To gain a better understanding of the mechanisms leading to the formation and progression of diseases, scientists utilize model cell systems in order to simulate physiological and pathological processes. While cell systems provide invaluable tools for researchers, cells by their inherent nature are complex. Having the appropriate molecular probes are crucial to being successful at interrogating complex cellular structures as well as gaining insight into molecule-molecule interactions, enzyme activity, and other processes. The advent of platforms such as fluorescence (or Förster) energy transfer (FRET) along with the abilities to fluorescently label peptides have enabled researchers to advance their studies of the cell. Moreover, this has led to cutting-edge applications related to medical research.
In this article, we will discuss some of the features and tips for working with fluorophores and running FRET analysis. We will also share some examples of how these tools enable researchers to gain a better understanding of the events related to many well-known diseases.
Fluorescent peptide labeling
Fluorophores absorb light in the ultraviolet or visible range and re-emit part of the energy as radiation (fluorescence). The emitted radiation has a longer wavelength, than the excitation light (Stokes shift). The emission wavelength furthermore does not depend on the wavelength of the excitation light.
Fluorescent labels (dyes) can be covalently bound to the amino-terminus of the peptides during solid phase synthesis, or, linked site-specifically to already existing peptides. Amine- or thiol-groups in the side-chains of the amino-acids can be used for the labeling, if this does not impair the biological function. Biotinylated peptides can be labeled in situ using fluorescent streptavidin- or avidin-conjugates. Carboxy-terminal labeling is often more difficult, and the feasibility should be clarified in advance. Capitalizing on these features, peptides can be fluorescently-labeled and used as reporters in imaging studies (confocal fluorescence microscopy) as well as cellular enzyme assays such as monitoring protease activity [1].
In the case of measuring protease activity, a peptide substrate is labeled at the amino-terminus with a fluorophore. In addition, the carboxy-terminus of the peptide can have a biotin moiety attached. After a proteolytic cleavage event, the uncut peptides and carboxy-terminal cleavage products are removed using streptavidin. The remaining fluorescence provides a measurement for the proteolytic activity of the enzyme [2]. For fluorescence microscopy, the fluorophores should have a high stability against photo-bleaching under the high light intensities applied. In general, the fluorophores should exhibit a strong fluorescence.
FRET analysis
FRET is an established experimental platform that takes advantage of the distance-dependent energy transfer from a donor molecule to an acceptor molecule. FRET relies on the ability to effectively label the molecules with a fluorophore. The fluorescence readout of an assay results from an excited fluorophore-labeled donor that emits energy, which is absorbed by a second dye label-acceptor. Donor and acceptor are also often referred to as “FRET-pair”. In contrast to collisional or dynamic quenching, a direct contact between fluorophore and quencher is not required. Since FRET typically takes place in a distal range between 1-10 nm, it can be employed to measure processes on a molecular scale. The acceptor can either act as a secondary fluorophore (ratiometric FRET), or, can abolish (quench) the donor fluorescence, the latter used for example in protease assays (Figure 1). In order to obtain an efficient FRET, the emission spectrum of the donor and the absorption spectrum of the acceptor label should overlap as complete as possible.
Figure 1: Monitoring protease activity using a FRET substrate. Substrate cleavage separates fluorophore (F) and quencher (Q), thus recovering the donor fluorescence.
Using fluorescent peptides in medical research
FRET analysis using fluorescently-labeled peptides has been utilized by researchers to examine a number of disease states such as Alzheimer’s disease, inflammation, degradation of bone, autoimmune disease and cancer. The methodology capitalizes on the ability to monitor protease or cleavage events in a very specific and defined manner, based on the options and availability of labeled peptides used in the reactions.
In the case of Alzheimer's disease research, being able to understand the formation of senile plaques is of central importance. A number of fluorescent Aβ peptides are available, representing the main research targets Aβ (1-42) and Aβ (1-40), but also a variety of truncated forms. Researchers have been able to examine phagocytosis events in microglia [3], cell-to-cell transfer of Aβ (1-42) [4], the interaction of Aβ (1-40) with lipid bilayers [5] as well as conformational changes that impact the onset of the disease [6]. The enzymes β- and γ-secretase take part in the processing of the amyloid precursor protein (APP) to the mature amyloid β peptide. FRET-labeled substrate peptides containing the wild-type or double-mutated (“Swedish”) cleavage site are used for activity assays; the Swedish mutation strongly influences the aggregation behavior of the amyloid β peptides and hence had been identified as a factor for the early onset of Alzheimer`s Disease [7].
Another class of targets that researchers are utilizing FRET analysis and fluorescently-labeled peptides for is the study of matrix-metalloproteases (MMPs). MMPs are enzymes that have been shown to have a role in inflammation, degradation of bone, autoimmune diseases, and cancer. Using FRET with labeled substrates [8], researchers have been able to gain insights into the catalytic efficiency and hydrolysis of MMPs as they relate to these diseases.
Faulty regulation in programmed cell death (apoptosis) processes may result in a number of severe diseases, and the activation of caspases (cysteine-dependent aspartate-specific protease) represents a point of no return in the regulatory cascade. Fluorescent- and FRET-labeled caspase substrates have been used by researchers to better understand the regulatory events [9].
Conclusion
Fluorescently labeled peptides are versatile and valuable tools for medical and biological research. Peptides can be labeled during or after synthesis. Important properties of the fluorophores are a strong fluorescence and, for fluorescence microscopy, a high photo-stability. With FRET-based experiments, the emission spectra of the donor and the excitation spectra of the acceptor should overlap as good as possible, which should be considered for the choice of the fluorescent labels. Taken together, these fluorescently labeled peptides and FRET analysis have enabled researchers to gain a better understanding of many cellular processes and regulatory events that are linked to disease states. In some cases, the research led to significant progress in the development of novel therapeutics.
References
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[2] D. Baechle et al., Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity, Journal of Peptide Science 11(3) 166-74 (2005)
[3] D. Ozawa et al., Shuttling protein nucleolin is a microglia receptor for amyloid beta
peptide 1-42, Biological and Pharmaceutical Bulletin 36(10) 1587–1593 (2013)
[4] J. Domert et al., Spreading of amyloid-β peptides via neuritic cell-to-cell transfer is
dependent on insufficient cellular clearance, Neurobiology of Disease 65 82–92 (2014)
[5] K. Ikeda and K. Matsuzaki, Driving force of binding of amyloid beta-protein to lipid bilayers, Bichemical and Biophysical Research Communications 370(3) 525-9 (2008)
[6] Y. Wang et al., Two-photon and time-resolved fluorescence conformational studies of aggregation in amyloid peptides, The Journal of Physical Chemistry B 114 7112–7120 (2010)
[7] M. Citron et al., Generation of amyloid beta protein from its precursor is sequence specific, Neuron 14(3) 661-700 (1995)
[8] D. M. Bickett et al., A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9), Analytical Biochemistry 212(1) 58-64 (1993)
[9] E. Schmitt et al., Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells, Cell Death and Differentiation 5(6) 506-516 (1998)
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