Jiying Zhang

Bio: Jiying Zhang is an academic researcher from University of Pittsburgh. The author has contributed to research in topics: Binding site & Binding domain. The author has an hindex of 1, co-authored 1 publications receiving 11 citations.

Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein.

Abstract: Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells.

Using peptides to study the interaction between the p53 tetramerization domain and HIV‐1 tat

Abstract: Peptides are valuable tools for studying protein–protein interactions, especially in cases of isolated protein domains and natively unfolded proteins. Here, we used peptides to quantitatively characterize the interaction between the natively unfolded HIV-1 Tat protein and the tetramerization domain of the cellular tumor suppressor protein p53. We used peptide mapping, fluorescence anisotropy, and NMR spectroscopy to perform a detailed structural and biophysical characterization of the interaction between the two proteins and elucidate its molecular mechanism, which have so far been studied using cell-based methods. We show that the p53 tetramerization domain, p53(326–355), binds directly to residues 1–35 and 47–57 in Tat. We have characterized the interaction between p53(326–355) and Tat(47–57) in detail. The p53 residues that are mainly involved in binding to Tat(47–57) are E343 and E349, which bind to the positively charged arginine-rich motif of Tat by a partly electrostatic mechanism. All oligomerization states of p53(326–355) bind Tat(47–57) without inhibiting p53 tetramerization, since the residues in p53(326–355) that bind Tat(47–57) face away from the tetramerization interface. We conclude that p53 is able to bind Tat as a transcriptionally active tetramer. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 105–116, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Altering the Tat-derived peptide bioactivity landscape by changing the arginine side chain length.

Abstract: Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.

Hurdles and Improvements in Therapeutic Gene Transfer for Cancer

Abstract: Over the past decades, gene therapy has emerged as representing a potential treatment modality for cancer. One promising cancer gene therapy approach is based on introducing a gene that encodes for an enzyme called a suicide protein into tumor. This enzyme converts a normally harmless prodrug into a toxic form that induces tumor cell death. Cell killing is observed also in the surrounding, non-transduced cells, which is a benefit since all tumor cells do not need contain the therapeutic gene. This phenomenon is called the bystander effect. Nevertheless, true success in clinical trials has not been achieved mainly due to insufficient gene delivery rate of the current vectors and inadequate bystander effect in many tumors. In the present study we evaluated various methods to overcome these problems; first by characterizing factors that may influence efficient therapeutic gene transfer and further by modifying viral vectors and the therapeutic gene with the aid of cell penetrating peptides. A number of factors, including host cell immune responses, can influence the gene transfer efficiency of viral and non-viral vectors. For that reason, we studied the contribution of the type I interferon response, an arm of innate immune system, to the therapeutic gene transfer. The commonly used viral vectors, with the exception of Semliki Forest virus, succeeded in avoiding the induction of the type I IFN response. However, the delivery of plasmid DNA and particularly most forms of RNA triggered the response in a variety of studied cell lines. In order to improve delivery of therapeutic gene into the tumor cells, we evaluated the feasibility of using cell penetrating peptides derived from Drosophila Antennapedia homeodomain and HIV-1 transactivator protein (TAT). These cationic peptides enhanced transduction efficiency of adenoand lentiviral vectors significantly in most of the tested human tumor cells. However, the property of a commonly used commercial transduction enhancer was found to be even better at boosting efficacy than the cell penetrating peptides. In another study included in this thesis, the cell penetrating peptide TAT was linked to suicide-marker fusion gene (TAT-TK-GFP and TK-GFP) to extend the cytotoxic impact of suicide gene therapy to adjacent cells and thus to compensate for the poor gene delivery rate. Against our original hypothesis, we found that the TAT containing fusion proteins were not trafficking between the cells. Despite the lack of intercellular movement, TAT-mediated increased cell killing was observed in some of the tested human tumor cell lines. However, in many cell lines the killing efficiencies of TAT-TK-GFP and TK-GFP were similar and in some cell lines the efficiency of TK-GFP was even better. In conclusion, these results indicate that the gene delivery can induce undesired immune responses in target cells and thus may represent a barrier against efficient therapeutic gene transfer. Although cell penetrating peptides improved the viral transduction rate, the utility of these peptides as general enhancers will most likely be limited by their high manufacturing costs compared to commercially available and clinically approved compound. Even though TAT -containing suicide fusion protein showed some enhancement of cell killing in certain tumor cell lines, no overall difference in efficacy between TAT-TK-GFP and TK-GFP was seen. Therefore, this concept needs to be further refined if it is to be considered as a potential supplement for cancer suicide gene therapy. National Library of Medicine Classification: QZ 52, QZ 266, QU 470, QU 475, QU 68 Medical Subject Headings: Neoplasms/therapy; Gene Therapy; Gene Transfer Techniques; Transduction, Genetic; Genetic Vectors; Viruses; Cell Death; Bystander Effect; Immunity; Interferon Type I; Drosophila Proteins; Antennapedia Homeodomain Protein; Gene Products, tat; Peptide Fragments; Transcription Factors; Genes, Transgenic, Suicide; Thymidine Kinase; Ganciclovir

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy.

Abstract: Cancer suicide gene therapy based on herpes simplex virus type I thymidine kinase (HSV-TK) and ganciclovir (GCV) suffers from the lack of efficacy in clinical use, which is mostly due to low gene-transfer efficiency and absence of bystander effect in tumors. We have previously demonstrated the enhancement of GCV cytotoxicity by fusing the HSV-TK with the cell penetrating peptide from HIV-1 transactivator protein transduction domain (TAT PTD). Despite the earlier promising results, we found that the triple fusion protein HIV-1 transactivator protein transduction domain-thymidine kinase suicide gene-green fluorescent protein marker gene (TAT-TK-GFP) increased GCV cytotoxicity only in 3/12 of different human tumor cell lines. Extended GCV exposure enhanced the cytotoxic effect of HSV-TK/GCV gene therapy, but the difference between TK-GFP and TAT-TK-GFP was not statistically significant. The modest improvement on cell killing mediated by TAT PTD in Chinese hamster ovary cells appeared to be associated with cell-surface heparan sulfate proteoglycan (HSPG) composition. However, TAT-mediated increased cell death did not correlate with the density of cell-surface HSPG expression in different tumor cell lines. In conclusion, although some degree of enhancement by TAT was shown in certain tumor cells in vitro, it is unlikely that TAT peptide linked to a suicide protein could be a useful booster of in vivo gene therapy trials.