In previous studies, we demonstrated the primary role of the polycationic polyamines in translational regulation by overexpression of a polyamine catabolic enzyme, spermidine/spermine acetyltransferase 1 (SAT1) in HEK293 cells. Upon rapid depletion of polyamines by transduction with the adeno SAT1 virus, protein synthesis was inhibited at the level of initiation, while no or little inhibition of synthesis of RNA or DNA occurred. In the current period, we have obtained evidence for enhanced protein synthesis in cells with increased cellular putrescine and spermidine, due to stabilization of ornithine decarboxylase (ODC) activity, the first step enzyme in polyamine biosynthesis. This finding resulted from a genome-wide siRNA screen that was designed to identify genes whose silencing would enhance recombinant protein production. Silencing of OAZ1, the gene that encodes the ODC antizyme 1, consistently increased production of recombinant proteins, including soluble, membrane-bound and secreted proteins. Silencing of OAZ1 increased the recombinant protein production at the translational level, not at the transcriptional level, underscoring the important role of polyamines in translational regulation. We also investigated the function of eIF5A by proteomic analyses of HeLa cells depleted of eIF5A by adeno-eIF5A shRNA transduction, to identify cellular pathways and proteins that are directly or indirectly influenced by eIF5A. We identified 3810, 1258 and 2750 unique proteins (with 2 unique peptides with >95% confidence level) in iTRAQ experiment 1, 2 and 3, respectively, and 972 proteins commonly detected in all three runs. Of these, 104 proteins with significantly altered levels (protein ratio 1.5 or 0.66, p value 0.05) at 72, and 96h of Ad-eIF5A-shRNA transduction were selected for further analyses for polyproline occurrence and functional ontology classification. No consistent relationship was observed between the content of polyproline motifs and the protein levels in eIF5A-depleted cells. Analyses of the functional ontology of the significantly altered proteins revealed specific biological processes that are prominently up-or down-regulated in eIF5A-depleted cells and identified protein folding as the major cellular process affected by the depletion of eIF5A. Activation of the unfolded protein response (UPR) was confirmed by increases in levels of UPR signaling molecules, especially p50-ATF6. Our data from these unbiased, quantitative, proteomic analyses demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells.

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23
Fiscal Year
2016
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Dental & Craniofacial Research
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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

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