Publications by authors named "Sushil Devkota"

14 Publications

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CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

Nat Commun 2021 05 11;12(1):2625. Epub 2021 May 11.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.
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http://dx.doi.org/10.1038/s41467-021-22927-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113449PMC
May 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Impaired AKT signaling and lung tumorigenesis by PIERCE1 ablation in KRAS-mutant non-small cell lung cancer.

Oncogene 2020 09 29;39(36):5876-5887. Epub 2020 Jul 29.

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Korea.

KRAS-mutant non-small cell lung cancer (NSCLC) is a major lung cancer subtype that leads to many cancer-related deaths worldwide. Although numerous studies on KRAS-mutant type NSCLC have been conducted, new oncogenic or tumor suppressive genes need to be detected because a large proportion of NSCLC patients does not respond to currently used therapeutics. Here, we show the tumor-promoting function of a cell cycle-related protein, PIERCE1, in KRAS-mutant NSCLC. Mechanistically, PIERCE1 depletion inhibits cell growth and AKT phosphorylation (pAKT) at S473, which is particularly observed in KRAS-mutant lung cancers. Analyses of AKT-related genes using microarray, immunoblotting, and real-time quantitative PCR indicated that PIERCE1 negatively regulates the gene expression of the AKT suppressor, TRIB3, through the CHOP pathway, which is a key regulatory pathway for TRIB3 expression. Similarly, in vivo analyses of PIERCE1 depletion in the KRAS mutation-related lung cancer mouse models revealed the suppressive effect of PIERCE1 knockout in urethane- and KRAS-induced lung tumorigenesis with decreased pAKT levels observed in the tumors. Tissue microarrays of human lung cancers indicated the expression of PIERCE1 in 83% of lung cancers and its correlation with pAKT expression. Thus, we illustrate how PIERCE1 depletion may serve as a therapeutic strategy against KRAS-mutant NSCLC and propose the clinical benefit of PIERCE1.
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http://dx.doi.org/10.1038/s41388-020-01399-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471098PMC
September 2020

DNAJC14 Ameliorates Inner Ear Degeneration in the DFNB4 Mouse Model.

Mol Ther Methods Clin Dev 2020 Jun 30;17:188-197. Epub 2019 Nov 30.

Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.

The His723Arg (H723R) mutation in , encoding pendrin, is the most prevalent mutation in East Asia, resulting in DFNB4, an autosomal recessive type of genetic hearing loss. Although the main pathological mechanism of H723R was identified as a protein-folding defect in pendrin, there is still no curative treatment for associated hearing loss. Here, we show that H723R-pendrin expression and activity are rescued by activation of the chaperonin DNAJC14. , DNAJC14 was activated via Japanese encephalitis virus (JEV) inoculation, and toxin-attenuated JEV rescued the surface expression and anion exchange activity of H723R-pendrin. Human H723R-pendrin transgenic mice (hH723R Tg) were established in a mouse knockout background, in which only hH723R-pendrin was expressed in the inner ear (Pax2-Cre dependent) to mimic human DFNB4 pathology. Crossing hH723R Tg with DNAJC14-overexpressing mice resulted in reduced cochlear hydrops and more preserved outer hair cells in the cochlea compared to those in hH723R Tg mice. Furthermore, the stria vascularis and spiral ligament were thicker and KCNJ10 expression was increased with DNAJC14 overexpression; however, hearing function and enlarged endolymphatic hydrops were not recovered. These results indicate that DNAJC14 overexpression ameliorates the cochlear degeneration caused by misfolded pendrin and might be a potential therapeutic target for DFNB4.
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http://dx.doi.org/10.1016/j.omtm.2019.11.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940655PMC
June 2020

The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis.

Authors:
Sushil Devkota

BMB Rep 2018 Sep;51(9):437-443

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA.

Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers. [BMB Reports 2018; 51(9): 437-443].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177507PMC
September 2018

Emerging Paradigm of Crosstalk between Autophagy and the Ubiquitin-Proteasome System.

Mol Cells 2017 Dec 12;40(12):897-905. Epub 2017 Dec 12.

Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center, Yonsei University, Seoul 03722, Republic of Korea.

Cellular protein homeostasis is maintained by two major degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Until recently, the UPS and autophagy were considered to be largely independent systems targeting proteins for degradation in the proteasome and lysosome, respectively. However, the identification of crucial roles of molecular players such as ubiquitin and p62 in both of these pathways as well as the observation that blocking the UPS affects autophagy flux and vice versa has generated interest in studying crosstalk between these pathways. Here, we critically review the current understanding of how the UPS and autophagy execute coordinated protein degradation at the molecular level, and shed light on our recent findings indicating an important role of an autophagy-associated transmembrane protein EI24 as a bridging molecule between the UPS and autophagy that functions by regulating the degradation of several E3 ligases with Really Interesting New Gene (RING)-domains.
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http://dx.doi.org/10.14348/molcells.2017.0226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750708PMC
December 2017

Hippocampal TERT Regulates Spatial Memory Formation through Modulation of Neural Development.

Stem Cell Reports 2017 08 27;9(2):543-556. Epub 2017 Jul 27.

Department of Clinical Pharmacology, Institution of Stem Cells and Neuroregeneration, Pharmacy College, Nanjing Medical University, Nanjing 211166, P.R. China. Electronic address:

The molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment.
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http://dx.doi.org/10.1016/j.stemcr.2017.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550029PMC
August 2017

The autophagy process.

Authors:
Sushil Devkota

Oncotarget 2017 Mar;8(12):18623

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

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http://dx.doi.org/10.18632/oncotarget.15951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386633PMC
March 2017

Functional characterization of EI24-induced autophagy in the degradation of RING-domain E3 ligases.

Autophagy 2016 11 19;12(11):2038-2053. Epub 2016 Aug 19.

a Department of Biochemistry, College of Life Science and Biotechnology and Yonsei Laboratory Animal Research Center , Yonsei University , Seoul , Republic of Korea.

Historically, the ubiquitin-proteasome system (UPS) and autophagy pathways were believed to be independent; however, recent data indicate that these pathways engage in crosstalk. To date, the players mediating this crosstalk have been elusive. Here, we show experimentally that EI24 (EI24, autophagy associated transmembrane protein), a key component of basal macroautophagy/autophagy, degrades 14 physiologically important E3 ligases with a RING (really interesting new gene) domain, whereas 5 other ligases were not degraded. Based on the degradation results, we built a statistical model that predicts the RING E3 ligases targeted by EI24 using partial least squares discriminant analysis. Of 381 RING E3 ligases examined computationally, our model predicted 161 EI24 targets. Those targets are primarily involved in transcription, proteolysis, cellular bioenergetics, and apoptosis and regulated by TP53 and MTOR signaling. Collectively, our work demonstrates that EI24 is an essential player in UPS-autophagy crosstalk via degradation of RING E3 ligases. These results indicate a paradigm shift regarding the fate of E3 ligases.
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http://dx.doi.org/10.1080/15548627.2016.1217371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103340PMC
November 2016

Telomerase reverse transcriptase induces basal and amino acid starvation-induced autophagy through mTORC1.

Biochem Biophys Res Commun 2016 09 18;478(3):1198-204. Epub 2016 Aug 18.

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea; Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea. Electronic address:

Telomerase is a reverse transcriptase that consists of the telomerase RNA component (TERC) and the reverse transcriptase catalytic subunit (TERT) and specializes in the elongation of telomere ends. New evidence suggests that beyond classical telomere maintenance, TERT also possesses telomere length-independent functions that are executed via interaction with other binding proteins. One such reported TERT-interacting proteins is mTOR, a master nutrient sensor that is upregulated in several cancers; however, the physiological implications of the TERT-mTOR interaction in normal cellular processes as well as in tumorigenesis are poorly understood. Here, we report that TERT inhibits the kinase activity of mTOR complex 1 (mTORC1) in multiple cell lines, resulting in the activation of autophagy under both basal and amino acid-deprived conditions. Furthermore, TERT-deficient cells display the inability to properly execute the autophagy flux. Functionally, TERT-induced autophagy provides a survival advantage to cells in nutrient-deprived conditions. Collectively, these findings support a model in which gain of TERT function modulates mTORC1 activity and induces autophagy, which is required for metabolic rewiring to scavenge the nutrients necessary for fueling cancer cell growth in challenging tumor microenvironments.
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http://dx.doi.org/10.1016/j.bbrc.2016.08.094DOI Listing
September 2016

EI24 regulates epithelial-to-mesenchymal transition and tumor progression by suppressing TRAF2-mediated NF-κB activity.

Oncotarget 2013 Dec;4(12):2383-96

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.

Tumor metastasis is a multistep process that requires the concerted activity of discrete biological functions. The epithelial-to-mesenchymal transition (EMT) is the most critical mechanism implicated in tumor progression that is controlled by the inflammatory microenvironment. Understanding how an inflammatory microenvironment is maintained and contributes to tumor progression will be crucial for the development of new effective therapies. Here, we report that etoposide induced 2.4 (EI24) has a multifaceted role against tumor progression that is regulated by both EMT and inflammation. Decreased expression levels of EI24 in epithelial tumor cells induced EMT in association with increased cell motility and invasiveness and resistance to anoikis. Overexpression of EI24 resulted in the opposite cell biological characteristics and suppressed in vivometastatic behavior. EI24 attenuated NF-κB activity by binding to the Complex I component TRAF2 and inducing its lysosome-dependent degradation, leading to transcriptional alterations of EMT- and inflammation-related genes. Analysis of clinical samples demonstrated that reduced EI24 expression and copy number was positively correlated with tumor malignancy and poor prognosis. Collectively, these findings establish EI24 as a critical suppressor of tumor progression and implicate EI24 expression level in malignant tumors as a useful therapeutic and diagnostic marker.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926834PMC
http://dx.doi.org/10.18632/oncotarget.1434DOI Listing
December 2013

Ei24, a novel E2F target gene, affects p53-independent cell death upon ultraviolet C irradiation.

J Biol Chem 2013 Oct 6;288(43):31261-7. Epub 2013 Sep 6.

From the Department of Biochemistry, College of Life Science and Biotechnology, and Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Korea and.

The deficiency of retinoblastoma (Rb) gene deregulates E2F transcription factors and thus induces E2F target genes directly or p53 target genes indirectly via mouse p19(Arf) (or p14(ARF) in humans), an E2F target gene. Here, we identified that etoposide-induced 2.4 mRNA (Ei24)/p53-induced gene 8 (Pig8), a p53 target gene involved in apoptosis and autophagy, was up-regulated in Rb(-/-) mouse embryonic fibroblasts (MEFs). The Ei24 promoter was activated by E2F1 via multiple E2F-responsive elements, independently of the previously reported p53-responsive element. Chromatin immunoprecipitation assays revealed that E2F1 directly acts on the mouse Ei24 promoter. We observed that Ei24 expression was suppressed in p53(-/-) MEFs upon UVC irradiation, which was exacerbated in p53(-/-) E2f1(-/-) MEFs, supporting the positive role of E2F1 on Ei24 transcription. Furthermore, Ei24 knockdown sensitized p53(-/-) MEFs against UVC irradiation. Together, our data indicate that Ei24 is a novel E2F target gene contributing to the survival of p53-deficient cells upon UVC irradiation and thus may have a potential significance as a therapeutic target of certain chemotherapy for treating p53-deficient tumors.
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http://dx.doi.org/10.1074/jbc.M113.477570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829436PMC
October 2013

Ei24-deficiency attenuates protein kinase Cα signaling and skin carcinogenesis in mice.

Int J Biochem Cell Biol 2012 Nov 6;44(11):1887-96. Epub 2012 Jul 6.

Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea.

Etoposide-induced gene 24 (Ei24) is a p53 target gene that inhibits growth, induces apoptosis and autophagy, as well as suppresses breast cancer. To evaluate the role of Ei24 in in vivo tumorigenesis, we generated an Ei24-deficient mouse model. Here, we report that, although Ei24 homozygous knockout mice are embryonic lethal, Ei24 heterozygous null mice are attenuated to DMBA/TPA-induced carcinogenesis with regard to the number and size of tumors but not the incidence. Ei24 contains a functional consensus motif, named as an R motif that is highly analogous to amino acids 105-110 of RINCK1, an E3 ligase for protein kinase C (PKC) proteins. We found that Ei24 stabilizes PKCαvia RINCK degradation and competition with RINCK for binding with the C1a domain of PKCα. We also found that Ei24 contributes to PKCα-mediated transactivation of EGFR by promoting PKCα membrane localization and interaction with EGFR. Finally, using Oncomine database we show that Ei24 and EGFR are upregulated in some subsets of human HNSCC. These results suggest that Ei24 is a regulator of the RINCK1-PKCα-EGFR signaling pathway in the development of skin-cancer.
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http://dx.doi.org/10.1016/j.biocel.2012.06.034DOI Listing
November 2012

Pierce1, a novel p53 target gene contributing to the ultraviolet-induced DNA damage response.

Cancer Res 2010 Dec;70(24):10454-63

Department of Biochemistry, College of Life Science and Biotechnology, and Laboratory Animal Research Center, Yonsei University, Seoul, Korea.

Retinoblastoma (Rb) and p53 genes are mutated or inactivated in most human cancers and mutually regulate each other. Recently, we reported that expression of diverse genes was altered in Rb-deficient mouse embryonic fibroblasts (MEF). In this study, we found that Pierce1, a novel transcript upregulated in Rb-deficient MEFs, is a transcriptional target of p53. Although Pierce1 promoter did not respond to the ectopic expression of E2F1, it was strongly activated by p53 via 2 cis-elements. Consistently, the expression of Pierce1 was induced by genotoxic stresses that activate p53 but was not detected in p53-deficient MEFs. Pierce1 was posttranslationally stabilized by ultraviolet C (UVC) irradiation, and UVC-activated ATR (ataxia telangiectasia-mutated and Rad3-related) signaling suppressed proteosomal degradation of Pierce1 protein. Furthermore, knockdown of Pierce1 compromised the checkpoint response of wild-type MEFs to UVC irradiation, accompanying the diminished expression of p53 target genes. Together, our data suggest that Pierce1 is an important p53 target gene contributing to normal DNA damage response and may play crucial roles in maintaining genomic integrity against genotoxic stresses, including UVC irradiation.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0031DOI Listing
December 2010