A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner

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Abstract

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

Introduction

Polyglutamine (polyQ) diseases are a group of familial neurodegenerative disorders, and nine diseases including Huntington's disease and six types of spinocerebellar ataxia (SCA), have been described for which the common mutation is a CAG trinucleotide expansion [1]. All these disorders, excepting spinal bulbar muscular atrophy, are dominantly inherited, progressive, usually begin in mid-life, and result in severe neuronal dysfunction and neuronal cell death in the selective region of the brain [2]. Misfolding and aggregation of polyQ-containing disease proteins is thought to be critical to pathogenesis. The molecular mechanisms by which the proteins implicated in the polyQ diseases aggregate are still unknown. Recent studies suggest that the age-dependent accumulation of the protein aggregates in neurodegenerative diseases reflects the progressive inability of the cellular quality control machinery to recognize and eliminate potentially toxic misfolded proteins. This raises the possibility that the cellular machinery for protein quality control may play an important role in polyQ disease.

Cellular machinery for protein quality control is constituted by a group of specialized proteins, known as heat shock proteins (HSPs), which promote the folding and assembly of mature protein complexes. There are several numbers of HSP families and they include HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40) and HSPB (small HSP) familes [3]. The most abundant family of HSP consists of the highly conserved 70-kDa heat shock proteins (HSP70), which were shown to be essential in numerous cellular processes. The apparent discrepancy between the broad substrate specificity of HSP70 on one hand and the high specialization of HSP70 function on the other has been explained by the discovery that HSP70 work in concert with partner co-chaperones, DNAJ proteins [4]. DNAJ proteins, as opposed to HSP70, constitute a heterogeneous group of multi-domain proteins defined by the highly conserved J-domain, which are necessary to stimulate ATPase activity of interacting HSP70 [5], [6]. And the heterogeneity of DNAJ proteins defines diversity of functional properties of HSP70, and DNAJ family proteins have broad biological functions [7], [8], [9], [10], [11]. Recent studies reveled that DNAJ proteins have roles in suppression of protein aggregation formation [12], [13], [14].

In the present study, we focused on nuclear DNAJ proteins, since polyQ aggregates are formed inside the nucleus in most polyQ diseases [15], and analyzed a novel DNAJ protein, DNAJC8.

Section snippets

Cell culture and transfection

A neuroblastoma cell line SH-SY5Y and a cervical carcinoma cell line HeLa were purchased from american type culture collection (ATCC; Manassas, VA, USA) and maintained. Plasmid transfection was performed by using Lipofectamine 2000 reagent (Thermo Fisher Scientific) according to the manufacture's protocol. For stable transfection, puromycin (3.5 μg/ml; Sigma–Aldrich, St Louis, MO, USA) was added to culture medium 48 h after transfection. For confocal microscopy, the cells were transferred into

Structure and gene expression of DNAJC8 protein

To identify a protein that is localized in the nucleus and contains J-domain, we searched the human expressed sequence tag (EST) database. We found a large number of proteins that contain amino acid sequences with similarity to J-domain of HSP40 (Caenorhabditis elegans). A DNAJ proteins, DNAJC8 was identified to contain nuclear localization signals [6] (Fig. 1A). DNAJC8 has the canonical J-domain (residue 68–127) including the conserved tripeptide, His-Pro-Asp, and the 50th Ala residue

Discussion

Nine neurodegenerative diseases have been known as polyglutamine diseases, in which certain proteins with polyQ repeat expansion forms aggregation and deposition in neuronal cells. The polyQ aggregates have neurotoxic property, therefore leading to neuronal dysfunction, degeneration and cell death. Though the precise mechanism of the aggregate formation in each polyQ-containing disease protein has not been well defined, polyQ expansion is thought to cause a conformational change that promotes

Declaration of financial disclosure

The authors have no financial conflict of interest.

Acknowledgment

We thank Dr. Takeshi Urano and Dr. Itaru Hirai for helpful discussions. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant no. 15H04722) (to T. T., Y. H. and N. S.) and program for developing the supporting system for upgrading education and research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N. S.). This study was supported in part by Grants-in-Aid

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