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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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The sacsin J-domain is functional. (A) Structure-aided alignment of the sacsin J-domain to four other J-domains of known structure and the HSJ1 J-domain. Comparison with a consensus of highly conserved residues derived from over 200 J-domain sequences is also shown. The J domains in the alignment are derived from Homo sapiens Sacsin, H. sapiens HSJ1, Escherichia coli DnaJ, H. sapiens HDJ1, the murine polyoma virus T antigen (TAg) and E. coli Hsc20. Those residues conserved in greater than 70% of all known J domains are indicated below the alignment as a consensus (Con). The position of each sequence is indicated at the start of each sequence. (B) Secondary structure of the sacsin J-domain (PDB code 1IUR) compared with the E. coli J-domain (PDB code 1XBL). The ribbon representations of the structures were rendered using PyMol. (C) The J-domain of sacsin was found to be functional using an in vivo complementation assay. Agt DnaJ (Agt) and Agt DnaJ with its J-domain swapped for that of sacsin (Sacsin) were able to replace the lack of chromosomally expressed DnaJ and CbpA in E. coli OD259 allowing growth at 37°C. Agt J-domain (H33Q) (Agt(H33Q)) and the Agt DnaJ-sacsin J-domain chimera with the H33Q substitution (Sacsin(H33Q)) were unable to replace DnaJ and CbpA in E. coli OD259, resulting in a lack of growth at 37°C. (D) Expression of Agt DnaJ and its chimeric derivatives was confirmed by western blot analysis.
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DDP067F4: The sacsin J-domain is functional. (A) Structure-aided alignment of the sacsin J-domain to four other J-domains of known structure and the HSJ1 J-domain. Comparison with a consensus of highly conserved residues derived from over 200 J-domain sequences is also shown. The J domains in the alignment are derived from Homo sapiens Sacsin, H. sapiens HSJ1, Escherichia coli DnaJ, H. sapiens HDJ1, the murine polyoma virus T antigen (TAg) and E. coli Hsc20. Those residues conserved in greater than 70% of all known J domains are indicated below the alignment as a consensus (Con). The position of each sequence is indicated at the start of each sequence. (B) Secondary structure of the sacsin J-domain (PDB code 1IUR) compared with the E. coli J-domain (PDB code 1XBL). The ribbon representations of the structures were rendered using PyMol. (C) The J-domain of sacsin was found to be functional using an in vivo complementation assay. Agt DnaJ (Agt) and Agt DnaJ with its J-domain swapped for that of sacsin (Sacsin) were able to replace the lack of chromosomally expressed DnaJ and CbpA in E. coli OD259 allowing growth at 37°C. Agt J-domain (H33Q) (Agt(H33Q)) and the Agt DnaJ-sacsin J-domain chimera with the H33Q substitution (Sacsin(H33Q)) were unable to replace DnaJ and CbpA in E. coli OD259, resulting in a lack of growth at 37°C. (D) Expression of Agt DnaJ and its chimeric derivatives was confirmed by western blot analysis.

Mentions: Sacsin was suggested to be a type III Hsp40 family protein based upon the presence of a putative J-domain near the C-terminus of its predicted amino acid sequence. Alignment of the human sacsin sequence with that of Hsp40/HDJ1 revealed approximately 60% identity over 30 residues (Fig. 4A) exhibiting more divergence than most mammalian J-domains. The structure of several J-domains has revealed the presence of four α-helices and a loop region between helices II and III (9), which contains the highly conserved histidine-proline-aspartic acid (HPD) motif. To further investigate if the sacsin J-domain was likely to be functional, we compared its structure (PDB:1IUR) to the structure of the Escherichia coli DnaJ J-domain (PDB: 1XBL) (15) (Fig. 4B). Helix II of the Sacsin J-domain is similar in size and structure to that of the J-domain of E. coli DnaJ with the histidine residue of the sacsin HPD motif projecting into the J-domain core in a comparable orientation (Fig. 4B). To test if the sacsin J-domain was able to function in the Hsp70 chaperone system, we utilized a bacterial in vivo complementation assay. E. coli strain OD259 contains disrupted genes for DnaJ and CbpA such that it will not grow at temperatures of 37°C and above (16) (Fig. 4C). Complementation with Agrobacterium tumefaciens (Agt) DnaJ and chimeric proteins containing J-domains from a wide range of organisms have been shown to compensate for the lack of chromosomal-encoded E. coli DnaJ and CbpA allowing growth at ≥37°C (17,18). To analyse the effect of sacsin's J-domain in this experimental system, the J-domain of Agt DnaJ was substituted with the J-domain of human sacsin to make a chimeric protein. The Agt DnaJ-sacsin was able to confer growth at ≥37°C (Fig. 4C). To confirm that the complementation was dependent on a DnaK (E. coli Hsp70)/sacsin J-domain interaction, the histidine residue in the HPD motif was mutated to glutamine. The modified Agt DnaJ-sacsin chimeric protein was unable to reverse the thermosensitivity of E. coli OD259 (Fig. 4C), consistent with previous studies that have also shown that the histidine to glutamine substitution in the HPD motif disrupted the ability of J-domains to functionally interact with Hsp70 (17). Western blot analysis confirmed that the chimeric proteins were produced at similar levels (Fig. 4D).


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)

The sacsin J-domain is functional. (A) Structure-aided alignment of the sacsin J-domain to four other J-domains of known structure and the HSJ1 J-domain. Comparison with a consensus of highly conserved residues derived from over 200 J-domain sequences is also shown. The J domains in the alignment are derived from Homo sapiens Sacsin, H. sapiens HSJ1, Escherichia coli DnaJ, H. sapiens HDJ1, the murine polyoma virus T antigen (TAg) and E. coli Hsc20. Those residues conserved in greater than 70% of all known J domains are indicated below the alignment as a consensus (Con). The position of each sequence is indicated at the start of each sequence. (B) Secondary structure of the sacsin J-domain (PDB code 1IUR) compared with the E. coli J-domain (PDB code 1XBL). The ribbon representations of the structures were rendered using PyMol. (C) The J-domain of sacsin was found to be functional using an in vivo complementation assay. Agt DnaJ (Agt) and Agt DnaJ with its J-domain swapped for that of sacsin (Sacsin) were able to replace the lack of chromosomally expressed DnaJ and CbpA in E. coli OD259 allowing growth at 37°C. Agt J-domain (H33Q) (Agt(H33Q)) and the Agt DnaJ-sacsin J-domain chimera with the H33Q substitution (Sacsin(H33Q)) were unable to replace DnaJ and CbpA in E. coli OD259, resulting in a lack of growth at 37°C. (D) Expression of Agt DnaJ and its chimeric derivatives was confirmed by western blot analysis.
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Related In: Results  -  Collection

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

DDP067F4: The sacsin J-domain is functional. (A) Structure-aided alignment of the sacsin J-domain to four other J-domains of known structure and the HSJ1 J-domain. Comparison with a consensus of highly conserved residues derived from over 200 J-domain sequences is also shown. The J domains in the alignment are derived from Homo sapiens Sacsin, H. sapiens HSJ1, Escherichia coli DnaJ, H. sapiens HDJ1, the murine polyoma virus T antigen (TAg) and E. coli Hsc20. Those residues conserved in greater than 70% of all known J domains are indicated below the alignment as a consensus (Con). The position of each sequence is indicated at the start of each sequence. (B) Secondary structure of the sacsin J-domain (PDB code 1IUR) compared with the E. coli J-domain (PDB code 1XBL). The ribbon representations of the structures were rendered using PyMol. (C) The J-domain of sacsin was found to be functional using an in vivo complementation assay. Agt DnaJ (Agt) and Agt DnaJ with its J-domain swapped for that of sacsin (Sacsin) were able to replace the lack of chromosomally expressed DnaJ and CbpA in E. coli OD259 allowing growth at 37°C. Agt J-domain (H33Q) (Agt(H33Q)) and the Agt DnaJ-sacsin J-domain chimera with the H33Q substitution (Sacsin(H33Q)) were unable to replace DnaJ and CbpA in E. coli OD259, resulting in a lack of growth at 37°C. (D) Expression of Agt DnaJ and its chimeric derivatives was confirmed by western blot analysis.
Mentions: Sacsin was suggested to be a type III Hsp40 family protein based upon the presence of a putative J-domain near the C-terminus of its predicted amino acid sequence. Alignment of the human sacsin sequence with that of Hsp40/HDJ1 revealed approximately 60% identity over 30 residues (Fig. 4A) exhibiting more divergence than most mammalian J-domains. The structure of several J-domains has revealed the presence of four α-helices and a loop region between helices II and III (9), which contains the highly conserved histidine-proline-aspartic acid (HPD) motif. To further investigate if the sacsin J-domain was likely to be functional, we compared its structure (PDB:1IUR) to the structure of the Escherichia coli DnaJ J-domain (PDB: 1XBL) (15) (Fig. 4B). Helix II of the Sacsin J-domain is similar in size and structure to that of the J-domain of E. coli DnaJ with the histidine residue of the sacsin HPD motif projecting into the J-domain core in a comparable orientation (Fig. 4B). To test if the sacsin J-domain was able to function in the Hsp70 chaperone system, we utilized a bacterial in vivo complementation assay. E. coli strain OD259 contains disrupted genes for DnaJ and CbpA such that it will not grow at temperatures of 37°C and above (16) (Fig. 4C). Complementation with Agrobacterium tumefaciens (Agt) DnaJ and chimeric proteins containing J-domains from a wide range of organisms have been shown to compensate for the lack of chromosomal-encoded E. coli DnaJ and CbpA allowing growth at ≥37°C (17,18). To analyse the effect of sacsin's J-domain in this experimental system, the J-domain of Agt DnaJ was substituted with the J-domain of human sacsin to make a chimeric protein. The Agt DnaJ-sacsin was able to confer growth at ≥37°C (Fig. 4C). To confirm that the complementation was dependent on a DnaK (E. coli Hsp70)/sacsin J-domain interaction, the histidine residue in the HPD motif was mutated to glutamine. The modified Agt DnaJ-sacsin chimeric protein was unable to reverse the thermosensitivity of E. coli OD259 (Fig. 4C), consistent with previous studies that have also shown that the histidine to glutamine substitution in the HPD motif disrupted the ability of J-domains to functionally interact with Hsp70 (17). Western blot analysis confirmed that the chimeric proteins were produced at similar levels (Fig. 4D).

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