We report the development of a β‐turn mimic that allows the direct formation of cyclic peptides through a spontaneous cyclisation under standard solid phase peptide synthesis (SPPS) cleavage ...conditions. The mimic is formed via an acylhydrazone, which is either reduced in situ by triisopropylsilane‐trifluoroacetic acid, or which can be isolated and reduced in a separate step. This method uses commercially available reagents and is compatible with manual and automated SPPS methods. The cyclisation is tolerant of polar residues at the C‐terminal position, with the exception of asparagine, for which a subsequent structural rearrangement similar to aspartimide formation was observed. The cyclisation method has been shown to tolerate ring sizes equivalent to 5–10 amino acid residues. We have used this method to design and synthesise potential selective integrin binding sequences with controlled conformations.
BACKGROUNDHepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular fecal microbial transplant (FMT) in HE, with favorable ...changes in microbial composition and cognition. However, microbial functional changes are unclear. The aim of this study was to determine the effect of FMT on the gut-brain axis compared with placebo, using microbial function based on bile acids (BAs), inflammation (serum IL-6, LPS-binding protein LBP), and their association with EncephalApp.METHODSTwenty cirrhotic patients were randomized 1:1 into groups that received 1-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae or placebo capsules, with 5-month follow-up for safety outcomes. Stool microbiota and BA; serum IL-6, BA, and LBP; and EncephalApp were analyzed at baseline and 4 weeks after FMT/placebo. Correlation networks among microbiota, BAs, EncephalApp, IL-6, and LBP were performed before/after FMT.RESULTSFMT-assigned participants had 1 HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared with baseline. No change was seen in placebo. Correlation networks showed greater complexity after FMT compared with baseline. Beneficial taxa, such as Ruminococcaceae, Verrucomicrobiaceae, and Lachnospiraceae, were correlated with cognitive improvement and decrease in inflammation after FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes.CONCLUSIONGut microbial function in cirrhosis is beneficially affected by capsular FMT, with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.TRIAL REGISTRATIONClinicaltrials.gov NCT03152188.FUNDINGNational Center for Advancing Translational Sciences NIH grant R21TR002024, VA Merit Review grant 2I0CX001076, the United Kingdom National Institute for Health Research Biomedical Facility at Imperial College London, the British Heart Foundation, Wellcome Trust, and King's College London.
Radiotracers labelled with technetium-99m (
Tc) enable accessible diagnostic imaging of disease, provided that radiotracer preparation is simple. Whilst
Tc radiopharmaceuticals for imaging perfusion ...are routinely prepared from kits, and regularly used in healthcare, there are no
Tc-labelled receptor-targeted radiopharmaceuticals in widespread clinical use. This is in part due to the multistep radiosyntheses required for the latter. We demonstrate that the diphosphine, 2,3-bis(diphenylphosphino)maleic anhydride (BMA), is an excellent platform for preparation of kit-based, receptor-targeted
Tc-labelled radiotracers: its conjugates are simple to prepare and can be easily labelled with
Tc using one-step, kit-based protocols. Here, reaction of BMA with the α
β
-integrin receptor targeted cyclic peptide, Arg-Gly-Asp-DPhe-Lys (RGD), provided the first diphosphine-peptide conjugate, DP-RGD. DP-RGD was incorporated into a "kit", and addition of a saline solution containing
TcO
to this kit, followed by heating, furnished the radiotracer
TcO
(DP-RGD)
in consistently high radiochemical yields (>90%). The analogous ReO
(DP-RGD)
compound was prepared and characterised, revealing that both
TcO
(DP-RGD)
and ReO
(DP-RGD)
consist of a mixture of
and
geometric isomers. Finally,
TcO
(DP-RGD)
exhibited high metabolic stability, and selectively targeted α
β
-integrin receptors, enabling
SPECT imaging of α
β
-integrin receptor expression in mice.
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.
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.
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.
Mice over-expressing the creatine transporter have elevated myocardial creatine levels Cr and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very ...high Cr develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for Cr levels: wildtype, moderately elevated, and high Cr (65–85; 100–135; 160–250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for Cr quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC–MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA
P
≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high Cr, with proteins showing Cr-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between Cr and key cardiac metabolites. For example, positive correlations with α-glucose (
r
² = 0.45;
P
= 0.002), acetyl-carnitine (
r
² = 0.50;
P
= 0.001), glutamine (
r
² = 0.59;
P
= 0.0002); and negative correlations with taurine (
r
² = 0.74;
P
< 0.0001), fumarate (
r
² = 0.45;
P
= 0.003), aspartate (
r
² = 0.59;
P
= 0.0002), alanine (
r
² = 0.66;
P
< 0.0001) and phosphocholine (
r
² = 0.60;
P
= 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.
Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are ...regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.
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•The structure of CsfB is unique among anti-sigma factors•CsfB assembles into a tight homodimer of treble-clef zinc finger domains•CsfB dimerization is essential for inhibition of two alternative sigma factors
Martínez-Lumbreras, Alfano et al. have solved the structure of the anti-sigma factor CsfB and explored its role in inhibiting two alternative sigma factors during Bacillus subtilis spore formation. The results provide insight into the molecular mechanism underlying a gene expression switch in bacteria.
Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency ...of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.
PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, ...and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of “high-risk” human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-T/S-X-L/V binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.
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► The solution structure of the second PDZ domain of the MAGI-1 protein has been solved both in its free form and in complex with a peptide from the E6 HPV oncoprotein. ► Peptide binding leads to a disorder-to-order transition at the C-terminal part of the PDZ domain. ► Mutagenesis of the C-terminal extension (outside the canonical PDZ domain boundaries) leads to a decrease in affinity for the E6 peptide.
In response to Krstajic’s letter to the editor concerning our published paper, we here take the opportunity to reply, to re-iterate that no errors in our work were identified, to provide further ...details, and to re-emphasise the outputs of our study. Moreover, we highlight that all of the data are freely available for the wider scientific community (including the aforementioned correspondent) to undertake follow-on studies and comparisons.
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