Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts

Zeitschrift:: Orphanet Journal of Rare Diseases > Ausgabe 1/2016

Autoren:: Urszula Liwak-Muir, Hapsatou Mamady, Turaya Naas, Quinlan Wylie, Skye McBride, Matthew Lines, Jean Michaud, Stephen D. Baird, Pranesh K. Chakraborty, Martin Holcik

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Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.1186/s13023-016-0466-3) contains supplementary material, which is available to authorized users.

Pranesh K. Chakraborty and Martin Holcik jointly directed this work.

Abstract

Background

SIFD (Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay) is a novel form of congenital sideroblastic anemia associated with B-cell immunodeficiency, periodic fevers, and developmental delay caused by mutations in the CCA-adding enzyme TRNT1, but the precise molecular pathophysiology is not known.

Results

We show that the disease causing mutations in patient-derived fibroblasts do not affect subcellular localization of TRNT1 and show no gross morphological differences when compared to control cells. Analysis of cellular respiration and oxidative phosphorylation (OXPHOS) complexes demonstrates that both basal and maximal respiration rates are decreased in patient cells, which may be attributed to an observed decrease in the abundance of select proteins of the OXPHOS complexes.

Conclusions

Our data provides further insight into cellular pathophysiology of SIFD.

Additional file 1: Figure S1. Immunofluorescent imaging of mitochondrial network in fibroblast cells. (Left) Nuclei are identified by Hoechst stain (red) and mitochondria are stained with an anti-Tom20 antibody (green). Representative images are shown. (Right) The mitochondria segment length was calculated as the pixel distance from between either the end of a mitochondria or a branch point or the crossing over of another mitochondria and is averaged for each cell using a custom Acapella script run in the Columbus software. From 100 to 200 cells were measured per well and averaged over 14 wells for three separate experiments. Figure S2. Electron microscopy images of additional control fibroblasts. The red box identifies the area enlarged on the right. Figure S3. Additional control fibroblasts for membrane potential assessment. Shown are immunofluorescence images of the mitochondrial membrane potential as monitored by the incorporation of the TMRE dye (red) and compared to the total mitochondrial stain as measured by MitoTracker (green). Nuclei are identified by Hoechst stain (blue).Figure S4. (A) Oxygen consumption rate (OCR) of three control fibroblasts as measured by micro-oximetry analysis. (B) Densitometry of western blot analysis of 5 OXPHOS proteins. The band intensities were determined using ImageJ software. The intensity of each band is shown relative to total protein (ns = not significant; * = p < 0.05; ** = p<0.01; student t-test). (PDF 2743 kb)

Shi PY, Maizels N, Weiner AM. CCA addition by tRNA nucleotidyltransferase: polymerization without translocation? EMBO J. 1998;17(11):3197–206. CrossRefPubMedPubMedCentral

Chakraborty PK et al. Mutations in TRNT1, encoding the CCA-adding enzyme, cause congenital sideroblastic anemia with B cell immunodeficiency, periodic fevers and developmental delay (SIFD). Blood. 2014;5:2014–08.

Sasarman F et al. The 3’ addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1. Hum Mol Genet. 2015;4.

Anderson S et al. Sequence and organization of the human mitochondrial genome. Nature. 1981;290(5806):457–65. CrossRefPubMed

Fleming MD. Congenital sideroblastic anemias: iron and heme lost in mitochondrial translation. Hematology Am Soc Hematol Educ Program. 2011;2011:525–31. doi: 10.1182/asheducation-2011.1.525. PubMed

Graber TE et al. NF45 functions as an IRES trans-acting factor that is required for translation of cIAP1 during the unfolded protein response. Cell Death Differ. 2010;17(4):719–29. CrossRefPubMed

Youle RJ, van der Bliek AM. Mitochondrial fission, fusion, and stress. Science. 2012;337(6098):1062–5. doi: 10.1126/science.1219855. CrossRefPubMedPubMedCentral

Norton M et al. ROMO1 is an essential redox-dependent regulator of mitochondrial dynamics. Sci Signal. 2014;7(310):ra10. doi: 10.1126/scisignal.2004374. CrossRefPubMed

Fernandez-Vizarra E, Tiranti V, Zeviani M. Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects. Biochim Biophys Acta. 2009;1793(1):200–11. doi: 10.1016/j.bbamcr.2008.05.028. Epub 2008 Jun 21. CrossRefPubMed

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DeLuca AP et al. Hypomorphic mutations in TRNT1 cause retinitis pigmentosa with erythrocytic microcytosis. Hum Mol Genet. 2016;25(1):44–56. doi: 10.1093/hmg/ddv446. Epub 2015 Oct 22. CrossRefPubMed

Titel: Impaired activity of CCA-adding enzyme TRNT1 impacts OXPHOS complexes and cellular respiration in SIFD patient-derived fibroblasts
Autoren:: Urszula Liwak-Muir
Hapsatou Mamady
Turaya Naas
Quinlan Wylie
Skye McBride
Matthew Lines
Jean Michaud
Stephen D. Baird
Pranesh K. Chakraborty
Martin Holcik
Publikationsdatum: 01.12.2016
DOI: https://doi.org/10.1186/s13023-016-0466-3
Verlag: BioMed Central
Zeitschrift: Orphanet Journal of Rare Diseases
Ausgabe 1/2016
Elektronische ISSN: 1750-1172

Springer Medizin

Electronic supplementary material

Abstract

Background

Results

Conclusions

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