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Generation of Recombinant Mammalian Selenoproteins Through Ge- Netic Code Expansion with Photocaged Selenocysteine

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Generation of Recombinant Mammalian Selenoproteins Through Ge- Netic Code Expansion with Photocaged Selenocysteine bioRxiv preprint doi: https://doi.org/10.1101/759662; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Generation of Recombinant Mammalian Selenoproteins through Ge- netic Code Expansion with Photocaged Selenocysteine. Jennifer C. Peeler, Rachel E. Kelemen, Masahiro Abo, Laura C. Edinger, Jingjia Chen, Abhishek Chat- terjee*, Eranthie Weerapana* Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States Supporting Information Placeholder ABSTRACT: Selenoproteins contain the amino acid sele- neurons susceptible to ferroptotic cell death due to nocysteine and are found in all domains of life. The func- overoxidation and inactivation of GPX4-Cys.4 This ob- tions of many selenoproteins are poorly understood, servation demonstrates a potential advantage conferred partly due to difficulties in producing recombinant sele- by the energetically expensive production of selenopro- noproteins for cell-biological evaluation. Endogenous teins. mammalian selenoproteins are produced through a non- Sec incorporation deviates from canonical protein canonical translation mechanism requiring suppression of translation, requiring suppression of the UGA stop codon. the UGA stop codon, and a selenocysteine insertion se- In eukaryotes, Sec biosynthesis occurs directly on the quence (SECIS) element in the 3’ untranslated region of suppressor tRNA (tRNA[Ser]Sec). Specifically, tRNA[Ser]Sec the mRNA. Here, recombinant selenoproteins are gener- is aminoacylated with serine by seryl-tRNA synthetase ated in mammalian cells through genetic code expansion, (SerS), followed by phosphorylation by phosphoseryl- circumventing the requirement for the SECIS element, tRNA kinase (PSTK), and subsequent Se incorporation and selenium availability. An engineered orthogonal E. by Sec synthase (SecS), to generate Sec-tRNA[Ser]Sec (Fig- coli leucyl-tRNA synthetase/tRNA pair is used to incor- ure 1a).5,6 Suppression of UGA by Sec-tRNA[Ser]Sec re- porate a photocaged selenocysteine (DMNB-Sec) at the quires a Sec insertion sequence (SECIS) element in the UAG amber stop codon. Recombinantly expressed sele- selenoprotein mRNA. The SECIS appears in the 3’ UTR noproteins can be photoactivated in living cells with spa- of the selenoprotein mRNA, and binding of the SECIS tial and temporal control. Using this approach, the native binding protein 2 (SBP2) and Sec-specific translation selenoprotein methionine-R-sulfoxide reductase 1 is gen- elongation factor (EFSec) are required for successful Sec erated and activated in mammalian cells. The ability to insertion (Figure 1a).7-9 site-specifically introduce selenocysteine directly in Due to the complexity of endogenous selenoprotein mammalian cells, and temporally modulate selenoprotein production in eukaryotes, recombinant expression has activity, will aid in the characterization of mammalian se- proven challenging. The low efficiency of traditional ma- lenoprotein function. nipulations, such as overexpression and mutation, has hindered the investigation of selenoproteins directly in mammalian cells. As a result, the biochemical and cell- Selenoproteins contain the amino acid selenocysteine biological functions of many human selenoproteins re- 1 (Sec), which is a cysteine (Cys) cognate with selenium main poorly characterized. (Se) in place of sulfur (S). In the majority of characterized Selenoproteins can be produced using expressed pro- selenoproteins, such as glutathione peroxidase and thiore- tein ligation, but is limited to in vitro studies, and neces- doxin reductases, Sec performs redox-catalytic func- sitates refolding of the protein.10 A number of strategies tions1. Selenoproteins are found throughout all three do- have been employed to enhance the recombinant expres- mains of life, with 25 selenoproteins in humans.2 How- sion of selenoproteins in E. coli, including the use of re- ever, some species express Cys-containing homologs of assigned sense codons, engineered tRNAs, and recoded selenoproteins, suggesting that Cys can nominally per- release factor 1 deficient E. coli.11-15 While useful, these form the same function as Sec. The stringent requirement prokaryotic expression systems are frequently poorly for Sec in certain organisms is attributed to the reversible suited for mammalian protein expression, and is not con- nature of Sec oxidation, which confers protection in con- ducive to studying mammalian selenoproteins in their na- trast to irreversible Cys oxidation.3 Replacement of Sec tive cellular environment. In eukaryotic cells, unique with Cys in glutathione peroxidase 4 (GPX4) renders bioRxiv preprint doi: https://doi.org/10.1101/759662; this version posted September 5, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. a cell, and the slow kinetics of the uncaging reaction. Fi- Ser pSer Sec nally, the necessary concentration of ASec in the culture media (0.2 mM) is toxic to mammalian cells, and neces- SerS PSTK SecS sitates supplementation with high concentration of cyste- 21 ACU ACU ACU ACU ine (3.2 mM) to mitigate toxicity. [Ser]Sec Sec-tRNA Here, we report a robust platform to incorporate photo- caged DMNB-Sec (Figure 1b) into native selenoproteins Sec EFSec in mammalian cells. This platform presents distinct ad- vantages, including: (1) generation of the native seleno- SBP2 SECIS protein directly in mammalian cells, instead of in model ACU 5’ UGA 3’ organisms, such as E. coli or yeast; (2) production of a protected selenoprotein that is not susceptible to oxida- tive damage and inactivation prior to biochemical inter- b rogation; (3) use of a minimally toxic DMNB-Sec amino DMNB-Sec Sec acid at low concentrations (12.5-100 µM) without cyste- O2N OMe ine supplementation; and, (4) rapid uncaging by 365 nm irradiation, which is minimally invasive, and provides ex- OMe cellent spatial and temporal control.23 Se HSe UV DMNB-Sec (Figure 1b) was synthesized as previously OH OH reported.22 Incorporation in mammalian cells was H2N H2N achieved using the E. coli LeuRS BH5 T252A/tRNA pair, O O which was originally evolved for photocaged serine (DMNB-Ser)24, and subsequently used for DMNB-Sec in Figure 1. Selenocysteine incorporation and DMNB-Sec 22 structure. (a) Endogenous eukaryotic Sec-incorporation yeast. LeuRS and 8 copies of the Leu tRNA were cloned mechanism. (b) Structures of DMNB-Sec and Sec. into the pAcBac2 vector containing an eGFP-39-TAG re- porter (Figure S1).25-27 Transient transfection into SECIS elements from Toxoplasma have been used to im- HEK293T cells was followed by growth in the presence prove selenoprotein production, but is dependent on Se or absence of 100 µM DMNB-Sec. Fluorescence of eGFP 16 availability. was 5-fold higher for lysates from cells exposed to Genetic code expansion (GCE) technology allows site- DMNB-Sec (Figure 2a), consistent with the yeast LeuRS specific incorporation of noncanonical amino acids using system with DMNB-Sec and DMNB-Cys.22 engineered aminoacyl-tRNA synthetase (RS)/tRNA eGFP from non-irradiated cells, and cells grown in the pairs.17-19 GCE offers an attractive alternative for Sec in- absence of DMNB-Sec, were purified via a C-terminal corporation that overrides many limitations of the endog- 6XHis tag (Figure 2b). To determine uncaging efficiency, enous pathway. Furthermore, protected analogs of Sec eGFP-DMNB-Sec39 expressing cells were irradiated can be incorporated to produce selenoproteins that can be (365 nm, 10 mins) (Figure 2b). Intact-protein elec- subsequently activated on demand. An allyl-protected trospray-ionization mass spectrometry (ESI-MS) of these Sec (ASec) and a photocaged (methyl-(6-nitropiper- purified proteins confirmed the presence of the expected onyloxymethyl), MeNPOM) Sec (PSc) have been genet- DMNB-Sec and Sec-containing eGFP species (Figures ically encoded in E. coli using the pyrrolysyl-tRNA syn- 2c, S2, S3). DMNB-Sec uncaging in yeast lysates re- thetase/tRNA pair, and can be uncaged to Sec using Pd- sulted in the formation of diselenide dimers as well as de- 20,21 catalysis and light, respectively. A second photo- hydroalanine species.22 However, only the expected mon- caged (4,5-dimethoxy-2-nitrobenzyl, DMNB) Sec omer was observed in ESI-MS analysis of mammalian- (DMNB-Sec) was incorporated in yeast using the E. coli expressed eGFP-Sec39. Analysis of eGFP from cells leucyl-tRNA synthetase/tRNA pair. 22 Of these, only grown in the absence of DMNB-Sec identified a mass ASec has been successful applied to mammalian cells. consistent with the incorporation of leucine (Leu) or iso- ASec incorporation in mammalian cells used the engi- leucine (Ile) (Figure S4), which likely explains the low neered pyrrolysyl pair, but was restricted to a resilient levels of eGFP fluorescence observed in the absence of model protein (EGFP); whether this incorporation system DMNB-Sec (Figure 2a). When eGFP was expressed in is sufficiently robust to express more sensitive endoge- the presence of 100 µM DMNB-Sec, eGFP-Leu/Ile39 nous selenoproteins is unclear. Furthermore, decaging was not detectable, suggesting that incorporation of these ASec requires treatment with toxic palladium species, natural amino acids is suppressed by the presence of which severely limits its applicability in live mammalian DMNB-Sec. cells. Palladium-mediated uncaging also affords poor
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