In vivo substrate diversity and preference of small heat shock protein IbpB as revealed by using a genetically incorporated photo-cross-linker

Abstract

Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones found in all forms of life, are known to be able to protect cells against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro conditions. Nevertheless, it is poorly understood what natural substrate proteins are protected by sHSPs in living cells. Here, by using a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we identified a total of 95 and 54 natural substrate proteins of IbpB (an sHSP from Escherichia coli) in living cells with and without heat shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins (e.g. ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic enzymes. Furthermore, these two classes of proteins were found to be more prone to aggregation and/or inactivation in cells lacking IbpB under stress conditions (e.g. heat shock). Together, our in vivo data offer novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential protection on the protein synthesis machine and metabolic enzymes may dominantly contribute to the well known protective effect of sHSPs on cell survival against stresses.

Keywords: Heat Shock Protein; Metabolic Enzymes; Molecular Chaperone; Photocross-linking; Protein Aggregation; Protein Folding; Ribosomes; Small Heat Shock Proteins; Translation; Unnatural Amino Acid.

FIGURE 1.

IbpB preferentially functions under heat shock conditions. A, immunoblotting results of endogenous IbpB in wild-type cells cultured under the indicated stress conditions using the anti-IbpB polyclonal antibody. TnaA was used as a loading control. B, immunoblotting results of in vivo photocross-linked products of Tyr-45 Bpa expressed in ΔibpB cells using the anti-His tag antibody. Cells were grown at 30 °C and then subjected to stress treatment for half an hour by heating at 50 °C or by adding to a final concentration of 5 mm H₂O₂ or 10% ethanol before further analysis.

FIGURE 2.

Identification of IbpB-bound proteins in living cells. SDS-PAGE analysis of the purified in vivo photocross-linked products of the Phe-16 (F16) Bpa, Asn-25 (N25) Bpa, Tyr-45 (Y45) Bpa, Arg-67 (R67) Bpa, and Ala-139 (A139) Bpa variants of IbpB (as visualized by Coomassie Blue staining). The protein bands except for IbpB monomers were subjected to protein identification by LC-MS/MS, with the results shown in supplemental Tables S1–S3. The Phe-16 Bpa protein without in vivo photocross-linking was also purified in parallel (lane 2) and used as a negative control.

FIGURE 3.

Validation of the interactions of representative IbpB-bound proteins with IbpB using multiple approaches. A, immunoblotting results of EF-Tu or TnaA in the in vivo photocross-linked products of the Bpa variants of IbpB (using polyclonal antibodies against EF-Tu or TnaA). B, immunoblotting results of EF-Tu and TnaA that were present in the protein aggregates isolated from wild-type and ΔibpB cells after being heat-shocked at 50 °C for 4 h. C, suppression of the in vitro thermal aggregation of purified TnaA at 50 °C by purified IbpB, as monitored by absorbance spectroscopic analysis at 360 nm. The inset shows the SDS-PAGE analysis results of proteins present in the soluble and pellet fractions after such thermal aggregation. D, SDS-PAGE analysis of the protein aggregates of the wild-type and ΔibpB cells, similarly to what described in B. Protein bands in the aggregates (lanes 3 and 6) were subjected to protein identification by LC-MS/MS, with the results shown in supplemental Table S4. B–D, T, total proteins; S, soluble proteins; P, protein aggregates (pellet).

FIGURE 4.

IbpB protects representative metabolic enzymes from stress-induced inactivation. A, relative levels of catalase activity of the wild-type and ΔibpB cells (from three independent assays). Cells were heat-shocked at 50 °C for 0.5 h and/or treated with 5 mm H₂O₂ for 0.5 h, sonicated, and centrifuged. Cell extracts were then subjected to catalase activity assay as described under “Experimental Procedures.” B, immunoblotting detection of carbonylated proteins in the wild-type and ΔibpB cells after heat shock at 50 °C for 0.5 h and/or treatment with 5 mm H₂O₂ for 0.5 h. Protein bands with a higher level of carbonylation signals in ΔibpB cells than that in wild-type cells are indicated by asterisks. EF-Tu was immunoblotted as a loading control.