Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides

Abstract

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO 2 Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c ) and tyrosine nitrated proteoforms (NO 2 Y74-Cyt c and NO 2 Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO 2 Y74- and NO 2 Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c . Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO 2 Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO 2 Y-containing analog, suc-LLVNO 2 Y-AMC (NO 2 Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO 2 Y-AMC. Moreover, NO 2 Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO 2 Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO 2 Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO 2 Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO 2 Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

High glucose leads to redistribution of the proteasomal system

“…Proteasomal activity was measured via a modified protocol according to 41 . Fibroblasts were cultured as described above.…”

“…Proteasomal activity was measured via a modified protocol according to. 41 Fibroblasts were cultured as described above. Both untreated control and cells after 5 days of HG-exposure were harvested by trypsination and lysed in 1 mM DTT, then passed 20 times through a 27-gauge needle attached to a 1 mL syringe, followed by centrifugation (12,000 g for 15 min at 4 C) in order to remove nonlysed cells and membrane debris.…”

Unveiling the human nitroproteome: Protein tyrosine nitration in cell signaling and cancer

“…However, the data remain incomplete as the definite documentation of a bonafide denitrase, that is, an enzyme that removes the nitro group without degrading the protein, awaits confirmation. Removal of nitrated proteins by various cellular proteolytic systems has been clearly demonstrated ( 125 , 126 , 127 ). Alternatively, nitrated proteins may be removed via immunological processes.…”

Protein Tyrosine Nitration in Plant Nitric Oxide Signaling

“…NRs are polyubiquitinated ( Costa-Broseta et al, 2021 ) and are translocated to the nucleus by the SUMO E3 ligase SIZ1 ( Kim et al, 2018 ). Remarkably, a recent report showed that proteasomal-mediated cleavage at C-terminal of nitroTyr residues in proteins was reduced when compared to the unmodified residues, making nitration a possible factor that decreases the turnover of oxidized proteins ( Ott et al, 2021 ), and casting doubts on the relationship between protein nitration and proteasome-mediated degradation. However, polyubiquitination is not the only PTM functionally connected to Tyr nitration.…”