Unremitting progresses for phosphoprotein synthesis

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Abstract

Phosphorylation, one of the important protein post-translational modifications, is involved in many essential cellular processes. Site-specifical and homogeneous phosphoproteins can be used as probes for elucidating the protein phosphorylation network and as potential therapeutics for interfering their involved biological events. However, the generation of phosphoproteins has been challenging owing to the limitation of chemical synthesis and protein expression systems. Despite the pioneering discoveries in phosphoprotein synthesis, over the past decade, great progresses in this field have also been made to promote the biofunctional exploration of protein phosphorylation largely. Therefore, in this review, we mainly summarize recent advances in phosphoprotein synthesis, which includes five sections: 1) synthesis of the nonhydrolyzable phosphorylated amino acid mimetic building blocks, 2) chemical total and semisynthesis strategy, 3) in-cell and in vitro genetic code expansion strategy, 4) the late-stage modification strategy, 5) nonoxygen phosphoprotein synthesis.

Introduction

Phosphorylation, one of the most important protein post-translational modifications (PTMs) [1], refers to the reversible covalent attachment of phosphate groups to serine, threonine, tyrosine, histidine, arginine, lysine, aspartic acid, glutamic acid and cysteine residues catalyzed by protein kinases, which can be removed by phosphatases. Protein phosphorylation plays an essential role in signal transduction and many pivotal cellular events, such as cell proliferation, cell differentiation, and cell cycle [2,3]. The aberrant regulation of phosphorylation is associated with the pathogenesis of various diseases, particularly cancers [4]. Thus, many protein kinases have become highly important anticancer drug targets, of which some inhibitors have been approved as effective therapeutics for clinical use [1]. Nevertheless, the understanding of protein phosphorylation remains limited, majorly in the aspects of the catalytic mechanism of many kinases and phosphatases, the regulation factors of these enzymes' activities, and the effects of site-specific phosphorylation on various proteins’ structure and function. Among diverse technological hindrances, limited access to useful amount of site-specifically and homogeneously phosphorylated proteins, which often serve as probes for elucidating the protein phosphorylation network and as potential therapeutics for interfering their involved biological events, has greatly impeded the development of this research field [5].

The eukaryotic expression system generally produces heterogeneously phosphorylated forms of target protein that are hardly separated, whereas the common prokaryotic system expresses target protein without PTMs because of lacking the PTM machine. As a result, using negatively charged aspartate or glutamate residue as replacement of phosphorylated amino acid (pAA) via site-directed mutagenesis has become a common way to produce the phosphoprotein mimetic that often maintains similar properties with the native one. Nevertheless, such kind of replacement still results in functional fail of many phosphoproteins, particularly in the cases of pAA-mediated interactions, owing to the different charge densities and fine structures between Asp/Glu and native pAAs [6]. In recent years, with the advance of knowledge and technology in both chemical synthesis and protein expression, a variety of novel methodologies to generate site-specifically phosphorylated proteins and their diverse mimetics have been well established, including 1) chemical total and semisynthesis of phosphoproteins based on solid-phase peptide synthesis (SPPS) and peptide fragment ligation, 2) in-cell or in vitro incorporating pAAs or their mimetics into proteins via genetic code expansion (GCE) strategy, 3) late-stage enzyme-catalyzed or chemical modification of proteins with specific tags via the ‘tag-and-modify’ strategy, which greatly facilitate the functional studies of diverse phosphoproteins involved in many essential cellular processes. Each strategy has its advantages in phosphoprotein synthesis. Actually, previous progresses in the generation of phosphoproteins and their functional application have been well presented in an important review that Chen and Cole [7] wrote in 2015 [7,8]. However, over the past several years, with the development of novel chemically synthetic methods and modified protein expression systems, synthesis of phosphoproteins have made significant progress, especially in the field of pAA incorporation via the GCE strategy. Therefore, in this review, we mainly focus on recent advances in the synthesis of phosphoproteins and their functional studies, consisting of 1) synthesis of pAA mimetics, 2) chemical total and semisynthesis strategy, 3) GCE strategy, 4) late-stage modification strategy, and 5) nonoxygen phosphoprotein synthesis.

Section snippets

Synthesis of pAA mimetics

The approaches accessing to site-specifically phosphorylated proteins including chemical synthesis, GCE strategy, and late-stage modification have to use the pAA or their mimetics as building blocks (Table 1), which are installed into the desired sites of target proteins. Because SPPS needs to consume an excessive amount of pAA building blocks and the in-cell GCE strategy requires a large amount of pAAs applied in the cell culture medium, it is very necessary to develop highly efficient methods

Genetically encoded phosphoprotein synthesis

Over the past three decades, GCE has become a more and more attractive and vital strategy for in-cell and in vitro (cell-free) synthesis of proteins with UAA mutagenesis, including the direct incorporation of pAAs into proteins [8,79, 80, 81, ••82]. Based on the natural translation machinery system, the GCE involves several key translational steps (Figure 3): (a) an UAA and a transfer RNA (tRNA) are recognized by their cognate aminoacyl-tRNA synthetase (aaRS) simultaneously, resulting in the

Late-stage modification

In addition to protein total or semisynthesis that depends on peptide/protein fragments ligation at specific sites, there is another strategy defined as late-stage modification, which is suitable for obtaining phosphoproteins based on native recombinant proteins. The key step of this strategy is the direct phosphorylation on specific residue or ‘tag’ of target protein via kinase-catalyzed reaction (Figure 5a) [124,125] or chemical modification (Figure 5b) [126, •127, 128], which largely expand

Nonoxygen phosphoprotein synthesis

Following by O-phosphoprotein synthesis, nonoxygen phosphoprotein synthesis has attracted increasing attention. As for N-phosphorylation, Kee et al. [39] incorporated the synthetic stable pHis mimetic 33 building block into the full-length histone H4 via EPL strategy, which could be selectively recognized by the first 3-pHis polyclonal antibody. Then, Fuhs et al. [137] ligated the 1- or 3-pHis mimetics 32 or 33 containing peptides with the carrier protein KLH respectively via NCL, of which the

Summary and perspectives

Over the past decade, an increasing demand for useful amount of site-specifical and homogeneous phosphoproteins as probes or potential therapeutics have stimulated the rapid development of various phosphoproteins synthetic strategies, which are beneficial from recent advances of protein chemistry and modified protein expression system. The strategies for phosphoprotein synthesis, mainly including classic chemical total synthesis and semisynthesis, in-cell GCE methodology and in vitro CFPS

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the Major State Basic Research Development Program of China (2018YFA050760) and the National Natural Science Foundation of China (91753122, 21672125).

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