Mimivirus is a parasite of Acanthamoeba polyphaga, and is the largest DNA virus known. Starting with its full GenBank sequence we synthesized the unique viral enzyme - APMV- topoisomerase. This new protein possessed extremely low DNA relaxation and strong and uncommon ligation activities. Opposite to all known DNA ligases, which connect DNA with 5'P ends, the recombinant protein catalyses ligation to 5'OH DNA ends. In cells these ends are exclusively generated during nucleolytic degradation of engulfed DNA and their presence indicates active phagocytic digestion of nuclear chromatin. In this project we will isolate this unique activity of APMV topoisomerase and will employ it in the first selective assay for efferocytosis - the process of removal of dying cells via their engulfment by other cells. The proposal will reach these Specific Aims: 1. To isolate the 5'OH DNA connecting activity of APMV-topoisomerase and use it for the detection of active efferocytes. To develop the APMV-based assay for ultra-fast and specific labeling of these cells in fresh-frozen and formalin-fixed tissue sections. 2. To test and optimize the APMV-based labeling approach in several models of active efferocytic reaction using tissue section formats. To validate it's specificity and ensure adequate sensitivity and high speed of detection. The new assay will permit rapid and comprehensive labeling of active efferocytes in various tissue section formats. It will be useful in molecular research of pathologies where assessment of efferocytic reaction is essential, such as ischemia, inflammation, oncologic and immune disorders.
The purpose of this project is to introduce an enabling technology with broad application in fluorescence microscopy. It will contribute to the development of more efficient therapies and advance biomedical research of cancer, stroke, heart disease and many others.
|Minchew, Candace L; Didenko, Vladimir V (2017) Dual Detection of Nucleolytic and Proteolytic Markers of Lysosomal Cell Death: DNase II-Type Breaks and Cathepsin D. Methods Mol Biol 1554:229-236|
|Didenko, Vladimir V (2017) Express FRET Labeling and Analysis of Phagocytic Clearance. Methods Mol Biol 1644:3-11|
|Minchew, Candace L; Didenko, Vladimir V (2017) Quick Detection of DNase II-Type Breaks in Formalin-Fixed Tissue Sections. Methods Mol Biol 1644:113-119|
|Didenko, Vladimir V (2017) Zebra Tail Amplification: Accelerated Detection of Apoptotic Blunt-Ended DNA Breaks by In Situ Ligation. Methods Mol Biol 1644:167-177|
|Hauser, Paul; Wang, Sha; Didenko, Vladimir V (2017) Apoptotic Bodies: Selective Detection in Extracellular Vesicles. Methods Mol Biol 1554:193-200|