Withaferin A and Celastrol Overwhelm Proteostasis

Abstract

Withaferin A (WA) and celastrol (CEL) are major bioactive components of plants that have been widely employed in traditional medicine. The pleiotropic activities of plant preparations and the isolated compounds in vitro and in vivo have been documented in hundreds of studies. Both WA and CEL were shown to have anticancer activity. Although WA and CEL belong to different chemical classes, our synthesis of the available information suggests that the compounds share basic mechanisms of action. Both WA and CEL bind covalently to numerous proteins, causing the partial unfolding of some of these proteins and of many bystander proteins. The resulting proteotoxic stress, when excessive, leads to cell death. Both WA and CEL trigger the activation of the unfolded protein response (UPR) which, if the proteotoxic stress persists, results in apoptosis mediated by the PERK/eIF-2/ATF4/CHOP pathway or another UPR-dependent pathway. Other mechanisms of cell death may play contributory or even dominant roles depending on cell type. As shown in a proteomic study with WA, the compounds appear to function largely as electrophilic reactants, indiscriminately modifying reachable nucleophilic amino acid side chains of proteins. However, a remarkable degree of target specificity is imparted by the cellular context.

Keywords: IHSF; Withaferin A; celastrol; protein aggregation; protein unfolding; proteostasis.

Figure 1

Structures of compounds.

Figure 2

Protein unfolding induced by WA, IHSF115, and IHSF058. (a) Inactivation of RLUC in M1 cells (HeLa cells stably transfected with a constitutively expressed RLUC gene) exposed for 6 h to vehicle (−) or compounds at the indicated concentrations. *: p ≤ 0.05 (compared to cells exposed to vehicle). (b) RLUC activities in M1 cells exposed for 6 h to compounds at 25 µM in the absence or presence of 15 mM NAC. *: p < 0.05 (compared to cells exposed to vehicle). #: p < 0.05 (compared to cells treated with a compound in the absence of NAC). (c) Viability of HeLa cells exposed for 96 h to the indicated concentrations of compounds in the presence or absence of 5 mM NAC. Viability was estimated with an alamar blue assay. *: p < 0.05 (compared to cells exposed to vehicle). #: p < 0.05 (compared to cells treated with the same dose of compound in the presence of NAC). (d) Western blots documenting the unfolding of selected proteins in M1 cells exposed for 6 h to compounds at the indicated concentrations. S: detergent-soluble extract fraction; I: detergent-insoluble extract fraction. The data shown were taken from ref. [120].

Figure 3

Selected results from a pathway analysis of proteins unfolded (accumulated in the detergent-insoluble fraction) in M1 cells after exposure to 12.5 µM WA. The underlying data were taken from ref. [120].

Figure 4

Selected results from a pathway analysis of direct CEL targets identified by Zhou et al. [122].

Figure 5

Western blot documenting the unfolding of selected proteins in M1 cells exposed for 6 h to vehicle (−) or CEL at the indicated concentrations. S: detergent-soluble extract fraction; I: detergent-insoluble extract fraction. The data shown were taken from ref. [120].

Figure 6

Correlation of the sensitivities of the indicated cell lines to WA (top graph) or CEL (bottom graph) and to bortezomib. Viabilities were estimated with an alamar blue assay. See the text for further details.