Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic*

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett’s esophagus

Background Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett’s esophagus. Methods Six L2-IL1B mice with advanced stage of disease (12–16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12–14 months old) by a novel imaging system with two L2-IL1B mice used as negative controls. Results Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells by confocal microscopy further confirmed the imaging results. Conclusions This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett’s esophagus. Further investigations are needed to assess its use in the clinical setting.

Synthesis and characterization of TEMPO-oxidized peptide-cellulose conjugate biosensors for detecting human neutrophil elastase

Here we describe the synthesis and characterization of a peptide-cellulose conjugate biosensor based on TEMPO-oxidized nanofibrillated cellulose (tNFC) for detecting elevated levels of human neutrophil elastase (HNE) in chronic wounds. The fluorescent peptide HNE substrate constructed from n-succinyl-Ala-Pro-Ala-7-amino-4-methyl-coumarin was attached to the TEMPO-oxidized cellulose surface via polyethylene glycol linker. The characterization of the biosensor conjugate shows a high degree of peptide incorporation onto the surface with the degree of substitution of 0.057. The relatively small crystallite size of 26.0 Å compared to other cellulose- and nanocellulose-based materials leads to a large specific surface area which can promote access of HNE to the enzyme substrates due to decreased steric interactions. Likewise, the porosity for tNFC was found to be higher than all other samples, including the nanocellulosic aerogel, lending to its hydrogel-like nature. The properties of tNFC were compared to other cellulose-based materials. The volume of each crystallite and volume ratio to the largest sample was calculated. tNFC was found to occupy the smallest space resulting in high amounts of sensors per crystallite unit volume. With a small crystallite volume and large number of sensors, the tNFC peptide-cellulose conjugate biosensor could provide a more sensitive system and is a good candidate for point of care diagnostic devices for detecting elevated protease levels in humans.

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Multivalent peptide ligands to probe the chromocenter microenvironment in living cells

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here, we present tools to interrogate the environment of chromatin sub-compartments in the form of modular, cell-permeable, multivalent and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. As a target, here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. However, image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

Evaluation and Selection of Potent Fluorescent Immunosensors by Combining Fluorescent Peptide and Nanobodies Displayed on Yeast Surface

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies have impelled researchers to reconsider the importance of choice for animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resolution.This study aimed to follow the dynamic fibrotic features of BLM (Bleomycin)-treated mouse lungs with extended durations through a combination of the latest technologies (micro-CT imaging and histological detection of degraded collagens) with traditional methods. In addition, we also applied immunohistochemistry to explore the distribution of all hydroxyproline-containing molecules.As determined by classical biochemical method, total lung hydroxyproline contents reached peak at 4-week after bleomycin injury and maintained a steady high level thereafter until the end of the experiments (16-week). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degradation of collagens and a recovery of lung aeration post the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and a fluorescent peptide staining against degraded collagen, together showing large amounts of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10- or 16-week, rather than at 4-week post the BLM-treatment. Hence, our present data not only offer respiratory researchers a new perspective towards the resolution nature of mouse lung fibrosis, but also remind them to be cautious while using hydroxyproline content assay to evaluate the severity of fibrosis.