Structural and functional changes in RNAse A originating from tyrosine and histidine cross-linking and oxidation induced by singlet oxygen and peroxyl radicals

Abstract

Oxidation can be induced by multiple processes in biological samples, with proteins being important targets due to their high abundance and reactivity. Oxidant reactions with proteins are not comprehensively understood, but it is known that structural and functional changes may be a cause, or a consequence, of disease. The mechanisms of oxidation of the model protein RNAse A by singlet oxygen (O) were examined and compared to peroxyl radical (ROO) oxidation, both common biological oxidants. This protein is a prototypic member of the RNAse family that exhibits antiviral activity by cleaving single-stranded RNA. RNAse A lacks tryptophan and cysteine residues which are major oxidant targets, but contains multiple histidine, tyrosine and methionine residues; these were therefore hypothesized to be the major sites of damage. O and ROO induce different patterns and extents of damage; both induce cross-links and side-chain oxidation, and O exposure modulates enzymatic activity. Multiple products have been characterized including methionine sulfoxide and sulfone, alcohols, DOPA, 2-oxohistidine, histidine-derived ring-opened species and inter- and intra-molecular cross-links (di-tyrosine, histidine-lysine, histidine-arginine, tyrosine-lysine). In addition to methionine modification, which appears not to be causative to activity loss, singlet oxygen also induces alteration to specific histidine, tyrosine and proline residues, including modification and cross-linking of the active site histidine, His12. The high homology among the RNAse family suggests that similar modifications may occur in humans, and be associated with the increased risk of viral infections in people with diabetes, as markers for O have been found in early stages of this pathology.

UV/enzyme dual responsive photosensitizer‐loaded 4‐(Phenylazo)benzoic Acid‐mPEG nanosystem for enhanced photodynamic insecticide efficacy

“…Photosensitizers can produce 1 O 2 under the irradiation of UV or visible light, and many studies have shown that L‐Tyrosine (L‐Tyr) is the main oxidation target of 1 O 2 40–42 . After L‐Tyr is oxidized, the fluorescence intensity at 305 nm will decrease.…”

“…Photosensitizers can produce 1 O 2 under the irradiation of UV or visible light, and many studies have shown that L-Tyrosine (L-Tyr) is the main oxidation target of 1 O 2 . [40][41][42] After L-Tyr is oxidized, the fluorescence intensity at 305 nm will decrease. Therefore, the photodynamic activity of photosensitizers can be characterized by measuring the change of the fluorescence intensity of L-Tyr in the sample solution.…”

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification

“…Di-Tyr crosslinks have also been detected on synthetic peptides exposed to peroxidase activity (e.g., hemoglobin and H 2 O 2 [ 141 ]), and a large number of isolated proteins including (amongst others): lipoproteins [ 61 , 70 , 71 ], glucose 6-phosphate dehydrogenase (G6PDH) [ 53 ], ribonuclease A (RNAse A) [ 86 ], multiple extracellular matrix proteins [ 57 , 58 , 76 ], calmodulin [ 72 ], lysozyme [ 54 , 79 ], myoglobin [ 142 , 143 ], insulin [ 48 , 73 , 74 ] and human Δ25 centrin 2 [ 75 ].…”

UV oxidation of cyclic AMP receptor protein, a global bacterial gene regulator, decreases DNA binding and cleaves DNA at specific sites

“…products was determined on peptides generated by tryptic digestion as described previously [31][32][33] . Samples (150 µg protein) were prepared as described above, then subjected to buffer exchange into 100 mM ammonium bicarbonate buffer (ABC buffer) using spin filters (10 kDa cut-off).…”

“…Carbamidomethylation of cysteine was used as a fixed modification. Data was filtered in order to extract peptide-spectrum matches corresponding to established oxidative modifications using a list of known oxidative modifications 36 , with changes at Met, His, Tyr and Pro as variable modifications 29,31 . The % modification at a particular site was estimated using label-free quantification ratios relative to the parent, as determined using Maxquant.…”