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Conversion of Optineurin into an Hsp72-Inducible protein by C-terminal addition of green fluorescent protein

Title
Conversion of Optineurin into an Hsp72-Inducible protein by C-terminal addition of green fluorescent protein
Authors
Suh, WoolSohn, SeongsooKim, Tae EunKee, Changwon
Ewha Authors
서울
SCOPUS Author ID
서울scopus
Issue Date
2024
Journal Title
MOLECULAR VISION
ISSN
1090-0535JCR Link
Citation
MOLECULAR VISION vol. 30, pp. 114 - 122
Publisher
MOLECULAR VISION
Indexed
SCIE; SCOPUS WOS
Document Type
Article
Abstract
Purpose: Optineurin is known to be associated with glaucoma. This protein has often been investigated as a form of fusion with green fluorescent protein (GFP), but there have been few reports describing any undesired effect of the tag on the normal expression of naive optineurin. We investigated whether GFP tagging potentially does not influence the characteristics of optineurin expression. Methods: Wild-type and mutant (E50K and M98K) optineurins were fused with GFP or yellow fluorescent protein (YFP) either at their N-terminus or C-terminus. The fusion constructs were loaded onto an adenoviral vector and analyzed by western blot analysis and immunocytochemistry. Results: In human trabecular meshwork cells, optineurins fused to GFP at their C-terminus (OPTN-GFP) were prone to aggregation and did not fluoresce as brightly as their counterparts fused to YFP did regardless of whether they were wild-type or mutant forms. In addition, their expression was accompanied by the apparent induction of heat shock protein 72 (Hsp72). Furthermore, OPTN-GFP appears to interact with Hsp72 and be co-aggregated into vesicle-like structures scattered throughout the cytoplasm. However, optineurin fused to GFP at its N-terminus was not aggregate and did not induce Hsp72 expression. Conclusions: It is well-known that misfolded or unfolded proteins are prone to aggregation and, if they are fused with GFP, their chromophores are not fully fluorescent. Additionally, it is also well-known that heat shock response is a key cellular processes against the accumulation of misfolded or unfolded proteins. Therefore, our data suggest that the addition of GFP to the C-terminus of optineurin might convert it into an unfolded or partially folded protein.
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의료원 > 의료원 > Journal papers
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