|
Advanced Molecular Tools for Specific Detection of Complex Molecules as Promising Plasma Biomarkers
JUNHONG YAN, Phd student, Uppsala University
In proximity ligation assay (PLA), affinity probes, such as antibodies, are attached to oligonucleotides to form PLA probes. Once a target molecule is recognized by a two proximity probes the DNA strands are connected to each other via enzymatic ligation to form a DNA template that can be amplified and quantified as the measure of the target molecule’s concentration. A new format of proximity ligation assay (4PLA) was developed for detection of complex target structures such as microvesicles in which the target is ?rst captured via an immobilized antibody and subsequently detected using four PLA probes. The requirement for coincident binding by ?ve antibodies to generate an ampli?able reporter results in both increased speci?city and sensitivity. The method was used to detect microvesicles such as prostasomes in blood samples, demonstrating the potential of the technology to screen for higher order molecules as a new class of biomarkers. Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal ?uid, to appear in interstitial space and therewith into peripheral circulation. Levels of prostasomes were found significantly elevated in blood samples from patients with prostate cancer using 4PLA.
Exosomes: vesicular carriers of autotaxin, a novel mechanism of LPA signalling
Susanna Jethwa, , Babraham Institute
Autotaxin (ATX) is a lysophospholipase D (lysoPLD) and the predominant enzyme responsible for the extracellular production of the bioactive signalling lipid- lysophosphatidic acid (LPA).
ATX is a critical protein throughout development, and is also key in several diseases including atheroschlerosis, obesity, neuropathic pain and cancer.
Previous work has established that the majority of ATX is secreted from the cell (>95%), following proteolytic processing, but interestingly work has shown that extracellular autotaxin exists in at least two forms, a soluble form and a vesicular form – isolatable by high-speed ultracentrifugation.
Experiments to categorize the nature of vesicular ATX have demonstrated they display characteristics of exosomes including size, morphology, density and protein content. However unlike other exosomal proteins, research suggests that ATX associates with exosomes post-secretion.
Investigation of the function of vesicular ATX has concluded that it is catalytically active and appears to utilize exosomal membrane lysophosphatidylcholine (LPC) as a substrate, in a regulated manner, to generate the bioactive agonist LPA. This mechanism allows LPA to be directly delivered to cognate GPCRs to initiate signaling events including increased mitogenic signals (?pERK and ?pAKT) and localized calcium responses, which can be abrogated by pre-treatment of recipient cells with the pan LPA receptor antagonist Ki14625.
|
