Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.
Institute of Physics, Experimental Polymer Physics, Albert-Ludwigs-University Freiburg, Hermann-Herder-Str. 3, 79104, Freiburg, Germany.
Cell Stress Chaperones. 2018 Jul;23(4):673-683. doi: 10.1007/s12192-018-0879-0. Epub 2018 Feb 5.
Hsp70-1A-the major stress-inducible member of the HSP70 chaperone family-is being implicated in cancer diseases with the development of resistances to standard therapies. In normal cells, the protein is purely cytosolic, but in a growing number of tumor cells, a significant fraction can be identified on to the cell surface. The anchoring mechanism is still under debate, as Hsp70-1A lacks conventional signaling sequences for translocation from the cytosol to exoplasmic leaflet of the plasma membrane and common membrane binding domains. Recent reports propose a lipid-mediated anchoring mechanism based on a specific interaction with charged, saturated lipids such as dipalmitoyl phosphatidylserine (DPPS). Here, we prepared planar supported lipid bilayers (SLBs) to visualize the association of Hsp70-1A directly and on the single molecule level by atomic force microscopy (AFM). The single molecule sensitivity of our approach allowed us to explore the low concentration range of 0.05 to 1.0 μg/ml of Hsp70-1A which was not studied before. We compared the binding of the protein to bilayers with 20% DPPS lipid content both in the absence and presence of cholesterol. Hsp70-1A inserted exclusively into DPPS domains and assembled in clusters with increasing protein density. A critical density was reached for incubation with 0.5 μg/ml (7 nM); at higher concentrations, membrane defects were observed that originated from cluster centers. In the presence of cholesterol, this critical concentration leads to the formation of membrane blebs, which burst at higher concentrations supporting a previously proposed non-classical pathway for the export of Hsp70-1A by tumor cells. In the discussion of our data, we attempt to link the lipid-mediated plasma membrane localization of Hsp70-1A to its potential involvement in the development of resistances to radiation and chemotherapy based on our own findings and the current literature.
热休克蛋白 70-1A（Hsp70-1A）是热休克蛋白 70 伴侣家族中的主要应激诱导成员，它与标准治疗的耐药性的发展有关，正在被牵连到癌症疾病中。在正常细胞中，该蛋白纯粹存在于细胞质中，但在越来越多的肿瘤细胞中，可以在细胞表面鉴定到相当一部分 Hsp70-1A。由于 Hsp70-1A 缺乏从细胞质转移到质膜外叶的常规信号序列和常见的膜结合结构域，因此其锚定机制仍存在争议。最近的报告提出了一种基于与带电荷的饱和脂质（如二棕榈酰磷脂酰丝氨酸（DPPS））的特定相互作用的脂质介导的锚定机制。在这里，我们通过原子力显微镜（AFM）制备了平面支撑脂质双层（SLB），以直接和单分子水平可视化 Hsp70-1A 的结合。我们的方法的单分子灵敏度使我们能够探索以前未研究过的 0.05 至 1.0μg/ml 的 Hsp70-1A 的低浓度范围。我们比较了该蛋白与含有 20% DPPS 脂质的双层的结合情况，无论是在没有胆固醇的情况下还是在存在胆固醇的情况下。Hsp70-1A 仅插入 DPPS 结构域，并随着蛋白密度的增加而组装成簇。当孵育浓度为 0.5μg/ml（7nM）时，达到临界密度；在更高的浓度下，观察到源自簇中心的膜缺陷。在胆固醇存在的情况下，该临界浓度导致膜泡的形成，当浓度更高时，膜泡会破裂，这支持了肿瘤细胞中 HSP70-1A 非经典输出途径的先前提议。在讨论我们的数据时，我们试图将 Hsp70-1A 的脂质介导的质膜定位与其在辐射和化疗耐药性发展中的潜在作用联系起来，这是基于我们自己的发现和当前文献。
