N‑Terminomics/TAILS Profiling of Macrophages after Chemical Inhibition of Legumain

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.