ROS-induced cleavage of NHLRC2 by caspase-8 leads to apoptotic cell death in the HCT116 human colon cancer cell line

Abstract

Excess production of reactive oxygen species (ROS) is known to cause apoptotic cell death. However, the molecular mechanisms whereby ROS induce apoptosis remain elusive. Here we show that the NHL-repeat-containing protein 2 (NHLRC2) thioredoxin-like domain protein is cleaved by caspase-8 in ROS-induced apoptosis in the HCT116 human colon cancer cell line. Treatment of HCT116 cells with the oxidant tert-butyl hydroperoxide (tBHP) induced apoptosis and reduced NHLRC2 protein levels, whereas pretreatment with the antioxidant N-acetyl-l-cysteine prevented apoptosis and the decrease in NHLRC2 protein levels seen in tBHP-treated cells. Furthermore, the ROS-induced decrease in NHLRC2 protein levels was relieved by the caspase inhibitor z-VAD-fmk. We found that the thioredoxin-like domain of NHLRC2 interacted with a proenzyme form of caspase-8, and that caspase-8 cleaved NHLRC2 protein at Asp580 in vitro. Furthermore, siRNA-mediated knockdown of caspase-8 blocked the ROS-induced decrease in NHLRC2 protein levels. Both shRNA and CRISPR-Cas9-mediated loss of NHLRC2 resulted in an increased susceptibility of HCT116 cells to ROS-induced apoptosis. These results suggest that excess ROS production causes a caspase-8-mediated decrease in NHLRC2 protein levels, leading to apoptotic cell death in colon cancer cells, and indicate an important role of NHLRC2 in the regulation of ROS-induced apoptosis.

Structural analysis of human NHLRC2, mutations of which are associated with FINCA disease

“…SEC-MALS analysis of the mixture resulted in two separate peaks with weight-averaged masses of 59 kDa (aa 1–575 fragment) and 15 kDa (aa 587–726 fragment), indicating that the C-terminal domain does not stably interact with the rest of the NHLRC2. This domain has been reported to be cleaved off by caspase-8 leading to apoptotic cell death [ 1 ]. Altogether, these results demonstrate that all three domains of NHLRC2 do not stably associate with one another in solution.…”

“…Previously, NHLRC2 homologs were thought to be limited to animalia [ 1 , 2 ]. However, homologs can be found across different kingdoms of life, including in prokaryotes.…”

“…NHLRC2 (NHL repeat containing protein 2) is an essential cytosolic protein of unknown function [ 1 ], mutation of which was recently identified in association with FINCA ( fi brosis, n eurodegeneration, c erebral a ngiomatosis) disease, a multi-organ condition which is fatal in early childhood [ 2 ]. The most prominent features of the disease were severe interstitial pulmonary fibrosis and early-onset, progressive neurodegeneration.…”

“…While mutations in NHLRC2 were associated with a severe disease state, the physiological function of NHLRC2 has not been previously determined. In addition to having an apparent role in early development, NHLRC2 has been suggested to be a novel serum biomarker for Alzheimer´s disease [ 5 ] and as having an as yet unresolved role in the regulation of ROS-induced apoptosis [ 1 ]. Homozygous deletion of the NHLRC2 gene leads to an embryonic lethality in mice [ 2 , 6 ] and a recently reported mutation in a conserved region of the β-propeller domain NHLRC2 was shown to cause neural tube defects in Angus cattle [ 7 ].…”

Zfat expression in ZsGreen reporter gene knock‑in mice: Implications for a novel function of Zfat in definitive erythropoiesis

“…Cell suspensions were filtered through a 100- μ m cell strainer. Flow cytometric analysis was performed using FACSAria II (BD Biosciences, Franklin Lakes, NJ, USA), as described previously ( 17 , 22 ). ZsGreen was excited with the 488 nm laser and detected using the 530/30 nm emission filter.…”