酶响应肽段的多态性自组装图谱#

响应型肽段纳米材料在复杂生物环境中的具体组装形态长期以来难以直接观测。研究团队设计合成了包含硝基苯并氧二唑荧光团、四聚亮氨酸序列与磷酸酪氨酸的响应型前体分子NBD-L4pY。如图1所示，通过无细胞冷冻电镜分析发现，该短肽展现出高度依赖浓度与动力学历史的多态性交叉-β组装特征。在5.0 mM浓度下短肽倾向于形成直径约为12.5 nm的空心纳米管。当快速稀释至1.25 mM时，体系转变为直径约5 nm的C2对称性纳米纤维。继续稀释至400 μM时，溶液中则混合存在5 nm纳米纤维与一种直径约为7 nm的C13对称性纳米管。值得注意的是，直接平衡配制的400 μM短肽溶液并未观察到明显纤维结构，这表明快速稀释过程捕获了组装路径上的亚稳态多态结构。

Fig.1 Assembly history encodes a polymorphic peptide landscape in cell-free conditions. (A) Molecular design of NBD-L4pY and schematic of its enzymatic dephosphorylation yielding NBD-L4Y. (B) Transmission electron microscopy images of NBD-L4pY (400 μM or 10 mM) incubated for 24 h in the absence or presence of ALP (1 U/mL). Cryo-EM structural determination of NBD-L4pY at (C) 5.0 mM, (D) 1.25 mM, and (E) 400 μM. From left to right: (i) Representative cryo-EM micrographs, (ii) 2D class averages, (iii) 3D helical reconstructions, (iv) fitted atomic models shown in cross section, and (v) arrays of NBD-L4pY molecules along the filament axis. Scale bars, 50 nm. Arrows in (i) indicate distinct nanofiber morphologies; arrow colors match the backgrounds in (C–E). One peptide subunit is highlighted in red in each structure in (iv), and dashed lines in (v) indicate hydrogen bonds.

细胞界面对C13纳米管形态的特定选择#

为了明确细胞微环境究竟选择何种超分子结构，研究团队将骨肉瘤细胞与短肽孵育并在5分钟内进行冷冻固化。如图2所示，原位单颗粒冷冻电镜重构显示细胞周缘富集了高度均一的纳米管结构，分辨率达到了3.4 Å。结构解析证实该原位超分子结构正是无细胞稀释体系中捕获的C13对称性纳米管，其螺旋上升距离为4.65 Å，螺旋扭转角为4.17°。细胞表面丰富的碱性磷酸酶通过局部去磷酸化作用降低了短肽间的静电排斥，促进了纳米管的进一步聚集与束状化。这一发现证明细胞界面并非被动接受溶液中的多态混合物，而是显著偏好并稳定了具有特定堆积方式的C13纳米管架构。

Fig.2 The pericellular niche selects a predominant ∼7 nm peptide nanotube in situ. (A) Schematic illustration of in situ cryo-EM imaging of peptide nanofilaments generated at the cell periphery. (B) A low-magnification cryo-EM image of the cell periphery in the treatment of NBD-L4pY. Red arrows indicate peptide filaments, which were selected as target regions for data collection. (C) A cryo-EM micrograph and (D) 2D class average of peptide filaments and bundles at the cell periphery. The orange arrow indicates peptide filament. The magenta arrows indicate filament bundles. (E) Side and cross-section views of the 3D reconstruction of the in situ filaments.

细胞膜局部扰动与微观摄取过程#

结合冷冻电子断层扫描技术，研究人员对短肽作用初期（2分钟）的细胞边缘进行了三维维度的结构追踪。如图3所示，原位断层成像清晰展现了C13纳米管与细胞质膜的直接接触过程。在纳米管附着与侵入位点，质膜出现了明显的纳米级电子密度降低与局部形态扰动，这与早期细胞膜通透性的改变高度吻合。此时细胞内部的线粒体、核糖体及微管系统等超微结构依然保持完整，表明质膜物理扰动是短肽引发细胞损伤的起始步骤。荧光成像实验进一步证实，400 μM浓度下的短肽能在短时间内高效进入细胞并在核周及细胞核内富集，而高浓度组由于缺少该特定组装态反倒无法实现有效摄取。

Fig.3 Cell-selected pericellular nanotubes engage and perturb the plasma membrane. (A,C) Reconstructed tomograms and (B,D) corresponding segmentations of the Saos-2 cell periphery after 2 min of NBD-L4pY treatment. (E) Tomogram slice showing a peptide filament at a plasma-membrane contact site. (F) Magnified view of the boxed region in (E). (G) Segmentation and (H) magnified 3D rendering of the peptide-membrane contact site shown in (E,F). Panel labels: P, peptide; R, ribosome; MT, microtubule; V, vesicle; F-actin, filamentous actin.

细胞内超分子重构与肌动蛋白网络破坏#

短肽进入细胞内部后展现出明显的超分子重构行为。通过冷冻聚焦离子束切割制备细胞内薄片，冷冻电子断层扫描结果表明细胞质内的短肽组装体形态发生了明显改变，转化为与肌动蛋白丝（F-actin） 及膜性细胞器密切缠绕的致密密集体。生化分级分离实验与免疫荧光结果如图4所示，细胞内组装体能够特异性整合Profilin-1（PFN1）蛋白。体外重组实验进一步证实，这种重构后的组装体能以剂量依赖的方式直接抑制肌动蛋白聚合过程。短肽通过这一机制破坏了细胞骨架的动态平衡，最终诱导癌细胞发生非凋亡性死亡。

Fig.4 Cellular entry of NBD-L4pY drives subcellular redistribution and cytoskeletal perturbation. Confocal images of Saos-2 cells treated with NBD-L4pY at (A) 400 μM or (B) 5 mM over the course of 30 min. (C) Confocal images of Saos-2 cells prestained with a plasma-membrane dye and then treated with NBD-L4pY for 2 to 15 min (D) SIM images of cells prestained with Actin Tracker and then treated with NBD-L4pY for 5 min. Orange arrows indicate the cortical-rim distribution of peptide assemblies; the cyan arrow indicates dispersed intracellular assemblies. (E) Confocal images of cells treated with NBD-L4pY for 15 or 30 min, followed by staining of F-actin and nuclei. (F) Confocal images of cells treated with NBD-L4pY for 5 to 15 min, followed by staining of vinculin, F-actin, and nuclei. (G) Immunofluorescence staining of PFN1, CFL1, and ACTN1 in cells treated with NBD-L4pY for 15 min [NBD-L4pY] = 400 μM. Fig.5 Intracellular peptide remodeling generates dense assemblies that interface with F-actin and membranous organelles. (A) Workflow for preparing Saos-2 cells on gold EM grids for cryo-FIB milling and cryo-ET. (B) Representative SEM image of a milled cellular region. (C) SEM image of a finished lamella. (D,H) Reconstructed tomograms from lamellae of cells treated with NBD-L4pY for 15 min. (E,F,I,J) Magnified views of the boxed regions in (D,H). (G,K) Corresponding segmentations showing interfaces between peptide assemblies and intracellular structures. Panel labels: P, peptide; R, ribosome; V, vesicle; F-actin, filamentous actin; Golgi/ER, Golgi apparatus/endoplasmic reticulum; Mito, mitochondrion. Fig.6 Biochemical validation links peptide assemblies to PFN1 redistribution and impaired actin polymerization. (A) Workflow for preparing supernatant and pellet fractions from Saos-2 cells for WB analysis. (B) Optical image of cells treated with peptide for the indicated durations, followed by lysis and centrifugation. (C) WB analysis of supernatant and pellet fractions from (B) across the time course. (D) Actin polymerization assay showing conversion of monomers in the presence of peptide (50–400 μM). Data were fitted with a one-phase association model to calculate rate constant (K) and half-time. (E) TEM images of actin-binding proteins (i) PFN1, CFL1, and ACTN1 alone (0.4 mg/mL), (ii) after coincubation with peptide (400 μM); and (iii) after coincubation with peptide (400 μM) and ALP (1 U/mL).

总结及展望#

该工作打破了传统仅关注酶催化活性而忽视结构选择的局限，建立了超分子结构选择与重构决定生物学功能的全新模型。研究展示的两阶段致死机制——即细胞外界面选择膜活性纳米管诱导膜通透化，细胞内重构为致密组装体干扰骨架蛋白——为后续设计高选择性、跨尺度作用的响应型抗肿瘤肽类药物提供了重要的物理结构依据与设计指南。