Nitrilase-Activatable Noncanonical Amino Acid Precursors for Cell

Letters pubs.acs.org/acschemicalbiology

Nitrilase-Activatable Noncanonical Amino Acid Precursors for CellSelective Metabolic Labeling of Proteomes Zefan Li,† Yuntao Zhu,† Yuting Sun,‡ Ke Qin,† Weibing Liu,† Wen Zhou,† and Xing Chen*,†,§,∥,⊥ †

College of Chemistry and Molecular Engineering, ‡Academy for Advanced Interdisciplinary Studies, §Peking-Tsinghua Center for Life Sciences, ∥Synthetic and Functional Biomolecule Center, and ⊥Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, 100871, China S Supporting Information *

ABSTRACT: Cell-selective protein metabolic labeling is of great interest for studying cell−cell communications and tissue homeostasis. We herein describe a nitrilase-activatable noncanonical amino acid tagging (NANCAT) strategy that exploits an exogenous nitrilase to enzymatically convert the nitrilesubstituted precursors to their corresponding noncanonical amino acids (ncAAs), L-azidohomoalanine (Aha) or homopropargylglycine (Hpg), in living cells. Only cells expressing the nitrilase can generate Aha or Hpg in cellulo and metabolically incorporate them into the nascent proteins. Subsequent clicklabeling of the azide- or alkyne-incorporated proteins with fluorescent probes or with affinity tags enables visualization and proteomic profiling of nascent proteomes, respectively. We have demonstrated that NANCAT can serve as a versatile strategy for cell-selective labeling of proteomes in both bacterial and mammalian cells.

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to different cell types and organisms, and engineering new MetRS mutants or mutants of other aminoacyl-tRNA synthetases is often needed on a case-by-case basis.19,20 For example, when expressed in mammalian cells, the NLL-MetRS charges Anl onto the mammalian initiator Met tRNA but not the elongator Met tRNA, resulting in incomplete coverage of the nascent proteins.21 To address this issue, a mutant murine MetRS, mMetRSL274G, was recently identified and enabled cellselective incorporation of Anl in mammalian cells.22,23 Complementary to methods based on mutants of endogenous aminoacyl-tRNA synthetases, the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair from Methanosarcina was engineered to decode sense condons and thus enabled stochastic incorporation of a cyclopropene-bearing pyrrolysine analog into newly synthesized proteins in mammalian cells and in the ovaries of Drosohpila melanogaster.24 So far, all the methods have been limited to engineered aminoacyl-tRNA synthetases with substrate specificity that is orthogonal to the endogenous aminoacyl-tRNA synthetases. Here, we describe nitrilase-activated noncanonical amino acid tagging (NANCAT) as an effective and general strategy for cellselective metabolic protein labeling, which does not rely on engineering of aminoacyl-tRNA synthetases. Considering the broad applicability of Aha and Hpg, we designed chemical

n multicellular organisms, tissue- and cell-specific proteomes are established during development, and the proteostasis network is tightly regulated during the life cycle.1−3 Noncononical amino acids (ncAAs) bearing a bioorthogonal chemical reporter have emerged as a powerful tool for probing protein synthesis in situ.4 L-Azidohomoalanine (Aha) and homopropargylglycine (Hpg), the two most commonly used ncAAs that serve as surrogates of methionine (Met), are effectively activated by the endogenous methionyl-tRNA synthetase (MetRS) and incorporated into the Met codons of nascent proteins. By using bioorthogonal chemistry (e.g., click chemistry),5 the proteins are conjugated with fluorophores for imaging6 or with affinity tags for enrichment and proteomic analysis.7 Pulse labeling with ncAAs has greatly enhanced our capability to visualize and profile protein synthesis in bacteria,8 mammalian cells,6,7,9,10 and living animals including zebrafish,11 Drosophila,12 and mice.13 One limitation of metabolic protein labeling using ncAAs is the lack of cell-selectivity, which limits its use in studying protein synthesis in specific cell types when the target cells are in low abundance or difficult to isolate. Developing strategies for cell-selective metabolic protein labeling has recently attracted great interest.14 A mutant Escherichia coli MetRS, NLL-MetRS, was identified to activate longer-chain Met analogs, such as azidonorleucine (Anl) and 2aminooctynoic acid (Aoa), which are excluded by the endogenous MetRS.15,16 Only the bacterial cells expressing NLL-MetRS can incorporate Anl or Aoa, in the presence of mammalian cells15−17 or distinct bacteria.18 However, the pair of Anl/Aoa and E. coli NLL-MetRS is not generally applicable © XXXX American Chemical Society

Received: September 1, 2016 Accepted: October 27, 2016

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DOI: 10.1021/acschembio.6b00765 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

labeling newly synthesized proteins, whereas the neighboring cells cannot produce Aha (or Hpg) and are therefore not labeled (Figure 1B). In search of an enzyme−precursor pair, we set the criteria to be as follows: (i) The enzymatic reaction is orthogonal to endogenous enzymes, without interfering with or getting interference from the cellular milieu. (ii) The precursor is stable and inert. (iii) The enzymatic conversion is kinetically not slower than the subsequent step of charging of Aha to Met tRNA catalyzed by MetRS. On the basis of these considerations, we opted to evaluate nitrilase-catalyzed conversion of nitriles to carboxylic acids. The nitrile group is abiotic in animals and is highly stable and inert under physiological conditions. Chemical hydrolysis of nitrile compounds demands harsh conditions such as strong acids or bases at high temperatures. Various organisms including bacteria, fungi, and plants produce nitrilases, which have been widely characterized and utilized as biocatalysts in organic synthesis.25−27 The specificity of a wide range of nitrilases has been determined, and nitrilases with substrate preference to aromatic nitriles, arylacetonitriles, or aliphatic nitriles have been identified.25,28 As a generic principle, the α-carboxylic acid of ncAAs is substituted with a nitrile group. Accordingly, (S)-2-amino-4azidobutanenitrile (Abn) serves as the nitrile precursor of Aha and (S)-2-amino-hex-5-ynenitrile (Ahn) as the precursor of Hpg (Figure 1A). Abn was chemically synthesized from Boc protected Aha with an overall yield of 30%, using a synthetic route that is different from the previously reported synthesis (Scheme S1).29 Ahn was synthesized according to a reported

precursors of ncAAs that can be enzymatically converted to Aha or Hpg by an exogenous enzyme (Figure 1A). Only cells

Figure 1. Nitrilase-activated noncanonical amino acid tagging (NANCAT) for cell-selective metabolic labeling of proteins. (A) Abn and Ahn are nitrile-substituted precursors of Aha and Hpg, respectively, and are activated by RrNIT. (B) The nitrilase-expressing cells are able to produce Aha or Hpg from precursors. Restricted expression of RrNIT in target cells therefore allows for cell-selective labeling of nascent proteins.

expressing the converting enzyme are capable of synthesizing Aha or Hpg from supplemented precursors and metabolically

Figure 2. NANCAT in bacterial and mammalian cells. (A) RrNIT+ E. coli cells or E. coli cells were incubated with 500 μM Abn, 1 mM Met, 1 mM Met +500 μM Abn, or 500 μM Abn. Cell lysates were reacted with alkyne-Cy5 and analyzed by in-gel fluorescence scanning. (B) Imaging analysis of RrNIT+ E. coli cells labeled with Abn. Scale bar, 2 μm. (C) In-gel fluorescence analysis of RrNit+ HeLa cells labeled with Abn. (D) Imaging analysis of RrNit+ HeLa cells labeled with Abn. The nuclei were stained with Hoechst 33342 (blue signal). Scale bar, 20 μm. B


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Figure 3. Proteomic analysis of nascent proteins in RrNit+ HeLa cells labeled with NANCAT. (A) Distribution of the identified proteins in various cellular components. (B) Representative MS2 spectrum in which DIETFYNTSIEEMPLNVADLI from eukaryotic initiation factor 4A-I (elF4A1) was identified as an Abn-modified peptide (Met-398).

procedure, in a racemic mixture.30 Since the presence of D-Hpg does not impair protein labeling,31 the racemic Ahn preparation was used. Abn and Ahn exhibited satisfactory stability in aqueous solution within 3 h (Supporting Information Figure 1). To identify a nitrilase with high activity, three nitrilases from Rhodococcus rhodochrous (RrNIT), Alcaligenes faecalis (Af NIT), and Comamonas testosteroni sp (CtNIT) were recombinantly expressed and purified using the Escherichia coli expression system. Abn was incubated with the purified nitrilases, and the enzymatic reactions were monitored by LC-MS analysis. All three nitrilases were able to hydrolyze Abn to Aha, and the catalytic constants were determined using the LC-MS assay (Supporting Information Table 1 and Figure 2). RrNIT has the highest kcat/KM value, 2.04 × 103 M−1 s−1. In comparison to the activation of Aha by EcMetRS (kcat/KM = 1.42 × 103 M−1 s−1),32 the RrNIT-catalyzed reaction is as fast as the subsequent step of charging ncAA onto Met tRNA, thus minimizing the overall time for protein labeling in NANCAT. Furthermore, RrNIT was able to convert Ahn to Hpg, but with a slower conversion rate (Supporting Information Figure 3). Therefore, we chose the enzyme−substrate pair of RrNIT and Abn for most of the experiments in this work. We first validated NANCAT in E. coli, which has no endogenous nitrilase. An E. coli strain constitutively expressing RrNIT (RrNIT+) was incubated with 500 μM Abn for 3 h in M9 minimal medium. Protein lysates were collected and reacted with alkyne-Cy5 via Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC, also termed click chemistry).5 In-gel fluorescence scanning showed that Abn effectively labeled nascent proteins in RrNIT+ E. coli, in a way similar to Aha (Figure 2A). In contrast, no labeling was observed in wild type E. coli (RrNIT−) cells treated with Abn. The Abn labeling in RrNIT+ E. coli was abolished by competition with 1 mM Met. Furthermore, the selectively labeled proteins in Abn-treated RrNIT+ E. coli were imaged using confocal fluorescence

microscopy (Figure 2B). These results indicate that Abn can be enzymatically converted to Aha for labeling newly synthesized proteins in a cell-selective manner by controlling the expression of RrNIT in E. coli. Similarly, Ahn resulted in selective incorporation of Hpg into nascent proteins for subsequent detection using azide-bearing probes in RrNIT+ E. coli, which demonstrates the versatility of NANCAT in using different ncAA precursors (Supporting Information Figure 4). One advantage of the NANCAT strategy is the general applicability in various cell types. We therefore sought to establish NANCAT in mammalian cells. RrNIT fused with an N-terminal EGFP was successfully expressed in HeLa cells (Supporting Information Figure 5). HeLa cells stably expressing EGFP-RrNIT (RrNIT+) were established via flow cytometry-assisted sorting. The RrNIT+ HeLa cells were incubated with 500 μM Abn for 3 h. Abn (as well as Ahn) did not cause significant cytotoxicity (Supporting Information Figures 6 and 7). Cell lysates were reacted with alkyne-Cy5, and in-gel fluorescence scanning showed that a variety of nascent proteins were labeled with Aha generated from Abn (Figure 2C, lane 3). The pattern of labeled bands was almost identical to that from cells treated with Aha (Figure 2C, lane 4). The Abn labeling was abolished by competition with Met (Figure 2C, lane 2), and the wild type (RrNIT−) HeLa cells were not labeled (Figure 2C, lane 1). The labeling with Abn was incubation time- and concentration-dependent (Supporting Information Figures 8 and 9). Selective protein labeling using Abn in RrNIT+ cells was also visualized by confocal fluorescence microscopy (Figure 2D). Furthermore, Ahn could also be used to metabolically label nascent proteins in RrNIT+ HeLa cells (Supporting Information Figure 10). Taken together, these results demonstrate NANCAT as a versatile strategy for cell-selective labeling of proteomes in both bacterial and mammalian cells. C


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the supplemented ncAA precursors. Furthermore, the NANCAT strategy is complementary to the MetRS mutant-based strategy, which should enable simultaneous tagging of multiple proteomes in complex systems. In addition, nitrilases may be explored for cell-selective activation of fluorescent dyes or small-molecule drugs.

To further confirm the conversion of Abn to Aha and the subsequent incorporation into the Met sites of nascent proteins, we performed a site-specific proteomic analysis. RrNIT+ HeLa cells were treated with Abn, and the lysates were reacted with a photocleavable alkyne-biotin probe, alkynePC-biotin. The biotin-modified proteins were captured and enriched by streptavidin beads, followed by on-bead trypsin digestion to remove the peptides with no azide labeling and biotin modification. The remaining modified peptides on the beads were subjected to UV cleavage to release the peptides, which were then subjected to tandem mass spectroscopy analysis. We identified 616 unique Abn-modified Methionine sites on 584 peptides, which corresponded to 400 unique newly synthesized proteins (Figure 3 and Supporting Information Table 2). The identified proteins were distributed throughout the cells. Finally, we demonstrated the application of NANCAT in cell-selective metabolic protein labeling in coculture systems. RrNIT+ HeLa cells cocultured with wild type HeLa cells were incubated with 500 μM Abn for 3 h, followed by click-labeling with alkyne-Cy5. Confocal fluorescence microscopy showed that only the nascent proteins in the RrNIT+ cells were selectively labeled, whereas the WT HeLa cells exhibited minimal fluorescence (Figure 4A and Supporting Information

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00765. Supporting Scheme S1, Supporting Figures S1−S11, Supporting Tables S1 and S2, and experimental procedures (PDF) Table of Abn sites (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank H. Luo for RrNIT and Af NIT plasmids. We thank the mass spectrometry facility of the National Center for Protein Sciences at Peking University for assistance with proteomic analysis. This work was supported by the National Natural Science Foundation of China (No. 21425204) and the National Key Research and Development Projects (No. 2016YFA0501500).

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REFERENCES

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Figure 4. NANCAT-enabled cell-selective metabolic protein labeling in cocultures of mammalian cells. RrNit+ HeLa cells cocultured with WT HeLa cells were incubated with 500 μM Abn (A) or Aha (B) for 3 h. After fixation and permealization, the cells were reacted with alkyneCy5 and visualized by confocal fluorescence microscopy. Scale bars, 50 μm.

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Nitrilase-Activatable Noncanonical Amino Acid Precursors for Cell

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