Dataset Information

Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.

ABSTRACT: Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which, in the filamentous form, regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins have different post-translational modifications (PTMs) in different organisms, including methionine, alanine, aspartate and glutamate N-acetylation, N-arginylation and the methylation of the histidine at residue 73 (His-73), with different organisms displaying a distinct signature of PTMs. Currently, methods are not available to produce actin isoforms with an organism-specific PTM profile. Here, we report the Pick-ya actin method, a method to express actin isoforms from any eukaryote with its own key characteristic PTM pattern. We achieve this using a synthetic biology strategy in a yeast strain that expresses, one, actin isoforms with the desired N-end via ubiquitin fusion and, two, mammalian enzymes that promote acetylation and methylation. Pick-ya actin should greatly facilitate biochemical, structural and physiological studies of the actin cytoskeleton and its PTMs.

SUBMITTER: Hatano T

PROVIDER: S-EPMC7615240 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.

Hatano Tomoyuki T Sivashanmugam Lavanya L Suchenko Andrejus A Hussain Hamdi H Balasubramanian Mohan K MK

Journal of cell science 20200130 2

Actin is one of the most abundant eukaryotic cytoskeletal polymer-forming proteins, which, in the filamentous form, regulates a number of physiological processes, ranging from cell division and migration to development and tissue function. Actins have different post-translational modifications (PTMs) in different organisms, including methionine, alanine, aspartate and glutamate N-acetylation, N-arginylation and the methylation of the histidine at residue 73 (His-73), with different organisms dis ...[more]

PMID: 31964701

Similar Datasets

Project description:The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits β2 and β3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of β3. Interestingly, succinylation of β6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the β3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

Dataset Information

Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.

Publications

Pick-ya actin - a method to purify actin isoforms with bespoke key post-translational modifications.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets