Lipid Peroxidation Products Influence Calpain-1 Functionality In Vitro by Covalent Binding

Abstract

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 μM MDA but not with 500 and 1000 μM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 μM. No difference in calpain-1 autolysis was observed between the control and 1000 μM MDA. However, 1000 μM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

Secondary Lipid Oxidation Products as Modulators of Calpain-2 Functionality In Vitro

“…This may suggest that 2-hexenal and 4-hydroxynonenal modify residues near or adjacent to the active site but do not directly modify the residues within the active site. Similar results with calpain-1 were observed, where incubation with malondialdehyde at 100, 500, and 1000 μM had minimal impact on calpain-1 activity and autolysis, while 2-hexenal and 4-hydroxynonenal decreased the activity at all concentration levels tested and slowed down calpain-1 autolysis …”

“…The reactivity and adduction of lipid oxidation products to proteins have been evaluated with mass spectrometry approaches over the last two decades. Specific oxidation product adduction sites on tryptic peptides have been identified in vitro , including 4-hydroxynonenal modifications on lactate dehydrogenase, cytochrome c, , cytochrome c oxidase, hemoglobin, myoglobin, ,,, apomyoglobin, ,, calpain-1, and soy protein isolate, malondialdehyde modifications on pyruvate kinase, calpain-1, and soy protein isolate, and 2-hexenal modifications on model peptides . Many of these studies aimed to explain the mechanisms of the observed changes in protein function due to incubation with lipid oxidation products by identifying and quantifying the location and number of lipid oxidation products.…”

“…Calpain-2 (5 μg) was incubated with 100, 500, or 1000 μM of malondialdehyde, 2-hexenal, or 4-hydroxynonenal, or equal volumes of TEM (controls) buffer, for 24 h at 4 °C. The concentrations utilized throughout this study were based on previous in vitro studies of purified proteins ,, and were exploratory and titrated across three different concentrations. These concentrations are likely greater than those achieved in vivo in living and post-mortem skeletal muscles, but further characterization of these lipid oxidation product concentrations in vivo is warranted.…”

“…The dynamic modifications were methionine oxidation (+15.995 Da), carbamidomethylation of cysteine residues (+57.021 Da), and acetylation of the protein N terminus (+42.011 Da). Two additional Sequest HT nodes were included to separately search for dynamic MDA, HXL, and HNE modifications of cysteine, histidine, lysine, glutamine, arginine, and asparagine residues, including various product masses as described previously . Peptide spectrum matches (PSM) were validated using the Percolator node with a false discovery rate (FDR) of ≤1%.…”

“…Compounds such as malondialdehyde, acrolein, 4-hydroxyhexanal, and 4-hydroxy-2-nonenal have been most extensively studied as protein lipoxidation modifications, and additional compounds, such as 2-hexenal, − hexanal, 4-oxo-2-nonenal, and others, have been less studied. Purified or isolated proteins, including lactate dehydrogenase, pyruvate kinase, cytochrome c, , cytochrome c oxidase, β-lactoglobulin B, , hemoglobin, ,, insulin, , myoglobin, − apomyoglobin, ,, and calpain-1, are modified by lipid oxidation products in in vitro systems. Functional protein analysis combined with the mass spectrometry approach has demonstrated almost exclusive negative impacts of lipid oxidation on protein functionality and has provided insights into mechanistic modifications, which partly explain the differences in protein functionality.…”