Tam T. T. Bui

Lipid-Peptide Vesicle Nanoscale Hybrids for Triggered Drug Release by Mild Hyperthermia in vitro and in vivo

The present study describes leucine zipper peptide-lipid hybrid nanoscale vesicles engineered by self-assembled anchoring of the amphiphilic peptide within the lipid bilayer. These hybrid vesicles aim to combine the advantages of traditional temperature-sensitive liposomes (TSL) with the dissociative, unfolding properties of a temperature-sensitive peptide to optimize drug release under mild hyperthermia, while improving in vivo drug retention. The secondary structure of the peptide and its thermal responsiveness after anchoring onto liposomes were studied with circular dichroism. In addition, the lipid-peptide vesicles (Lp-peptide) showed a reduction in bilayer fluidity at the inner core, as observed with DPH anisotropy studies, while the opposite effect was observed with an ANS probe, indicating peptide interactions with both the headgroup region and the hydrophobic core. A model drug molecule, doxorubicin, was successfully encapsulated in the Lp-peptide vesicles at higher than 90% efficiency following the remote loading, pH-gradient methodology. The release of doxorubicin from Lp-peptide hybrids in vitro indicated superior serum stability at physiological temperatures compared to lysolipid-containing temperature-sensitive liposomes (LTSL) without affecting the overall thermo-responsive nature of the vesicles at 42 °C. A similar stabilizing effect was observed in vivo after intravenous administration of the Lp-peptide vesicles by measuring (14)C-doxorubicin blood kinetics that also led to increased tumor accumulation after 24 h. We conclude that Lp-peptide hybrid vesicles present a promising new class of TSL that can offer previously unexplored opportunities for the development of clinically relevant mild hyperthermia-triggered therapeutic modalities.

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein.

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3′ oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3′ UUUOH trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3′ UUUOH trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3′ oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation.

Observation of unphosphorylated STAT3 core protein binding to target dsDNA by PEMSA and X-ray crystallography

pSTAT3βtc and pSTAT3βtc bind by molecular sieving (View interaction)

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

Background: Antimicrobial peptides (AMPs) have the potential to act against multiple pathogenic targets. Results: AMPs that maintain conformational flexibility are more potent against multiple pathogens and less hemolytic. Conclusion: Antimicrobial action and hemolysis proceed via differing mechanisms. Significance: The potency, selectivity, and ability of AMPs to reach intracellular pathogens can be modulated using general principles. We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.

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. Chirality 25:288–293, 2013.

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.

TOAC: a useful Cα-tetrasubstituted α-amino acid for peptide conformational analysis by CD spectroscopy in the visible region. Part I

Doubly labelled 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TOAC)-containing trichogin analogues showed a correlation between the CD intensity of the TOAC transition and their conformation. The helical-inducing property of the TOAC residue is position dependent and, apart from the N-terminal position, better than that of Aib.

Synergic interplay of the La motif, RRM1 and the interdomain linker of LARP6 in the recognition of collagen mRNA expands the RNA binding repertoire of the La module

The La-related proteins (LARPs) form a diverse group of RNA-binding proteins characterized by the possession of a composite RNA binding unit, the La module. The La module comprises two domains, the La motif (LaM) and the RRM1, which together recognize and bind to a wide array of RNA substrates. Structural information regarding the La module is at present restricted to the prototypic La protein, which acts as an RNA chaperone binding to 3′ UUUOH sequences of nascent RNA polymerase III transcripts. In contrast, LARP6 is implicated in the regulation of collagen synthesis and interacts with a specific stem-loop within the 5′ UTR of the collagen mRNA. Here, we present the structure of the LaM and RRM1 of human LARP6 uncovering in both cases considerable structural variation in comparison to the equivalent domains in La and revealing an unprecedented fold for the RRM1. A mutagenic study guided by the structures revealed that RNA recognition requires synergy between the LaM and RRM1 as well as the participation of the interdomain linker, probably in realizing tandem domain configurations and dynamics required for substrate selectivity. Our study highlights a considerable complexity and plasticity in the architecture of the La module within LARPs.