Biosynthesis of Macrocyclic Peptides with C-Terminal ß-Amino-α-keto Acid Groups by Three Different Metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new compound class involving modifications installed by a cytochrome P450, a multinuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-l-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C cross-link between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid, while the methyltransferase acted on the ß-carbon of this α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configuration of the atropisomer formed upon biaryl cross-linking. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to isolate new macrocyclic RiPPs biosynthesized via previously undiscovered enzyme chemistry.

Biomimetic-Membrane-Protected Plasmonic Nanostructures as Dual-Modality Contrast Agents for Correlated Surface-Enhanced Raman Scattering and Photoacoustic Detection of Hidden Tumor Lesions.

Optical imaging and spectroscopic modalities are of considerable current interest for in vivo cancer detection and image-guided surgery, but the turbid or scattering nature of biomedical tissues has severely limited their abilities to detect buried or occluded tumor lesions. Here we report the development of a dual-modality plasmonic nanostructure based on colloidal gold nanostars (AuNSs) for simultaneous surface-enhanced Raman scattering (SERS) and photoacoustic (PA) detection of tumor phantoms embedded (hidden) in ex vivo animal tissues. By using red blood cell membranes as a naturally derived biomimetic coating, we show that this class of dual-modality contrast agents can provide both Raman spectroscopic and PA signals for the detection and differentiation of hidden solid tumors with greatly improved depths of tissue penetration. Compared to previous polymer-coated AuNSs, the biomimetic coatings are also able to minimize protein adsorption and cellular uptake when exposed to human plasma without compromising their SERS or PA signals. We further show that tumor-targeting peptides (such as cyclic RGD) can be noncovalently inserted for targeting the ανß3-integrin receptors expressed on metastatic cancer cells and tracked via both SERS and PA imaging (PAI). Finally, we demonstrate image-guided resections of tumor-mimicking phantoms comprising metastatic tumor cells buried under layers of skin and fat tissues (6 mm in thickness). Specifically, PAI was used to determine the precise tumor location, while SERS spectroscopic signals were used for tumor identification and differentiation. This work opens the possibility of using these biomimetic dual-modality nanoparticles with superior signal and biological stability for intraoperative cancer detection and resection.

Biosynthesis of macrocyclic peptides with C-terminal ß-amino-α-keto acid groups by three different metalloenzymes.

Advances in genome sequencing and bioinformatics methods have identified a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By mining genomes for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. All enzymes encoded by the BGC were functionally expressed in Burkholderia sp. FERM BP-3421. Structural characterization with 2D-NMR and Marfey's method on the resulting RiPP demonstrated that the P450 enzyme catalyzed the formation of a biaryl C-C crosslink between two Tyr residues with the B12-rSAM generating ß-methyltyrosine. The MNIO transformed a C-terminal Asp residue into aminopyruvic acid while the methyltransferase acted on the ß-carbon of the α-keto acid. Exciton-coupled circular dichroism spectroscopy and microcrystal electron diffraction (MicroED) were used to elucidate the stereochemical configurations of the atropisomer that formed upon biaryl crosslinking. The conserved Cys residue in the precursor peptide was not modified as in all other characterized MNIO-containing BGCs; However, mutational analyses demonstrated that it was essential for the MNIO activity on the C-terminal Asp. To the best of our knowledge, the MNIO featured in this pathway is the first to modify a residue other than Cys. This study underscores the utility of genome mining to discover new macrocyclic RiPPs and that RiPPs remain a significant source of previously undiscovered enzyme chemistry.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυß3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

Biomimetic Surface-Enhanced Raman Scattering Nanoparticles with Improved Dispersibility, Signal Brightness, and Tumor Targeting Functions.

The development of biocompatible and nontoxic surface-enhanced Raman scattering (SERS) nanoparticles is of considerable current interest because of their attractive biomedical applications such as ultrasensitive in vitro diagnostics, in vivo tumor imaging, and spectroscopy-guided cancer surgery. However, current SERS nanoparticles are prepared and stored in aqueous solution, have limited stability and dispersibility, and are not suitable for lyophilization and storage by freeze-drying or other means. Here, we report a simple but robust method to coat colloidal SERS nanoparticles by naturally derived biomimetic red blood cell membranes (RBCM), leading to a dramatic improvement in stability and dispersibility under freeze-thawing, lyophilization, heating, and physiological conditions. The results demonstrate that the lyophilized SERS nanoparticles in the solid form can be readily dissolved and dispersed in physiological buffer solutions. A surprising finding is that the RBCM-coated SERS particles are considerably brighter (by as much as 5-fold) than PEGylated SERS particles under similar experimental conditions. This additional enhancement is believed to arise from the hydrophobic nature of RBCM's hydrocarbon chains, which is known to reduce electronic dampening and boost electromagnetic field enhancement. A further advantage in using biomimetic membrane coatings is that the bilayer membrane structure allows nonvalent insertion of molecular ligands for tumor targeting. In particular, we show that cyclic-RGD, a tumor-targeting peptide, can be efficiently inserted into the membrane coatings of SERS nanoparticles for targeting the ανß3 integrin receptors expressed on cancer cells. Thus, biomimetic RBCMs provide major advantages over traditional polyethylene glycols for preparing SERS nanoparticles with improved dispersibility, higher signal intensity, and more efficient biofunctionalization.

Epidermal Remodeling in Caenorhabditis elegans Dauers Requires the Nidogen Domain Protein DEX-1.

Phenotypic plasticity is a critical component of an organism's ability to thrive in a changing environment. The free-living nematode Caenorhabditis elegans adapts to unfavorable environmental conditions by pausing reproductive development and entering a stress-resistant larval stage known as dauer. The transition into dauer is marked by vast morphological changes, including remodeling of epidermis, neurons, and muscle. Although many of these dauer-specific traits have been described, the molecular basis of dauer-specific remodeling is still poorly understood. Here we show that the nidogen domain-containing protein DEX-1 facilitates stage-specific tissue remodeling during dauer morphogenesis. DEX-1 was previously shown to regulate sensory dendrite formation during embryogenesis. We find that DEX-1 is also required for proper remodeling of the stem cell-like epidermal seam cells. dex-1 mutant dauers lack distinct lateral cuticular alae during dauer and have increased sensitivity to sodium dodecyl sulfate. Furthermore, we find that DEX-1 is required for proper dauer mobility. We show that DEX-1 is secreted from the seam cells during dauer, but acts locally in a cell-autonomous manner. We find that dex-1 expression during dauer is regulated through DAF-16/FOXO-mediated transcriptional activation. Finally, we show that dex-1 acts with a family of zona pellucida domain-encoding genes to regulate dauer-specific epidermal remodeling. Taken together, our data indicate that DEX-1 is an extracellular matrix component that plays a central role in C. elegans epidermal remodeling during dauer.

Epithelial Shaping by Diverse Apical Extracellular Matrices Requires the Nidogen Domain Protein DEX-1 in Caenorhabditis elegans.

The body's external surfaces and the insides of biological tubes, like the vascular system, are lined by a lipid-, glycoprotein-, and glycosaminoglycan-rich apical extracellular matrix (aECM). aECMs are the body's first line of defense against infectious agents and promote tissue integrity and morphogenesis, but are poorly described relative to basement membranes and stromal ECMs. While some aECM components, such as zona pellucida (ZP) domain proteins, have been identified, little is known regarding the overall composition of the aECM or the mechanisms by which different aECM components work together to shape epithelial tissues. In Caenorhabditis elegans, external epithelia develop in the context of an ill-defined ZP-containing aECM that precedes secretion of the collagenous cuticle. C. elegans has 43 genes that encode at least 65 unique ZP proteins, and we show that some of these comprise distinct precuticle aECMs in the embryo. Previously, the nidogen- and EGF-domain protein DEX-1 was shown to anchor dendrites to the C. elegans nose tip in concert with the ZP protein DYF-7 Here, we identified a new, strong loss-of-function allele of dex-1, cs201dex-1 mutants die as L1 larvae and have a variety of tissue distortion phenotypes, including excretory defects, pharyngeal ingression, alae defects, and a short and fat body shape, that strongly resemble those of genes encoding ZP proteins. DEX-1 localizes to ZP-containing aECMs in the tissues that show defects in dex-1 mutants. Our studies suggest that DEX-1 is a component of multiple distinct embryonic aECMs that shape developing epithelia, and a potential partner of multiple ZP proteins.