scopus (272) Google Scholar, 32Peng D.Q. Brubaker G. Wu Z. Zheng L. Willard B. Kinter M. Hazen S.L. Smith J.D. Apolipoprotein A-I tryptophan substitution leads to resistance to myeloperoxidase-mediated loss of function.Arterioscler. Thromb. Vasc. Biol. 2008; 28 (18688016): 2063-207010.1161/ATVBAHA.108.173815Crossref PubMed Scopus (84) Google Scholar). ApoA-I oxidized on Trp72 (oxTrp72-apoA-I) is present in plasma of patients with coronary artery disease (CAD), is highly enriched in atherosclerotic lesions, and is associated with increased cardiovascular disease risk (31Huang Y. DiDonato J.A. Levison B.S. Schmitt D. Li L. Wu Y. Buffa J. Kim T. Gerstenecker G.S. Gu X. Kadiyala C.S. Wang Z. Culley M.K. Hazen J.E. Didonato A.J. et al.An abundant dysfunctional apolipoprotein A1 in human atheroma.Nat. Med. 2014; 20 (24464187): 193-20310.1038/nm.3459Crossref PubMed Scopus (272) Google Scholar). In this study, we sought to address the absolute effect of oxidation at Trp72 in the absence of any other modification in apoA-I. Genetic code expansion (GCE) has emerged as a powerful method to site-specifically incorporate noncanonical amino acids (ncAAs) into proteins in living cells (33Porter J.J. Mehl R.A. Genetic code expansion: a powerful tool for understanding the physiological consequences of oxidative stress protein modifications.Oxid. Med. Cell Longev. 2018; 2018 (29849913): 760746310.1155/2018/7607463Crossref PubMed Scopus (8) Google Scholar). Specifically, engineered aminoacyl-tRNA synthetase:tRNA pairs are used to deliver a desired ncAA in response to a nonsense or frameshift codon. Herein, we used an evolved tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair from Saccharomyces cerevisiae (34Ellefson J.W. Meyer A.J. Hughes R.A. Cannon J.R. Brodbelt J.S. Ellington A.D. Directed evolution of genetic parts and circuits by compartmentalized partnered replication.Nat. Biotechnol. 2014; 32 (24185096): 97-10110.1038/nbt.2714Crossref PubMed Scopus (94) Google Scholar) to insert oxTrp at position 72 in apoA-I in Escherichia coli. We now report a significant impairment of in vitro cholesterol acceptor activity and in vivo HDL biogenesis function observed with 5-OHTrp72 apoA-I relative to WT apoA-I. To examine the specific functional consequences of a single-site oxTrp at position 72 in apoA-I, we used GCE to cotranslationally incorporate a noncanonical amino acid in vivo (33Porter J.J. Mehl R.A. Genetic code expansion: a powerful tool for understanding the physiological consequences of oxidative stress protein modifications.Oxid. Med. Cell Longev. 2018; 2018 (29849913): 760746310.1155/2018/7607463Crossref PubMed Scopus (8) Google Scholar). We used an engineered Trp-RS:suppressor tRNA pair (engineered machinery) from S. cerevisiae, which incorporates 5-OHTrp in E. coli in response to a genetically programmed amber nonsense codon (TAG) (34Ellefson J.W. Meyer A.J. Hughes R.A. Cannon J.R. Brodbelt J.S. Ellington A.D. Directed evolution of genetic parts and circuits by compartmentalized partnered replication.Nat. Biotechnol. 2014; 32 (24185096): 97-10110.1038/nbt.2714Crossref PubMed Scopus (94) Google Scholar). We first explored the possibility that this machinery might exhibit substrate promiscuity, thereby allowing us the opportunity to insert different oxidized tryptophans. The experimental approach is schematically depicted in Fig. 1A, where amber suppression in superfolder GFP (sfGFP) with a 150TAG leads to fluorescence of intact bacterial cells due to expression of full-length protein. We first examined the specificity of this orthogonal pair for oxidized tryptophan by co-transforming the plasmid encoding the engineered machinery with either one of two reporters encoding sfGFP-150TAG (Fig. 1, A and B), or apoA-I-72TAG (Fig. 1C), as described under “Experimental procedures.” Transformed E. coli BL21ai cells were grown on defined autoinduction media in the presence of the indicated oxidized tryptophan (oxTrp) forms without or with added lactose to induce Trp-RS R313 (see “Experimental procedures”). A robust amber suppression resulting in full-length sfGFP protein expression was observed only in the presence of 5-OHTrp, exhibiting an 8.8 ± 3.8-fold increase over lactose-induced control culture (Fig. 1B). This suggests that Trp-RS R313 selectively aminoacylates its cognate tRNA (tRNATrpCUA40A) with 5-OHTrp and not with 2-OHTrp, 2,3-Dioxindolyl alanine (2,3-diOHTrp) or kynurenine. There was a modest induction of full-length sfGFP in lactose-induced cultures in the absence of added oxTrp, which suggested that Trp-RS R313 may weakly aminoacylate its cognate suppressor tRNA with an endogenous (nonoxidized) Trp, which was present in the media at low levels, because Trp is an essential amino acid and needed to be present for incorporation into other sites in sfGFP. Notably, yield of the mutant sfGFP in response to 5-OHTrp was 50% of the WT sfGFP reporter (no stop codon) (Fig. 1B). This suggests that the Trp-RS R313:tRNATrpCUA40A orthogonal pair is quite efficient at suppressing the nonsense codon, at least with respect to position 150 in sfGFP under the experimental conditions used. We next proceeded to test whether we could insert 5-OHTrp at position 72 in apoA-I. Because WT apoA-I has an innate ability to bind lipopolysaccharide (35Ma J. Liao X.L. Lou B. Wu M.P. Role of apolipoprotein A-I in protecting against endotoxin toxicity.Acta Biochim. Biophys. Sin. 2004; 36 (15188057): 419-42410.1093/abbs/36.6.419Crossref Scopus (80) Google Scholar, 36Beck W.H. Adams C.P. Biglang-Awa I.M. Patel A.B. Vincent H. Haas-Stapleton E.J. Weers P.M. Apolipoprotein A-I binding to anionic vesicles and lipopolysaccharides: role for lysine residues in antimicrobial properties.Biochim. Biophys. Acta. 2013; 1828 (23454085): 1503-151010.1016/j.bbamem.2013.02.009Crossref PubMed Scopus (47) Google Scholar, 37Guo L. Ai J. Zheng Z. Howatt D.A. Daugherty A. Huang B. Li X.A. High density lipoprotein protects against polymicrobe-induced sepsis in mice.J. Biol. Chem. 2013; 288 (23658016): 17947-1795310.1074/jbc.M112.442699Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), we used the host strain ClearColi BL21 (DE3) (Lucigen, Madison, WI), which has been genetically engineered to express a modified lipopolysaccharide that does not trigger the endotoxic response (38Mamat U. Wilke K. Bramhill D. Schromm A.B. Lindner B. Kohl T.A. Corchero J.L. Villaverde A. Schaffer L. Head S.R. Souvignier C. Meredith T.C. Woodard R.W. Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.Microb. Cell Fact. 2015; 14 (25890161): 5710.1186/s12934-015-0241-5Crossref PubMed Scopus (141) Google Scholar), to express our recombinant apoA-I proteins, as described under “Experimental procedures.” Analysis of E. coli crude extracts by SDS-PAGE followed by Western blotting with antibodies specific for apoA-I and His tag showed that full-length His-tagged apoA-I protein was expressed only in the presence of 5-OHTrp (Fig. 1C). To confirm that the expressed full-length recombinant apoA-I contained 5-OHTrp at position 72, His-tagged WT (lacking amber (TAG) codon but otherwise having identical DNA sequence to apoA-I-72TAG) and the mutant protein were expressed in ClearColi, purified by nickel-nitrilotriacetic acid chromatography, and processed for proteomic analysis (see “Experimental procedures” for details). Equivalent amounts of each protein were fractionated by gradient (4–20%) reducing SDS-PAGE and visualized by Coomassie Blue (Fig. 2A). The mutant protein exhibited similar purity and equivalent gel fractionation properties compared with WT His10-apoA-I (Fig. 2A). Native peptides from WT and 5-OHTrp72 apoA-I variant, generated by trypsin digestion, were identified by nano-LC-MS/MS. Whereas detectable oxidation at Trp50 and Trp108 was minimal, the degree of oxidation at Trp72 in the mutant protein was 85% with the remaining 15% being WT tryptophan (Fig. 2B). This suggests that the incorporation fidelity of 5-OHTrp by the engineered orthogonal pair is 85% in 5-OHTrp-supplemented media in response to amber codon. Analysis of the peptide spanning residues 62–77 revealed a mass increase of 16 Da on all y ions present after the oxTrp72 in the noncanonical amino acid incorporated peptide (62EQLGPVTQEFW(ox)DNLEK77) spectra compared with the same y ions in WT apoA-I (Fig. 2C). This mass differential is consistent with single incorporation of an oxygen (5-OH moiety) on Trp72 of apoA-I, corresponding to the location of the amber codon (TAG). We previously identified two regions in apoA-I, P1 (38LGKQLNLKL46) and P2 (201STLSEKAK208), as potential contact sites with PON1 that promote PON1 thermal stability and activation (39Huang Y. Wu Z. Riwanto M. Gao S. Levison B.S. Gu X. Fu X. Wagner M.A. Besler C. Gerstenecker G. Zhang R. Li X.M. DiDonato A.J. Gogonea V. Tang W.H. et al.Myeloperoxidase, paraoxonase-1, and HDL form a functional ternary complex.J. Clin. Invest. 2013; 123 (23908111): 3815-382810.1172/JCI67478Crossref PubMed Scopus (201) Google Scholar). Because our mutation (5-OHTrp72) is outside these regions, we reasoned that PON1 stabilization/activation by our mutant protein would not be affected. Indeed, 5-OHTrp72 apoA-I was as effective as reconstituted HDL (containing WT-apoA-I) and modestly better than WT apoA-I at enhancing the arylesterase activity of recombinant PON1 (average PON1 arylesterase activities 30 min after protein additions to PON1 (expressed as μmol/min/mg PON1) were as follows: PON1 alone, 154.8 ± 3.5; PON1 + HDL, 361.3 ± 15.3; PON1 + WT apoA-I, 299.1 ± 5.8; PON1 + 5OHTrp72, 364.3 ± 0.01; PON1 + BSA, 194.3 ± 7.0; Fig. 3). This result demonstrates that 5-OHTrp72 apoA-I is fully functional, at least with respect to PON1 stabilization/activation. ABCA1-dependent cholesterol efflux activity from peripheral tissue and atherosclerotic lesions is an important atheroprotective function of apoA-I and is impaired through oxidation by MPO in vivo (30Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.J. Clin. Invest. 2004; 114 (15314690): 529-54110.1172/JCI200421109Crossref PubMed Scopus (636) Google Scholar, 31Huang Y. DiDonato J.A. Levison B.S. Schmitt D. Li L. Wu Y. Buffa J. Kim T. Gerstenecker G.S. Gu X. Kadiyala C.S. Wang Z. Culley M.K. Hazen J.E. Didonato A.J. et al.An abundant dysfunctional apolipoprotein A1 in human atheroma.Nat. Med. 2014; 20 (24464187): 193-20310.1038/nm.3459Crossref PubMed Scopus (272) Google Scholar, 32Peng D.Q. Brubaker G. Wu Z. Zheng L. Willard B. Kinter M. Hazen S.L. Smith J.D. Apolipoprotein A-I tryptophan substitution leads to resistance to myeloperoxidase-mediated loss of function.Arterioscler. Thromb. Vasc. Biol. 2008; 28 (18688016): 2063-207010.1161/ATVBAHA.108.173815Crossref PubMed Scopus (84) Google Scholar, 40Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. Green P.S. McDonald T.O. Brunzell J. Chait A. Oram J.F. O'Brien K. Geary R.L. Heinecke J.W. Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species.J. Biol. Chem. 2004; 279 (15292228): 42977-4298310.1074/jbc.M406762200Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 41Zheng L. Settle M. Brubaker G. Schmitt D. Hazen S.L. Smith J.D. Kinter M. Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dep" @default.
• W3008848398 created "2020-03-06" @default.
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• W3008848398 date "2020-04-01" @default.
• W3008848398 modified "2023-10-18" @default.
• W3008848398 title "Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis" @default.
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