Limits...
ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus

Identification of ANKS3, a partner of ANKS6.(A) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (B) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (C) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (D) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556665&req=5

pone.0136781.g001: Identification of ANKS3, a partner of ANKS6.(A) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (B) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (C) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (D) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.

Mentions: To identify protein partners of ANKS6, we used a yeast two hybrid system and screened a mouse 17-day embryo cDNA library. The screen was performed using the C-terminal extremity of the rat protein Anks6 harboring the SAM domain as the bait protein, which was expressed from the pGBD-B plasmid (Fig 1A). From the mating of 1x109 bait and 1x109 prey transformants, 384 hybrids were obtained and 25 were the result of positive protein interaction. From these, 4 clones were fully sequenced. All of them corresponded to partial cDNA sequences of the geneAnks3. The predicted ANKS3 protein is 655 aa long and presents organization of domains similar to that of ANKS6 (Fig 1B). Three of the four preys encoded the C-terminal extremity of ANKS3 and contained the SAM domain (aa 424–630) (Preys 1, 2, 4). The fourth sequence encoded the central region of ANKS3 containing the Poly Serine region and the SAM domain (aa 260–560) (Prey 3) (Fig 1B).


ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Identification of ANKS3, a partner of ANKS6.(A) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (B) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (C) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (D) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556665&req=5

pone.0136781.g001: Identification of ANKS3, a partner of ANKS6.(A) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (B) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (C) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (D) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.
Mentions: To identify protein partners of ANKS6, we used a yeast two hybrid system and screened a mouse 17-day embryo cDNA library. The screen was performed using the C-terminal extremity of the rat protein Anks6 harboring the SAM domain as the bait protein, which was expressed from the pGBD-B plasmid (Fig 1A). From the mating of 1x109 bait and 1x109 prey transformants, 384 hybrids were obtained and 25 were the result of positive protein interaction. From these, 4 clones were fully sequenced. All of them corresponded to partial cDNA sequences of the geneAnks3. The predicted ANKS3 protein is 655 aa long and presents organization of domains similar to that of ANKS6 (Fig 1B). Three of the four preys encoded the C-terminal extremity of ANKS3 and contained the SAM domain (aa 424–630) (Preys 1, 2, 4). The fourth sequence encoded the central region of ANKS3 containing the Poly Serine region and the SAM domain (aa 260–560) (Prey 3) (Fig 1B).

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus