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Endofin, a novel BMP-SMAD regulator of the iron-regulatory hormone, hepcidin.

Goh JB, Wallace DF, Hong W, Subramaniam VN - Sci Rep (2015)

Bottom Line: We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling.Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression.In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.

ABSTRACT
BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor, and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7. We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.

No MeSH data available.


Mutations disrupting the conserved FYVE domain of endofin cause mislocalisation.Plasmid constructs containing six naturally occurring endofin SNPs identified to be potentially deleterious were generated through site-directed mutagenesis and transfected into C3A cells. After 48 hours, cells were immunostained with anti-HA antibodies (first column). Cells were also immunostained with organelle markers, EEA1 and ERp57 as represented in the second column. The nuclei were stained with DAPI as represented in the third column which is an overlay of the first and second panels. (a) Schematic diagram of endofin protein structure with the FYVE domain (red rectangle), and mapped non-synonymous (boxes) and frameshift (triangles) mutations. (b) Western blotting showing the corresponding sizes of proteins containing non-synonymous and frameshift SNPs with premature stop codons. (c) Panels show C3A cells transfected with endofin SNP variants without an affected FYVE domain. (d) Panels show C3A cells transfected with SNP variants affecting the FYVE domain. Data are representative of three independent biological experiments. Scale bar represents 20 μm.
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f5: Mutations disrupting the conserved FYVE domain of endofin cause mislocalisation.Plasmid constructs containing six naturally occurring endofin SNPs identified to be potentially deleterious were generated through site-directed mutagenesis and transfected into C3A cells. After 48 hours, cells were immunostained with anti-HA antibodies (first column). Cells were also immunostained with organelle markers, EEA1 and ERp57 as represented in the second column. The nuclei were stained with DAPI as represented in the third column which is an overlay of the first and second panels. (a) Schematic diagram of endofin protein structure with the FYVE domain (red rectangle), and mapped non-synonymous (boxes) and frameshift (triangles) mutations. (b) Western blotting showing the corresponding sizes of proteins containing non-synonymous and frameshift SNPs with premature stop codons. (c) Panels show C3A cells transfected with endofin SNP variants without an affected FYVE domain. (d) Panels show C3A cells transfected with SNP variants affecting the FYVE domain. Data are representative of three independent biological experiments. Scale bar represents 20 μm.

Mentions: As decreased endofin expression modulated hepcidin levels, which in turn would result in increased body iron levels, we reasoned that single nucleotide polymorphisms (SNPs) which affected endofin localisation or function might have a similar effect. We identified naturally occurring endofin SNP variants through SNP database searches and analysed using the SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 (Polymorphism Phenotyping v2) protein prediction tools to determine potentially deleterious mutations and generated them through site-directed mutagenesis of HA-epitope tagged endofin constructs. These were transiently transfected into C3A cells (Fig. 5a,b).


Endofin, a novel BMP-SMAD regulator of the iron-regulatory hormone, hepcidin.

Goh JB, Wallace DF, Hong W, Subramaniam VN - Sci Rep (2015)

Mutations disrupting the conserved FYVE domain of endofin cause mislocalisation.Plasmid constructs containing six naturally occurring endofin SNPs identified to be potentially deleterious were generated through site-directed mutagenesis and transfected into C3A cells. After 48 hours, cells were immunostained with anti-HA antibodies (first column). Cells were also immunostained with organelle markers, EEA1 and ERp57 as represented in the second column. The nuclei were stained with DAPI as represented in the third column which is an overlay of the first and second panels. (a) Schematic diagram of endofin protein structure with the FYVE domain (red rectangle), and mapped non-synonymous (boxes) and frameshift (triangles) mutations. (b) Western blotting showing the corresponding sizes of proteins containing non-synonymous and frameshift SNPs with premature stop codons. (c) Panels show C3A cells transfected with endofin SNP variants without an affected FYVE domain. (d) Panels show C3A cells transfected with SNP variants affecting the FYVE domain. Data are representative of three independent biological experiments. Scale bar represents 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: Mutations disrupting the conserved FYVE domain of endofin cause mislocalisation.Plasmid constructs containing six naturally occurring endofin SNPs identified to be potentially deleterious were generated through site-directed mutagenesis and transfected into C3A cells. After 48 hours, cells were immunostained with anti-HA antibodies (first column). Cells were also immunostained with organelle markers, EEA1 and ERp57 as represented in the second column. The nuclei were stained with DAPI as represented in the third column which is an overlay of the first and second panels. (a) Schematic diagram of endofin protein structure with the FYVE domain (red rectangle), and mapped non-synonymous (boxes) and frameshift (triangles) mutations. (b) Western blotting showing the corresponding sizes of proteins containing non-synonymous and frameshift SNPs with premature stop codons. (c) Panels show C3A cells transfected with endofin SNP variants without an affected FYVE domain. (d) Panels show C3A cells transfected with SNP variants affecting the FYVE domain. Data are representative of three independent biological experiments. Scale bar represents 20 μm.
Mentions: As decreased endofin expression modulated hepcidin levels, which in turn would result in increased body iron levels, we reasoned that single nucleotide polymorphisms (SNPs) which affected endofin localisation or function might have a similar effect. We identified naturally occurring endofin SNP variants through SNP database searches and analysed using the SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 (Polymorphism Phenotyping v2) protein prediction tools to determine potentially deleterious mutations and generated them through site-directed mutagenesis of HA-epitope tagged endofin constructs. These were transiently transfected into C3A cells (Fig. 5a,b).

Bottom Line: We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling.Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression.In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Australia.

ABSTRACT
BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor, and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7. We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway, and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.

No MeSH data available.