Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, ...the chemical nature of the anti-inflammatory compounds has not yet been determined.
Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice.
The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice.
A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.
We apply the Iterative Feedback Tuning (IFT) method to the tuning of PID parameters in applications where the objective is to achieve a fast response to set point changes. We compare the performance ...of these IFT-tuned PID controllers with the performance achieved by four classical PID tuning schemes that are widely used in industry. Our simulations show that IFT always achieves a performance that is at least as good as that of the classical PID tuning schemes, and often dramatically better: faster settling time and less overshoot. In addition, IFT is also optimal with respect to the presence of noise, whereas the other schemes are designed for noise-free conditions. The IFT method used here is a variant of the initial IFT scheme, in which no weighting is applied to the control error during a time window that corresponds to the transient response, and where the length of this window is progressively reduced. This method was initially proposed in Lequin (CD-ROM of European Control Conference, Paper TH-A-H6, Brussels, Belgium, 1997) and elaborated on in Lequin et al. (Proceedings of the 14th IFAC World Congress, Paper I-3b-08-3, Beijing, People's Republic of China, 1999, pp. 433–437).
We have examined an optimization approach to iterative control design. The important ingredient is that the gradient of the design criterion is computed from measured closed loop data. The approach ...is thus not model-based. The scheme converges to a stationary point of the design criterion under the assumption of boundedness of the signals in the loop. From a practical viewpoint, the scheme offers several advantages. It is straightforward to apply. It is possible to control the rate of change of the controller in each iteration. The objective can be manipulated between iterations in order to tighten or loosen performance requirements. Certain frequency regions can be emphasized if desired. This direct optimal tuning algorithm is particularly well suited for the tuning of the basic control loops in the process industry, which are typically PID loops. These primary loops are often very badly tuned, making the application of more advanced (for example, multivariable) techniques rather useless. A first requirement in the successful application of advanced control techniques is that the primary loops be tuned properly. This new technique appears to be a very practical way of doing this, with an almost automatic procedure.
The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide ...cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 Å), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 Å apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.
Fibronectin (FN) forms fibrillar networks coupling cells to the extracellular matrix. The formation of FN fibrils, fibrillogenesis, is a tightly regulated process involving the exposure of cryptic ...binding sites in individual FN type III (FN-III) repeats presumably exposed by mechanical tension. The FN-III 1 module has been previously proposed to contain such cryptic sites that promote the assembly of extracellular matrix FN fibrils. We have combined NMR and steered molecular dynamics simulations to study the structure and mechanical unfolding pathway of FN-III 1 . This study finds that FN-III 1 consists of a β-sandwich structure that unfolds to a mechanically stable intermediate about four times the length of the native folded state. Considering previous experimental findings, our studies provide a structural model by which mechanical stretching of FN-III 1 may induce fibrillogenesis through this partially unfolded intermediate.
Numerous backbone constraints can be used to develop pseudopeptides or pseudomimetics of biologically active peptides. Among those, N- and Cα-methyl amino acids that can be incorporated by solidphase ...peptide synthesis in a bioactive sequence represent important tools to restrictφ and ψ angles of peptide backbone. This review will focus on the chemical syntheses of N- and Cα-methyl amino acids, their effects on peptide conformation and structure, and their role on the peptide stability towards enzymatic degradation and on the biological activities of the resulting analogues.
The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are ...polycationic (Lys-rich), α-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic α-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6−15 (mean hydrophobicity, +4.40, determined by the Liu−Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41−62. Dermaseptin S9 is a potent antibacterial, acting on Gram-positive and Gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic α-helical conformation, mostly in residues 6−21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic α-helical peptides.
Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the ...effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn2+ as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI−TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.
Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in ...TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3−18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11−31 connected to a more flexible helical segment spanning residues 1−8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1−11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure−activity studies on dermaseptin B2 analogues showed that the N-terminal 1−11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10−33 region is also important for full antibiotic activity.
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