Development and characterization of fluorescent cholesteryl probes with enhanced solvatochromic and pH-sensitive properties for live-cell imaging.

We present novel fluorescent cholesteryl probes (CNDs) with a modular design based on the solvatochromic 1,8-phthalimide scaffold. We have explored how different modules-linkers and head groups-affect the ability of these probes to integrate into lipid membranes and how they distribute intracellularly in mouse astrocytes and fibroblasts targeting lysosomes and lipid droplets. Each compound was assessed for its solvatochromic behavior in organic solvents and model membranes.

Modular Fluorescent Cholesterol Naphthalimide Probes And Their Application For Cholesterol Trafficking Studies In Cells.

Development of fluorescent cholesterol analogs to better understand subcellular cholesterol trafficking is of great interest for cell biology and medicine. Our approach utilizes a bifunctional 1,8-naphthalimide scaffold with a push-pull character, modified on one side with a head group and a linker on the other side connecting it to cholesterol an ester bond. Through structure-function studies, we've explored how different substituents-linkers and head groups-affect the ability of these fluorescent cholesterol naphthalimide analogs (CNDs) to mimic natural cholesterol behavior at both molecular and cellular levels.

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from β cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology.

Solvatochromic and pH-Sensitive Fluorescent Membrane Probes for Imaging of Live Cells.

Membrane trafficking is essential for all cells, and visualizing it is particularly useful for studying neuronal functions. Here we report the synthesis, characterization, and application of several membrane- and pH-sensitive probes suitable for live-cell fluorescence imaging. These probes are based on a 1,8-naphthalimide fluorophore scaffold.

Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells.

Background: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.

Tricine as a convenient scaffold for the synthesis of -terminally branched collagen-model peptides.

A novel and convenient method for the synthesis of -terminally branched collagen-model peptides has been achieved using tricine (-[tris(hydroxymethyl)methyl]glycine) as a branching scaffold and 1,2-diaminoethane or 1,4-diaminobutane as a linker. The peptide sequence was incorporated directly onto the linker and scaffold during solid-phase synthesis without additional manipulations. The resulting branched triple-helical peptides exhibited comparable thermal stabilities to the parent, unbranched sequence, and served as substrates for matrix metalloproteinase-1 (MMP-1).

Determining the Substrate Specificity of Matrix Metalloproteases using Fluorogenic Peptide Substrates.

A continuous assay method, such as the one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors toward native substrates, a variety of fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET) triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. Results of these studies have been valuable for providing insights into (a) the relative triple-helical peptidase activities of the various collagenolytic MMPs, (b) the collagen preferences of these MMPs, and (c) the relative roles of MMP domains and specific residues in efficient collagenolysis.

Imaging Matrix Metalloproteinase Activity Implicated in Breast Cancer Progression.

Proteolysis has been cited as an important contributor to cancer initiation and progression. One can take advantage of tumor-associated proteases to selectively deliver imaging agents. Protease-activated imaging systems have been developed using substrates designed for hydrolysis by members of the matrix metalloproteinase (MMP) family.

Identification of activators of methionine sulfoxide reductases A and B.

The methionine sulfoxide reductase (Msr) family of enzymes has been shown to protect cells against oxidative damage. The two major Msr enzymes, MsrA and MsrB, can repair oxidative damage to proteins due to reactive oxygen species, by reducing the methionine sulfoxide in proteins back to methionine. A role of MsrA in animal aging was first demonstrated in Drosophila melanogaster where transgenic flies over-expressing recombinant bovine MsrA had a markedly extended life span.

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP.

Glycosylation modulates melanoma cell α2β1 and α3β1 integrin interactions with type IV collagen.

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater.

Structure of a proteolytically resistant analogue of (NLys)5SFTI-1 in complex with trypsin: evidence for the direct participation of the Ser214 carbonyl group in serine protease-mediated proteolysis.

Peptide-peptoid hybrids are found to be potent inhibitors of serine proteases. These engineered peptidomimetics benefit from both types of units of the biopolymeric structure: the natural inhibitor part serves as a good binding template, while the P1-positioned peptoid component provides complete resistance towards proteolysis. In this report, the mechanism of proteolytic resistance of a P1 peptoid-containing analogue is postulated based on the crystal structure of the (NLys)(5)-modified sunflower trypsin inhibitor SFTI-1 in complex with bovine trypsin solved at 1.

Monitoring and Inhibiting MT1-MMP during Cancer Initiation and Progression.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion. Numerous substrates and binding partners have been identified for MT1-MMP, and its role in collagenolysis appears crucial for tumor invasion. However, development of MT1-MMP inhibitors must consider the substantial functions of MT1-MMP in normal physiology and disease prevention.

Peptoids and peptide-peptoid hybrid biopolymers as peptidomimetics.

Peptoids (oligomers of N-substituted glycine residues) and peptide-peptoid hybrid polymers (peptomers) are interesting classes of compounds mimicking structure and function of biologically active peptides. The oligomeric peptidomimetics such as peptoids are particularly important compounds since they provide access to an enormous molecular diversity, by variation of the building blocks. The modular structure of peptoids, ease of synthesis, and high compatibility with existing peptide chemistry synthetic protocols, make peptoids and peptoid-containing peptidomimetics ideal tools for structure-activity and drug discovery related studies.