Journal: Nature Communications

Article Title: Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

doi: 10.1038/ncomms10640

Figure Lengend Snippet: ( a ) Immunostaining of PAM (green) in a chicken spinal cord (HH stage 20 embryo). Traverse sections of chick embryos were analysed. Differentiated cells in marginal zones were identified by postmitotic marker isl1/2 (red). Scale bar, 50 μm. ( b ) Intensity profile along the line indicated by the arrow in a shows overlap between PAM and Isl1/2 expression. Medial and lateral borders are represented as M and L, respevtively. ( c ) Schematic of differentiation of SH-SY5Y cells by sequential treatments with retinoic acid (DIF RA) and BDNF (DIF BDNF). ( d ) Differentiation is associated with upregulation of genes involved in copper fluxes to mitochondria and, especially, the secretory pathway. The mRNA levels were determined by ΔΔCt analysis using GAPDH as a reference gene and normalized to non-differentiated cells (red line). Each value is presented as mean±s.d. ( n =3). ( e ) Protein levels for ATP7A and Atox1 increase upon differentiation. Equal amount of protein was loaded to each lane. Neuronal differentiation was verified by upregulation of MAP2. ( f ) Cellular copper content in differentiated SH-SY5Y cells (BDNF) is higher than in non-differentiated (ND) cells. Copper amounts were determined by atomic absorption and normalized to protein amounts. Data from three independent measurements. ( g , h ) ATP7A is localized within TGN and vesicular structures which are distinct from endosomal or lysosomal compartments. Coimmunostaining with TGN was shown as a representative image (see for other images). Nucleus was stained with DAPI (blue). Scale bar, 10 μm. Colocalization of ATP7A and various markers were evaluated using Pearson's product-moment coefficients. Three replicate samples were prepared and analysed. Each value is presented as mean±s.d. ( n =3).

Article Snippet: Primary antibodies used in cell staining were mouse monoclonal anti-FLAG M2 clone (Sigma, F1804), rabbit polyclonal anti-ATP7A (Hycult Biotech, HP8040), mouse monoclonal anti-ATP7A (Santa Cruz, 376467), mouse monoclonal anti-EEA1 (BD Biosciences, 610456), mouse monoclonal anti-Rab11 (BD Biosciences, 610656), mouse monoclonal anti-Lamp1 (developmental studies hybridoma bank, H4A3-s), and sheep monoclonal anti-TGN46 (Gene Tex, GTX74290).

Techniques: Immunostaining, Marker, Expressing, Staining

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Topology and reaction cycle of ATP7A. ( a ) Topology of ATP7A. ATP7A, and other P-type ATPases consist of three cytoplasmic domains, nucleotide binding (N, red), phosphorylation (P, blue) and dephosphorylation/actuation (A, yellow). The transmembrane domain encompasses eight membrane-spanning segments (TM), two class I specific (TMA-TMB, cyan) and six conserved segments (TM1-6, wheat). The N-terminus contains six class-specific metal-binding domains (H1-H6, cyan). The copper-donating chaperone ATOX1 (green) and conserved motifs are shown. The non-cytosolic part of ATP7A is located in the TGN or in the extracellular milieu, due to copper-dependent trafficking. ( b ) The Albers-Post (E1-E2) reaction cycle of ATP7A and other Cu-transporting P-type ATPases. The domains are colored as described above, and copper ions are shown in green. Phosphorylation events in the intracellular domains drive large conformational changes that permit alternating access to transport sites in the membrane about 50 Å from the ATP-targeted catalytic aspartate. A high-affinity state (E1) binds copper and enters an occluded state, which then undergoes phosphorylation (E1.Pi-ADP). Completion of this event (E1P) triggers release of the ion, establishing an outward-facing, low-affinity state (E2P). Release of inorganic phosphate (E2.Pi) yields the fully dephosphorylated conformation (E2), which is followed by restoration of the inward-facing conformation (E1) that initiates a new reaction cycle. ( c ) Proposed cellular trafficking of ATP7A as an effect of copper concentration. At low cellular copper concentrations the wild-type ATP7A is located in the Trans-Golgi Network (TGN), whereas at higher intracellular copper levels, the steady state distribution of ATP7A shifts to the plasma membrane (and cytosolic vesicles, not shown).

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Binding Assay, De-Phosphorylation Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Summary. The color represents the mutational effect on ATP7A localization: Normal trafficking of the protein (blue); retention in post-Golgi compartments (grey); retention in the trans-Golgi network (orange); not certain (green); no detectable protein (purple). “A” denote atypical, “O” OHS, and “C” classical phenotype. Pos: indicates affected region (MBD, TM, A, P). Protein: Indicates whether protein was detectable with IF “Yes” or WB only “Yes WB ”. Localization: Location of ATP7A always in TGN even in the presence of Cu is designated by “TGN(+Cu)”. Never located in TGN is designated by “not TGN”; Location in the TGN only the absence of copper is designated by “normal”. No detectable protein “n.d”. ATP7A-mRNA: indicates % transcript level compared to transcript level in control fibroblasts. Splicing: indicates normal splicing “Normal” or aberrant splicing “All mal”. Empty boxes, indicate “not investigated”. Compl: indicates ability to complement the ccc2Δ yeast strain. “Some 56 ” and “Some 80 ”, indicate some complementation after 56 hours and 80 hours, respectively. “Yes 80 ” indicates full complementation after 80 hours. If no hours are indicated, it means >56 hours. Uptake/retention: Uptake indicates the amount of radioactive copper accumulated after 20 hours incubation ( 64 Cu/mg protein/20 hours). Retention indicated the amount in % of total accumulated 64 Cu retained after subsequent 24 hours in the absence of radioactive copper.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Incubation

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Cellular investigation of endogenous ATP7A protein in MD fibroblasts by indirect immunofluorescence (IF). MD fibroblasts with various missense mutations were stained with primary antibodies against ATP7A (green; position 1) and the Golgi specific marker GS28 (red; position 2), respectively. Also a merge picture is shown (position 3). The nuclei were counterstained with DAPI. Copper-dependent trafficking was investigated in the presence of BCS (upper panel) and CuCl 2 (lower panel), respectively. The pictures are divided into groups defined by the effect of the mutation on cellular location of the ATP7A protein. P1: Normal control cells (C+). P2: ATP7A negative control cells (C−). P3–P4: Copper dependent trafficking. P5-P17: No ATP7A signal was detected. P18–P38: Hampered copper-induced trafficking from TGN.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Immunofluorescence, Staining, Marker, Mutagenesis, Negative Control

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Western blots of lysates from MD fibroblasts. Western blots of lysates from MD fibroblasts with no detectable ATP7A protein when analysed by Immunofluorescence (IF). The specific ATP7A missense mutations are indicated above the lanes. Control + lysate from normal fibroblasts. Control − lysate from MD fibroblasts containing a big deletion of the ATP7A gene (exon 3–23 deleted). The protein product of the housekeeping gene GAPDH was used as an internal control for correct loading of 25 μg protein in each lane. The staining of ATP7A and GAPDH was performed on one membrane cut in two. Gaps indicate separate gels. Small gaps indicate that the gel was cut in producing the figure.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: ( A ) RT-PCR investigation of ATP7A transcript. RNA from MD fibroblasts were subjected to RT-PCR spanning the exons with the missense mutations. Control samples are from a normal control. Size (in bp) of marker DNA fragments are indicated. The PCR products were separated by gel electrophoresis, purified and sequenced (Table ). Primers are given in Supplementary Table . (I) No detectable ATP7A protein and a reduced amount of ATP7A transcript. A: PCR amplification of exons 7–14. E628V C and K633R C (mutations in exon 8); skipping of exon 8, exons 8–9 or exons 8–10. G876R C (mutation in exon 12); skipping of exon 12, exons 11–13 or exons 10–13. R844H C indistinguishable from the wild-type sample. (II) No detectable ATP7A protein and a normal amount of ATP7A transcript. B: PCR amplification of exons 7–14. K802N C (mutation in exon 10); skipping of exon 10. C: PCR amplification of exons 12–23. K1037N C (mutation in exon 15); skipping of exon 15. D: PCR amplification of exons 19–23. G1369R C (mutation in exon 21); skipping of exon 21 or exons 20–21, and A1373P C (mutation in exon 21); normal transcripts or skipping of exon 21 or exons 20–21. (III) Reduced amount of ATP7A protein and normal amount of ATP7A transcript. E: PCR amplification of exons 7–14. G860V C indistinguishable from the wild-type sample F: PCR amplification of exons 17–23. G1255R C indistinguishable from the wild-type sample. ( B ) Mutations presumed to affect donor splice sites. According to “human-Splicing Finder” ( http://www.umd.be/HSF/ ) splice sites with consensus values (CV)’s higher than 80 are strong splice sites, whereas splice sites with CV’s between 65 and 70 are weak . A relative change in CV (∆CV) of 10 percentage points relative to wild-type sites is predicted to attenuate splicing. ( C ) Mutations presumed to affect ESS/ESE sites. Disruption of ESE sequences and/or creation of an ESS sequences may lead to exon skipping. In agreement with the observed splicing pattern, the four base pair substitutions are predicted by the web-tools EX-SKIP ( http://ex-skip.ing.cas.cz/ ) to lead to exon skipping as a result of increased ESS/ESE values. Selected motifs are illustrated.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Nucleic Acid Electrophoresis, Purification, Amplification, Mutagenesis

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Sequence of PCR products obtained by spanning RT-PCR.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: WB of lysates isolated from MD fibroblast cultures cultured at 30 °C and 37 °C, respectively. The MD fibroblasts contain different missense mutations as indicated. Control + is lysate from normal fibroblasts. Control − is lysate from MD fibroblast, containing a big deletion of the ATP7A gene (exon 3–23 deleted). The blots are representative for Western blots of three separate cultures that were harvested and prepared individually for each mutation and control. The Western blots were performed on separate occasions; therefore band intensity cannot be compared between different gels. ( a ) Mutations that lead to low levels of ATP7A in this study. ( b ) Mutations that are similar to or resemble the mutations analysed by Vonk et al . . Gaps indicate that the gel was cut in producing the figure.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Isolation, Cell Culture, Western Blot, Mutagenesis

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: WB of lysates from MD fibroblasts cultured in the absence or presence of 25 µM Bortezomib, respectively. The MD fibroblasts contain different missense mutations as indicated. Control + is lysate from normal fibroblasts. Control − is lysate from MD fibroblast, containing a big deletion of the ATP7A gene (exon 3–23 deleted). The protein product of the housekeeping gene Alfa-Tubulin was used as an internal control for the correct loading of 25 μg protein in each lane.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Cell Culture

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Complementation of the ccc2Δ iron requiring phenotype on agar plates. All 36 variants were investigated for their ability to complement the high iron requirement of a ccc2Δ yeast strain by plating cells on agar plates containing the iron chelator Ferrozine. WT, ccc2Δ yeast cells producing wild-type ATP7A; EV, ccc2Δ yeast cells expressing no ATP7A (empty vector). Mutants are spotted according to their amino acid substitution. To control for cell viability, each yeast strain was also spotted on iron-containing agar plates.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Complementation of the ccc2Δ iron requiring phenotype in liquid culture. The mutants R844H, G860V and G1255R leading to no, or only little protein accumulation in patient fibroblasts and the frequent mutant G727R were investigated for their ability to complement the high iron requirement of a ccc2Δ yeast strain in liquid cultures containing 1 mM Ferrozine and increasing amounts of Fe 2+ , as indicated. Wild-type, ccc2Δ yeast cells producing wild-type ATP7A; Empty vector, ccc2Δ yeast cells expressing no ATP7A.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Mutagenesis, Plasmid Preparation, Expressing

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: WB of crude lysates from yeast transformed with ATP7A expression plasmids. Crude membrane proteins were isolated from ccc2Δ yeast cells, transformed with plasmids encoding G727R, R844H, G860V, G1255R, WT, or no ATP7A (empty vector, EV). 25 μg crude membrane proteins were analyzed by Western blotting as described in Materials and Methods.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Transformation Assay, Expressing, Isolation, Plasmid Preparation, Western Blot

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: Distribution and effect of ATP7A disease-causing missense mutations. The mutations, presented in this study, are plotted as spheres in a previously established homology model of ATP7A , based on the crystal structure of the homologous protein LpCopA from Legionella pneumophila . The various domains of ATP7A are colored as in Fig. . However, metal-binding domains 1 to 4 (H1–H4) are not depicted in the figure, as their structural localization is unknown, and regions with major insertions relative to LpCopA are shown in black. The approximate position of the (TGN- or plasma-) membrane is shown in wheat. The color of spheres represent the mutational effect on ATP7A localization: Copper dependent trafficking of the protein (blue); retention in post-Golgi compartments (grey); retention in the trans-Golgi network (orange); not certain (green); no detectable protein (purple). Underlined missense mutations have been reported to confer a non-classical phenotype. A1325V leads to classical MD in some patients (see Fig. ).

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

doi: 10.1038/s41598-017-00618-6

Figure Lengend Snippet: The post-Golgi arrested mutations G666R A and G876E A . The domains of ATP7A are colored as in Fig. , and functionally relevant residues shown as sticks using a previously established homology model of ATP7A (6) based on the crystal structure of the homologous protein LpCopA from Legionella pneumophila (2). (A) close-view of G666R at the possible release pathway from the CPC-motif (C1000 and C1002) and via D782 to the non-cytosolic side. The mutation may directly prevent copper passage through sterical hindrance or influence of the local environment at the membrane interface. ( b ) Close-view of G876E at the catalytic Aspartate (D1044). G876 is located in the TGE-loop of the A-domain which is responsible for dephosphorylation (AlF 4 − is a phosphate mimic used for structure determination of LpCopA). The mutation is likely to prevent dephosphorylation and turn-over.

Article Snippet: A Taq-Man 6-carboxy-fluorescein (FAM) labeled probe and primer pairs against the boundary between exon 1 and exon 2 (part number Hs00921963_m1) or against the boundary between exon 22 and exon 23 (part number Hs00921966_m1) in ATP7A cDNA were used to detect the total amount of ATP7A transcript.

Techniques: Mutagenesis, De-Phosphorylation Assay