Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity

Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and associated with accumulation of high levels porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about cytotoxic effects these porphyrin nanostructures. Previously, we demonstrated unique ability fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), cause organelle-selective protein aggregation, which posited be a major mechanism exerts effect. Herein, tested hypothesis PP-IX-mediated aggregation modulated PP-IX nanostructures via depends on oxidizing potential protein-binding ability. UV–visible spectrophotometry showed pH-mediated reversible transformations Biochemical analysis nanostructure size PP-IX-induced oxidation aggregation. Furthermore, albumin, most abundant serum protein, preferentially binds dimers enhances binding quenched albumin intrinsic fluorescence oxidized His-91 residue Asn/Asp, likely previously described photo-oxidation for other proteins. Extracellular protected from intracellular porphyrinogenic stress acting as sponge. This work highlights importance context porphyrias offers insights novel therapeutic approaches. Protoporphyrin-IX (PP-IX) nature’s template several essential biomolecules heme, chlorophyll, coenzyme F430 (in methanogenic bacteria), vitamin B12 (1Dayan F.E. Dayan E.A. Porphyrins: One ring colors life: A class pigment molecules king george iii, vampires herbicides.Am. Scientist. 2011; 99: 236-243Crossref Scopus (19) Google Scholar). The macrocycle consists four pyrrole rings connected methene bridges two ionizable propioniate side chains. highly conjugated 18 ?-electrons confers distinct UV-visible absorbance properties (2Gotardo F. Cocca L.H.Z. Acunha T.V. Longoni A. Toldo J. Gonçalves P.F.B. Iglesias B.A. De Boni L. Investigating intersystem crossing rate triplet quantum yield protoporphyrin ix means pulse train technique.Chem. Phys. Lett. 2017; 674: 48-57Crossref (20) Due its amphiphilic tends aggregate aqueous solution (3Scolaro L.M. Castriciano M. Romeo Patanè S. Cefalì E. Allegrini Aggregation behavior solutions: Clear evidence vesicle formation.J. Chem. B. 2002; 106: 2453-2459Crossref (126) Indeed, has been reported exist detergent micellarized 0.56 kDa monomers (4Isamu I. Kazutomo U. Association water poly(n-vinylpyrrolidone) solutions. Interaction between poly(n-vinylpyrrolidone).Bull. Soc. Jpn. 1991; 64: 2005-2007Crossref Scholar), (5Margalit R. Shaklai N. Cohen Fluorimetric studies dimerization equilibrium haemato derivative.Biochem. 1983; 209: 547-552Crossref PubMed (92) tetramers (6Seo Jang Warnke Gewinner Schöllkopf W. von Helden G. Stacking geometries early aggregates revealed gas-phase infrared spectroscopy.J. Am. 2016; 138: 16315-16321Crossref (18) higher-order structures >70 spontaneous self-association occurs combination hydrogen bonding (between propionate carboxylate) ?-? stacking porphine Scholar, 6Seo 7Hunter C.A. Sanders J.K.M. nature .Pi.-.Pi. Interactions.J. 1990; 112: 5525-5534Crossref (4832) resulting face-to-face or “H-aggregates” have chains adjacent “head-to-tail” orientation These supramolecular offer advantages over constituent (8Zhou Li Du X. Xu Supramolecular biofunctional materials.Biomaterials. 129: 1-27Crossref (121) 9Lehn chemistry.Science. 1993; 260: 1762-1763Crossref (594) self-assembling property similar compounds being explored variety applications (10Magna Monti D. Di Natale C. Paolesse Stefanelli assembly systems well-defined nanostructures: An update.Molecules. 2019; 24Crossref (23) 11Elemans J.A.A.W. van Hameren Nolte R.J.M. Rowan A.E. Molecular materials self-assembly phthalocyanines, perylenes.Adv. Mater. 2006; 18: 1251-1266Crossref (619) 12Wurthner Kaiser T.E. Saha-Moller C.R. J-aggregates: From serendipitous discovery engineering functional dye materials.Angew. Int. Ed. Engl. 50: 3376-3410Crossref (1595) e.g., synthetic photodynamic therapy (13Jin C.S. Lovell J.F. Chen Zheng Ablation hypoxic tumors dose-equivalent photothermal, not photodynamic, using nanostructured assembly.ACS Nano. 2013; 7: 2541-2550Crossref (290) 14Constantin Neagu Ion R.M. Gherghiceanu Stavaru Fullerene-porphyrin therapy.Nanomedicine (Lond.). 2010; 5: 307-317Crossref (56) 15Jin Cui Wang Targeting-triggered porphysome disruption activatable therapy.Adv. Healthc. 2014; 3: 1240-1249Crossref (112) photovoltaic cells (16Imahori H. Fukuzumi Porphyrin- fullerene-based molecular devices.Adv. Funct. 2004; 14: 525-536Crossref (438) 17Hasobe T. Imahori Kamat P.V. Ahn T.K. Kim S.K. Fujimoto Hirakawa Photovoltaic composite nanoclusters fullerenes gold nanoparticles.J. 2005; 127: 1216-1228Crossref (456) 18Walter M.G. Rudine A.B. Wamser C.C. Porphyrins phthalocyanines solar cells.J. Phthalocyanines. 759-792Crossref (544) photocatalysis (19Zhang Cao Bai Fan Self-assembled one-dimensional enhanced photocatalytic generation.Nano 2018; 560-566Crossref (107) 20Wang Z. Ho K.J. Medforth C.J. Shelnutt J.A. Porphyrin nanofiber bundles phase-transfer ionic self-metallization.Adv. 2557-2560Crossref (108) 21La D.D. Bhosale S.V. Jones L.A. Arginine-induced porphyrin-based self-assembled under simulated sunlight irradiation.Photochem. Photobiol. Sci. 16: 151-154Crossref 22Chen Y. Huang Z.-H. Yue Kang Integrating nanoparticles 2d graphene matrix free-standing nanohybrid films visible-light activity.Nanoscale. 6: 978-985Crossref 23Medforth Martin K.E. Song Jacobsen J.L. nanostructures.Chem. Commun. 2009; : 7261-7277Crossref (246) sensor (23Medforth 24Dini Martinelli Pomarico Filippini D'Amico Lundstrom Chemical sensitivity nano-aggregates.Nanotechnology. 20055502Crossref (34) 25Wang nanotubes self-assembly.J. 126: 15954-15955Crossref (368) Although macromolecular species extensively studied, role porphyria known. include eight mutations any enzymes (26Puy Gouya Deybach J.-C. Porphyrias.Lancet. 375: 924-937Abstract Full Text PDF (464) 27Maitra Bragazzi Cunha Elenbaas J.S. Bonkovsky H.L. Shavit Omary M.B. Porphyrin-induced porphyria-associated cell injury.Cell Mol. Gastroenterol. Hepatol. 8: 535-548Abstract (16) Heme biosynthesis starts mitochondrion where aminolevulinic acid (ALA) synthase catalyzes rate-limiting conversion glycine succinyl Co-A ?-ALA (27Maitra 28Schultz I.J. Paw B.H. Hamza Iron trafficking biogenesis.J. Biol. 285: 26753-26759Abstract 29Montgomery Bissell Chapter 66 - porphyrias.in: Rosenberg R.N. Pascual J.M. Rosenberg's Genetic Basis Neurological Psychiatric Disease. 5th Academic Press, Boston, MA2015: 731-749Crossref (0) 30Ajioka R.S. Phillips J.D. Kushner J.P. Biosynthesis mammals.Biochim. Biophys. Acta. 1763: 723-736Crossref (330) Upon translocation cytosol, ALA converted steps first cyclic tetrapyrrole, uroporphyrinogen, then coproporphyrinogen Coproporphyrinogen enters mitochondria, it formation protoporphyrinogen Porphyrinogens colorless, nonfluorescent (31Badminton M.N. Elder G.H. 28 porphyrias: Inherited haem synthesis.in: Marshall W.J. Lapsley Day A.P. Ayling Clinical Biochemistry: Metabolic Aspects. 3rd Churchill Livingstone, London, UK2014: 533-549Crossref (2) Scholar) compounds, auto-oxidized more stable toxic metabolites, tightly regulated. In porphyrias, blockages enzyme lead intermediates consequent organ tissue damage Porphyrin-mediated proposed occur through reactive oxygen (ROS) generated type I/II photosensitized reactions (32Foote Definition i ii oxidation.Photochem. 54: 659Crossref (906) 33Takeshita K. Takajo Hirata Ono Utsumi vivo radical generation skin protoporphyria model mouse visible light exposure: l-band esr study.J. Invest. Dermatol. 122: 1463-1470Abstract (62) 34Brun Sandberg Mechanisms photosensitivity porphyric patients special emphasis erythropoietic protoporphyria.J. Photochem. B 10: 285-302Crossref (38) However, precise ROS well specific targets porphyrin-generated poorly understood Accumulating findings though involves “porphyrination-deporphyrination” cycle 35Singla Griggs N.W. Kwan Snider N.T. Maitra Ernst S.A. Herrmann Lamin an porphyria-induced liver injury.J. Cell 3105-3112Crossref (21) 36Saggi Jiang Zhang P. Cornuet Singh Locker Ma Dailey Abrams Monga S.P. Nejak-Bowen Loss hepatocyte beta-catenin protects mice experimental 70: 108-117Abstract (9) 37Elenbaas Liu Lentz S.I. Nelson Hoenerhoff M.J. precursor-inducible zebrafish acute hepatic multiorganelle stress.FASEB 30: 1798-1810Crossref (12) 38Zhang Ramakrishnan Triner Centofanti Gyorffy Sebolt-Leopold Dame M.K. Varani Brenner D.E. Fearon E.R. Shah Y.M. Tumor-selective proteotoxicity verteporfin inhibits colon cancer progression independently yap1.Sci. Signal. 2015; ra98Crossref (111) 39Maitra Whitesall S.E. Basrur V. D'Alecy L.G. Ambient promotes selective subcellular after endogenous exogenous stress.J. 290: 23711-23724Abstract (15) this cycle, target proteins (porphyrination) induces localized unfolding conformational change (40Fernandez N.F. Sansone Mazzini Brancaleon Irradiation causes ix/?-lactoglobulin noncovalent complex.J. 2008; 7592-7600Crossref (22) 41Belcher Fernandez Haskins W.E. Photoinduced ?-lactoglobulin mediated water-soluble porphyrin.J. 113: 6020-6030Crossref Photosensitization protein-bound generates singlet (1O2), methionines methionine sulfone sulfoxide 42Maitra Carter E.L. Richardson Rittie Nesvizhskii A.I. Osawa Wolf M.W. Ragsdale S.W. Lehnert Oxygen conformation dependent hepatocytes light-exposed cells.Cell 659-682.e651Abstract (5) Subsequent interactions protein-PP-IX lattice-like formation. During deporphyrination, acidic pH high-salt conditions release disaggregation We posit proteotoxic involve accumulation. speciation differential binding. results emission signatures 4Isamu 5Margalit 43Teng K.W. Lee S.H. Characterization vitro spectroscopy polar plot analysis.J. 123: 5832-5840Crossref (4) solution, strength principal modulators investigated at 7.4 (physiological), 4.5 (lysosomal), 9, spectra assignments. were collected diluting freshly prepared stock solutions indicated buffers (Fig. 1, B). 100 mM HCl, exists monomers, characterized sharp Soret band 409 nm 1A, Table 1). At 4.5, form H-aggregates (higher-order structures), broad shoulders 356 466 nm. 9 NaOH, exclusively dimers, judged characteristic peak 380 Notably, spectrum shows features both displaying slightly red-shifted (379 469 nm) compared suggests 7.4, mixture dimers. addition changes band, Q-bands 500–700 region observed. nitrogens expected protonated, observed (Table number Q bands increases 3 (pH 7.4) 4 9) extent protonation decreases 1).Table 1Summary UV-vis spectral nanostructuresAbsorbance peaks (nm)Fluorescence (nm)PP-IX speciationSoretQ4Q3Q2Q1[NaOH], mM381516554579633630, 687DimerpH 9384517555587640641, 685pH 7.4379–469-542595648631, 670Dimer, structurespH 4.5356, 466-539597647662, 720Higher-order structures[HCl], mM409-556597-610, 665MonomerpH 9+Emp408507541581632638, 702pH 7.4+Emp408508541580633638, 4.5+Emp408505543579635639, 703BSA+PP-IX, 4.5355, 469-539597649638, 684BSA+PP-IX structuresBSA+PP-IX, 7.4391516553585629642, 683BSA+PP-IX dimerBSA+PP-IX, 9391509553576627642, 682BSA+PP-IX Emp406504539585629636, 700MonomerThe table plots shown Figure A, B, E, 4, A–D. Open new tab Fluorescence 1B) significant quenching PP-IX. Thus, displays minimal signal 1B). also pH-induced C D). stru ctures increased, albeit incomplete 1C, see shoulder arrow). transition reverse direction (dimers ? structures) was when progressively acidified 1D). Earlier had containing detergents (e.g., Empigen BB, NP-40) (36Saggi Given profound effect speciation, w