Sumita Raha

Peptide-mediated cancer targeting of nanoconjugates

Targeted use of nanoparticles in vitro, in cells, and in vivo requires nanoparticle surface functionalization. Moieties that can be used for such a purpose include small molecules as well as polymers made of different biological and organic materials. Short amino acid polymers, peptides, can often rival target binding avidity of much larger molecules. At the same time, peptides are smaller than most nanoparticles and thus allow for multiple nanoparticle modifications and creation of pluripotent nanoparticles. Most nanoparticles provide multiple binding sites for different cargo and targeting peptides which can be used for the development of novel approaches for cancer targeting, diagnostics, and therapy. In this review, we will focus on peptides which have been used for the preparation of different nanoparticles designed for cancer research.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco

Key messagePhylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen.AbstractTobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene—as a fusion with the GFP gene—targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.

Production of xylanase in transgenic tobacco for industrial use in bioenergy and biofuel applications

Xylanases are used in various agricultural and industrial applications. A synthetic, modified, codon-optimized xylanase gene (XynZ) from Clostridium thermocellum was expressed in transgenic tobacco plants. The coding sequence of XynZ was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. Three constructs were developed to evaluate XynZ expression levels by targeting gene products into the cytosol, intercellular space, or endoplasmic reticulum in transgenic plants. These chimeric genes, expressed in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) were stably inherited in successive plant generations (R0, R1, and R2 progeny; primary, second, and third generation) as shown by molecular characterization (RT-PCR and qRT-PCR) and enzymatic assays. A Western blot analysis of plant extracts showed presence of a polypeptide of the expected size that cross-reacted with xylanase-specific antibodies. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effect due to transgene expression. The expressed xylanase was heat-stable, having optimum activity between 55°C and 75°C over a pH range of 5 to 5.6.

A Region Containing an as-1 Element of Dahlia Mosaic Virus (DaMV) Subgenomic Transcript Promoter Plays a Key Role in Green Tissue- and Root-Specific Expression in Plants

A subgenomic transcript (Sgt) promoter was isolated from the genomic clone of dahlia mosaic virus (DaMV), which is a double-stranded DNA virus of the Caulimoviridae family. The DaMVSgt promoter, which is linked to the heterologous β-glucuronidase (GUS) reporter gene, was characterized in transient protoplasts and in transgenic tobacco, as well as in Arabidopsis plants. The 5′- and 3′-deletion analysis of a 591-bp DaMVSgt promoter fragment indicated that a 441-bp promoter fragment (−372 to +69 from the transcription start site; TSS) was sufficient for maximal promoter activity. A 141-bp promoter fragment (−72 to +69 from TSS) was the minimal promoter region that also showed relatively strong activity. The three activation sequence-1 (as-1) elements and the border regions were primarily responsible for the promoter activity, as revealed by a finer internal deletion and mutation analysis of the cis-elements and of the immediate border sequence of the activation domain. Electrophoretic mobility shift assay (EMSA), supershift EMSA, DNase I footprinting, Southwestern blotting, and UV cross-linking studies demonstrated the binding of a tobacco transcription factor, TGA1a, that correlated with 2,4-dichlorophenylacetic acid (2,4D)-induced transcriptional activity of the DaMVSgt promoter. Histological GUS staining and the GUS enzymatic assay demonstrated that the 441-bp DaMVSgt4 promoter and 141-bp minimal DaMVSgt4F are 5.5 and 4.6 times, respectively, stronger than the CaMV 35S promoter. The minimal DaMVSgt4F promoter is more active than CaMV 35S in all types of green tissues and roots, without any detectable expression in reproductive tissues and seeds. The DaMVSgt4F promoter may be useful for transgene containment applications.

Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.)

To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells

The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5′ AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

The Ubiquitin-Proteasome System and DNA Repair

The 2004 Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose for their work in discovering the ubiquitin-proteasome system (UPS), as reviewed by Herrmann and others (Herrmann et al., 2007). The mechanisms by which proteolysis occurs had remained elusive until the late 1970s, when a series of key experiments paved the way for a new area of research (Ciechanover et al., 1978; Ciechanover et al., 1980b; Hershko et al., 1980). These studies revealed that the majority of protein degradation is nonlysosomal and adenosine triphosphate (ATP)-dependent. Most importantly, it was also demonstrated that this proteolysis requires at least two components: one with protease activity and another in the form of an 8.5-kDa heat-stable protein. These elements were later identified as the proteasome and ubiquitin, respectively (Ciechanover et al., 1980a; Wilkinson et al., 1980; Hough et al., 1986; Waxman et al., 1987; Arrigo et al., 1988). Substrates of the UPS include many short-lived regulatory proteins in addition to misfolded and defective proteins (Dahlmann, 2007; Naiki & Nagai, 2009; Xie, 2010). Conserved from Archaea to humans, the UPS is thought to be responsible for degrading approximately 90% of nuclear and cytoplasmic proteins (Magill et al., 2003). Through regulation of protein expression, the UPS controls processes such as protein homeostasis, cell-cycle, cell division, cellular differentiation, apoptosis, signal transduction, gene expression, immunity, and DNA repair (Magill et al., 2003; Finley, 2009; Liggett et al., 2010; Shabek & Ciechanover, 2010; Xie, 2010). Although much focus on this system revolves around its proteolytic function and regulation, members of the UPS also play non-proteolytic roles in transcription, membrane trafficking, protein kinase activation, chromatin dynamics, and DNA repair (Chen & Sun, 2009; Xie, 2010). The UPS plays one of the central roles in pathology and disease, and it has become the target of several newer therapeutic modalities. In patients with some forms of cardiac dysfunction, neurodegeneration, autoimmune disease, and viral infections, proteasome activity and/or expression is diminished (Magill et al., 2003; Dahlmann, 2007; Naiki & Nagai, 2009). Conversely, in some cancer patients and patients with cachexia, an increase in proteasome expression has been observed. According to the idea that this increase in proteosome activity is a potential therapeutic target, proteosome inhibitors are developed and isolated from natural products (Orlowski & Kuhn, 2008; Groll et al., 2009; Huang & Chen, 2009).