Marcos Pires

Silver lining: A new method for peptide macrocyclization is described, which employs the Ag^I‐promoted transformation of peptide thioamides. The Ag^I chemoselectively activates the thioamide and tethers the N‐terminal thioamide to the C‐terminal carboxylate. Using this method, cyclic peptides are produced in high yields within 1 hour, free of epimerization and cyclodimerization.

Abstract

Peptide macrocyclization is often a slow process, plagued by epimerization and cyclodimerization. Herein, we describe a new method for peptide macrocyclization employing the Ag^I‐promoted transformation of peptide thioamides. The Ag^I has a dual function: chemoselectively activating the thioamide and tethering the N‐terminal thioamide to the C‐terminal carboxylate. Extrusion of Ag₂S generates an isoimide intermediate, which undergoes acyl transfer to generate the native cyclic peptide, resulting in a rapid, traceless macrocylization process. Cyclic peptides are furnished in high yields within 1 hour, free of epimerization and cyclodimerization.

Controlled interactions: Genetically encoded biotin and 2‐iminobiotin lysine provide stringent control over the site of modification and stoichiometry. Well‐defined biotinylated proteins can potentially simplify protein investigation, protein immobilization, and the production of protein–drug conjugates through the strong interaction to streptavidin.

Abstract

The biotin–streptavidin interaction is among the strongest known in nature. Herein, the site‐directed incorporation of biotin and 2‐iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2‐Iminobiotin lysine was employed for protein purification based on the pH‐dependent dissociation constant to streptavidin. By using the high‐affinity binding of biotin lysine, the bacterial protein RecA could be specifically isolated and its interaction partners analyzed. Furthermore, the biotinylation approach was successfully transferred to mammalian cells. Stringent control over the biotinylation site and the tunable affinity between ncAAs and streptavidin of the different biotin analogues make this approach an attractive tool for protein interaction studies, protein immobilization, and the generation of well‐defined protein–drug conjugates.

Excessive consumption of beverages sweetened with high-fructose corn syrup (HFCS) is associated with obesity and with an increased risk of colorectal cancer. Whether HFCS contributes directly to tumorigenesis is unclear. We investigated the effects of daily oral administration of HFCS in adenomatous polyposis coli (APC) mutant mice, which are predisposed to develop intestinal tumors. The HFCS-treated mice showed a substantial increase in tumor size and tumor grade in the absence of obesity and metabolic syndrome. HFCS increased the concentrations of fructose and glucose in the intestinal lumen and serum, respectively, and the tumors transported both sugars. Within the tumors, fructose was converted to fructose-1-phosphate, leading to activation of glycolysis and increased synthesis of fatty acids that support tumor growth. These mouse studies support the hypothesis that the combination of dietary glucose and fructose, even at a moderate dose, can enhance tumorigenesis.

Zhou et al. reported a penicillin binding protein (PBP)-like thioesterase catalyzing peptide chain termination in surugamide non-ribosomal peptide (NRP) biosynthesis. The family of enzymes is widely distributed in NRP biosynthetic pathways in bacteria, either as standalones or C-terminal domains of multidomain NRPSs, suggesting a general mechanism in Nature.

Structures of a bacterial extracellular contractile injection system, the Photorhabdus virulence cassette (PVC), reveal its assembly pathway and unique features compared to other phage tail-like complexes

A molecule counter, added at the channel exit of a bacterial cell membrane pore creates an additional energy barrier to quantify molecules that permeate. The efficiency with charged peptides was tested. In a second series the antibiotic norfloxacin was tested. Introducing the partial channel blocker allows to distinguish binding from translocation for a broad range of molecules.

Abstract

Biological channels facilitate the exchange of molecules across membranes, but general tools to quantify transport are missing. Electrophysiology is the method of choice to study the functional properties of channels. However, analyzing the current fluctuation of channels typically does not identify successful transport, that is, distinguishing translocation from binding. To distinguish both processes, we added an additional barrier at the channel exit acting as a molecular counter. To identify permeation, we compare the molecule residence time in the native channel with one that is chemically modified at the exit. We use the well‐studied outer membrane channel from E. coli, OmpF. Position 181, which is below the constriction region, was subsequently mutated into cysteine (E181C) in an otherwise cysteine‐free system, then functionalized by covalent binding with one of the two blockers MTSES or GLT. We measured the passage of model peptides, mono‐, tri‐, hepta‐arginine and of norfloxacin, as an example for antibiotic permeation.

α‐Gal–antibody (Ab) conjugates have been developed that can dramatically increase cellular cytotoxicity by recruiting natural Abs through the interaction between α‐gal and anti‐gal Abs. The potency of the α‐gal–Ab conjugates increased with the amount of α‐gal conjugated to the Ab. The method developed here will enable the re‐development of Abs to improve their potency.

Abstract

Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α‐gal–Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α‐gal and anti‐gal Abs. The potency of the α‐gal–Ab conjugates depended on the amount of α‐gal conjugated to the antibody: the larger the amount of α‐gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α‐gal dendrimer allowed the introduction of large amounts of α‐gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re‐development of Abs to improve their potency.

Modifying mycobacteria by mycoloylation: A suite of trehalose monomycolate (TMM)‐based metabolic reporters provides versatility and specificity for analyzing and engineering the outer membrane of living mycobacteria. These compounds gave insight into the substrate tolerance of mycoloyltransferases and allow one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling through tetrazine ligation.

Abstract

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)‐catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne‐modified TMM analogue (O‐AlkTMM‐C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM‐based reporters bearing alkyne, azide, trans‐cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell‐surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole‐cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

The repertoire of maternal anti-viral antibodies in human newborns

The repertoire of maternal anti-viral antibodies in human newborns, Published online: 18 March 2019; doi:10.1038/s41591-019-0392-8

A longitudinal survey of maternal antiviral antibodies in newborns provides a blueprint for understanding infectious disease susceptibility and vaccine development, and inform vaccine scheduling in newborn children.

S(−S)nap! Intermolecular pairs of vicinal amino acids in ligand–receptor complexes can be conveniently identified by using the “thiol trapping” method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine. The method provides outcomes comparable to those of classical disulfide trapping and is compatible with the presence of reducing reagents during electrophoretic analysis.

Abstract

Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the “thiol trapping” method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin‐releasing factor receptor and either α‐chloro‐ or α‐bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical “disulfide trapping”. The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini‐Gs, GTPγS, and Gαs‐depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

The molecular design principles that guide development of synthetic antimicrobial polymers are discussed with a particular focus on the principle of “amphiphilic balance” as it relates to some recently developed polyphosphonium compounds with somewhat atypical structure. It is found that the fundamental concept of amphiphilic balance is still applicable to these new polymers.

Abstract

The purpose of this Viewpoint is to discuss the molecular design principles that guide development of synthetic antimicrobial polymers, especially those intended to mimic the structure of host defense peptides (HDPs). In particular, we focus on the principle of “amphiphilic balance” as it relates to some recently developed polyphosphoniums with somewhat atypical structure. We find that the fundamental concept of amphiphilic balance is still applicable to these new polymers, but that the method to achieve such balance is somewhat unique. We then briefly outline the future challenges and opportunities in this field.

We explored a novel bioorthogonal approach to release on demand the content of nanoparticles. Exploiting our recently described click‐and‐release technology, we developed a new generation of cleavable micelles able to disassemble through a sequential enzymatic and bioorthogonal activation process. Proof of concept experiments showed that this new approach could be successfully used to deliver the substances encapsulated into micelles in living cells as well as in mice via two complementary targeted strategies.

Copper is a critical enzyme cofactor in the body but also a potent cellular toxin when intracellularly unbound. Thus, there is a delicate balance of intracellular copper, maintained by a series of complex interactions between the metal and specific copper transport and binding proteins. The gastrointestinal (GI) tract is the...

Zhang et al. developed potent persulfide donors consisting of sulfane sulfur atoms stabilized by N-acetyl-L-cysteine (NAC polysulfides) via disulfide bonds at both sides. Strong anti-inflammatory activity of NAC polysulfides was demonstrated in cultured macrophage models and a mouse endotoxin shock model.

ABSTRACT

High-level resistance often evolves when populations of bacteria are exposed to antibiotics, by either mutations or horizontally acquired genes. There is also variation in the intrinsic resistance levels of different bacterial strains and species that is not associated with any known history of exposure. In many cases, evolved resistance is costly to the bacteria, such that resistant types have lower fitness than their progenitors in the absence of antibiotics. Some longer-term studies have shown that bacteria often evolve compensatory changes that overcome these tradeoffs, but even those studies have typically lasted only a few hundred generations. In this study, we examine changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any antibiotic. On average, the evolved bacteria were more susceptible to most antibiotics than was their ancestor. The bacteria at 50,000 generations tended to be even more susceptible than after 2,000 generations, although most of the change occurred during the first 2,000 generations. Despite the general trend toward increased susceptibility, we saw diverse outcomes with different antibiotics. For streptomycin, which was the only drug to which the ancestral strain was highly resistant, none of the evolved lines showed any increased susceptibility. The independently evolved lineages often exhibited correlated responses to the antibiotics, with correlations usually corresponding to their modes of action. On balance, our study shows that bacteria with low levels of intrinsic resistance often evolve to become even more susceptible to antibiotics in the absence of corresponding selection.

IMPORTANCE Resistance to antibiotics often evolves when bacteria encounter antibiotics. However, bacterial strains and species without any known exposure to these drugs also vary in their intrinsic susceptibility. In many cases, evolved resistance has been shown to be costly to the bacteria, such that resistant types have reduced competitiveness relative to their sensitive progenitors in the absence of antibiotics. In this study, we examined changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any drug. The evolved bacteria tended to become more susceptible to most antibiotics, with most of the change occurring during the first 2,000 generations, when the bacteria were undergoing rapid adaptation to their experimental conditions. On balance, our findings indicate that bacteria with low levels of intrinsic resistance can, in the absence of relevant selection, become even more susceptible to antibiotics.

Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity

Antibody neutralization of microbiota-derived circulating peptidoglycan dampens inflammation and ameliorates autoimmunity, Published online: 04 March 2019; doi:10.1038/s41564-019-0381-1

Monoclonal antibody neutralization of circulating microbiota-derived peptidoglycan subunits alleviates autoimmune disease.

Helical antimicrobial peptides assemble into protofibril scaffolds that present ordered dsDNA to TLR9

Helical antimicrobial peptides assemble into protofibril scaffolds that present ordered dsDNA to TLR9, Published online: 04 March 2019; doi:10.1038/s41467-019-08868-w

Amphihelical antimicrobial peptides (AMPs) are bactericidal host defense factors, but their function as immunomodulators is emerging. Here the authors show that several AMPs organize DNA into periodic nanocrystals by self-assembling into superhelical protofibril scaffolds, which potentiates DNA sensing by TLR9.

Antigen-specific therapeutic approaches for autoimmunity

Antigen-specific therapeutic approaches for autoimmunity, Published online: 25 February 2019; doi:10.1038/s41587-019-0015-4

Pere Santamaria and Pau Serra review the state of the art in antigen-specific therapies for autoimmune disease, discussing mechanism of action and clinical efficacy of each strategy.

Listeria hijacks host mitophagy through a novel mitophagy receptor to evade killing

<i>Listeria</i> hijacks host mitophagy through a novel mitophagy receptor to evade killing, Published online: 25 February 2019; doi:10.1038/s41590-019-0324-2

Removal of damaged mitochondria maintains cellular homeostasis and regulates inflammation. Qian and colleagues describe a mechanism by which intracellular bacteria such as Listeria can elicit mitophagy to enable their survival.

Fluorogenic d-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays

Fluorogenic <span class="small-caps">d</span>-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays, Published online: 25 February 2019; doi:10.1038/s41557-019-0217-x

Biosynthesis of peptidoglycan requires carefully orchestrated transpeptidation reactions to maintain the structural integrity of this essential component of the bacterial cell wall. Now, rotor-fluorescent d-amino acids have been shown to enable real-time tracking of these transpeptidation reactions in live bacterial cells. These powerful tools allow visualization of peptidoglycan biosynthesis with high spatiotemporal resolution.

Horizontal gene transfer of a full operon encoding siderophore biosynthesis genes from bacteria to a group of budding yeasts was followed by acquisition of eukaryotic genomic and transcriptional features.

Extreme slow growth as alternative strategy to survive deep starvation in bacteria

Extreme slow growth as alternative strategy to survive deep starvation in bacteria, Published online: 21 February 2019; doi:10.1038/s41467-019-08719-8

Bacteria can become dormant or form spores when starved for nutrients. Here, Gray et al. describe an alternative strategy, or ‘oligotrophic growth state’, showing that non-sporulating Bacillus subtilis cells can survive deep starvation conditions by adopting an almost coccoid shape and extremely low growth rates.

Multi-omic measurements of heterogeneity in HeLa cells across laboratories

Multi-omic measurements of heterogeneity in HeLa cells across laboratories, Published online: 18 February 2019; doi:10.1038/s41587-019-0037-y

Systems-wide analysis of HeLa cell lines from 13 labs identifies substantial molecular and phenotypic variability.

Siderophore-inspired chelator hijacks uranium from aqueous medium

Siderophore-inspired chelator hijacks uranium from aqueous medium, Published online: 18 February 2019; doi:10.1038/s41467-019-08758-1

Development of simple uranyl recognition motifs possessing siderophore-like binding strength and selectivity presents a challenge. Here the authors show a comprehensive theoretical and experimental study on uranyl binding with a polymeric adsorbent material decorated with a non-toxic siderophore inspired small molecule chelator.