Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability

Abstract

Spastin is one of the proteins which lead to hereditary spastic paraplegia (HSP), whose dysfunction towards microtubule severing and membrane transporting is critically important. The present study is to elucidate the mechanisms of the protein stability regulation of spastin. The ubiquitin encoding plasmids are transfected into COS-7 cells with different fusion tags including Green Fluorescent Protein (GFP), mCherry and Flag. The expression level of spastin was detected, microtubule severing activity and neurite outgrowth were quantified. The data showed that ubiquitin overexpression significantly induced the decreased expression of spastin, suppressed the activity of microtubule severing in COS-7 cells and inhibited the promoting effect on neurite outgrowth in cultured hippocampal neurons. Furthermore, when modulating the overexpression experiments of ubiquitin, it was found that relatively small tag like Flag, but not large tags such as GFP or mCherry fused with ubiquitin, retained the activity on spastin stability. The present study investigated the effects of small/large tags addition to ubiquitin and the novel mechanisms of post-transcriptional modifications of spastin on regulating neurite outgrowth, in the attempt to experimentally elucidate the mechanisms that control the level or stability of spastin in hereditary spastic paraplegia.

Phosphorylation mutation impairs the promoting effect of spastin on neurite outgrowth without affecting its microtubule severing ability

“… 29 , 30 Spastin is highly expressed during development and promotes axon elongation by severing long microtubules to short segments. 18 When the expression of spastin is disturbed, significant defects can occur in the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. 31 Spastin promotes microtubule amplification by increasing the rate at which shrinking microtubules are converted into growing ones (called rescue) so that the microtubule fragments created by severing act as seeds for the growth of new microtubules.…”

“…Overexpression of microtubule severing proteins, such as spastin or katanin, within COS7 cells and staining of microtubules are currently commonly used to quantify microtubule severing activity, as previously reported. 18,24 The results showed that compared to the fluorescence intensity of microtubules in COS7 cells in the GFP vector control group, both GFP-spastin WT and GFPspastin 11A significantly reduced the fluorescence intensity of microtubules (Figure 3 A,B). Interestingly, the difference between the GFP-spastin 11A group and the GFP-spastin WT group was not statistically significant (p>0.05) (Figure 3B).…”

“…Our previous studies have demonstrated that spastin can effectively promote neurite outgrowth by regulating microtubule dynamics, 16 , 17 and the protein stability of spastin is essential for neurite outgrowth. 18 However, the regulation of protein function is not only related to the amount of protein expression but also closely related to the post-translational modification of proteins. Phosphorylation is a common post-translational modification of proteins, 19 which can regulate the transduction of cell signaling pathways, gene expression, cell cycle, and other cellular activities.…”