https://doi.org/10.4081/ejh.2021.3352
Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability
Submitted: 25 October 2021
Accepted: 23 November 2021
Published: 7 December 2021
Accepted: 23 November 2021
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All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
Authors
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.
Blackstone C. Hereditary spastic paraplegia. Handb Clin Neurol 2018;148:633-52.
DOI: https://doi.org/10.1016/B978-0-444-64076-5.00041-7
Zhu Z, Zhang C, Zhao G, Liu Q, Zhong P, Zhang M, et al. Novel mutations in the SPAST gene cause hereditary spastic paraplegia. Parkinsonism Relat Disord 2019;69:125-33.
DOI: https://doi.org/10.1016/j.parkreldis.2019.11.007
Solowska JM, Baas PW. Hereditary spastic paraplegia SPG4: what is known and not known about the disease. Brain 2015;138:2471-84.
DOI: https://doi.org/10.1093/brain/awv178
Claudiani P, Riano E, Errico A, Andolfi G, Rugarli EI. Spastin subcellular localization is regulated through usage of different translation start sites and active export from the nucleus. Exp Cell Res 2005;309:358-69.
DOI: https://doi.org/10.1016/j.yexcr.2005.06.009
Roll-Mecak A, Vale RD. Structural basis of microtubule severing by the hereditary spastic paraplegia protein spastin. Nature 2008;451:363-7.
DOI: https://doi.org/10.1038/nature06482
Solowska JM, D'Rozario M, Jean DC, Davidson MW, Marenda DR, Baas PW. Pathogenic mutation of spastin has gain-of-function effects on microtubule dynamics. J Neurosci 2014;34:1856-67.
DOI: https://doi.org/10.1523/JNEUROSCI.3309-13.2014
Kuo YW, Trottier O, Mahamdeh M, Howard J. Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules. Proc Natl Acad Sci USA 2019;116:5533-41.
DOI: https://doi.org/10.1073/pnas.1818824116
Allison R, Lumb JH, Fassier C, Connell JW, Ten Martin D, Seaman MN, et al. An ESCRT-spastin interaction promotes fission of recycling tubules from the endosome. J Cell Biol 2013;202:527-43.
DOI: https://doi.org/10.1083/jcb.201211045
Connell JW, Allison RJ, Rodger CE, Pearson G, Zlamalova E, Reid E. ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic. Cell Mol Life Sci 2020;77:2641-58.
DOI: https://doi.org/10.1007/s00018-019-03313-z
Chang CL, Weigel AV, Ioannou MS, Pasolli HA, Xu CS, Peale DR, et al. Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III. J Cell Biol 2019;218:2583-99.
Lopes AT, Hausrat TJ, Heisler FF, Gromova KV, Lombino FL, Fischer T, et al. Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits. PLoS Biol 2020;18:e3000820.
DOI: https://doi.org/10.1371/journal.pbio.3000820
Zhang C, Li D, Ma Y, Yan J, Yang B, Li P, et al. Role of spastin and protrudin in neurite outgrowth. J Cell Biochem 2012;113:2296-307.
DOI: https://doi.org/10.1002/jcb.24100
Jiang T, Cai Z, Ji Z, Zou J, Liang Z, Zhang G, et al. The lncRNA MALAT1/miR-30/spastin axis regulates hippocampal neurite outgrowth. Front Cell Neurosci 2020;14:555747.
DOI: https://doi.org/10.3389/fncel.2020.555747
Wood JD, Landers JA, Bingley M, McDermott CJ, Thomas-McArthur V, Gleadall LJ, et al. The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo. Hum Mol Genet 2006;15:2763-71.
DOI: https://doi.org/10.1093/hmg/ddl212
Pisciottani A, Biancolillo L, Ferrara M, Valente D, Sardina F, Monteonofrio L, et al. HIPK2 phosphorylates the microtubule-severing enzyme spastin at S268 for abscission. Cells 2019;8:684.
DOI: https://doi.org/10.3390/cells8070684
Sardina F, Pisciottani A, Ferrara M, Valente D, Casella M, Crescenzi M, et al. Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation. Life Sci Alliance 2020;3:e202000799.
DOI: https://doi.org/10.26508/lsa.202000799
Swatek KN, Komander D. Ubiquitin modifications. Cell Res 2016;26:399-422.
DOI: https://doi.org/10.1038/cr.2016.39
Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu Rev Biochem 2009;78:399-434.
DOI: https://doi.org/10.1146/annurev.biochem.78.101807.093809
Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol 2009;10:319-31.
DOI: https://doi.org/10.1038/nrm2673
Ye Y, Rape M. Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 2009;10:755-64.
DOI: https://doi.org/10.1038/nrm2780
Wang HL, He CY, Chou AH, Yeh TH, Chen YL, Li AH. Polyglutamine-expanded ataxin-7 decreases nuclear translocation of NF-kappaB p65 and impairs NF-kappaB activity by inhibiting proteasome activity of cerebellar neurons. Cell Signal 2007;19:573-81.
DOI: https://doi.org/10.1016/j.cellsig.2006.08.006
Bragoszewski P, Turek M, Chacinska A. Control of mitochondrial biogenesis and function by the ubiquitin - proteasome system. Open Biology 2017;7:13.
DOI: https://doi.org/10.1098/rsob.170007
Geng F, Wenzel S, Tansey WP. Ubiquitin and proteasomes in transcription. Annu Rev Biochem 2012;81:177-201.
DOI: https://doi.org/10.1146/annurev-biochem-052110-120012
Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Anal Bioanal Chem 2010;397:3173-8.
DOI: https://doi.org/10.1007/s00216-010-3821-6
Vandemoortele G, Eyckerman S, Gevaert K. Pick a tag and explore the functions of your pet protein. Trends Biotechnol 2019;37:1078-90.
DOI: https://doi.org/10.1016/j.tibtech.2019.03.016
Zhao X, Li G, Liang S. Several affinity tags commonly used in chromatographic purification. J Anal Methods Chem 2013;2013:581093.
DOI: https://doi.org/10.1155/2013/581093
Hoffmann C, Gaietta G, Bünemann M, Adams SR, Oberdorff-Maass S, Behr B, et al. A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods 2005;2:171-6.
DOI: https://doi.org/10.1038/nmeth742
Zhang JF, Yin YC, Ji ZS, Cai ZB, Zhao B, Li J, et al. Endophilin2 interacts with GluA1 to mediate AMPA receptor endocytosis induced by oligomeric amyloid-beta. Neural Plasticity 2017;2017:8197085.
DOI: https://doi.org/10.1155/2017/8197085
Ji ZS, Zhang GW, Chen L, Li J, Yang YH, Cha CH, et al. Spastin interacts with CRMP5 to promote neurite outgrowth by controlling the microtubule dynamics. Dev Neurobiol 2018;78:1191-205.
Cai ZB, Zhu XN, Zhang GW, Wu FM, Lin HS, Tan MH. Ammonia induces calpain-dependent cleavage of CRMP-2 during neurite degeneration in primary cultured neurons. Aging (Albany NY) 2019;11:4354-66.
DOI: https://doi.org/10.18632/aging.102053
Ji Z, Zhang G, Chen L, Li J, Yang Y, Cha C, et al. Spastin interacts with CRMP5 to promote neurite outgrowth by controlling the microtubule dynamics. Dev Neurobiol 2018;78:1191-205.
DOI: https://doi.org/10.1002/dneu.22640
Chang CL, Weigel AV, Ioannou MS, Pasolli HA, Xu CS, Peale DR, et al. Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III. J Cell Biol 2019;218:2583-99.
DOI: https://doi.org/10.1083/jcb.201902061
Plaud C, Joshi V, Marinello M, Pastre D, Galli T, Curmi PA, et al. Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons. Biochim Biophys Acta Mol Basis Dis 2017;1863:1666-77.
DOI: https://doi.org/10.1016/j.bbadis.2017.04.007
Park SH, Zhu PP, Parker RL, Blackstone C. Hereditary spastic paraplegia proteins REEP1, spastin, and atlastin-1 coordinate microtubule interactions with the tubular ER network. J Clin Invest 2010;120:1097-110.
DOI: https://doi.org/10.1172/JCI40979
Grumati P, Dikic I. Ubiquitin signaling and autophagy. J Biol Chem 2018;293:5404-13.
DOI: https://doi.org/10.1074/jbc.TM117.000117
Landgraf D, Okumus B, Chien P, Baker TA, Paulsson J. Segregation of molecules at cell division reveals native protein localization. Nat Methods 2012;9:480-2.
DOI: https://doi.org/10.1038/nmeth.1955
Majorek KA, Kuhn ML, Chruszcz M, Anderson WF, Minor W. Double trouble-buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci 2014;23:1359-68.
DOI: https://doi.org/10.1002/pro.2520
Song J, Markley JL. Cautionary tail: the presence of an N-terminal tag on dynein light-chain Roadblock/LC7 affects its interaction with a functional partner. Protein Pept Lett 2007;14:265-8.
DOI: https://doi.org/10.2174/092986607780090801
Biancucci M, Dolores JS, Wong J, Grimshaw S, Anderson WF, Satchell KJF, et al. New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag. BMC Biotechnol 2017;17:1.
DOI: https://doi.org/10.1186/s12896-016-0323-4
Einhauer A, Schuster M, Wasserbauer E, Jungbauer A. Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion. Protein Expr Purif 2002;24:497-504.
DOI: https://doi.org/10.1006/prep.2001.1595
How to Cite
Zou, J. ., Cai, Z. ., Liang , Z. ., Liang, Y. ., Zhang, G. ., Yang, J. ., … Tan, M. (2021). Different fusion tags affect the activity of ubiquitin overexpression on spastin protein stability. European Journal of Histochemistry, 65(4). https://doi.org/10.4081/ejh.2021.3352
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