{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:17:11Z","timestamp":1760242631857,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2017,12,8]],"date-time":"2017-12-08T00:00:00Z","timestamp":1512691200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003329","name":"Ministerio de Econom\u00eda y Competitividad","doi-asserted-by":"publisher","award":["SAF-2012-34556"],"award-info":[{"award-number":["SAF-2012-34556"]}],"id":[{"id":"10.13039\/501100003329","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100015496","name":"CIBERNED","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100015496","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMN\u03947) by alternative splicing. To study SMN and SMN\u03947 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMN\u03947 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMN\u03947 in the cell.<\/jats:p>","DOI":"10.3390\/ijms18122667","type":"journal-article","created":{"date-parts":[[2017,12,8]],"date-time":"2017-12-08T11:37:40Z","timestamp":1512733060000},"page":"2667","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMN\u03947 Proteins Are Degraded by the Proteasome Pathway"],"prefix":"10.3390","volume":"18","author":[{"given":"Ra\u00fal","family":"S\u00e1nchez-Lanzas","sequence":"first","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d (UAM-CSIC), Centro de Investigaci\u00f3n Biom\u00e9dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina de la Universidad Aut\u00f3noma de Madrid (UAM), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6579-8442","authenticated-orcid":false,"given":"Jos\u00e9 G.","family":"Casta\u00f1o","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica, Instituto de Investigaciones Biom\u00e9dicas \u201cAlberto Sols\u201d (UAM-CSIC), Centro de Investigaci\u00f3n Biom\u00e9dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Facultad de Medicina de la Universidad Aut\u00f3noma de Madrid (UAM), 28029 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2017,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"974","DOI":"10.1177\/0883073807305670","article-title":"Modern management of spinal muscular atrophy","volume":"22","author":"Iannaccone","year":"2007","journal-title":"J. Child Neurol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1038\/nrn2670","article-title":"Spinal muscular atrophy: Why do low levels of survival motor neuron protein make motor neurons sick?","volume":"10","author":"Burghes","year":"2009","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1177","DOI":"10.1093\/hmg\/8.7.1177","article-title":"A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2","volume":"8","author":"Monani","year":"1999","journal-title":"Hum. Mol. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1016\/j.neuint.2004.04.005","article-title":"Degradation of survival motor neuron (SMN) protein is mediated via the ubiquitin\/proteasome pathway","volume":"45","author":"Chang","year":"2004","journal-title":"Neurochem. Int."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.1128\/MCB.01262-08","article-title":"Regulation of SMN protein stability","volume":"29","author":"Burnett","year":"2009","journal-title":"Mol. Cell. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1101\/gad.1884910","article-title":"A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity","volume":"24","author":"Cho","year":"2010","journal-title":"Genes Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.1016\/j.cca.2010.07.035","article-title":"Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) regulates the level of SMN expression through ubiquitination in primary spinal muscular atrophy fibroblasts","volume":"411","author":"Hsu","year":"2010","journal-title":"Clin. Chim. Acta"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1083\/jcb.200805092","article-title":"Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes","volume":"183","author":"Boulon","year":"2008","journal-title":"J. Cell Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"43741","DOI":"10.1074\/jbc.M112.372318","article-title":"Ubiquitin Specific Protease 9X deubiquitinates and stabilizes the spinal muscular atrophy protein-survival motor neuron","volume":"287","author":"Han","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1863","DOI":"10.1091\/mbc.e13-01-0042","article-title":"The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein","volume":"24","author":"Kwon","year":"2013","journal-title":"Mol. Biol. Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.1093\/hmg\/ddw021","article-title":"Monoubiquitination of survival motor neuron regulates its cellular localization and Cajal body integrity","volume":"25","author":"Han","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Alvarez-Castelao, B., Ruiz-Rivas, C., and Castano, J.G. (2012). A critical appraisal of quantitative studies of protein degradation in the framework of cellular proteostasis. Biochem. Res. Int., 2012.","DOI":"10.1155\/2012\/823597"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.molcel.2011.08.025","article-title":"Systematic and quantitative assessment of the ubiquitin-modified proteome","volume":"44","author":"Kim","year":"2011","journal-title":"Mol. Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1074\/mcp.M111.016857","article-title":"Methods for quantification of in vivo changes in protein ubiquitination following proteasome and deubiquitinase inhibition","volume":"11","author":"Udeshi","year":"2012","journal-title":"Mol. Cell Proteom."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1038\/nature10098","article-title":"Global quantification of mammalian gene expression control","volume":"473","author":"Schwanhausser","year":"2011","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5275","DOI":"10.1021\/pr101183k","article-title":"Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover","volume":"10","author":"Cambridge","year":"2011","journal-title":"J. Proteome Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3028","DOI":"10.1016\/j.celrep.2016.07.088","article-title":"FOXO\/DAF-16 activation slows down turnover of the majority of proteins in C. elegans","volume":"16","author":"Dhondt","year":"2016","journal-title":"Cell Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1081","DOI":"10.1016\/0309-1651(80)90045-4","article-title":"Kinetics of degradation of \u201cshort-\u201d and \u201clong-lived\u201d proteins in cultured mammalian cells","volume":"4","author":"Wheatley","year":"1980","journal-title":"Cell Biol. Int. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"770","DOI":"10.1038\/35008096","article-title":"Rapid degradation of a large fraction of newly synthesized proteins by proteasomes","volume":"404","author":"Schubert","year":"2000","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"E1591","DOI":"10.1073\/pnas.1321902111","article-title":"The nature and extent of contributions by defective ribosome products to the HLA peptidome","volume":"111","author":"Bourdetsky","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1126\/science.aaf4384","article-title":"A large fraction of HLA class I ligands are proteasome-generated spliced peptides","volume":"354","author":"Liepe","year":"2016","journal-title":"Science"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"6140","DOI":"10.1128\/MCB.24.14.6140-6150.2004","article-title":"N-Terminal ubiquitination of extracellular signal-regulated kinase 3 and p21 directs their degradation by the proteasome","volume":"24","author":"Coulombe","year":"2004","journal-title":"Mol. Cell. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1862","DOI":"10.1101\/gad.1213904","article-title":"N-terminal polyubiquitination and degradation of the Arf tumor suppressor","volume":"18","author":"Kuo","year":"2004","journal-title":"Genes Dev."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"41414","DOI":"10.1074\/jbc.M407201200","article-title":"The tumor suppressor protein p16INK4a and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue","volume":"279","author":"Fajerman","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"32614","DOI":"10.1074\/jbc.M403794200","article-title":"Ubiquitin-Proteasome-mediated degradation of Id1 is modulated by MyoD","volume":"279","author":"Lingbeck","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"15690","DOI":"10.1073\/pnas.0808373105","article-title":"The N-terminal domain of MyoD is necessary and sufficient for its nuclear localization-dependent degradation by the ubiquitin system","volume":"105","author":"Sadeh","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"40192","DOI":"10.1074\/jbc.M110.131615","article-title":"Ubiquitin proteasome-dependent degradation of the transcriptional coactivator PGC-1\u03b1 via the N-terminal pathway","volume":"285","author":"Leone","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"18784","DOI":"10.1074\/jbc.C113.477596","article-title":"The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N-terminus of substrates","volume":"288","author":"Scaglione","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1833","DOI":"10.1016\/j.biocel.2013.05.026","article-title":"Non-canonical ubiquitylation: Mechanisms and consequences","volume":"45","author":"McDowell","year":"2013","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"12449","DOI":"10.1073\/pnas.1210303109","article-title":"N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB","volume":"109","author":"Lasa","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1126\/science.1183147","article-title":"N-terminal acetylation of cellular proteins creates specific degradation signals","volume":"327","author":"Hwang","year":"2010","journal-title":"Science"},{"key":"ref_32","first-page":"153","article-title":"Ubiquitin-independent degradation of proteins by the proteasome","volume":"1786","author":"Bossis","year":"2008","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/j.bbamcr.2013.05.008","article-title":"Ubiquitin-independent proteasomal degradation","volume":"1843","author":"Erales","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1140","DOI":"10.3390\/biom4041140","article-title":"Proteins directly interacting with mammalian 20S proteasomal subunits and ubiquitin-independent proteasomal degradation","volume":"4","author":"Castano","year":"2014","journal-title":"Biomolecules"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"862","DOI":"10.3390\/biom4030862","article-title":"Regulating the 20S proteasome ubiquitin-independent degradation pathway","volume":"4","author":"Sharon","year":"2014","journal-title":"Biomolecules"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"546","DOI":"10.1002\/wrna.76","article-title":"SMN in spinal muscular atrophy and snRNP biogenesis","volume":"2","author":"Coady","year":"2011","journal-title":"Wiley Interdiscip. Rev. RNA"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.bbagrm.2016.12.008","article-title":"Diverse role of survival motor neuron protein","volume":"1860","author":"Singh","year":"2017","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1038\/sj.embor.7400941","article-title":"Chaperoning ribonucleoprotein biogenesis in health and disease","volume":"8","author":"Pellizzoni","year":"2007","journal-title":"EMBO Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1038\/nature19947","article-title":"Proteome complexity and the forces that drive proteome imbalance","volume":"537","author":"Harper","year":"2016","journal-title":"Nature"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"8691","DOI":"10.1523\/JNEUROSCI.0417-15.2015","article-title":"Disease mechanisms and therapeutic approaches in spinal muscular atrophy","volume":"35","author":"Tisdale","year":"2015","journal-title":"J. Neurosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. 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