Chemical biology approaches for studying posttranslational modifications

Abstract

Posttranslational modification (PTM) is a key mechanism for regulating diverse protein functions, and thus critically affects many essential biological processes. Critical for systematic study of the effects of PTMs is the ability to obtain recombinant proteins with defined and homogenous modifications. To this end, various synthetic and chemical biology approaches, including genetic code expansion and protein chemical modification methods, have been developed. These methods have proven effective for generating site-specific authentic modifications or structural mimics, and have demonstrated their value for in vitro and in vivo functional studies of diverse PTMs. This review will discuss recent advances in chemical biology strategies and their application to various PTM studies.

Keywords: Chemical biology approach; genetic code expansion; posttranslational modification (PTM); protein chemical modification.

Figure 1.

Types of PTMs accessed by chemical biology approaches. Chemical structures of the modified amino acids are shown. 1, phospho-Ser; 2, phospho-Tyr; 3, sulfo-Tyr; 4, nitro-Tyr; 5, monomethyl-Lys; 6, dimethyl-Lys; 7, trimethyl-Lys; 8, formyl-Lys; 9, acetyl-Lys; 10, propionyl-Lys; 11, butyryl-Lys; 12, crotonyl-Lys; 13, succinyl-Lys; 14, monomethyl-Arg; 15, dimethyl-Arg; 16, O-GlcNAc mimic; 17, O-GlcNAc homo-Ser; 18, N-GlcNAc mimic; 19, mono Ub; 20, di Ub; 21, homotypic Ub chain; 22, mixed Ub chain; 23, branched Ub chain.

Figure 2.

Schematics of chemical biology methodologies for site-specific installation of PTMs in proteins. (A) Native chemical ligation (right) and Expressed protein ligation (left) methods. (B) Genetic code expansion (right) and genetic code reprogramming (left) methods. (C) Chemical modification methods; thiol-based chemistry (top) and a three-step chemoselective carbon-carbon bond conjugation approach (bottom).

Figure 3.

Functional studies of serine phosphorylation using the genetic phosphoserine-incorporation system. (A) Phosphorylation of H3S10 induces enhanced H3 acetylation by Gcn5-SAGA complex in yeast. (B) H3S28 phosphorylation facilitates p300/CBP-mediated H3K27 acetylation and activates transcription. (C) Phosphorylated TRIM9 negatively regulates NF-kB pathway by stabilizing NF-kB inhibitory proteins. (D) S291 phosphorylation in cGAS reduces its enzymatic activity to regulate innate immune DNA-sensing pathway. (E) PINK1-mediated S65 phosphorylation of Ub stimulates E3 ligase activity of PARKIN. (F) Phosphorylation in Ub controls the specificity of E3 ligase and DUB activities.

Figure 4.

Functional studies of lysine modifications using the site-specific lysine acetylation and methylation systems. (A) Nucleosomes carrying acetylated H3 histones can be used to determine the specificity of NAD+-dependent histone deacetylases (SIRTs). (B) K403 acetylation in glucose-6-phosphate dehydrogenase negatively regulates its enzymatic activity. (C) K178 acetylation in DnaA blocks DNA replication initiation by blocking its binding to ATP or oriC in E. coli. (D) K412 acetylation in Cdc11 controls its function and localization via acetylation-SUMOylation switch during the cell cycle in yeast. (E) Transgenic mouse with expanded genetic code with acetyllysine allows temporal and spatial control of protein acetylation and facilitates systematic in vivo acetylome studies. (F) K79 methylation in histone H3 induces chromatin transcription through enhanced p300-mediated histone acetylation.

Figure 5.

Functional studies of other modifications. (A) Site-specific sulfation in hirudin results in highly enhanced binding toward thrombin. (B) Nitration in Hsp90 induces motor neuron death through the Fas pathway. (C) LacZ-type reporter enzyme SSβG carrying mimics of glycosylation and sulfation can be used to probe brain inflammation in vivo. (D) K120 mono-ubiquitination in H2B enhances hDot1L-mediated H3K79 methylation.