Involvement of ALADIN in adrenal cell function and hormone and response
Zusammenfassung der Projektergebnisse
The goal of this project was the investigation of the involvement of ALADlN in adrenal cell function and hormone response. ALADIN, encoded by the AAAS gene, is dysfunctional in triple A syndrome, which is associated with severe adrenal insufficiency. The protein is a component of the nuclear pore complex (NPC), which is the exclusive gateway allowing the regulated trafficking of macromolecules between the nucleus and cytoplasm. In addition NPCs are also involved in chromatin organization, gene regulation and cell cycle progression. The diversity of symptoms observed in patients suggests that different functional cellular pathways are involved in the pathogenesis of the disorder. In the second funding period of the KFO252 we focussed on the role of ALADIN in oxidative stress response and steroidogenesis. We examined the impact of ALADIN on cell viability, oxidative stress response and nuclear import of aprataxin, DNA ligase 1 and ferritin heavy chain 1. These proteins possess protective roles in the nucleus during oxidative stress and are involved in DNA single strand break repair. We could show that ALADIN deficiency in human adrenocortical cells leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after treatment with superoxide anions producing agents. Furthermore we observed significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. Our findings indicate that AAAS knock-down induces a significant down-regulation of the enzymes involved in the glucocorticoid pathway. Moreover, on cell metabolite level we revealed that ALADIN knock-down cells produce a significant lower amount of precursor metabolites of the glucocorticoid pathway whereas over-expression of AAAS did not result in significant differences of steroid levels. We observed no rescue of steroidogenesis after recovery of ALADIN in the down-regulated human adrenal cell line NCI-H295R. Using ALADIN null mice we attempted to verify the critical role of ALADIN in the cellular redox regulation. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but with a lack of a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN. In collaboration with other groups we discovered that ALADIN is required for the production of fertile mouse oocytes. We found out that ALADIN-knockout-mouse oocytes have defects in polar body ejection and chromosome alignment, indicating that ALADIN has a pivotal role in centrosomal division. Moreover, in somatic cells ALADIN is involved via Aurora A in cell cycle regulation by maintaining optimal force balancing within mitotic spindles. When ALADIN is depleted, a delay in chromosome alignment, hyperstretching of paired kinetochores, and destabilized K-fibres that are fluxing at three times the normal rate is seen. In an attempt to find new interaction partners of ALADIN by proteome analysis we identified the microsomal protein Progesterone Receptor Membrane Component 2 (PGRMC2) as novel interactor. Moreover, depletion of ALADIN results in mislocalization of PGRMC1 in metaphase cells. PGRMC1 and PGRMC2 are reported to interact and furthermore, to bind to each other during metaphase, thereby suppressing entry into cell cycle. We suggest that loss of the regulatory association between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibres. In summary, our investigations provided an important contribution to the further understanding of the cellular role of ALADIN and hence of the pathogenesis of triple A syndrome.
Projektbezogene Publikationen (Auswahl)
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Changes in differential gene expression in fibroblast cells from patients with triple A syndrome under oxidative stress. Horm Metab Res. 2013 Feb;45(2):102-8
Koehler K, End K, Kind B, Landgraf D, Mitzscherling P, Huebner A
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Mutations in GMPPA cause a glycosylation disorder characterized by intellectual disability and autonomic dysfunction. Am J Hum Genet. 2013 Oct 3;93(4):727-34
Koehler K, Malik M, Mahmood S, Gießelmann S, Beetz C, Hennings JC, Huebner AK, Grahn A, Reunert J, Nürnberg G, Thiele H, Altmüller J, Nürnberg P, Mumtaz R, Babovic-Vuksanovic D, Basel-Vanagaite L, Borck G, Brämswig J, Mühlenberg R, Sarda P, Sikiric A, Anyane-Yeboa K, Zeharia A, Ahmad A, Coubes C, Wada Y, Marquardt T, Vanderschaeghe D, Van Schaftingen E, Kurth I, Huebner A, Hübner CA
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Organ-specific Neurodegeneration in Triple A syndrome-related achalasia. Am J Med. 2015 Sep;128(9):e9-12
Zimmer V, Vanderwinden JM, Zimmer A, Ostertag D, Strittmatter M, Koehler K, Huebner A, Lammert F
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Role of ALADIN in human adrenocortical cells for oxidative stress response and steroidogenesis. PLoS One. 2015 Apr 13;10(4):e0124582
Jühlen R, Idkowiak J, Taylor AE, Kind B, Arlt W, Huebner A, Koehler K
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The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation. Mol Biol Cell. 2015 Oct 1;26(19):3424-38
Carvalhal S, Ribeiro SA, Arocena M, Kasciukovic T, Temme A, Koehler K, Huebner A, Griffis ER
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Identification of a novel putative interaction partner of the nucleoporin ALADIN. Biol Open. 2016 Nov 15;5(11):1697-1705
Jühlen R, Landgraf D, Huebner A, Koehler K
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NCI H295R cells following angiotensin II, forskolin and abiraterone treatment. J Steroid Biochem Mol Biol. 2016 Jan;155(Pt A):67-75
Mangelis A, Dieterich P, Peitzsch M, Richter S, Jühlen R, Hübner A, Willenberg HS, Deussen A, Lenders JW, Eisenhofer G
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A novel TRAPPC11 mutation in two Turkish families associated with cerebral atrophy, global retardation, scoliosis, achalasia and alacrima. J Med Genet. 2017 Mar;54(3):176-185
Koehler K, Milev MP, Prematilake K, Reschke F, Kutzner S, Jühlen R, Landgraf D, Utine E, Hazan F, Diniz G, Schuelke M, Huebner A, Sacher M
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ALADIN is required for the production of fertile mouse oocytes. Mol Biol Cell. 2017;28:2470-2478
Carvalhal S, Stevense M, Koehler K, Naumann R, Huebner A, Jessberger R, Griffis E
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Triple A Syndrome: Preliminary response to the antioxidant N-acetylcysteine treatment in a child. Horm Res Paediatr. 2017;88(2):167-171
Fragoso MCBV, Albuquerque EVA, Cardoso ALA, da Rosa PWL, de Paulo RB, Schimizu MHM, Seguro AC, Passarelli M, Koehler K, Huebner A, Almeida MQ, Latronico AC, Arnhold IJP, Mendonca BB
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Compensation for chronic oxidative stress in ALADIN null mice. Biol Open. 2018;7
Jühlen R, Peitzsch M, Gärtner S, Landgraf D, Eisenhofer G, Huebner A, Koehler K
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Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers. Cell Div. 2018;13:8
Jühlen R, Landgraf D, Huebner A, Koehler K
