Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival

Fengjuan Wang,Sylviane Muller,Jean-Baptiste Madinier,Philippe Hammann,Nicolas Schall,Srinivasa Reddy Bonam,Nicolas Page,Jean-Paul Briand,Lauriane Kuhn,Olivier Chaloin

doi:10.1038/s41598-018-34887-6

Abstract

The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.

Highlights

In contrast to cytoplasmic HSP70s that are generated in response to stress, proteins of the HSPA8/HSC70 family are constitutively expressed
We found that one of the serious consequences of this defective relocation of HSPA8 into the cytoplasm was the inability of heat-shocked cells to survive after a second type of aggression, e.g. a mild oxidative stress that virtually mimics a state of inflammation
Crosslinking experiments followed by proteomics studies allowed us to demonstrate that these effects of P140 likely result from its binding to regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure

Summary

Introduction

In contrast to cytoplasmic HSP70s that are generated in response to stress, proteins of the HSPA8/HSC70 family are constitutively expressed They exhibit pleiotropic properties and are crucial for cell survival[1,2]. When the physiological situation returns to normal (recovery phase from stress), HSPA8 proteins are released from nuclear and nucleolar anchors and its shuttling to cytoplasm is restored. The duration of this process (in the range of several hours) varies according to the type of cells and stress. We found that according to the type of injury that was applied to cells (HS or oxidative stress), the cytoplasmic-nuclear shuttling capacities of HSPA8 were differently regulated. Crosslinking experiments followed by proteomics studies allowed us to demonstrate that these effects of P140 likely result from its binding to regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure

Methods

Results

Conclusion