Abstract

As hypusine biosynthetic steps present unique target for intervention in aberrant cell proliferation, we need to develop new inhibitors of deoxyhypusine synthase (DHS) through high throughput screening (HTS) of small molecule libraries. However, the conventional DHS assay measuring incorporation of radioactivity from spermidine into eIF5A protein is not suitable for HTS. In this period, we have concentrated our efforts on the development of a new DHS assay that is compatible with HTS. This was accomplished by incubation of DHS with spermidine and NAD in the absence of eIF5A precursor protein to generate NADH and by detection of NADH using NADH-Glo assay. Robust luminescence signals were generated in the DHS/NADH-Glo coupled reaction. The reaction is sensitive and reproducible. Using this assay, we have initiated HTS and will continue screening of several chemical libraries. Since HTS requires a large amount of DHS, we also devised a new method to purify recombinant DHS protein from E. coli BL21(DE3) cells in high yield, by codon optimization. Although majority of the recombinant enzymes resulted in inclusion bodies, the enzyme was solubilized from inclusion bodies using high pH Tris buffer containing 2M urea.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIADE000608-24
Application #
9555612
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2017
Total Cost
Indirect Cost

  Institution

Name
Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
Zip Code

  Publications

Park, Myung Hee; Mandal, Ajeet; Mandal, Swati et al. (2017) A new non-radioactive deoxyhypusine synthase assay adaptable to high throughput screening. Amino Acids 49:1793-1804
Mandal, Ajeet; Mandal, Swati; Park, Myung Hee (2016) Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells. Sci Rep 6:25795
Saxena, Deepti; Spino, Michael; Tricta, Fernando et al. (2016) Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial. PLoS One 11:e0154842
Xiao, Su; Chen, Yu Chi; Buehler, Eugen et al. (2016) Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol Bioeng 113:2403-15
Jasniewski, Andrew J; Engstrom, Lisa M; Vu, Van V et al. (2016) X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a. J Biol Inorg Chem 21:605-18
Mandal, Swati; Mandal, Ajeet; Park, Myung Hee (2015) Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells. Biochem J 468:435-47
Frey, Avery G; Nandal, Anjali; Park, Jong Hwan et al. (2014) Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci U S A 111:8031-6
Mittal, Nimisha; Morada, Marie; Tripathi, Pankaj et al. (2014) Cryptosporidium parvum has an active hypusine biosynthesis pathway. Mol Biochem Parasitol 195:14-22
Mandal, Ajeet; Mandal, Swati; Park, Myung Hee (2014) Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution. PLoS One 9:e111800
Beninati, Simone; Park, Myung Hee; Wolff, Edith et al. (2013) In memoriam: John E. Folk (1925-2010). Amino Acids 44:11-8

Showing the most recent 10 out of 30 publications