Expression of selenoproteins via genetic code expansion in mammalian cells.

Selenoproteins, which contain the 21st amino acid selenocysteine, play roles in maintaining cellular redox homeostasis. Many open questions remain in the field of selenoprotein biology, including the functions of a number of uncharacterized human selenoproteins, and the properties of selenocysteine compared to its analogous amino acid cysteine. The mechanism of selenocysteine incorporation involves an intricate machinery that deviates from the mechanism of incorporation for the canonical 20 amino acids. As a result, recombinant expression of selenoproteins has been historically challenging, and has hindered a deeper evaluation of selenoprotein biology. Genetic code expansion methods, which incorporate protected analogs of selenocysteine, allow the endogenous selenocysteine incorporation mechanism to be bypassed entirely to facilitate selenoprotein expression. Here we present a method for incorporating a photocaged selenocysteine amino acid (DMNB-Sec) into human selenoproteins directly in mammalian cells. This approach offers the opportunity to study human selenoproteins in their native cellular environment and should advance our understanding of selenoprotein biology.

Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine.

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Genetically encoded protein sulfation in mammalian cells.

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.

Chemical Biology Approaches to Interrogate the Selenoproteome.

Selenoproteins are the family of proteins that contain the amino acid selenocysteine. Many selenoproteins, including glutathione peroxidases and thioredoxin reductases, play a role in maintaining cellular redox homeostasis. There are a number of examples of homologues of selenoproteins that utilize cysteine residues, raising the question of why selenocysteines are utilized. One hypothesis is that incorporation of selenocysteine protects against irreversible overoxidation, typical of cysteine-containing homologues under high oxidative stress. Studies of selenocysteine function are hampered by challenges both in detection and in recombinant expression of selenoproteins. In fact, about half of the 25 known human selenoproteins remain uncharacterized. Historically, selenoproteins were first detected via labeling with radioactive 75Se or by use of inductively coupled plasma-mass spectrometry to monitor nonradioactive selenium. More recently, tandem mass-spectrometry techniques have been developed to detect selenocysteine-containing peptides. For example, the isotopic distribution of selenium has been used as a unique signature to identify selenium-containing peptides from unenriched proteome samples. Additionally, selenocysteine-containing proteins and peptides were selectively enriched using thiol-reactive electrophiles by exploiting the increased reactivity of selenols relative to thiols, especially under low pH conditions. Importantly, the reactivity-based enrichment of selenoproteins can differentiate between oxidized and reduced selenoproteins, providing insight into the activity state. These mass spectrometry-based selenoprotein detection approaches have enabled (1) production of selenoproteome expression atlases, (2) identification of aging-associated changes in selenoprotein expression, (3) characterization of selenocysteine reactivity across the selenoprotein family, and (4) interrogation of selenoprotein targets of small-molecule drugs. Further investigations of selenoprotein function would benefit from recombinant expression of selenoproteins. However, the endogenous mechanism of selenoprotein production makes recombinant expression challenging. Primarily, selenocysteine is biosynthesized on its own tRNA, is dependent on multiple enzymatic steps, and is highly sensitive to selenium concentrations. Furthermore, selenocysteine is encoded by the stop codon UGA, and suppression of that stop codon requires a selenocysteine insertion sequence element in the selenoprotein mRNA. In order to circumvent the low efficiency of the endogenous machinery, selenoproteins have been produced in vitro through native chemical ligation and expressed protein ligation. Attempts have also been made to engineer the endogenous machinery for increased efficiency, including recoding the selenocysteine codon, and engineering the tRNA and the selenocysteine insertion sequence element. Alternatively, genetic code expansion can be used to generate selenoproteins. This approach allows for selenoprotein production directly within its native cellular environment, while bypassing the endogenous selenocysteine incorporation machinery. Furthermore, by incorporating a caged selenocysteine by genetic code expansion, selenoprotein activity can be spatially and temporally controlled. Genetic code expansion has allowed for the expression and uncaging of human selenoproteins in E. coli and more recently in mammalian cells. Together, advances in selenoprotein detection and expression should enable a better understanding of selenoprotein function and provide insight into the necessity for selenocysteine production.

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5.

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.

Isopeptide and ester bond ubiquitination both regulate degradation of the human dopamine receptor 4.

How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.

Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes.

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic, and chemoselective reagents. To overcome these limitations, we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Site-specific incorporation of unnatural amino acids as probes for protein conformational changes.

Site-specific in vivo incorporation of unnatural amino acids provides powerful tools for the study of protein interaction and dynamics. Here, we provide a protocol for the incorporation of six such UAA probes into a GFP reporter system, expressed in Escherichia coli from both arabinose and lactose-inducible expression plasmids using an autoinduction media.

Genetically encoded initiator for polymer growth from proteins.

Despite the importance of protein-polymer bioconjugates, there is no general method for producing homogeneous recombinant protein that contains polymer initiators at defined sites. To address this deficiency, we designed the amino acid 4-(2'-bromoisobutyramido)phenylalanine (1) as an initiator in atom-transfer radical polymerization (ATRP) that would provide a stable linkage between the protein and growing polymer. We synthesized 1 and evolved a Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA(CUA) pair to genetically encode this initiator in response to an amber codon. To demonstrate the utility of this initiator, we produced green fluorescent protein (GFP) with 1 site-specifically incorporated on its surface (GFP-1). Purified GFP-1 was then used as an initiator under standard ATRP conditions with a monomer, oligo(ethylene oxide) monomethyl ether methacrylate, efficiently producing a polymer-GFP bioconjugate where the polymer is connected at our selected site on GFP.

Enhancing the utility of unnatural amino acid synthetases by manipulating broad substrate specificity.

Many unnatural amino acid synthetases have been evolved to enable the site-specific in vivo incorporation of many useful functionalities into proteins. While these unnatural amino acid-tRNA synthetase-tRNA(CUA) pairs do not incorporate endogenous amino acids, their substrate specificity has not been assessed for other unnatural amino acids. Here we demonstrate that the unnatural synthetases can be permissive to many unnatural amino acid substrates. The utility of unnatural synthetases can be further expanded by manipulating the synthetase active sites by mutagenesis. Here we have also shown that an l-2-naphthylalanine synthetase can be converted into a permissive l-4-benzoylphenylalanine synthetase with a single mutation without compromising fidelity. Permissive unnatural amino acid synthetases should significantly expand the tool set available for manipulation of proteins.

Probing protein folding using site-specifically encoded unnatural amino acids as FRET donors with tryptophan.

The experimental study of protein folding is enhanced by the use of nonintrusive probes that are sensitive to local conformational changes in the protein structure. Here, we report the selection of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of two unnatural amino acids that can function as fluorescence resonance energy transfer (FRET) donors with Trp to probe the disruption of the hydrophobic core upon protein unfolding. l-4-Cyanophenylalanine (pCNPhe) and 4-ethynylphenylalanine (pENPhe) were incorporated into the hydrophobic core of the 171-residue protein, T4 lysozyme. The FRET donor ability of pCNPhe and pENPhe is evident by the overlap of the emission spectra of pCNPhe and pENPhe with the absorbance spectrum of Trp. The incorporation of both unnatural amino acids in place of a phenylalanine in the hydrophobic core of T4 lysozyme was well tolerated by the protein, due in part to the small size of the cyano and ethynyl groups. The hydrophobic nature of the ethynyl group of pENPhe suggests that this unnatural amino acid is a more conservative substitution into the hydrophobic core of the protein compared to pCNPhe. The urea-induced disruption of the hydrophobic core of the protein was probed by the change in FRET efficiency between either pCNPhe or pENPhe and the Trp residues in T4 lysozyme. The methodology for the study of protein conformational changes using FRET presented here is of general applicability to the study of protein structural changes, since the incorporation of the unnatural amino acids is not inherently limited by the size of the protein.