Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

Marianne Paulsen; Connie Lund; Zarqa Akram; Jakob R Winther; Nina Horn; Lisbeth Birk Møller

doi:10.1086/505407

Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

Marianne Paulsen, Connie Lund, Zarqa Akram, Jakob R Winther, Nina Horn, Lisbeth Birk Møller

Biomolecular Sciences

47 Citations (Scopus)

Abstract

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.

Original language	English
Journal	American Journal of Human Genetics
Volume	79
Issue number	2
Pages (from-to)	214-29
Number of pages	15
ISSN	0002-9297
DOIs	https://doi.org/10.1086/505407
Publication status	Published - 2006

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@article{6c6a39c0c78d11dd9473000ea68e967b,

title = "Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites",

abstract = "Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.",

author = "Marianne Paulsen and Connie Lund and Zarqa Akram and Winther, {Jakob R} and Nina Horn and M{\o}ller, {Lisbeth Birk}",

note = "Keywords: Adenosine Triphosphatases; Adolescent; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; CHO Cells; Cation Transport Proteins; Cell-Free System; Cells, Cultured; Child; Child, Preschool; Copper; Cricetinae; Fibroblasts; Frameshift Mutation; Humans; Infant; Male; Menkes Kinky Hair Syndrome; Molecular Sequence Data; Peptide Chain Initiation, Translational; Sequence Deletion; Survivors",

year = "2006",

doi = "10.1086/505407",

language = "English",

volume = "79",

pages = "214--29",

journal = "American Journal of Human Genetics",

issn = "0002-9297",

publisher = "Cell Press",

number = "2",

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TY - JOUR

T1 - Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

AU - Paulsen, Marianne

AU - Lund, Connie

AU - Akram, Zarqa

AU - Winther, Jakob R

AU - Horn, Nina

AU - Møller, Lisbeth Birk

N1 - Keywords: Adenosine Triphosphatases; Adolescent; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; CHO Cells; Cation Transport Proteins; Cell-Free System; Cells, Cultured; Child; Child, Preschool; Copper; Cricetinae; Fibroblasts; Frameshift Mutation; Humans; Infant; Male; Menkes Kinky Hair Syndrome; Molecular Sequence Data; Peptide Chain Initiation, Translational; Sequence Deletion; Survivors

PY - 2006

Y1 - 2006

N2 - Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.

AB - Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.

U2 - 10.1086/505407

DO - 10.1086/505407

M3 - Journal article

C2 - 16826513

SN - 0002-9297

VL - 79

SP - 214

EP - 229

JO - American Journal of Human Genetics

JF - American Journal of Human Genetics

IS - 2

ER -

Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

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