315 - CRISPR-Cas9 knockout of lanthionine synthase-like protein-1 (LanCL1) in HeLa cells renders the cells hypersensitive to oxidative stress despite inducing an upregulation of glutathione-dependent antioxidant defense mechanisms

Abstract

Lanthionine cyclase-like protein-1 (LanCL1) is a glutathione-binding Zn2+ metalloprotein present ubiquitously in mammalian cells and expressed heavily in brain and kidney. LanCl1 function has not been exactly elucidated. Recent reports contend that LanCL1 genetic ablation induced age-related neurodegeneration in mice. Some evidence has been published that LanCL1 has glutathione-S-transferase (GST)-like antioxidant activity, while other recent claims have been made that LanCL1 suppresses phospho-activation of c-Jun amino-terminal kinase (JNK) and other mitogen-activated kinase (MAPK) cascades; but none of these prior studies has been independently replicated, to date. In this study, CRISPR-Cas9 technology was used to completely and stably ablate LanCL1 from HeLa cells and the cells were then phenotyped with respect to their behavior in the face of an oxidative challenge. LanCL1-KO cells appeared grossly normal but were hypersensitive to H2O2 toxicity (LD50% = 171 μM for wild-type vs 87 μM for LanCL1-KO cells). Cellular [GSH], [GSSG], and [GSH]/[GSH] were unaltered by LanCL1 knockout; however, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were significantly elevated and GST activity was non-significantly elevated in LanCL1 KO cultures. LanCL1 KO cells showed no alterations in basal or H2O2 –induced phosphorylation of p38-MAPK, ERK p42/p44 or JNK. There was, however, a significant 52% decrease in total c-Jun and a 28% decrease in NFKB proteins associated with LanCL1-KO. Taken together these data suggest that LanCL1 likely is not a GST-like antioxidant enzyme itself, but works to regulate redox-sensitive transcription factor activation downstream from classic MAPK cascades. The data emphasize that LanCL1 expression is vital to maintenance of cell homeostasis in the face of a potentially toxic redox challenge, and underscore the importance of elucidating the exact function of this novel glutathione-binding protein. Research supported in part by National Institutes of Health (NS093594; NS082283) and the Degen Foundation.