title: Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.
abstract: Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.
authors.affiliation: Department of Neuroscience, Santa Fe Health Alzheimer's Disease Research Center, University of Florida, Gainesville, FL, USA. xugl@ufl.edu
authors.forename: Guilian
authors.initials: G
authors.lastname: Xu
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Stevens
authors.forename: Firas
authors.initials: F
authors.lastname: Kobeissy
authors.forename: Firas
authors.initials: F
authors.lastname: Kobiessy
authors.forename: Hilda
authors.initials: H
authors.lastname: Brown
authors.forename: Scott
authors.initials: S
authors.lastname: McClung
authors.forename: Mark S
authors.initials: MS
authors.lastname: Gold
authors.forename: David R
authors.initials: DR
authors.lastname: Borchelt
mesh_terms: D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism / Q000502:physiology; D009474:Neurons / Q000378:metabolism; D025821:Polyubiquitin / Q000378:metabolism; D014176:Protein Biosynthesis; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D054875:Ubiquitination / Q000502:physiology
chemical_list: D006360:Heat-Shock Proteins; D020543:Proteome; D025821:Polyubiquitin