POTRA domains of the TamA insertase interact with the outer membrane and modulate membrane properties.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Insights into Molecular Interactions between a GAPDH-Related Fish Antimicrobial Peptide, Analogs Thereof, and Bacterial Membranes.

Interactions between SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four analogs thereof with model bacterial membranes were studied using Fourier-transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. MD trajectory analyses showed that the N-terminal segment of the peptide analogs has many contacts with the polar heads of membrane phospholipids, while the central α helix interacts strongly with the hydrophobic core of the membranes. The peptides also had a marked influence on the wave numbers associated with the phase transition of phospholipids organized as liposomes in both the interface and aliphatic chain regions of the infrared spectra, supporting the interactions observed in the MD trajectories. In addition, interesting links were found between peptide interactions with the aliphatic chains of membrane phospholipids, as determined by FTIR and from the MD trajectories, and the membrane permeabilization capacity of these peptide analogs, as previously demonstrated. To summarize, the combined experimental and computational efforts have provided insights into crucial aspects of the interactions between the investigated peptides and bacterial membranes. This work thus makes an original contribution to our understanding of the molecular interactions underlying the antimicrobial activity of these GAPDH-related antimicrobial peptides from Scombridae.

Novel filamin C (FLNC) variant causes a severe form of familial mixed hypertrophic-restrictive cardiomyopathy.

Variants of filamin C (FLNC) have been identified as rare genetic substrate for hypertrophic cardiomyopathy (HCM). Data on the clinical course of FLNC-related HCM are conflicting with some studies suggesting mild phenotypes whereas other studies have reported more severe outcomes. In this study, we present a novel FLNC variant (Ile1937Asn) that was identified in a large family of French-Canadian descent with excellent segregation data. FLNC-Ile1937Asn is a novel missense variant characterized by full penetrance and poor clinical outcomes. End stage heart failure requiring transplantation occurred in 43% and sudden cardiac death in 29% of affected family members. Other particular features of FLNC-Ile1937Asn include an early disease onset (mean age of 19 years) and the development of a marked atrial myopathy (severe biatrial dilatation with remodeling and multiple complex atrial arrhythmias) that was present in all gene carriers. The FLNC-Ile1937Asn variant is a novel, pathogenic mutation resulting in a severe form of HCM with full disease penetrance. The variant is associated with a high proportion of end-stage heart failure, heart transplantation, and disease-related mortality. Close follow-up and appropriate risk stratification of affected individuals at specialized heart centers is recommended.

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

Structural insights into the putative bacterial acetylcholinesterase ChoE and its substrate inhibition mechanism.

Mammalian acetylcholinesterase (AChE) is well-studied, being important in both cholinergic brain synapses and the peripheral nervous systems and also a key drug target for many diseases. In contrast, little is known about the structures and molecular mechanism of prokaryotic acetylcholinesterases. We report here the structural and biochemical characterization of ChoE, a putative bacterial acetylcholinesterase from Pseudomonas aeruginosa Analysis of WT and mutant strains indicated that ChoE is indispensable for P. aeruginosa growth with acetylcholine as the sole carbon and nitrogen source. The crystal structure of ChoE at 1.35 Å resolution revealed that this enzyme adopts a typical fold of the SGNH hydrolase family. Although ChoE and eukaryotic AChEs catalyze the same reaction, their overall structures bear no similarities constituting an interesting example of convergent evolution. Among Ser-38, Asp-285, and His-288 of the catalytic triad residues, only Asp-285 was not essential for ChoE activity. Combined with kinetic analyses of WT and mutant proteins, multiple crystal structures of ChoE complexed with substrates, products, or reaction intermediate revealed the structural determinants for substrate recognition, snapshots of the various catalytic steps, and the molecular basis of substrate inhibition at high substrate concentrations. Our results indicate that substrate inhibition in ChoE is due to acetate release being blocked by the binding of a substrate molecule in a nonproductive mode. Because of the distinct overall folds and significant differences of the active site between ChoE and eukaryotic AChEs, these structures will serve as a prototype for other prokaryotic acetylcholinesterases.

Extended Linkers Improve the Detection of Protein-protein Interactions (PPIs) by Dihydrofolate Reductase Protein-fragment Complementation Assay (DHFR PCA) in Living Cells.

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.

Reduced Susceptibility to Neuraminidase Inhibitors in Influenza B Isolate, Canada.

We identified an influenza B isolate harboring a Gly407Ser neuraminidase substitution in an immunocompromised patient in Canada before antiviral therapy. This mutation mediated reduced susceptibility to oseltamivir, zanamivir, and peramivir, most likely by preventing interaction with the catalytic Arg374 residue. The potential emergence of such variants emphasizes the need for new antivirals.

CRISPR-Induced Deletion with SaCas9 Restores Dystrophin Expression in Dystrophic Models In Vitro and In Vivo.

Duchenne muscular dystrophy (DMD), a severe hereditary disease affecting 1 in 3,500 boys, mainly results from the deletion of exon(s), leading to a reading frameshift of the DMD gene that abrogates dystrophin protein synthesis. Pairs of sgRNAs for the Cas9 of Staphylococcus aureus were meticulously chosen to restore a normal reading frame and also produce a dystrophin protein with normally phased spectrin-like repeats (SLRs), which is not usually obtained by skipping or by deletion of complete exons. This can, however, be obtained in rare instances where the exon and intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion) are at similar positions in the SLR. We used pairs of sgRNAs targeting exons 47 and 58, and a normal reading frame was restored in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. Restoration of the DMD reading frame and restoration of dystrophin expression were also obtained in vivo in the heart of the del52hDMD/mdx. Our results provide a proof of principle that SaCas9 could be used to edit the human DMD gene and could be considered for further development of a therapy for DMD.

Diversity-Oriented Synthesis of Diol-Based Peptidomimetics as Potential HIV Protease Inhibitors and Antitumor Agents.

Peptidomimetic HIV protease inhibitors are an important class of drugs used in the treatment of AIDS. The synthesis of a new type of diol-based peptidomimetics is described. Our route is flexible, uses d-glucal as an inexpensive starting material, and makes minimal use of protection/deprotection cycles. Binding affinities from molecular docking simulations suggest that these compounds are potential inhibitors of HIV protease. Moreover, the antiproliferative activities of compounds 33 a, 35 a, and 35 b on HT-29, M21, and MCF7 cancer cell lines are in the low micromolar range. The results provide a platform that could facilitate the development of medically relevant asymmetrical diol-based peptidomimetics.

Lobaric acid and pseudodepsidones inhibit NF-κB signaling pathway by activation of PPAR-γ.

Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1ß and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists. These findings suggest that lobaric acid and pseudodepsidones reduce the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.

Synthesis of novel substituted pyrimidine derivatives bearing a sulfamide group and their in vitro cancer growth inhibition activity.

The synthesis of two series of novel substituted pyrimidine derivatives bearing a sulfamide group have been described and their in vitro cancer growth inhibition activities have been evaluated against three human tumour cell lines (HT-29, M21, and MCF7). In general, growth inhibition activity has been enhanced by the introduction of a bulky substituent on the aromatic ring with the best compound having GI50<6µM for all the human tumour cell lines. The MCF7 selective compounds were evaluated on four additional human invasive breast ductal carcinoma cell lines (MDA-MB-231, MDA-MB-468, SKBR3, and T47D) and were selective against T47D cell line in all cases except one, suggesting a potential antiestrogen activity.

Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids.

Molecular model of hemoglobin N from Mycobacterium tuberculosis bound to lipid bilayers: a combined spectroscopic and computational study.

A singular aspect of the 2-on-2 hemoglobin structures of groups I and II is the presence of tunnels linking the protein surface to the distal heme pocket, supporting the storage and the diffusion of small apolar ligands to/from the buried active site. As the solubility of apolar ligands is greater in biological membranes than in solution, the association of these proteins with biological membranes may improve the efficiency of ligand capture. As very little is known on this subject, we have investigated the interactions between hemoglobin N (HbN), a group I 2-on-2 hemoglobin from the pathogenic Mycobacterium tuberculosis (Mtb), and biological membranes using both experimental techniques and MD simulations. HbN has a potent nitric oxide dioxygenase activity (HbN-Fe²âº-O2 + •NO + H2O → HbN-Fe³âº-OH2 + NO3⁻) that is thought to protect the aerobic respiration of Mtb from inhibition by •NO. Three different membrane compositions were chosen for the studies, representative of the mycobacterial plasma membrane and the mammalian cell membranes. Both the experimental and the modeling results agreed with each other and allow for a detailed molecular description of HbN in association with membranes of different compositions. The results indicated that HbN is a peripheral protein, and the association with the membranes occurred via the pre-A, G, and H helices. In addition, HbN would be allowed to modulate the binding to the membranes via electrostatic interactions between the lipid membranes and the Asp100 residue. In its membrane-bound form the short tunnel of HbN is oriented toward the membrane interior and the other tunnels point toward the solvent. Such protein orientation would facilitate the uptake of nonpolar substrates from the membrane and the release of products to the solvent. It is interesting to note that the pre-A, G, and H helices are conserved among HbN from a few other Mycobacteria.

The influenza fusion peptide promotes lipid polar head intrusion through hydrogen bonding with phosphates and N-terminal membrane insertion depth.

Influenza infection requires fusion between the virus envelope and a host cell endosomal membrane. The influenza hemagglutinin fusion peptide (FP) is essential to viral membrane fusion. It was recently proposed that FPs would fuse membranes by increasing lipid tail protrusion, a membrane fusion transition state. The details of how FPs induce lipid tail protrusion, however, remain to be elucidated. To decipher the molecular mechanism by which FPs promote lipid tail protrusion, we performed molecular dynamics simulations of the wild-type (WT) FP, fusogenic mutant F9A, and nonfusogenic mutant W14A in model bilayers. This article presents the peptide-lipid interaction responsible for lipid tail protrusion and a related lipid perturbation, polar head intrusion, where polar heads are sunk under the membrane surface. The backbone amides from the four N-terminal peptide residues, deeply inserted in the membrane, promoted both perturbations through H bonding with lipid phosphates. Polar head intrusion correlated with peptides N-terminal insertion depth and activity: the N-termini of WT and F9A were inserted deeper into the membrane than nonfusogenic W14A. Based on these results, we propose that FP-induced polar head intrusion would complement lipid tail protrusion in catalyzing membrane fusion by reducing repulsions between juxtaposed membranes headgroups. The presented model provides a framework for further research on membrane fusion and influenza antivirals.

Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system.

The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.

Assessing the Activity under Different Physico-Chemical Conditions, Digestibility, and Innocuity of a GAPDH-Related Fish Antimicrobial Peptide and Analogs Thereof.

The antimicrobial activity of SJGAP (skipjack tuna GAPDH-related antimicrobial peptide) and four chemical analogs thereof was determined under different physicochemical conditions, including different pH values, the presence of monovalent and divalent cations, and after a heating treatment. The toxicity of these five peptides was also studied with hemolytic activity assays, while their stability under human gastrointestinal conditions was evaluated using a dynamic in vitro digestion model and chromatographic and mass spectrometric analyses. The antibacterial activity of all analogs was found to be inhibited by the presence of divalent cations, while monovalent cations had a much less pronounced impact, even promoting the activity of the native SJGAP. The peptides were also more active at acidic pH values, but they did not all show the same stability following a heat treatment. SJGAP and its analogs did not show significant hemolytic activity (except for one of the analogs at a concentration equivalent to 64 times that of its minimum inhibitory concentration), and the two analogs whose digestibility was studied degraded very rapidly once they entered the stomach compartment of the digestion model. This study highlights for the first time the characteristics of antimicrobial peptides from Scombridae or homologous to GAPDH that are directly related to their potential clinical or food applications.

Insight into the Binding Mechanisms of Quartz-Selective Peptides: Toward Greener Flotation Processes.

Mining practices, chiefly froth flotation, are being critically reassessed to replace their use of biohazardous chemical reagents in favor of biofriendly alternatives as a path toward green processes. In this regard, this study aimed at evaluating the interactions of peptides, as potential floatation collectors, with quartz using phage display and molecular dynamics (MD) simulations. Quartz-selective peptide sequences were initially identified by phage display at pH = 9 and further modeled by a robust simulation scheme combining classical MD, replica exchange MD, and steered MD calculations. Our residue-specific analyses of the peptides revealed that positively charged arginine and lysine residues were favorably attracted by the quartz surface at basic pH. The negatively charged residues at pH 9 (i.e., aspartic acid and glutamic acid) further showed affinity toward the quartz surface through electrostatic interactions with the positively charged surface-bound Na+ ions. The best-binding heptapeptide combinations, however, contained both positively and negatively charged residues in their composition. The flexibility of peptide chains was also shown to directly affect the adsorption behavior of the peptide. While attractive intrapeptide interactions were dominated by a weak peptide-quartz binding, the repulsive self-interactions in the peptides improved the binding propensity to the quartz surface. Our results showed that MD simulations are fully capable of revealing mechanistic details of peptide adsorption to inorganic surfaces and are an invaluable tool to accelerate the rational design of peptide sequences for mineral processing applications.

The influenza fusion peptide adopts a flexible flat V conformation in membranes.

Knowledge about the influenza fusion peptide (FP) membrane insertion mode is crucial for understanding its fusogenic mechanism. NMR and electron paramagnetic resonance experiments showed that in micelles, the FP inserted as a fixed-angle inverted V. In membranes, however, it was shown to insert as a straight α-helix (by molecular-dynamics simulations) and to adopt multiple kinked conformations (by solid-state NMR). In this work we performed explicit-solvent molecular-dynamics simulations of the influenza FP, and its F9A and W14A mutants, in POPC membranes. The Hα1 chemical shifts predicted from the molecular-dynamics structures are in excellent agreement with the experimental values obtained for the three peptides. The peptide orientation and conformations observed from the simulations lead to a flexible flat-V model in which the peptide lies almost flat on the membrane surface and alternates between kinked and straight-helix conformations.