HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase

Abstract

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H2O2 resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H2O2 led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.

A peptide methionine sulfoxide reductase highly expressed in photosynthetic tissue in Arabidopsis thaliana can protect the chaperone‐like activity of a chloroplast‐localized small heat shock protein

“…Previously described forms of PMSR also depend on a reductant for activity, with thioredoxin most likely to be the endogenous reductant (Brot et al ., 1981; Moskovitz et al ., 1996). A functional restoration of activity, as seen for the chaperone‐like activity of Hsp21 in Figure 4, has previously been shown for α‐1‐proteinase inhibitor (Abrams et al ., 1981), calmodulin (Sun et al ., 1999) and the HIV‐2 protease (Davis et al ., 2000).…”

“…For example, the accumulation of methionine sulfoxides in calmodulin from brains of aging rats is accompanied by a decrease in the ability of calmodulin to activate the plasma membrane Ca‐ATPase (Gao et al ., 1998). Another interesting example is the oxidation‐induced inactivation of a protease in two different forms of the human infectious virus (HIV), HIV‐1 and HIV‐2 (Davis et al ., 2000). In the HIV‐1 protease, inactivation is mediated by glutathionylation of cysteine residue 95, an oxidative cross‐linking reaction between glutathione and the cysteine residue, forming a disulfide bridge.…”

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase

“…Oxidized β-casein Measured 1.6 ± 0.2 18.6 ± 6.0 90,000 Corrected 2 37.2 ± 12.0 45,000 1 Considering that only the S-diastereomer serve as substrates for DorA reductase [28] and assuming that the R-diastereomer does not act as inhibitor, the K M values were divided by 2. 2 As the ESI-MS analysis indicated that 2 MetO are available substrate for DorA (Figure 1), the K M value was multiplied by 2.…”

“…Either free or included in proteins, the sulfur containing amino acid methionine (Met) can be oxidized to methionine sulfoxide (MetO) which exists as two diastereomers, methionine-S-sulfoxide (Met-S-O) and methionine-R-sulfoxide (Met-R-O) [1]. Formation of MetO in proteins was shown to alter their function in numerous ways, from damageable effects for their activity and stability, to controlled regulation of enzyme activity and protein-protein interactions [2][3][4][5][6]. Contrary to oxidative modifications of most other amino acids, the oxidation of Met into MetO in proteins is reversible thanks to methionine sulfoxide reductase (Msr) enzymes, which are present in all organisms across the tree of life, with few exceptions [7,8].…”

Resolving oxidative damage to methionine by an unexpected membrane‐associated stereoselective reductase discovered using chiral fluorescent probes

“…Sulfur‐containing methionine residues in living organisms are susceptible to oxidation and can be transformed by exogenous and endogenous reactive oxygen species (ROS) to the corresponding methionine sulfoxide . This oxidative damage to methionine can disrupt the function of a variety of proteins . On the other hand, specific oxidation of methionine to methionine sulfoxide in proteins can also act as a gain‐of‐function post‐translational modification .…”