SIRT5 catalytic activity is necessary to interact with Nsp14.

Abstract

A. Cartoon representation of the protein structure of SIRT5 catalytic site in complex with cofactor NAD and succinylated lysine substrate (SuK), showing conserved residues mutated in panel B. B. Strep-tag affinity-purification and western blot after transfection of Sirt5-KD HEK293T cells with Nsp14-strep and SIRT5 catalytic mutants, showing that the interaction with Nsp14 is lost in several mutants. C. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with increasing concentrations of SIRT5 inhibitor Sirt5-i. High concentrations of Sirt5-i prevent the interaction. D. Strep-tag affinity-purification and western blot after transfection with Nsp14-strep and SIRT5, in SIRT5-KD HEK293T cells incubated with NAMPT FK866 inhibitor (low cellular NAD), FK866 and NMN, or NMN alone (high cellular NAD). SIRT5 binding strength correlated with NAD levels. E. Pan-acetylation, malonylation and succinylation in SIRT5-KD HEK293T total or Strep-purified proteins, after transfection with Nsp14-Strep, GFP-strep control and/or SIRT5. No specific lysine modifications could be detected. F. Summary of mass spectrometry experiments. Nsp14-strep proteins purified from SIRT5-KD HEK293T, with or without co-transfection with SIRT5, were analyzed by mass spectrometry. No acetylation, malonylation, succinylation or glutarylation modifications could be detected.</p