bims-lycede 2024-12-15 papers

STAR Protoc. 2024 Dec 10. pii: S2666-1667(24)00648-8. [Epub ahead of print]5(4): 103483

Protocol for purifying and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes.

Felichi Mae Arines, Aleksander Wielenga, Randy B Stockbridge, Ming Li.

Studying the biochemical function of membrane transporters is important in understanding the biology of transporter-laden organelles such as lysosomes and vacuoles. We present a protocol for overexpressing, purifying, and reconstituting a vacuole membrane transporter Ypq1 into proteoliposomes and describe steps to measure transport activity using radioactive substrates. The protocols established here can be used to study other vacuolar or lysosomal membrane transporters. For complete details on the use and execution of this protocol, please refer to Arines et al.1.

Keywords: Cell Membrane; Protein Biochemistry; Protein expression and purification

DOI: https://doi.org/10.1016/j.xpro.2024.103483

Autophagy. 2024 Dec 11.

Lysosomal damage due to cholesterol accumulation triggers immunogenic cell death.

Karla Alvarez-Valadez, Allan Sauvat, Julien Diharce, Marion Leduc, Gautier Stoll, Lionel Guittat, Flavia Lambertucci, Juliette Paillet, Omar Motino, Lucille Ferret, Alexandra Muller, Sabrina Forveille, Maria Chiara Maiuri, Oliver Kepp, Alexandre G de Brevern, Harald Wodrich, Jonathan G Pol, Guido Kroemer, Mojgan Djavaheri-Mergny.

Cholesterol serves as a vital lipid that regulates numerous physiological processes. Nonetheless, its role in regulating cell death processes remains incompletely understood. In this study, we investigated the role of cholesterol trafficking in immunogenic cell death. Through cell-based drug screening, we identified two antidepressants, sertraline and indatraline, as potent inducers of the nuclear translocation of TFEB (transcription factor EB). Activation of TFEB was mediated through the autophagy-independent lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3). Both compounds promoted cholesterol accumulation within lysosomes, resulting in lysosomal membrane permeabilization, disruption of autophagy and cell death that could be reversed by cholesterol depletion. Molecular docking analysis indicated that sertraline and indatraline have the potential to inhibit cholesterol binding to the lysosomal cholesterol transporters, NPC1 (NPC intracellular cholesterol transporter 1) and NPC2. This inhibitory effect might be further enhanced by the upregulation of NPC1 and NPC2 expression by TFEB. Both antidepressants also upregulated PLA2G15 (phospholipase A2 group XV), an enzyme that elevates lysosomal cholesterol. In cancer cells, sertraline and indatraline elicited immunogenic cell death, converting dying cells into prophylactic vaccines that were able to confer protection against tumor growth in mice. In a therapeutic setting, a single dose of each compound was sufficient to significantly reduce the outgrowth of established tumors in a T-cell-dependent manner. These results identify sertraline and indatraline as immunostimulatory agents for cancer treatment. More generally, this research shed light on novel therapeutic avenues harnessing lysosomal cholesterol transport to regulate immunogenic cell death.

Keywords: Autophagy; NPC intracellular cholesterol transporter 1 and 2; TFEB; cancer; lipid transport; lysosomal membrane permeabilization

DOI: https://doi.org/10.1080/15548627.2024.2440842

Biochem Pharmacol. 2024 Dec 04. pii: S0006-2952(24)00695-6. [Epub ahead of print]232 116694

The TFEB activator clomiphene citrate ameliorates lipid metabolic syndrome pathology by activating lipophagy and lipolysis.

Lu Li, Jieru Lin, Chunhuan Huang, Jiamiao Liu, Yi Yuan, Zhenxing Liu, Yuyin Li, Wei Li, Aipo Diao.

The balance between lipid synthesis and lipid catabolism is critical to maintain energy homeostasis. Lipophagy and lipolysis are two important pathways for lipid selective catabolism. Defects in lipophagy and lipolysis are linked to lipid metabolic diseases. Transcription factor EB (TFEB) is a master regulator of autophagy and lysosome biogenesis, as well as lipid metabolism by promoting expression of genes encoding fat catabolic lipases. Therefore, targeting TFEB provides a novel potential strategy for the treatment of lipid metabolic diseases. In this study, we showed that the TFEB activator clomiphene citrate (CC) activated the autophagy-lysosome and lipolysis pathways, and promoted degradation of lipid droplets induced by the free fatty acids oleate and palmitate in HepG2 cells. Moreover, CC treatment promoted lipid catabolism and attenuated obesity, restored lipid levels, blood glucose levels and insulin resistance, hepatocellular injury and hepatic steatosis, as well as liver inflammation in the HFD fed mice. In addition, we found that En-CC, a trans-isomer of CC, displayed less toxicity and more efficient activation of TFEB. Consistent with CC, En-CC treatment improved lipid metabolic syndrome pathology. These findings demonstrate that CC promotes clearance of lipids and ameliorates HFD-induced lipid metabolic syndrome pathology through activating TFEB-mediated lipophagy and lipolysis, indicating that CC has the potential to be used to treat lipid metabolic diseases.

Keywords: Autophagy; Clomiphene citrate; Hepatic steatosis; Lipolysis; Obesity; TFEB

DOI: https://doi.org/10.1016/j.bcp.2024.116694

Biochem Biophys Res Commun. 2024 Dec 02. pii: S0006-291X(24)01638-3. [Epub ahead of print]742 151102

ESCRT mediates micronucleophagy and macronucleophagy in yeast.

Most Naoshia Tasnin, Yuka Takahashi, Tsuneyuki Takuma, Takashi Ushimaru.

Endosomal sorting complex required for transport (ESCRT) is required for maintenance of nuclear functions and prevention of neurodegenerative diseases. The budding yeast Saccharomyces cerevisiae is an ideal model for studying ESCRT-dependent diseases. Nucleolar proteins are degraded by macronucleophagy and micronucleophagy after nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) kinase. Here, we show that ESCRT is critical for micronucleophagic degradation of nucleolar proteins upon TORC1 inactivation. In addition, ESCRT was also critical for rDNA condensation and nucleolar remodeling, which is necessary for proper micronucleophagic degradation of nucleolar proteins after TORC1 inactivation. On the other hand, ESCRT was dispensable for bulk macroautophagy, whereas it was also critical for macronucleophagy. Thus, ESCRT has an important role for elimination of nucleolar proteins in response to nutrient deprivation.

Keywords: Autophagy; ESCRT; Micronucleophagy; Nucleophagy; TORC1; macronucleophagy

DOI: https://doi.org/10.1016/j.bbrc.2024.151102

Trends Cell Biol. 2024 Dec 10. pii: S0962-8924(24)00230-7. [Epub ahead of print]

Death by ribosome.

Anna Constance Vind, Franklin L Zhong, Simon Bekker-Jensen.

Next to their essential role as protein production factories, ribosomes serve as molecular sensors of cell stress. Stalled and collided ribosomes trigger specific stress signaling, including the ribotoxic stress response (RSR). The RSR is initiated by the mitogen-activated protein (MAP)-3 kinase ZAKα in response to a plethora of translational aberrations, leading to activation of the stress-activated MAP kinases p38 and jun N-terminal kinase (JNK). Recent insights have highlighted an important role for the RSR pathway in triggering programmed cell death processes, including apoptosis and pyroptosis, in a broad range of physiologically relevant conditions. In this review, we summarize recent work on known links between programmed and accidental ribosome toxicity, RSR signaling, and cell death.

Keywords: ZAKα; apoptosis; pyroptosis; ribotoxic stress response; self-inflicted ribotoxicity

DOI: https://doi.org/10.1016/j.tcb.2024.10.013