Browsing by Author "Chen, Yi-Lin"

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Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli
(2009)
Chiu, Hsien-Tai
;
Lin, Yu-Chin
;
Lee, Meng-Na
;
Chen, Yi-Lin
;
Wang, Mei-Sin
The indolocarbazole family of natural products has attracted great attention because of their unique structural features and potential therapeutic applications. Structurally distinct in the family, K-252a is characterized by an unusual dihydrostreptose moiety cross-bridged to K-252c aglycone with two C-N linkages. K-252a has served as a valuble lead for treatments of various cancers and neurodegenerative disorders. Recent cloning of the nok gene cluster for biosyntheses of K-252a and its analogs from Nocardiopsis sp. K-252 (NRRL15532) has revealed the nokABCD genes indispensible for K-252c biosynthesis and the key gene (nokL) coding for N-glycosylation. Herein, we report the first, successful demonstration of in vitro sugar transferase activity of indolocarbazole N-glycosyltransferase (NokL) by use of soluble protein expressed from Escherichia coli. Notably, NokL was found to exhibit peculiar mode of substrate promiscuity. Moreover, NokA and NokB reactions were biochemically characterized thoroughly by natual and alternative (e. g. fluoro-) substrates and by ammonium hydroxide (NH(4)OH). Interestingly, the in vitro expression of NokA revealed high substrate stereoselectivity, giving several indole-3-pyruvic acid-derived compounds, including indol-3-carboxaldehyde (ICA) and indole-3-acetic acid. The use of NH(4)OH successfully dissected the in vitro NokA/NokB coupled reaction, revealing mechanistic insight into the enzymes and their cross-talking relationship. Also, a simple, useful method to synthesize K-252d, ICA and chromopyrrolic acid (the NokB product) was developed by the E. coli expression systems of NokL, NokA and NokA/NokB, respectively. Together with NokA and NokB, NokL may serve as a useful tool for combinatorial engineering of K-252a and its analogs for improved therapeutic values.
Butylidenephthalide Suppresses Human Telomerase Reverse Transcriptase (TERT) in Human Glioblastomas
(2011-11-01)
Lin, Po-Cheng
;
Lin, Shinn-Zong
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Chen, Yi-Lin
;
Chang, Jeng-Shou
;
Ho, Li-Ing
Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo. We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined. Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence. These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.
CARB 126-Combinatorial biosynthesis of NDP-deoxysugars and spinosyn glycosides by in vitro recombination of secondary metabolite biosynthetic pathways
(2007-03-25)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
生物科技學系
;
Department of Biological Science and Technology
Exploring combinatorial potential of K-252c biosynthesis by precursor-directed enzyme-coupling reactions
(2011-08-28)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Lin, Yu-Chin
;
Lee, Meng-Na
;
生物科技學系
Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs
(2009)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Chen, Chien-Yu
;
Jin, Chyn
;
Lee, Meng-Na
Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazole glycoside and exhibits potent neuroprotective and broad anticancer activities. K-252a consists of K-252c and the unusual dihydrostreptose moiety, linked together with oxidative and glycosidic C-N bonds. Herein, we reported a complete sequence of an approximately 45 kb genomic fragment harboring the gene cluster for the biosynthesis of indolocarbazole alkaloids in Nocardiopsis sp. K-252 (NRRL15532). The sequence of 35 open reading frames discovered several new, critical genes, hence shedding new light on biosynthesis, resistance and regulation of K-252a and its analogs. To functionally characterize the gene cluster in vitro and in enzyme level, a multigene expression cassette containing the K-252c biosynthetic genes was constructed and successfully overexpressed in Escherichia coli to yield soluble proteins for cell-free tandem enzymatic assays. Consequently, the heterologous expression with soluble NokA and NokB led to in vitro production of chromopyrrolic acid (CPA), thereby providing functional evidence for K-252c biosynthesis. Moreover, a facile production of CPA in culture broth was successfully accomplished by using an in vivo biotransformation of L-tryptophan with E. coli harboring the gene cassette. Importantly, by sequence analysis and the functional characterization here and in the companion paper, biosynthetic pathways leading to formation of K-252a and its analogs were hence proposed. Together, the results provide critical information and materials useful for combinatorial biosynthesis of K-252a and its analogs for therapeutic applications.
Molecular insight into catalytic mechanism of spinosyn rhamnosyltransferase by in vitro combinatorial reactions with NDP-sugar-modifying enzymes
(2009-08-16)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Lin, Yu-Chin
;
Chen, Yi-Hsine
;
生物科技學系
Sudden rupture with internal bleeding and shock following torsion and necrosis of a large uterine leiomyoma
(2019-01-01)
Chen, Yi-Lin
;
Chen, Li-Ru
;
Chen, Kuo-Hu
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機械工程學系
;
Department of Mechanical Engineering
Tandem enzymatic syntheses of TDP-deoxysugars and spinosyn analogs by in vitro recombination of natural product biosynthetic pathways
(2007-03-25)
Chiu, Hsien-Tai
;
Chen, Yi-Lin
;
Wu, Chih Chien
;
生物科技學系
;
Department of Biological Science and Technology
Trichodiene synthase: Synthesis and inhibition kinetics of 12-fluoro-farnesylphosphonophosphate for sesquiterpene cyclases
(2006-10-01)
Chen, Yi-Lin
;
Chiu, Hsien-Tai
;
生物科技學系
;
Department of Biological Science and Technology
trans, trans-Farnesyl diphosphate (FPP) serves as a universal substrate for a large family of sesquiterpene cyclases that are responsible for biosynthesis of more than 300 structurally diverse sesquiterpenes in nature. A new FPP substrate analogue, 12-fluoro-farnesylphosphonophosphate (12-F-F-CH2PP), was synthesized in this paper for applications on kinetic and mechanistic studies of the enzyme family. Trichodiene synthase (TS), a sesquiterpene cyclase, catalyzes the conversion of trans,transfarnesyl diphosphate (FPP) to trichodiene. 12-F-F-CH2PP was tested as a potential inhibitor of TS. Inactivation and inhibition kinetic experiments showed that 12-F-F-CH2PP was not a mechanism-based inactivator for TS; instead, a mixed-type reversible inhibition was observed with inhibition constants K-i1 = 2.33 +/- 0.50 mu M and K-i2 25.80 +/- 7.70 mu M, values close to those previously determined for farnesylphosphonophosphate, K-i1 3.25 mu M and K-i2 = 9-10 mu M Although 12-F-F-CH2PP did not irreversibly inactivate TS, this new analogue serves as a potential active-site directed inactivator and mechanistic probe of other sesquiterpene cyclases and FPP-utilizing enzymes, which utilize FPP as a common acyclic substrate.
Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis
(2016-09-19)
Wu, Shou-Cheng
;
Lu, Chung-Yen
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Chen, Yi-Lin
;
Lo, Feng-Chun
;
Wang, Ting-Yin
Nitric oxide (NO) is an important cellular signaling molecule that modulates various physiological activities. Angiogenesis-promoting activities of NO-donor drugs have been explored in both experimental and clinical studies. In this study, a structurally well characterized and water-soluble neutral {Fe-(NO)(2)}(9) DNIC [(S(CH2)(2)OH)(S(CH2)(2)NH3)Fe(NO)(2)] (DNIC 2) was synthesized to serve as a NO-donor species. The antitumor activity of DNIC 2 was determined by MTT assay, confocal imaging, and Annexin-V/PI staining. The IC50 values of DNIC 2 were 18.8, 42.9, and 38.6 mu M for PC-3, SKBR-3, and CRLS866 tumor cells, respectively. Moreover, DNIC 2 promoted apoptotic cell death via activation of apoptosis-associated proteins and inhibition of survival associated proteins. In particular, DNIC 2 treatment suppressed PC-3 tumor growth by 2.34- and 19.3-fold at 7 and 21 days, in comparison with the control group. These results indicate that water-soluble DNIC 2 may serve as a promising drug for cancer therapy.