tetano
Editor, Senior Moderator
mBio
. 2021 Aug 17;e0209421.
doi: 10.1128/mBio.02094-21. Online ahead of print.
Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300
David A Davis[SUP] 1 [/SUP], Haydar Bulut[SUP] 1 [/SUP], Prabha Shrestha[SUP] 1 [/SUP], Amulya Yaparla[SUP] 1 [/SUP], Hannah K Jaeger[SUP] 1 [/SUP], Shin-Ichiro Hattori[SUP] 2 [/SUP], Paul T Wingfield[SUP] 3 [/SUP], John J Mieyal[SUP] 4 [/SUP], Hiroaki Mitsuya[SUP] 1 2 [/SUP], Robert Yarchoan[SUP] 1 [/SUP]
Affiliations
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (M[SUP]pro[/SUP]), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. M[SUP]pro[/SUP] is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show M[SUP]pro[/SUP] is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated M[SUP]pro[/SUP] could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M[SUP]pro[/SUP] primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated M[SUP]pro[/SUP] exists as a monomer. Tryptic and chymotryptic digestions of M[SUP]pro[/SUP] as well as experiments using a C300S M[SUP]pro[/SUP] mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon M[SUP]pro[/SUP] glutathionylation. These findings indicate that M[SUP]pro[/SUP] dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for M[SUP]pro[/SUP] activity, and provides a novel target for the development of agents to block M[SUP]pro[/SUP] dimerization and activity. This feature of M[SUP]pro[/SUP] may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (M[SUP]pro[/SUP]), a dimeric enzyme necessary for viral replication. Most work thus far developing M[SUP]pro[/SUP] inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for M[SUP]pro[/SUP] activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits M[SUP]pro[/SUP] activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.
Keywords: COVID-19; SARS-CoV-2; dimerization; drug targets; glutaredoxin; glutathionylation; main protease; oxidative stress; thioltransferase.
. 2021 Aug 17;e0209421.
doi: 10.1128/mBio.02094-21. Online ahead of print.
Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300
David A Davis[SUP] 1 [/SUP], Haydar Bulut[SUP] 1 [/SUP], Prabha Shrestha[SUP] 1 [/SUP], Amulya Yaparla[SUP] 1 [/SUP], Hannah K Jaeger[SUP] 1 [/SUP], Shin-Ichiro Hattori[SUP] 2 [/SUP], Paul T Wingfield[SUP] 3 [/SUP], John J Mieyal[SUP] 4 [/SUP], Hiroaki Mitsuya[SUP] 1 2 [/SUP], Robert Yarchoan[SUP] 1 [/SUP]
Affiliations
- PMID: 34399606
- DOI: 10.1128/mBio.02094-21
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (M[SUP]pro[/SUP]), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. M[SUP]pro[/SUP] is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show M[SUP]pro[/SUP] is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated M[SUP]pro[/SUP] could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M[SUP]pro[/SUP] primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated M[SUP]pro[/SUP] exists as a monomer. Tryptic and chymotryptic digestions of M[SUP]pro[/SUP] as well as experiments using a C300S M[SUP]pro[/SUP] mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon M[SUP]pro[/SUP] glutathionylation. These findings indicate that M[SUP]pro[/SUP] dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for M[SUP]pro[/SUP] activity, and provides a novel target for the development of agents to block M[SUP]pro[/SUP] dimerization and activity. This feature of M[SUP]pro[/SUP] may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (M[SUP]pro[/SUP]), a dimeric enzyme necessary for viral replication. Most work thus far developing M[SUP]pro[/SUP] inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for M[SUP]pro[/SUP] activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits M[SUP]pro[/SUP] activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.
Keywords: COVID-19; SARS-CoV-2; dimerization; drug targets; glutaredoxin; glutathionylation; main protease; oxidative stress; thioltransferase.