Report
Teaser, summary, work performed and final results
Periodic Reporting for period 1 - NterAct (Discovery and functional significance of post-translational N-terminal acetylation)
Teaser
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...
Summary
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), which for many years have been considered an extended part of the cellular translation machinery. Since Nt-acetylation was thought to be unregulated and irreversible, it has remained vastly understudied. However, there are several examples of post-translationally Nt-acetylated proteins and peptides for which the functional consequences at the cellular and organismal levels are highly significant.
In the last decade, we revealed 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.
The peptide hormones α-melanocyte stimulating hormone (α-MSH) and β-endorphin (β-END) regulate appetite, metabolism, sexual behaviour and pain, and their activity has long been known to be critically modulated by post-translational Nt-acetylation. The identity of the NAT catalysing these acetylations, here denoted HormNat, remains unknown, despite its significance and the evidence of its existence. Since HormNat has a key role in physiological processes, its identification is crucial for a detailed understanding of how the body regulates these circuits.
Another example is the unique post-translational Nt-acetylation of actin conserved among animal species with potential major impacts on the actin cytoskeleton. Also this NAT, here denoted ActNat, has remained unidentified for decades.
This proposal aims to identify and characterise all NATs acting post-translationally as well as enzymes reversing this modification, Nt-deacetylases (NDACs).
The proposed project has three main objectives:
I)To identify all human NATs acting post-translationally with emphasis on ActNat and HormNat.
II)To elucidate the molecular mechanisms, regulation and impact of the novel NATs.
III)To define the dynamics and reversibility of Nt-acetylation and potential Nt-deacetylases (NDACs).
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.
Work performed
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 this period (month 1-18), we have obtained further data establishing that NAA80 knockout (KO) cells display an increased migration speed (Figure 1) (PMID:30534344 and unpublished data). Also, we have revealed that actin Nt-acetylation impacts cell adhesion and potentially invasiveness as well as other phenotypes (unpublished).
Several additional cell models including muscle cells have been developed for the study of actin Nt-acetylation (NAA80 KO) and are under investigation.
Mechanistically, we have verified that Profilins may stably interact with NAA80 and that such an interaction may play a functional role in NAA80-mediated actin Nt-acetylation (unpublished).
Several candidate proteins with an undefined function are currently under assessment as novel NATs and HormNat, but we have no concluding data so far.
Our studies on the dynamics of Nt-acetylation have progressed. Regarding actin Nt-acetylation and actin Nt-arginylation, we are soon ready to conclude on the abundancy and potential interplay between these two mutually exclusive modifications.
Final results
We have made significant progress on defining the mechanisms for NAA80 mediated actin Nt-acetylation.
In the next period, we anticipate to complete several milestones related to this including the trimeric structure of Actin-NAA80-Profilin and the impact of Profilins on NAA80 function.
The NAA80 enzyme is unique in many aspects among the human NATs, and its mechanism of operation has revealed important insights of conceptual interest.
Further we have expanded our knowledge beyond state of the art concerning the cellular impact of protein Nt-acetylation, by in depth studies of actin Nt-acetylation. We expect to publish several novel phenotypes until the end of the project, including some with a potential clinical impact.
Data on HormNat and other novel NATs as well as potential NDACs are not conclusive, but we remain optimistic that we in the project period may be able to define some of these factors.
Website & more info
More info: https://www.uib.no/en/rg/nat.