Towards a Functional Understanding of Protein N-Terminal Acetylation

Protein N-terminal acetylation is a major modification of eukaryotic proteins. Its functional implications include regulation of protein–protein interactions and targeting to membranes, as demonstrated by studies of a handful of proteins. Fifty years after its discovery, a potential general function of the N-terminal acetyl group carried by thousands of unique proteins remains enigmatic. However, recent functional data suggest roles for N-terminal acetylation as a degradation signal and as a determining factor for preventing protein targeting to the secretory pathway, thus highlighting N-terminal acetylation as a major determinant for the life and death of proteins. These contributions represent new and intriguing hypotheses that will guide the research in the years to come

Setup and Integration of the Readout Electronics for the Pixel Layers of the New ALICE FoCal Detector

Masteroppgave i fysikkPHYS399MAMN-PHY

A synopsis of eukaryotic Nα-terminal acetyltransferases: nomenclature, subunits and substrates

We have introduced a consistent nomenclature for the various subunits of the NatA-NatE N-terminal acetyltransferases from yeast, humans and other eukaryotes

"The role of ICT in the teaching of English as a Foreign Language in Norwegian lower secondary schools : a study of ICT use and patterns of associated factors"

Studien set søkelyset på engelsklærarar sin bruk av IKT og kva som skil engelsklærarar med lågfrekvent og høgfrekvent bruk. Datamateriale frå engelsklærarar ved eit tilfeldig utval ungdomsskular frå heile landet vart samla inn ved hjelp av ein webbasert survey med 248 spørsmål. Spørsmåla om lærarane sin generelle og faglege bakgrunn, samt erfaringar og syn på IKT, vart plukka ut på bakgrunn av funn frå tidlegare forsking, læreplanmål, tilbakemeldingar frå piloteringa og eigne erfaringar som ungdomsskulelærar. Data vart analyserte ved hjelp av deskriptiv statistikk, korrelasjonsanalysar, analysar av varians (ANOVA), faktoranalysar og regresjonsanalysar. Analysane viser at det i første rekke er bruk av tekstbehandlingsprogram, presentasjonsverktøy og Internett for informasjonsinnhenting som engelsklærarar bruker meir enn "av og til". Også bruk av Internettbaserte oppgåver knytt til læreverk og tilsvarande fagspesifikke verktøy er vanlege i engelskundervisinga. Digital kommunikasjon er berre sjeldan brukt. Faktorar som viser seg å henge saman med IKT-bruk inkluderer m.a: stor vektlegging av den digitale basiskompetansen som uttrykt i læreplanen; set stor pris på det at IKT gjev tilgang til ekstra innhald og varierte undervisingsmåtar; har stor tru på positive effektar av IKT på elevane si læring; legg stor vekt på faginnhald som handlar om det å utvikle elevane sine meta-kognitive ferdigheiter; har tru på ei tilretteleggande lærarrolle; og har stor uformell IKT-kompetanse. Analysane viser at det er heile ti fagspesifikke faktorar knytt til innhald, undervisingsorganisering eller sjølvopplevd kompetanse som har innverknad på grad av IKT bruk. Dette tyder på at framtidige analysar av situasjonen i skulen må i større grad ta omsyn til den faglege konteksten IKT-bruken er ein del av. På basis av funna i denne undersøkinga bør framtidige kurs for lærarar ha ei tydeleg fagdidaktisk vinkling med vekt på erfaringstileigning og utveksling, og der former for fagspesifikk læringsutbytte er utgangspunkt for vurdering av verdien av ulik teknologibruk

A role for human N-alpha acetyltransferase 30 (Naa30) in maintaining mitochondrial integrity

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC N-tacetylates a large variety of proteins and is essential for mitochondrial integrity and function

NAA10 p.(D10G) and NAA10 p.(L11R) variants hamper formation of the NatA N-terminal acetyltransferase complex

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.publishedVersio

Optimized bisubstrate inhibitors for the actin N-terminal acetyltransferase NAA80

Acetylation of protein N-termini is one of the most common protein modifications in the eukaryotic cell and is catalyzed by the N-terminal acetyltransferase family of enzymes. The N-terminal acetyltransferase NAA80 is expressed in the animal kingdom and was recently found to specifically N-terminally acetylate actin, which is the main component of the microfilament system. This unique animal cell actin processing is essential for the maintenance of cell integrity and motility. Actin is the only known substrate of NAA80, thus potent inhibitors of NAA80 could prove as important tool compounds to study the crucial roles of actin and how NAA80 regulates this by N-terminal acetylation. Herein we describe a systematic study toward optimizing the peptide part of a bisubstrate-based NAA80 inhibitor comprising of coenzyme A conjugated onto the N-terminus of a tetrapeptide amide via an acetyl linker. By testing various combinations of Asp and Glu which are found at the N-termini of β- and γ-actin, respectively, CoA-Ac-EDDI-NH2 was identified as the best inhibitor with an IC50 value of 120 nM

N-terminal acetylation of actin by NAA80 is essential for structural integrity of the Golgi apparatus

N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization.publishedVersio

HIV-1 Rev oligomerization is not obligatory in the presence of an extra basic domain

BACKGROUND: The HIV-1 Rev regulatory protein binds as an oligomeric complex to viral RNA mediating nuclear export of incompletely spliced and non-spliced viral mRNAs encoding the viral structural proteins. However, the biological significance of the obligatory complex formation of Rev upon the viral RNA is unclear. RESULTS: The activity of various fusion proteins based on the negative oligomerization-defect Rev mutant M4 was tested using Rev dependent reporter constructs. An artificial M4 mutant dimer and an M4 mutant containing an extra basic domain from the HTLV-I Rex protein exhibited nearly full activity when compared to wild type Rev. CONCLUSION: Rev dimerization appears to be required to expose free basic domains whilst the Rev oligomeric complex remains bound to viral RNA via other basic domains

Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK

The human N-terminal acetyltransferase E (NatE) contains NAA10 and NAA50 catalytic, and NAA15 auxiliary subunits and associates with HYPK, a protein with intrinsic NAA10 inhibitory activity. NatE co-translationally acetylates the N-terminus of half the proteome to mediate diverse biological processes, including protein half-life, localization, and interaction. The molecular basis for how NatE and HYPK cooperate is unknown. Here, we report the cryo-EM structures of human NatE and NatE/HYPK complexes and associated biochemistry. We reveal that NAA50 and HYPK exhibit negative cooperative binding to NAA15 in vitro and in human cells by inducing NAA15 shifts in opposing directions. NAA50 and HYPK each contribute to NAA10 activity inhibition through structural alteration of the NAA10 substrate-binding site. NAA50 activity is increased through NAA15 tethering, but is inhibited by HYPK through structural alteration of the NatE substrate-binding site. These studies reveal the molecular basis for coordinated N-terminal acetylation by NatE and HYPK.publishedVersio