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The C175R mutation alters nuclear localization and transcriptional activity of the nephronophthisis NPHP7 gene product.

Ramachandran H, Yakulov TA, Engel C, Müller B, Walz G - Eur. J. Hum. Genet. (2015)

Bottom Line: Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children.Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment.Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Freiburg University Medical Center, Freiburg, Germany.

ABSTRACT
Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7(C175R) point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7(C175R), prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions.

No MeSH data available.


Related in: MedlinePlus

The GLIS2/NPHP7C175R mutation does not disrupt the interaction with known interacting proteins. (a) The GLIS2/NPHP7C175R mutation abolishes the prediction of the first zinc-finger (SMART analysis tool). (b) Interaction of V5-tagged wild-type GLIS2/NPHP7 (V5.NPHP7.WT) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. HC indicates heavy chain of antibodies. The additional bands in lanes 2 and 4 are likely non-specific background due cross-reactivity of the anti-Flag antibody and slightly higher protein concentrations in these lanes. (c) Interaction of V5-tagged mutant GLIS2/NPHP7C175R (V5.NPHP7C175R) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. There was no detectable difference regarding the interaction of these three candidate proteins. IP, immunopricipitation; WB, western blot.
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fig1: The GLIS2/NPHP7C175R mutation does not disrupt the interaction with known interacting proteins. (a) The GLIS2/NPHP7C175R mutation abolishes the prediction of the first zinc-finger (SMART analysis tool). (b) Interaction of V5-tagged wild-type GLIS2/NPHP7 (V5.NPHP7.WT) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. HC indicates heavy chain of antibodies. The additional bands in lanes 2 and 4 are likely non-specific background due cross-reactivity of the anti-Flag antibody and slightly higher protein concentrations in these lanes. (c) Interaction of V5-tagged mutant GLIS2/NPHP7C175R (V5.NPHP7C175R) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. There was no detectable difference regarding the interaction of these three candidate proteins. IP, immunopricipitation; WB, western blot.

Mentions: The comparison of wild-type and mutant GLIS2/NPHP7 using the SMART analysis tool (http://smart.embl-heidelberg.de/) revealed that the C175R point mutation abolished the prediction of the first zinc-finger (Figure 1a). Several GLIS2/NPHP7-binding proteins have been identified that appear to affect the function and/or localization of this transcriptional repressor. Importantly, binding of β-catenin and canonical Wnt signaling appears to involve the first zinc-finger, the site of the C175R mutation, while binding of CtBP1, a transcriptional co-repressor involved in HDAC3-mediated gene silencing complex seem to occur within the C-terminal domain of GLIS2/NPHP7. To determine whether the C175R mutation disrupts the interaction with important binding partners, we tested the interaction with CtBP1, HDAC3 and β-catenin. However, we observed no differences between wild-type and mutant GLIS2/NPHP7 with these three proteins (Figure 1b and c), suggesting that the C175R mutation does not compromise the overall conformation of GLIS2/NPHP7, or abrogate the interaction with defined binding partners.


The C175R mutation alters nuclear localization and transcriptional activity of the nephronophthisis NPHP7 gene product.

Ramachandran H, Yakulov TA, Engel C, Müller B, Walz G - Eur. J. Hum. Genet. (2015)

The GLIS2/NPHP7C175R mutation does not disrupt the interaction with known interacting proteins. (a) The GLIS2/NPHP7C175R mutation abolishes the prediction of the first zinc-finger (SMART analysis tool). (b) Interaction of V5-tagged wild-type GLIS2/NPHP7 (V5.NPHP7.WT) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. HC indicates heavy chain of antibodies. The additional bands in lanes 2 and 4 are likely non-specific background due cross-reactivity of the anti-Flag antibody and slightly higher protein concentrations in these lanes. (c) Interaction of V5-tagged mutant GLIS2/NPHP7C175R (V5.NPHP7C175R) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. There was no detectable difference regarding the interaction of these three candidate proteins. IP, immunopricipitation; WB, western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4930099&req=5

fig1: The GLIS2/NPHP7C175R mutation does not disrupt the interaction with known interacting proteins. (a) The GLIS2/NPHP7C175R mutation abolishes the prediction of the first zinc-finger (SMART analysis tool). (b) Interaction of V5-tagged wild-type GLIS2/NPHP7 (V5.NPHP7.WT) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. HC indicates heavy chain of antibodies. The additional bands in lanes 2 and 4 are likely non-specific background due cross-reactivity of the anti-Flag antibody and slightly higher protein concentrations in these lanes. (c) Interaction of V5-tagged mutant GLIS2/NPHP7C175R (V5.NPHP7C175R) with Flag-tagged CtBP1, HDAC3 and β-catenin. CD2AP was used as a negative control. There was no detectable difference regarding the interaction of these three candidate proteins. IP, immunopricipitation; WB, western blot.
Mentions: The comparison of wild-type and mutant GLIS2/NPHP7 using the SMART analysis tool (http://smart.embl-heidelberg.de/) revealed that the C175R point mutation abolished the prediction of the first zinc-finger (Figure 1a). Several GLIS2/NPHP7-binding proteins have been identified that appear to affect the function and/or localization of this transcriptional repressor. Importantly, binding of β-catenin and canonical Wnt signaling appears to involve the first zinc-finger, the site of the C175R mutation, while binding of CtBP1, a transcriptional co-repressor involved in HDAC3-mediated gene silencing complex seem to occur within the C-terminal domain of GLIS2/NPHP7. To determine whether the C175R mutation disrupts the interaction with important binding partners, we tested the interaction with CtBP1, HDAC3 and β-catenin. However, we observed no differences between wild-type and mutant GLIS2/NPHP7 with these three proteins (Figure 1b and c), suggesting that the C175R mutation does not compromise the overall conformation of GLIS2/NPHP7, or abrogate the interaction with defined binding partners.

Bottom Line: Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children.Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment.Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein.

View Article: PubMed Central - PubMed

Affiliation: Renal Division, Freiburg University Medical Center, Freiburg, Germany.

ABSTRACT
Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7(C175R) point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7(C175R), prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions.

No MeSH data available.


Related in: MedlinePlus