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.

Vulnerability of newly synthesized proteins to proteostasis stress

“…Given that the HEK293 cell is a workhorse cell line in these types of studies, we sought to identify proteins in HEK293 cells that might be vulnerable to proteostatic stress. Following protocols established in our previous study of human SH-SY5Y and CCF-STTG1 cells (Xu et al, 2012), we conducted a liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of proteins in HEK293 cells that lose solubility after thermal stress. For comparison, as an alternative means to produce proteostatic stress, cells were also treated with MG132 to inhibit the proteasome, causing an accumulation of misfolded proteins that would normally have been degraded (for a review, see Balch et al, 2008).…”

“…For comparison, as an alternative means to produce proteostatic stress, cells were also treated with MG132 to inhibit the proteasome, causing an accumulation of misfolded proteins that would normally have been degraded (for a review, see Balch et al, 2008). Sequential detergent extraction was used to separate proteins by solubility in phosphate-buffered saline (PBS), then Nonidet P-40 (NP40), then sodium deoxycholate (DOC), with a final extraction of the DOCinsoluble fraction in SDS (Xu et al, 2012). This sequential approach of fractionation was used to identify the detergent that provided the best separation of soluble and insoluble proteins.…”

“…Among the proteins identified as sensitive to thermal proteostatic stress were several that emerged as possible biomarkers that consistently lose solubility, including ubiquitin, CDK1, FEN1 and TDP-43 (also known as TARDBP). In cells held at 37°C, these proteins are predominantly, sometimes exclusively, found in PBS soluble fractions (Xu et al, 2012), whereas in the heat-stressed cells these proteins are detected in detergent-insoluble fractions. In the present study, we used human HEK293 cell models to compare the expression of mutant huntingtin (Q103) as a proteostasis stressor to thermal stress.…”

“…In a previously published proteomic analysis of two human neural cell lines, we embarked on an effort to identify human proteins that might be vulnerable to proteostatic stress (Xu et al, 2012). Using a thermal stress paradigm in two independently derived human cell lines, we identified ∼30 cytosolic and nuclear proteins that lose solubility upon heat shock (Xu et al, 2012), indicating that vulnerability to proteostatic stress is not restricted to rare-variant proteins but applicable to a larger population of normal proteins.…”

“…Using a thermal stress paradigm in two independently derived human cell lines, we identified ∼30 cytosolic and nuclear proteins that lose solubility upon heat shock (Xu et al, 2012), indicating that vulnerability to proteostatic stress is not restricted to rare-variant proteins but applicable to a larger population of normal proteins. Among the proteins identified as sensitive to thermal proteostatic stress were several that emerged as possible biomarkers that consistently lose solubility, including ubiquitin, CDK1, FEN1 and TDP-43 (also known as TARDBP).…”