Uwada Junsuke, Nakazawa Hitomi, Kiyoi Takeshi, Yazawa Takashi, Muramatsu Ikunobu, Masuoka Takayoshi
Department of Pharmacology, School of Medicine, Kanazawa Medical University, Ishikawa 920-0293, Japan.
Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.
J Cell Sci. 2025 Jan 1;138(1). doi: 10.1242/jcs.262236. Epub 2025 Jan 10.
FYVE-type zinc finger-containing phosphoinositide kinase (PIKFYVE), which is essential for phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] production, is an important regulator of lysosomal homeostasis. PIKFYVE dysfunction leads to cytoplasmic vacuolization; however, the underlying mechanism remains unknown. In this study, we explored the cause of vacuole enlargement upon PIKFYVE inhibition in DU145 prostate cancer cells. Enlargement of vacuoles upon PIKFYVE inhibition required glutamine and its metabolism by glutaminases. Addition of ammonia, a metabolite of glutamine, was sufficient to enlarge vacuoles via PIKFYVE inhibition. Moreover, PIKFYVE inhibition led to intracellular ammonium accumulation. Endosome-lysosome permeabilization resulted in ammonium leakage from the cells, indicating ammonium accumulation in the endosomes and lysosomes. Ammonium accumulation and vacuole expansion were suppressed by the lysosomal lumen neutralization. It is therefore assumed that PIKFYVE inhibition interferes with the efflux of NH4+, which formed through protonation of NH3 in the lysosomal lumen, leading to osmotic swelling of vacuoles. Notably, glutamine or ammonium is required for PIKFYVE inhibition-induced suppression of lysosomal function and autophagic flux. In conclusion, this study shows that PIKFYVE inhibition disrupts lysosomal homeostasis via ammonium accumulation.
含FYVE结构域锌指的磷酸肌醇激酶（PIKFYVE）对磷脂酰肌醇3,5-二磷酸[PtdIns(3,5)P2]的产生至关重要，是溶酶体稳态的重要调节因子。PIKFYVE功能障碍会导致细胞质空泡化；然而，其潜在机制仍不清楚。在本研究中，我们探究了DU145前列腺癌细胞中PIKFYVE抑制后液泡增大的原因。PIKFYVE抑制后液泡的增大需要谷氨酰胺及其通过谷氨酰胺酶的代谢。添加氨（谷氨酰胺的一种代谢产物）足以通过PIKFYVE抑制使液泡增大。此外，PIKFYVE抑制导致细胞内铵积累。内体-溶酶体通透性增加导致铵从细胞中泄漏，表明铵在内体和溶酶体中积累。溶酶体腔中和抑制了铵的积累和液泡扩张。因此推测，PIKFYVE抑制会干扰NH4+的外流，NH4+是由溶酶体腔内NH3质子化形成的，导致液泡渗透性肿胀。值得注意的是，PIKFYVE抑制诱导的溶酶体功能抑制和自噬流需要谷氨酰胺或铵。总之，本研究表明PIKFYVE抑制通过铵积累破坏溶酶体稳态。
