Publications by authors named "Po-Hao Hsu"

5 Publications

  • Page 1 of 1

Identification of MKRN1 as a second E3 ligase for Eag1 potassium channels reveals regulation via differential degradation.

J Biol Chem 2021 Feb 26:100484. Epub 2021 Feb 26.

Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; Brain Research Center, National Yang-Ming University, Taipei, Taiwan. Electronic address:

Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K channels, however, remains unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent non-glycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, non-glycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.
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http://dx.doi.org/10.1016/j.jbc.2021.100484DOI Listing
February 2021

Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.

Sci Rep 2017 01 18;7:40825. Epub 2017 Jan 18.

Institute of Anatomy and Cell Biology, School of Medicine, National  Yang-Ming University, Taipei, Taiwan.

Mammalian Eag1 (Kv10.1) potassium (K) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels.
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http://dx.doi.org/10.1038/srep40825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241692PMC
January 2017

Ca(2+)-binding protein centrin 4 is a novel binding partner of rat Eag1 K(+) channels.

FEBS Open Bio 2016 04 7;6(4):349-57. Epub 2016 Mar 7.

Institute of Anatomy and Cell Biology School of Medicine National Yang-Ming University Taipei Taiwan; Brain Research Center National Yang-Ming University Taipei Taiwan.

Eag1 is neuron-specific K(+) channel abundantly expressed in the brain and retina. Subcellular localization and physiological analyses in neurons reveal that Eag1 may participate in Ca(2+)-signaling processes in the synapse. Here, we searched for rat Eag1 (rEag1)-binding proteins that may contribute to Ca(2+) regulation of the K(+) channel. Yeast two-hybrid screening identified centrin 4, a member of the centrin family of Ca(2+)-binding proteins. GST pull-down and immunoprecipitation assays in brain and retina lysates confirm the interaction of centrin with rEag1 in neurons. Centrin 4 binds to rEag1 in the absence of Ca(2+). Raising Ca(2+) concentration enhances the association efficiency of centrin 4 and rEag1, and is required for the suppression of rEag1 currents by centrin 4. Altogether, our data suggest that centrin 4 is a novel binding partner that may contribute to Ca(2+) regulation of rEag1 in neurons.
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http://dx.doi.org/10.1002/2211-5463.12045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821352PMC
April 2016

Densin-180 is not a transmembrane protein.

Cell Biochem Biophys 2013 Nov;67(2):773-83

Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

In the central nervous system, densin-180 (densin) is one of the major components of the post-synaptic density (PSD) of excitatory synapses. Through its intricate interaction with various post-synaptic proteins, this scaffold protein may play a key role in synaptic regulation. Initial structural analyses suggest that densin is a transmembrane protein and may participate in cell-adhesion function between pre- and post-synaptic membranes. Whereas recent biochemical and mass spectrometry studies indicate that densin may instead be a membrane-associated protein with no extracellular domain. To further investigate the structural topology of densin, we began with examining the extracellular accessibility of multiple epitopes in densin. We have provided immunofluorescence evidence showing that none of the tested epitope sites in densin was accessible to extracellularly applied antibodies. In addition, both protease digestion and surface biotinylation data failed to affirm the presence of extracellular domain for densin. However, protein extraction experiments indicated that densin exhibited a significant hydrophobic interaction with the cell membrane that was not expected of cytosolic proteins. Our data therefore do not support the transmembrane model, but rather are consistent with the idea that the topology of densin involves the membrane association configuration.
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http://dx.doi.org/10.1007/s12013-013-9570-3DOI Listing
November 2013

14-3-3θ is a binding partner of rat Eag1 potassium channels.

PLoS One 2012 20;7(7):e41203. Epub 2012 Jul 20.

Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

The ether-à-go-go (Eag) potassium (K(+)) channel belongs to the superfamily of voltage-gated K(+) channel. In mammals, the expression of Eag channels is neuron-specific but their neurophysiological role remains obscure. We have applied the yeast two-hybrid screening system to identify rat Eag1 (rEag1)-interacting proteins from a rat brain cDNA library. One of the clones we identified was 14-3-3θ, which belongs to a family of small acidic protein abundantly expressed in the brain. Data from in vitro yeast two-hybrid and GST pull-down assays suggested that the direct association with 14-3-3θ was mediated by both the N- and the C-termini of rEag1. Co-precipitation of the two proteins was confirmed in both heterologous HEK293T cells and native hippocampal neurons. Electrophysiological studies showed that over-expression of 14-3-3θ led to a sizable suppression of rEag1 K(+) currents with no apparent alteration of the steady-state voltage dependence and gating kinetics. Furthermore, co-expression with 14-3-3θ failed to affect the total protein level, membrane trafficking, and single channel conductance of rEag1, implying that 14-3-3θ binding may render a fraction of the channel locked in a non-conducting state. Together these data suggest that 14-3-3θ is a binding partner of rEag1 and may modulate the functional expression of the K(+) channel in neurons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041203PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3401112PMC
April 2013