Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin

Abstract

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.

Keywords: 4-hydroxy-2-nonenal; Alzheimer's disease; oxidative stress; photocrosslinking; tandem mass spectrometry; ubiquitin.

Figure 1

+ve ESI mass spectra for Ub solution at the indicated concentrations.

Figure 2

+ve ESI mass spectra for Ub solution 100µM after incubation with HNE 400 µM for 24 h in HEPES buffer without (a) and with (b) the addition of Guanidine-HCl Solution (final concentration 6M).

Figure 3

Kinetics of HNE (400 µM) addition to Ub (40 µM) in HEPES as measured by MRM/MS. The ratio between the Ub, Ub·HNE₁ and Ub·HNE₂ peaks, respectively black, blue and grey and spiked bradykinin was monitored as a function of reaction time. Error bars have been calculated from the standard deviations obtained from 3 different measurements.

Figure 4

Ub sequence showing consistent trypsin cleavage sites (solid arrows) and inconsistent cleavage sites (open arrows). Asterisks indicate sites of HNE modification. Low: Ub ≤ 5 µM or Ub ≤ 15 µM + HNE. High: Ub > 15 µM.

Figure 5

SDS-PAGE using 10–20% Tricine gels and silver-stained. Oligomeric species detected for Ub solutions at the indicated conditions. Splitting of the bands is likely due to alternative crosslink structures. The more intense monomeric and dimeric bands in the sixth line are due to sample overload. Experiment was performed in duplicate.

Figure 6

Crystal structure (1UBQ) of Ub showing the location of ⁶Lys and ⁶⁸His side chains.