Microvesicles are small membrane-enclosed sacs that bud from a range of cells following membrane fission. During the budding process, lipid rearrangement takes place causing an overexpression of phosphatidylserine on the outer leaflet of these vesicles, ranging in diameters in the magnitude starting from ~100 nm. Recent studies suggested that these shedding vesicles are a direct indication of metastatic cancerous cells. This exciting finding implied a novel strategy to develop non-invasive, readily available biomarkers to detect metastatic cancers, which is still a challenging goal using the current methodology. Our overall goal of the proposed studies is to select and optimize peptides as biological probes to target small vesicles with high membrane curvature. This idea is inspired from naturally-occurring proteins that are known to perform specific functions by sensing and/or inducing membrane curvature in important biological processes. i.e. endocytosis, membrane fusion, cell signaling, etc. Identifying peptides that can sense curvature would be a novel approach. Chosen from a focused peptide library that were previously selected for the desired properties for curvature sensing, the MARCKS peptide provides a promising lead that has rendered promising preliminary results. The MARKS peptides specifically interact with the phosphatidylserine that is found in highly curved membrane bilayers. Based on current results and published biophysical binding models, we are able to propose our own model as to how this peptide would preferentially bind to certain vesicle sizes as well as confidently propose two goals for this research. This proposal has two specific aims (1) to characterize the MARCKS peptide as a peptide curvature sensor in biophysical membrane models;(2) to use the MARCKS peptide to detect microvesicles in in vitro and ex vivo systems.
Specific Aim 1 will be accomplished through the experimental methods of fluorescence, isothermal calorimetry (ITC), proton nuclear magnetic resonance (NMR) and other biophysical assays.
Specific Aim 2 will be accomplished through fluorescence enhancement assays, confocal fluorescence imaging, and the state-of-the-art Nanoparticle Tracking Analysis technology. Successful completion of the proposed studies will establish a new method to use readily modifiable peptide probes to detect microvesicles, laying the groundwork for developing the next generation of diagnostic tools and biomarkers to detect metastatic cancers.
Microvesicles with highly curved membranes have been reported to shed from metastatic cancer cells, implying a novel strategy to develop inexpensive, non-invasive biomarkers for metastasis that account for the majority of cancer-related deaths. Designing detection probes that target shedding vesicles using small peptides will positively impact cancer diagnosis technologie
|Morton, Leslie A; Tamura, Ryo; de Jesus, Armando J et al. (2014) Biophysical investigations with MARCKS-ED: dissecting the molecular mechanism of its curvature sensing behaviors. Biochim Biophys Acta 1838:3137-44|
|Morton, Leslie A; Yang, Hengwen; Saludes, Jonel P et al. (2013) MARCKS-ED peptide as a curvature and lipid sensor. ACS Chem Biol 8:218-25|
|Saludes, Jonel P; Morton, Leslie A; Coulup, Sara K et al. (2013) Multivalency amplifies the selection and affinity of bradykinin-derived peptides for lipid nanovesicles. Mol Biosyst 9:2005-9|
|Saludes, Jonel P; Morton, Leslie A; Ghosh, Nilanjan et al. (2012) Detection of highly curved membrane surfaces using a cyclic peptide derived from synaptotagmin-I. ACS Chem Biol 7:1629-35|
|Morton, Leslie A; Saludes, Jonel P; Yin, Hang (2012) Constant pressure-controlled extrusion method for the preparation of Nano-sized lipid vesicles. J Vis Exp :|