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

Abstract

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.

Extra-mitochondrial mouse frataxin and its implications for mouse models of Friedreich’s ataxia

“…Without the protection of mitochondrial GRP75, cytosolic mature mouse frataxin could then be a substrate for XPNPEP3 action. Removal of this N-terminal leucine residue would then generate truncated mature mouse frataxin (79-207) 29 , the major proteoform that we found in mouse tissues. Subsequent sequential hydrolysis of single N-terminal residues would then result in additional truncated proteoforms.…”

“…To date, a non-specific protease capable of truncating frataxin in mammalian cells has not yet been identified. However, legumain, an asparaginyl peptidase recently identified in macrophages, can hydrolyze the full-length mouse frataxin at arginine-77 29 .…”

“…Therefore, it is conceivable that other frataxin proteoforms can be processed in mammalian cells without requiring the action of mitochondrial MPP. In fact, the asparaginyl endopeptidase, legumain, was shown recently to cleave full-length mouse frataxin at asparagine-77, producing mature mouse frataxin (78-207) 29 . Without the protection of mitochondrial GRP75, cytosolic mature mouse frataxin could then be a substrate for XPNPEP3 action.…”

“…To date, a non-specific protease capable of truncating frataxin in mammalian cells has not yet been identified. However, legumain, an asparaginyl peptidase recently identified in macrophages, can hydrolyze the full-length mouse frataxin at arginine-77 29 . Human mature SILAC-frataxin (81-210) is 98.5% homologous and 92.3% identical with the predicted sequence of mouse mature frataxin (78-207).…”

Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

“…Our current proteomic analysis has revealed enrichment of several newly associated but also previously reported proteases and protease inhibitors in SS. The activity of cysteine proteases (cathepsins) and serine proteases (neutrophil elastase, prostasin, proteinase-3/myeloblastin) could be further investigated using activity-based probes in SS ( Edgington-Mitchell et al, 2017 ; Anderson et al, 2019 , 2020 ; Mainoli et al, 2020 ; Mountford et al, 2020 ; Tu et al, 2021 ). The upregulation of cathepsin G in the saliva of patients with SS is likely associated with an increase in neutrophils and corresponds to an elevation in synovial fluid of RA patients ( Gao, 2018 ).…”