Engineering bioactive peptide-based therapeutic molecules.

Peptides are increasingly emerging as human therapeutic drugs. By screening very large phage display libraries, novel bioactive peptides that bind to the target of interest with desired biological properties can be identified. Peptides that are obtained in this fashion become the basis for therapeutic molecule development. However, naked peptides are usually not sufficient to be therapeutic molecules by themselves. They need to be chemically modified or conjugated to other molecules to obtain desired physicochemical and in vivo properties. In this chapter, we describe a general methodology of identifying bioactive peptides by biopanning of peptide phage libraries. As an example of therapeutic peptide modifications, we also describe a method for fusing the peptides to the Fc portion of antibody molecule to increase in vivo stability and activity.

The homeodomain-leucine zipper ATHB23, a phytochrome B-interacting protein, is important for phytochrome B-mediated red light signaling.

Phytochromes are red (R)/far-red (FR) photoreceptors that are central to the regulation of plant growth and development. Although it is well known that photoactivated phytochromes are translocated into the nucleus where they interact with a variety of nuclear proteins and ultimately regulate genome-wide transcription, the mechanisms by which these photoreceptors function are not completely understood. In an effort to enhance our understanding of phytochrome-mediated light signaling networks, we attempted to identify novel proteins interacting with phytochrome B (phyB). Using affinity purification in Arabidopsis phyB overexpressor, coupled with mass spectrometry analysis, 16 proteins that interact with phyB in vivo were identified. Interactions between phyB and six putative phyB-interacting proteins were confirmed by bimolecular fluorescence complementation (BiFC) analysis. Involvement of these proteins in phyB-mediated signaling pathways was also revealed by physiological analysis of the mutants defective in each phyB-interacting protein. We further characterized the athb23 mutant impaired in the homeobox protein 23 (ATHB23) gene. The athb23 mutant displayed altered hypocotyl growth under R light, as well as defects in phyB-dependent seed germination and phyB-mediated cotyledon expansion. Taken together, these results suggest that the ATHB23 transcription factor is a novel component of the phyB-mediated R light signaling pathway.

Auxin response factor 2 (ARF2) plays a major role in regulating auxin-mediated leaf longevity.

Auxin regulates a variety of physiological and developmental processes in plants. Although auxin acts as a suppressor of leaf senescence, its exact role in this respect has not been clearly defined, aside from circumstantial evidence. It was found here that ARF2 functions in the auxin-mediated control of Arabidopsis leaf longevity, as discovered by screening EMS mutant pools for a delayed leaf senescence phenotype. Two allelic mutations, ore14-1 and 14-2, caused a highly significant delay in all senescence parameters examined, including chlorophyll content, the photochemical efficiency of photosystem II, membrane ion leakage, and the expression of senescence-associated genes. A delay of senescence symptoms was also observed under various senescence-accelerating conditions, where detached leaves were treated with darkness, phytohormones, or oxidative stress. These results indicate that the gene defined by these mutations might be a key regulatory genetic component controlling functional leaf senescence. Map-based cloning of ORE14 revealed that it encodes ARF2, a member of the auxin response factor (ARF) protein family, which modulates early auxin-induced gene expression in plants. The ore14/arf2 mutation also conferred an increased sensitivity to exogenous auxin in hypocotyl growth inhibition, thereby demonstrating that ARF2 is a repressor of auxin signalling. Therefore, the ore14/arf2 lesion appears to cause reduced repression of auxin signalling with increased auxin sensitivity, leading to delayed senescence. Altogether, our data suggest that ARF2 positively regulates leaf senescence in Arabidopsis.

CRY1 inhibits COP1-mediated degradation of BIT1, a MYB transcription factor, to activate blue light-dependent gene expression in Arabidopsis.

Cryptochromes (CRY) are one of the two major classes of photoreceptors that perceive light stimuli in the UV-A to blue light region and they are involved in multiple aspects of plant growth and development. However, knowledge regarding their signaling transduction components and mechanisms remains limited. Here, we report that a MYB transcription factor Blue Insensitive Trait 1 (BIT1), plays an important role in controlling blue light responses. Hypocotyl growth responses indicate that BIT1 functions as a positive element in blue light signaling, since BIT1 antisense and knock-out lines show a reduced light response in blue light. BIT1 controls blue light-dependent expression of various genes such as PsbS, a member of the light-harvesting complex gene family. A transactivation assay showed that BIT1 regulates promoter activity of PsbS in a blue light-dependent manner and that it requires CRY1 for activation of the PsbS promoter. BIT1 undergoes degradation in darkness and CRY1 functions to stabilize BIT1 in a blue light-dependent manner. In contrast, COP1 binds to BIT1 and mediates its degradation. We propose that the PsbS promoter is activated in blue light via the blue light-dependent stabilization of BIT1 by CRY1, while in darkness BIT1 is degraded by COP1-mediated proteolysis.

Overexpression of a chromatin architecture-controlling AT-hook protein extends leaf longevity and increases the post-harvest storage life of plants.

Leaf senescence is the final stage of leaf development and is finely regulated via a complex genetic regulatory network incorporating both developmental and environmental factors. In an effort to identify negative regulators of leaf senescence, we screened activation-tagged Arabidopsis lines for mutants that exhibit a delayed leaf senescence phenotype. One of the mutants (ore7-1D) showed a highly significant delay of leaf senescence in the heterozygous state, leading to at least a twofold increase in leaf longevity. The activated gene (ORE7/ESC) encoded a protein with an AT-hook DNA-binding motif; such proteins are known to co-regulate transcription of genes through modification of chromatin architecture. We showed that ORE7/ESC, in addition to binding to a plant AT-rich DNA fragment, could also modify the chromatin architecture, as illustrated by an altered distribution of a histone-GFP fusion protein in the nucleus of the mutant. Globally altered gene expression, shown by microarray analysis, also indicated that activation of ORE7/ESC results in a younger condition in the mutant leaves. We propose that ectopically expressed ORE7/ESC is negatively regulating leaf senescence and suggest that the resulting chromatin alteration may have a role in controlling leaf longevity. Interestingly, activation of ORE7/ESC also led to a highly extended post-harvest storage life.

Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer.

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.