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A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

Min W, Angileri F, Luo H, Lauria A, Shanmugasundaram M, Almerico AM, Cappello F, de Macario EC, Lednev IK, Macario AJ, Robb FT - Sci Rep (2014)

Bottom Line: These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog.The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective.Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore; and Institute of Marine and Environmental Technology (IMET); Columbus Center, Baltimore, MD 21201, USA.

ABSTRACT
Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

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Related in: MedlinePlus

Oligomeric states of purified PfCD, I138H, and I138R Cpns determined by gel filtration chromatography (top panel) and corresponding SDS-PAGE analysis (bottom panel).PfCD, or I138H, or I138R purified by gel filtration chromatography followed by anion exchange chromatography were pooled together for ATPase activity and MDH heat-protection activity assays. The same amounts of these pooled pure Cpn proteins were subjected to 4–9% gradient native-PAGE analysis (top panel) and 12% SDS-PAGE analysis (bottom panel) and then stained with Coomassie brilliant blue G-250. The figures to the right, 16 on top and 1 at the bottom, indicate hexadecamer and monomer, respectively, while the other oligomers are distributed in between them as shown by the gel bands. The thick arrow at the very bottom indicates the monomeric band of the purified Cpns run with same amount of protein in SDS-PAGE. St, molecular weight standard.
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f4: Oligomeric states of purified PfCD, I138H, and I138R Cpns determined by gel filtration chromatography (top panel) and corresponding SDS-PAGE analysis (bottom panel).PfCD, or I138H, or I138R purified by gel filtration chromatography followed by anion exchange chromatography were pooled together for ATPase activity and MDH heat-protection activity assays. The same amounts of these pooled pure Cpn proteins were subjected to 4–9% gradient native-PAGE analysis (top panel) and 12% SDS-PAGE analysis (bottom panel) and then stained with Coomassie brilliant blue G-250. The figures to the right, 16 on top and 1 at the bottom, indicate hexadecamer and monomer, respectively, while the other oligomers are distributed in between them as shown by the gel bands. The thick arrow at the very bottom indicates the monomeric band of the purified Cpns run with same amount of protein in SDS-PAGE. St, molecular weight standard.

Mentions: Fig. 4 shows the wild type, I138H and I138R complexes separated on a 4–9% gradient native-PAGE gel. Group II Cpns typically form double-ring hexadecamers, the quaternary structures of group II Cpn, consisting of a double toroid cylinder assembled into two rings of subunits stacked back-to-back. This conformation is optimal for promoting correct folding of non-native cellular proteins22. Whereas there was no clear difference in molar concentration ratios of hexadecamer between PfCD and I138H, a relatively lower concentration of double-ringed Cpn was observed in the case of I138R (Fig. 4, top panel). Densitometric analysis revealed that I138 formed double ring approximately 4–6 fold less than PfCD or I138H, indicating a functional deficit in maintaining the double ring structure (results not shown).


A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model.

Min W, Angileri F, Luo H, Lauria A, Shanmugasundaram M, Almerico AM, Cappello F, de Macario EC, Lednev IK, Macario AJ, Robb FT - Sci Rep (2014)

Oligomeric states of purified PfCD, I138H, and I138R Cpns determined by gel filtration chromatography (top panel) and corresponding SDS-PAGE analysis (bottom panel).PfCD, or I138H, or I138R purified by gel filtration chromatography followed by anion exchange chromatography were pooled together for ATPase activity and MDH heat-protection activity assays. The same amounts of these pooled pure Cpn proteins were subjected to 4–9% gradient native-PAGE analysis (top panel) and 12% SDS-PAGE analysis (bottom panel) and then stained with Coomassie brilliant blue G-250. The figures to the right, 16 on top and 1 at the bottom, indicate hexadecamer and monomer, respectively, while the other oligomers are distributed in between them as shown by the gel bands. The thick arrow at the very bottom indicates the monomeric band of the purified Cpns run with same amount of protein in SDS-PAGE. St, molecular weight standard.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4209464&req=5

f4: Oligomeric states of purified PfCD, I138H, and I138R Cpns determined by gel filtration chromatography (top panel) and corresponding SDS-PAGE analysis (bottom panel).PfCD, or I138H, or I138R purified by gel filtration chromatography followed by anion exchange chromatography were pooled together for ATPase activity and MDH heat-protection activity assays. The same amounts of these pooled pure Cpn proteins were subjected to 4–9% gradient native-PAGE analysis (top panel) and 12% SDS-PAGE analysis (bottom panel) and then stained with Coomassie brilliant blue G-250. The figures to the right, 16 on top and 1 at the bottom, indicate hexadecamer and monomer, respectively, while the other oligomers are distributed in between them as shown by the gel bands. The thick arrow at the very bottom indicates the monomeric band of the purified Cpns run with same amount of protein in SDS-PAGE. St, molecular weight standard.
Mentions: Fig. 4 shows the wild type, I138H and I138R complexes separated on a 4–9% gradient native-PAGE gel. Group II Cpns typically form double-ring hexadecamers, the quaternary structures of group II Cpn, consisting of a double toroid cylinder assembled into two rings of subunits stacked back-to-back. This conformation is optimal for promoting correct folding of non-native cellular proteins22. Whereas there was no clear difference in molar concentration ratios of hexadecamer between PfCD and I138H, a relatively lower concentration of double-ringed Cpn was observed in the case of I138R (Fig. 4, top panel). Densitometric analysis revealed that I138 formed double ring approximately 4–6 fold less than PfCD or I138H, indicating a functional deficit in maintaining the double ring structure (results not shown).

Bottom Line: These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog.The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective.Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Maryland at Baltimore; and Institute of Marine and Environmental Technology (IMET); Columbus Center, Baltimore, MD 21201, USA.

ABSTRACT
Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

Show MeSH
Related in: MedlinePlus