Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis

Abstract

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Stem Cell Quiescence: Dynamism, Restraint, and Cellular Idling

“…The incorporation of amino acids into proteins requires processing through the cell machinery; specific tRNA synthetase, amino acid, tRNA pairs are needed to incorporate an amino acid into a growing peptide, making biorthogonal labeling strategies very suitable for genetic control ( Figure 4E). The expression of a methionyl-tRNA synthetase (MetRS) with an expanded amino acid binding site (L274G) enables the methionine tRNA to be loaded with the non-canonical amino acid, azidonorleucine (ANL) (Mahdavi et al, 2016). A gene encoding MetRS L274G can be expressed in a cell-specific manner, allowing for cell-type-specific incorporation of ANL into nascent polypeptides, which can be isolated and analyzed.…”

Spatially and temporally regulating translation via mRNA‐binding proteins in cellular and neuronal function

“…Using this approach, it was shown that the mRNA-binding protein hnRNPQ regulates the translation of Gap43 mRNA [173]. Additionally, a novel labeling strategy will also likely allow cell type-specific protein labeling in vivo [174]. Utilizing a modified version of methionyl-tRNA synthetase that can charge tRNA to a noncanonical amino acid azidonorleucine, this study was able to demonstrate robust protein labeling in cultured cells, opening the door to possible tissue-specific expression and cell type-specific labeling of proteins in vivo.…”

Proteomic identification ofCoxiella burnetiieffector proteins targeted to the host cell mitochondria during infection

“…The ability to cross-link effectorhost protein interactions in-organello could be particularly advantageous to the study of effector interactions without the need for overexpression systems. Conversely, incorporation of a noncanonical amino acid into bacterial proteins allows the specific labelling of bacterial proteins, distinguishable from the host cell proteome (88)(89)(90). The click-chemistry based enrichment of bacterial proteins during infection could greatly assist in identifying effector proteins in low abundance during intracellular infection.…”