N-Terminomics/TAILS Profiling of Macrophages after Chemical Inhibition of Legumain
| Autor: | Luiz Gustavo de Almeida, Antoine Dufour, Henna Sekhon, Laura E. Edgington-Mitchell, Daniel Young, Bethany M. Anderson |
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| Rok vydání: | 2019 |
| Předmět: |
Proteomics
Iron medicine.medical_treatment Mitochondrion Legumain Biochemistry Mice 03 medical and health sciences 0302 clinical medicine medicine Animals Humans Asparagine Shotgun proteomics 030304 developmental biology 0303 health sciences Protease biology Chemistry Macrophages Endopeptidase Mitochondria 3. Good health Cell biology Cysteine Endopeptidases RAW 264.7 Cells Gene Expression Regulation Isotope Labeling 030220 oncology & carcinogenesis biology.protein Frataxin Peptides |
| Zdroj: | Biochemistry. 59:329-340 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b00821 |
| Popis: | Legumain (asparaginyl endopeptidase) is the only protease with a preference for cleavage after asparagine residues. Increased legumain activity is a hallmark of inflammation, neurodegenerative diseases, and cancer, and legumain inhibitors have exhibited therapeutic effects in mouse models of these pathologies. Improved knowledge of its substrates and cellular functions is a requisite to further validation of legumain as a drug target. We, therefore, aimed to investigate the effects of legumain inhibition in macrophages using an unbiased and systematic approach. By shotgun proteomics, we identified 16 094 unique peptides in RAW264.7 cells. Among these, 326 unique peptides were upregulated in response to legumain inhibition, while 241 were downregulated. Many of these proteins were associated with mitochondria and metabolism, especially iron metabolism, indicating that legumain may have a previously unknown impact on related processes. Furthermore, we used N-terminomics/TAILS (terminal amine isotopic labeling of substrates) to identify potential substrates of legumain. We identified three new proteins that are cleaved after asparagine residues, which may reflect legumain-dependent cleavage. We confirmed that frataxin, a mitochondrial protein associated with the formation of iron-sulfur clusters, can be cleaved by legumain. This further asserts a potential contribution of legumain to mitochondrial function and iron metabolism. Lastly, we also identified a potential new cleavage site within legumain itself that may give rise to a 25 kDa form of legumain that has previously been observed in multiple cell and tissue types. Collectively, these data shed new light on the potential functions of legumain and will be critical for understanding its contribution to disease. |
| Databáze: | OpenAIRE |
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