We have been studying RNA-processing proteins RNase III (model system for a family of dsRNA-specific endonucleases exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer), KsgA (universally conserved methyltransferase that functions as a ribosomal biogenesis factor), and Era (conserved GTPase that couples cell growth with cell division). Previously, we made pioneering contribution to the mechanism of RNase III action, significant progress in KsgA-RNA interactions, and a breakthrough advance in the structure and function of Era. This year, we have completed our structural studies of bacterial Era. Era, essential for bacterial cell viability, is composed of a GTPase domain and an RNA-binding KH domain. We have characterized the functional cycle of Era, providing structural basis for its essential roles in the maturation of 16S rRNA and assembly of 30S ribosomal subunit. We have shown that Era recognizes 10 nucleotides (nt 1530-1539, GAUCACCUCC in Escherichia coli) near the 3'end of 16S rRNA, and that this recognition stimulates the GTP-hydrolyzing activity of the protein. The GAUCA sequence and the upstream helix 45 (h45, nt 1506-1529) are highly conserved in all three kingdoms of life. We have shown that Era also binds h45. Among the 10 nt, however, G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the GTPase activity, its interaction with Era is essential for the protein to function, leading to the discovery of a cold-sensitive phenotype of the protein. Present in nearly every bacterial species and essential for both cell growth and cell division, Era is unique among all other known protein functions of bacteria. Inhibition of Era function will likely stop the synthesis of bacterial ribosome. Therefore, Era is a potential target for the development of novel antibiotics to fight the worldwide crisis of antibiotic resistance. The eukaryotic homologs of Era (EraL1) is an attractive candidate for a tumor suppressor. It is located in the small subunit of mitochondrial ribosome and interacts with the 12S rRNA, playing important roles in mitochondrial ribosome assembly and cell viability. Currently, structural and functional studies of human and mouse EraL1 proteins are undertaken. We have been working on RNA polymerase (RNAP)-associated transcription factors SspA (stringent starvation protein A), RapA (ATP-dependent dsDNA translocase that recycles RNAP during transcription), and N-utilizing substances A, B, E, and G (NusA, NusB, NusE, and NusG). Previously, we determined the crystal structure of SspA, RapA, and NusG. This year, we have provided structural insight into phage lambda N protein-mediated transcription antitermination. Processive transcription antitermination requires the assembly of a complete antitermination complex, which is initiated by the formation of the ternary NusB-NusE-BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that BoxA is composed of eight nt that are recognized by the NusB-NusE heterodimer. Functional data support the structural observations and establish the relative significance of key protein-protein and protein-RNA interactions. Further crystallographic investigation of a NusB-NusE-dsRNA complex reveals a heretofore unobserved dsRNA-binding site, which is contiguous with BoxA-binding site. We propose that the observed dsRNA represents the BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex. Our effort in structure-based drug development has been focused on Glutathione S-transferase (GST) and 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK). GST represents a superfamily of detoxification enzymes, represented by GST-alpha, -mu, -pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. HPPK is a key enzyme in the folate biosynthetic pathway. Folate cofactors are essential for life. Mammals derive folates from their diet, whereas most microorganisms must synthesize folates de novo. In addition, HPPK is unique for microorganisms and is not the target for any existing antibiotics. Therefore, it is an attractive target for developing novel antimicrobial agents. Previously, our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. We also designed, synthesized, and characterized a group of HPPK inhibitors as lead compounds for novel antibiotics. These inhibitors are linked purine pterin compounds. They bind to HPPK with high affinity and specificity. This year, we have optimized the synthetic route of HPPK inhibitors, leading to the invention of a novel intermediate (6-carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin) and a new method for the synthesis of a known intermediate (6-aldehyde-7,7-dimethyl-7,8-dihydropterin) with a yield of 95%. The derivatives of these two compounds can be used as antifolate agents (antibacterials, antimalarials, and anticancer drugs), as nitric oxide synthase activators for the treatment of cardiovascular diseases, and as pteridine reductase inhibitors targeting African sleeping sickness and the Leishmaniases. The method can also be applied to other systems from methyl heteroaromatic and aromatic compounds to carboxylic ester heteroaromatic and aromatic compounds.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010326-12
Application #
8348975
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2011
Total Cost
$1,350,391
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Song, He; Fang, Xianyang; Jin, Lan et al. (2017) The Functional Cycle of Rnt1p: Five Consecutive Steps of Double-Stranded RNA Processing by a Eukaryotic RNase III. Structure 25:353-363
Ji, Xinhua (2016) Structural insights into cell cycle control by essential GTPase Era. Postepy Biochem 62:335-342
Kumari, Vandana; Dyba, Marzena A; Holland, Ryan J et al. (2016) Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 11:e0163821
Kakar, Smita; Fang, Xianyang; Lubkowska, Lucyna et al. (2015) Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL STUDIES. J Biol Chem 290:23656-69
Li, Shengjian; Liang, Yu-He; Mariano, Jennifer et al. (2015) Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B. J Biol Chem 290:30225-39
Blaszczyk, Jaroslaw; Lu, Zhenwei; Li, Yue et al. (2014) Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase. Cell Biosci 4:52
Antony, Marie L; Lee, Joomin; Hahm, Eun-Ryeong et al. (2014) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of ?-tubulin. J Biol Chem 289:1852-65
Maciag, Anna E; Holland, Ryan J; Kim, Youseung et al. (2014) Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells. J Med Chem 57:2292-302
Liang, Yu-He; Lavoie, Mathieu; Comeau, Marc-Andre et al. (2014) Structure of a eukaryotic RNase III postcleavage complex reveals a double-ruler mechanism for substrate selection. Mol Cell 54:431-44
Court, Donald L; Gan, Jianhua; Liang, Yu-He et al. (2013) RNase III: Genetics and function; structure and mechanism. Annu Rev Genet 47:405-31

Showing the most recent 10 out of 28 publications