【Angew.Chem.】湖南大学蒋健晖|半合成LC3相互作用降解剂：实现宏自噬活性可视化与高达85%的靶向蛋白降解#

文章标题：Semi-Synthetic LC3-Interacting Degrader for Visualizing Macroautophagic Activity and Targeted Degradation 文章作者：Ke-Ke Zhang, Ji Cheng, Jing-Yi Zhang, Li-Juan Tang, Fenglin Wang, Xia Chu, Jian-Hui Jiang 文章链接：https://doi.org/10.1002/anie.7042721

研发背景与工具创新：打造能发光示警的“细胞垃圾导航仪”#

细胞内部时刻都在运转着一套名为“宏自噬”的垃圾清理与回收系统，它就像细胞内部的垃圾清运车，将受损的细胞器、有害蛋白包裹起来并送入强酸性的“溶酶体处理厂”中降解消化。然而，现有的检测手段往往容易干扰细胞原有的正常代谢，且难以同时做到实时动态监测与精准引导降解。

为了解决这一难题，研究团队设计出一种名为 SLID（半合成LC3相互作用降解剂） 的多功能分子工具。该工具巧妙整合了三个核心模块：用于精准识别自噬垃圾车标记物（LC3蛋白）的导航序列（LIR）、用于增强结合抓力的疏水与多聚基团（HyD与HOTag3），以及一个可挂载荧光分子的自标化标签（HaloTag）。配合特制的环境pH敏感荧光探针（Rho-HaLo），该探针在中性细胞质中不发光，一旦进入强酸性的溶酶体，在 pH 4.5 环境下荧光信号可暴增8倍，从而以极高对比度清晰点亮整个自噬清理过程。研究人员进一步从13种候选序列中筛选出亲和力最佳的 TP53INP2 来源识别片段，在饥饿诱导约 30分钟内 即可实现对细胞自噬全流程的高灵敏度动态追踪。

Fig.1 Design and characterization of SLID platform for fluorogenic imaging of macroautophagic activities and selection of high-affinity LIRs. (a) Schematic of the SLID platform for fluorogenic imaging of macroautophagic activities and visualization of targeted degradation. The SLID platform comprises an LIR for LC3 binding and a HaloTag for labeling with a pH-sensitive fluorophore Rho-HaLo. An HyD and an oligomeric HOTag3 motif are appended to enhance LC3 binding. For chemically inducible degradation, SLID is fused with a targeting binding domain for POI recruitment through a dimerization-induced proximity system. (b) Confocal images and line-scan analysis of GFP and Rho-HaLo fluorescence for transfected cells cultured in DMEM and EBSS. Data from three independent experiments. Scale bar: 10 µm. Relative fluorescence intensities (RFI) of Rho-HaLo (c) and autophagosome numbers (d) for transfected cells cultured in DMEM, EBSS, EBSS pretreated with Baf-A1 or CQ. Rho-HaLo fluorescence intensities were normalized to those of cells cultured in DMEM. (e) RFI of Rho-HaLo fluorescence intensities for cells expressing SLID without HyD or HOTag3 after starvation induction. Rho-HaLo fluorescence intensities were normalized to those of cells transfected with HyD–LIR-HaloTag–GFP–HOTag3 cultured in DMEM. (f) High-affinity LIR selection as indicated by Rho-HaLo fluorescence intensities for cells transfected with SLID using different LIRs. (c–f) Data were represented as mean ± s.d. from three independent experiments. (g) Ratios of mCherry to GFP fluorescence in cells co‑expressing the macroautophagic sensor mCherry–GFP–LC3 together with: SLID with LIR; SLID with LIR–HOTag3; or LC3 alone. Ctrl: cells co‑expressing mCherry–GFP–LC3 and an empty vector. Ratio values were normalized to those of the control (Ctrl) group. The solid black lines denote medians, dashed lines represent quartiles. (n = 3). Statistical comparison was performed using one-way ANOVA (****p < 0.0001).#

靶向降解与实测突破：瓦解疾病蛋白聚集体并清除85%以上的大块沉淀#

除了作为自噬监测的“指示灯”，研究人员还通过引入小分子诱导的临近系统，将 SLID 升级为可控的靶向降解平台。该系统能够像分子机械臂一样，在特定化学信号（AP21967）的指令下，将指定的有害目标蛋白强行拉拽至自噬体中消灭。

在性能验证中，该平台展现出优异且广谱的清除能力：

基础模型蛋白清除：对不同表达丰度的目标蛋白实现了 72%至81%的高效降解。
攻克非溶性蛋白聚集体：针对传统降解途径束手无策的大颗粒沉淀，诱导活化后的 SLID 成功将大型蛋白聚集体消减了 85%。
瓦解重大疾病致病蛋白：在神经退行性疾病模型中，SLID 成功特异性清除了导致亨廷顿舞蹈症的高突变聚谷氨酰胺聚集体（HTT Q50、HTT Q80），以及与阿尔茨海默病密切相关的Tau蛋白致病突变聚集体（P301L/S320F）；同时还成功降解了由激酶异常激活引发的相分离无膜凝聚体。

Fig.2 SLID for fluorogenic imaging of overall macroautophagic activities and dissection of the molecular players involved in macroautophagy. Confocal images of cells expressing SLID incubated with Rho-HaLo or Rho-Tpp upon mitophagy induction by CCCP treatment (a), lipophagy induction by oleic acid and starvation (b), and pexophagy induction by 2,2’-dipyridyl (c). (d) Quantitative analysis of probe fluorescence intensities for images shown in (a–c). Probe fluorescence intensities were normalized to those of cells cultured in DMEM. (e) Fluorescence intensities of Rho-HaLo in cells expressing SLID following hypoxia induction for various time intervals. (f) Schematic of the roles of several molecular players in macroautophagy, including isolation membrane elongation, phagophore closure, and lysosome fusion, along with confocal images for SLID-expressing cells co-transfected with NC siRNA or siRNAs for knockdown (KD) of ATG2A, CHMP2A or RAB7. (g) Quantitative analysis of Rho-HaLo fluorescence intensities in the images shown in (f). Values were normalized to the Rho-HaLo fluorescence intensity for cells cultured in DMEM. (h) qRT-PCR analysis of ATG2A, CHMP2A, and RAB7 mRNAs levels in cells co‑transfected with cognate siRNAs or NC siRNA. (a–c, f) Data from three independent experiments. Scale bar: 5 µm. (d, e, f) Data were presented as mean ± s.d. from three independent experiments. Statistical comparison was performed using one-way ANOVA (****p < 0.0001; n.s., p ≥ 0.05).#

Fig.3 Chemically inducible SLID for self-indicating and macroautophagy-targeted degradation. (a) Schematic of SLIDGFP for GFP degradation. Upon macroautophagy induction, GFP captured by GBP1 of SLIDGFP was degraded. (b) Relative fluorescence intensities (RFI) of GFP and RFP for cells expressing SLIDGFP and GFP‒T2A‒RFP cultured in DMEM, EBSS, or EBSS pretreated with Baf-A1 and CQ. GFP and RFP fluorescence intensities were normalized to those of cells cultured in DMEM. Box plots show the distribution of measured RFI values; internal horizontal lines denote medians, box limits mark the 25th and 75th percentiles, and whiskers span values within 1.5 × interquartile range. (n = 3). (c) Self‑indicating degradation of GFP by SLIDGFP. Time-dependent GFP and Rho-HaLo fluorescence in cells expressing SLIDGFP and GFP upon starvation induction, along with confocal fluorescence images for transfected cells before and 24 h after starvation induction. Scale bar: 5 µm. (d) RFI of GFP for cells expressing SLIDGFP and GFP driven by promoters of different strengths cultured in DMEM or EBSS as determined by flow cytometry. GFP fluorescence was normalized to that of cells with miniCMV-driven GFP expression in DMEM. (e) Schematic of the SLID platform for inducible GFP degradation via AP-mediated split GBP1 complementation and GFP binding, alongside flow cytometry profiles of GFP in transfected cells cultured in DMEM or EBSS in the presence or absence of AP. (f) Schematic of the inducible SLID platform based on chemical dimerization-induced proximity of the target-binding domain and LIR. (g) Flow cytometry profiles of GFP for cells expressing the SLID platform with chemical dimerization-induced proximity of the target-binding domain and LIR, in DMEM or EBSS with or without AP. (h) RFI of GFP for cells expressing inducible SLID with or without HOTag3, in DMEM or EBSS with or without AP. GFP fluorescence was normalized to that for transfected cells in DMEM without AP. Data are presented as mean value ± s.d. from three independent experiments.#

Fig.4 Chemically inducible SLID platform for targeted degradation of aggregates and phase-separated condensates. (a) Schematic of SLIDGFP for inducible degradation of GFP puncta. GFP fused with LZ and HOTag6 formed aggregates, which were degraded upon macroautophagy induction. Confocal images (b) and flow cytometry profiles (c) of GFP fluorescence for cells cultured in DMEM or EBSS in the presence or absence of AP. (d) Schematic of SLID for inducible degradation of pathological HTTpolyQ and Tau aggregates. (e) Confocal images of cells co-expressing SLID and GFP-fused HTTQ50 or Tau (P301L, S320F) cultured in DMEM or EBSS with or without AP. (f) Violin plot of GFP fluorescence for cells co-expressing SLID and HTT with different polyQ lengths under various treatment conditions as determined by flow cytometry. Values were normalized to those of cells cultured in DMEM without AP. (g) Schematic of SLIDPKA for degradation of phase-separated condensates using one of the phase components FHA1 as the targeting module, which specifically recognizes phosphorylated substrate sequences. (h) Confocal fluorescence images of HeLa (upper row) or HEK293T (lower row) cells expressing SLIDPKA under different treatments, showing specific degradation of phase separation triggered by PKA activation in HeLa (upper row) and HEK293T (lower row) cells. From left to right in each row: cells were cultured in DMEM, DMEM supplemented with AP, EBSS medium, and AP-containing EBSS medium. (i) Violin plot of GFP fluorescence for cells co-expressing SLIDPKA and modified SPARK system under various treatment conditions as determined by flow cytometry. GFP fluorescence was normalized to that of transfected HeLa or HEK293T cells cultured in DMEM without AP. (f, i) The solid black lines denote medians, dashed lines represent quartiles. (n = 3). Statistical comparison was performed using one-way ANOVA (****p < 0.0001; n.s., p ≥ 0.05). Data from three independent experiments. Scale bar: 5 µm.#

Fig.5 SLID platform to image macroautophagic activities in young and senescent primary fibroblast cells. (a) Schematic of imaging macroautophagic activities in MRC-5 cells transduced with SLID and treated with drug for senescence induction. (b) Confocal images of SLID-transduced young and senescent MRC-5 induced by different stimuli and treated with Rho-HaLo. (c) SA‑β‑Gal staining and confocal images of young and senescent MRC-5 induced by different stimuli and treated with Rho-Tpp. (d) Quantitative analysis of Rho-HaLo or Rho-Tpp fluorescence for cells in (b and c). Fluorescence values were normalized to those of young MRC-5 cells treated with Rho-Tpp. Data are presented as mean value ± s.d. from three independent experiments. (e) Schematic of changes in macroautophagy and mitophagy activities in primary young and senescent fibroblasts. In young fibroblasts, the level of mitophagy is high, whereas in senescent cells, mitochondria tend to aggregate and mitophagy is attenuated. As a compensatory mechanism, senescent cells upregulate macroautophagy to clear damaged mitochondria and protein aggregates. Data from three independent experiments. Scale bar: 10 µm.#

Fig. 6 SLID platform enables protein degradation and senolysis in senescent cells. (a) Schematic of the lentiviral vector framework used to deliver inducible SLIDFOXO4 for targeted degradation of FOXO4 in MRC-5 cells, triggering apoptosis in senescent cells. (b) Confocal images of FOXO4 immunofluorescence in SLIDFOXO4-transduced cells cultured in DMEM or EBSS and treated with or without H2O2 (for senescence induction) and AP (for dimerization induction). Data from three independent experiments. Scale bar: 10 µm. (c) Confocal images of Annexin V and PI staining for SLIDFOXO4-transduced cells under different treatment conditions. Senescent cells treated with AP and cultured in EBSS were used as a positive control. Data from three independent experiments. Scale bar: 100 µm. (d) Quantitative analysis of Annexin V and PI fluorescence intensities in cells under different treatments in (c). Fluorescence was normalized to that in transduced cells cultured in DMEM without AP and H2O2 treatment. (e) Schematic of SLIDHSP90 for targeted degradation of HSP90 in MRC-5 cells, triggering apoptosis in senescent cells. (f) Quantitative analysis of Annexin V and PI fluorescence intensities for SLIDHSP90-transduced cells under different treatments. Fluorescence was normalized to that in transduced cells cultured in DMEM without AP and H2O2 treatment . ( d, f) Data are presented as mean value ± s.d. from three independent experiments.#

衰老细胞精准识别与定向清除：开辟抗衰老治疗新途径#

衰老细胞在机体内往往表现出特殊的自噬代谢特征：其线粒体特异性自噬能力明显减弱，但为了清除受损物质，整体宏自噬清理通路却出现显著上调。基于这一生物学特性，研究团队借助病毒载体将 SLID 系统导入原代肺成纤维衰老模型中，精准捕获并证实了衰老细胞中自噬流显著活跃的特征。

研究团队顺势将该工具转化为一种新型抗衰老靶向清除方案（Senolytics）：利用衰老细胞内高度活跃的自噬流水线，定向降解维持衰老细胞存活的关键“保护伞”蛋白——阻止细胞凋亡的 FOXO4 蛋白以及稳定促存活通路的 HSP90 分子伴侣。实验证实，通过小分子激活降解后，衰老细胞内的促存活蛋白被快速破坏，成功诱导了衰老细胞发生特异性凋亡，而正常年轻细胞则不受明显损伤。

该项研究不仅攻克了细胞自噬过程“难追踪、难利用”的痛点，更为清除致病蛋白聚集体与衰老细胞提供了高特异性的全新分子工具箱。

【Angew.Chem.】湖南大学蒋健晖|半合成LC3相互作用降解剂：实现宏自噬活性可视化与高达85%的靶向蛋白降解

【Angew.Chem.】湖南大学蒋健晖|半合成LC3相互作用降解剂：实现宏自噬活性可视化与高达85%的靶向蛋白降解#

研发背景与工具创新：打造能发光示警的“细胞垃圾导航仪”#

靶向降解与实测突破：瓦解疾病蛋白聚集体并清除85%以上的大块沉淀#

衰老细胞精准识别与定向清除：开辟抗衰老治疗新途径#

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