scholarly journals Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia

2016 ◽  
Vol 310 (5) ◽  
pp. C329-C336 ◽  
Author(s):  
N. Wang ◽  
H. S. Kang ◽  
G. Ahmmed ◽  
S. A. Khan ◽  
V. V. Makarenko ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Intermittent Hypoxia ◽  
Neuroblastoma Cells ◽  
Herg Channel ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Related Gene ◽  
Similar Reduction ◽  
Channel Protein ◽  
And Function

Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K+ current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K+ current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca2+ concentration ([Ca2+]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca2+]i mediates hERG protein degradation by IH.

scholarly journals KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

2012 ◽  
Vol 302 (4) ◽  
pp. H910-H922 ◽  
Author(s):  
Mei Zhang ◽  
Yuhong Wang ◽  
Min Jiang ◽  
Dimitar P. Zankov ◽  
Sabeeha Chowdhury ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Degradation ◽  
Spontaneously Hypertensive Rat ◽  
Current Amplitude ◽  
Delayed Rectifier ◽  
Hcn Channels ◽  
Dependent Manner ◽  
Spontaneously Hypertensive ◽  
Delayed Rectifier Current ◽  
Underlying Mechanisms

KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.

How to Fluorescently Label the Potassium Channel: A Case in hERG

2020 ◽  
Vol 27 (18) ◽  
pp. 3046-3054
Author(s):  
Xiaomeng Zhang ◽  
Beilei Wang ◽  
Zhenzhen Liu ◽  
Yubin Zhou ◽  
Lupei Du
Keyword(s):  
Potassium Channel ◽  
Herg Channel ◽  
Pore Channel ◽  
Design Rationale ◽  
Related Gene ◽  
Open Conformation ◽  
Cardiac Action ◽  
Qt Syndrome ◽  
Herg Channels ◽  
Action Potential Repolarization

hERG (Human ether-a-go-go-related gene) potassium channel, which plays an essential role in cardiac action potential repolarization, is responsible for inherited and druginduced long QT syndrome. Recently, the Cryo-EM structure capturing the open conformation of hERG channel was determined, thus pushing the study on hERG channel at 3.8 Å resolution. This report focuses primarily on summarizing the design rationale and application of several fluorescent probes that target hERG channels, which enables dynamic and real-time monitoring of potassium pore channel affinity to further advance the understanding of the channels.

scholarly journals Laminin γ1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve

2003 ◽  
Vol 163 (4) ◽  
pp. 889-899 ◽  
Author(s):  
Zu-Lin Chen ◽  
Sidney Strickland
Keyword(s):  
Schwann Cells ◽  
Peripheral Nerves ◽  
Matrix Proteins ◽  
Myelin Proteins ◽  
Sciatic Nerve Crush ◽  
Similar Reduction ◽  
Nerve Crush ◽  
And Function ◽  
Schwann Cell Differentiation

Laminins are heterotrimeric extracellular matrix proteins that regulate cell viability and function. Laminin-2, composed of α2, β1, and γ1 chains, is a major matrix component of the peripheral nervous system (PNS). To investigate the role of laminin in the PNS, we used the Cre–loxP system to disrupt the laminin γ1 gene in Schwann cells. These mice have dramatically reduced expression of laminin γ1 in Schwann cells, which results in a similar reduction in laminin α2 and β1 chains. These mice exhibit motor defects which lead to hind leg paralysis and tremor. During development, Schwann cells that lack laminin γ1 were present in peripheral nerves, and proliferated and underwent apoptosis similar to control mice. However, they were unable to differentiate and synthesize myelin proteins, and therefore unable to sort and myelinate axons. In mutant mice, after sciatic nerve crush, the axons showed impaired regeneration. These experiments demonstrate that laminin is an essential component for axon myelination and regeneration in the PNS.

scholarly journals Deficiency of decay-accelerating factor and complement receptor 1–related gene/protein y on murine platelets leads to complement-dependent clearance by the macrophage phagocytic receptor CRIg

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1109-1119 ◽  
Author(s):  
David D. Kim ◽  
Takashi Miwa ◽  
Yuko Kimura ◽  
Reto A. Schwendener ◽  
Menno van Lookeren Campagne ◽  
...  
Keyword(s):  
Human Platelets ◽  
Transfer Model ◽  
Complement Receptor ◽  
Dependent Manner ◽  
Complement Receptors ◽  
Related Gene ◽  
Decay Accelerating Factor ◽  
Gene Ablation ◽  
Complement Receptor 1

Abstract Complement activation on human platelets is known to cause platelet degranulation and activation. To evaluate how normal platelets escape complement attack in vivo, we studied the fate of murine platelets deficient in 2 membrane complement regulatory proteins using an adoptive transfer model. We show here that deficiency of either decay-accelerating factor (DAF) or complement receptor 1–related gene/protein y (Crry) on murine platelets was inconsequential, whereas DAF and Crry double deficiency led to rapid clearance of platelets from circu-lation in a complement- and macrophage-dependent manner. This finding contrasted with the observation on erythrocytes, where Crry deficiency alone resulted in complement susceptibility. Quantitative flow cytometry revealed that DAF and Crry were expressed at similar levels on platelets, whereas Crry expression was 3 times higher than DAF on erythrocytes. Antibody blocking or gene ablation of the newly identified complement receptor CRIg, but not complement receptor 3 (CR3), rescued DAF/Crry-deficient platelets from complement-dependent elimination. Surprisingly, deficiency of CRIg, CR3, and other known complement receptors failed to prevent Crry-deficient erythrocytes from complement-mediated clearance. These results show a critical but redundant role of DAF and Crry in platelet survival and suggest that complement-opsonized platelets and erythrocytes engage different complement receptors on tissue macrophages in vivo.

scholarly journals High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

2021 ◽  
pp. 247255522098504
Author(s):  
Jeffrey R. Simard ◽  
Linda Lee ◽  
Ellen Vieux ◽  
Reina Improgo ◽  
Trang Tieu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rapid Development ◽  
Dependent Manner ◽  
Western Blots ◽  
Advantages And Disadvantages ◽  
Degradation Rates

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

scholarly journals The Expression and Distributions of ANP32A in the Developing Brain

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Shanshan Wang ◽  
Yunliang Wang ◽  
Qingshan Lu ◽  
Xinshan Liu ◽  
Fuyu Wang ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Fetal Brain ◽  
Tissue Expression ◽  
Developing Brain ◽  
Adult Brain ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Growing Up ◽  
Embryonic Mouse ◽  
And Function

Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A (ANP32A), has multiple functions involved in neuritogenesis, transcriptional regulation, and apoptosis. However, whether ANP32A has an effect on the mammalian developing brain is still in question. In this study, it was shown that brain was the organ that expressed the most abundant ANP32A by human multiple tissue expression (MTE) array. The distribution of ANP32A in the different adult brain areas was diverse dramatically, with high expression in cerebellum, temporal lobe, and cerebral cortex and with low expression in pons, medulla oblongata, and spinal cord. The expression of ANP32A was higher in the adult brain than in the fetal brain of not only humans but also mice in a time-dependent manner. ANP32A signals were dispersed accordantly in embryonic mouse brain. However, ANP32A was abundant in the granular layer of the cerebellum and the cerebral cortex when the mice were growing up, as well as in the Purkinje cells of the cerebellum. The variation of expression levels and distribution of ANP32A in the developing brain would imply that ANP32A may play an important role in mammalian brain development, especially in the differentiation and function of neurons in the cerebellum and the cerebral cortex.

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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