scholarly journals CMT2N-causing aminoacylation domain mutants enable Nrp1 interaction with AlaRS

2021 ◽  
Vol 118 (13) ◽  
pp. e2012898118
Author(s):  
Litao Sun ◽  
Na Wei ◽  
Bernhard Kuhle ◽  
David Blocquel ◽  
Scott Novick ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Hydrodynamic Diameter ◽  
Mutant Proteins ◽  
X Ray ◽  
Trna Synthetases ◽  
X Ray Crystallography ◽  
Charcot Marie Tooth Disease ◽  
Neuropilin 1 ◽  
Editing Domain

Through dominant mutations, aminoacyl-tRNA synthetases constitute the largest protein family linked to Charcot-Marie-Tooth disease (CMT). An example is CMT subtype 2N (CMT2N), caused by individual mutations spread out in AlaRS, including three in the aminoacylation domain, thereby suggesting a role for a tRNA-charging defect. However, here we found that two are aminoacylation defective but that the most widely distributed R329H is normal as a purified protein in vitro and in unfractionated patient cell samples. Remarkably, in contrast to wild-type (WT) AlaRS, all three mutant proteins gained the ability to interact with neuropilin 1 (Nrp1), the receptor previously linked to CMT pathogenesis in GlyRS. The aberrant AlaRS-Nrp1 interaction is further confirmed in patient samples carrying the R329H mutation. However, CMT2N mutations outside the aminoacylation domain do not induce the Nrp1 interaction. Detailed biochemical and biophysical investigations, including X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange (HDX), switchSENSE hydrodynamic diameter determinations, and protease digestions reveal a mutation-induced structural loosening of the aminoacylation domain that correlates with the Nrp1 interaction. The b1b2 domains of Nrp1 are responsible for the interaction with R329H AlaRS. The results suggest Nrp1 is more broadly associated with CMT-associated members of the tRNA synthetase family. Moreover, we revealed a distinct structural loosening effect induced by a mutation in the editing domain and a lack of conformational impact with C-Ala domain mutations, indicating mutations in the same protein may cause neuropathy through different mechanisms. Our results show that, as with other CMT-associated tRNA synthetases, aminoacylation per se is not relevant to the pathology.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain fromAeropyrum pernix

2012 ◽  
Vol 68 (11) ◽  
pp. 1390-1393 ◽  
Author(s):  
Sadeem Ahmad ◽  
Antony S. K. Sravankumar ◽  
Shobha P. Kruparani ◽  
Rajan Sankaranarayanan
Keyword(s):  
Trna Synthetase ◽  
Asymmetric Unit ◽  
Crystallographic Investigation ◽  
X Ray ◽  
Trna Synthetases ◽  
Aeropyrum Pernix ◽  
Substrate Analogues ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Evolutionary Aspects

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeonPyrococcus abysiiin complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeonAeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase,i.e.ThrRS1 and ThrRS2, encoded by two different genes,thrS1andthrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 fromA. pernixis reported. The crystals belong to either theP41212 orP43212 space group and consist of one monomer per asymmetric unit.

A novel palladium (II) complex with a ferrocene-based ligand: Synthesis, X-ray crystallography and in vitro anticancer activity study

2021 ◽  
Vol 126 ◽  
pp. 108448
Author(s):  
Guoyuan Du ◽  
Zhonghui Zhang ◽  
Xiangyu Lu ◽  
Wentao Cai ◽  
Liji Wu ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ligand Synthesis

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity

2012 ◽  
Vol 443 (2) ◽  
pp. 477-484 ◽  
Author(s):  
Min Tan ◽  
Wei Yan ◽  
Ru-Juan Liu ◽  
Meng Wang ◽  
Xin Chen ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Trna Synthetase ◽  
Modular Construction ◽  
Aquifex Aeolicus ◽  
Trna Synthetases ◽  
Naturally Occurring ◽  
Aminoacyl Trna Synthetases ◽  
Editing Domain ◽  
Editing Activity ◽  
Shed Light

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.

scholarly journals Nitro/Nitrosyl-Ruthenium Complexes Are Potent and Selective Anti-Trypanosoma cruzi Agents Causing Autophagy and Necrotic Parasite Death

2014 ◽  
Vol 58 (10) ◽  
pp. 6044-6055 ◽  
Author(s):  
Tanira M. Bastos ◽  
Marília I. F. Barbosa ◽  
Monize M. da Silva ◽  
José W. da C. Júnior ◽  
Cássio S. Meira ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Inhibitory Concentration ◽  
Content Type ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ruthenium Nitrosyl ◽  
Index Analysis ◽  
Antiparasitic Drug ◽  
Drug Complex

ABSTRACTcis-[RuCl(NO2)(dppb)(5,5′-mebipy)] (complex 1),cis-[Ru(NO2)2(dppb)(5,5′-mebipy)] (complex 2),ct-[RuCl(NO)(dppb)(5,5′-mebipy)](PF6)2(complex 3), andcc-[RuCl(NO)(dppb)(5,5′-mebipy)](PF6)2(complex 4), where 5,5′-mebipy is 5,5′-dimethyl-2,2′-bipyridine and dppb is 1,4-bis(diphenylphosphino)butane, were synthesized and characterized. The structure of complex 2 was determined by X-ray crystallography. These complexes exhibited a higher anti-Trypanosoma cruziactivity than benznidazole, the current antiparasitic drug. Complex 3 was the most potent, displaying a 50% effective concentration (EC50) of 2.1 ± 0.6 μM against trypomastigotes and a 50% inhibitory concentration (IC50) of 1.3 ± 0.2 μM against amastigotes, while it displayed a 50% cytotoxic concentration (CC50) of 51.4 ± 0.2 μM in macrophages. It was observed that the nitrosyl complex 3, but not its analog lacking the nitrosyl group, releases nitric oxide into parasite cells. This release has a diminished effect on the trypanosomal protease cruzain but induces substantial parasite autophagy, which is followed by a series of irreversible morphological impairments to the parasites and finally results in cell death by necrosis. In infected mice, orally administered complex 3 (five times at a dose of 75 μmol/kg of body weight) reduced blood parasitemia and increased the survival rate of the mice. Combination index analysis of complex 3 indicated that itsin vitroactivity against trypomastigotes is synergic with benznidazole. In addition, drug combination enhanced efficacy in infected mice, suggesting that ruthenium-nitrosyl complexes are potential constituents for drug combinations.

Synthesis, structure, and in vitro anti-lung cancer activity on an In-based nanoscale coordination polymer

2018 ◽  
Vol 41 (3-4) ◽  
pp. 129-133 ◽  
Author(s):  
De-Gui Shu ◽  
Wen-Yu Chen
Keyword(s):  
Coordination Polymer ◽  
Solvothermal Reaction ◽  
X Ray Diffraction ◽  
Anticancer Activities ◽  
X Ray ◽  
X Ray Crystallography ◽  
Accessible Volume ◽  
Nanoscale Coordination Polymer ◽  
Template Complex

Abstract Here, a new indium (In)-based coordination polymer [In(hip)](DMF)2(H2O)3 (1, DMF=N,N-dimethylformamide) was successfully prepared by a solvothermal reaction of In(NO3)3·6H2O and 5-hydroxyisophthalic acid (H3hip) in a mixed solvent of DMF and H2O with the presence of NaCl as a template. Complex 1 was characterized by elemental analysis (EA), single-crystal X-ray crystallography, and powder X-ray diffraction (PXRD), and the results reveal that complex 1 shows a two-dimensional (2D) grid-like network with considerable solvent accessible volume that was generated from the packing of the 2D layers via the AB pattern. Furthermore, complex 1 could be downsized into nanoscale particles with the aid of polyvinylpyrrolidone (PVP). In addition, the anticancer activities of 1 and the nanoscale 1 were probed via the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals Sesquiterpene Lactones from Inula oculus-christi

2010 ◽  
Vol 5 (4) ◽  
pp. 1934578X1000500 ◽  
Author(s):  
Mahmoud Mosaddegh ◽  
Maryam Hamzeloo Moghadam ◽  
Saeedeh Ghafari ◽  
Farzaneh Naghibi ◽  
Seyed Nasser Ostad ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Crystal Structures ◽  
Spectroscopic Data ◽  
Sesquiterpene Lactones ◽  
X Ray ◽  
X Ray Crystallography ◽  
First Time

Inula oculus-christi L. (Compositae) extract was chromatographed and three sesquiterpene lactones ergolide, gaillardin and pulchellin C were isolated. The structures of these compounds were determined by analysis of their spectroscopic data, and their crystal structures were defined using X-ray crystallography; the isolation of ergolide and pulchellin C is reported for the first time from this species. These three compounds were evaluated for their in vitro cytotoxic activity against MDBK, MCF7 and WEHI164 cells; ergolide and gaillardin exhibited lower and significantly different IC50 values compared with pulchellin C ( p<0.001).

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