Protein labelling

The functional diversity of the proteome in both eukaryotic and prokaryotic systems is elaborated through a variety of posttranslational modifications (PTMs) that allow for structural diversities and reactivity profiles far more complex than what is attainable through the use of the 20 canonical amino acids.¹ These numerous PTMs are installed, decoded and removed by dedicated writers, readers and erasers, respectively, empowering the complexity that is inherent to biological signaling networks.² In order to understand this biological complexity and engineer novel function into proteins, it is imperative that the community of chemists and biologists implement innovative platforms to covalently modify proteins, in a manner similar to the PTMs adapted by nature. Methods to incorporate new functionality that either mimics or expands upon what is present within these complex biological systems have a plethora of applications in understanding and exploiting biological systems with eventual therapeutic gain. This themed issue on “Protein Labelling” specifically focuses on the covalent modification of proteins with functionalities outside of that provided by the canonical amino acids. The contributions within this collection serve to elegantly showcase the methods available for protein labelling and the application of these diverse protein labelling tools to investigate the intricate biology of PTMs, inhibit or monitor protein activity through covalent probes, and engineer novel function into proteins for improved therapeutic targeting and delivery.

Covalent modification of proteins can be achieved through a number of strategies. One commonly used approach is to exploit the inherent reactivity of the canonical amino acid side-chains through targeted electrophile–nucleophile pairings. In an opinion piece included within this themed issue, Jones provides an overview of this chemically-reactive proteome with a focus on recent studies that take advantage of the nucleophilicity of amino acids such as cysteine, serine, tyrosine and lysine for small-molecule attachment (DOI: 10.1039/C5MB00760G). These chemical methods for protein modification have facilitated advancements in inhibitor development, and target identification. A second strategy for incorporating unnatural functionality into proteins involves the genetic encoding of a short peptide that is subsequently appended to a small molecule or metal ion through enzymatic or non-enzymatic means. In a comprehensive review on this topic, Seitz and Beck-Sickinger et al. provide insight into the diverse peptide tags available for protein labelling both in vitro and in vivo (DOI: 10.1039/C6MB00023A). One such peptide tag that can be enzymatically functionalized for site-selective labelling is the Sortase A (SrtA) system. Webb and Beales et al. highlight a general approach for nanodisc labelling using the SrtA system (DOI: 10.1039/C6MB00126B). Briefly, the membrane scaffold protein (MSP) of the nanodisc was chemically modified using the SrtA system, and used for imaging nanodisc uptake into cells. This method is highly generalizable and can be applied to incorporate a diverse array of functionalities onto nanodiscs. Lastly, unnatural functionality can be genetically encoded into proteins through site-specific incorporation of noncanonical amino acids (ncAAs). This is achieved through adaptation of an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair that specifically recognizes a select ncAA and incorporates it into a protein at a designated site. Typically, ncAA incorporation is genetically encoded at a UAG nonsense codon, whereby the orthogonal tRNA competes with the release factor RF-1 for binding to the UAG codon at the ribosome. In an elegant study, Chatterjee et al. show that in an E. coli strain devoid of UAG stop codons and RF-1, the efficiency of incorporation of ncAAs at multiple sites within a protein, is significantly improved (DOI: 10.1039/C6MB00070C). These contributions serve to highlight the current state-of-the-art, cutting-edge advancements and innovative applications of methods to incorporate unnatural functionality into proteins.

As mentioned previously, a diverse array of PTMs serve to mediate protein localization, signaling networks and protein–protein interactions within cells. Understanding the protein targets of these PTMs, as well as characterizing the activity and substrate repertoire of the enzymes that install and delete these modifications is critical to furthering our knowledge of the biological complexity imparted by these PTMs. Several contributions within this issue focus on protein labelling within the context of studying protein PTMs. To identify sites of protein modification through distinct PTMs, it is necessary to introduce unnatural functionalities that allow for enrichment of modified proteins for identification by immunoblotting and mass spectrometry (MS). To identify sites of protein modification by O-GlcNAc, Hsieh-Wilson et al. utilize a two-step chemoenzymatic approach (DOI: 10.1039/C6MB00138F). In the first step, the endogenous O-GlcNAc modification is tagged with an unnatural azido sugar (GalNAz). The GalNAz-modified proteins are then conjugated to a MS-compatible cleavable enrichment tag using the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC), for downstream release of O-GlcNAc modified peptides. They apply this cleavable-linker strategy to identify known and previously uncharacterized sites of O-GlcNAc modification on alpha-crystallin and O-GlcNAc transferase. Similarly, by applying protein labelling methods to study protein palmitoylation, Martin et al. show that expression of the Snail transcription factor that drives epithelial–mesenchymal transition (EMT), results in global changes in cellular palmitoylation (DOI: 10.1039/C6MB00019C). These studies into global palmitoylation changes were enabled by the use of an alkynyl fatty acid analog 17-ODYA, which can be conjugated to reporter tags using CuAAC for enrichment and subsequent MS-based identification of palmitoylated proteins potentially involved in tumour suppression. In a similar vein, Chen et al. describe the dual functionalization of ubiquitin to allow for FRET-based monitoring of ubiquitin C-terminal hydrolase, UCH-L3, activity. They utilize two consecutive native-chemical ligation methods together with chemoselective reactions to incorporate two unnatural functionalities into the N- and C-termini of ubiquitin. The resulting UCH-L3 activity sensor is a valuable tool for screening for inhibitors of this ubiquitin hydrolase (DOI: 10.1039/C6MB00165C). Another focus in this issue is on oxidative PTMs that occur in the presence of reactive oxygen species (ROS). One of these highly functionally relevant modes of protein oxidation is the formation of cysteine sulfenic acids. In particular, sulfenic-acid modification of protein tyrosine phosphatases (PTPs) are known to inhibit PTP activity and modulate signaling cascades. Carroll et al. present an immunochemical strategy to detect PTP oxidation by generating an antibody to specifically recognize the active-site cysteine-containing peptide of PTP modified by dimedone, which is a chemical probe selective for sulfenic acids (DOI: 10.1039/C5MB00847F). This innovative method elegantly combines the chemoselectivity of dimedone with the extended epitope selectivity of antibodies to enable the targeted interrogation of PTP oxidation in proteomes. The ability to selectively covalently modify protein PTMs or adapt endogenous pathways to incorporate unnatural PTM analogs, have facilitated our understanding of the underlying biology of diverse PTMs.

The covalent modification of proteins by small molecules serves as the basis of covalent inhibitor design and activity-based protein profiling (ABPP). Several contributions in this issue describe protein labelling by small-molecule probes either through direct conjugation to a reactive amino acid within the protein active site, or through incorporation of photoaffinity labels for covalent crosslinking. Albrow and Storer et al. developed small molecule probes to monitor histone deacetylase (HDAC) activity (DOI: 10.1039/C6MB00109B). These probes incorporate photoreactive groups onto known HDAC inhibitor scaffolds to generate class IIa and pan-HDAC probes that enabled HDAC-interacting proteins to be isolated and identified using MS. In a similar approach, Overkleeft et al. describe the incorporation of photoreactive groups into the scaffold of the H89 protein kinase inhibitor to generate a chemical probe for activity profiling of clinically relevant kinases (DOI: 10.1039/C6MB00257A). These photoreactive probes are shown to react with PKA and AKT1 in an activity-based manner. In an interesting application of covalent small-molecule probes, Li et al. developed benzimidazole probes targeting the H⁺/K⁺-ATPase, ATP4A, to explore the efficiency of bioorthogonal reactions (DOI: 10.1039/C6MB00024J). Specifically, by incorporating bioorthogonal handles to known benzimidazole scaffolds that covalently bind to ATP4A, they demonstrate that the tetrazine ligation has superior efficiency relative to the strain promoted azide–alkyne cycloaddition. Lastly, Weerapana et al. describe a tyrosine-reactive inhibitor of GSTP1 that incorporates a dichlorotriazine electrophile (DOI: 10.1039/C6MB00250A). This covalent GSTP1 inhibitor, LAS17, provides a unique mode of GSTP1 inhibition through covalent modification of a reactive tyrosine proximal to the glutathione-binding site of this enzyme. In summary, these contributions to the protein labelling issue highlight the incorporation of photoreactive groups and electrophiles into small-molecule scaffolds to generate ABPP probes for diverse protein classes, and irreversible inhibitors for therapeutically relevant enzymes.

The last application of protein labelling that is highlighted within this issue, is the chemical modification of proteins for improved therapeutic delivery. Hackenberger and Rubini et al. describe the use of unnatural amino acid incorporation coupled with chemoselective reactions to generate erythropoietin (EPO) analogs modified by branched polyethylene glycol (PEG) chains (DOI: 10.1039/C5MB00857C). These engineered EPO derivatives are expected to display improved pharmacokinetic properties. Similar methods are also utilized to chemically modify antibodies for targeted delivery of small molecules, which forms the basis of the antibody drug conjugate field. The application of diverse protein labelling strategies to protein therapeutics and antibodies, can serve to improve in vivo stability and targeting.

We are extremely grateful to all of the contributors to this themed issue on protein labelling. As highlighted in this editorial, these contributions reflect the diversity of methods available for protein labelling, as well as the application of these methods to understand biological processes and generate improved therapeutics.

References

1. C. T. Walsh, S. Garneau-Tsodikova and G. J. Gatto Jr., Protein posttranslational modifications: the chemistry of proteome diversifications, Angew. Chem., Int. Ed., 2005, 44, 7342–7372.
2. P. Beltrao, P. Bork, N. J. Krogan and V. van Noort, Evolution and functional cross-talk of protein post-translational modifications, Mol. Syst. Biol., 2013, 9, 714.

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