听觉脑干依靠精确的电路来促进声源定位。在小鸡身上,这种特殊电路的发展需要caspase-3的非凋亡活性,我们先前鉴定了数百种蛋白水解底物。在这里,我们测试了半胱天冬酶切割位点的序列是否在凋亡和非凋亡环境中差异编码蛋白水解偏好。我们构建了非凋亡小鸡听觉脑干中半胱天冬酶活性的共有序列,该序列包含切割位点N末端的四个残基:IX(G/R)D丨其中X代表无显着富集,而丨代表切割位点。我们鉴定了在上述共有序列中发现的含有基序的半胱天冬酶底物中显著富集的GO术语。(G/R)D÷与术语“细胞骨架的结构成分”(SCoC)相关,提示SCoC蛋白在非凋亡发育过程中可能被caspase活性特异性靶向。为了确定该共有序列是否特异于胚胎第(E)10天的非凋亡听觉脑干,我们使用在听觉脑干神经元经历凋亡细胞死亡(E13)时收获的脑干的蛋白质质谱。凋亡基序VD在E13裂解位点中显著富集,表明P2亚位点的基序偏好已向规范的caspase共有序列转移。此外,蒙特卡罗模拟显示,只有GD基序与凋亡的听觉脑干中的SCoC底物相关,表明GD在非凋亡和凋亡背景下编码SCoC蛋白的特异性,尽管在后者中不受欢迎。最后,为了鉴定候选人类非凋亡共有序列,我们使用蒙特卡罗分析来确定与Degrab酶中SCoCcaspase底物相关的基序和基序对,人类凋亡细胞系中切割位点的数据库。我们发现了11个与SCoC蛋白水解显著相关的基序,包括IXXD和GD。我们采用了逐步方法来选择基序对,该基序对针对给定的SCoC裂解事件覆盖率优化了SCoC特异性,产生11个基序对可能在SCoC指导的人类非凋亡半胱天冬酶共有序列中是优选的。GD+IXXD是这些图案对之一,表明脊椎动物之间非凋亡共识位点的保守性。
The auditory brainstem relies on precise circuitry to facilitate sound source localization. In the chick, the development of this specialized circuitry requires non-apoptotic activity of caspase-3, for which we previously identified several hundred proteolytic substrates. Here we tested whether the sequence of the caspase cleavage site differentially encodes proteolytic preference in apoptotic and non-apoptotic contexts. We constructed a
consensus sequence for
caspase activity in the non-apoptotic chick auditory brainstem comprising the four residues N-terminal to the cleavage site: IX(G/R)D↓ where X represents no significant enrichment and ↓ represents the cleavage site. We identified GO terms significantly enriched among caspase substrates containing motifs found in the above
consensus sequence. (G/R)D↓ was associated with the term \"Structural Constituent of Cytoskeleton\" (SCoC), suggesting that SCoC proteins may be specifically targeted by caspase activity during non-apoptotic developmental processes. To ascertain whether this
consensus sequence was specific to the non-apoptotic auditory brainstem at embryonic day (E) 10, we used protein mass spectrometry of brainstems harvested at a time when auditory brainstem neurons undergo apoptotic cell death (E13). The apoptotic motif VD was significantly enriched among E13 cleavage sites, indicating that motif preference at the P2 subsite had shifted toward the canonical caspase
consensus sequence. Additionally, Monte Carlo simulations revealed that only the GD motif was associated with SCoC substrates in the apoptotic auditory brainstem, indicating that GD encodes specificity for SCoC proteins in both non-apoptotic and apoptotic contexts, despite not being preferred in the latter. Finally, to identify candidate human non-apoptotic consensus sequences, we used Monte Carlo analyses to determine motifs and motif pairs associated with SCoC caspase substrates in the Degrabase, a database of cleavage sites in human apoptotic cell lines. We found 11 motifs significantly associated with SCoC proteolysis, including IXXD and GD. We employed a stepwise method to select motif pairs that optimized SCoC specificity for a given coverage of SCoC cleavage events, yielding 11 motif pairs likely to be preferred in SCoC-directed human non-apoptotic caspase
consensus sequences. GD + IXXD was among these motif pairs, suggesting a conservation of non-apoptotic consensus sites among vertebrates.