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6. A CRISPR RNA Is Closely Related With the Size of the Cascade Nucleoprotein Complex

Author

Do-Heon Gu, Sung Chul Ha, and Jeong-Sun Kim

Subjects
CRISPR, cascade, Csy3, helical backbone, crRNA, Microbiology, QR1-502
Abstract

The currently known prokaryotic adaptive immune system against mobile genetic elements is based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) proteins and the transcribed short CRISPR RNA (crRNA) molecule form a heterologous ribonucleoprotein complex that neutralizes invading foreign nucleic acids, wherein the crRNA molecule base-pairs with the exogenous genetic elements. In the ribonucleoprotein complexes of the type I CRISPR system, a helical backbone of six identical subunits is commonly found. However, it is not clear how this ribonucleoprotein complex is assembled and what is the determinant factor for its size. We elucidated the crystal structure of the Csy3 subunit of the type I-F ribonucleoprotein complex from Zymomonas mobilis (ZmCsy3), in which seven ZmCsy3 protomers in the asymmetric unit form a molecular helix that is part of a filamentous structure in the entire crystal system. This ZmCsy3 helical structure is remarkably similar to the crRNA-bound hexameric Csy3 backbone from Pseudomonas aeruginosa, with conserved interactions between neighboring subunits. The monomeric ZmCsy3 in solution is transformed into different oligomeric states depending on the added crRNAs. These results suggest that a crRNA and Csy3 subunit play a determinant role in the stepwise formation of the functional Cascade ribonucleoprotein complex and the recruitment of other subunits, and crRNA functions as a molecular ruler for determining the size of the Cascade silencing complex.

Published
2019
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13. BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II

Author

Chae Soon Lee, Sung Chul Ha, Jinhong Kim, Suk Youl Park, Cheolsoo Eo, Seungyu Rah, Hyo-Yun Kim, Do Heon Gu, Jeong-Sun Kim, Jun Lim, Seung A Hwangbo, Young Duck Yun, Hyeongju Choi, Jangwoo Kim, Jie Oh Lee, In Deuk Seo, W. Lee, and Yeon Gil Kim

Subjects
Nuclear and High Energy Physics, Materials science, Macromolecular Substances, 030303 biophysics, Legionella, Crystal structure, Neisseria meningitidis, Crystallography, X-Ray, 03 medical and health sciences, Optics, macromolecular crystallography, serial crystallography, Carbon Radioisotopes, Instrumentation, 030304 developmental biology, Zymomonas, 0303 health sciences, Radiation, business.industry, Detector, Proteins, Beamlines, Equipment Design, Undulator, Beamline, microfocus, Goniometer, protein structures, X-ray crystallography, Muramidase, Protein Structural Elements, business, Protein crystallization, Synchrotrons, Beam (structure)
Abstract

A new protein crystallography beamline (BL-11C) has been constructed at Pohang Light Source II. The BL-11C beamline allows routine protein-structure determinations under a robot sample-mounting system and room-temperature structure determinations through synchrotron serial crystallography experiments., BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0–20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s−1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

Published
2021
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14. Dual Oxidase 2 (DUOX2) as a Proteomic Biomarker for Predicting Treatment Response to Chemoradiation Therapy for Locally Advanced Rectal Cancer: Using High-Throughput Proteomic Analysis and Machine Learning Algorithm

Author

Lee, Hyebin, primary, Ryu, Han Suk, additional, Park, Hee Chul, additional, Yu, Jeong Il, additional, Yoo, Gyu Sang, additional, Choi, Changhoon, additional, Nam, Heerim, additional, Lee, Jason Joon Bock, additional, Do, In-Gu, additional, Han, Dohyun, additional, and Ha, Sang Yun, additional

Published
2022
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21. Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes

Author

Cristescu, Razvan, Lee, Jeeyun, Nebozhyn, Michael, Kim, Kyoung-Mee, Ting, Jason C., Wong, Swee Seong, Liu, Jiangang, Yue, Yong Gang, Wang, Jian, Yu, Kun, Ye, Xiang S., Do, In-Gu, Liu, Shawn, Gong, Lara, Fu, Jake, Jin, Jason Gang, Choi, Min Gew, Sohn, Tae Sung, Lee, Joon Ho, Bae, Jae Moon, Kim, Seung Tae, Park, Se Hoon, Sohn, Insuk, Jung, Sin-Ho, Tan, Patrick, Chen, Ronghua, Hardwick, James, Kang, Won Ki, Ayers, Mark, Hongyue, Dai, Reinhard, Christoph, Loboda, Andrey, Kim, Sung, and Aggarwal, Amit

Subjects
Gene expression -- Health aspects, Tumor proteins -- Identification and classification, Stomach cancer -- Genetic aspects -- Development and progression, Biological sciences, Health
Abstract

Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease. We aim to establish clinically relevant molecular subtypes that would encompass this heterogeneity and provide useful clinical information. We use gene expression data to describe four molecular subtypes linked to distinct patterns of molecular alterations, disease progression and prognosis. The mesenchymal-like type includes diffuse-subtype tumors with the worst prognosis, the tendency to occur at an earlier age and the highest recurrence frequency (63%) of the four subtypes. Microsatellite-unstable tumors are hyper-mutated intestinal-subtype tumors occurring in the antrum; these have the best overall prognosis and the lowest frequency of recurrence (22%) of the four subtypes. The tumor protein 53 (TP53)-active and [TP53.sup.-]inactive types include patients with intermediate prognosis and recurrence rates (with respect to the other two subtypes), with the [TP53.sup.-]active group showing better prognosis. We describe key molecular alterations in each of the four subtypes using targeted sequencing and genome-wide copy number microarrays. We validate these subtypes in independent cohorts in order to provide a consistent and unified framework for further clinical and preclinical translational research., Gastric cancer (GC) is the fourth-most-common cancer globally and the second-leading cause of cancer deaths (1). It accounts for 989,600 cases annually, with East Asia accounting for more than half [...]

Published
2015
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31. Preexisting oncogenic events impact trastuzumab sensitivity in ERBB2-amplified gastroesophageal adenocarcinoma

Author

Kim, Jihun, Fox, Cameron, Peng, Shouyong, Pusung, Mark, Pectasides, Eirini, Matthee, Eric, Hong, Yong Sang, Do, In-Gu, Jang, Jiryeon, Thorner, Aaron R., Van Hummelen, Paul, Rustgi, Anil K., Wong, Kwok-Kin, Zhou, Zhongren, Tang, Ping, Kim, Kyoung-Mee, Lee, Jeeyun, and Bass, Adam J.

Subjects
Oncogenes -- Physiological aspects -- Research, Esophageal cancer -- Drug therapy -- Genetic aspects -- Research, Health care industry
Abstract

Patients with gastric and esophageal (GE) adenocarcinoma tumors in which the oncogene ERBB2 has been amplified are routinely treated with a combination of cytotoxic chemotherapy and the ERBB2-directed antibody trastuzumab; however, the addition of trastuzumab, even when tested in a selected biomarker-positive patient population, provides only modest survival gains. To investigate the potential reasons for the modest impact of ERBB2-directed therapies, we explored the hypothesis that secondary molecular features of ERBB2-amplified GE adenocarcinomas attenuate the impact of ERBB2 blockade. We analyzed genomic profiles of ERBB2-amplified GE adenocarcinomas and determined that the majority of ERBB2-amplified tumors harbor secondary oncogenic alterations that have the potential to be therapeutically targeted. These secondary events spanned genes involved in cell-cycle regulation as well as phosphatidylinositol-3 kinase and receptor tyrosine kinase signaling. Using ERBB2-amplified cell lines, we demonstrated that secondary oncogenic events could confer resistance to ERBB2-directed therapies. Moreover, this resistance could be overcome by targeting the secondary oncogene in conjunction with ERBB2-directed therapy. EGFR is commonly coamplified with ERBB2, and in the setting of ERBB2 amplification, higher EGFR expression appears to mark tumors with greater sensitivity to dual EGFR/ERBB2 kinase inhibitors. These data suggest that combination inhibitor strategies, guided by secondary events in ERBB2-amplified GE adenocarcinomas, should be evaluated in clinical trials., Introduction Gastric adenocarcinoma is the fourth most common cancer and the third leading cause of cancer deaths worldwide, and the incidence of esophageal adenocarcinoma is increasing dramatically in many Western [...]

Published
2014
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33. Effect of simvastatin plus cetuximab/irinotecan for KRAS mutant colorectal cancer and predictive value of the RAS signature for treatment response to cetuximab

Author

Lee, Jeeyun, Hong, Yong Sang, Hong, Jung Yong, Han, Se Won, Kim, Tae Won, Kang, Hye Jin, Kim, Tae You, Kim, Kyu-pyo, Kim, Suk Hyung, Do, In-Gu, Kim, Kyoung-Mee, Sohn, Insuk, Park, Se Hoon, Park, Joon Oh, Lim, Ho Yeong, Cho, Yong Beom, Lee, Woo Yong, Yun, Seong Hyeon, Kim, Hee Cheol, Park, Young Suk, and Kang, Won Ki

Published
2014
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36. Analysis of Trends in Regulatory Science and Regulatory Science Experts Training Projects: US, Japan, Singapore, and Korea

Author

Park, Jaehong, primary, Shin, Hocheol, additional, Kim, Jiwon, additional, Kim, Minsu, additional, Do, In Gu, additional, Lim, Heeyeon, additional, Lee, Jiwon, additional, Lee, Yun-ji, additional, Jung, Sun-Young, additional, Kang, Wonku, additional, Kim, Hahyung, additional, Choi, Young Wook, additional, and Kim, Eunyoung, additional

Published
2021
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39. Structural basis for the selective addition of an oxygen atom to cyclic ketones by Baeyer-Villiger monooxygenase from Parvibaculum lavamentivorans

Author

Do Heon Gu, Tien Duc Nguyen, Pil Won Seo, Ji Won Kim, Goeun Choi, Jeong-Sun Kim, and Jin Byung Park

Subjects
Models, Molecular, 0301 basic medicine, Protein Conformation, Stereochemistry, Biophysics, Crystallography, X-Ray, Biochemistry, Mixed Function Oxygenases, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Bacterial Proteins, Oxidoreductase, Catalytic Domain, Moiety, Amino Acid Sequence, Molecular Biology, Alphaproteobacteria, chemistry.chemical_classification, Flavin adenine dinucleotide, biology, Active site, Substrate (chemistry), Regioselectivity, Cell Biology, Ketones, Oxygen, 030104 developmental biology, chemistry, Cyclization, 030220 oncology & carcinogenesis, biology.protein, Parvibaculum lavamentivorans, NADP, Methyl group
Abstract

Baeyer-Villiger monooxygenase (BVMO) catalyzes insertion of an oxygen atom into aliphatic or cyclic ketones with high regioselectivity. The BVMOs from Parvibaculum lavamentivorans (BVMOParvi) and Oceanicola batsensis (BVMOOcean) are interesting because of their homologies, with >40% sequence identity, and reaction with the same cyclic ketones with a methyl moiety to give different products. The revealed BVMOParvi structure shows that BVMOParvi forms a two-domain structure like other BVMOs. It has two inserted residues, compared with BVMOOcean, that form a bulge near the bound flavin adenine dinucleotide in the active site. Furthermore, this bulge is linked to a nearby α-helix via a disulfide bond, probably restricting access of the bulky methyl group of the substrate to this bulge. Another sequence motif at the entrance of the active site (Ala-Ser in BVMOParvi and Ser-Thr in BVMOOcean) allows a large volume in BVMOParvi. These minute differences may discriminate a substrate orientation in both BVMOs from the initial substrate binding pocket to the final oxygenation site, resulting in the inserted oxygen atom being in different positions of the same substrate.

Published
2019
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