Bioluminescent Annexin Fusion Proteins (AFPs) for Atherosclerosis Detection

Abstract

The process of controlled cell death known as apoptosis plays a critical role in the homeostatic tissue maintenance of multicellular organisms under normal, healthy conditions. However, this process (and its dysregulation) has also been implicated in a number of pathologies such as cancer, neurodegenerative diseases, autoimmune diseases, and cardiovascular disease (CVD). This broad involvement of apoptosis in these conditions has led to a sizable amount of research focused on techniques capable of selectively detecting apoptotic cells. Many such techniques rely on the exposure of the specific marker phosphatidylserine (PS) on the surface of apoptotic cells. The protein Annexin V has been shown to selectively bind to exposed PS in a specific manner with high affinity. Detection of apoptosis using this specific interaction has previously been performed utilizing fluorescence or isotopic radio-labeling. In contrast to these approaches, the use of bioluminescence is proven to be an effective means of signal generation in vivo, and provides a number of benefits over both fluorescence and isotopic labeling. Detection via bioluminescence has been shown to exhibit lower background and higher signal-to-noise ratios when compared to fluorescence, and does not carry restrictive handling and disposal regulations encountered when utilizing isotopic labeling. We have begun the development of a library of fusion proteins incorporating both Annexin V and bioluminescent reporters, providing the capability of targeting and binding apoptotic cells as well as generating a detectable bioluminescent signal. The first AFP, containing the bioluminescent reporter Renilla luciferase (RLuc8), was successfully utilized in the detection of atherosclerosis both in vitro as well as in vivo, and was shown to have potential in identifying atherosclerotic plaques associated with CVD). We are now expanding the AFP library to generate a set of unique and tunable AFPs combining Annexin V with a variety of robust bioluminescent reporters. These AFP reporters include: Gaussia luciferase (GLuc), one of the smallest and brightest known luciferases; Aequorin, previously utilized for site-selective and random mutagenesis by our lab resulting in an extensive library of reporters with tunable emission wavelengths and kinetics; and a truncated Vargula luciferase variant (tVLuc), exhibiting glow-type emission kinetics and first reported expression in a bacterial system by our lab. Support or Funding Information This work was supported in part by grants from the National Institutes of Health (1R01GM114321 and R01GM047915). Dr. Sylvia Daunert is grateful to the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in the Department of Biochemistry and Molecular Biology. Apoptosis can be initiated via extrinsic (blue box) or intrinsic (orange box) pathways. However, both pathways converge at the activation of caspases which, in turn, lead to the exposure of phosphatidylserine (PS) on the surface of the apoptotic cell. Under apoptotic conditions, the Annexin Fusion Proteins (AFPs) can bind exposed PS and generate a bioluminescent signal in the presence of their luminescent substrate. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.