Testing the length limit of loop grafting in a helical repeat protein.

Alpha-helical repeat proteins such as consensus-designed tetratricopeptide repeats (CTPRs) are exceptionally stable molecules that are able to tolerate destabilizing sequence alterations and are therefore becoming increasingly valued as a modular platform for biotechnology and biotherapeutic applications. A simple approach to functionalize the CTPR scaffold that we are pioneering is the insertion of short linear motifs (SLiMs) into the loops between adjacent repeats. Here, we test the limits of the scaffold by inserting 17 highly diverse amino acid sequences of up to 58 amino acids in length into a two-repeat protein and examine the impact on protein folding, stability and solubility. The sequences include three SLiMs that bind oncoproteins and eleven naturally occurring linker sequences all predicted to be intrinsically disordered but with conformational preferences ranging from compact globules to expanded coils. We show that the loop-grafted proteins retain the native CTPR structure and are thermally stable with melting temperatures above 60 ​°C, despite the longest loop sequence being almost the same size as the CTPR scaffold itself (68 amino acids). Although the main determinant of the effect of stability was found to be loop length and was relatively insensitive to amino acid composition, the relationship between protein solubility and the loop sequences was more complex, with the presence of negatively charged amino acids enhancing the solubility. Our findings will help us to fully realize the potential of the repeat-protein scaffold, allowing a rational design approach to create artificial modular proteins with customized functional capabilities

Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.BBSRC Doctoral Training Programme (DTP) scholarship Oliver Gatty Studentship AstraZeneca PhD studentship. UK Medical Research Foundation. CRUK Pioneer Award (C17838/A22676) CRUK BTERP Award (C17838/A27225) Leverhulme Trust (RPG-2014-089) Cambridge Newton Trust BBSRC project grant (BB/T002697/1)

ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors

Abstract Background Intrinsically fluorescent glucose derived carbon nanospheres (CSP) efficiently enter mammalian cells and also cross the blood brain barrier (BBB). However, the mechanistic details of CSP entry inside mammalian cells and its specificity are not known. Results In this report, the biochemical and cellular mechanism of CSP entry into the living cell have been investigated. By employing confocal imaging we show that CSP entry into the mammalian cells is an ATP-dependent clathrin mediated endocytosis process. Zeta potential studies suggest that it has a strong preference for cells which possess high levels of glucose transporters such as the glial cells, thereby enabling it to target individual organs/tissues such as the brain with increased specificity. Conclusion The endocytosis of Glucose derived CSP into mammalian cells is an ATP dependent process mediated by clathrin coated pits. CSPs utilize the surface functional groups to target cells containing glucose transporters on its membrane thereby implicating a potential application for specific targeting of the brain or cancer cells.</p

Multifunctional carbon nanospheres with magnetic and luminescent probes: probable brain theranostic agents

Multi-functional carbon nanospheres with magnetic Prussian blue nanoparticles and luminescent lanthanide ions have been prepared. The negatively charged surface of the glucose derived carbon sphere facilitates the nucleation of Prussian blue nanoparticles on its surface. The luminescent lanthanide probes were attached to the surface of the carbon sphere through a benzene tricarboxylic acid linker. These multifunctional hybrid organic–inorganic composites are superparamagnetic and show enhanced luminescent properties. Their ability to cross the blood–brain barrier (enter the brain cell nucleus with no animal toxicity) in a mouse model indicate that these nanocomposites are promising theranostic agents for the treatment of brain diseases