Site‐Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover

Abstract

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio‐orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence‐based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse‐chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells

“…In comparison to the incorporation of BocK in R26 wtRS and DiazK in R26 RS_DiazK , BcnK was less efficiently incorporated in R26 RS_BcnK , indicated by the lower correlation between mCh and eGFP fluorescence. This difference may be attributable to the reduced PylRS aminoacylation activity of BcnK compared to BocK or DiazK ( 99 ). Furthermore, the transfection efficiency of stable R26 RS clones was generally low (∼25%, Supplementary Figure S2B ).…”

“…Next, we wondered to what extent the nucleotide composition around UAG determines ncAA incorporation efficiencies. Genetic code expansion with PylS / PylT in HEK293T cells has been reported to suppress endogenous amber stop codons resulting in off-target labeling of the cellular proteome ( 25 , 99 ). In line with these studies, we observed widespread amber suppression of endogenous proteins in the stable R26 RS_BcnK mESC clone by in-gel fluorescence analysis of BcnK harboring proteins with a silicon rhodamine-tetrazine conjugate (SiR-Tet) ( Supplementary Figure S1E, S3A ).…”

Insights into receptor structure and dynamics at the surface of living cells

“…The ncAA bicyclo[6.1.0]nonyne-lysine (BCNK) was inserted by overriding a stop-codon (TAG) in the coding sequence with BCNK-loaded tRNA UAG . For loading, modified pyrrolysyl-tRNA synthethase and four copies of tRNA UAG were co-expressed from a concomitant plasmid 35 , 36 . We selected 14 solvent-exposed side chains in the extracellular domain for BCNK replacement, henceforth referred to as receptor mutants (Fig.…”

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines

“…Amber suppression sites were inserted in IL-12α/23α constructs by site-directed mutagenesis PCR using Pfu (Promega) DNA polymerase in a pSVL vector backbone. “TAG”-replaced coding sequences were subcloned into the pPB vector as reported previously ( 56 ) in-frame downstream of the EF-1 promoter. Constructs equipped with a C-terminal FLAG tag were subcloned in a similar approach separated by four (IL-12α) or five (IL-23α) GS-linker repeats.…”

“…Constructs equipped with a C-terminal FLAG tag were subcloned in a similar approach separated by four (IL-12α) or five (IL-23α) GS-linker repeats. Other plasmids used in this study were an IL-12β construct in the pcDNA3.1(+) vector ( 56 ) and immunoglobulin γ 1 heavy chain in pSVL, a kind gift from Linda M. Hendershot, St Jude Children’s Research Hospital. All constructs were verified by sequencing.…”