时间标签：数据日期 2026-10-31 · 生成时间 2026-11-01 05:30（Asia/Shanghai）｜ 检索窗口 PubMed edat 2026/10/28–2026/10/31 ｜ 情报发生日期 2026-10-31
研究问题
Notably, Ctsb remains catalytically active at both acidic and neutral pH [, ], indicating it can maintain proteolytic function within nonlysosomal environments.；Despite detailed in vitro characterization of its substrate specificity [], how Ctsb contributes to endogenous protein processing in intact cells remains poorly understood.；However, as TAILS‐based analysis exclusively captures N‐terminal proteolytic events, it cannot detect proteolytically generated C‐termini, particularly those generated near the native protein terminus.
方法
Cell lysates were clarified by centrifugation (21,000 × g, 5 min, 4 °C) and protein concentrations were determined using Pierce BCA protein assay kits (Thermo Fisher Scientific #23223, #23224) for downstream analysis (Sections , , and ).；For pulldown samples, 500 µL of immunoprecipitation buffer (PBS, pH 7.4, 0.5% NP‐40, 1 mM EDTA) was added on ice, followed by antibodies against proteins of interest: 10 µL goat anti‐mouse Ctsx (R&D #AF1033), 10 µL goat anti‐mouse Ctsb (R&D #AF965), 20 µL goat anti‐human Ctss (R&D #AF1183), and 10 µL goat anti‐mouse Ctsl (R&D #AF1515).；Agarose beads were pelleted by brief centrifugation and washed four times with immunoprecipitation buffer, followed by a final wash with 0.9% NaCl.；Cells were subsequently labeled with 50 nM LysoTracker Red (Invitrogen #L7528) for 1 h, followed by nuclear staining with 10 µg/mL Hoechst 33 258 (Sigma‐Aldrich #14530) for 5 m
核心结论
Complete loss of Ctsb labeling was observed following treatment with 5 or 10 µM Z‐RK‐AOMK, indicating near‐complete inhibition of intracellular Ctsb activity (Figure ).；Although this value is higher than that previously reported in human neuroblastoma cells (50 nM) [], complete inhibition of intracellular Ctsb activity was achieved at 5 µM in RAW264.7 cells.；This difference may reflect cell‐specific factors, including variation in Ctsb expression levels, compound uptake or accumulation, or differences in assay readouts. ‖ 结论：Collectively, these findings refine the functional view of Ctsb in macrophages, positioning it as a regulated protease with selective processing activity rather than a broadly degradative protease under basal conditions.
局限
Consistent with this, sequence motif analysis [] of differentially enriched neo‐N‐termini did not reveal a robust or biologically interpretable consensus cleavage signature (Figure S3 and Table S6), likely reflecting the limited number of regulated N‐terminal cleavage events and the heterogeneity of contributing proteases.；Residual fluorescence persisted following pretreatment with 5 µM Z‐RK‐AOMK prior to probe incubation (Figure , bottom panel).；This residual signal may reflect a combination of low‐level off‐target probe retention, including labeling of other cathepsins, ornon‐specific intracellular accumulation of the hydrophobic BODIPY fluorophore, which can contribute to background fluor
对本项目的价值
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全文来源
PMC JATS 全文（正文 59,580 字符，已过标题/DOI 逐字归属校验）
原文题录
- 英文标题：Integration of Chemical Probes and Degradomics Reveals That Cathepsin B Functions Primarily as a Dipeptidyl Carboxypeptidase in Macrophages.
- 期刊与标识：ChemMedChem · PMID 42956330 · DOI 10.1002/cmdc.70530
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