Oppstalling av laboratoriemus i et naturlig habitat : virkning på immunsystem, tarmmikrobiota og utvikling av kolorektal kreft

The natural habitat for the house mouse is on the ground, typically close to humans and their livestock and hence surrounded by a rich microbial diversity that throughout evolutionary history has driven the adaptation of the house mouse. It is thus paradoxical that almost without exception, experimental disease studies using this mammalian model take place in perfect isolation from the outer microbial world. The end goal for preclinical research, humans, rarely live in microbial isolation, although lifestyles can arguably be said to vary on a scale. To develop a preclinical model that better resemble realistic lifestyles of mammals, we have established a system where laboratory mice are raised under a full set of environmental conditions present in a typical farmyard habitat for the house mouse. We call the process feralization, and the first paper covered by this thesis show the resulting mammal display more functionally mature states of immune cells and a diverse gut microbiota, likely surpassing conventional laboratory mice in resembling responses of free-living mice. Furthermore, we demonstrated the use of this animal modelling approach that recapitulates realistic disease responses in a naturalized mammal. We first established a protocol of the AOM/DSS model for colorectal cancer (CRC) induction in mice using a lower-than-usual dose of DSS that is presented in paper II. In paper III, we employed the AOM/DSS model, as well as a previously established genetic Min/+ model of CRC, in a feralization system. We showed that the mice feralized in a farmyard-type habitat were protected against colorectal carcinogenesis compared to conventionally reared laboratory mice. Moreover, our feralization model allows for full control of the timing of microbial exposure. We took advantage of this by including groups of mice that were either born in the farmyard habitat or introduced to it in later life, demonstrating that neonatal microbial exposure was not essential for the CRC protection. The findings were supported by changes in gut microbiota profiles, as well as immunophenotypes indicative of antigenic experience in the feralized mice. In currently unpublished work, we aimed to narrow in on mechanisms for the protective effects conveyed by feralization in the intestines. Assays investigating mucus layer properties showed no differences following feralization, yet a few genes in the colon mucosa related to barrier function were found to be significantly upregulated between feralized and conventional laboratory mice. Further assessments are needed to elaborate on the mechanisms underlying the beneficial effects of feralization.Det naturlige habitatet for husmus er typisk nær mennesker og deres husdyr, og husmusa er gjennom evolusjon tilpasset et slikt rikt mikrobielt levemiljø. Det er derfor paradoksalt at eksperimentelle studier der mus brukes for å studere sykdomsmekanismer, omtrent uten unntak foregår i perfekt isolasjon fra den ytre, mikrobielle verden. Målet for prekliniske studier ved bruk av forsøksmus er typisk å overføre funnene til relevans for mennesker, men mennesker lever sjeldent i mikrobiell isolasjon, selv om individuelle livsstiler varierer stort. For å utvikle en preklinisk musemodell som mer realistisk representerer naturlige livstiler hos pattedyr, har vi etablert et system der laboratoriemus fostres opp i en mer naturlig situasjon, der habitatet deres er beriket med elementer som er tilstede i et typisk gårdsmiljø. Vi har kalt denne prosessen «feralisering», og den første artikkelen som omfattes av denne avhandlingen demonstrerer at de «feraliserte» musene viser tegn til mer funksjonelt modne immunceller og en rikere tarmmikrobiota sammenliknet med laboratoriemus oppstallet under tradisjonelle, rene forhold. Videre har vi demonstrert bruken av feraliserings-systemet som kan benyttes til å studere realistiske sykdomsresponser i et naturalisert pattedyr. Først etablerte vi en protokoll for kjemisk induksjon av kolorektalkreft hos mus, ved å bruke en lavere-enn-normal dose av DSS i en AOM/DSS modell. Dette arbeidet er presentert i artikkel II. I artikkel III benyttet vi AOM/DSS modellen, så vel som en tidligere etablert genetisk Min/+ musemodell for kolorektalkreft, i feraliseringssystemet. Vi demonstrerte at musene som ble feralisert i et naturalistisk gårdsmiljø var beskyttet mot tykktarmskreft, sammenliknet med laboratoriemus oppstallet under tradisjonelle, rene forhold. Videre tillater feraliserings-systemet full kontroll av timingen for mikrobiell eksponering. Vi utnyttet dette ved å inkludere en gruppe av mus som enten var født i gårdsmiljøet, eller introdusert dit senere i livet. Med dette viste vi at neonatal mikrobiell eksponering ikke var essensiell for beskyttelse mot kolorektal kreft. Funnene ble støttet av endringer i tarmmikrobiotaprofiler, samt immunofenotyper som kan indikere at de har blitt eksponert for antigener. I nåværende upublisert arbeid hadde vi som mål å undersøke mulige mekanismer for hvordan feralisering i et gårdsmiljø kunne gi beskyttelse mot kolorektal kreft. Undersøkelser av slimlaget i tarm viste ingen forskjeller etter feralisering. Likevel var det none få gener i tykktarmsslimhinnen relatert til barrierefunksjon som var oppregulert i feraliserte mus sammenliknet med konvensjonelt oppstallede laboratoriemus. Videre undersøkelser er nødvendig for å kunne utdype mer når det gjelder mekanismene bak feraliserings beskyttende effekt

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

N-terminal acetylation levels are maintained during acetyl-CoA deficiency in Saccharomyces cerevisiae

N-terminal acetylation (Nt-acetylation) is a highly abundant protein modification in eukaryotes and impacts a wide range of cellular processes, including protein quality control and stress tolerance. Despite its prevalence, the mechanisms regulating Nt-acetylation are still nebulous. Here, we present the first global study of Nt-acetylation in yeast cells as they progress to stationary phase in response to nutrient starvation. Surprisingly, we found that yeast cells maintain their global Nt-acetylation levels upon nutrient depletion, despite a marked decrease in acetyl-CoA levels. We further observed two distinct sets of protein N termini that display differential and opposing Nt-acetylation behavior upon nutrient starvation, indicating a dynamic process. The first protein cluster was enriched for annotated N termini showing increased Nt-acetylation in stationary phase compared with exponential growth phase. The second protein cluster was conversely enriched for alternative nonannotated N termini (i.e. N termini indicative of shorter N-terminal proteoforms) and, like histones, showed reduced acetylation levels in stationary phase when acetyl-CoA levels were low. Notably, the degree of Nt-acetylation of Pcl8, a negative regulator of glycogen biosynthesis and two components of the pre-ribosome complex (Rsa3 and Rpl7a) increased during starvation. Moreover, the steady-state levels of these proteins were regulated both by starvation and NatA activity. In summary, this study represents the first comprehensive analysis of metabolic regulation of Nt-acetylation and reveals that specific, rather than global, Nt-acetylation events are subject to metabolic regulation

NAA80 is actin’s N-terminal acetyltransferase and regulates cytoskeleton assembly and cell motility

Actin, one of the most abundant proteins in nature, participates in countless cellular functions ranging from organelle trafficking and pathogen motility to cell migration and regulation of gene transcription. Actin's cellular activities depend on the dynamic transition between its monomeric and filamentous forms, a process exquisitely regulated in cells by a large number of actin-binding and signaling proteins. Additionally, several posttranslational modifications control the cellular functions of actin, including most notably N-terminal (Nt)-acetylation, a prevalent modification throughout the animal kingdom. However, the biological role and mechanism of actin Nt-acetylation are poorly understood, and the identity of actin's N-terminal acetyltransferase (NAT) has remained a mystery. Here, we reveal that NAA80, a suggested NAT enzyme whose substrate specificity had not been characterized, is Nt-acetylating actin. We further show that actin Nt-acetylation plays crucial roles in cytoskeletal assembly in vitro and in cells. The absence of Nt-acetylation leads to significant differences in the rates of actin filament depolymerization and elongation, including elongation driven by formins, whereas filament nucleation by the Arp2/3 complex is mostly unaffected. NAA80-knockout cells display severely altered cytoskeletal organization, including an increase in the ratio of filamentous to globular actin, increased filopodia and lamellipodia formation, and accelerated cell motility. Together, the results demonstrate NAA80's role as actin's NAT and reveal a crucial role for actin Nt-acetylation in the control of cytoskeleton structure and dynamics

Protein Termini 2022: central roles of protein ends

International audienceAlthough locating at the protein ends, N- and C-termini are at the center of numerous cellular functions. This topic engages an increasing number of scientists, recently forming the International Society of Protein Termini (ISPT). Protein Termini 2022 gathered this interdisciplinary community to discuss how protein ends may steer protein functionality

Co-translational, post-translational, and non-catalytic roles of N-terminal acetyltransferases

Recent studies of N-terminal acetylation have identified new N-terminal acetyltransferases (NATs) and expanded the known functions of these enzymes beyond their roles as ribosome-associated co-translational modifiers. For instance, the identification of Golgi- and chloroplast-associated NATs shows that acetylation of N termini also happens post-translationally. In addition, we now appreciate that some NATs are highly specific; for example, a dedicated NAT responsible for post-translational N-terminal acetylation of actin was recently revealed. Other studies have extended NAT function beyond Nt acetylation, including functions as lysine acetyltransferases (KATs) and non-catalytic roles. Finally, emerging studies emphasize the physiological relevance of N-terminal acetylation, including roles in calorie-restriction-induced longevity and pathological α-synuclein aggregation in Parkinson’s disease. Combined, the NATs rise as multifunctional proteins, and N-terminal acetylation is gaining recognition as a major cellular regulator

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin

Actin is the most abundant protein in our cells, and also one of the most studied. Nevertheless, an important modifier of actin, the N-terminal acetyltransferase (NAT) for actin, remained unknown until now. The recent identification of the enzyme that catalyzes actin acetylation, has opened up for functional studies of unacetylated actin using knockout cells. This enzyme, called NAA80 (Nα-acetyltransferase 80) or NatH, belongs to the NAT family of enzymes, which together provides N-terminal acetylation for around 80 % of the human proteome. In many cases, N-terminal acetylation is essential. In the case of actin, the acetyl group that NAA80 attaches to actin plays an important role in actin’s polymerization properties as well as in actin’s function in cell migration

N-Terminal Acetylation by NatC Is Not a General Determinant for Substrate Subcellular Localization in <i>Saccharomyces cerevisiae</i>

<div><p>N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in <i>Saccharomyces cerevisiae</i>. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.</p></div

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