Department of Microbiology, University of Calcutta, 35 B.C Road, Kolkata, 700019, India.
Department of Biochemistry, Bose Institute, P1/12 CIT Scheme, VII M, Kolkata, 700054, India.
Toxicol Lett. 2021 Mar 1;338:97-104. doi: 10.1016/j.toxlet.2020.12.008. Epub 2020 Dec 9.
Cigarette smoking is a risk factor for developing chronic obstructive pulmonary disease and protein aggresome formation is considered to be a hallmark event for the disease. Since dysfunction of lysosome-mediated protein degradation leads to enhanced accumulation of misfolded proteins and subsequent aggresome formation, we examined the effect of cigarette smoke extract (CSE) on ESCRT-mediated sorting in S. cerevisiae as this process is necessary for the functioning of the vacuole, the lysosomal equivalent in yeast. An operational ESCRT pathway is essential for ion homeostasis and our observation that exposure to CSE caused increased sensitivity to LiCl indicated CSE-induced impairment of ESCRT function. To confirm the inhibition of ESCRT function, the targeting of carboxypeptidase S (CPS), which reaches the vacuole lumen via the ESCRT pathway, was examined. Treatment with CSE resulted in the mislocalization of GFP-tagged CPS to the vacuolar membrane, instead of the vacuolar lumen, confirming defective functioning of the ESCRT machinery in CSE-treated cells. Further analysis revealed that CSE-treatment inhibited the recruitment of the ESCRT-0 component, Vps27, to the endosome surface, which is a key event is for the functioning of the ESCRT pathway. This lack of endosomal recruitment of Vps27 most likely results from a depletion of the endosomally-enriched lipid, phosphatidylinositol 3-phosphate (PI3-P), which is the target of Vps27. This is supported by our observation that the presence of excess leucine, a known activator of the lipid kinase responsible for the generation of PI3-P, Vps34, in the medium can rescue the CSE-induced ESCRT misfunctioning. Thus, the current study provides an insight into CSE-induced aggresome formation as it documents that CSE treatment compromises vacuolar degradation due to an impairment of the ESCRT pathway, which likely stems from the inhibition of Vps34. It also indicates that leucine has the potential to attenuate the CSE-induced accumulation of misfolded proteins.
吸烟是导致慢性阻塞性肺疾病的一个危险因素，而蛋白质聚集物的形成被认为是该疾病的一个标志性事件。由于溶酶体介导的蛋白质降解功能障碍会导致错误折叠的蛋白质积累增加，并随后形成聚集物，因此我们研究了香烟烟雾提取物 (CSE) 对酿酒酵母中 ESCRT 介导的分选的影响，因为这个过程对于液泡（酵母中的溶酶体等效物）的功能是必要的。ESCRT 途径的有效运作对于离子平衡至关重要，我们观察到暴露于 CSE 会导致对 LiCl 的敏感性增加，这表明 CSE 诱导的 ESCRT 功能障碍。为了确认 ESCRT 功能的抑制，研究了靶向到达液泡腔的羧肽酶 S（CPS）的 ESCRT 途径。用 CSE 处理会导致 GFP 标记的 CPS 错误定位到液泡膜，而不是液泡腔，这证实了 CSE 处理细胞中的 ESCRT 机制功能障碍。进一步的分析表明，CSE 处理抑制了 ESCRT-0 成分 Vps27 到内体表面的募集，这是 ESCRT 途径运作的关键事件。Vps27 在内体表面的募集缺乏很可能是由于富含内体的脂质磷脂酰肌醇 3-磷酸（PI3-P）的耗尽，而 Vps27 是其靶标。这一观点得到了我们的观察结果的支持，即在培养基中存在过量的亮氨酸（一种已知的激活负责产生 PI3-P 的脂质激酶的物质）可以挽救 CSE 诱导的 ESCRT 功能障碍。因此，本研究提供了对 CSE 诱导的聚集物形成的深入了解，因为它记录了 CSE 处理会由于 ESCRT 途径的功能障碍而导致液泡降解受损，这可能源于 Vps34 的抑制。它还表明亮氨酸有可能减轻 CSE 诱导的错误折叠蛋白质的积累。
