• Mass Spectrometry Letters
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• 제15권1호
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• pp.1-25
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• 2024

Protein glycosylation, a highly significant and ubiquitous post-translational modification (PTM) in eukaryotic cells, has attracted considerable research interest due to its pivotal role in a wide array of essential biological processes. Conducting a comprehensive analysis of glycoproteins is imperative for understanding glycoprotein bio-functions and identifying glycosylated biomarkers. However, the complexity and heterogeneity of glycan structures, coupled with the low abundance and poor ionization efficiencies of glycopeptides have all contributed to making the analysis and subsequent identification of glycans and glycopeptides much more challenging than any other biopolymers. Nevertheless, the significant advancements in enrichment techniques, chromatographic separation, and mass spectrometric methodologies represent promising avenues for mitigating these challenges. Numerous substrates and multifunctional materials are being designed for glycopeptide enrichment, proving valuable in glycomics and glycoproteomics. Mass spectrometry (MS) is pivotal for probing protein glycosylation, offering sensitivity and structural insight into glycopeptides and glycans. Additionally, enhanced MS-based glycopeptide characterization employs various separation techniques like liquid chromatography, capillary electrophoresis, and ion mobility. In this review, we highlight recent advances in enrichment methods and MS-based separation techniques for analyzing different types of protein glycosylation. This review also discusses various approaches employed for glycan release that facilitate the investigation of the glycosylation sites of the identified glycoproteins. Furthermore, numerous bioinformatics tools aiding in accurately characterizing glycan and glycopeptides are covered.

• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6429-6436
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• 2014

Poly-ADP-ribosylation (PAR) is a post-translational modification of mainly chromosomal proteins. It is known to be strongly involved in several molecular events, including nucleosome-remodelling and carcinogenesis. In this investigation, it was attempted to evaluate PAR level as a reliable biomarker for early detection of cancer in blood lymphocyte histones. PAR of isolated histone proteins was monitored in normal and dimethylnitrosamine (DMN)-exposed mice tissues using a novel ELISA-based immuno-probe assay developed in our laboratory. An inverse relationship was found between the level of PAR and period of DMN exposure in various histone proteins of blood lymphocytes and spleen cells. With the increase in the DMN exposure period, there was reduction in the PAR level of individual histones in both cases. It was also observed that the decrease in the level of PAR of histones resulted in progressive relaxation of genomic DNA, perhaps triggering activation of genes that are involved in initiation of transformation. The observed effect of carcinogen on the PAR of blood lymphocyte histones provided us with a handy tool for monitoring biochemical or physiological status of individuals exposed to carcinogens without obtaining biopsies of cancerous tissues, which involves several medical and ethical issues. Obtaining blood from any patient and separating blood lymphocytes are routine medical practices involving virtually no medical intervention, post-procedure medical care or trauma to a patient. Moreover, the immuno-probe assay is very simple, sensitive, reliable and cost-effective. Therefore, combined with the ease of preparation of blood lymphocytes and the simplicity of the technique, immuno-probe assay of PAR has the potential to be applied for mass screening of cancer. It appears to be a promising step in the ultimate goal of making cancer detection simple, sensitive and reliable in the near future.

• KSBB Journal
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• 제24권3호
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• pp.217-226
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• 2009

p53은 전사인자로서 세포의 사멸이나 세포주기 조절 등 다양한 세포 활성을 보이기 때문에 일반적인 환경에서는 매우 낮은 수준으로 단백질 양이 확인된다. p53의 단백질 양과 활성은 다양한 세포 내 신호에 의하여 이루어지는 후전사 변형을 통하여 조절 받는다. 이중 유비퀴틴화는 세포 내에서 p53 단백질의 발현 수준이 낮게 유지되는 것이 가능하게 하는 대표적인 기전이다. 이러한 기전을 일으키는 대표적인 p53의 E3 ligase로는 mdm2, Pirh2, COP1, ARF-BP1 등이 보고되어 있으며, 각각 negative feedback loop나 다른 기전을 통하여 p53 단백질의 분해를 유도하여 세포의 항상성을 조절한다. 이 밖에도 p53은 mdm2나 WWP1, UBC13, MSL2와 같은 E3 ligase로 인해서 모노 유비퀴틴화 되고, p53의 세포 내 위치가 조절되어 전사인자로서의 활성이 억제된다. p53의 세포 내 위치와는 관계없이 p53의 전사인자로써의 활성 또한 아세틸화와 유비퀴틴화의 경쟁적 반응으로 인해 조절 될 수 있다. E4F1에 의한 유비퀴틴화는 세포주기와 관련된 유전자의 발현을 증가시키되 세포사멸 관련 유전자의 발현은 감소시키는 것으로 보아 p53의 수많은 downstream gene의 발현 또한 유비퀴틴화를 통해 조절 될 수 있음이 제시되었다. 앞으로의 연구는 신규 E3 ligase에 의한 p53의 유비퀴틴화 기전 연구 뿐 아니라 이와 관련된 다른 변형과의 관계에 대한 연구 또한 매우 중요하게 부각되어 질 것으로 예상된다.

• 생명과학회지
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• 제23권4호
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• pp.586-594
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• 2013

단백질 번역 후 O-GlcNAc 수식은 단백질 조절의 새로운 기전으로 대두되고 있다. 전통적인 당수식과 달리 O-GlcNAc 수식은 단 한번의 O-GlcNAc 전달로 이루어지며, 핵 및 세포질단백질 모두에 수식될 수 있다. O-GlcNAc은 이 분자를 끝으로 하는 최종수식으로 생각되어 왔으나, 최근의 논문(J Proteome Res. 2011 10:2725-2733)은 AP180 단백질에 O-GlcNAc-P가 존재함을 보고하였다. 이 논문은 O-GlcNAc-P가 일반적인 단백질수식인지에 대한 중요한 질문을 던진다. 이에 답하고자 저자들은 HEK293T 세포에 O-GlcNAc 인산화효소 NAGK를 DsRed2에 연결한 DsRed2-$NAGK_{WT}$ 혹은 효소활성이 없는 돌연변이 NAGK를 표현하는 DsRed2-$NAGK_{D107A}$를 표현시키고, 단백질 추출물을 얻어 2D-PAGE로 분리한 후 인산화 정도를 측정하여, $NAGK_{WT}$에 의하여 인산화가 증가되는 15개의 단백질 스폿을 선별하였다. 이 가운데 7개 스팟을 동정한 결과 2개의 스폿은 O-GlcNAc 수식 단백질인 $HSP90{\beta}$, 다른 2개의 스폿도 O-GlcNAc 수식 단백질인 ENO1로 동정되었으며, 나머지(dUTP nucleotidohydrolase mitochondrial isoform 2, glutathione S-transferase P, grp94)는 O-GlcNAc 수식 여부를 아직 모르는 단백질이였다. NAGK에 의하여 O-GlcNAc 단백질의 인산화가 증가된다는 사실은 O-GlcNAc이 인산화되어 O-GlcNAc-P로 수식됨을 시사하며, 따라서 본 연구의 결과는 O-GlcNAc이 최종 수식이 아님을 지지한다.

• Journal of Photoscience
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• 제9권2호
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• pp.229-232
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• 2002

There are two distinct UV-responsive signaling pathways in UV-irradiated mammalian cells, i.e., the DNA damage-dependent and -independent pathways. The former occurs in nucleus and results in growth arrest and apoptosis via post-translational modification of p53. The latter is initiated by oxidative stress and/or by damages in cell membrane or cytoplasm, which activate signaling cascade through intracellular molecules including mitogen activated protein kinases (MAPK). In normal human fibroblastic cells, all of MAPK family members, extracellular signal-related kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, were rapidly phosphorylated following UV-irradiation. ERK phosphorylation was suppressed by an inhibitor of receptor tyrosine kinases (RTK). As ERK usually responds to mitogenic stimuli from RTK ligands, UV-induced ERK phosphorylation may be linked to the proliferation of survived cells. In contrast, phosphorylation of JNK and p38, as well as apoptosis, were modulated by the level of UV-generated oxidative stress Therefore, JNK and p38 may take part in oxidative stress-mediated apoptosis. Phosphorylation of p53 at Ser and Thr residues are essential for stabilization and activation of p53. Among several sites reported, we confirmed phosphorylation at Ser-15 and Ser-392 after UV-irradiation. Both of these were inhibited by a phosphoinositide 3-kinase inhibitor, presumably due to the shutdown of signals from DNA damage to p53. Phosphorylation at Ser-392 was also sensitive to an antioxidant and a p38 inhibitor, suggesting that Ser-392 of p53 is one of the possible points where DNA damage-dependent and -independent apoptic signals merge. Thus, MAPK pathway links UV-induced intracellular signals to the nuclear responses and modifies DNA damage-dependent cellular outcome, resulting in the determination of cell death.

• BMB Reports
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• 제48권6호
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• pp.354-359
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• 2015

Vascular smooth muscle cells (VSMCs) undergo death during atherosclerosis, a widespread cardiovascular disease. Recent studies suggest that oxidative damage occurs in VSMCs and induces atherosclerosis. Here, we analyzed oxidative damage repair in VSMCs and found that VSMCs are hypersensitive to oxidative damage. Further analysis showed that oxidative damage repair in VSMCs is suppressed by a low level of poly (ADP-ribosyl)ation (PARylation), a key post-translational modification in oxidative damage repair. The low level of PARylation is not caused by the lack of PARP-1, the major poly(ADP-ribose) polymerase activated by oxidative damage. Instead, the expression of poly(ADP-ribose) glycohydrolase, PARG, the enzyme hydrolyzing poly(ADP-ribose), is significantly higher in VSMCs than that in the control cells. Using PARG inhibitor to suppress PARG activity facilitates oxidative damage-induced PARylation as well as DNA damage repair. Thus, our study demonstrates a novel molecular mechanism for oxidative damage-induced VSMCs death. This study also identifies the use of PARG inhibitors as a potential treatment for atherosclerosis. [BMB Reports 2015; 48(6): 354-359]

• Journal of Korean Neurosurgical Society
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• 제29권2호
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• pp.155-166
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• 2000

This study was designed to investigate the underlying mechanisms for the temporal changes of the striatal dopamine D2 receptors in the rat model of parkinsonism. After injection of the 6-hydroxydopamine into the substantia nigra of adult rats, we measured the receptor binding capacity(Bmax), mRNA and protein of the D2 receptor at 2, 4 and 8 weeks. Following the lesion, mRNA and protein were elevated simultaneously on both sides of the striata. They showed more increase on the normal side at 2 and 4 weeks, and then they were almost equally abundant on both sides at 8 weeks. We also observed their increased production in the diffuse cortical and subcortical regions. The Bmax value also increased bilaterally in both striata, and was higher on the normal side at 2 weeks and then on the lesioned side at 4 and 8 weeks. These findings suggest that production of the striatal D2 receptor is regulated at the transcriptional level in this animal model. They also imply that this control may be mediated through a pathway which can have influence on the whole brain, rather than the local control of the dopamine content alone. The measured functional activity(Bmax) of the D2 receptor was not proportional to the amount of the receptor mRNA and proteins produced. This difference may be explained by the post-translational modification of the receptor proteins, which may be controlled by such factor as the local concentration of dopamine.

• 한국환경농학회지
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• 제25권4호
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• pp.371-381
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• 2006

New proteomic techniques have been pioneered extensively in recent years, enabling the high-throughput and systematic analyses of cellular proteins in combination with bioinformatic tools. Furthermore, the development of such novel proteomic techniques facilitates the elucidation of the functions of proteins under stress or disease conditions, resulting in the discovery of biomarkers for responses to environmental stimuli. The ultimate objective of proteomics is targeted toward the entire proteome of life, subcellular localization biochemical activities, and the regulation thereof. Comprehensive analysis strategies of proteomics can be classified into three categories: (i) protein separation via 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification via either Edman sequencing or mass spectrometry (MS), and (iii) proteome quantitation. Currently, MS-based proteomics techniques have shifted from qualitative proteome analysis via 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, toward quantitative proteome analysis. In vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes. protein-labeling tagging with isotope-coded affinity tags, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope-labeled amino acids can be in vivo labeled into live culture cells via metabolic incorporation. MS-based proteomics techniques extend to the detection of the phosphopeptide mapping of biologically crucial proteins, which ale associated with post-translational modification. These complementary proteomic techniques contribute to our current understanding of the manner in which life responds to differing environment.

• The Korean Journal of Physiology and Pharmacology
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• 제22권6호
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• pp.617-625
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• 2018

Neddylation is a post-translational protein modification process. MLN4924 is a newly discovered pharmaceutical neddylation inhibitor that suppresses cancer growth with several cancer types. In our study, we first investigated the effect of MLN4924 on colon cancer cells (HCT116 and HT29). MLN4924 significantly inhibited the neddylation of cullin-1 and colon cancer cell growth in a time and dose-dependent manner. MLN4924 induced G2/M cell cycle arrest and apoptosis in HCT116 and HT29 cells. Moreover, MLN4924 also triggered autophagy in HCT116 and HT29 cells via suppressing the PI3K/AKT/mTOR pathway. Inhibiting autophagy by autophagy inhibitor 3-MA or ATG5 knockdown reversed the function of MLN4924 in suppressing colon cancer cell growth and cell death. Interestingly, MLN4924 suppresses colon cell growth in a xenograft model. Together, our finding revealed that blocking neddylation is an attractive colon cancer therapy strategy, and autophagy might act as a novel anti-cancer mechanism for the treatment of colon cancer by MLN4924.

• KSBB Journal
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• 제30권3호
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.