Novel human cell expression method reveals the role and prevalence of posttranslational modification in non-muscle tropomyosins
| Autor: | Kyle R. Barrie, Roberto Dominguez, Peter J. Carman |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Gene isoform
WPRE woodchuck hepatitis virus posttranscriptional regulatory element FDR false discovery rate Gene Expression Tropomyosin macromolecular substances intein Proteomics Biochemistry Cell Line HEK human embryonic kidney posttranslational modification (PTM) Humans Tpm tropomyosin Cytoskeleton protein expression Molecular Biology Actin Coiled coil chemistry.chemical_classification Chemistry HEK 293 cells cytoskeleton Cell Biology Recombinant Proteins Amino acid Cell biology Acetylation HCD higher-energy collisional dissociation PTM posttranslational modification Intein actin Protein Processing Post-Translational CMV cytomegalovirus promoter Function (biology) Research Article CBD chitin-binding domain |
| Zdroj: | The Journal of Biological Chemistry |
| DOI: | 10.1101/2021.04.05.438513 |
| Popis: | Biochemical studies require large quantities of proteins, which are typically obtained using bacterial overexpression. However, the folding machinery in bacteria is inadequate for expressing many mammalian proteins, which additionally undergo posttranslational modifications (PTMs) that bacteria, yeast, or insect cells cannot perform. Many proteins also require native N- and C-termini and cannot tolerate extra tag amino acids for proper function. Tropomyosin (Tpm), a coiled coil protein that decorates most actin filaments in cells, requires both native N- and C-termini and PTMs, specifically N-terminal acetylation (Nt-acetylation), to polymerize along actin filaments. Here, we describe a new method that combines native protein expression in human cells with an intein-based purification tag that can be precisely removed after purification. Using this method, we expressed several nonmuscle Tpm isoforms (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2) and the muscle isoform Tpm1.1. Proteomics analysis revealed that human-cell-expressed Tpms present various PTMs, including Nt-acetylation, Ser/Thr phosphorylation, Tyr phosphorylation, and Lys acetylation. Depending on the Tpm isoform (humans express up to 40 Tpm isoforms), Nt-acetylation occurs on either the initiator methionine or on the second residue after removal of the initiator methionine. Human-cell-expressed Tpms bind F-actin differently than their Escherichia coli-expressed counterparts, with or without N-terminal extensions intended to mimic Nt-acetylation, and they can form heterodimers in cells and in vitro. The expression method described here reveals previously unknown features of nonmuscle Tpms and can be used in future structural and biochemical studies with Tpms and other proteins, as shown here for α-synuclein. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|