A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation
(A) Schematic representation of FLAG-TEV-SNAP-Ago2 immunopurified on magnetic beads. (B) FLAG-TEV-SNAP-Ago2 was immunopurified onto magnetics beads via anti-FLAG antibody, and then the FLAG-tag was cleaved off by TEV protease in buffer, crude lysates, or their boiled supernatants from fly S2 or human HEK293T cells. PK indicates that the boiled supernatant had been mostly deproteinized by proteinase K in advance. BZ indicates that DNA and RNA in boiled supernatant had been degraded by benzonase. BZ/PK indicates that DNA and RNA had been degraded by benzonase, followed by the deproteinization by proteinase K. Eluted Ago2 (top) and Ago2 still remaining on the beads (bottom) were visualized by a red fluorescent dye covalently attached to the SNAP tag. The values on the top indicate relative amounts of eluted Ago2 normalized to that with S2 crude cell lysate. Proteins remaining in the boiled supernatants have an activity to promote Ago2 elution. (C) Small RNA pull-down assay for pre-Ago2-RISC and mature Ago2-RISC assembled in the reconstitution system, containing a 32P-radiolabeled small RNA duplex, Ago2, Dicer-2/R2D2, and the Hsp70/Hsp90 chaperone machinery. Supplementation of the boiled supernatants from S2 or HEK293T cells promoted the formation of both pre- and mature Ago2-RISC. (D) Quantification of (C). Data represent means ± SD from 3 independent experiments. (E) Heat-soluble proteins in the boiled supernatants protect the LDH activity from desiccation. LDH mixed with the indicated boiled supernatants or buffer was dried up overnight, and the remaining LDH activities were measured. Data represent means ± SD from 3 independent experiments. The numerical data pertaining to this figure can be found in S1 Data file. Ago, Argonaute; GST, glutathione S-transferase; Hsp, heat shock protein; LDH, lactate dehydrogenase; RISC, RNA-induced silencing complex; TEV, tobacco etch virus.