Control of Cellular Bcl-x_L Levels by Deamidation-Regulated Degradation

Dho, So Hee,Deverman, Benjamin E.,Lapid, Carlo,Manson, Scott R.,Gan, Lu,Riehm, Jacob J.,Aurora, Rajeev,Kwon, Ki-Sun,Weintraub, Steven J. Public Library of Science 2013 PLoS biology Vol.11 No.6

<P>The cellular concentration of Bcl-x<SUB>L</SUB> is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-x<SUB>L</SUB> undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-x<SUB>L</SUB> is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-x<SUB>L</SUB> for degradation. Additionally, we show that degradation of deamidated Bcl-x<SUB>L</SUB> is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-x<SUB>L</SUB>, underscoring its importance in Bcl-x<SUB>L</SUB> regulation. Our findings strongly suggest that deamidation-regulated Bcl-x<SUB>L</SUB> degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.</P><P><B>Author Summary</B></P><P>Cellular levels of the pro-survival protein Bcl-x<SUB>L</SUB> are an important determinant of cellular susceptibility to many death stimuli, including most cancer therapies. We previously showed that human Bcl-x<SUB>L</SUB> undergoes deamidation – the conversion of two neutral asparaginyl side-chains into negatively charged aspartyl side-chains – a process that occurs spontaneously but is accelerated by the treatment of tumor cells with DNA-damaging agents. Here, we show that deamidation activates a hitherto undetected signal sequence within Bcl-x<SUB>L</SUB> that targets it for degradation by a pathway involving the proteolytic enzyme calpain. This increased degradation of Bcl-x<SUB>L</SUB>, and the consequent enhanced cellular susceptibility to programmed cell death, may contribute to the ability of DNA-damaging agents to kill tumors. We also demonstrate that deamidation of Bcl-x<SUB>L</SUB> has likely been conserved from the simplest metazoans to humans, underscoring the importance of deamidation in the regulation of Bcl-x<SUB>L</SUB>.</P>

Beyond the Role of CD55 as a Complement Component

So Hee Dho,Jae Cheong Lim,Lark Kyun Kim 대한면역학회 2018 Immune Network Vol.18 No.1

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Lee Yeongun,Dho So Hee,Lee Jiyeon,Hwang Ji-Hyun,Kim Minjung,Choi Won-Young,Lee Jin-Young,Lee Jongwon,Chang Woochul,Lee Min Young,Choi Jungmin,Kim Tae-You,Kim Lark Kyun 생화학분자생물학회 2022 Experimental and molecular medicine Vol.54 No.-

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

Lee, Seung-Min,Dho, So Hee,Ju, Sung-Kyu,Maeng, Jin-Soo,Kim, Jeong-Yoon,Kwon, Ki-Sun Kluwer Academic Publishers 2012 Biogerontology Vol.13 No.5

<P>Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated β-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.</P>

Functional Magnetic Resonance Imaging and Diffusion Tensor Imaging for Language Mapping in Brain Tumor Surgery: Validation With Direct Cortical Stimulation and Cortico–Cortical Evoked Potential

Kang Koung Mi,Kim Kyung Min,Kim In Seong,Kim Joo Hyun,Kang Ho,Ji So Young,Dho Yun-Sik,Oh Hyongmin,Park Hee-Pyoung,Seo Han Gil,Kim Sung-Min,Choi Seung Hong,Park Chul-Kee 대한영상의학회 2023 Korean Journal of Radiology Vol.24 No.6

Objective: Functional magnetic resonance imaging (fMRI) and diffusion tensor imaging-derived tractography (DTI-t) contribute to the localization of language areas, but their accuracy remains controversial. This study aimed to investigate the diagnostic performance of preoperative fMRI and DTI-t obtained with a simultaneous multi-slice technique using intraoperative direct cortical stimulation (DCS) or corticocortical evoked potential (CCEP) as reference standards. Materials and Methods: This prospective study included 26 patients (23–74 years; male:female, 13:13) with tumors in the vicinity of Broca’s area who underwent preoperative fMRI and DTI-t. A site-by-site comparison between preoperative (fMRI and DTI-t) and intraoperative language mapping (DCS or CCEP) was performed for 226 cortical sites to calculate the sensitivity and specificity of fMRI and DTI-t for mapping Broca’s areas. For sites with positive signals on fMRI or DTI-t, the true-positive rate (TPR) was calculated based on the concordance and discordance between fMRI and DTI-t. Results: Among 226 cortical sites, DCS was performed in 100 sites and CCEP was performed in 166 sites. The specificities of fMRI and DTI-t ranged from 72.4% (63/87) to 96.8% (122/126), respectively. The sensitivities of fMRI (except for verb generation) and DTI-t were 69.2% (9/13) to 92.3% (12/13) with DCS as the reference standard, and 40.0% (16/40) or lower with CCEP as the reference standard. For sites with preoperative fMRI or DTI-t positivity (n = 82), the TPR was high when fMRI and DTI-t were concordant (81.2% and 100% using DCS and CCEP, respectively, as the reference standards) and low when fMRI and DTI-t were discordant (≤ 24.2%). Conclusion: fMRI and DTI-t are sensitive and specific for mapping Broca’s area compared with DCS and specific but insensitive compared with CCEP. A site with a positive signal on both fMRI and DTI-t represents a high probability of being an essential language area.