Porphyrin Nanostructures Modulates Its Protein Aggregation Ability via Differential Oxidation and Protein Binding
Authors: Pinsky Benjamin Michael , Maitra Dhiman , Omary M. Bishr 
Published: June 2021

BackgroundPorphyrias are a group of diseases that associate with the accumulation of high levels of porphyrins due to well‐defined defect in the heme biosynthetic pathway. Porphyrins are known to spontaneously associate to form higher order structures. Prior studies from our lab demonstrated the unique abilities of fluorescent biological porphyrins to mediate protein aggregation in an organelle‐selective fashion. This protein aggregation provides a potential mechanism for cell and tissue damage that afflicts patients with porphyria. We hypothesize that the proteotoxicity of porphyrins is modulated by its supramolecular structures.MethodsUV‐visible and fluorescent spectrophotometry were utilized to study the speciation of protoporphyrin‐IX (PP‐IX) as a function of pH. The oxidizing potential of different PP‐IX species towards C=C containing biologic chromophores, including flavin mononucleotide (FMN) and free thiols, were tested. The differential binding of PP‐IX species to bovine serum albumin (BSA), and PP‐IX species aggregating ability towards glyceraldehyde 3‐phoshate dehydrogenase (GAPDH), were tested using spectrophotometry and gel electrophoresis followed by immunoblotting.ResultsAt lysosomal pH 4.5, PP‐IX (monomer ~ 0.5 kDa) was predominantly found as higher ordered species (~ 70 kDa). By contrast, PP‐IX was completely dimerized at pH 9 and formed a mixture of higher structures and dimers at pH 7.4. The dimers had significantly higher quantum yield than higher order structures, which manifested in their increased oxidizing ability towards FMN. Notably, PP‐IX dimers and higher structures were equally efficient in oxidizing free thiols. BSA, an abundant serum porphyrin binding protein, had two classes of PP‐IX binding sites, one for higher structures, and another for dimers. BSA preferentially bound PP‐IX dimers and had the ability to break‐up higher structures into dimers, but did not bind monomeric PP‐IX. While BSA‐PP‐IX interaction did not cause protein aggregation, GAPDH underwent aggregation upon interacting with PP‐IX, with a unique gel shift for higher order structures versus dimers and/or mixture of dimers/higher order structures.ConclusionOur results demonstrate that PP‐IX self‐associates to provide different porphyrin species depending on pH. Low pH4.5 leads to large PP‐IX structures while high pH leads to PP‐IX dimers and neutral pH7.4 leads to a mix of dimeric and larger structures. Differences in PP‐IX speciation have a profound effect on the ability of PP‐IX to bind to proteins and thereby induce protein oxidation and aggregation. These findings are likely to have important consequences in the pathogenesis of porphyrias, and offers insights into potential therapeutic approaches that target porphyrin speciation.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) grant R01 DK116548 (M.B.O.); and by a Summer Undergraduate Research Fellowship, Department of Molecular and Integrative Physiology, University of Michigan (B.M.P.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.