Dataset Information

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

ABSTRACT: Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown to be cleaved by caspase-3, especially in non-apoptotic contexts. Functional annotation analysis revealed that our caspase-3 substrates were also enriched for proteins associated with several protein categories, including proteins found in extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem was highly enriched for our caspase-3 substrates. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of extracellular vesicle (EV) proteins.

SUBMITTER: Weghorst F

PROVIDER: S-EPMC7689216 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

Weghorst Forrest F Mirzakhanyan Yeva Y Samimi Kian K Dhillon Mehron M Barzik Melanie M Cunningham Lisa L LL Gershon Paul D PD Cramer Karina S KS

Frontiers in cellular neuroscience 20201112

Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown ...[more]

PMID: 33281555

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Project description: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.

Dataset Information

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

Publications

Caspase-3 Cleaves Extracellular Vesicle Proteins During Auditory Brainstem Development.

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