Browsing by Author "Wang, Hung-Jung"

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Core Site-Moiety Maps Reveal Inhibitors and Binding Mechanisms of Orthologous Proteins by Screening Compound Libraries
(2012-02-29)
Hsu, Kai-Cheng
;
Cheng, Wen-Chi
;
Chen, Yen-Fu
;
Wang, Hung-Jung
;
Li, Ling-Ting
Members of protein families often share conserved structural subsites for interaction with chemically similar moieties despite low sequence identity. We propose a core site-moiety map of multiple proteins (called CoreSiMMap) to discover inhibitors and mechanisms by profiling subsite-moiety interactions of immense screening compounds. The consensus anchor, the subsite-moiety interactions with statistical significance, of a CoreSiMMap can be regarded as a "hot spot" that represents the conserved binding environments involved in biological functions. Here, we derive the CoreSiMMap with six consensus anchors and identify six inhibitors (IC50 < 8.0 mu M) of shikimate kinases (SKs) of Mycobacterium tuberculosis and Helicobacter pylori from the NCI database (236,962 compounds). Studies of site-directed mutagenesis and analogues reveal that these conserved interacting residues and moieties contribute to pocket-moiety interaction spots and biological functions. These results reveal that our multi-target screening strategy and the CoreSiMMap can increase the accuracy of screening in the identification of novel inhibitors and subsite-moiety environments for elucidating the binding mechanisms of targets.
Crystal Structure of Shikimate Dehydrogenase from Helicobacter Pylori.
(2009-01-01)
Cheng, Wen-Chi
;
Lin, Shuang-Chih
;
Wang, Hung-Jung
;
Yang, Jinn-Moon
;
Lin, Jong-Yih
Identification of the PCa28 gene signature as a predictor in prostate cancer
(2018-01-01)
Lee, Jung-Yu
;
Lin, Si-Yu
;
Chuang, Yi-Hsuan
;
Huang, Sing-Han
;
Tseng, Yu-Yao
Prostate cancer (PCa) is the second-leading cause of cancer death among men in the worldwide. Most PCa is slowly growing and usually early symptomless. About 70% of PCa patients were diagnosed at later stage and metastasis has been observed. Additionally, the cure rate of PCa closely relies on the early diagnosis with biomarkers. Prostatic Specific Antigen (PSA) is currently the only clinical biomarker for PCa diagnosis. However, the PSA test has inherent limitations and has about 75% of false-positive results. The identification of a set of genes (as biomarkers) for diagnosis and prognosis is an urgent clinical issue for PCa. Here, we integrated genome-wide analysis and protein-protein interaction network to identify potential genes for early diagnostic biomarkers of PCa. First, we collected gene expression datasets of 145 PCa samples, consisting of both tumor and corresponding normal tissues, from two different sources in Gene Expression Omnibus (GEO). We found 158 and 268 significantly highly and lowly expressed genes, respectively, in tumor samples. Moreover, we proposed cluster score (CS) and predicting score (PS) to select 28 prostate cancer-related genes (called PCa28). The results indicate that PCa28 can discriminate between the normal/tumor tissues and are specific for prostate cancer. Finally, we examined 8 genes in PCa28 on four PCa cell lines by real time quantitative polymerase chain reaction (RT-qPCR). Experimental results show that up-regulated genes have higher expression level in tumor cells in comparison to normal cells, and down-regulated genes have lower expression level in tumor cells. We believe that our method is useful and PCa28 are potential biomarkers that provide the clues to develop targeting therapy for PCa.
Identification of the PCA29 gene signature as a predictor in prostate cancer
(2019-06-01)
Lee, Jung-Yu
;
Lin, Si-Yu
;
Lin, Chun-Yu
;
Chuang, Yi-Huan
;
Huang, Sing-Han
Prostate cancer (PCa) is the second leading cause of cancer death among men worldwide. About 70% of PCa patients were diagnosed at later stage, and metastasis has been observed. Additionally, the cure rate of PCa closely relies on the early diagnosis with biomarkers. The identification of biomarkers for diagnosis and prognosis is an urgent clinical issue for PCa. Here, we developed a novel scoring strategy, including cluster score (CS) and predicting score (PS), to identify 29 PCa genes (called PCa29) for early diagnostic biomarkers from two datasets in Gene Expression Omnibus. The result indicates that PCa29 can discriminate between normal and tumor tissues and are specific for prostate cancer. To validate PCa29, we found that 97% of PCa29 were consistently significant with these gene expressions in The Cancer Genome Atlas; furthermore, similar to 70% of PCa29 are consensus to the protein expression in The Human Protein Atlas. Finally, we examined 10 genes in PCa29 on three PCa cell lines by real-time quantitative polymerase chain reaction. The experimental results show that the trend of the differential PCa29 expression is consistent with the analyzed results from our novel scoring method. We believe that our method is useful and PCa29 are potential biomarkers that provide the clues to develop targeting therapy for PCa.
Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing beta-1,3-Glucanase
(2009-09-25)
Wu, Hsin-Mao
;
Liu, Sheng-Wen
;
Hsu, Ming-Tsung
;
Hung, Chiu-Lien
;
Lai, Chun-Chieh
Laminaripentaose-producing beta-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide beta-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 angstrom resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (alpha/beta) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 angstrom, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu(154) and Asp(170)) and four other sugar-binding residues (Thr156, Asn(158), Trp(163), and Thr(167)). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu(154) and Asp(170) as acid and base catalysts, respectively, whereas the side chains of Thr(156), Asn(158), and Trp(163) demarcate subsite +5. Site-directed mutagenesis of Glu(154) and Asp(170) confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu(154) and Asp(170) to cleave a beta-1,3-glucan into specific alpha-pentasaccharide oligomers.
Structures of Helicobacter pylori Shikimate Kinase Reveal a Selective Inhibitor-Induced-Fit Mechanism
(2012-03-16)
Cheng, Wen-Chi
;
Chen, Yen-Fu
;
Wang, Hung-Jung
;
Hsu, Kai-Cheng
;
Lin, Shuang-Chih
Shikimate kinase (SK), which catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP, is the enzyme in the fifth step of the shikimate pathway for biosynthesis of aromatic amino acids. This pathway is present in bacteria, fungi, and plants but absent in mammals and therefore represents an attractive target pathway for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here we investigated the detailed structure-activity relationship of SK from Helicobacter pylori (HpSK). Site-directed mutagenesis and isothermal titration calorimetry studies revealed critical conserved residues (D33, F48, R57, R116, and R132) that interact with shikimate and are therefore involved in catalysis. Crystal structures of HpSK center dot SO4, R57A, and HpSK center dot shikimate-3-phosphate center dot ADP show a characteristic three-layer architecture and a conformationally elastic region consisting of F48, R57, R116, and R132, occupied by shikimate. The structure of the inhibitor complex, E114A.162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form. These results reveal considerable insight into the active-site chemistry of SKs and a selective inhibitor-induced-fit mechanism.