Receptor-Mediated Mitophagy Rescues Cancer Cells under Hypoxic Conditions.

Targeting mitochondria with thenoyltrifluoroacetone (TTFA), an inhibitor of Complex II in the respiratory chain, stimulated cisplatin-induced apoptosis in various cell lines in normoxia but not in hypoxia. This can be explained by the elimination of mitochondria involved in triggering apoptotic cell death by mitophagy, either Parkin-dependent or receptor-mediated. Treatment with TTFA alone or in combination with cisplatin did not cause accumulation of PINK1, meaning that under hypoxic conditions cells survive through activation of a receptor-mediated pathway. Hypoxia triggers the accumulation of BNIP3 and BNIP3L (also known as NIX), key participants in receptor-mediated mitophagy. Under hypoxic conditions, stimulation of autophagy, as assessed by the accumulation of lipidated form of LC3 (LC3II), was observed. To exclude the contribution of canonical macroautophagy in LC3II accumulation, experiments were performed using U1810 cells lacking ATG13, a key enzyme of macroautophagy. Despite the absence of ATG13, hypoxia-mediated accumulation of LC3II was not affected, underlying the importance of the receptor-mediated pathway. In order to prove the protective role of BNIP3 against cisplatin-induced apoptosis, BNIP3-deficient A549 cells were used. Surprisingly, a BNIP3 knockout did not abolish hypoxia-induced protection; however, in cells lacking BNIP3, a compensatory upregulation of BNIP3L was detected. Thus, in the absence of BNIP3, mitophagy could be maintained by BNIP3L and lead to cell death suppression due to the elimination of proapoptotic mitochondria. When both BNIP3 and BNIP3L were knocked out, the inhibitory effect of hypoxia on apoptosis was diminished, although not abolished completely. Undoubtedly, receptor-mediated mitophagy is likely to be one of the mechanisms responsible for cell death suppression under hypoxic conditions.

Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module.

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena sp. PCC 7120 (termed AnaCTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples. We characterize the spectroscopic properties of the obtained pigment-protein complexes and the thermodynamics of liposome-protein carotenoid transfer and demonstrate the delivery of carotenoid echinenone from AnaCTDH into liposomes with an efficiency of up to 70 ± 3%. Most importantly, we show efficient carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species (ROS). Incubation of neuroblastoma cell line Tet21N in the presence of 1 µM AnaCTDH binding echinenone decreased antimycin A ROS production by 25% (p < 0.05). The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

For cells to survive they need to be able to remove faulty or damaged components. The ability to recycle faulty parts is so crucial that some of the molecular machinery responsible is the same across the plant and animal kingdoms. One of the major recycling pathways cells use is autophagy, which labels damaged proteins with molecular tags that say 'eat-me'. Proteins called receptors then recognize these tags and move the faulty component into vesicles that transport the cargo to a specialized compartment that recycles broken parts. Cells make and fold around 40% of their proteins at a site called the endoplasmic reticulum, or ER for short. However, the process of folding and synthesizing proteins is prone to errors. For example, when a cell is under stress this can cause a 'stall' in production, creating a build-up of faulty, partially constructed proteins that are toxic to the cell. There are several quality control systems which help recognize and correct these errors in production. Yet, it remained unclear how autophagy and these quality control mechanisms are linked together. Here, Stephani, Picchianti et al. screened for receptors that regulate the recycling of faulty proteins by binding to the 'eat-me' tags. This led to the identification of a protein called C53, which is found in both plant and animal cells. Microscopy and protein-protein interaction tests showed that C53 moves into transport vesicles when the ER is under stress and faulty proteins start to build-up. Once there, C53 interacts with two proteins embedded in the wall of the endoplasmic reticulum. These proteins form part of the quality control system that senses stalled protein production, labelling the stuck proteins with 'eat-me' tags. Together with C53, they identify and remove half-finished proteins before they can harm the cell. The fact that C53 works in the same way in both plant and human cells suggests that many species might use this receptor to recycle stalled proteins. This has implications for a wide range of research areas, from agriculture to human health. A better understanding of C53 could be beneficial for developing stress-resilient crops. It could also aid research into human diseases, such as cancer and viral infections, that have been linked to C53 and its associated proteins.

To Eat or to Die: Deciphering Selective Forms of Autophagy.

Autophagy is an evolutionarily conserved process whereby damaged and redundant components of the cell are degraded in structures called autophagolysosomes. Currently, three main types of autophagy are recognized: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). However, we still know little about some specific types of autophagy that are linked to various intracellular compartments and their roles in the physiology of the whole organism and connections to various diseases. Here, we aim to shed light on the latest insights on and mechanisms of several selective forms of autophagy.

Distinct effects of etoposide on glutamine-addicted neuroblastoma.

The majority of anticancer drugs are DNA-damaging agents, and whether or not they may directly target mitochondria remains unclear. In addition, tumors such as neuroblastoma exhibit addiction to glutamine in spite of it being a nonessential amino acid. Our aim was to evaluate the direct effect of widely used anticancer drugs on mitochondrial activity in combination with glutamine withdrawal, and possible apoptotic effects of such interaction. Our results revealed that etoposide inhibits mitochondrial respiratory chain Complex I causing the leakage of electrons and the superoxide radical formation. However, it was not sufficient to induce apoptosis, and apoptotic manifestation was detectable only alongside the withdrawal of glutamine, a precursor for antioxidant glutathione. Thus, the simultaneous depletion of glutathione and destabilization of mitochondria by ROS can compromise the barrier properties of the mitochondrial membrane, leading to cytochrome c release and the activation of the mitochondrial apoptotic pathway. Thus, the depletion of antioxidants or the inhibition of the pathways responsible for cellular antioxidant response can enhance mitochondrial targeting and strengthen antitumor therapy.

Involvement of mitophagy in cisplatin-induced cell death regulation.

Mitophagy, the selective degradation of mitochondria via the autophagic pathway, is a vital mechanism of mitochondrial quality control in cells. The removal of malfunctioning or damaged mitochondria is essential for normal cellular physiology and tissue development. Stimulation of mitochondrial permeabilization and release of proapoptotic factors from the intermembrane space is an essential step in triggering the mitochondrial pathway of cell death. In this study, we analyzed the extent to which mitophagy interferes with cell death, attenuating the efficiency of cancer therapy. We show that stimulation of mitophagy suppressed cisplatin-induced apoptosis, while mitophagy inhibition stimulates apoptosis and autophagy. Suppression of mitophagy involved production of reactive oxygen species, and the fate of cell was dependent on the interplay between endoplasmic reticulum stress and autophagy.

2-Deoxy-D-glucose has distinct and cell line-specific effects on the survival of different cancer cells upon antitumor drug treatment.

The dependence of tumors on glycolysis for ATP generation offers a rationale for therapeutic strategies aimed at selective inhibition of the glycolytic pathway. Analysis of tumor cell responses to anticancer drugs revealed that inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) generally augmented the apoptotic response; however, in HCT116 human colon carcinoma cells, apoptosis was suppressed. A comparison of neuroblastoma SK-N-BE(2) and HCT116 cells revealed, that in contrast to HCT116, in SK-N-BE(2) cells 2-DG alone was able to induce cell death. In SK-N-BE(2) cells the decrease in ATP levels upon treatment with 2-DG was more prominent because in HCT116 cells mitochondria compensated for the loss of ATP caused by glycolysis suppression. In both cells lines 2-DG triggered endoplasmic reticulum (ER) stress, assessed by the accumulation of the marker protein GRP78/BiP. Suppression of ER stress by mannose attenuated the 2-DG-induced apoptotic response in SK-N-BE(2) cells, implying that apoptosis in these cells is a consequence of ER stress induction. In HCT116 cells, ER stress stimulated autophagy, assessed by the accumulation of the lipidated form of LC3. The inhibitor of ER stress mannose attenuated autophagy and reversed 2-DG-mediated suppression of cisplatin-induced apoptosis. When autophagy in HCT116 cells was suppressed by bafilomycin, cisplatin-induced apoptosis was decreased. At the same time, stimulation of autophagy in SK-N-BE(2) cells suppressed cell death. Thus, successful treatment of tumors with conventionally used anticancer drugs should be combined with targeting metabolic pathways involved in the regulation of apoptosis, autophagy, and cellular bioenergetics.