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The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.

Parfitt DA, Michael GJ, Vermeulen EG, Prodromou NV, Webb TR, Gallo JM, Cheetham ME, Nicoll WS, Blatch GL, Chapple JP - Hum. Mol. Genet. (2009)

Bottom Line: Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional.We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1.Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK.

ABSTRACT
An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

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Sacsin has a predominantly cytoplasmic localization with partial mitochondrial overlap. Immunofluorescent detection of sacsin in SH-SY5Y cells using r-sacsin1–17 (A, B) and r-sacsin4489–4503 (C, D). The antibodies were competed with immunizing peptide in (B and D). (E–G) Sacsin distribution (red) partially overlapped with a fluorescent mitochondria marker (green). Cells were imaged by laser scanning confocal microscopy. Microscope settings were constant for acquisition in the peptide competition experiments. Scale bar = 10 µm.
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DDP067F2: Sacsin has a predominantly cytoplasmic localization with partial mitochondrial overlap. Immunofluorescent detection of sacsin in SH-SY5Y cells using r-sacsin1–17 (A, B) and r-sacsin4489–4503 (C, D). The antibodies were competed with immunizing peptide in (B and D). (E–G) Sacsin distribution (red) partially overlapped with a fluorescent mitochondria marker (green). Cells were imaged by laser scanning confocal microscopy. Microscope settings were constant for acquisition in the peptide competition experiments. Scale bar = 10 µm.

Mentions: SACS is expressed in the human neuroblastoma derived cell line SH-SY5Y (Supplementary Material, Fig. S5). We examined the subcellular localization of endogenous sacsin in SH-SY5Y cells by staining with r-sacsin1–17 and r-sacsin4489–4503. Both N- and C-terminal sacsin antibodies produced the same staining pattern (Fig. 2A–D). The protein had a predominantly cytoplasmic localization with some punctate staining also visible. A small proportion of sacsin immunoreactivity appeared nuclear (Supplementary Material, Fig. S3). Interestingly, bioinformatics analysis revealed that sacsin contains multiple putative nuclear localization signals (Supplementary Material, Table S1). To further investigate the subcellular distribution of sacsin we tested for co-localization between sacsin and specific organelle markers. Sacsin immunoreactivity partially overlapped with the distribution of MitoTracker (Fig. 2E–G) a mitochondrial fluorescent probe, and the mitochondrial protein Hsp60 (Supplementary Material, Fig. S3). The overlap between sacsin localization and MitoTracker was quantified using MetaMorph 7.5 image analysis software. About 30% ± 3% of sacsin staining showed co-localization with MitoTracker fluorescence. Significantly, 40% ± 2% of MitoTracker staining did not overlap with sacsin immunoreactivity (similar values were obtained for the overlap between sacsin and Hsp60 immunoreactivity). Analyses of the sacsin protein sequence did not reveal the presence of a mitochondrial leader sequence. Together, these observations suggest that a proportion of sacsin localizes at or near the cytoplasmic face of the mitochondria.


The ataxia protein sacsin is a functional co-chaperone that protects against polyglutamine-expanded ataxin-1.

Parfitt DA, Michael GJ, Vermeulen EG, Prodromou NV, Webb TR, Gallo JM, Cheetham ME, Nicoll WS, Blatch GL, Chapple JP - Hum. Mol. Genet. (2009)

Sacsin has a predominantly cytoplasmic localization with partial mitochondrial overlap. Immunofluorescent detection of sacsin in SH-SY5Y cells using r-sacsin1–17 (A, B) and r-sacsin4489–4503 (C, D). The antibodies were competed with immunizing peptide in (B and D). (E–G) Sacsin distribution (red) partially overlapped with a fluorescent mitochondria marker (green). Cells were imaged by laser scanning confocal microscopy. Microscope settings were constant for acquisition in the peptide competition experiments. Scale bar = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667285&req=5

DDP067F2: Sacsin has a predominantly cytoplasmic localization with partial mitochondrial overlap. Immunofluorescent detection of sacsin in SH-SY5Y cells using r-sacsin1–17 (A, B) and r-sacsin4489–4503 (C, D). The antibodies were competed with immunizing peptide in (B and D). (E–G) Sacsin distribution (red) partially overlapped with a fluorescent mitochondria marker (green). Cells were imaged by laser scanning confocal microscopy. Microscope settings were constant for acquisition in the peptide competition experiments. Scale bar = 10 µm.
Mentions: SACS is expressed in the human neuroblastoma derived cell line SH-SY5Y (Supplementary Material, Fig. S5). We examined the subcellular localization of endogenous sacsin in SH-SY5Y cells by staining with r-sacsin1–17 and r-sacsin4489–4503. Both N- and C-terminal sacsin antibodies produced the same staining pattern (Fig. 2A–D). The protein had a predominantly cytoplasmic localization with some punctate staining also visible. A small proportion of sacsin immunoreactivity appeared nuclear (Supplementary Material, Fig. S3). Interestingly, bioinformatics analysis revealed that sacsin contains multiple putative nuclear localization signals (Supplementary Material, Table S1). To further investigate the subcellular distribution of sacsin we tested for co-localization between sacsin and specific organelle markers. Sacsin immunoreactivity partially overlapped with the distribution of MitoTracker (Fig. 2E–G) a mitochondrial fluorescent probe, and the mitochondrial protein Hsp60 (Supplementary Material, Fig. S3). The overlap between sacsin localization and MitoTracker was quantified using MetaMorph 7.5 image analysis software. About 30% ± 3% of sacsin staining showed co-localization with MitoTracker fluorescence. Significantly, 40% ± 2% of MitoTracker staining did not overlap with sacsin immunoreactivity (similar values were obtained for the overlap between sacsin and Hsp60 immunoreactivity). Analyses of the sacsin protein sequence did not reveal the presence of a mitochondrial leader sequence. Together, these observations suggest that a proportion of sacsin localizes at or near the cytoplasmic face of the mitochondria.

Bottom Line: Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional.We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1.Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1.

View Article: PubMed Central - PubMed

Affiliation: William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, UK.

ABSTRACT
An extensive protein-protein interaction network has been identified between proteins implicated in inherited ataxias. The protein sacsin, which is mutated in the early-onset neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay, is a node in this interactome. Here, we have established the neuronal expression of sacsin and functionally characterized domains of the 4579 amino acid protein. Sacsin is most highly expressed in large neurons, particularly within brain motor systems, including cerebellar Purkinje cells. Its subcellular localization in SH-SY5Y neuroblastoma cells was predominantly cytoplasmic with a mitochondrial component. We identified a putative ubiquitin-like (UbL) domain at the N-terminus of sacsin and demonstrated an interaction with the proteasome. Furthermore, sacsin contains a predicted J-domain, the defining feature of DnaJ/Hsp40 proteins. Using a bacterial complementation assay, the sacsin J-domain was demonstrated to be functional. The presence of both UbL and J-domains in sacsin suggests that it may integrate the ubiquitin-proteasome system and Hsp70 function to a specific cellular role. The Hsp70 chaperone machinery is an important component of the cellular response towards aggregation prone mutant proteins that are associated with neurodegenerative diseases. We therefore investigated the effects of siRNA-mediated sacsin knockdown on polyglutamine-expanded ataxin-1. Importantly, SACS siRNA did not affect cell viability with GFP-ataxin-1[30Q], but enhanced the toxicity of GFP-ataxin-1[82Q], suggesting that sacsin is protective against mutant ataxin-1. Thus, sacsin is an ataxia protein and a regulator of the Hsp70 chaperone machinery that is implicated in the processing of other ataxia-linked proteins.

Show MeSH
Related in: MedlinePlus