Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

Kasson, Samuel; Dharmapriya, Nuwani; Kim, In‐Kwon

doi:10.1371/journal.pone.0254022

Cited by 10 publications

(9 citation statements)

References 53 publications

(90 reference statements)

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“…MARylated PARP10 (0.5 μM) was incubated with Mac1 variants (20 μM) for 1 hour at room temperature prior to SDS-PAGE and transfer to nitrocellulose membrane and blotting with anti-MAR/PAR antibody (1:1000 dilution, Cell Signaling, 83732). G) Kinetic measurements of MARylated PARP10 hydrolysis by Mac1 variants (concentration indicated between parentheses) using NudT5/AMP-Glo to detect ADP-ribose [ 51 , 59 ]. H) Catalytic efficiency ( k cat / K M ) was determined by linear regression of data in G using GraphPad Prism.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo

Taha,

Suryawanshi,

Chen

et al. 2023

PLoS Pathog

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Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.

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Section: Resultsmentioning

confidence: 99%

“…Human NUDT5 (residues 1–219) was expressed from a pET21b vector containing a 3C-protease cleavage N-terminal His 6 tag [ 59 ] using BL21(DE3) E . coli cells co-transformed with a pKJE7 plasmid expressing the chaperones DnaK, DnaJ and GrpE (Takara, 3340).…”

Section: Methodsmentioning

confidence: 99%

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo

Taha,

Suryawanshi,

Chen

et al. 2023

PLoS Pathog

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“…The ADP-ribosyl hydrolase activity of macrodomains was determined using the previously described method (54) with auto-MARylated PARP10 as substrate (53). ADP-ribose produced by Mac1 is hydrolyzed by the NUDT5 phosphodiesterase to AMP, which is detected using the AMP-Glo assay kit (Promega, V5011).…”

Section: Parp10 Solution Assaymentioning

confidence: 99%

“…MARylated PARP10 (0.5 µM) was incubated with Mac1 variants (20 µM) for 1 hour at room temperature prior to SDS-PAGE and transfer to nitrocellulose membrane and blotting with anti-MAR/PAR antibody (1:1000 dilution, Cell Signaling, 83732). G) Kinetic measurements of MARylated PARP10 hydrolysis by Mac1 variants (concentration indicated between parentheses) using NudT5/AMP-Glo to detect ADP-ribose (53,54). Catalytic efficiency (kcat/KM) was determined by linear regression using GraphPad Prism.…”

Section: E)mentioning

confidence: 99%

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesisin vivo

Taha

Suryawanshi

Chen

et al. 2023

Preprint

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Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the therapeutic potential of Mac1 inhibition, we generated recombinant viruses and replicons encoding catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced activity by ~10-fold, mutations to aspartic acid (N40D) reduced the catalytic activity by ~100-fold relative to wildtype activity. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, N40D replicated at >1000-fold lower levels while inducing a robust interferon response, and all infected animals survived infection with the mutant, but not the wildtype virus. Our data validate SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a reasonable target to develop antivirals, while emphasizing the importance of amino acid identity in viral mutagenesis studies and underscoring the limitations of solely relying on in vitro viral replication studies for target validation.

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“…ADP-ribose can also be converted enzymatically to AMP that can be subsequently detected. [58,59,69,70] ADP-ribose is released from the MARylated protein by the macrodomain and the assay utilizes a NudF or a Nudt5 ADPribose pyrophosphatase that cleave ADP-ribose to AMP and ribose 5phosphate. The amount of AMP can be measured with a commercial AMP-Glo reagent.…”

Section: Activity-based Assays For Adp-ribosylation Erasersmentioning

confidence: 99%

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

2021

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ADP-ribosylation is a post-translational modification catalyzed by writer enzymes -ADP-ribosyltransferases. The modification is part of many signaling events, can modulate the function and stability of target proteins, and often results in the recruitment of reader proteins that bind to the ADP-ribosyl groups. Erasers are integral actors in these signaling events and reverse the modification. ADP-ribosylation can be targeted with therapeutics and many inhibitors against writers exist, with some being in clinical use. Inhibitors against readers and erasers are sparser and development of these has gained momentum only in recent years. Drug discovery has been hampered by the lack of specific tools, however many significant advances in the methods have recently been reported. We discuss assays used in the field with a focus on methods allowing efficient identification of small molecule inhibitors and profiling against enzyme families. While human proteins are focused, the methods can be also applied to bacterial toxins and virus encoded erasers that can be targeted to treat infectious diseases in the future.

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Selective monitoring of the protein-free ADP-ribose released by ADP-ribosylation reversal enzymes

Cited by 10 publications

References 53 publications

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication in vivo

A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesisin vivo

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

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