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Hongyu Ying, Xiang Shen, Minghua Wang, Beatrice Yue; Biophysical Properties of Human Wild Type Optineurin Protein . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3652.
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© ARVO (1962-2015); The Authors (2016-present)
To study biophysical properties of human wild type optineurin protein including posttranslational modifications and aggregation status. Optineurin is a gene linked to normal tension glaucoma, amyotrophic lateral sclerosis, and other diseases.
The posttranslational modification sites were identified by liquid chromatography-tandem mass spectrometry. For thioflavin T (ThT) assay, purified optineurin incubated at 37°C in physiological buffer for up to 4 days was mixed with ThT solution. The resulting fluorescence was determined at 450 nm Ex/485 nm Em. For ionic strength effects, the buffer contained 0 to 2M concentrations of NaCl. For pH effects, purified optineurin was incubated in buffers containing 150 mM NaCl and pH ranging from 2.3 to10. For atomic force microscopy (AFM), freshly purified optineurin (10 µg/ml) was incubated at either 4°C or 37°C for 4 days. The AFM images were acquired by an Agilent 5400 scanning probe microscope. For protein-protein interactions, bimolecular fluorescence complementation (BiFc) plasmid pairs (pOPTN-YN+pOPTN-YC, pYN-OPTN+pOPTN-YC, or pb-Jun-YN + pb-Fos-YC) were co-transfected into mouse neuroblastoma Neuro2A cells for 20 h. Cells were subsequently fixed and imaged.
Five phosphorylation sites (Ser173, 174, 177, 526 and 528) and one acetylation site (Ser 2) in optineurin were identified. By ThT assay, purified optineurin incubated in physiological buffer resulted in heightened ThT fluorescence intensity in a time-dependent manner. The ThT fluorescence was also increased with increasing ionic strength. For pH effects, the highest fluorescence was observed at pH 6.5. By AFM, optineurin was found to form aggregates after incubation for 4 days. The aggregates formed at 4°C were fewer and smaller than those at 37°C. By BiFc assay, green fluorescence was observed in the cytoplasm of cells co-transfected with optineurin BiFc plasmid pairs indicating that optineurin interacted with itself to form dimers/oligomers.
New phosphorylation and acetylation sites were identified in optineurin, which might be important for its functions. Optineurin was also shown to be an aggregate prone protein. It interacted with itself as detected by BiFc analysis, and formed aggregates as evidenced by ThT assay and AFM. The baseline biophysical information provided in the present study will be of value, facilitating elucidation of pathophysiological pathways triggered by optineurin mutants.
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