Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2.

Katharina Reglinski,Wolfgang Schliebs,Dominic Waithe,Sabrina Altendorf,Ralf Erdmann,Marina Keil,Christian Eggeling

doi:10.1371/journal.pone.0140685

Katharina Reglinski, Wolfgang Schliebs + Show 5 more

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https://doi.org/10.1371/journal.pone.0140685

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Abstract

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.

Highlights

Dynamic ubiquitination is involved in the regulation of many cellular processes such as cell proliferation, differentiation and apoptosis
No punctate pattern but a diffuse background staining was observed for all ubiquitin-specific protease 2 (USP2)-GFP isoforms in PEX5-deficient cells, indicative of a cytosolic localization and demonstrating that the peroxisomal localization depends on a functional peroxisome targeting signal of type 1 (PTS1) receptor (Fig 2, ΔPEX5T)
Replacement of the -SRM sequence by -SKL increased the amount of particulate USP2-3 to approximately 50%. These findings indicate that wild-type USP2-3 is mostly located in the cytosol, where it can interact with its various substrates, but a part of the expressed protein is transported into peroxisomes by the PTS1 receptor PEX5

Summary

Introduction

Dynamic ubiquitination is involved in the regulation of many cellular processes such as cell proliferation, differentiation and apoptosis. Ubiquitin (Ub), a protein of 76 amino acids, is covalently attached to target proteins by E3 Ub-ligases. Poly-ubiquitination leads to degradation of these proteins by the Ubiquitin-Proteasome pathway [1]. Removal of the ubiquitin moiety by specific hydrolyzing enzymes called DUBs (deubiquitinating enzymes) increases the steady-state level of the target protein and stimulates the activity of the cellular pathway. In which the ubiquitinated protein is involved. Many USPs have a regulatory function in cell death or growth signaling cascades and are involved in the pathogenesis of human diseases [2]

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