Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein.
Phosphatidylinositol Monophosphates Regulate the Membrane Localization of HSPA1A, a Stress-Inducible 70-kDa Heat Shock Protein.
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DOI：
10.3390/biom12060856
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发表时间：
2022-06-20
期刊：
影响因子：
5.5
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中科院分区：
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HSPA1A 是一种分子伴侣，可调节应激细胞和癌细胞的存活。除了其细胞质促生存功能外，HSPA1A 还定位并嵌入应激细胞和肿瘤细胞的质膜 (PM) 中。膜相关的 HSPA1A 发挥免疫调节功能并使肿瘤对标准疗法产生耐药性。因此，了解和操纵 HSPA1A 的表面呈现是一种有前途的治疗方法。然而，HSPA1A 通往细胞表面的途径仍然是个谜，因为这种蛋白质缺乏已知的膜定位信号。考虑到 HSPA1A 与脂质结合，如磷脂酰丝氨酸 (PS) 和单磷酸化磷酸肌醇 (PIP)，我们假设这种相互作用调节 HSPA1A 的 PM 定位和锚定。为了检验这一假设，我们对人类细胞系进行热休克，耗尽特定的脂质靶标，并使用共聚焦显微镜和细胞表面生物素化来量化 HSPA1A 的 PM 定位。这些实验表明，HSPA1A 与掩盖 PI(4)P 和 PI(3)P 的脂质生物传感器的共转染显着减少了 HSPA1A 的热诱导表面呈现。接下来，我们使用离子霉素、氧化苯胂 (PAO)、GSK-A1 和渥曼青霉素操纵细胞脂质含量。这些实验表明，HSPA1A 的 PM 定位不受离子霉素影响，但被 PAO、GSK-A1 和渥曼青霉素显着降低，证实了共转染实验获得的结果。我们通过使用雷帕霉素诱导的磷酸酶系统选择性耗尽 PI(4)P 和 PI(4,5)P2 来验证这些结果。我们的研究结果强烈支持这样的观点，即 HSPA1A 的表面呈现是一种多方面的脂质驱动现象，由伴侣与特定内体和 PM 脂质的结合控制。
HSPA1A is a molecular chaperone that regulates the survival of stressed and cancer cells. In addition to its cytosolic pro-survival functions, HSPA1A also localizes and embeds in the plasma membrane (PM) of stressed and tumor cells. Membrane-associated HSPA1A exerts immunomodulatory functions and renders tumors resistant to standard therapies. Therefore, understanding and manipulating HSPA1A’s surface presentation is a promising therapeutic. However, HSPA1A’s pathway to the cell surface remains enigmatic because this protein lacks known membrane localization signals. Considering that HSPA1A binds to lipids, like phosphatidylserine (PS) and monophosphorylated phosphoinositides (PIPs), we hypothesized that this interaction regulates HSPA1A’s PM localization and anchorage. To test this hypothesis, we subjected human cell lines to heat shock, depleted specific lipid targets, and quantified HSPA1A’s PM localization using confocal microscopy and cell surface biotinylation. These experiments revealed that co-transfection of HSPA1A with lipid-biosensors masking PI(4)P and PI(3)P significantly reduced HSPA1A’s heat-induced surface presentation. Next, we manipulated the cellular lipid content using ionomycin, phenyl arsine oxide (PAO), GSK-A1, and wortmannin. These experiments revealed that HSPA1A’s PM localization was unaffected by ionomycin but was significantly reduced by PAO, GSK-A1, and wortmannin, corroborating the findings obtained by the co-transfection experiments. We verified these results by selectively depleting PI(4)P and PI(4,5)P2 using a rapamycin-induced phosphatase system. Our findings strongly support the notion that HSPA1A’s surface presentation is a multifaceted lipid-driven phenomenon controlled by the binding of the chaperone to specific endosomal and PM lipids.
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