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A common trait of conformational disease causing proteins is "?-strand promiscuity",where conversion of a loop to a ?-strand through interactions with a pre-existing ?-sheet leads to loop-sheet polymerization and formation of the characteristic fibers, amyloids, and plaques. In spongiform encephalopathies conversion of cellular prion PrPc to the pathogenic isoform PrPsc is accompanied by a marked diminution in ?-helical content (from 42% to 30%) and a dramatic increase in ?-sheet structure (from 3% to 43%). <P>Our overarching objective is to develop nanoscale approaches to control protein secondary structure to address conformational diseases relevant to agriculture and medicine. Our research aims to quantify the binding affinities (Aim-1) and inhibitory / reverse-fibrillation activities (Aim-2) of functionalized nanoparticles with model pathogenic proteins. Undergraduate involvement in research will be a key aspect to this work and student participation in local research conferences (e.g. NanoUtah) will provide students the opportunity to present their research. Nanoparticle cytotoxicity will be assessed as a new laboratory module in BE 5850/6850 Biomedical Engineering. The project will likely lead to several papers addressing nanoparticle biological activity. Findings will be disseminated through conferences and peer reviewed manuscripts throughout the timeframe of this research.
Non-Technical Summary:<br/>
Amyloid based diseases affect humans, cattle, sheep, and elk. Amyloid etiology involves recruitment and self-assembly of proteins into insoluble fibrils and plaques, leading to a variety of diseases including Alzheimer's, Parkinson's, cataracts, dialysis related amyloidosis, and type II diabetes in humans, and spongiform encephalopathies in cattle, sheep, and elk. The proposed research addresses the fundamental aspects of protein-protein interactions to better understand how pathogenic proteins recruit native proteins into pathogenic conformations that self-assemble to form fibrils and plaques. From this understanding we will develop nano-scale therapeutics to inhibit protein assembly into fibrils and plaques associated with these diseases.
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Approach:<br/>
The fluoro-silane, 3F, will be employed as a functional monomer that stabilizes ?-helical secondary structure as a polymerizable analog to hexafluoroisopropanol (a known helix inducer). The alkyl-silane, nPM, will be investigated as a co-monomer with 3F to construct low MW diblock copolymers and mixed functionality NPs. We will develop nanotherapeutics (low MW polymers, functionalized nanoparticles) against several proteins modeling conformational diseases. The first protein is ?2-microglobulin that self-assembles into fibrils in dialysis related amyloidosis. The amyloid-? peptide implicated in plaque formation in Alzheimer's disease will also be studied. These proteins, like prion, are switch-proteins, that can adopt two stable conformations, one of them being pathogenic with a tendency recruit and self-assemble. The library of 3F/nPM low MW compounds will be prepared through controlled condensation polymerization and purification on HPLC. Silica nanoparticles prepared by the Stober method will be functionalized with the 3F/nPM compounds exhibiting the most activity against target protein conformation. Conformational changes will be addressed using fluorescence spectroscopy and confirmed using circular dichroism. Preformed fibrils will be attacked with the developed compounds and defibrillation will be assessed in situ using atomic force microscopy.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: "The PI taught methods of sol-gel nanoparticle synthesis developed for this project to over 80 undergraduate students in BENG 1890, Introduction to Undergraduate Research and Design, Fall 2012. The students learned fundamental methods to prepare zinc oxide nanoparticles, vary reaction parameters to tune particle size, and use modern spectroscopic methods to characterize the nanoparticle properties. The PI and his student researchers designed, conducted and analyzed experiments testing ability of nanoparticles to inhibit model protein (lysozyme, albumin) aggregation into amyloid fibrils. The PI and students presented this research as two poster and two podium presentations locally and internationally at NanoUtah2012, Salt Lake City, October 2012, and Colloids and Nanomedicine, Amsterdam, The Netherlands, July 2012. The importance of this research was recognized through a podium presentation at Colloids and Nanomedicine 2012 in Amsterdam (just one of 8 presentations accepted from over 300 submitted). The PI, his former Ph.D. student (Y. Peng) and USU collaborators (M. Walsh, T. Doyle) published a book chapter on nanoparticle approaches to controlling protein conformation as part of a Biomedical Nanotechnology Series."
<br/>PARTICIPANTS: USU: Dr. Christian Dimkpa, Dr. Marie Walsh, Dr. Timothy Doyle. Undergraduate Researchers: Rachael Johnson, Seth Hansen, Kyle Isaacson, Arther Hart, Joshua Israelsen.
<br/>TARGET AUDIENCES: Academia, Agricultural Community, Medical Community, General Public.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: "The project resulted in several improvements in methods for applying native and silane coated zinc oxide nanoparticles to restrict protein aggregation into amyloid fibrils. Inhibiting this process is important as these fibrils are associated with neurodegenerative diseases in animals and humans. The uncontrolled association of proteins such as prion is a hallmark of deadly neuroligical diseases such as spongiform encephalopathy in cattle. Our findings using a model protein system for these diseases demonstrates restricted protein self-assembly into amyloid-like fibrils in the presence of zinc oxide nanoparticles synthesized in our laboratory. The PI and student researchers have developed novel methods to deposit organo-silane coatings on these zinc oxide nanoparticles. These thin ""glass-like"" coatings create a semi-permeable shell around the nanoparticle, allowing us to control their activity and lifetime in protein solutions (such as blood or cerebrospinal fluid). This is a key step necessary to advance this research toward development of nanoparticle based therapeutic against neuroligical diseases arising from protein misfolding and aggregation."