Characterization of TAMRA- and biotin-conjugated peptide arrays for on-chip matrix metalloproteinase activity assay

공덕훈,Mahendra Prasad Bhatt,이승택,김영명,하권수 한국바이오칩학회 2012 BioChip Journal Vol.6 No.4

Peptide arrays have been widely used for high-throughput determination of protease activities in cells and tissues because specific peptides have high binding affinity for the active site of enzymes. Designing peptide substrate probes for enzyme activity assays have been considered to be important; however, the significance of its reporter tag for detecting enzymatic reactions is relatively underestimated. Thus, we investigated the effect of the reporter tag of peptide substrate probes on on-chip protease activity assays. We optimized and characterized proteolytic activity assay of matrix metalloproteinase-3 using direct and indirect substrate probes, tetramethyl-6-carboxyrhodamine (TAMRA)- and biotin-conjugated peptide arrays, respectively. Proteolytic activity assays using both substrate probes demonstrated similar sensitivity, ratio of maximal to minimal FI, IC50 of GM6001, and interarray reproducibility. However, biotin-conjugated substrate arrays showed a wider dynamic range than TAMRA-conjugated substrate arrays. Thus, this comparative study provides a wealth of information for developing optimal probes necessary for effective analysis of enzyme activity and kinetics.

C-reactive Protein as a Parameter for Defining Normal Blood Samples in Identification and Evaluation of Serological Biomarkers

전혜윤,공덕훈,김수현,서인범,한은택,김영명,하권수 한국바이오칩학회 2015 BioChip Journal Vol.9 No.1

Selecting normal control samples is criticalfor the identification and evaluation of serum biomarkerproteins; however, currently few inclusion parametershave been identified. In this paper, we investigatedwhether serum C-reactive protein (CRP) levelsare a suitable inclusion parameter to define normalcontrols. We analyzed serum α-fetoprotein (AFP), cytokeratin19 fragment (CYFRA 21-1), intercellular adhesionmolecule 1 (ICAM-1), and fibronectin expressionlevels from normal individuals (n=97) and patientswith hepatocellular carcinoma (n=36), lung cancer(n=50), and colorectal cancer (n=30). Receiveroperating characteristic (ROC) analysis of the biomarkerproteins was performed using four differentcontrol groups (C1-C4) with increasing serum CRPlevels. The biomarker expression levels were higher inall three sets of cancer patients when compared withcontrol groups C1 through C3. The AUC, sensitivity,and specificity values of the biomarkers dramaticallydecreased with increasing CRP levels in the controlgroups. These results suggest that the serum CRP levelaffects downstream ROC analysis, and that CRPlevels could be used as a control inclusion parameterto define normal samples for identifying and evaluatingserum cancer biomarkers.

DWN12088, A Prolyl-tRNA Synthetase Inhibitor, Alleviates Hepatic Injury in Nonalcoholic Steatohepatitis

이동건,조수호,이은수,하경봉,박나원,공덕훈,박상인,박준석,Chung Choon Hee 대한당뇨병학회 2024 Diabetes and Metabolism Journal Vol.48 No.1

Background: Nonalcoholic steatohepatitis (NASH) is a liver disease caused by obesity that leads to hepatic lipoapoptosis, resulting in fibrosis and cirrhosis. However, the mechanism underlying NASH is largely unknown, and there is currently no effective therapeutic agent against it. DWN12088, an agent used for treating idiopathic pulmonary fibrosis, is a selective prolyl-tRNA synthetase (PRS) inhibitor that suppresses the synthesis of collagen. However, the mechanism underlying the hepatoprotective effect of DWN12088 is not clear. Therefore, we investigated the role of DWN12088 in NASH progression.Methods: Mice were fed a chow diet or methionine-choline deficient (MCD)-diet, which was administered with DWN12088 or saline by oral gavage for 6 weeks. The effects of DWN12088 on NASH were evaluated by pathophysiological examinations, such as real-time quantitative reverse transcription polymerase chain reaction, immunoblotting, biochemical analysis, and immunohistochemistry. Molecular and cellular mechanisms of hepatic injury were assessed by <i>in vitro</i> cell culture.Results: DWN12088 attenuated palmitic acid (PA)-induced lipid accumulation and lipoapoptosis by downregulating the Rho-kinase (ROCK)/AMP-activated protein kinase (AMPK)/sterol regulatory element-binding protein-1c (SREBP-1c) and protein kinase R-like endoplasmic reticulum kinase (PERK)/α subunit of eukaryotic initiation factor 2 (eIF2α)/activating transcription factor 4 (ATF4)/C/EBP-homologous protein (CHOP) signaling cascades. PA increased but DWN12088 inhibited the phosphorylation of nuclear factor-κB (NF-κB) p65 (Ser536, Ser276) and the expression of proinflammatory genes. Moreover, the DWN12088 inhibited transforming growth factor β (TGFβ)-induced pro-fibrotic gene expression by suppressing TGFβ receptor 1 (TGFβR1)/Smad2/3 and TGFβR1/glutamyl-prolyl-tRNA synthetase (EPRS)/signal transducer and activator of transcription 6 (STAT6) axis signaling. In the case of MCD-diet-induced NASH, DWN12088 reduced hepatic steatosis, inflammation, and lipoapoptosis and prevented the progression of fibrosis.Conclusion: Our findings provide new insights about DWN12088, namely that it plays an important role in the overall improvement of NASH. Hence, DWN12088 shows great potential to be developed as a new integrated therapeutic agent for NASH.

Aster tataricus 물 추출물의 mitogen-activated protein kinase 신호 전달 경로를 통한 면역 조절 효과

이채연(Chea Yeon Lee),박효성(Hyo Sung Park),공덕훈(Deok-Hoon Kong),김영관(Young Kwan Kim),조화정(Whajung Cho) 한국영양학회 2020 Journal of Nutrition and Health Vol.53 No.5

본 연구는 AT의 뿌리를 제외한 전체 AT의 에탄올 및 물 추출물의 면역 조절 효과를 비교하고 THP-1의 cytokine 분비를 조절하는 분자 메커니즘을 조사하였다. AT의 물 추출물 및 에탄올 추출물은 THP-1 세포에 독성이 없으며 세포 증식을 증가키는 것을 확인하였다. 에탄올 추출물은 영향이 없는데 반해, 물 추출물은 THP-1의 IL-1β의 분비를 증가시켰으며 COX-2 및 iNOS 단백질의 발현을 증가시켰다. 또한, MAPK 및 Akt의 인산화와 IkBα의 분해를 유도하는 것을 확인하였다. AT에 의한 IL-1β 분비는 ERK 및 JNK 억제제에 의해 감소되었으며, TNF-α의 분비는 ERK, p38 MAPK 및 JNK 억제제에 의해 감소되었다. 흥미롭게도, p38 MAPK 억제제는 AT에 의한 IL-1β의 생성을 추가로 증가시켰다. 이 결과는 AT 지상부의 물 추출물에 MAPK 신호 전달 경로를 통해 면역 세포를 자극하여 cytokine의 생산을 유도하는 생리활성물질이 존재한다는 것을 의미한다. 따라서, AT 지상부는 면역력 강화제의 천연 소재로써 이용될 수 있을 것으로 사료된다. Purpose: Aster tataricus (AT) is one of the Asteraceae perennial herbs used in traditional Chinese medicine. The herb contains various bioactive substances, such as flavonoids, isoflavonoids, and phenolic compounds in the roots, and exhibits a range of effects including anti-bacterial, anti-oxidant, and anti-inflammatory activities. This study compared the immunomodulatory effects of ethanol and water extracts of whole AT, except the roots, and analyzed the molecular mechanisms for the regulatory effects on cytokine secretion from THP-1 cells. Methods: The effects of AT extract on the cell viability and proliferation of THP-1 cells were analyzed using the Cell Counting Kit-8 method. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture supernatant of the AT-treated THP-1 cells were measured using an enzyme-linked immunosorbent assay. The protein levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor kappa B (IκBα), and mitogen-activated protein kinase (MAPK) phosphorylation in the cell lysates were determined by western blotting. Results: The water extract and the ethanol extract of AT did not affect the cell viability, and increased the proliferation of THP-1 cells significantly compared to the vehicle. The water extract increased the secretion of IL-1β from THP-1 cells in a dose-dependent manner, but the ethanol extract had no effect. The expression of COX-2 and iNOS protein and the phosphorylation of MAPK and Akt were induced in AT-treated cells. In addition, IκBα was degraded by AT in a concentration-dependent manner. IL-1β secretion by AT was reduced by extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors, while TNF-α secretion was decreased by inhibitors of ERK, p38 MAPK, and JNK. Interestingly, the p38 MAPK inhibitor increased the production of IL-1β by AT further. Conclusion: The water extract of the above-ground parts of AT contains immunomodulatory bioactive substances that stimulate immune cells through the MAPK signaling pathway.