Before project start-up we were able to define the identity of the actin N-terminal acetyltransferase (NAA80/ActNat) (PMID: 29581253) and solve its crystal structure (PMID: 29581307). We also managed to perform an initial characterization of the impact of actin Nt-acetylation on cell motility and cytoskeletal dynamics (PMID: 29581253).
In addition to defining the identity of the actin NAT, NAA80, several candidate proteins with an undefined function have been assessed as novel NATs, but we have no concluding data so far on additional human NAT enzymes.
However, functional and structural data uncovered that one particular candidate enzyme acetylates a specific amino acid in human cells (manuscript in preparation).
Another candidate enzyme harbored acetyltransferase activity towards lysine residues and further experiments are likely to validate this as a lysine acetyltransferase (KAT) (unpublished data).
We have obtained further data establishing that NAA80 knockout (KO) cells display an increased migration speed (Figure 1) (PMID:30534344 and unpublished data).
Furthermore, we established that the structure of the Golgi apparatus depends on NAA80 and proper actin Nt-acetylation, thus connecting the modification state of actin to the organization of the Golgi (PMID: 32209306). This effect was observed in both migrating and non-migrating cells and may be connected to an increased fraction of actin filaments (F-actin) relative to monomeric actin (G-actin).
In terms of cellular phenotypes, we have revealed that actin Nt-acetylation impacts initial cell spreading area and cell adhesion (unpublished data).
NAA80 KO zebrafish are viable and have a hearing defect similar to what is observed in patients with pathogenic NAA80 variants (manuscript in preparation).
Mechanistically, we have found that Profilins may stably interact with NAA80 and that such an interaction may play a functional role in NAA80-mediated actin Nt-acetylation.
A trimeric structure of Actin-Profilin-NAA80 revealed an extensive and conserved interaction interface between NAA80 and its substrate actin, and represented the first structure of a NAT enzyme bound to its entire substrate (PMID: 32284999). This interaction was essential for cellular acetylation of actin by NAA80. It was also established that NAA80 acetylated monomeric actin (G-actin) and not filamentous actin (F-actin). Cellular studies interestingly revealed that NAA80 strongly preferred complexing with the less abundant profilin, PFN2, and not the major PFN1 and that this interaction occurs without the presence of actin (PMID: 32978259). Our data propose a model where PFN2 stably associate with NAA80 to create activated NAA80-PFN2 dimers which may acetylate actin monomers before these are available for filament formation. In sum, our studies have established the molecular mechanism for actin N-terminal acetylation.
Bisubstrate NAA80 inhibitors have been optimized to facilitate further development of drug-like molecules (
https://doi.org/10.3389/fchem.2023.1202501(se abrirá en una nueva ventana)).
Regarding the mutually exclusive actin modifications Nt-acetylation and Nt-arginylation, we conclude that in mammalian cells the majority of endogenous actin is Nt-acetylated, while no Nt-arginylated actin is detected.
Lack of NAA80 shifts the bulk of actin from an acetylated state to an unacetylated state, and only a very small fraction of the available actin becomes arginylated (PMID: 34896361).
On the impact of Nt-acetylation, we defined in yeast that NatA was important for the integrity of the ribosome, implying that protein Nt-acetylation is important for protein-protein interactions and protein stability (Guzman et al. submitted, Preprint:
https://doi.org/10.1101/2022.10.17.512508(se abrirá en una nueva ventana)).
In human cells, we revealed that NatC-mediated Nt-acetylation confers protein stability. Nt-acetylation shields Methionine starting proteins from the recognition by specific Ubiquitin E3 ligases KCMF1-UBR4 thus preventing their proteasomal degradation (Varland et al. submitted, Preprint: doi:
https://doi.org/10.1101/2022.09.01.505523(se abrirá en una nueva ventana)). NatC KO-induced protein degradation and phenotypes are reversed by Ub E3 ligase knockdown, demonstrating the central cellular role of this interplay. In fruit fly, we find that this pathway is important for organismal longevity and motility.
In terms of human pathophysiology, we recently uncovered that several individuals with primary familial brain calcification harbored pathogenic NAA60 variants, thus causally linking NatF mediated post-translational Nt-acetylation to neurological disease (Chelban et al., submitted).
