Giglione, C. | bioRxiv Science

bioRxiv2023

NatB-dependent acetylation protects procaspase-8 from UBR4-mediated degradation and is required for full induction of the extrinsic apoptosis pathway

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB-mediated acetylation serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to extrinsic pro-apoptotic stimuli, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway impacts apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

biochemistry↗

bioRxiv2021

Proteome-wide probing of the dual NMT-dependent myristoylation tradeoff unveils potent, mechanism-based suicide inhibitors

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a major and ubiquitous lipid modification. Originally thought to modify only N-terminal glycine -amino groups (G-myristoylation), NMTs are now known to modify lysine {varepsilon}-amino groups (K-myristoylation), the significance of which is uncertain. Here we exploited systematic structural proteomics analyses and a novel pipeline involving the Shigella IpaJ protease to discriminate K- and G-myristoylation with unprecedented accuracy and identify the specific features driving each modification. NMT-dependent K-myristoylation occurs post-translationally and only on lysines 1, 2, or 3 following G-myristoylation or caspase cleavage. Direct interactions between the substrates reactive amino group and the NMT catalytic base slow K-myristoylation catalysis. IpaJ unmasked novel K-myristoylation sites in a dozen human proteins. The unique properties of NMT-driven K-myristoylation allowed us to design potent, mechanism-based suicide NMT inhibitors. These analyses unravel the respective paths towards K-myristoylation, G-myristoylation, or NMT inhibition, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group. SIGNIFICANCE STATEMENTWe report the specific and unique elements guiding N-myristoyltransferase to either alpha or epsilon myristoylation, allowing us to establish the post-translational nature of N-myristoyltransferase-dependent lysine myristoylation and design novel, potent N-myristoyltransferase inhibitors.

biochemistry↗