A Splice Isoform of DNedd4, DNedd4-Long, Negatively Regulates Neuromuscular Synaptogenesis and Viability in Drosophila

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

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PPARγΔ5, a Naturally Occurring Dominant-Negative Splice Isoform, Impairs PPARγ Function and Adipocyte Differentiation

Cell Reports ◽

10.1016/j.celrep.2018.10.035 ◽

2018 ◽

Vol 25 (6) ◽

pp. 1577-1592.e6 ◽

Cited By ~ 10

Author(s):

Marianna Aprile ◽

Simona Cataldi ◽

Maria Rosaria Ambrosio ◽

Vittoria D’Esposito ◽

Koini Lim ◽

...

Keyword(s):

Adipocyte Differentiation ◽

Dominant Negative ◽

Naturally Occurring ◽

Splice Isoform

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NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis

PLoS Genetics ◽

10.1371/journal.pgen.1009402 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009402

Author(s):

Kimia Ghaffari ◽

Lain X. Pierce ◽

Maria Roufaeil ◽

Isabel Gibson ◽

Kevin Tae ◽

...

Keyword(s):

Epithelial Cells ◽

Network Formation ◽

Stop Codon ◽

Cell Lineage ◽

Adaptor Protein ◽

Specific Protein ◽

Chemical Mutagenesis ◽

Biliary Epithelial Cells ◽

Regulatory Complex ◽

Splice Isoform

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in thenckap1lgene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediatednckap1ldeletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver ofnckap1lmutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show thatnckap1lgenetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that althoughnckap1lwas previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

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Regulation of SH3PX1 by dNedd4-long at the Drosophila neuromuscular junction

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005161 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1739-1752 ◽

Cited By ~ 2

Author(s):

Samantha S. Wasserman ◽

Alina Shteiman-Kotler ◽

Kathryn Harris ◽

Konstantin G. Iliadi ◽

Avinash Persaud ◽

...

Keyword(s):

Neurotransmitter Release ◽

Neuromuscular Junctions ◽

Cell Periphery ◽

Middle Region ◽

Binding Partners ◽

Direct Binding ◽

Specific Regulation ◽

Neuromuscular Synaptogenesis

Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic–specific regulation of SH3PX1 by dNedd4Lo.

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