Publications by authors named "Hyunwoo Choi"

25 Publications

  • Page 1 of 1

Integrated Quantitative Phosphoproteomics and Cell-based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-related Phosphorylation Sites.

Mol Cells 2021 Jun 23. Epub 2021 Jun 23.

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Korea.

Cardiac hypertrophic signaling cascades resulting in heart failure diseases are mediated by protein phosphorylation. Recent developments in mass spectrometry-based phosphoproteomics have led to the identification of thousands of differentially phosphorylated proteins and their phosphorylation sites. However, functional studies of these differentially phosphorylated proteins have not been conducted in a large-scale or high-throughput manner due to a lack of methods capable of revealing the functional relevance of each phosphorylation site. In this study, an integrated approach combining quantitative phosphoproteomics and cell-based functional screening using phosphorylation competition peptides was developed. A pathological cardiac hypertrophy model, junctate-1 transgenic mice and control mice, were analyzed using label-free quantitative phosphoproteomics to identify differentially phosphorylated proteins and sites. A cell-based functional assay system measuring hypertrophic cell growth of neonatal rat ventricle cardiomyocytes (NRVMs) following phenylephrine treatment was applied, and changes in phosphorylation of individual differentially phosphorylated sites were induced by incorporation of phosphorylation competition peptides conjugated with cell-penetrating peptides. Cell-based functional screening against 18 selected phosphorylation sites identified three phosphorylation sites (Ser-98, Ser-179 of Ldb3, and Ser-1146 of palladin) displaying near-complete inhibition of cardiac hypertrophic growth of NRVMs. Changes in phosphorylation levels of Ser- 98 and Ser-179 in Ldb3 were further confirmed in NRVMs and other pathological/physiological hypertrophy models, including transverse aortic constriction and swimming models, using site-specific phospho-antibodies. Our integrated approach can be used to identify functionally important phosphorylation sites among differentially phosphorylated sites, and unlike conventional approaches, it is easily applicable for large-scale and/or high-throughput analyses.
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http://dx.doi.org/10.14348/molcells.2021.4002DOI Listing
June 2021

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

J Clin Invest 2021 Feb;131(4)

Acceleron Pharma Inc., Cambridge, Massachusetts, USA.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-β superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.
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http://dx.doi.org/10.1172/JCI138634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880416PMC
February 2021

Aberrant role of ALK in tau proteinopathy through autophagosomal dysregulation.

Mol Psychiatry 2021 Jan 15. Epub 2021 Jan 15.

School of Biological Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.

Proteinopathy in neurodegenerative diseases is typically characterized by deteriorating activity of specific protein aggregates. In tauopathies, including Alzheimer's disease (AD), tau protein abnormally accumulates and induces dysfunction of the affected neurons. Despite active identification of tau modifications responsible for tau aggregation, a critical modulator inducing tau proteinopathy by affecting its protein degradation flux is not known. Here, we report that anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase, is crucial for the tau-mediated AD pathology. ALK caused abnormal accumulation of highly phosphorylated tau in the somatodendritic region of neurons through its tyrosine kinase activity. ALK-induced LC3-positive axon swelling and loss of spine density, leading to tau-dependent neuronal degeneration. Notably, ALK activation in neurons impaired Stx17-dependent autophagosome maturation and this defect was reversed by a dominant-negative Grb2. In a Drosophila melanogaster model, transgenic flies neuronally expressing active Drosophila Alk exhibited the aggravated tau rough eye phenotype with retinal degeneration and shortened lifespan. In contrast, expression of kinase-dead Alk blocked these phenotypes. Consistent with the previous RNAseq analysis showing upregulation of ALK expression in AD [1], ALK levels were significantly elevated in the brains of AD patients showing autophagosomal defects. Injection of an ALK.Fc-lentivirus exacerbated memory impairment in 3xTg-AD mice. Conversely, pharmacologic inhibition of ALK activity with inhibitors reversed the memory impairment and tau accumulation in both 3xTg-AD and tauC3 (caspase-cleaved tau) transgenic mice. Together, we propose that aberrantly activated ALK is a bona fide mediator of tau proteinopathy that disrupts autophagosome maturation and causes tau accumulation and aggregation, leading to neuronal dysfunction in AD.
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http://dx.doi.org/10.1038/s41380-020-01003-yDOI Listing
January 2021

Ascorbic acid-mediated reductive disassembly of Fe-tannic acid shells in degradable single-cell nanoencapsulation.

Chem Commun (Camb) 2020 Nov 1;56(89):13748-13751. Epub 2020 Oct 1.

Center for Cell-Encapsulation Research, Department of Chemistry, KAIST, Daejeon 34141, Korea.

Rapid degradation of Fe-tannic acid films is achieved under mild conditions via ascorbic acid-mediated Fe reduction, which overcomes the problems in the disassembly of a metal-organic complex including slow reaction rates and reaction incompatibility with living cells. The strategy of reductive disassembly is applied to degradable single-cell nanoencapsulation, providing an advanced tool for tightly controlling and manipulating the cell-material interface.
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http://dx.doi.org/10.1039/d0cc05856dDOI Listing
November 2020

Fabrication and Characterization of Neurocompatible Ulvan-Based Layer-by-Layer Films.

Langmuir 2020 10 23;36(39):11610-11617. Epub 2020 Sep 23.

Department of Chemistry, KAIST, Daejeon 34141, Korea.

Construction of extracellular matrix-mimetic nanofilms has considerable potential in biomedical and nanomedicinal fields. In this work, we fabricated neurocompatible layer-by-layer (LbL) films based on ulvan (ULV), a highly sulfated polysaccharide having compositional similarity to glycosaminoglycans that play important functional roles in the brain. ULV was durably assembled as a film with chitosan, another marine-derived polysaccharide, and the film enabled the stable adhesion of primary hippocampal neurons with high viability, comparable to the conventional poly-d-lysine surface. Notably, the ULV-based LbL films accelerated neurite outgrowth and selectively suppressed the adhesion of astrocytes, highlighting its potential as an advanced platform for neural implants and devices.
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http://dx.doi.org/10.1021/acs.langmuir.0c02173DOI Listing
October 2020

Enzymatically degradable, starch-based layer-by-layer films: application to cytocompatible single-cell nanoencapsulation.

Soft Matter 2020 Jul;16(26):6063-6071

Center for Cell-Encapsulation Research, Department of Chemistry, KAIST, Daejeon 34141, Korea.

The build-up and degradation of cytocompatible nanofilms in a controlled fashion have great potential in biomedical and nanomedicinal fields, including single-cell nanoencapsulation (SCNE). Herein, we report the fabrication of biodegradable films of cationic starch (c-ST) and anionic alginate (ALG) by electrostatically driven layer-by-layer (LbL) assembly technology and its application to the SCNE. The [c-ST/ALG] multilayer nanofilms, assembled either on individual Saccharomyces cerevisiae or on the 2D flat gold surface, degrade on demand, in a cytocompatible fashion, via treatment with α-amylase. Their degradation profiles are investigated, while systematically changing the α-amylase concentration, by several surface characterization techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. DNA incorporation in the LbL nanofilms and its controlled release, upon exposure of the nanofilms to an aqueous α-amylase solution, are demonstrated. The highly cytocompatible nature of the film-forming and -degrading conditions is assessed in the c-ST/ALG-shell formation and degradation of S. cerevisiae. We envisage that the cytocompatible, enzymatic degradation of c-ST-based nanofilms paves the way for developing advanced biomedical devices with programmed dissolution in vivo.
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http://dx.doi.org/10.1039/d0sm00876aDOI Listing
July 2020

Neuro-taxis: Neuronal movement in gradients of chemical and physical environments.

Dev Neurobiol 2020 09 6;80(9-10):361-377. Epub 2020 May 6.

Department of Chemistry, KAIST, Daejeon, Korea.

Environmental chemical and physical cues dynamically interact with migrating neurons and sprouting axons, and in particular, the gradients of environmental cues are regarded as one of the factors intimately involved in the neuronal movement. Since a growth cone was first described by Cajal, more than one century ago, chemical gradients have been suggested as one of the mechanisms by which the neurons determine proper paths and destinations. However, the gradients of physical cues, such as stiffness and topography, which also interact constantly with the neurons and their axons as a component of the extracellular environments, have rarely been noted regarding the guidance of neurons, despite their gradually increasingly reported influences in the case of nonneuronal-cell migration. In this review, we discuss chemical (i.e., chemo- and hapto-) and physical (i.e., duro-) taxis phenomena on the movement of neurons including axonal elongation. In addition, we suggest topotaxis, the most recently proposed physical-taxis phenomenon, as another potential mechanism in the neuronal movement, based on the reports of neuronal recognition of and responses to nanotopography.
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http://dx.doi.org/10.1002/dneu.22749DOI Listing
September 2020

Single-Cell Nanoencapsulation: From Passive to Active Shells.

Adv Mater 2020 Sep 7;32(35):e1907001. Epub 2020 Apr 7.

Center for Cell-Encapsulation Research, Department of Chemistry, KAIST, Daejeon, 34141, South Korea.

Single-cell nanoencapsulation is an emerging field in cell-surface engineering, emphasizing the protection of living cells against external harmful stresses in vitro and in vivo. Inspired by the cryptobiotic state found in nature, cell-in-shell structures are formed, which are called artificial spores and which show suppression or retardation in cell growth and division and enhanced cell survival under harsh conditions. The property requirements of the shells suggested for realization of artificial spores, such as durability, permselectivity, degradability, and functionalizability, are demonstrated with various cytocompatible materials and processes. The first-generation shells in single-cell nanoencapsulation are passive in the operation mode, and do not biochemically regulate the cellular metabolism or activities. Recent advances indicate that the field has shifted further toward the formation of active shells. Such shells are intimately involved in the regulation and manipulation of biological processes. Not only endowing the cells with new properties that they do not possess in their native forms, active shells also regulate cellular metabolism and/or rewire biological pathways. Recent developments in shell formation for microbial and mammalian cells are discussed and an outlook on the field is given.
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http://dx.doi.org/10.1002/adma.201907001DOI Listing
September 2020

Astrocyte-Encapsulated Hydrogel Microfibers Enhance Neuronal Circuit Generation.

Adv Healthc Mater 2020 03 20;9(5):e1901072. Epub 2020 Jan 20.

Center for Cell-Encapsulation Research, Department of Chemistry, KAIST, Daejeon, 34141, Korea.

Astrocytes, the most representative glial cells in the brain, play a multitude of crucial functions for proper neuronal development and synaptic-network formation, including neuroprotection as well as physical and chemical support. However, little attention has been paid, in the neuroregenerative medicine and related fields, to the cytoprotective incorporation of astrocytes into neuron-culture scaffolds and full-fledged functional utilization of encapsulated astrocytes for controlled neuronal development. In this article, a 3D neurosupportive culture system for enhanced induction of neuronal circuit generation is reported, where astrocytes are confined in hydrogel microfibers and protected from the outside. The astrocyte-encapsulated microfibers significantly accelerate the neurite outgrowth and guide its directionality, and enhance the synaptic formation, without any physical contact with the neurons. This astrocyte-laden system provides a pivotal culture scaffold for advanced development of cell-based therapeutics for neural injuries, such as spinal cord injury.
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http://dx.doi.org/10.1002/adhm.201901072DOI Listing
March 2020

Correcting Smad1/5/8, mTOR, and VEGFR2 treats pathology in hereditary hemorrhagic telangiectasia models.

J Clin Invest 2020 02;130(2):942-957

Litwin-Zucker Center for Alzheimer's Disease and Memory Disorders and.

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1/ENG/Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K/Akt/mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs - including in HHT patient blood outgrowth ECs - and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in patients with HHT.
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http://dx.doi.org/10.1172/JCI127425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994128PMC
February 2020

Highlights of the 13th International Hereditary Hemorrhagic Telangiectasia Scientific conference.

Angiogenesis 2019 11;22(4):583-584

The Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis Children's Hospital, St. Louis, MO, USA.

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http://dx.doi.org/10.1007/s10456-019-09685-xDOI Listing
November 2019

Application of intraoperative lung-protective ventilation varies in accordance with the knowledge of anaesthesiologists: a single-Centre questionnaire study and a retrospective observational study.

BMC Anesthesiol 2018 04 2;18(1):33. Epub 2018 Apr 2.

Department of anesthesiology and Pain Medicine, anaesthesia and Pain Research Institute, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, South Korea.

Background: The benefits of lung-protective ventilation (LPV) with a low tidal volume (6 mL/kg of ideal body weight [IBW]), limited plateau pressure (< 28-30 cm HO), and appropriate positive end-expiratory pressure (PEEP) in patients with acute respiratory distress syndrome have become apparent and it is now widely adopted in intensive care units. Recently evidence for LPV in general anaesthesia has been accumulated, but it is not yet generally applied by anaesthesiologists in the operating room.

Methods: This study investigated the perception about intraoperative LPV among 82 anaesthesiologists through a questionnaire survey and identified the differences in ventilator settings according to recognition of lung-protective ventilation. Furthermore, we investigated the changes in the trend for using this form of ventilation during general anaesthesia in the past 10 years.

Results: Anaesthesiologists who had received training in LPV were more knowledgeable about this approach. Anaesthesiologists with knowledge of the concept behind LPV strategies applied a lower tidal volume (median (IQR [range]), 8.2 (8.0-9.2 [7.1-10.3]) vs. 9.2 (9.1-10.1 [7.6-10.1]) mL/kg; p = 0.033) and used PEEP more frequently (69/72 [95.8%] vs. 5/8 [62.5%]; p = 0.012; odds ratio, 13.8 [2.19-86.9]) for laparoscopic surgery than did those without such knowledge. Anaesthesiologists who were able to answer a question related to LPV correctly (respondents who chose 'height' to a multiple choice question asking what variables should be considered most important in the initial setting of tidal volume) applied a lower tidal volume in cases of laparoscopic surgery and obese patients. There was an increase in the number of patients receiving LPV (V < 10 mL/kgIBW and PEEP ≥5 cm HO) between 2004 and 2014 (0/818 [0.0%] vs. 280/818 [34.2%]; p <  0.001).

Conclusions: Our study suggests that the knowledge of LPV is directly related to its implementation, and can explain the increase in LPV use in general anaesthesia. Further studies should assess the impact of using intraoperative LPV on clinical outcomes and should determine the efficacy of education on intraoperative LPV implementation.
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http://dx.doi.org/10.1186/s12871-018-0495-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879938PMC
April 2018

The Effect of Electrical Muscle Stimulation and In-bed Cycling on Muscle Strength and Mass of Mechanically Ventilated Patients: A Pilot Study.

Acute Crit Care 2018 Feb 14;33(1):16-22. Epub 2017 Feb 14.

Department of Anesthesiology and Pain Medicine, Anesthesia and Pain Research Institute, Yonsei University College of Medicine, Seoul, Korea.

Background: Critically ill patients experience muscle weakness, which leads to functional disability. Both functional electrical stimulation (FES) and in-bed cycling can be an alternative measure for intensive care unit (ICU) patients who are not feasible for active exercise. The aim of this study was to examine whether FES and in-bed cycling have a positive effect on muscle mass in ICU patients.

Methods: Critically ill patients who received mechanical ventilation for at least 24 hours were included. After passive range of motion exercise, in-bed cycling was applied for 20 minutes, and FES was applied for 20 minutes on the left leg. The right leg received in-bed cycling and the left leg received both FES and in-bed cycling. Thigh circumferences and rectus femoris cross-sectional area (CSA) were assessed with ultrasonography before and after the intervention. Muscle strength was assessed by Medical Research Council scale.

Results: A total of 10 patients were enrolled in this study as a pilot study. Before and after the intervention, the CSA of right rectus femoris increased from 5.08 ± 1.51 cm to 6.01 ± 2.21 cm , which was statistically significant (P = 0.003). The thigh circumference was also increased and statistically significant (P = 0.006). There was no difference between left and right in regard to FES application. There is no significant change in muscle strength before and after the intervention (right and left, P = 0.317 and P = 0.368, respectively).

Conclusions: In-bed cycling increased thigh circumferences rectus femoris CSA. Adding FES did not show differences.
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http://dx.doi.org/10.4266/acc.2017.00542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849008PMC
February 2018

PKCθ-Mediated PDK1 Phosphorylation Enhances T Cell Activation by Increasing PDK1 Stability.

Mol Cells 2017 Jan 26;40(1):37-44. Epub 2017 Jan 26.

School of Life Sciences and Cell Logistics Research Center, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Korea.

PDK1 is essential for T cell receptor (TCR)-mediated activation of NF-κB, and PDK1-induced phosphorylation of PKCθ is important for TCR-induced NF-κB activation. However, inverse regulation of PDK1 by PKCθ during T cell activation has not been investigated. In this study, we found that PKCθ is involved in human PDK1 phosphorylation and that its kinase activity is crucial for human PDK1 phosphorylation. Mass spectrometry analysis of wild-type PKCθ or of kinase-inactive form of PKCθ revealed that PKCθ induced phosphorylation of human PDK1 at Ser-64. This PKCθ-induced PDK1 phosphorylation positively regulated T cell activation and TCR-induced NF-κB activation. Moreover, phosphorylation of human PDK1 at Ser-64 increased the stability of human PDK1 protein. These results suggest that Ser-64 is an important phosphorylation site that is part of a positive feedback loop for human PDK1-PKCθ-mediated T cell activation.
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http://dx.doi.org/10.14348/molcells.2017.2236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303887PMC
January 2017

Caspase-cleaved tau exhibits rapid memory impairment associated with tau oligomers in a transgenic mouse model.

Neurobiol Dis 2016 Mar 17;87:19-28. Epub 2015 Dec 17.

Global Research Laboratory, School of Biological Sciences, Seoul National University, Seoul 151-747, Republic of Korea. Electronic address:

In neurodegenerative diseases like AD, tau forms neurofibrillary tangles, composed of tau protein. In the AD brain, activated caspases cleave tau at the 421th Asp, generating a caspase-cleaved form of tau, TauC3. Although TauC3 is known to assemble rapidly into filaments in vitro, a role of TauC3 in vivo remains unclear. Here, we generated a transgenic mouse expressing human TauC3 using a neuron-specific promoter. In this mouse, we found that human TauC3 was expressed in the hippocampus and cortex. Interestingly, TauC3 mice showed drastic learning and spatial memory deficits and reduced synaptic density at a young age (2-3months). Notably, tau oligomers as well as tau aggregates were found in TauC3 mice showing memory deficits. Further, i.p. or i.c.v. injection with methylene blue or Congo red, inhibitors of tau aggregation in vitro, and i.p. injection with rapamycin significantly reduced the amounts of tau oligomers in the hippocampus, rescued spine density, and attenuated memory impairment in TauC3 mice. Together, these results suggest that TauC3 facilitates early memory impairment in transgenic mice accompanied with tau oligomer formation, providing insight into the role of TauC3 in the AD pathogenesis associated with tau oligomers and a useful AD model to test drug candidates.
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http://dx.doi.org/10.1016/j.nbd.2015.12.006DOI Listing
March 2016

ENC1 Modulates the Aggregation and Neurotoxicity of Mutant Huntingtin Through p62 Under ER Stress.

Mol Neurobiol 2016 12 5;53(10):6620-6634. Epub 2015 Dec 5.

Global Research Laboratory, School of Biological Science/Bio-MAX Institute, Seoul National University, Seoul, 151-742, South Korea.

Huntington's disease (HD) is a devastating neurodegenerative disorder, which is caused by the expression and aggregation of polyQ-expanded mutant huntingtin protein (mtHTT). While toxic mtHTT aggregates are primarily eliminated through autophagy, autophagy dysfunction is often observed in HD pathogenesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.
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http://dx.doi.org/10.1007/s12035-015-9557-8DOI Listing
December 2016

OCIAD2 activates γ-secretase to enhance amyloid β production by interacting with nicastrin.

Cell Mol Life Sci 2014 Jul 24;71(13):2561-76. Epub 2013 Nov 24.

Creative Research Initiative (CRI)-Acceleration Research Laboratory, School of Biological Science/Bio-MAX Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Korea.

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aβ), a key molecule that causes major neurotoxicity during the early stage of Alzheimer's disease (AD). However, despite its important role in Aβ production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aβ production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aβ production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aβ generation.
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http://dx.doi.org/10.1007/s00018-013-1515-xDOI Listing
July 2014

Fabrication of vertically well-aligned P(VDF-TrFE) nanorod arrays.

Adv Mater 2012 Nov 20;24(42):5708-12. Epub 2012 Aug 20.

Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Korea.

By combining the merits of traditional template-assisted methods for polymer nanostructure fabrication, we demonstrate an immersion crystallization process that combines features of polymer crystallization and template removal simultaneously. Well-aligned poly(vinylidene fluoride-trifluoroethylene) copolymer nanorod arrays are prepared for the first time via this simple and convenient new method.
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http://dx.doi.org/10.1002/adma.201201940DOI Listing
November 2012

Neuropathogenic role of adenylate kinase-1 in Aβ-mediated tau phosphorylation via AMPK and GSK3β.

Hum Mol Genet 2012 Jun 13;21(12):2725-37. Epub 2012 Mar 13.

Global Research Laboratory, School of Biological Science/Bio-MAX Institute, Seoul National University, Seoul, South Korea.

Abnormally hyperphosphorylated tau is often caused by tau kinases, such as GSK3β and Cdk5. Such occurrence leads to neurofibrillary tangle formation and neuronal degeneration in tauopathy, including Alzheimer's disease (AD). However, little is known about the signaling cascade underlying the pathologic phosphorylation of tau by Aβ(42). In this study, we show that adenylate kinase 1 (AK1) is a novel regulator of abnormal tau phosphorylation. AK1 expression is markedly increased in the brains of AD patients and AD model mice and is significantly induced by Aβ(42) in the primary neurons. Ectopic expression of AK1 alone augments the pathologic phosphorylation of tau at PHF1, CP13 and AT180 epitopes and enhances the formation of tau aggregates. Inversely, downregulation of AK1 alleviates Aβ(42)-induced hyperphosphorylation of tau. AK1 plays a role in Aβ(42)-induced impairment of AMPK activity and GSK3β activation in the primary neurons. Pharmacologic studies show that treatment with an AMPK inhibitor activates GSK3β, and a GSK3β inhibitor attenuates AK1-mediated tau phosphorylation. In a Drosophila model of human tauopathy, the retinal expression of human AK1 severely exacerbates rough eye phenotype and increases abnormal tau phosphorylation. Further, neural expression of AK1 reduces the lifespan of tau transgenic files. Taken together, these observations indicate that the neuronal expression of AK1 is induced by Aβ(42) to increase abnormal tau phosphorylation via AMPK-GSK3β and contributes to tau-mediated neurodegeneration, providing a new upstream modulator of GSK3β in the pathologic phosphorylation of tau.
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http://dx.doi.org/10.1093/hmg/dds100DOI Listing
June 2012

Quantitative measurement of in-plane cantilever torsion for calibrating lateral piezoresponse force microscopy.

Rev Sci Instrum 2011 Nov;82(11):113706

Materials Science Division, Argonne National Laboratory, Lemont, Illinois 60439, USA.

A simple quantitative measurement procedure of in-plane cantilever torsion for calibrating lateral piezoresponse force microscopy is presented. This technique enables one to determine the corresponding lateral inverse optical lever sensitivity (LIOLS) of the cantilever on the given sample. Piezoelectric coefficient, d(31) of BaTiO(3) single crystal (-81.62 ± 40.22 pm/V) which was calculated using the estimated LIOLS was in good agreement with the reported value in literature.
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http://dx.doi.org/10.1063/1.3660806DOI Listing
November 2011

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification.

J Chromatogr B Analyt Technol Biomed Life Sci 2011 Oct 31;879(28):2991-7. Epub 2011 Aug 31.

School of Life Sciences, Gwangju Institute of Science & Technology, 1 Oryong-Dong, Buk-Gu, Gwangju 500-712, Republic of Korea.

Immobilized metal affinity chromatography (IMAC) and metal oxide type affinity chromatography (MOAC) techniques have been widely used for mass spectrometry-based phosphorylation analysis. Unlike MOAC techniques, IMAC requires rather complete removals of buffering reagents, salts and high concentrations of denaturant prior to sample loading in order for the successful enrichment of phosphopeptides. In this study, a simple off-line capillary column-based IMAC phosphopeptide enrichment method can shorten sample preparation time by eliminating the speed-vac step from the desalting process. Tryptic digest peptide samples containing 2M urea can be directly processed and the entire IMAC procedure can be completed within 6 h. When tryptic digest peptide samples prepared from mouse whole brain tissues were analyzed using our method, an average of 249 phosphoproteins and 463 unique phosphopeptides were identified from single 2-h RPLC-MS/MS analysis (~88% specificity). An additional advantage of this method is the significantly improved reproducibility of the phosphopeptide enrichment results. When four independent phosphopeptide enrichment experiments were carried out, the peak areas of phosphopeptides identified among four enrichment experiments were relatively similar (less than 16.2% relative standard dev.). Because of this increased reproducibility, relative phosphorylation quantification analysis of major phosphoproteins appears to be feasible without the need for stable isotope labeling techniques.
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http://dx.doi.org/10.1016/j.jchromb.2011.08.035DOI Listing
October 2011

Lithium rescues the impaired autophagy process in CbCln3(Δex7/8/Δex7/8) cerebellar cells and reduces neuronal vulnerability to cell death via IMPase inhibition.

J Neurochem 2011 Feb 19;116(4):659-68. Epub 2011 Jan 19.

Creative Research Initiative (CRI)-Acceleration Research Laboratory, School of Biological Science/Bio-MAX Institute, Seoul National University, Seoul, Korea.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a neurodegenerative disorder caused by mutation in CLN3. Defective autophagy and concomitant accumulation of autofluorescence enriched with mitochondrial ATP synthase subunit c were previously discovered in Cln3 mutant knock-in mice. In this study, we show that treatment with lithium reduces numbers of LC3-positive autophagosomes and accumulation of LC3-II in Cln3 mutant knock-in cerebellar cells (CbCln3(Δex7/8/Δex7/8) ). Lithium, an inhibitor of GSK3 and IMPase, reduces the accumulation of mitochondrial ATP synthase subunit c and autofluorescence in CbCln3(Δex7/8/Δex7/8) cells, and mitigates the abnormal subcellular distribution of acidic vesicles in the cells. L690,330, an IMPase inhibitor, is as effective as lithium in restoring autophagy in CbCln3(Δex7/8/Δex7/8) cells. Moreover, lithium or down-regulation of IMPase expression protects CbCln3(Δex7/8/Δex7/8) cells from cell death induced by amino acid deprivation. These results suggest that lithium overcomes the autophagic defect in CbCln3(Δex7/8/Δex7/8) cerebellar cells probably through IMPase, thereby reducing their vulnerability to cell death.
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http://dx.doi.org/10.1111/j.1471-4159.2010.07158.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517618PMC
February 2011

Detection of multiphosphorylated peptides in LC-MS/MS analysis under low pH conditions.

Anal Chem 2008 Apr 18;80(8):3007-15. Epub 2008 Mar 18.

Department of Life Sciences, Gwangju Institute of Science & Technology 1 Oryong-Dong, Buk-Gu, Gwangju, Korea 500-712.

An improved method of detection of multiphosphorylated peptides by RPLC-MS/MS analysis under low pH conditions (pH approximately 1.7, 3% formic acid) is demonstrated for the model phosphoproteins, bovine alpha- and beta-casein. Changes in the pH conditions from normal (pH approximately 3.0, 0.1% formic acid) to low (pH approximately 1.7, 3% formic acid) significantly improved the detection limit of multiphosphorylated peptides carrying negative (-) solution charge states. In particular, bovine beta-casein tetraphosphorylated peptide, was detected with a loading amount of only 50 fmol of trypsin-digested bovine beta-casein under low pH conditions, which is 200 times lower than necessary to detect the peptide under normal pH conditions. In order to understand the low pH effect, various loading amounts of trypsin-digested bovine alpha- and beta-caseins were analyzed by RPLC-MS/MS analyses under two different pH conditions. The question of whether the low pH condition improves the detection of multiphosphorylated peptides by increasing ionization efficiencies could not be proven in this study because synthetic multiphosphorylated peptides could not be easily obtained by peptide synthesis. Interestingly, increased hydrophilicity resulting from multiple phosphorylation events is shown to negatively affect the peptide retention on reversed-phase column material. It was also demonstrated that the low pH condition could effectively enhance the retention of multiphosphorylated peptides on reversed-phase column material. The usefulness of low pH RPLC analysis was tested using an actual phosphopeptide-enriched sample prepared from mouse brain tissues. Previously, low pH solvents have been used in SCX fractionation and TiO2 enrichment processes to selectively enrich phosphopeptides during the phosphopeptide enrichment procedure, but the improved detection of multiphosphorylated peptides in RPLC-MS/MS analysis under low pH conditions has not been reported before (Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P. Mol. Cell. Proteomics 2004, 3, 1093-1101. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1488-1493. Schlosser, A.; Vanselow, J. T.; Kramer, A. Anal. Chem. 2005, 77, 5243-5250.).
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http://dx.doi.org/10.1021/ac7023393DOI Listing
April 2008

Improved detection of multi-phosphorylated peptides by LC-MS/MS without phosphopeptide enrichment.

Mol Cells 2007 Jun;23(3):340-8

Department of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi- phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.
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June 2007

Neuronal vulnerability of CLN3 deletion to calcium-induced cytotoxicity is mediated by calsenilin.

Hum Mol Genet 2007 Feb 22;16(3):317-26. Epub 2006 Dec 22.

Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.

Calsenilin/DREAM/KChIP3, a neuronal Ca(2+)-binding protein, has multifunctions in nucleus and cytosol. Here, we identified CLN3 as a calsenilin-binding partner whose mutation or deletion is observed in Batten disease. In vitro binding and immunoprecipitation assays show that calsenilin interacts with the C-terminal region of CLN3 and the increase of Ca(2+) concentration in vitro and in cells causes significant dissociation of calsenilin from CLN3. Ectopic expression of CLN3 or its deletion mutant containing only the C-terminus (153-438) and capable of binding to calsenilin suppresses thapsigargin or A23187-induced death of neuronal cells. In contrast, CLN3 deletion mutant containing the N-terminus (1-153) or (1-263), which is frequently found in Batten disease, induces the perturbation of Ca(2+) transient and fails to inhibit the cell death. In addition, the expression of calsenilin is increased in the brain tissues of CLN3 knock-out mice and SH-SY5Y/CLN3 knock-down cells. Down-regulation of CLN3 expression sensitizes SH-SY5Y cells to thapsigargin or A23187. However, additional decrease of calsenilin expression rescues the sensitivity of SH-SY5Y/CLN3 knock-down cells to Ca(2+)-mediated cell death. These results suggest that the vulnerability of CLN3 knock-out or CLN3 deletion (1-153)-expressing neuronal cells to Ca(2+)-induced cell death may be mediated by calsenilin.
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http://dx.doi.org/10.1093/hmg/ddl466DOI Listing
February 2007
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