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Abstract: A novel lipopeptide C16KTTβAH was designed that incorporates the KTT tripeptide sequence from “Matrixyl” lipopeptides along with the bioactive βAH (β-alanine-histidine) carnosine dipeptide motif, attached to a C16 hexadecyl lipid chain. We show that this peptide amphiphile self-assembles above a critical aggregation concentration into β-sheet nanotape structures in water, PBS and cell culture media. Nanotape bundle structures were imaged in PBS, the bundling resulting from nanotape associations due to charge screening in the buffer. In addition, hydrogelation was observed and the gel modulus was measured in different aqueous media conditions, revealing tunable hydrogel modulus depending on concentration and nature of the aqueous phase. Stiff hydrogels were observed by direct dissolution in PBS and it was also possible to prepare hydrogels with unprecedented high modulus from low concentration solutions by injection of dilute aqueous solutions into PBS. These hydrogels have exceptional stiffness compared to previously reported β-sheet peptide-based materials. In addition, macroscopic soft threads can be drawn from concentrated aqueous solutions of the lipopeptides which contain aligned nematic structures. The anti-cancer activity of the lipopeptide was assessed using two model breast cancer cell lines, compared to two fibroblast cell line controls. These studies revealed selective concentration-dependent cytotoxicity against MCF-7 cancer cells in a mM concentration range. It was shown that this occurs below the onset of lipopeptide aggregation (i.e. below the critical aggregation concentration), indicating that the cytotoxicity is not related to self-assembly but is an intrinsic property of C16KTTβAH. Finally, hydrogels of this lipopeptide demonstrated slow uptake and release of the dye Congo red, a model diagnostic compound.

Open Access

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Abstract: Natural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 2 EBV is less transforming in vitro due to sequence differences in the EBV transcription factor EBNA2. This correlates with reduced activation of the EBV oncogene LMP1 and some cell genes. Transcriptional activation by type 1 EBNA2 can be suppressed through the binding of two PXLXP motifs in its transactivation domain (TAD) to the dimeric coiled-coil MYND domain (CC-MYND) of the BS69 repressor protein (ZMYND11). We identified a third conserved PXLXP motif in type 2 EBNA2. We found that type 2 EBNA2 peptides containing this motif bound BS69CC-MYND efficiently and that the type 2 EBNA2TAD bound an additional BS69CC-MYND molecule. Full-length type 2 EBNA2 also bound BS69 more efficiently in pull-down assays. Molecular weight analysis and low-resolution structures obtained using small-angle X-ray scattering showed that three BS69CC-MYND dimers bound two molecules of type 2 EBNA2TAD, in line with the dimeric state of full-length EBNA2 in vivo. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved B-cell growth maintenance and the transcriptional activation of the LMP1 and CXCR7 genes. Our data indicate that increased association with BS69 restricts the function of type 2 EBNA2 as a transcriptional activator and driver of B cell growth and may contribute to reduced B-cell transformation by type 2 EBV.

Open Access

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Abstract: The self-assembly and biocatalytic activity of the proline-functionalized lipopeptide PRW-NH-C16 are examined, and compared to that of the related PRW-O-C16 lipopeptide, which differs in having an ester linker between the lipid chain and tripeptide headgroup instead of an amide linker. Lipopeptide PRW-NH-C16 self-assembles into spherical micelles above a critical aggregation concentration, similar to the behaviour of PRW-O-C16 reported previously [B.M. Soares et al. Phys. Chem. Chem. Phys., 2017, 19, 1181—1189]. However, PRW-NH-C16 shows improved catalytic activity in a model aldol reaction. In addition, we explore incorporation of the biocatalytic lipopeptide into lipid cubosomes. SAXS shows that increasing lipopeptide concentration leads to an expansion of the monoolein cubosome lattice spacing, and loss of long-range cubic order as the lipopeptide is encapsulated in the cubosomes. At higher loadings of lipopeptide, reduced cubosome formation is observed at the expense of vesicle formation. Our results show that the peptide-lipid chain linker does not influence self-assembly but does impart improved biocatalytic activity. Furthermore, we show that lipopeptides can be incorporated into lipid cubosomes, leading to restructuring into vesicles at high loadings. These findings point the way towards the future development of bioactive lipopeptide assemblies and slow release cubosome-based delivery systems.

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Abstract: UNC-6 (UNCoordinated-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1 it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40 specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution X-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.

Open Access

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Abstract: The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.