measuring-membrane-lipid-turnover-with-ph-sensitive-fluorescent-lipid

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 ...

Florida Atlantic University

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. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes. 

Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

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. Subsequently, MsrA was also shown to be involved in the life span extension in Caenorhabditis elegans. These results supported other studies that indicated up-regulation, or activation, of the normal cellular protective mechanisms that cells use to defend against oxidative damage could be an approach to treat age related diseases and slow the aging process. In this study we have identified, for the first time, compounds structurally related to the natural products fusaricidins that markedly activate recombinant bovine and human MsrA and human MsrB. 

Identification of activators of methionine sulfoxide reductases A and B.

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. We presently describe approaches by which one can optically image matrix metalloproteinase activity implicated in breast cancer progression, with consideration of selective versus broad protease probes. 

Imaging Matrix Metalloproteinase Activity Implicated in Breast Cancer Progression.

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. The present chapter provides an overview of MMP FRET triple-helical substrates and describes how to construct and utilize these substrates.

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

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). The tricine-based branch reported herein represents the simplest synthetic scaffold for the convenient synthesis of covalently linked homomeric collagen-model triple-helical peptides.

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

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 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvβ3 integrins for in vitro assays.

Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in 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. They exhibit a solvatochromic effect, and one of them, ND6, shows a substantial fluorescence difference between pH 6 and 7. The solvatochromic effect and pH-sensitivity of those probes are explained using quantum chemical calculations, and molecular dynamics simulation confirms their integration and interaction with membrane lipids. For live-cell fluorescence imaging, we tested those probes in a cancer cell line (MCF7), cancer spheroids (MDA-MB-468), and cultured hippocampal neurons. Confocal imaging showed an excellent signal-to-noise ratio from 400:1 to about 1300:1 for cell membrane labeling. We applied ND6 during stimulation to label nerve terminals via dye uptake during evoked synaptic vesicle turnover. By ND6 imaging, we revealed cholesterol's multifaced role in replenishing synaptic vesicle pools. Our results demonstrate these fluorescent probes' great potential in studying membrane dynamic and synaptic functions in neurons and other secretory cells and tissues.

Maciej J. Stawikowski

Department of Chemistry and Biochemistry

Florida Atlantic University

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