Comparative biochemical studies of ATPases in cells from patients with the T8993G or T8993C mitochondrial DNA mutations

Vázquez-Memije, Martha Elisa; Shanske, Sara; Santorelli, Filippo M.; Kranz-Eble, Pamela; DeVivo, Darryl C.; DiMauro, Salvatore

doi:10.1023/a:1005418718299

Cited by 42 publications

(32 citation statements)

References 25 publications

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“…However, the L156R mutant had a trend to show a higher oligomycin sensitivity that is more evident at oligomycin amounts around 0.02 g/mg protein. This tendency is in consonance with the higher oligomycin sensitivity reported previously for ATP synthesis in fibroblasts with the same mutations (8). A similar trend was also observed with mitochondria isolated from human fibroblasts harboring the L156R/L156P mutations (not shown).…”

Section: T8993g Mutationsupporting

confidence: 91%

“…A similar inhibition is observed when these mutations are modeled in Escherichia coli (13)(14)(15). The substitution of Leu-156 by arginine (mutation T8993G) is more deleterious to ATP synthesis than its substitution by proline (mutation T8993C) (60 versus 25% inhibition in human mitochondria) (6,8) and therefore leads to more severe NARP/MILS manifestations (16 -19). In addition, mitochondria bearing the T8993G mutation are hyperpolarized (9,20) and have an increased production of reactive oxygen species (20).…”

mentioning

confidence: 71%

“…Submitochondrial particles (EDTA-SMP) were prepared by sonication of isolated mitochondria as in Ref. 8. F 1 F 0 immunoprecipitation was carried out from 3 mg of human mitochondria solubilized with n-dodecyl-␤-D-maltoside (Sigma), as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively (see Fig. 5A), the phosphorylation of ADP by inorganic phosphate ( 32 P i ) was used to determine ATP synthesis (8). ATP hydrolysis rates were measured spectrophotometrically with mitochondrial membranes obtained by osmotic shock, using an ATP-regenerating system, as described before (9).…”

Section: Methodsmentioning

confidence: 99%

“…To verify IF 1 release, membranes were pelleted by centrifugation at 15,000 ϫ g, and supernatants were assayed by Western blot for the presence of IF 1 . ATP hydrolysis carried out by SMP was measured by the colorimetric determination of P i released from ATP, as reported (8).…”

Section: Methodsmentioning

confidence: 99%

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ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

Cortés-Hernández

Vázquez-Memije

Garcia

2007

Journal of Biological Chemistry

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The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F 1 F 0 -ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F 1 F 0 -ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F 1 and F 0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F 1 F 0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F 1 F 0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.

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Section: T8993g Mutationsupporting

confidence: 91%

mentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

Cortés-Hernández

Vázquez-Memije

Garcia

2007

Journal of Biological Chemistry

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Biochemical-Clinical Correlation in Patients With Different Loads of the Mitochondrial DNA T8993G Mutation

Carelli¹,

Baracca²,

Barogi³

et al. 2002

Arch Neurol

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This study indicates a close relationship between tissue heteroplasmy, expression of the biochemical defect in platelets, and clinical involvement. The biochemical defect was greater than previously reported, and we found no evidence of a biochemical threshold. The uniform distribution of high mutant loads among our patients' tissues suggests a differential tissue-specific reliance on mitochondrial ATP synthesis.

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Long‐term outcome of Leigh syndrome caused by the NARP‐T8993C mtDNA mutation

Debray

Lambert

Lortie

et al. 2007

American J of Med Genetics Pt A

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Mutations at mitochondrial DNA (mtDNA) nucleotide 8993 can cause neurogenic weakness, ataxia and retinitis pigmentosa (NARP syndrome), or maternally inherited Leigh syndrome (LS), with a correlation between the amount of mutant mtDNA and the severity of the neurological disease. The T8993C mutation is generally considered to be clinically milder than the T8993G mutation but when the level of heteroplasmy exceeds 90%, progressive neurodegeneration has been found. We report on a long-term follow-up of a patient who presented at 4 years of age with typical LS but showed an unexpected resolution of his symptoms and a favorable outcome. At 18 years of age, his neurological examination was near normal, with neither peripheral neuropathy nor retinopathy. mtDNA analysis identified the presence of T8993C mutation at high level (>95%) in the patient's blood leukocytes. This case report and literature review emphasizes the variability of the phenotypic expression of the T8993C mutation and the need for caution in predictive counseling in such patients. (c) 2007 Wiley-Liss, Inc.

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Comparative biochemical studies of ATPases in cells from patients with the T8993G or T8993C mitochondrial DNA mutations

Cited by 42 publications

References 25 publications

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

ATP6 Homoplasmic Mutations Inhibit and Destabilize the Human F1F0-ATP Synthase without Preventing Enzyme Assembly and Oligomerization

Biochemical-Clinical Correlation in Patients With Different Loads of the Mitochondrial DNA T8993G Mutation

Long‐term outcome of Leigh syndrome caused by the NARP‐T8993C mtDNA mutation

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