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Overlap of NatA and IAP substrates implicates N-terminal acetyltion in protein stabilization

ABSTRACT: SMAC/DIABLO and HTRA2 are mitochondrial proteins whose N-termina sequences, known as inhibitor of apoptosis binding motifs (IBMs), bind and activate ubiquitin ligases knwon as inhibitor of apoptosis proteins (IAPs), unleashing a cell´s poteintial.IBMs comprise a 4-residue, loose consensus sequence, and binding to IAPs requires an unmodified N-terminus. Closely related, IBM-like N-termini, are present in approximately 5% of human proteins. We show that suppression of the N-alpha-acetytransferase NatA truns these cryptic IBM-like sequences into very efficient IAP binders in cell lysates and in vitro, and ultimately also triggers apoptosis. Thus, N-terminal acetylation of IBM-like motifs in NatA substrates shields them from IAPs. This previously unrecognized relatonship suggests that N-terminal acetylation is generally protective against protein degradation in human cells. It also identifies IAPs as agents of a general quality control mechanism targeting unacetylated rogues in metazoans

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Tanja Bange

LAB HEAD: Andrea Musacchio

PROVIDER: PXD021815 | Pride | 2021-09-09

REPOSITORIES: Pride

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Project description:Metastatic malignant melanoma is the deadliest skin cancer type, and it is characterized by its high resistance to apoptosis. The main driving mutations in melanoma cause hyperactivation of genes within the ERK pathway, with BRAF mutations being the most frequent ones, followed by NRAS, NF1 and MEK mutations. Increasing evidence shows that the MST2/Hippo pathway is also deregulated in this cancer type. While mutations are rare, MST2/Hippo pathway core proteins expression levels are often dysregulated in melanoma. The expression of the tumour suppressor RASSF1A, a bona fide activator of the pro-apoptotic MST2 pathway, is silenced by promoter methylation in over half of melanomas and correlates with poor prognosis. Here, using mass spectrometry-based interaction proteomics we identified Second Mitochondria-derived Activator of Caspases (SMAC) as a novel LATS1 interactor. We show that RASSF1A-dependent activation of the pro-apoptotic MST2 pathway promotes LATS1-SMAC interaction and negatively regulates the anti-apoptotic signal mediated by the members of the IAP family. Moreover, proteomic experiments identified a common cluster of the apoptotic regulator that bind to SMAC and LATS1. Mechanistic analysis show that the LATS1-SMAC complex promotes XIAP ubiquitination and its subsequent degradation which ultimately results apoptosis. Importantly, we show that the oncogenic BRAFV600E mutant prevents the pro-apoptotic signal mediated by the LAST1-SMAC complex while treatment of melanoma cell lines with BRAF inhibitors promotes this complex, indicating that inhibition of the LATS1-SMAC might be necessary for BRAFV600E-driven melanoma. Finally, we show that LATS1-SMAC interaction is regulated by the SMAC mimetic Birinapant which requires the inhibition of C-IAP1 and the degradation of XIAP, indicating that the MST2/Hippo pathway is part of the mechanism of action of Birinapant. Overall, the current work shows SMAC-dependent apoptosis is regulated by the LATS1 tumour suppressor and supports the idea that LATS1 is a signalling hub that regulates the crosstalk between the MST2 pathway, the apoptotic network and the RAF/ERK pathway.

Project description:Rationale- NAA15 is a component of the N-terminal (Nt) acetyltransferase complex, NatA. The mechanism by which NAA15 haploinsufficiency causes congenital heart disease (CHD) remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective- We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity, and identifying proteins dependent upon a full amount of NAA15.Methods and Results - We introduced heterozygous LoF, compound heterozygous and missense residues (R276W) in iPS cells using CRISPR/Cas9. Haploinsufficient NAA15 iPS cells differentiate into cardiomyocytes, unlike NAA15-null iPS cells, presumably due to altered composition of NatA. Mass spectrometry (MS) analyses reveal ~80% of identified iPS cell NatA targeted proteins displayed partial or complete Nt-acetylation. Between null and haploinsufficient NAA15 cells Nt-acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W iPSCs. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; eighteen were ribosomal-associated proteins. At least four proteins were encoded by genes known to cause autosomal dominant CHD. Conclusions - These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and Nt-acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated CHD. Additionally, genetically engineered iPS cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.

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Overlap of NatA and IAP substrates implicates N-terminal acetyltion in protein stabilization

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