A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation
(A) Heat solubility of the six Hero proteins selected. Each Hero protein, GFP, or GST was fused with a FLAG-tag and expressed in HEK293T cells. The cell lysates were prepared with (+) or without (−) boiling, and then the proteins in the lysates or their supernatants were detected by anti-FLAG western blotting. The result was confirmed by two independent experiments. (B) Hero proteins protect the LDH activity from desiccation. LDH mixed with the indicated Hero protein or controls was dried up overnight and the remaining LDH activities were measured. Note that much higher concentrations of arginine (500-fold) and trehalose (100-fold) were used, compared to those of the proteins. Data were normalized to the activity of LDH incubated on ice overnight and represent means ± SD from 3 independent experiments. (C) Hero proteins protect GFP in organic solvent. GFP mixed with the indicated Hero protein or controls was exposure to chloroform and the remaining GFP fluorescence was measured. Data were normalized to the activity of GFP without the exposure to chloroform and represent means ± SD from 3 independent experiments. (D) Hero proteins protect the luciferase activity from heat shock. Firefly luciferase was transfected into HEK293T cells, together with each Hero protein or chaperone factor, and then the cells were exposed to heat shock. Remaining luciferase activities after heat shock were measured. Data were normalized to the activity of luciferase in cells without the exposure to heat shock, and represent means ± SD from 5 independent experiments. The numerical data pertaining to this figure can be found in S1 Data file. BSA, bovine serum albumin; GFP, green fluorescent protein; GST, glutathione S-transferase; Hero, heat-resistant obscure; Hsp, heat shock protein; LDH, lactate dehydrogenase.