# Transmission of Non-Constitutive Proteasomes Between Cells via Extracellular Vesicles

## TL;DR

This study shows that non-constitutive proteasomes can be transferred between cells through extracellular vesicles, a process that increases with IFN-γ stimulation.

## Contribution

The study demonstrates direct transmission of non-constitutive proteasomes between cells via extracellular vesicles for the first time.

## Key findings

- EVs containing non-constitutive proteasomes were isolated and characterized from various cell lines.
- IFN-γ stimulation increased the content of non-constitutive proteasomes in EVs.
- Recipient cells internalized EVs containing β5i-mCherry proteasomes, confirming intercellular transfer.

## Abstract

Most intracellular proteins are degraded by the ubiquitin–proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs.