Fig. 2: Conversion of PR-Ub into ADPR-Ub by LnaB requires Actin as a co-factor.

a Native lysates of mammalian cells activate LnaB. Native (N) or boiled (B) lysates of 293HEK cells were added to reactions containing PR-Ub, MavK, or LnaB, and the production of ADPR-Ub was detected by immunoblotting. b Identification of Actin as a LnaB-binding protein. Flag-LnaB expressed in HEK293T cells was isolated by immunoprecipitation and the bound proteins were identified by mass spectrometry. Similarly obtained samples with Flag-RavN were used as a control. alpha Actin (ACTC1) and beta Actin-like 2 (ACTBL2) were among the most abundant proteins identified. c Interactions between LnaB and Actin measured by ITC. Raw ITC curves (top panel) and binding isotherms with fitting curves (bottom panel) of LnaB titration by Actin. The thermogram is a monophasic curve with an inflection point at a molar ratio of 0.84. The binding affinity is approximately 1.24 μM, and the stoichiometry is 1:1 of Actin:LnaB. The thermodynamic parameters were also shown, ΔH: − 6.88 kJ•mol⁻¹ and ΔS: 88.75 J•mol⁻¹•K⁻¹. d LnaB and actin utilize ATP to convert PR-Ub into ADPR-Ub. Actin was added to a subset of reactions containing LnaB and PR-Ub. Samples separated by SDS-PAGE were probed for ADPR-Ub (upper panel), ubiquitin, LnaB, or Actin by immunoblotting with antibodies specific to each protein or its epitope tag. e, f. LnaB transfers the AMP moiety of ATP to PR-Ub. The chemical structure of ADPR-Ub with the AMP moiety added to the phosphate group on PR-Ub being highlighted (dashed box) (e). ³²P-α-ATP was added to the indicated reactions and incubated at 37 ^oC for 1 h. Samples separated by SDS-PAGE were detected by CBB staining (left) and autoradiograph, respectively. Note the presence of self-modified LnaB in the reaction without PR-Ub (f). g ATP analogs with a cleavable α phosphate support LnaB activity. Samples of reactions receiving the indicated ATP analogs were resolved by SDS-PAGE and ADPR-Ub and the reactants were detected by immunoblotting by antibodies specific for ADPR, Ub, LnaB, or Actin. Note that ApCpp is uncleavable at the α position and thus did not support the activity of LnaB. In each case, similar results were obtained in at least three independent experiments.