Study of nano-hydroxyapatite tagged alkaline protease isolated from Himalayan sub-alpine Forest soil bacteria and role in recalcitrant feather waste degradation.

Purified calcium serine metalloprotease from Stenotrophomonas maltophilia strain SMPB12 exhibits highest enzyme activity at pH 9 and temperature range between 15 °C-25 °C. Enzyme supplemented with 40 µM Ca-Hap-NP (NP-protease) showed maximum elevated activity of 17.29 µmole/min/ml (1.9-fold of original protease activity). The thermostability of the enzyme was maintained for 1 h at 60 °C over an alkaline pH range 7.5-10, as compared to the NP untreated enzyme whose activity was of 8.97 µmole/min/ml. A significant loss of activity with EDTA (1.05 µmole/min/ml, 11.75 %), PMSF (0.93 µmole/min/ml, 10.46 %) and Hg2+ (3.81 µmole/min/ml, 42.49 %) was also observed. Kinetics study of NP-protease showed maximum decreases in Km (28.11 %) from 0.28 mM (NP untreated enzyme) to 0.22 mM (NP-protease) along with maximum increase in Vmax (42.88 %) from 1.25 µmole/min/ml to 1.79 µmole/min/ml at varying temperatures. The enhanced activity of NP-protease was able to efficiently degrade recalcitrant solid wastes like feather to produce value-added products like amino acids and helps in declogging recalcitrant solid wastes. The nano-enabled protease may be utilized in a smaller amount for degrading in bulk recalcitrant solid proteinaceous waste at 15 °C temperature as declogging agents providing an eco-friendly efficient process.

Constrained α-Helical Peptides as Inhibitors of Protein-Protein and Protein-DNA Interactions.

Intracellular regulatory pathways are replete with protein-protein and protein-DNA interactions, offering attractive targets for therapeutic interventions. So far, most drugs are targeted toward enzymes and extracellular receptors. Protein-protein and protein-DNA interactions have long been considered as "undruggable". Protein-DNA interactions, in particular, present a difficult challenge due to the repetitive nature of the B-DNA. Recent studies have provided several breakthroughs; however, a design methodology for these classes of inhibitors is still at its infancy. A dominant motif of these macromolecular interactions is an α-helix, raising possibilities that an appropriate conformationally-constrained α-helical peptide may specifically disrupt these interactions. Several methods for conformationally constraining peptides to the α-helical conformation have been developed, including stapling, covalent surrogates of hydrogen bonds and incorporation of unnatural amino acids that restrict the conformational space of the peptide. We will discuss these methods and several case studies where constrained α-helices have been used as building blocks for appropriate molecules. Unlike small molecules, the delivery of these short peptides to their targets is not straightforward as they may possess unfavorable cell penetration and ADME properties. Several methods have been developed in recent times to overcome some of these problems. We will discuss these issues and the prospects of this class of molecules as drugs.

Mutant and Wild-Type Tumor Suppressor p53 Induces p300 Autoacetylation.

The transcriptional co-activator p300 is essential for p53 transactivation, although its precise role remains unclear. We report that p53 activates the acetyltransferase activity of p300 through the enhancement of p300 autoacetylation. Autoacetylated p300 accumulates near the transcription start sites accompanied by a similar enrichment of activating histone marks near those sites. Abrogation of p53-p300 interaction by a site-directed peptide inhibitor abolished p300-mediated histone acetylation, suggesting a crucial role played by the activation in p53-mediated gene regulation. Gain-of-function mutant p53, known to impart aggressive proliferative properties in tumor cells, also activates p300 autoacetylation. The same peptide abolished many of the gain-of-function properties of mutant p53 as well. Reversal of gain-of-function properties of mutant p53 suggests that molecules targeting the p53-p300 interface may be good candidates for anti-tumor drugs.

A peptide-based synthetic transcription factor selectively activates transcription in a mammalian cell.

A peptide-based cell permeable synthetic transcription factor is reported, which binds to its target site with high affinity and specificity. When linked to a HAT-binding peptide, it causes significant upregulation of gene expression in a mammalian cell. Such molecules may be developed for selectively activating repressed genes in mammalian cells.

Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.