quantitative-analysis-thermogravimetry-mass-spectrum-analysis-for

Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and ...

Chinese Academy of Sciences

LeuRS (leucyl-tRNA synthetase) catalyses the esterification of tRNAsLeu with leucine. This family of enzymes is divided into prokaryotic and eukaryal/archaeal groups according to the presence and position of specific insertions and extensions. In the present study, we investigated the function of LSD1 (leucine-specific domain 1), which is naturally present in eukaryal/archaeal LeuRSs, but absent from prokaryotic LeuRSs. When mutated in their common domain, the eukaryal and archaeal LeuRSs exhibited defects in the first reaction step of amino acid activation with variations of leucine or ATP-binding strength, whereas the tRNA aminoacylation was moderately affected. When the eukaryal extension was mutated, severe tRNA charging defects were observed, suggesting that eukaryotes evolved this LSD1 extension in order to improve the aminoacylation reaction step. The results also showed that the LSD1s from organisms of both groups are dispensable for post-transfer editing. Together, the data provide us with a further understanding of the organization and structure of LeuRS domains.

Functional characterization of leucine-specific domain 1 from eukaryal and archaeal leucyl-tRNA synthetases.

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNALeu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNALeu knockout strains carrying deletions of the genes for tRNALeu(GAG) and tRNALeu(UAG). Disrupting the single gene encoding tRNALeu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNALeu(UAG) had a lethal effect on the yeast strain, indicating that tRNALeu(UAG) decoding capacity could not be compensated by another tRNALeu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNALeu(UAG), a selection to identify critical tRNALeu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.

In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain.

Aminoacyl-tRNA synthetase (aaRS) catalyzes the first step of protein synthesis, producing aminoacyl-tRNAs as building blocks. Eukaryotic aaRS differs from its prokaryotic counterpart in terminal extension or insertion. Moreover, the editing function of aaRSs is an indispensable checkpoint excluding non-cognate amino acids at a given codon and ensuring overall translational fidelity. We found higher eukaryotes encode two cytoplasmic threonyl-tRNA synthetases (ThrRSs) with difference in N-terminus. The longer isoform is more closely related to the ThrRSs of higher eukaryotes than to those of lower eukaryotes. A yeast strain was generated to include deletion of the thrS gene encoding ThrRS. Combining in vitro biochemical and in vivo genetic data, ThrRSs from eukaryotic cytoplasm were systematically analyzed, and role of the eukaryotic cytoplasmic ThrRS-specific N-terminal extension was elucidated. Furthermore, the mechanisms of aminoacylation and editing activity mediated by Saccharomyces cerevisiae ThrRS (ScThrRS) were clarified. Interestingly, yeast cells were tolerant of variation at the editing active sites of ScThrRS without significant Thr-to-Ser conversion in the proteome even under significant environmental stress, implying checkpoints downstream of aminoacylation to provide a further quality control mechanism for the yeast translation system. This study has provided the first comprehensive elucidation of the translational fidelity control mechanism of eukaryotic ThrRS.

Translational fidelity maintenance preventing Ser mis-incorporation at Thr codon in protein from eukaryote.

The C-terminal extension of prokaryotic leucyl-tRNA synthetase (LeuRS) has been shown to make contacts with the tertiary structure base pairs of tRNA(Leu) as well as its long variable arm. However, the precise role of the flexibly linked LeuRS C-terminal domain (CTD) in aminoacylation and editing processes has not been clarified. In this study, we carried out aspartic acid scanning within the CTD of Mycobacterium tuberculosis LeuRS (MtbLeuRS) and studied the effects on tRNA(Leu)-binding capacity and enzymatic activity. Several critical residues were identified to impact upon the interactions between LeuRS and tRNA(Leu) due to their contributions in the maintenance of structural stability or a neutral interaction interface between the CTD platform and tRNA(Leu) elbow region. Moreover, we propose Arg921 as a crucial recognition site for the tRNA(Leu) long variable arm in aminoacylation and tRNA-dependent pre-transfer editing. We also show here the CTD flexibility conferred by Val910 in regulation of LeuRS-tRNA(Leu) interaction. Taken together, our results suggest the structural importance of the CTD in modulating precise interactions between LeuRS and tRNA(Leu) during the quality control of leucyl-tRNA(Leu) synthesis. This system for the investigation of the interactions between MtbLeuRS and tRNA(Leu) provides a platform for the development of novel antitubercular drugs.

Crucial role of the C-terminal domain of Mycobacterium tuberculosis leucyl-tRNA synthetase in aminoacylation and editing.

Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNA(Leu). LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain-domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS. 

A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity.

A phase of beryllium fluoride borate Be2BO3F (BBF) was successfully developed and grown by spontaneous nucleation from high temperature solution. The crystal belongs to the trigonal space group of R3̅c (No. 167), with lattice parameters a = 4.442(1) Å, c = 24.956(5) Å, and Z = 2. It is constructed by the infinite planar [Be2BO3F2]∞ layers, in which the planar triangle [BO3](3-) and the tetrahedral [BeO3F](5-) anionic groups are arranged in parallel via corner-sharing O atoms in each ab plane. BBF is an incongruent compound and decomposes at about 650 °C. The deep-ultraviolet (DUV) transmittance spectrum reveals that its UV cutoff wavelength is down to ∼150 nm. Theoretical calculations show that BBF has a large birefringence (Δn = 0.13 at 200 nm), which mainly originates from the infinite planar [Be2BO3F2]∞ layers. In conclusion, BBF may be served as a potential DUV birefringent material. 

Be2BO3F: A Phase of Beryllium Fluoride Borate Derived from KBe2BO3F2 with Short UV Absorption Edge.

A series of a novel beryllium-free KBBF family of nonlinear-optical materials AZnBOX (A = K, Rb and X = Cl; A = Na, K, Rb and X = Br) were successfully synthesized through molecular engineering design, and single crystals of AZnBOCl (A = K, Rb) were grown by a spontaneous nucleation technique from self-flux systems. As a representative for the halogen KBBF family of crystals, KZnBOCl features the infinite lattice layer [ZnBOCl] made up of BO and ZnOCl anionic groups, and the in-layer BO groups are completely coplanar and well-aligned. Besides, KZnBOCl exhibits high transmittance in the range of 300-2000 nm with a UV-transmission cutoff of around 200 nm according to transmission spectra. The compounds of AZnBOCl (A = K, Rb) are both phase-matchable with powder second-harmonic-generation efficiencies of 1.3 and 1.17 times that of KHPO for KZnBOCl and RbZnBOCl, respectively, which are similar to that of KBBF.

Beryllium-Free KBBF Family of Nonlinear-Optical Crystals: AZnBOX (A = Na, K, Rb; X = Cl, Br).

A new perovskite stoichiometric alkali tin dihalides compound, CsSnFCl, is synthesized by a hydrothermal method. This compound belongs to the monoclinic space group of P21/c with cell parameters of a = 9.5645(4) Å, b = 14.2057(7) Å, c = 13.5828(6) Å, and β = 93.2450(10)°. Unlike the common perovskites in which octahedra are interconnected to be a three-dimensional network, CsSnFCl possesses a zero-dimensional structure consisting of Cs cations, isolated [SnCl] trigonal pyramids, and dimer structural units [SnFCl]; the latter microscopic unit is found for the first time. The thermal stability and UV-vis-NIR diffuse reflectance spectroscopy in CsSnFCl are measured, and the electronic structure is calculated. Interestingly, the 5s lone-pair electrons on Sn cations are stereochemically active, which results in a pretty good photocatalytic activity of the title compound.

Structure and Characterization of a Zero-Dimensional Alkali Tin Dihalides Compound CsSnFCl with the [SnFCl] Clusters.

Molecular structural design is a compelling strategy to develop new compounds and optimize the crystal structure by atomic-scale manipulation. Herein, two fluorine-beryllium borates, BaMgBe(BO)F and BaCaBe(BO)F, have been rationally designed to overcome the structural instability problems of SrBeBO (SBBO). When relatively large Ba atoms were introduced, the [BeBO] double layers of SBBO were successfully broken, generating flexible [BeBOF] single layers. Also, the strategy adopted in this work has many implications in understanding the structural chemistry and designing novel optical functional materials in a beryllium borate system.

Structural Design of Two Fluorine-Beryllium Borates BaMBe(BO)F (M = Mg, Ca) Containing Flexible Two-Dimensional [BeBOF] Single Layers without Structural Instability Problems.

Two new polyborates, BaBeBO and SrBeBO, in a three-dimensional (BeBO) network featuring KBBF-type two-dimensional planes are synthesized. Compared with KBBF, both compounds possess comparable optical birefringence and deep-ultraviolet (deep-UV) cutoff edges and exhibit better bulk growth habits owing to their three-dimensional networks, which make them applicable deep-UV optical materials.

Two KBBF-Type Beryllium Borates MBeBO (M = Sr, Ba) with a Three-Dimensional (BeBO) Network.

TARS and TARS2 encode cytoplasmic and mitochondrial threonyl-tRNA synthetases (ThrRSs) in mammals, respectively. Interestingly, in higher eukaryotes, a third gene, TARSL2, encodes a ThrRS-like protein (ThrRS-L), which is highly homologous to cytoplasmic ThrRS but with a different N-terminal extension (N-extension). Whether ThrRS-L has canonical functions is unknown. In this work, we studied the organ expression pattern, cellular localization, canonical aminoacylation and editing activities of mouse ThrRS-L (mThrRS-L). Tarsl2 is ubiquitously but unevenly expressed in mouse tissues. Different from mouse cytoplasmic ThrRS (mThrRS), mThrRS-L is located in both the cytoplasm and nucleus; the nuclear distribution is mediated via a nuclear localization sequence at its C-terminus. Native mThrRS-L enriched from HEK293T cells was active in aminoacylation and editing. To investigate the in vitro catalytic properties of mThrRS-L accurately, we replaced the N-extension of mThrRS-L with that of mThrRS. The chimeric protein (mThrRS-L-NT) has amino acid activation, aminoacylation and editing activities. We compared the activities and cross-species tRNA recognition between mThrRS-L-NT and mThrRS. Despite having a similar aminoacylation activity, mThrRS-L-NT and mThrRS exhibit differences in tRNA recognition and editing capacity. Our results provided the first analysis of the aminoacylation and editing activities of ThrRS-L, and improved our understanding of Tarsl2.

A threonyl-tRNA synthetase-like protein has tRNA aminoacylation and editing activities.

Latest studies have indicated that microRNAs (miRNAs) play important roles in defending against bacterial and viral infections in marine crustacean, whereas little is known regarding the immunological roles of crustacean miRNAs in response to parasitic infection. To further reveal the host-parasite interactions between the parasitic dinoflagellate Hematodinium and its crustacean hosts, we applied the high-throughput sequencing technology to identify and characterize miRNAs in the Chinese swimming crab Portunus trituberculatus challenged with the Hematodinium parasite at a timescale of 16 days (d). A total of 168 miRNAs were identified and 51 miRNAs were differentially expressed in the hepatopancreas tissues of affected hosts. Eleven of the differentially expressed miRNAs were selected and verified by the quantitative real-time RT-PCR (qRT-PCR), manifesting the consistency between the high throughout sequencing and qRT-PCR assays. Further analysis of the putative target genes indicated that various immune-related pathways (e.g. endocytosis, Fc gamma R-mediated phagocytosis, lysosome, ECM-receptor interaction, complement and coagulation cascades, antigen processing and presentation, focal adhesion, etc.) and signal transduction pathways (e.g. JAK-STAT signaling pathway, MAPK signaling pathway, p53 signaling pathway, etc.) were mediated by the differentially expressed miRNAs. The results presented fundamental knowledge on the immunological roles of crustacean miRNAs and contributed to the better understanding of hosts' miRNAs-mediated immunity against the parasitic infection.

Differential expression of microRNAs in Portunus trituberculatus in response to Hematodinium parasites.

Candida albicans is a major fungal pathogen of humans, causing both superficial and life-threatening systemic infections in immunocompromised people. The conserved Ras/cAMP/PKA pathway plays a key role in regulating multiple traits important for the virulence of C. albicans such as cell growth, yeast-hyphae transition, white-opaque switching, sexual reproduction, and biofilm development. Diverse external signals influence cell physiology by activating this signaling pathway. The key components of the Ras/cAMP/PKA pathway include two Ras GTPases (Ras1 and Ras2), an adenylyl cyclase (Cyr1, also known as Cdc35), two cyclic nucleotide phosphodiesterases (Pde1 and Pde2), and the catalytic (Tpk1 and Tpk2) and regulatory (Bcy1) subunits of PKA kinase. Activation of this pathway dramatically alters the gene expression profile via several transcription factors, leading to the activation of specific biological processes. Here, we review the progress made in the past two decades to elucidate the molecular mechanisms by which the Ras/cAMP/PKA pathway senses diverse environmental cues and controls specific cellular responses and its connection with other signaling pathways in C. albicans. This article is protected by copyright. All rights reserved.

Article	Year
Functional characterization of leucine-specific domain 1 from eukaryal and archaeal leucyl-tRNA synthetases. The Biochemical journal\| PubMed ID: 20482517	2010
In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain. Nucleic acids research\| PubMed ID: 22917587	2012
Translational fidelity maintenance preventing Ser mis-incorporation at Thr codon in protein from eukaryote. Nucleic acids research\| PubMed ID: 23093606	2013
Crucial role of the C-terminal domain of Mycobacterium tuberculosis leucyl-tRNA synthetase in aminoacylation and editing. Nucleic acids research\| PubMed ID: 23268443	2013
A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity. RNA (New York, N.Y.)\| PubMed ID: 25051973	2014
Be2BO3F: A Phase of Beryllium Fluoride Borate Derived from KBe2BO3F2 with Short UV Absorption Edge. Inorganic chemistry\| PubMed ID: 27332696	2016
Beryllium-Free KBBF Family of Nonlinear-Optical Crystals: AZnBOX (A = Na, K, Rb; X = Cl, Br). Inorganic chemistry\| PubMed ID: 27989204	2016
Structure and Characterization of a Zero-Dimensional Alkali Tin Dihalides Compound CsSnFCl with the [SnFCl] Clusters. Inorganic chemistry\| PubMed ID: 28225268	2017
Structural Design of Two Fluorine-Beryllium Borates BaMBe(BO)F (M = Mg, Ca) Containing Flexible Two-Dimensional [BeBOF] Single Layers without Structural Instability Problems. Inorganic chemistry\| PubMed ID: 28885824	2017
Two KBBF-Type Beryllium Borates MBeBO (M = Sr, Ba) with a Three-Dimensional (BeBO) Network. Inorganic chemistry\| PubMed ID: 28945347	2017
A threonyl-tRNA synthetase-like protein has tRNA aminoacylation and editing activities. Nucleic acids research\| PubMed ID: 29579307	2018
Differential expression of microRNAs in Portunus trituberculatus in response to Hematodinium parasites. Fish & shellfish immunology\| PubMed ID: 30195909	2018
Multiple roles and diverse regulation of the Ras/cAMP/Protein Kinase A pathway in Candida albicans. Molecular microbiology\| PubMed ID: 30299574	2018

Qian Huang

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