Molecular Mechanism of a Temperature-Sensitive Phenotype in Peroxisomal Biogenesis Disorder

ATP(CTP) : tRNA nucleotidyltransferase catalyses the posttranscriptional addition of cytidine, cytidine and adenosine to the 3 ends of tRNAs. Previously, a temperaturesensitive phenotype in Saccharomyces cerevisiae resulting from a mutation in the CCA1 gene coding for this enzyme was identified. Here, we show that a single guanineto-adenine transition in cca1-1 generates the temperature-sensitive phenotype. Alignment of the amino acid sequence of S. cerevisiae tRNA nucleotidyltransferase with other tRNA nucleotidyltransferases for which crystal structures have been solved suggests that the resulting Glu-to-Lys substitution is in a ß-turn connecting the structurally and functionally important head and neck domains of the protein. Proteins containing Gln, His or Phe at this position were constructed to further characterize the importance of this residue in enzyme structure and function. As with the Lys variant, the Phe and His variants generate a temperature-sensitive phenotype in isogenic yeast strains, further supporting the role of this position in maintaining the structure and function of this enzyme. Comparative biophysical and biochemical characterization of both the wild-type and variant proteins indicates that amino acid substitutions at this position can result in a structural change in the protein that reduces enzyme activity (both at the permissive and non-permissive temperatures), decreases the melting temperature of the protein and alters its stability at the non-permissive temperature (37 • C).

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“…This is consistent with what has been observed for other proteins generating ts phenotypes (see e.g. Jona et al, 2002;Hashimoto et al, 2005).…”

Section: Resultssupporting

confidence: 92%

Characterization of a temperature‐sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase

Shan

Russell

Paul

et al. 2008

Yeast

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“…In the human population, temperature-sensitive mutations of hemoglobin, antithrombin Rouen-VI, xerodermal pigmentosum group D helicase subunit of transcription factor II H (TFIIH) and SH3 domain of peroxin (PEX13) protein with an associated feverish crisis have been reported sporadically [Zinkham et al, 1979;Bruce et al, 1994;Vermeulen et al, 2001;Hashimoto et al, 2005]. In the present study, we found decreased thermal stability of CPT II variants in IAE patients with the number of cases more than 100 children annually in Japan [Yokota et al, 2000;Morishima et al, 2002].…”

Section: Discussionsupporting

confidence: 56%

Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy

et al. 2008

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Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylcarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of carnitine palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid beta-oxidation decreased to 30-59% and intracellular ATP levels to 48-79%, and a marked reduction of mitochondrial membrane potential at 41 degrees C, compared with control cells transfected with WT at 37 degrees C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE.

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“…The subcellular localization (diffuse cytosolic or punctate peroxisomal) was visualized with fluorescence microscopy studies (Mandel et al 1994;Roels et al 2003). The second is temperature sensitivity, the restoration of morphological peroxisome formation and peroxisomal biochemical function in PBD fibroblasts cultured at 30°C rather than 37°C (Hashimoto et al 2005;Imamura et al 1998;Osumi et al 2000). It has been suggested that temperature sensitivity is a consequence of an imbalance of folding and unfolding kinetics of mutant proteins, resulting in a reduction of correctly folded protein as temperature increases (Hashimoto et al 2005).…”

Section: Discussionmentioning

confidence: 99%

A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 °C

et al. 2007

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Mutations in 12 different PEX genes can cause a generalized peroxisomal biogenesis disorder with clinical phenotypes ranging from Zellweger syndrome to infantile Refsum disease. To identify the specific PEX gene to be sequenced, complementation analysis is first performed in fibroblasts using catalase immunofluorescence. A patient with a relatively mild phenotype of infantile cholestasis, hypotonia and motor delay had elevated plasma very longchain fatty acids and bile acid precursors, but fibroblast studies revealed normal or only mildly abnormal peroxisomal parameters and mosaic catalase immunofluorescence. This mosaicism persisted even when the incubation temperature was increased from 37°C to 40°C, a maneuver previously shown to abolish mosaicism by exacerbating peroxisomal dysfunction. As mosaicism precludes complementation analysis, a candidate gene approach was employed. After PEX1 sequencing was unrewarding, PEX12 sequencing revealed homozygosity for a novel c.102A>T (p.R34S) missense mutation affecting a partially conserved residue in the N-terminal region important for localization to peroxisomes. Transfection of patient fibroblasts with wild-type PEX12 cDNA confirmed that a PEX12 defect was the basis for the PBD. Homozygosity for c.102A>T was identified in a second patient of similar ethnic origin also presenting with a mild phenotype. PEX12 is a highly probable candidate gene for direct sequencing in the context of a mild clinical phenotype with mosaicism and minimally abnormal peroxisomal parameters in fibroblasts.

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Molecular Mechanism of a Temperature-Sensitive Phenotype in Peroxisomal Biogenesis Disorder

Cited by 15 publications

References 29 publications

Characterization of a temperature‐sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase

Characterization of a temperature‐sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase

Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy

A novel PEX12 mutation identified as the cause of a peroxisomal biogenesis disorder with mild clinical phenotype, mild biochemical abnormalities in fibroblasts and a mosaic catalase immunofluorescence pattern, even at 40 °C

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