N-terminal acetylation is one of the most abundant co- and posttranslational protein modifications, conserved from prokaryotes to eukaryotes. The functional consequences of this modification are ...manifold, ranging from protein folding, stability, and interaction to subcellular localization. We describe here an isotope-labeled
C-acetyl-Coenzyme A-based acetylation assay, allowing the determination of weak catalytic activities of NATs in vitro. It allows the use of purified recombinant enzymes from Escherichia coli, or co-immunoprecipitated enzymes from various organisms, as well as the determination of the in vitro activity of various cell lysates. Although marked as an old-fashioned biochemical approach, it is the ideal method to hunt for catalytic activities and defining peptide specificities of new potential N-terminal acetyltransferase candidates.
Protein Termini 2022: central roles of protein ends Arnesen, Thomas; Aksnes, Henriette; Giglione, Carmela
Trends in biochemical sciences (Amsterdam. Regular ed.),
06/2023, Letnik:
48, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Although 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.
Protein N-terminal acetylation is catalyzed by N-terminal acetyltransferases and represents one of the most common protein modifications in eukaryotes. An increasing number of studies report on the ...importance of N-terminal acetylation for protein degradation, complex formation, subcellular targeting, and protein folding. N-terminal acetyltransferases are recognized to play important roles in a diversity of cellular processes like apoptosis, cell proliferation, sister chromatid cohesion, and chromatin silencing and are even linked to the development of rare genetic disorders and cancer. This article summarizes our current knowledge on the implications of N-terminal acetylation at the protein, cellular, and physiological levels.
Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is ...catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate=MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix Formula: see text that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Primary aldosteronism (PA) is a frequent cause (about 10 %) of hypertension. Some cases of PA were recently found to be caused by mutations in the potassium channel KCNJ5. Our objective ...was to determine the mutation status of
KCNJ5
and seven additional candidate genes for tumorigenesis:
YY1
,
FZD4
,
ARHGAP9
,
ZFP37
,
KDM5C
,
LRP1B
, and
PDE9A
and, furthermore, the surgical outcome of PA patients who underwent surgery in Western Norway.
Methods
Twenty-eight consecutive patients with aldosterone-producing adrenal tumors (20 patients with single adenoma, 8 patients with unilateral multiple adenomas or hyperplasia) who underwent surgery were included in this study. All patients were operated on by uncomplicated laparoscopic total adrenalectomy. Genomic DNA was isolated from tumor and non-tumor adrenocortical tissue, and DNA sequencing revealed the mutation status.
Results
Ten out of 28 (36 %) patients with PA displayed tumor mutations in
KCNJ5
(p. G151R and L168R) while none were found in the corresponding non-tumor samples. No mutations were found in the other seven candidate genes screened. The presence of
KCNJ5
mutations was associated with lower blood pressure and a higher chance for cure by surgery when compared to patients harboring the
KCNJ5
wild type.
Conclusions
KCNJ5
mutations are associated with a better surgical outcome. Preoperative identification of the mutation status might have impact on surgical strategy (total vs. subtotal adrenalectomy).
In school settings, self-control is central to the ability of learners to complete their academic work successfully. Learners’ self-control is directly influenced by the ways in which educators ...execute their work, including their instructional explanations, their classroom management, and the expectations that they express to their learners. Our research on this phenomenon investigated Finnish and Swedish learners in upper secondary schools. Not only is the use of digital technology very different in these two countries; the autonomy and status of educators are as well. This article compares the empirical significance of antecedents of learners’ academic self-control in the two national settings by surveying 2191 learners in Swedish and Finnish schools. Our analysis applies structural equation modeling to two cross-sectional datasets, and the results reveal that the associations between educators’ instructional explanations, classroom management, and their high expectations on the one hand and learners’ academic self-control on the other are stronger overall among Finnish students than among Swedish students. Furthermore, the association between digital technology use and learners’ perceptions of conflict between school norms and Internet opportunities are much stronger in the Swedish sample than the Finnish sample. Lastly, we discuss the meaning of these results and their possible implications for research and practice.
School learners can struggle with setting and striving for objectives that require sustained academic self‐discipline. We believe that teachers’ instructional qualities and school culture are factors ...that can successfully elicit students’ self-discipline in their academic work. This study explores and compares factors related to students’ academic self-discipline among Norwegian and Finnish youths at the upper secondary level. The Finnish students’ excellent results on international comparative tests have led many commentators to consider the Finnish model of education very worthy of emulation. Another reason to com-pare these two groups is that Finnish and Norwegian classrooms differ in their levels of in-class Internet access and computer use for learning purposes. From this perspective, it is interesting to compare empirical associations between instructional factors (as well as students’ school appreciation) and students’ academic self-discipline. The instructional factors in our theoretical model were teachers’ classroom management, teaching quality, teachers’ expressed expectations and the value students placed on the school as an institution. A total of 1433 urban Finnish and Norwegian upper secondary students in general study programmes participated in our cross-sectional questionnaire. We used structural equation modelling for our analysis, and the results show that the associations between instructional qualities (quality instruction, classroom management and high expectations) and academic self-discipline are overall stronger in the Finnish sample than the Norwegian sample. However, students’ appreciation for school was more highly associated with academic self-discipline in Norway than in Finland. Furthermore, the associations between in-class Internet access and motivational conflict were clearly higher in the Norwegian sample than in the Finnish sample. In both samples, we found strong associations between motivational conflict and academic self-discipline. We also discuss the meaning of these results and their implications for research and practice.
Protein N-terminal acetylation is a co- and posttranslational modification, conserved among eukaryotes. It determines the functional fate of many proteins including their stability, complex ...formation, and subcellular localization. N-terminal acetyltransferases (NATs) transfer an acetyl group to the N-termini of proteins, and the major NATs in yeast and humans are NatA, NatB, and NatC. In this study, we characterized the Trypanosoma cruzi (T. cruzi) NatC and NatA protein complexes, each consisting of one catalytic subunit and predicted auxiliary subunits. The proteins were found to be expressed in the three main life cycle stages of the parasite, formed stable complexes in vivo, and partially cosedimented with the ribosome in agreement with a cotranslational function. An in vitro acetylation assay clearly demonstrated that the acetylated substrates of the NatC catalytic subunit from T. cruzi were similar to those of yeast and human NatC, suggesting evolutionary conservation of function. An RNAi knockdown of the Trypanosoma brucei (T. brucei) NatC catalytic subunit indicated that reduced NatC-mediated N-terminal acetylation of target proteins reduces parasite growth.
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