Mitochondrial heat shock protein 70 HspA9 participates in the quality control of a model nuclear misfolded protein

Abstract

To maintain protein homeostasis (proteostasis), cells have evolved a highly sophisticated protein quality control system for the handling of misfolded, mislocalized, as well as other aberrant proteins. Depending on their folding states and localisation, aberrant proteins may be subjected to refolding, degradation, or active deposition into defined aggregation sites. Previous research in this area has been mostly focusing on the handling of misfolded proteins in the cytosol or endoplasmic reticulum. However, their handling in the nucleus also deserves similar attention, as it is where the genetic materials are kept and some essential gene expression processes such as transcription, splicing, and ribosome biogenesis are taking place. Many transcription and splicing factors contain intrinsically disordered regions causing them to be conformationally unstable, and therefore an efficient nuclear protein quality control system may be required to maintain their fully functional three-dimensional structure. Moreover, it has been recently shown that cytosolic misfolded proteins are actively transported into the nucleus for degradation and aggregate deposition, and that excess misfolded proteins can be stored at the nucleolus in a refolding-competent state, suggesting that the nucleus may be the major site for the execution and coordination of different protein quality control strategies. In fact, some neurodegenerative diseases, such as Huntington's disease, are characterised by the formation of intra-nuclear aggregates, which implies a causal relationship between failure of nuclear protein quality control and proteopathies.

In this study, we used the human HEK293T cell line to perform a quantitative proteomic identification of the interactors of a model nuclear misfolded protein, which may represent potential nuclear protein quality control factors. The model protein used was the nuclear localisation signal (NLS)-tagged wild-type firefly luciferase with C-terminal GFP (NLS-WT-Fluc-GFP), which showed increased misfolding during heat shock. The protein was shown to be efficiently targeted to the nucleus by the NLS. Upon immunoprecipitation and quantitative mass spectrometry, we identified 755 proteins that showed enriched binding to NLS-WT-Fluc-GFP during heat shock as compared to the control without heat shock. Over 50% of these proteins were nuclear proteins, which had diverse cellular functions ranging from unfolded protein binding, ubiquitin protein ligase binding, to DNA and RNA metabolism as revealed by GO term analysis. Among these, we have selected 4 potential candidates for further characterisation, including the chaperones HspA8 and HspA9, the sentrin-specific protease SENP3, as well as the melanoma-associated antigen MAGED4 that may enhance the activity of some E3 ligases.

After validation by co-immunoprecipitation, mitochondrial heat shock protein 70 — HspA9 was selected, on the basis of its relatively strong binding strength to the model misfolded protein, for further characterisation on its roles in nuclear protein quality control. Knocking down HspA9 was shown to cause faster unfolding of NLS-WT-Fluc-GFP during heat shock and slower refolding during recovery. The result indicated that HspA9 may be involved in nuclear protein quality control by preventing misfolding of correctly folded proteins and facilitating refolding of already misfolded proteins.