• Journal of Microbiology and Biotechnology
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• 제34권6호
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• pp.1348-1355
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• 2024

The eukaryotic translation initiation factor eIF5B is a bacterial IF2 ortholog that plays an important role in ribosome joining and stabilization of the initiator tRNA on the AUG start codon during the initiation of translation. We identified the fluorophenyl oxazole derivative 2,2-dibromo-1-(2-(4-fluorophenyl)benzo[d]oxazol-5-yl)ethanone quinolinol as an inhibitor of fungal protein synthesis using an in vitro translation assay in a fungal system. Mutants resistant to this compound were isolated in Saccharomyces cerevisiae and were demonstrated to contain amino acid substitutions in eIF5B that conferred the resistance. These results suggest that eIF5B is a target of potential antifungal compound and that mutation of eIF5B can confer resistance. Subsequent identification of 16 other mutants revealed that primary mutations clustered mainly on domain 2 of eIF5B and secondarily mainly on domain 4. Domain 2 has been implicated in the interaction with the small ribosomal subunit during initiation of translation. The tested translation inhibitor could act by weakening the functional contact between eIF5B and the ribosome complex. This data provides the basis for the development of a new family of antifungals.

• Journal of Microbiology and Biotechnology
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• 제30권8호
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• pp.1261-1271
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• 2020

Cytochrome c_L (Cytc_L) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between Cytc_L and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing Cytc_L from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MP^T at 2.13 Å resolution. The crystal structure of Ma-Cytc_L revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na⁺, Ca⁺, and Fe²⁺ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-Cytc_L, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to Cytc_L within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

• Journal of Genetic Medicine
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• 제11권1호
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• pp.22-26
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• 2014

Maple syrup urine disease (MSUD) is a disorder that involves the metabolism of branched chain amino acids, arising from a defect in branched-chain ${\alpha}$-keto acid dehydrogenase complex. Mutations have been identified in the BCKDHA, BCKDHB, or DBT genes, which encode different subunits of the BCKDH complex. Although encephalopathy and progressive neurodegeneration are its major manifestations, the severity of the disease may range from the severe classic type to milder intermediate variants. We report two Korean siblings with the milder intermediate MSUD who were diagnosed with MSUD by a combination of newborn screening tests using tandem mass spectrometry and family genetic screening for MSUD. At diagnosis, the patients' plasma levels were elevated for leucine, isoleucine, valine, and alloisoleucine, and branched-chain ${\alpha}$-keto acids and branched-chain ${\alpha}$-hydroxy acids were detected in their urine. BCKDHA, BCKDHB, and DBT analysis was performed, and two novel mutations were identified in BCKDHB. Our patients were thought to have the milder intermediate variant of MSUD, rather than the classic form. Although MSUD is a typical metabolic disease with poor prognosis, better outcomes can be expected if early diagnosis and prompt management are provided, particularly for milder forms of the disease.

• Genomics & Informatics
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• 제3권2호
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• pp.61-65
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• 2005

Comparing 231 genes on chimpanzee chromosome 22 with their orthologous on human chromosome 21, we have found that 15 orthologs have indels within their coding sequences. It was rather surprising that significant number of genes have changed by indel, despite the shorter time since their divergence and led us hypothesize that indels and structural changes may represent one of the major mechanism of proteome evolution in the higher primates. Human T-complex protein 10 like (TCP 10L) is a representative having indel within its coding sequence. Gene structure of human TCP10L compared with chimpanzee TCP10L gene showed 16 base pair difference in genomic DNA. As a result of the indel, frame shift mutation occurs in coding sequence (CDS) and human TCP10L express longer polypeptide of 21 amino acid residues than that of chimpanzee. Our prediction found that the indel may affect to dramatic change of secondary protein structure between human and chimpanzee TCP10L. Especially, the structural changes in the C-terminal region of TCP10L protein may affect on the interacting potential to other proteins rather than DNA binding function of the protein. Through these changes, TCP10L might influence gene expression profiles in liver and testis and subsequently influence the physiological changes required in primate evolution.

• Animal cells and systems
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• 제9권3호
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• pp.161-171
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• 2005

Integrin-linked kinase 1 (ILK1) regulates several protein kinases, including PKB/Akt kinase and glycogen synthase kinase ${\beta}$. ILK1 is also involved distinctively in the cell morphological and structural functions by interacting with the components of the extracellular matrix or integrin. According to the information of serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity (R-X-R-X-X(S/T)-${\phi};{\phi}$ indicates a hydrophobic amino acid), two putative phosphorylation sites, $Thr^{181}\;and\;Ser^{246}$, were found in ILK1. We showed that ILK1 fusion protein and two fluorescein-labeled ILK1 peptides, $FITC-^{174}RTRPRNGTLN^{183}$ and $FITC-^{239}CPRLRIFSHP^{248}$, were phosphorylated by SGK1 in vitro. We also identified that 14-3-3 ${\theta}\;{\varepsilon}\;and\;{\xi}$, among several 143-3 isotypes $({\beta},\;{\gamma},\;{\varepsilon},\;{\eta},\;{\sigma},\;{\theta},\;{\tau}\;and\;{\xi})$ formed protein complex with ILK1 in COS-1 cells. Furthermore, the phosphorylation of $Ser^{246}$ by SGK1 induced the binding with 14-3-3. It was also demonstrated that 14-3-3-bound ILK1 has reduced kinase activity. Thus, these data suggest that SGK1 phosphorylates $Thr^{181}\;and\;Ser^{246}$ of ILK1 and the phosphorylation of its $Ser^{246}$ makes ILK1 bind to 14-3-3, resulting in the inhibition of ILK1 kinase activity.

• Journal of Animal Science and Technology
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• 제64권5호
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• pp.885-896
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• 2022

This experiment was conducted to investigate the effects of three different copper (Cu) sources (one inorganic and two organics) and levels (0, 50, and 100 mg/kg) on the growth performance, Cu digestibility, fecal mineral excretion, serum mineral concentration, jejunal morphology, and serum biochemical profile of growing pigs. A total of 42 male, growing pigs (31.08 ± 1.82 kg) were randomly assigned to seven treatments consisting of one negative control (0 mg/kg of added Cu level) and treatments with copper sulfate (CuSO4), Cu-amino acid complex (CuAA), and Cu-hydroxy-4-methylthio butanoate chelate complex (CuHMB) at 50 and 100 mg/kg each for 28 d. Pigs fed 50 or 100 mg/kg of Cu showed improved (p < 0.05) average daily gain and feed intake. Although Cu excretion decreased (p < 0.01) in pigs fed 100 mg/kg of organic Cu sources compared to those fed CuSO4, there was no difference between the Cu sources in pigs fed 50 mg/kg. However, the apparent total tract digestibility of Cu increased (p < 0.01) in pigs fed organic Cu sources compared with that in pigs fed CuSO4. The addition of CuHMB increased (p < 0.01) serum phosphorus and sulfur concentrations; however, there were no effects of source and level on jejunal morphology and serum biochemical profile. These results suggest that the inclusion (50 mg/kg) of organic Cu sources (CuAA and CuHMB) in the growing pig diet could be beneficial for growth performance and Cu availability and may reduce environmental pollution.

• Asian-Australasian Journal of Animal Sciences
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• 제18권5호
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• pp.747-754
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• 2005

Nutritional regulation of gene expression associated with growth and feeding behavior in avian species can become an important technique to improve poultry production according to the supply of nutrients in the diet. Insulin-like growth factor-I (IGF-I) found in chickens has been characterized to be a 70 amino acid polypeptide and plays an important role in growth and metabolism. Although it is been well known that IGF-I is highly associated with embryonic development and post-hatching growth, changes in the distribution of IGF-I gene expression throughout early- to late-embryogenesis have not been studied so far. We revealed that the developmental pattern of IGF-I gene expression during embryogenesis differed among various tissues. No bands of IGF-I mRNA were detected in embryonic liver at 7 days of incubation, and thereafter the amount of hepatic IGF-I mRNA was increased from 14 to 20 days of incubation. In eyes, a peak in IGF-I mRNA levels occurred at mid-embryogenesis, but by contrast, IGF-I mRNA was barely detectable in the heart throughout all incubation periods. In the muscle, no significant difference in IGF-I gene expression was observed during different stages of embryogenesis. After hatching, hepatic IGF-I gene expression as well as plasma IGF-I concentration increases rapidly with age, reaches a peak before sexual maturity, and then declines. The IGF-I gene expression is very sensitive to changes in nutritional conditions. Food-restriction and fasting decreased hepatic IGF-I gene expression and refeeding restored IGF-I gene expression to the level of fed chickens. Dietary protein is also a very strong factor in changing hepatic IGF-I gene expression. Refeeding with dietary protein alone successfully restored hepatic IGF-I gene expression of fasted chickens to the level of fed controls. In most circumstances, IGF-I makes a complex with specific high-affinity IGF-binding proteins (IGFBPs). So far, four different IGFBPs have been identified in avian species and the major IGFBP in chicken plasma has been reported to be IGFBP-2. We studied the relationship between nutritional status and IGFBP-2 gene expression in various tissues of young chickens. In the liver of fed chickens, almost no IGFBP-2 mRNA was detected. However, fasting markedly increased hepatic IGFBP-2 gene expression, and the level was reduced after refeeding. In the gizzard of well-fed young chickens, IGFBP-2 gene expression was detected and fasting significantly elevated gizzard IGFBP-2 mRNA levels to about double that of fed controls. After refeeding, gizzard IGFBP-2 gene expression decreased similar to hepatic IGFBP-2 gene expression. In the brain, IGFBP-2 mRNA was observed in fed chickens and had significantly decreased by fasting. In the kidney, IGFBP-2 gene expression was observed but not influenced by fasting and refeeding. Recently, we have demonstrated in vivo that gizzard and hepatic IGFBP-2 gene expression in fasted chickens was rapidly reduced by intravenous administration of insulin, as indicated that in young chickens the reduction in gizzard and hepatic IGFBP-2 gene expression in vivo stimulated by malnutrition may be, in part, regulated by means of the increase in plasma insulin concentration via an insulin-response element. The influence of dietary protein source (isolated soybean protein vs. casein) and the supplementation of essential amino acids on gizzard IGFBP-2 gene expression was examined. In both soybean protein and casein diet groups, the deficiency of essential amino acids stimulated chickens to increase gizzard IGFBP-2 gene expression. Although amino acid supplementation of a soybean protein diet significantly decreased gizzard IGFBP-2 mRNA levels, a similar reduction was not observed in chickens fed a casein diet supplemented with amino acids. This overview of nutritional regulation of IGF-I and IGFBP-2 gene expression in young chickens would serve for the establishment of the supply of nutrients to diets to improve poultry production.

• 미생물학회지
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• 제48권2호
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• pp.66-72
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• 2012

Nup188 단백질은 진화적으로 보존된 가장 큰 핵공단백질 중의 하나로 핵공복합체의 inner ring을 구성하는 인자이다. Nup188의 이종상동체인 분열효모 S. pombe의 Nup184 단백질은 영양분이 풍부한 완전배지(YES 배지)에서 정상적인 생장과 mRNA의 핵에서 세포질로의 이동에 필요하다. 본 연구에서는 ${\Delta}nup184$ 결실돌연변이를 YES 배지에서 배양할 때 보이는 생장지체와 mRNA export 결함을 상보하기 위해서 Nup184의 카르복시 부위(아미노산 잔기482에서 1628까지)가 필요함을 알아내었다. 또한 이 부위는 GFP-Nup184 융합단백질이 핵막에 위치하기 위해서도 필요하였다. 이 과정에서 S. pombe GeneDB (Sanger 연구소, 영국)에 등록되어 있는 Nup184의 열린읽기틀 (1564개의 아미노산 잔기로 된 단백질로 예측)이 우리가 얻은 염기서열 데이터에 비해 66개의 아미노산 잔기가 짧다는 것을 발견하였다. 이 카르복시-말단 부위는 Nup184의 기능에 반드시 필요하였다. 이외에도 Nup184 단백질이 세포 안에서 SUMO 변형되어 있음을 보였다.

• 한국버섯학회지
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• 제13권3호
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• pp.207-211
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• 2015

Kenaf(Hibiscus cannabinus L.)의 버섯 배지원으로서의 활용 가능성을 판단하고자 물리성과 영양원 함유량 등을 분석한 결과 배지의 형태를 유지하고 영양원의 공급기지 역할을 하는 주재료로서 활용 가능성이 높았으며 탄소, 질소 함량과 C/N율, 일반성분, 미량원소 그리고 아미노산 함량 등도 일정 수준을 유지하고 있어서 배지 영양원으로서 역할도 기대할 수 있었다. 실제로 Kenaf는 다공성 세포구조 특성으로 높은 함수능력을 가지고 있었는데 bast 부위의 경우 수분 흡수율이 578%로 수분 조절제와 산소 공급자 역할을 충실히 할 것으로 판단되었다. 총 탄소 함량은 91.4%로 포플러톱밥이나 밀기울, 미강에 비해 아주 높았다. 총 질소는 1.76%로 배지 주재료로는 높은 특성을 유지하였으며 C/N율은 51.9 였다. 가용성무질소물은 46.6%로 미강 정도의 수준을 유지하였다. 탄소원 구성을 보면 cellulose 함량은 포플러에 비해 상대적으로 높았고 lignin 함량은 낮았는데, 비교적 원활하게 사용할 수 있는 탄소원인 hemicellulose+pectin 혼합물이 포플러에 비해 3.7% 많았다. 무기이온 함량도 케나프가 포플러톱밥에 비해 전체적으로 높았는데 Fe은 4.2배, P은 3.2배, K는 2.2배 많았고 Ca는 16 mg/kg 더 많았다. 아미노산 함량은 전체적으로 포플러톱밥보다 많았으며 미강보다는 함량이 낮았다.

• 미생물학회지
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• 제39권3호
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• pp.148-154
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• 2003

Moraxella sp. CK-1는 남조류 Anabaena cylindrica의 생장을 억제한다고 알려져 있다. Moraxella sp. CK-1의 세포외 autolysin의 분리는 다음과 같은 방법으로 분리를 시도하였다. 흡광도 660 nm에서 0.7～0.8이 되도록 BGC-11세포배양액에서 키우고, 원심분리로 균주인 Moraxella sp. CK-1를 제거한 뒤 세포배양액을 Amicon ultrafiltration으로 농축을 하였다. 농축한 세포배양액을 $(NH_{4})_{2}SO_{4}$ 로 0～20%, 20～40%, 40～60%, 60～80%로 분획하여 단백질을 14,000${\times}$g로 침전 시켰다. 침전된 단백질을 20 mM Tris-HC1, pH 8.0완충용액으로 현탁시킨 뒤, 동일 완충용액을 이용하여 투석을 하였다. $(NH_{4})_{2}SO_{4}$로 분획한 뒤 활성확인을 위해 Anabaena Cylindrica가 도포된 평판배지에서 활성을 확인한 결과, 40～60%, 60～80%에서 활성이 있음을 확인하였다. 이들을 각각 Mono-$Q^{TM}$ HR 5/5 (column volume 1 ml) column을 이용하여 FPLC에서 단백질을 분리하였다. $(NH_{4})_{2}SO_{4}$ 로 분획한 40～60%에서는 major peak 3개, 60～80% 분획에서는 2개의 major peak가 분리되었다. Mono-$Q^{TM}$ HR 5/5 column에서 분리되어 나온 major peak 5개를 투석한 뒤 Anabaena cylindrica lawn에서 활성을 확인하였으나 확인이 되지 않았다. PVDF membrane을 이용하여 transfer하여 40～60% 침전에서 17 kDa의 단백질을 얻어냈다. 이를 N-terminal amino acid sequence를 하여 serine pretense의 계열임을 확인하였다.