Human Cytomegalovirus의 UL97 단백질 인산화 효소의 기질 특이성 연구를 위한 Peptide Library의 스크리닝

백문창(Moon-Chang Baek) 대한미생물학회 2006 Journal of Bacteriology and Virology Vol.36 No.2

KESYSVYVYKV로부터 변형된 펩타이드 기질을 이용한 항바이러스제의 타깃이 되는 UL97 단백질 인산화 효소의 기질 특이성

백문창,Baek, Moon-Chang 대한약학회 2008 약학회지 Vol.52 No.6

Human cytomegalovirus expresses an unusual protein kinase UL97, a member of ${H_V}{U_L}$ family of protein kinase. UL97 can phosphorylate nucleoside analogs such as ganciclovir as well as protein/peptide. It has previously been reported that UL97 is able to phosphorylate a KESYSVYVYKV peptide and that P+5 position (K) is important. We examined the extent of contribution of other positions (P-4 through P+6) of the peptide to be substrate of UL97 using alanine substituted peptides (Ala scanning) and deleted peptides. The result suggested that the E (P-2) is negative effect and P+5 (K) is still important. The peptide YSVYVYK is the shortest substrate enough to show high activity, which could be a starting point to develop peptidomimetic drug. This study would give important information to deeply understand the substrate specificity of UL97 and develop an antiviral drug using the small peptide identified here.

Spot Assay를 통한 Human Cytomegalovirus의 UL97 단백질 인산화 효소의 기질 특이성

백문창,Baek, Moon-Chang 대한약학회 2006 약학회지 Vol.50 No.4

Protein kinase UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs as well as protein/peptide. Previously we found a H2B-derived peptide, KESYSVYVYKV and reported that the P+5 position (K) is important. To further understand the substrate specificity at the P+5 position, we introduced spot assay system and showed that a peptide containing K residue among other amino acids at the P+5 position is the best substrate. Also other residues such as M, I, L, or G are good enough to be substrate of UL97. This result may aid the discovery of a new antiviral inhibitor.

세포내 총체적인 인산화 펩타이드 및 인산화 위치 규명을 위해 질량분석기 전 단계의 C4 및 양이온 교환수지 칼럼 이용 방법의 비교

김혜정,백문창,Kim, Hye-Jeong,Baek, Moon-Chang 대한약학회 2015 약학회지 Vol.59 No.3

Protein phosphorylation is one of most important post-translational modifications (PTMs) and plays an important role in regulation of protein function. Here we develop a method for a global identification of phosphopeptides and phosphorylation sites using nano-LC MS/MS. We compared two separation methods, C4 and strong cation ion exchange (SCX). Before phosphopeptides enrichment with $TiO_2$, total proteins from Rat 1 cells have been separated using C4 column or tryptic peptides of proteins from the cells have been separated using SCX column. Finally, we have detected 52 phosphorylation sites on 41 proteins from SCX method and 375 phosphorylation sites on 252 proteins from C4 method, and determined the function and localization of identified phosphoproteins using DAVID software. In particular, we showed new phosphorylation sites from membrane proteins related to various cell signaling mechanisms. This method may contribute to study global signal networks induced by various signals including ligands and drugs.

${\apha}$-Casein의 인산화 위치 규명을 위한 티타늄 다이옥사이드($TiO_2$) 방법의 최적화

김혜정,박자혜,백문창,Kim, Hye-Jeong,Park, Ja-Hye,Baek, Moon-Chang 대한약학회 2008 약학회지 Vol.52 No.5

Phosphorylation plays the most important role in cell signaling mechanism. Various methods to identify the phosphorylation sites of proteins using tandem mass spectrometry (MS/MS) have been reported recently. Furthermore, the enrichment strategy such as Titanium dioxide ($TiO_2$) method should be combined with MS/MS analysis to effectively identify phosphorylation sites. It is necessary to optimize phosphopeptide-enrichment strategy, $TiO_2$ method in this study, due to the low amount of phosphorylated form followed by analyzing them by MS/MS. To evaluate the several conditions to enrich phosphopeptides using $TiO_2$ method, we used ${\apha}$-casein as a standard phosphoprotein and analyzed a representative phosphopeptide (VPQLEIVPNpSAEER) peak of MS spectrum. Batch is better than column method for binding and 300 g/l DHB in loading buffer is better than lower concentration of DHB. 3% TFA and pH 10.5 shows high efficiency of phosphopeptide-enrichment for washing and elution steps, respectively. Finally we identified various efficient conditions of phosphopeptide-enrichment method using $TiO_2$. This optimized method would assist in reliable identifying thousands of phosphorylation sites existed in low abundance from various complex proteins.

단백체 분석을 위한 일차원 및 이차원 역상크로마토그래피의 비교

문평곤(Pyong-Gon Moon),조영은(Young-Eun Cho),백문창(Moon-Chang Baek) 大韓藥學會 2010 약학회지 Vol.54 No.5

Single-dimensional (1-D) and two-dimensional (2-D) LC methods were utilized to separate peptides from various sources followed by MS/MS analysis. Two-dimensional ultra-high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than 1-D LC. The most popular 2-D LC approach used today for proteomic research combines strong cation exchange and reversed-phase LC. We have evaluated an alternative mode for 2-D LC of peptides using 2-D RP-RP nano UPLC Q-TOF Mass Spectrometry, employing reversed-phase columns in both separation dimensions. As control experiments, we identified 129 proteins in 1-D LC and 322 proteins in 2-D LC from E.coli extract peptides. Furthermore, we applied this method to rat primary hepatocyte and a total of 170 proteins were identified from 1-D LC, and 527 proteins were identified from all 2-D LC system. The in-depth protein profiling established by this 2-D LC MS/MS from rat primary hepatocyte could be a very useful reference for future applications in regards to drug induced liver toxicity.

젤 전기영동 및 액체 크로마토그래피 분리 방법을 이용하여 지방 세포로부터 분비되는 단백질들에 대한 프로테오믹스 연구 방법

황현호(Hyun-Ho Hwang),백문창(Moon-Chang Baek) 대한약학회 2011 약학회지 Vol.55 No.3

Adipocytes have been known to secrete a number of important proteins called adipokines with roles in energy metabolism, reproduction, cardiovascular function and immunity. In this study we have attempted to identify intensively secretory proteins from 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into mature adipocytes and then the cells were left in serum-free medium. The supernatant was filtrated and dialyzed. Lyophilized secretome was fractionated by two different methods, 1-D SDS PAGE and RP-FPLC. The tryptic peptides from the gel slices and the FPLC fractions were analyzed by nanoLC/ESI-MS/MS. We identified a total of 303 identical proteins from two methods, 251 proteins from 1-D gel and 184 proteins from RP-FPLC. 86 of them were listed as a secretory protein Finally, we identified many known or unknown secreted proteins existed in the low level including adiponectin, angiotensinogen, bone morphogenetic protein-1 (BMP-1), macrophage migration inhibitory factor (MIF), insulin like growth factor-II (IGF-II), interleukin-6 (IL-6), follistatin-related protein-1, minecan, and resistin. The existence of some of secreted proteins has been confirmed in RNA level. This proteomic experiment is useful for the intensive screening of secretory proteins in many kinds of other cells.

에리스로마이신 고도내성 대장균 209K 유래 마크로라이드-포스포트란스페라제 K의 정제 및 특성

김숙경(Sook Kyung Kim),오태권(Tae Gwon Oh),백문창(Moon Chang Baek),홍종수(Jong Soo Hong),김병각(Byong Kak Kim),최응칠(Eung Chil Choi) 대한약학회 1997 약학회지 Vol.41 No.3

Resistance gene mphK was cloned from Escherichia coli 209K strain which is highly resistant to erythromycin (EM). By using the cloned plasmid pGE64, E. coli NM522 was transformed. The comparison of macrolide-phosphotransferase K [MPH(K)] activity between E. coli 209K and E. coli NM522(pGE64) showed that the total enzyme activity of MN522(pGE64) was fifty-fole higher than that of 209K. To identify characteristics of MPH(K) more precisely. MPH(K) was isolated and purified from the NM522 (pGE64). The final purification f MPH(K) through several stages of purification process was 89 fole and the overall recovery was 11%. This enzyme was monomer with the molecular weight of 34 kDa and its isoelectric point (pI) was 5.0. The optimal pH and temperature for activity were 8.0 and 40oC, respectively.

MC3T3-E1 조골세포의 분화와 무기질화에 대한 우유 유래 Extracellular Vesicles의 효과

권재희(JaeHee Kwon),서현주(Hyun-Ju Seo),권인숙(In-Sook Kwun),백문창(Moon-Chang Baek),조영은(Young-Eun Cho) 한국식품영양과학회 2022 한국식품영양과학회지 Vol.51 No.11

현재 골다공증 예방 및 치료에 사용되는 bisphosphonate와 estrogen은 파골세포의 활성을 지연시켜 뼈 재흡수를 억제하는 약물로써는 유효하나 이미 소실된 뼈 질량을 회복시킬 수는 없다. 또한 만성 질환인 골다공증에 약리학적 치료를 위한 약물보다는 천연 및 무독성의 식품 단백질에 의한 치료가 골다공증의 예방 및 치료에 더 유효한 방법이 될 수 있다. 따라서 본 연구에서는 MC3T3-E1 전조골세포을 이용하여 우유 유래 extracellular vesicles(EVs)가 조골세포의 분화 및 무기질화에 미치는 영향을 확인하였고, 골다공증과 같은 골질환을 예방 및 치료할 수 있는 효과적이고 안전한 소재임을 조사하였다. 우유 유래 EVs를 분화배지에 0, 1, 5, 10 μg/mL 농도가 되도록 제조하여 MC3T3-E1 세포에 처리하여 3일 및 7일 동안 배양하였고 ALP 활성도, Von Kossa 및 alizarin red 염색법 등에 의해 조골세포의 활성 변화를 분석하였다. 초원심분리에 의해 분리된 우유 유래 EVs의 평균 직경은 156±2.5 nm였으며, EV 관련 마커 단백질인 CD63 및 유청단백질 특이적 마커인 락토페린의 존재를 immunoblot 분석을 통해 확인하였다. MTT 분석 결과는 우유 유래 EVs가 3일째에 조골세포 증식을 증가시키는 것으로 나타났다. 세포 외 ALP 활성은 3일째에 우유 유래 EVs 농도가 증가함에 따라 유의하게 증가하였다. Von Kossa 및 alizarin red 염색 결과 우유 유래 EVs 처리 후 3일 및 7일에 용량 의존적 방식으로 광물화된 결절이 유의하게 증가하였다. 본 연구 결과에서 우유 유래 EVs가 조골세포의 증식, 분화 및 광물화를 자극하여 골다공증과 같은 골 질환을 예방 및 치료할 수 있는 안전한 소재로서의 가능성을 확인하였다. Osteoporosis, which is caused by the structural weakness of bone tissue, is a systemic skeletal disease that decreases bone mass and increases the risk of fractures. For the aging population, osteoporosis is a major cause of morbidity and health expenditure. The objective of the present study was to investigate the potential anti-osteoporotic properties of cow milk-derived extracellular vesicles (EVs) on the differentiation and mineralization in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in 0, 1, 5, and 10 μg/mL cow milk-derived EVs for 3 and 7 days. It was observed that the average diameter of EVs from cow milk isolated by ultracentrifugation was 156±2.5 nm, and the presence of EV-associated marker protein CD63 and whey protein-specific marker lactoferrin was confirmed by immunoblot analysis. Our results determined that cow milk-derived EVs were increased osteoblastic cell proliferation and extracellular alkaline phosphatase (ALP) activity. Markedly, cow milk-derived EVs were significantly elevated the mineralized nodules in a does dependent manner for 3 and 7 days. Taken together, our results clearly demonstrated that cow milk-derived EVs may be useful in preventing osteoporosis by stimulating osteoblastic differentiation and mineralization.

Bifidobacterium bifidum에서 리팜피신에 대한 내성기전

정영자(Young Ja Chung),박성수(Seong Soo Park),백문창(Moon Chang Baek),김병각(Byong Kak Kim),최응칠(Eung Chil Choi) 대한약학회 1998 약학회지 Vol.42 No.2

Bifidobacterium bifidum OFR9 that exhibits acquired resistance to rifampicin and fluoroquinolones was selected by MNNG and multi-step mutation method. To investigate the resistance mechanism to rifampicin in the strain, RNA polymerase from B. bifidum parent strain and rifampicin-resistance OFR9 was partially purified and its sensitivity to rifampicin was assayed. The profile of RNA polymerase preparation of B. bifidum parent and B. bifidum OFR9 is similar to that of E. coli RNA polymerase that includes the basic subunits of beta`, beta, sigma, alpha but which are a little different in size when they are compared with E. coli RNA polymerase subunits. RNA polymerase isolated from the parent strain was inhibited by 1mcg/ml rifampicin but that from B. bifidum OFR9 was not affected by 100mcg/ml concentration of rifampicin. RNA polymerase activity of B. bifidum OFR9 was maintained over 90% through that rifampicin concentration. This result is consistent with MIC values of in vitro test. It can be concluded that the mechanism of rifampicin resistance in B. bifidum OFR9 is due to an alteration of RNA polymerase.