Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.
We demonstrate the use of luminescent QDs conjugated to antibody fragments to develop solution-phase nanoscale sensing assemblies, based on fluorescence resonance energy transfer (FRET) for the specific detection of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments. The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue prebound in the antibody binding site quenches the QD photoluminescence via proximity-induced FRET. Analysis of the data collected at increasing dye-labeled analogue to QD ratios provided an insight into understanding how the antibody fragments self-assemble on the QD. Addition of soluble TNT displaces the dye-labeled analogue, eliminating FRET and resulting in a concentration-dependent recovery of QD photoluminescence. Sensor performance and specificity were evaluated.
A successful structure-based design of a class of non-peptide small-molecule MDM2 inhibitors targeting the p53-MDM2 protein-protein interaction is reported. The most potent compound 1d binds to MDM2 protein with a Ki value of 86 nM and is 18 times more potent than a natural p53 peptide (residues 16-27). Compound 1d is potent in inhibition of cell growth in LNCaP prostate cancer cells with wild-type p53 and shows only a weak activity in PC-3 prostate cancer cells with a deleted p53. Importantly, 1d has a minimal toxicity to normal prostate epithelial cells. Our studies provide a convincing example that structure-based strategy can be employed to design highly potent, non-peptide, cell-permeable, small-molecule inhibitors to target protein-protein interaction, which remains a very challenging area in chemical biology and drug design.
SUMMARY Obesity-related leptin resistance manifests in loss of leptin’s ability to reduce appetite and increase energy expenditure. Obesity is also associated with increased activity of the endocannabinoid system, and CB1 receptor (CB1R) inverse agonists reduce body weight and the associated metabolic complications, although adverse neuropsychiatric effects halted their therapeutic development. Here we show that in mice with diet-induced obesity (DIO), the peripherally restricted CB1R inverse agonist JD5037 is equieffective with its brain-penetrant parent compound in reducing appetite, body weight, hepatic steatosis, and insulin resistance, even though it does not occupy central CB1R or induce related behaviors. Appetite and weight reduction by JD5037 are mediated by resensitizing DIO mice to endogenous leptin through reversing the hyperleptinemia by decreasing leptin expression and secretion by adipocytes and increasing leptin clearance via the kidney. Thus, inverse agonism at peripheral CB1R not only improves cardiometabolic risk in obesity but has antiobesity effects by reversing leptin resistance.
The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core-shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein-nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.maltose-binding protein ͉ three-dimensional structure ͉ nanotechnology ͉ nanocrystal T he burgeoning field of nanotechnology promises to revolutionize many scientific fields, and the first generation of functional hybrid nanomaterials exploring the interface between biology and materials science is now being developed and prototyped (1-3). One exciting avenue of biomaterials research involves protein-nanomaterial composites (2-4). Proteins lend many of their unique properties to these hybrid materials, such as: assisting in ordered self-assembly processes such as that of Pd nanoparticles assembled on tubulin or viral assembly of orientated nanowires (5, 6), engendering exquisite biorecognition properties such as the receptors used in hybrid nanocrystal biosensors (7), and catalyzing useful electrochemical and cleavage reactions (2, 8). Of critical importance in developing these materials is a fundamental understanding of how proteins or bioreceptors interact with inorganic nanomaterials.The unique properties of luminescent colloidal semiconductor nanocrystals or quantum dots (QDs) have recently been incorporated into hybrid functional nanoassemblies. Cadmium selenide-zinc sulfide (CdSe-ZnS) core-shell QDs, in particular, have exceptional photochemical stability and relatively high quantum yields, as well as broad excitation and size-tunable photoluminescence spectra with narrow emission bandwidt...
Trypsin from bovine pancreas (MW 23.8 kDa, 12 705 BAEE units/mg protein, 12 705 BAEE units/mg solid, foreign chymotrypsin activity 0.67 BTEE units/mg protein; EC number 3.4.21.4; derived from New Zealand sourced pancreas, ethanol precipitate) was purchased from Sigma-Aldrich (St. Louis, MO). Note: BAEE is N α -benzoyl-L-arginine ethyl ester; BTEE is N α -tyrosine-L-arginine ethyl ester. InstrumentsPL measurements were made using either a Tecan (Durham, NC) Sapphire or Tecan Infinite M1000 fluorescence multifunction plate reader. Peptide LabelingA mass of ca. 1 mg of peptide was dissolved in 1 mL of phosphate buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4), and combined with excess Cy3maleimide monoreactive dye (GE Healthcare, Piscataway, NJ) or A594-maleimide (Invitrogen by Life Technologies, Carlsbad, CA) at room temperature for 1-2 h and then left overnight at 4°C. Excess unreacted dye was initially removed by loading the reaction onto three consecutive 0.5 mL columns of Ni 2+ -nitrilotriacetic acid (Ni-NTA) agarose media (Qiagen Valencia, CA). Columns with bound peptide were washed with PBS, and the labeled peptide eluted with 300 mM imidazole in PBS. Cy3-labeled peptide was desalted and the imidazole removed using a reverse-phase oligonucleotide purification cartridge (OPC; Applied Biosystems, Foster City, CA) and washes with 0.1 M triethylamine acetate buffer. Labeled peptide was eluted using 1 mL of 70% acetonitrile in doubly-distilled deionized H 2 O. Purified Cy3-peptide was quantitated by UV-visible spectroscopy using the Cy3 (ε = 150 000 M −1 cm −1 at 550 nm) and A594 (ε = 73 000 M −1 cm −1 at 590 nm) absorbance peaks, then aliquoted, dried, and stored at −20 °C prior to use. This protocol has been described in detail elsewhere. 1 Preparation of 2-naphthylamine calibration samples2-Naphthylamine samples were prepared from the hydrolysis of the corresponding concentration of BANA using 1.4 µM trypsin at 37 ˚C for 4 h, and then left at 22 ˚C for 1.5 h. Preparation of pre-digested peptide (product fragment) samplesEqual volumes of Cy3/A594-labeled peptide substrate (50 µM) and trypsin (8.6 µM) were mixed and left overnight at room temperature. These solutions were used for calibrations with only digested product fragments. For the mixed calibration with both A594-labeled substrate and digested product fragment (vide infra), residual trypsin activity in the solution (800 µL, 25 µM)
We demonstrate the use of a hybrid fluorescent protein semiconductor quantum dot (QD) sensor capable of specifically monitoring caspase 3 proteolytic activity. mCherry monomeric red fluorescent protein engineered to express an N-terminal caspase 3 cleavage site was ratiometrically selfassembled to the surface of QDs using metal-affinity coordination. The proximity of the fluorescent protein to the QD allows it to function as an efficient fluorescent resonance energy transfer acceptor. Addition of caspase 3 enzyme to the QD-mCherry conjugates specifically cleaved the engineered mCherry linker sequence altering energy transfer with the QD and allowing quantitative monitoring of proteolytic activity. Inherent advantages of this sensing approach include bacterial expression of the protease substrate in a fluorescently-appended form, facile self-assembly to QDs, and the ability to recombinantly modify the substrate to target other proteases of interest.The creation of hybrid biological-inorganic nanomaterials capable of enhanced sensing, catalysis, or actuation is a major goal of nanotechnology 1 . Sensors consisting of nanoparticlebioconjugates in particular are predicted to find utility in medicine, bioresearch, security, and defense applications. Amongst the challenges in creating these materials are efficiently interfacing the biological elements (proteins, peptides, DNA) with the nanoparticle surface. Chemistries for accomplishing this should be facile, allow both participants to function in concert, and should be amenable to creating a wide variety of other functional nanomaterials 1-3 . We have shown that polyhistidine appended proteins, peptides, and even DNA can self-assemble to CdSe-ZnS core-shell semiconductor quantum dots (QDs) via metalaffinity coordination 2 . This rapid, high-affinity interaction allows control over the ratio of attached biological moiety per QD and can even allow for control over protein orientation 2 . Bioconjugation using this strategy allows utilization of the QD as both a central nanoscaffold and exciton donor for self-assembling a variety of QD-protein, peptidyl, and DNA nanoconjugates capable of sensing nutrients, explosives, DNA, and enzymatic activity via fluorescence resonance energy transfer (FRET) 1-3 . Use of QDs as FRET donors provides inherent photophysical benefits cumulatively unavailable to organic dyes including: the ability to optimize spectral overlap by size-tuning the QD photoluminescence (PL), control over intra-assembly FRET by arraying multiple acceptors around the QD, reduced direct excitation of the acceptor and access to multiplex FRET configurations. 2c These properties have led a growing number of groups to adopt QD-FRET as the signal transduction modality for sensors targeting pH changes, HIV-related peptides, nucleic acids, sugars, β-lactamase activity and antibiotics. 2,3 Here, we demonstrate that the fluorescent protein mCherry modified to express a caspase 3 cleavage site can be ratiometrically self-assembled to QDs to create a sensitive and ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.