Oxygen and Conformation Dependent Protein Oxidation and Aggregation by Porphyrins in Hepatocytes and Light-Exposed CellsSummary
| Autor: | Nicolai Lehnert, Dhiman Maitra, Stephen W. Ragsdale, Laure Rittié, M. Bishr Omary, Alexey I. Nesvizhskii, Haoming Zhang, Matthew W. Wolf, Harald Herrmann, Venkatesha Basrur, Rani Richardson, Yoichi Osawa, Eric L. Carter |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Male Protein Conformation Protoporphyrins BCA Bicinchoninic acid Protein aggregation Protein oxidation chemistry.chemical_compound Mice 0302 clinical medicine Met methionine RPN1 26S proteasome regulatory subunit RPN1 polycyclic compounds Heme Original Research DFO deferoxamine DMA dimethylacetamide IF intermediate filament Photosensitizing Agents Chemistry Liver Neoplasms Gastroenterology K keratin DDC 3 5-diethoxycarbonyl-1 4-dihydrocollidine EPR electron paramagnetic resonance spectroscopy Liver ALA δ-aminolevulinic acid Zn-PP zinc protoporphyrin-IX 030211 gastroenterology & hepatology Phototoxicity 1O2 singlet oxygen Copro coproporphyrin PP-IX protoporphyrin-IX SDS sodium dodecyl sulfate Carcinoma Hepatocellular Porphyrins O2- superoxide PP-Dim protoporphyrin-IX dimethyl ester PBS phosphate-buffered saline HMW high molecular weight Deferoxamine Uro uroporphyrin Cell Line ER endoplasmic reticulum 03 medical and health sciences Porphyrias Protein Aggregates GOX glucose oxidase ROS reactive oxygen species medicine Animals Humans Photosensitivity Disorders NP-40 Nonidet P-40 lcsh:RC799-869 Cell damage PAGE polyacrylamide gel electrophoresis Hepatology Porphyria TMPD N N N′ N′-tetramethyl-p-phenylenediamine Aminolevulinic Acid medicine.disease Porphyrin POBN α-(4-pyridyl-1-oxide)-N-tert-butylnitrone Mice Inbred C57BL Oxygen Oxidative Stress 030104 developmental biology Proteotoxicity MS mass spectrometry Biophysics Hepatocytes lcsh:Diseases of the digestive system. Gastroenterology Amino Acid Oxidation |
| Zdroj: | Cellular and Molecular Gastroenterology and Hepatology, Vol 8, Iss 4, Pp 659-682.e1 (2019) Cellular and Molecular Gastroenterology and Hepatology |
| Popis: | Background & Aims Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent. Methods Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed. Results Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source. Conclusions Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias. Graphical abstract |
| Databáze: | OpenAIRE |
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