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。 2022Jan 27;59(1):2100267.
doi：10.1183/13993003.00267-2021。 印刷2022年1月。

通过对致病突变体的研究揭示了对表面活性剂蛋白C运输的新见解

Jennifer A Dickens1，，，， Eimear N Rutherford2，，，， Susana Abreu2，，，， Joseph E Chambers2，，，， Matthew O Ellis2，，，， Annemarie van Schadewijk3，，，， Pieter S Hiemstra3，，，， Stefan J Marciniak2
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通过对致病突变体的研究揭示了对表面活性剂蛋白C运输的新见解

Jennifer A Dickens等。 EUR RESSIR j。 。
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Abstract

Background:肺泡上皮细胞功能障碍起着不rtant role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but remains incompletely understood. Some monogenic forms of pulmonary fibrosis are associated with expression of mutant surfactant protein C (SFTPC). The commonest pathogenic mutant, I73T, mislocalises to the alveolar epithelial cell plasma membrane and displays a toxic gain of function. Because the mechanisms explaining the link between this mutant and IPF are incompletely understood, we sought to interrogate SFTPC trafficking in health and disease to understand the functional significance of SFTPC-I73T relocalisation.

Methods:我们在细胞模型中对SFTPC运输进行了机械分析，该模型重现了in vivophenotype and validated findings in human primary alveolar organoids.

Results:We show that wild-type SFTPC takes an unexpected indirect trafficking route通过the plasma membrane and undergoes the first of multiple cleavage events before reaching the multivesicular body (MVB) for further processing. SFTPC-I73T takes this same route, but its progress is retarded both at the cell surface and due to failure of trafficking into the MVB. Unable to undergo onward trafficking, it is recycled to the plasma membrane as a partially cleaved intermediate.

Conclusion:这se data show for the first time that all SFTPC transits the cell surface during normal trafficking, and the I73T mutation accumulates at the cell surface through both retarded trafficking and active recycling. This understanding of normal SFTPC trafficking and how the I73T mutant disturbs it provides novel insight into SFTPC biology in health and disease, and in the contribution of the SFTPC mutant to IPF development.

Conflict of interest statement

Conflict of interest: J.A. Dickens has nothing to disclose. Conflict of interest: E.N. Rutherford has nothing to disclose. Conflict of interest: S. Abreu has nothing to disclose. Conflict of interest: J.E. Chambers has nothing to disclose. Conflict of interest: M.O. Ellis has nothing to disclose. Conflict of interest: A. van Schadewijk has nothing to disclose. Conflict of interest: P.S. Hiemstra has nothing to disclose. Conflict of interest: S.J. Marciniak has nothing to disclose.

Figures

FIGURE 1
FIGURE 1
表面活性剂蛋白C（SFTPC）-I73T异常位置并处理in vivoandin vitro。a）SFTPC描述结构和致病性I73T突变的位置以及域以及近似报道的切割位点的示意图（虚线，编号为1-4）。b）从有条件表达SFTPC-WT（野生型）或SFTPC-I73T的转基因小鼠中对肺泡组织免疫染色，揭示了SFTPC-WT的点状细胞内染色，并将SFTPC-I73T重新分布到Apical Plasmammbrane。c）当绿色荧光蛋白（GFP）-SFTPC-WT在HeLa细胞中表达IT在细胞内点状结构中的位置，而GFP-SFTPC-I73T重新定位到质膜和细胞内的细胞内结构。在表达相等量的GFP（右上图）的细胞中，通过Brichos结构域抗体（右下图）测量的细胞表面SFTPC-I73T过多。d）使用NPROSFTPC抗体从载体控制或GFP-SFTPC表达细胞的裂解物的免疫印迹显示SFTPC-I73T的处理发生了改变。TM：跨膜。比例尺=10μm或5μm（缩放图像）。
FIGURE 2
FIGURE 2
All surfactant protein C (SFTPC) is tracked from early compartments to the plasma membrane. a) Schematic of Retention Using Selective Hooks (RUSH) system, in which proteins are held in early tracking compartments using a streptavidin “hook” before release after addition of biotin. b) Localisation of SFTPC in HeLa cells expressing the RUSH-green fluorescent protein (GFP)-SFTPC fusion protein treated±biotin for 16 h confirms they traffic normally to post-Golgi compartments. c) Real-time tracking of GFP-SFTPC with transferrin receptor (TfnR) or CDMPR reveals co-localisation of SFTPC with TfnR in tubular structures, but failure of co-localisation beyond the Golgi with CDMPR which is seen in vesicles. d) After 2 h of biotin exposure, GFP-SFTPC-wild type (WT) is visible at the plasma membrane; this is reflected in increased BRICHOS domain presence at the plasma membrane by flow cytometry. SBP: streptavidin-binding protein, mCh: mCherry. Scale bars=10 μm.
FIGURE 3
FIGURE 3
来自质膜的表面活性剂蛋白C（SFTPC）内在化是AP2的，并且受I73T突变阻碍。a）定量SFTPC内部化测定法。表达绿色荧光蛋白（GFP）-SFTPC野生型（WT）或-I73T的细胞被标记为冰上的Brichos结构域抗体，然后在指定的时间内允许蛋白质在37°C下内化。将细胞放回冰上，固定但未透化，并在通过流式细胞仪分析之前用二抗标记。n = 3。数据表示为平均值± SEM。b) and c) HeLa cells stably expressing GFP-SFTPC-WT or -I73T were treated with 30 µM bafilomycin for 16 h to inhibit clathrin-mediated endocytosis. This results in cell-surface SFTPC accumulation as seen by confocal microscopy and measured by flow cytometry. d) AP2-deficient cells were used to assess localisation of Retention Using Selective Hooks (RUSH)ed GFP-SFTPC-WT and -I73T after 2 h of biotin treatment. AP2M1 CRISPR knockout cells were immunoblotted for the AP2α-subunit. Although some α-subunit remains, this complex is nonfunctional. Scale bars=10 μm.
FIGURE 4
FIGURE 4
Retardation of surfactant protein C (SFTPC)-I73T internalisation is not due to failed oligomerisation or ubiquitination. a) HeLa cells were transfected with haemagglutinin (HA)-tagged and green fluorescent protein (GFP)-tagged SFTPC (wild type (WT) or I73T) and lysates subjected to anti-GFP immunoprecipitation before immunoblotting for the HA tag and GFP. Both SFTPC-WT and SFTPC-I73T are equally able to oligomerise. b) Lysates from HeLa cells expressing SFTPC-WT or SFTPC-I73T were subjected to SFTPC immunoprecipitation and immunoblotted for ubiquitin. Note the presence of oligoubiquitinated SFTPC-WT (arrows) which is not seen in I73T-expressing cells. c) Quantitative SFTPC internalisation assay of cells expressing GFP-SFTPC-WT, I73T or K6R. n=3. Data are presented as mean± SEM。
FIGURE 5
FIGURE 5
在K63链中进行泛素化的表面活性剂蛋白C（SFTPC）泛素化是需要跟踪多囊体（MVB）的。a）表达绿色荧光蛋白（GFP）-SFTPC-wild类型（WT），泛素化缺陷的K6R和I73T的HeLa细胞的共聚焦成像表明，像I73T一样，SFTPC-K6R像i73t一样，将其重新分配到细胞表面并重新分布为中层（中等底层），插图）。b）用炒（SCR）或ESCRT0蛋白HRS siRNA转染HeLa细胞48小时。免疫印迹确认敲低和共聚焦成像显示GFP-SFTPC-WT的重新分布急剧，向质膜。流式细胞仪证实，在HRS siRNA存在下，总GFP的总GFP和SFTPC的部分重新分布略有增加。c）制造了一个UBE2N CRISPR淘汰池，并由免疫印迹确认了成功。共聚焦显微镜揭示了GFP信号的总体增加以及GFP-SFTPC-WT的重新分布到这些细胞中的质膜，通过流式细胞仪证实。 ns：非重要性；L.C。：加载控制。比例尺=10μm。
FIGURE 6
FIGURE 6
表面活性剂蛋白C（SFTPC）C末端裂解发生后发生通过the plasma membrane, but prior to multivesicular body (MVB) internalisation. a) HeLa cells expressing the Retention Using Selective Hooks (RUSH)-SFTPC fusion protein were allowed to traffic for the times indicated before lysates were subjected to green fluorescent protein (GFP) immunoprecipitation and NproSFTPC immunoblotting. The intermediate forms (* and arrowhead) appear contemporaneously for SFTPC-wild type (WT) and -I73T, but the first I73T intermediate (*) accumulates as the WT intermediate is cleared. b) HeLa cells deficient in Ube2N or Hrs in which SFTPC cannot enter MVBs accumulate SFTPC*. c) HeLa cells were transfected with GFP-SFTPC-Halo and the Halotag labelled with TMR ligand for 15 min before fixing. d) Treatment of GFP-SFTPC-WT expressing HeLa cells with 100 nM bafilomycin for 16 h results in preferential accumulation of full-length proprotein. e) HeLa cells stably expressing GFP-SFTPC-WT or I73T were incubated with proteinase K to digest exposed proteins at the plasma membrane before being lysed and subjected to immunoblot for SFTPC, human leukocyte antigen (HLA) and GAPDH. The full-length (WT and I73T) and intermediate (SFTPC*) species are digested by proteinase K, suggesting that they reside at least partially at the cell surface. Lysed: lysis before proteinase K treatment. Scale bars=10 μm or 5 μm (zoomed images).
FIGURE 7
FIGURE 7
Z-VLL-CHO抑制表面活性剂蛋白C（SFTPC）C末端裂解。a）稳定表达绿色荧光蛋白（GFP）-SFTPC-wild类型（WT）或-I73T的HeLa细胞用5μMZ-VLL-CHO处理16小时，并用于NPROSFTPC的裂解物。用Z-VLL-CHO处理的细胞会产生过量的早期C末端加工中间体，总体SFTPC（通过GFP测量）增加了SFTPC，并将SFTPC重新分布在质膜上，如B）共焦显微镜和C）流动细胞仪。比例尺=10μm。
FIGURE 8
FIGURE 8
跟踪表面活性剂蛋白C（SFTPC）通过在人类肺泡瀑样细胞表面。一)Human alveolar type 2 (AT2) cell organoids were treated with bafilomycin or Z-VLL-CHO and the localisation of SFTPC determined by immunohistochemistry. E-cadherin (E-Cad) was used to delineate the basolateral membranes. Note that both compounds redistribute SFTPC to the apical plasma membrane. Scale bars=10 μm or 5 μm (zoomed images). b) Proposed model of SFTPC tracking. SFTPC pro-protein is tracked to the plasma membrane from the Golgi apparatus before AP2-dependent endocytosis in clathrin-coated vesicles. Initial C-terminal cleavage occurs in an early endocytic compartment before further cleavage in a later compartment by a membrane protease. This facilitates ubiquitination and allows internalisation into multivesicular bodies (MVBs) for onward cleavage and packaging into lamellar bodies. In the presence of the I73T mutation, endocytosis is reduced, early intermediates accumulate and are recycled due to a later block in tracking mediated by failure of later cleavage, ubiquitination and MVB internalisation.
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