bims-ershed 2022-09-11 papers

Methods Mol Biol. 2022 ;2543 155-166

Cell Cycle Analysis of ER Stress and Autophagy.

Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.

Keywords: Autophagy; ER stress; Misfolded proteins; PERK; Reticulophagy

DOI: https://doi.org/10.1007/978-1-0716-2553-8_13

Front Cell Dev Biol. 2022 ;10 915065

BOK controls ER proteostasis and physiological ER stress responses in neurons.

Franziska Walter, Beatrice D'Orsi, Anagha Jagannathan, Heiko Dussmann, Jochen H M Prehn.

The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.

Keywords: Bcl-2 family; ER stress; ER stress reporters; calcium signaling; live-cell imaging; proteostasis; unfolded protein response

DOI: https://doi.org/10.3389/fcell.2022.915065

Hum Mol Genet. 2022 Sep 06. pii: ddac218. [Epub ahead of print]

Prdx5 regulates DNA damage response through autophagy-dependent Sirt2-p53 axis.

Ewud Agborbesong, Julie X Zhou, Linda X Li, Peter C Harris, James P Calvet, Xiaogang Li.

DNA damage response (DDR) is an important signaling-transduction network that promotes the repair of DNA lesions which can induce and/or support diseases. However, the mechanisms involved in its regulation are not fully understood. Recent, studies suggest that the peroxiredoxin 5 (Prdx5) enzyme, which detoxifies reactive oxygen species is associated to genomic instability and signal transduction. Its role in the regulation of DDR, however, is not well characterized. In this study, we demonstrate a role of Prdx5 in the regulation of the DDR signaling pathway. Knockdown of Prdx5 resulted in DNA damage manifested by the induction of phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1). We show that Prdx5 regulates DDR through: 1) polo-like kinase 1 (Plk1) mediated phosphorylation of ataxia telangiectasia mutated (ATM) kinase to further trigger downstream mediators Chek1 and Chek2; 2) the increase of the acetylation of p53 at lysine 382, stabilizing p53 in the nucleus and enhancing transcription; 3) the induction of autophagy, which regulates the recycling of molecules involved in DDR. We identified Sirt2 as a novel deacetylase of p53 at lysine 382, and Sirt2 regulated the acetylation status of p53 at lysine 382 in a Prdx5-dependent manner. Furthermore, we found that exogenous expression of Prdx5 decreased DNA damage and the activation of ATM in Pkd1 mutant renal epithelial cells, suggesting that Prdx5 may play a protective role from DNA damage in cystic renal epithelial cells. This study identified a novel mechanism of Prdx5 in the regulation of DDR through the ATM/p53/Sirt2 signaling cascade.

DOI: https://doi.org/10.1093/hmg/ddac218