Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado–Joseph disease gene product with an expanded polyglutamine stretch
Introduction
Machado–Joseph disease (MJD) is an inherited neurodegenerative disorder caused by the CAG expansion in the coding region of the MJD1 gene (Kawaguchi et al., 1994). The normal gene product ataxin-3 is reported to be localized in the cytoplasm and nucleus (Paulson et al., 1997a, Tait et al., 1998). The pathological variant of ataxin-3 possessing an expanded polyglutamine stretch causes nuclear inclusions and cell death in vivo and in vitro (Evert et al., 1999, Ikeda et al., 1996, Jackson et al., 1998, Paulson et al., 1997b, Warrick et al., 1998, Yoshizawa et al., 2000). Misfolding of the protein fragment containing an expanded polyglutamine stretch is considered significant in producing aggregates and cell death (Kakizuka, 1998, Paulson, 1999, Perutz, 1999). The inhibition of protein misfolding is thus a hypothetical target for effective therapy in polyglutamine diseases. The overexpression of chaperone proteins, which recognizes misfolded proteins and suppresses protein aggregation, has been shown to reduce the rate of cell death in cultured cells and transgenic animals (Chai et al., 1999, Chan et al., 2000, Cummings et al., 1998, Kazemi-Esfarjani and Benzer, 2000, Kobayashi et al., 2000, Stenoien et al., 1999, Warrick et al., 1999). We previously studied the effects of low molecular weight compounds, such as the organic solvent dimethyl sulfoxide (DMSO), cellular osmolytes glycerol, and trimethylamine N-oxide (TMAO), which stabilize proteins in their native conformation, on aggregate formation and cell death induced by the truncated ataxin-3 with an expanded polyglutamine stretch in vitro (Yoshida et al., 2002). These reagents effectively suppressed aggregate formation and cell death, suggesting a rationale for the inhibition of protein misfolding. Our findings and those of others prompted us to explore the effects of other molecules that potentially influence the protein folding in polyglutamine-induced toxicity.
One such candidate is 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine, or ectoine. Ectoine, originally identified in halophilic eubacteria as a compatible solute, is a nonionic organic molecule of low molecular mass that serves as an osmoprotectant (Galinski et al., 1985). Ectoine was shown to be widely distributed among different bacteria and to be useful in preserving enzymatic activity against freeze–thawing, freeze–drying, and heat treatment through protein stabilization (Lippert and Galinski, 1992).
In this study, we examine the effects of several compatible solutes, including ectoine, on mutant ataxin-3 fragment-induced aggregate formation and apoptotic cell death. In visually examining cells expressing truncated ataxin-3 with 77 glutamines (Q77), we noted that ectoine reduced large cytoplasmic inclusions and increased the frequency of nuclear inclusions. Immunoblot analysis demonstrated a reduction in the total amount of aggregates composed of the mutant ataxin-3 fragment. Despite the increased frequency of nuclear inclusions, the integrity of nuclei appeared to be maintained. Judging from nuclear morphology, ectoine decreased apoptotic cell death. Annexin V staining confirmed the result obtained from nuclear morphology. Neither hydroxyectoine nor betaine showed similar effects. These results suggest that ectoine, a widely distributed natural osmoprotectant in bacteria, functions as a novel molecule protecting cells from polyglutamine-induced toxicity.
Section snippets
Reagents and plasmids
Ectoine, hydroxyectoine, betaine, and staurosporin were purchased from Sigma (Sigma, St. Louis, MO, USA). Sucrose was obtained from Wako (Wako, Osaka, Japan). Plasmid encoding N-terminal-truncated ataxin-3 with Q77 together with C-terminal myc and His epitopes was as described elsewhere (Yoshizawa et al., 2000). In this plasmid, 286 amino acid residues of ataxin-3 were deleted from the N-terminal side. The resultant protein was designated ΔN286 (Q77). The GFP tagged-ΔN286 (Q77)-expressing
Ectoine did not demonstrate cytotoxicity up to a concentration of 150 mM
We first studied the toxic effects of ectoine on neuro2a cells. As shown in Fig. 1 (A–H), ectoine did not demonstrate cytotoxicity up to a concentration of 150 mM. Cells in the presence of 150 mM of ectoine were slightly rounder than those without ectoine, but morphological changes were very subtle, so we decided to examine the effect of ectoine up to this concentration.
Ectoin increases inclusion frequency but decreases total aggregates produced by the ataxin-3 fragment with an expanded polyglutamine stretch
We used a plasmid encoding ΔN286(Q77), an ataxin-3 fragment with 77 polyglutamines together with an N-terminal deletion of 286
Discussion
Using the in vitro cellular model for MJD pathology, we demonstrated that ectoine reduced apoptotic features without decreasing inclusion frequency. Total aggregates decreased in immunoblot analysis. We previously reported that chemical chaperones-low molecular weight compounds that stabilize proteins in their native conformation-reduced aggregate formation and cytotoxicity induced by truncated expanded ataxin-3 in vitro (Yoshida et al., 2002). These compounds included the organic solvent
Acknowledgments
We thank Ms. Sumiko Nissato for her technical assistance. This study was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the University of Tsukuba, Japan.
References (30)
- et al.
Inhibition of insulin amyloid formation by small stress molecules
FEBS Lett.
(2004) - et al.
Polyglutamine expansions: proteolysis, chaperones, and the dangers of promiscuity
Neuron
(2000) - et al.
Polyglutamine-expanded human Huntington transgenes induce degeneration of Drosophila photoreceptor neurons
Neuron
(1998) Protein precipitation: a common etiology in neurodegenerative disorders?
Trends Genet.
(1998)- et al.
Chaperones HSP70 and HSP40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract
J. Biol. Chem.
(2000) - et al.
Effect of tetrahydroprimidine derivatives on protein-nucleic acids interaction: type II restriction endonucleases as a model system
J. Biol. Chem.
(1999) Protein fate in neurodegenarative proteinopathies: polyglutamine diseases join the (mis)fold
Am. J. Hum. Genet.
(1999)- et al.
Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3
Neuron
(1997) Glutamine repeats and neurodegenerative diseases: molecular aspects
Trends Biochem. Sci.
(1999)- et al.
Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila
Cell
(1998)
Chemical chaperones reduce aggregate formation and cell death caused by the truncated Machado–Joseph disease gene product with an expanded polyglutamine stretch
Neurobiol. Dis.
Differential susceptibility of cultured cell lines to aggregate formation and cell death produced by the truncated Machado–Joseph disease gene product with an expanded polyglutamine stretch
Brain Res. Bull.
Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase
Biotechnol. Appl. Biochem.
Analysis of the role of heat shock protein (HSP) molecular chaperones in polyglutamine disease
J. Neurosci.
Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila
Hum. Mol. Genet.
Cited by (41)
Strategies for the biological production of ectoine by using different chassis strains
2024, Biotechnology AdvancesTMAO to the rescue of pathogenic protein variants
2022, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :Literature data depicting the effect of various osmolytes in polyQ-mediated pathologies have indicated reduction of aggregate formation or increase in solubilization of the preformed aggregates [92]. On the other hand, osmolytes could also alter subcellular localization of aggregates [93] and enhance mTOR-independent autophagy thereby accelerating the clearance of polyQ aggregates [94]. In a systematic study carried out on huntingtin exon 1 protein with 53Q (Htt53Q) and 20Q (Htt20Q), it was observed that TMAO induces the formation of unstructured, bulky amorphous assemblies which had no cytotoxic effects as compared to the control aggregates [33].
Genome analysis provides insights into crude oil degradation and biosurfactant production by extremely halotolerant Halomonas desertis G11 isolated from Chott El-Djerid salt-lake in Tunisian desert
2019, GenomicsCitation Excerpt :Gaboyer et al. [26] reported that Halomonas lionensis sp. nov., an extremely halotolerant bacterium isolated from a Mediterranean Sea sediment, has developed adaptive strategies, based notably on ectoine accumulation, to overcome extreme environmental conditions. Ectoine possesses additional protective properties, compared with other compatible solutes, and stabilizes even whole cells against stresses such as UV radiation [12,25,39,44]. The ectC gene encoding the key enzyme (ectoine synthase) involved in ectoine biosynthesis seems to be absent in PBN3 genome based on comparative analysis using the SEED Viewer computational tool [63] (Supplementary Fig. S2).
Roles of osmolytes in protein folding and aggregation in cells and their biotechnological applications
2018, International Journal of Biological MacromoleculesCitation Excerpt :Inhibition of aggregation by osmolytes may be due to solubilization of the native state [55] or because of changes in the stabilities of aggregation-prone species or in the aggregation pathway. Ameliorative effects have been associated with osmolyte-induced formation of nontoxic aggregated species [63–65]. The impacts of osmolytes on aggregation can be extremely complex and subtle, for example, trehalose suppresses the toxicity of Aβ40 promotes toxic oligomer formation by Aβ42 [61].
Ectoines in cell stress protection: Uses and biotechnological production
2010, Biotechnology AdvancesCitation Excerpt :Ectoine reduces apoptotic features by reducing the total amount of aggregates and changing its intracellular distribution. Thus, it decreases cytotoxicity increasing frequency of nuclear inclusions (Furusho et al., 2005). Ectoine administration inhibited nanoparticle-induced signaling, which is known to be responsible for proinflammatory reactions in lung epithelial cells (Sydlik et al., 2009) (Table 2).
Metabolic Engineering of Escherichia coli for Ectoine Production With a Fermentation Strategy of Supplementing the Amino Donor
2022, Frontiers in Bioengineering and Biotechnology