Abstract
Here, we report that minimal functional gelsolin i.e. fragment 28–161 can display F-actin depolymerizing property even after heating the protein to 80 °C. Small angle X-ray scattering (SAXS) data analysis confirmed that under Ca2+-free conditions, 28–161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca2+, it forms dimers which proceed to non-characterizable aggregates. The dimeric association also explained the observed decrease in ellipticity in circular dichroism experiments with increase in temperature. Importantly, SAXS data based models correlated well with our crystal structure of dimeric state of 28–161. Characterization of higher order association by electron microscopy, Congo red and ThioflavinT staining assays further confirmed that only in absence of Ca2+ ions, heating transforms 28–161 into β-amyloids. Gel filtration and other experiments showed that β-amyloids keep leaching out the monomer, and the release rates could be enhanced by addition of L-Arg to the amyloids. F-actin depolymerization showed that addition of Ca2+ ions to released monomer initiated the depolymerization activity. Overall, we propose a way to compose a supramolecular assembly which releases functional protein in sustained manner which can be applied for varied potentially therapeutic interventions.
Highlights
A rescue for the depleting levels of pGSN was experimented by repletion with exogenous recombinant human gelsolin and based on remarkable positive outcome compared to placebo; the approach has been termed as gelsolin replacement therapy (GRT)[6]
The cytokine analysis of the LPS challenged mice treated with recombinant human gelsolin (rGSN) and bonsai gelsolin showed that the immune response of mice treated with bonsai gelsolin shifted from pro-inflammatory to anti-inflammatory[6]
28–161 was heated to elevated temperatures, cooled to room temperature followed by addition of Ca2+ ions in sample and the protein solution was added to pyrene-labelled F-actin
Summary
A rescue for the depleting levels of pGSN was experimented by repletion with exogenous recombinant human gelsolin (rGSN) and based on remarkable positive outcome compared to placebo; the approach has been termed as gelsolin replacement therapy (GRT)[6]. Our shape-function data showed that this bonsai version requires free Ca2+ ions or low pH to open up to bind actin and effect its function. The cytokine analysis of the LPS challenged mice treated with rGSN and bonsai gelsolin showed that the immune response of mice treated with bonsai gelsolin shifted from pro-inflammatory to anti-inflammatory[6]. All these indicate towards a possible role of the bonsai rGSN i.e. 28–161 as a substitute in the therapeutic potential of GRT. Though we had seen Ca2+ ions or pH induced activation of this bonsai gelsolin, there was no information on influence of temperature on the function of this fragment of gelsolin. We present here a way to compose self-releasing higher order assembly of this potentially therapeutic protein which may aid in its application in scenarios requiring sustained release of this functional protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
