A Global Ligandability Map of Tryptoline Butynamide Stereoprobes Identifies Covalent Inhibitors of the Actin Maturation Protease.
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Pathway order: ACTMAP removes the N-terminally acetylated methionine of beta- and gamma-actin post-translationally, and NAA80 then re-acetylates the new N-terminus.
"protein identified in a haploid genetic screen to function as a protease that post-translationally cleaves the N-terminally acetylated methionine from β- and γ-actin, which are then reacetylated by N-acetyltransferase NAA80 to generate mature actins."
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The fold is that of a bacterial cysteine protease and Cys132 is the catalytic nucleophile.
"AlphaFold predictions revealed that ACTMAP has structural similarity to bacterial cysteine proteases with C132 representing the catalytic nucleophile."
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Covalent stereoprobes react with WT ACTMAP but not with the C132A mutant.
"we confirmed the stereoselective reactivity of WX-02-570 with recombinant WT-ACTMAP, but not a C132A mutant, both in HEK293T cells and in cell lysates by gel-ABPP"
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Specificity control on the residue assignment: two other cysteines remain reactive.
"Other representative cysteine mutants of ACTMAP (C119A and C271A) retained reactivity with WX-02-570"
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Genetic disruption of ACTMAP in human cells causes immature actin to accumulate, assayed with an antibody specific for the unprocessed beta-actin N-terminus.
"genetic disruption of ACTMAP by CRISPR/Cas9 gene editing resulted in substantial accumulation of immature actin as measured by Western blotting with an antibody recognizing the N-terminus of β-actin"
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Pharmacological engagement of Cys132 reproduces the genetic phenotype in human cancer cells.
"which produced a concentration-dependent increase in immature β-actin"
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Profilin 1 and 2 are established ACTMAP interactors and are recovered in ACTMAP immunoprecipitates.
"These IP-MS experiments also revealed stereoselective reductions in the enrichment of established ACTMAP-interacting proteins, such as profilin 1 and 2 (PFN1/2)"