The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis

Abstract

Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.

Chlorpromazine induces cytotoxic autophagy in glioblastoma cells via endoplasmic reticulum stress and unfolded protein response

“…Among these ATP- or GTP-binding proteins, we focused our attention on the following: ER chaperone BiP [ 38 – 40 ]; endoplasmin [ 41 , 42 ]; heat shock protein HSP 90-beta [ 43 ]; members of T-complex protein 1 (TRiC) [ 44 , 45 ], i.e. TCP1, CCT5 and CCT6; heat shock protein 75 kDa [ 46 ]; EF1-α1 [ 47 ]; EF2 [ 48 ], substrate of eEF2K [ 49 , 50 ]; and TER ATPase [ 51 ].…”

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines

“…Recently, Jin X et al, considering the interaction of PHBs with proteins that participate in the regulation of translation, analyzed the effect of different PHB-binding compounds on the translation machinery [ 64 ]. These authors found a reduction in new protein synthesis upon fluorizoline treatment in human cell lines, caused by inhibition of eIF2α and eEF2.…”

Chlorpromazine Induces Cytotoxic Autophagy in Glioblastoma Cells via Endoplasmic Reticulum Stress and Unfolded Protein Response

“…Among these ATP-or GTP-binding proteins, we focused our attention on the following: ER chaperone BiP [38][39][40]; endoplasmin [41,42]; heat shock protein HSP 90-beta [43]; members of T-complex protein 1 (TRiC) [44,45], i.e. TCP1, CCT5 and CCT6; heat shock protein 75 kDa [46]; EF1-α1 [47]; EF2 [48], substrate of eEF2K [49,50]; and TER ATPase [51].…”