A Versatile Virus-Mimetic Engineering Approach for Concurrent Protein Nanocage Surface-Functionalization and Cargo Encapsulation.

Naturally occurring protein nanocages like ferritin are self-assembled from multiple subunits. Because of their unique cage-like structure and biocompatibility, there is a growing interest in their biomedical use. A multipurpose and straightforward engineering approach does not exist for using nanocages to make drug-delivery systems by encapsulating hydrophilic or hydrophobic drugs and developing vaccines by surface functionalization with a protein like an antigen. Here, a versatile engineering approach is described by mimicking the HIV-1 Gap polyprotein precursor. Various PREcursors of nanoCages (PREC) are designed and created by linking two ferritin subunits via a flexible linker peptide containing a protease cleavage site. These precursors can have additional proteins at their N-terminus, and their protease cleavage generates ferritin-like nanocages named protease-induced nanocages (PINCs). It is demonstrated that PINC formation allows concurrent surface decoration with a protein and hydrophilic or hydrophobic drug encapsulation up to fourfold more than the amount achieved using other methods. The PINCs/Drug complex is stable and efficiently kills cancer cells. This work provides insight into the precursors' design rules and the mechanism of PINCs formation. The engineering approach and mechanistic insight described here will facilitate nanocages' applications in drug delivery or as a platform for making multifunctional therapeutics like mosaic vaccines.

Binding Interactions of Peptide Aptamers.

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

15-deoxy-Δ12,14-Prostaglandin J2 inhibits human soluble epoxide hydrolase by a dual orthosteric and allosteric mechanism.

Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.

Consequences of point mutations in melanoma-associated antigen 4 (MAGE-A4) protein: Insights from structural and biophysical studies.

The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.

Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs) of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

Spectroscopic studies on nicotine and nornicotine in the UV region.

The UV absorption and electronic circular dichroism (ECD) spectra of (R)- and (S)-nicotine and (S)-nornicotine in aqueous solution were measured to a significantly lower wavelength range than previously reported, allowing the identification of four previously unobserved electronic transitions. The ECD spectra of the two enantiomers of nicotine were equal in magnitude and opposite in sign, while the UV absorption spectra were coincidental. In line with previous observations, (S)-nicotine exhibited a negative cotton effect centered on 263 nm with vibronic structure (π-π1 * transition) and a broad, positive ECD signal at around 240 nm associated with the n-π1 * transition. As expected this band disappeared when the pyridyl aromatic moiety was protonated. Four further electronic transitions are reported between 215 and 180 nm; it is proposed the negative maxima around 206 nm is either an n-σ* transition or a charge transfer band resulting from the movement of charge from the pyrrolidyl N lone pair to the pyridyl π* orbital. The pyridyl π-π2* transition may be contained within the negative ECD signal envelope at around 200 nm. Another negative maximum at 188 nm is thought to be the pyridyl π-π3 * transition, while the lowest wavelength end-absorption and positive ECD may be associated with the π-π4 * transition. The UV absorption spectra of (S)-nornicotine was similar to that of (S)-nicotine in the range 280-220 nm and acidification of the aqueous solution enhanced the absorption. The ECD signals of (S)-nornicotine were considerably less intense compared to (S)-nicotine and declined further on acidification; in the far UV region the ECD spectra diverge considerably.

Observation of a single-stranded DNA/pyrrolobenzodiazepine adduct.

Pyrrolobenzodiazepine (PBD) antitumor agents have, to date, only been observed to bind to duplex DNA, apparently requiring a minor groove environment for covalent bond formation between their C11-position and the C2-NH(2) functionality of a guanine base. Using an HPLC/MS assay we have now observed and isolated for the first time PBD adducts with single-stranded DNA fragments. Surprisingly, these adducts could only be formed through dissociation of duplex DNA adducts and not by direct interaction of PBDs with single-stranded DNA. They were sufficiently stable for characterization by MALDI-TOF-MS and remained intact after storing at -20 °C for at least 20 days, although the PBD became detached from the DNA within 7 days if stored at room temperature. Furthermore, addition of a complementary strand allowed the duplex adduct to reform. The relative stability of single-stranded PBD/DNA adducts despite a complete loss of minor groove structure was further confirmed by CD spectroscopic analysis. The CD signal induced by the presence of a PBD molecule in the single-stranded adducts remained prominent despite heating for 2 h at 50-60 °C, thus indicating their relatively robust nature.

Mutants of the tumour suppressor p53 L1 loop as second-site suppressors for restoring DNA binding to oncogenic p53 mutations: structural and biochemical insights.

To assess the potential of mutations from the L1 loop of the tumour suppressor p53 as second-site suppressors, the effect of H115N and S116M on the p53 'hot spot' mutations has been investigated using the double-mutant approach. The effects of these two mutants on the p53 hot spots in terms of thermal stability and DNA binding were evaluated. The results show that: (i) the p53 mutants H115N and S116M are thermally more stable than wild-type p53; (ii) H115N but not S116M is capable of rescuing the DNA binding of one of the most frequent p53 mutants in cancer, R248Q, as shown by binding of R248Q/H115N to gadd45 (the promoter of a gene involved in cell-cycle arrest); (iii) the double mutant R248Q/H115N is more stable than wild-type p53; (iv) the effect of H115N as a second-site suppressor to restore DNA-binding activity is specific to R248Q, but not to R248W; (v) molecular-dynamics simulations indicate that R248Q/H115N has a conformation similar to wild-type p53, which is distinct from that of R248Q. These findings could be exploited in designing strategies for cancer therapy to identify molecules that could mimic the effect of H115N in restoring function to oncogenic p53 mutants.

The p73 DNA binding domain displays enhanced stability relative to its homologue, the tumor suppressor p53, and exhibits cooperative DNA binding.

The p53 protein family is involved in the control of an intricate network of genes implicated in cell cycle, through to germ line integrity and development. Although the role of p53 is well-established, the intrinsic nature of its homologue p73 has yet to be fully elucidated. Here, the biochemical characterization and homology-based modeling of the p73 protein is presented and the implications for its function(s) examined. The DNA binding domains (DBDs) of p53, p63, and p73 bind to the specific target site of a 30-mer gadd45 dsDNA, as tested by EMSA. The monomeric DBDs bind cooperatively forming tetrameric complexes. However, a larger construct consisting of p73 DBD plus TET domain (p73 CT) and the corresponding p53 DBD plus TET domain (p53 CT) bind gadd45 differently than the respective DBDs. Significantly, p73 DBD exhibited enhanced thermodynamic stability relative to the p53 DBD but not compared to p63 DBD as shown by DSC, CD, and equilibrium unfolding. The p73 CT is less stable than p73 DBD. The modeling data show distinct electrostatic surfaces of p73 and p53 dimers when bound to DNA. Specifically, the p73 surface is less complementary for DNA binding, which may account for the differences in affinity and specificity for p53 REs. These stability and DNA binding data for p73 in vitro enhance and complement our understanding of the role of the p73 protein in vivo and could be exploited in designing strategies for cancer therapy in places where p53 is mutated.

Multiple protein interactions involving proposed extracellular loop domains of the tight junction protein occludin.

Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101-121 and O-B:210-228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101-121 and O-B:210-228 showed them to have dissimilar solution conformation characteristics. Although O-A:101-121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210-228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210-228, but not O-A:101-121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210-228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures.