Periodic Reporting for period 4 - NterAct (Discovery and functional significance of post-translational N-terminal acetylation)
Período documentado: 2022-12-01 hasta 2023-05-31
In mammalian cells, around 80% of cellular proteins are subjected to N-terminal (Nt) acetylation, thus making this one of the most abundant protein modifications.
For most Nt-acetylated proteins the acetyl group is added co-translationally by the action of ribosome-associated N-terminal acetyltransferases (NATs).
In the last decade, we defined the presumed complete human co-translational NAT-machinery, NatA-NatE, and the first NAT acting post-translationally, NatF. However, significant regulatory NATs remain to be discovered and the roles of Nt-acetylation at the biochemical and physiological levels are not understood.
There are several examples of Nt-acetylated proteins and peptides for which the functional consequences at the cellular and organismal levels are highly significant.
One example is the most abundant intracellular protein in human cells, the cytoskeletal protein actin involved in numerous cellular functions such as cell motility and intracellular transport.
This proposal aimed to identify and characterize NAT enzymes, including their molecular mechanisms, regulation and impact.
The output will be the identification of key regulatory switches and insights beyond state-of-the-art at the molecular, cellular and organism levels.
The potential societal impact includes knowledge on essential regulatory switches in human physiology and the ability to regulate these to fight disease and improve health.
For most Nt-acetylated proteins the acetyl group is added co-translationally by the action of ribosome-associated N-terminal acetyltransferases (NATs).
In the last decade, we defined the presumed complete human co-translational NAT-machinery, NatA-NatE, and the first NAT acting post-translationally, NatF. However, significant regulatory NATs remain to be discovered and the roles of Nt-acetylation at the biochemical and physiological levels are not understood.
There are several examples of Nt-acetylated proteins and peptides for which the functional consequences at the cellular and organismal levels are highly significant.
One example is the most abundant intracellular protein in human cells, the cytoskeletal protein actin involved in numerous cellular functions such as cell motility and intracellular transport.
This proposal aimed to identify and characterize NAT enzymes, including their molecular mechanisms, regulation and impact.
The output will be the identification of key regulatory switches and insights beyond state-of-the-art at the molecular, cellular and organism levels.
The potential societal impact includes knowledge on essential regulatory switches in human physiology and the ability to regulate these to fight disease and improve health.
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).
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).
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). 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).
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).
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).
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). 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).
We have made significant progress on defining the novel N-terminal acetyltransferase NAA80, including the mechanisms for NAA80 mediated actin Nt-acetylation as well as elucidating the cellular and organismal impact of this actin modification.
This importantly includes a trimeric structure of NAA80-Actin-Profilin, the first structure of a NAT enzyme bound to its entire substrate.
These studies represent a novel concept in the field of protein N-terminal acetylation since it stresses how this modification may operate post-translationally as compared to the established co-translational mode.
In our survey of uncharacterized putative human acetyltransferases, we have also uncovered a novel amino acid acetyltransferase and a lysine acetyltransferase of which the impact pends further investigation.
We have defined that acetylation is the dominant actin Nt-modification, while actin Nt-arginylation is not detectable in cells.
Further we have expanded our knowledge beyond state of the art concerning the cellular and organismal impact of protein Nt-acetylation.
For actin, lack of Nt-acetylation impacts cell motility, cell adhesion, actin polymerization, the Golgi apparatus, and impairs hearing in animals.
In a more general perspective, we have established the role of protein Nt-acetylation in promoting protein stability and its involvement in neurological disease.
This importantly includes a trimeric structure of NAA80-Actin-Profilin, the first structure of a NAT enzyme bound to its entire substrate.
These studies represent a novel concept in the field of protein N-terminal acetylation since it stresses how this modification may operate post-translationally as compared to the established co-translational mode.
In our survey of uncharacterized putative human acetyltransferases, we have also uncovered a novel amino acid acetyltransferase and a lysine acetyltransferase of which the impact pends further investigation.
We have defined that acetylation is the dominant actin Nt-modification, while actin Nt-arginylation is not detectable in cells.
Further we have expanded our knowledge beyond state of the art concerning the cellular and organismal impact of protein Nt-acetylation.
For actin, lack of Nt-acetylation impacts cell motility, cell adhesion, actin polymerization, the Golgi apparatus, and impairs hearing in animals.
In a more general perspective, we have established the role of protein Nt-acetylation in promoting protein stability and its involvement in neurological disease.