Within this research, the CrdA protein from
(
CrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity ...toward copper was experimentally determined. Incubation of
CrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled
CrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from
and from
, respectively. Within the conserved amino acid motif,
CrdA contains two additional methionines and two glutamic acid residues (
MX
MPGMXX
X
M) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the
cell and that the affinity of the interaction corresponds to a middle or strong metal-protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for
.
Seryl-tRNA synthetases (SerRSs), members of the aminoacyl-tRNA synthetase family, interact with diverse proteins, enabling SerRSs to enhance their role in the translation of the genetic message or to ...perform alternative functions in cellular processes beyond translation. Atypical archaeal SerRS interacts with arginyl-tRNA synthetase and proteins of the ribosomal P-stalk to optimize translation through tRNA channeling. The complex between yeast SerRS and peroxin Pex21p provides a connection between translation and peroxisome function. The partnership between
SerRS and BEN1 indicates a link between translation and brassinosteroid metabolism and may be relevant in plant stress response mechanisms. In
, the unusual heterodimeric mitochondrial SerRS coordinates mitochondrial translation and replication via interaction with LON protease. Evolutionarily conserved interactions of yeast and human SerRSs with m
C32 tRNA methyltransferases indicate coordination between tRNA modification and aminoacylation in the cytosol and mitochondria. Human cytosolic SerRS is a cellular hub protein connecting translation to vascular development, angiogenesis, lipogenesis, and telomere maintenance. When translocated to the nucleus, SerRS acts as a master negative regulator of
gene expression. SerRS alone or in complex with YY1 and SIRT2 competes with activating transcription factors NFκB1 and c-Myc, resulting in balanced
expression important for proper vascular development and angiogenesis. In hypoxia, SerRS phosphorylation diminishes its binding to the
promoter, while the lack of nutrients triggers SerRS glycosylation, reducing its nuclear localization. Additionally, SerRS binds telomeric DNA and cooperates with the shelterin protein POT1 to regulate telomere length and cellular senescence. As an antitumor and antiangiogenic factor, human cytosolic SerRS appears to be a promising drug target and therapeutic agent for treating cancer, cardiovascular diseases, and possibly obesity and aging.
Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion ...primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of
Arabidopsis thaliana
seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAs
Ser
, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNA
Ser
specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.
We have previously identified a unique disulfide bond in the crystal structure of Arabidopsis cytosolic seryl‐tRNA synthetase involving cysteines evolutionarily conserved in all green plants. Here, ...we discovered that both cysteines are important for protein stability, but with opposite effects, and that their microenvironment may promote disulfide bond formation in oxidizing conditions. The crystal structure of the C244S mutant exhibited higher rigidity and an extensive network of noncovalent interactions correlating with its higher thermal stability. The activity of the wild‐type showed resistance to oxidation with H2O2, while the activities of cysteine‐to‐serine mutants were impaired, indicating that the disulfide link may enable the protein to function under oxidative stress conditions which can be beneficial for an efficient plant stress response.
We have examined the role of the disulfide link between evolutionarily conserved cysteines in plant cytosolic seryl‐tRNA synthetase. We have identified features of the protein microenvironment which may promote disulfide bond formation in oxidizing conditions. Activity assays showed that the disulfide link is important for protein resistance to oxidation, which may be beneficial for translation during oxidative stress conditions in plants.
We have recently identified BEN1 as a protein interactor of seryl-tRNA synthetase (SerRS) from model plant Arabidopsis thaliana. BEN1 contains an NADP+ binding domain and possesses acidic N-terminal ...extension essential for interaction with A. thaliana SerRS. This extension, specific for BEN1 homologues from Brassicaceae family, is solvent-exposed and distant to the nucleotide-binding site. We prepared a truncated BEN1 variant ΔN17BEN1 lacking the first 17 amino acid of this N-terminal extension as well as full-length BEN1 to investigate how the truncation affects the binding affinity towards coenzyme NADP+. By performing microscale thermophoresis (MST) experiments we have shown that both BEN1 variants bind the NADP+ cofactor, however, truncated BEN1 showed 34-fold higher affinity towards NADP+ indicating that its core protein structure is not just preserved but it binds NADP+ even stronger. To further corroborate the obtained results, we opted for a computational approach based on classical molecular dynamics simulations of both complexes. Our results have shown that both truncated and intact BEN1 variants form the same number of interactions with the NADP+ cofactor; however, it was the interaction occupancy that was affected. Namely, three independent MD simulations showed that the ΔN17BEN1 variant in complex with NADP+ has significantly higher interaction occupancy thus binds NADP+ with more than one order of magnitude higher affinity. Contrary to our expectations, the truncation of this distant region that does not communicate with the nucleotide-binding site didn't result in the gain of interaction but affected the intrinsic conformational dynamics which in turn fine-tuned the binding affinity by increasing the interaction occupancy and strength of the key conserved cation-π interaction between Arg69 and adenine of NADP+ and hydrogen bond between Ser244 and phosphate of NADP+.
•Intact and truncated BEN1 lacking 17 amino acids at the N-terminus were heterologously expressed and purified from E. coli.•The binding affinity between two variants of BEN1 protein and NADP+ cofactor was assessed by microscale thermophoresis (MST).•Truncated BEN1 showed 34-fold higher affinity towards NADP+ in comparison to full-length BEN1 protein.•Transient nature of non-covalent interactions and the binding affinity were examined by molecular dynamics simulations.•Higher binding affinity is the result of the increased interaction occupancy and strength of the key conserved interactions.
The rules of the genetic code are established by aminoacyl‐tRNA synthetases (aaRSs) enzymes, which covalently link tRNA with the cognate amino acid. Many aaRSs are involved in diverse cellular ...processes beyond translation, acting alone, or in complex with other proteins. However, studies of aaRS noncanonical assembly and functions in plants are scarce, as are structural studies of plant aaRSs. Here, we have solved the crystal structure of Arabidopsis thaliana cytosolic seryl‐tRNA synthetase (SerRS), which is the first crystallographic structure of a plant aaRS. Arabidopsis SerRS displays structural features typical of canonical SerRSs, except for a unique intrasubunit disulfide bridge. In a yeast two‐hybrid screen, we identified BEN1, a protein involved in the metabolism of plant brassinosteroid hormones, as a protein interactor of Arabidopsis SerRS. The SerRS:BEN1 complex is one of the first protein complexes of plant aaRSs discovered so far, and is a rare example of an aaRS interacting with an enzyme involved in primary or secondary metabolism. To pinpoint regions responsible for this interaction, we created truncated variants of SerRS and BEN1, and identified that the interaction interface involves the SerRS globular catalytic domain and the N‐terminal extension of BEN1 protein. BEN1 does not have a strong impact on SerRS aminoacylation activity, indicating that the primary function of the complex is not the modification of SerRS canonical activity. Perhaps SerRS performs as yet unknown noncanonical functions mediated by BEN1. These findings indicate that – via SerRS and BEN1 – a link exists between the protein translation and steroid metabolic pathways of the plant cell.
Database
Structural data are available in the PDB under the accession number PDB ID 6GIR.
Here, we determine the first crystal structure of plant aminoacyl‐tRNA synthetase, seryl‐tRNA synthetase (SerRS) from Arabidopsis thaliana, and show that it interacts with BEN1, a protein involved in the metabolism of plant brassinosteroid hormones. Our findings thus link translation and steroid metabolic pathways in plants.
Osnovna zadaća seril-tRNA-sintetaze (SerRS) je vezanje serina na tRNASer pripremajući tako podlogu za biosintezu proteina. U ovom radu pokazano je kako citosolna SerRS iz biljke uročnjaka ...(Arabidopsis thaliana, (L.) Heynh) ostvaruje i nekanonske funkcije ulazeći u dosad nepoznatu interakciju s proteinom BEN1 koji sudjeluje u metabolizmu brasinosteroida, spojeva koji među ostalim sudjeluju u moderiranju staničnog odgovora na stres što potencijalno uključuje SerRS u stanični odgovor na stres. Nadalje, pokazano je in vitro kako istraživana SerRS gotovo jednako učinkovito aminoacilira bakterijske i eukariotske tRNASer supstrate. S obzirom da je slično ponašanje uočeno i za kukuruznu SerRS, izgleda da biljne SerRS pokazuju neobično široku tRNASer specifičnost, za razliku od SerRS iz drugih organizama. Lokalizacijski eksperimenti u uvjetima stabilne transformacije u transgeničnim biljkama A. thaliana nisu pokazali prisutnost SerRS u jezgri stanice, dok se u uvjetima tranzijentne transformacije SerRS u jezgrama protoplasta biljke A. thaliana pronalazi u 10-20% slučajeva. Konačno, utvrđena je statistički značajna razlika u duljini korijena transgeničnih biljaka A. thaliana koje nadeksprimiraju SerRS u odnosu na divlji tip u abiotičkom stresu izazvanom ionskim i osmotskim stresorima, te kadmijem kada su biljke kontinuirano uzgajane na podlogama s navedenim stresorima.
Primary function of seryl-tRNA synthetase (SerRS) is attachment of serine to tRNASer preparing a foundation for successful protein biosynthesis. It is shown in this dissertation that cytosolic SerRS from plant Arabidopsis thaliana (L.) Heynh, also performs non-canonical functions by establishing so far the unknown interaction with protein BEN1 which is involved in metabolism of brassinosteroids, compounds important in cell stress response what potentially implicates involvement of SerRS in cell stress response. Furthermore, in vitro experiments showed that SerRS almost equally well catalyzes aminoacylation of bacterial and eukaryotic tRNASer substrates. Given the fact that the same behaviour was previously shown for monocot maize SerRS, it seems that plant SerRSs exibit unusually broad tRNASer specificity, unlike SerRSs from other organisms. Localization experiments under conditions of stable transformation in transgenic A. thaliana plants did not detect SerRS in the cell nucleus. Under transient transformation of A. thaliana protoplasts SerRS was detected in cell nucleus in 10-20 % of calls. Finally, statistically significant difference in root length of transgenic A. thaliana plants overexpressing SerRS in comparison with wild type was found under conditions of abiotic stress caused by ionic and osmotic stressors and cadmium in plants grown continuously on stress growth medium.
Benigne bolesti anorektalne regije, u koje ubrajamo analne apscese, analne fistule, analne fisure i hemoroidalnu bolest, česta su stanja koja susreću liječnici obiteljske medicine kao i bolnički ...specijalisti. Unatoč rasprostranjenosti patologije, još uvijek ne postoje jasne smjernice za adekvatno liječenje navedenih stanja, koje se još uvijek često svodi na preporuke koje nemaju uporište u medicini temeljenoj na dokazima. Zbog toga je ekspertna skupina, u kojoj su sudjelovali članovi Hrvatskog društva za digestivnu kirurgiju kojima je uže područje interesa koloproktologija, odlučili sastaviti ove smjernice s namjerom da se standardizira liječenje benignih bolesti anorektalne regije, kako na primarnoj tako i na tercijarnoj razini.
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