Stable Dimer Research Articles

Dynamic stability of salt stable cowpea chlorotic mottle virus capsid protein dimers and pentamers of dimers

Intermediates of the self-assembly process of the salt stable cowpea chlorotic mottle virus (ss-CCMV) capsid can be modelled atomistically on realistic computational timescales either by studying oligomers in equilibrium or by focusing on their dissociation instead of their association. Our previous studies showed that among the three possible dimer interfaces in the icosahedral capsid, two are thermodynamically relevant for capsid formation. The aim of the current study is to evaluate the relative structural stabilities of the three different ss-CCMV dimers and to find and understand the conditions that lead to their dissociation. Long timescale molecular dynamics simulations at 300 K of the various dimers and of the pentamer of dimers underscore the importance of large contact surfaces on stabilizing the capsid subunits within an oligomer. Simulations in implicit solvent show that at higher temperature (350 K), the N-terminal tails of the protein units act as tethers, delaying dissociation for all but the most stable interface. The pentamer of dimers is also found to be stable on long timescales at 300 K, with an inherent flexibility of the outer protein chains.

Force generation of KIF1C is impaired by pathogenic mutations

Intracellular transport is essential for neuronal function and survival. The most effective plus-end-directed neuronal transporter is the kinesin-3 KIF1C, which transports large secretory vesicles and endosomes.1-4 Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients.5-8 In contrast to other kinesin-3s, KIF1C is a stable dimer and a highly processive motor in its native state.9,10 Here, we establish a baseline for the single-molecule mechanics of Kif1C. We show that full-length KIF1C molecules can processively step against the load of an optical trap and reach average stall forces of 3.7 pN. Compared with kinesin-1, KIF1C has a higher propensity to slip backward under load, which results in a lower maximal single-molecule force. However, KIF1C remains attached to the microtubule while slipping backward and re-engages quickly, consistent with its super processivity. Two pathogenic mutations, P176L and R169W, that cause hereditary spastic paraplegia in humans7,8 maintain fast, processive single-molecule motility invitro but with decreased run length and slightly increased unloaded velocity compared with the wild-type motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles. Our results show how its mechanics supports KIF1C's role as an intracellular transporter and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.

Structural Analysis and Conformational Dynamics of Short Helical Hyperphosphorylated Segments of Tau Protein (Sequence 254-290) in Alzheimer's Disease: A Molecular Dynamics Simulation Study.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder whose early diagnosis leads to a chance for successful treatment and decreases the side effects. Hyperphosphorylation of tau proteins is a pathological hallmark of AD that causes it to lose its attachment ability to the microtubules. Alteration of tau structure due to its hyperphosphorylation is an exciting challenge regarding AD treatments. Here, we aimed to examine the structural alterations of short helical segments of tau protein with one to three phosphorylated sites by molecular dynamics simulation. Results indicated that the interaction of two similar segments with three phosphorylated sites (P-Ser262, 285, and 289) formed a compact and more stable structure than the one phosphorylated site complex (P-Ser262). Moreover, due to the high dynamics of the P-Ser262 complex, several structures were made with different conformational dynamics, but there was only one stable cluster of the P-Ser262, 285, and 289 complex during simulation. It seems that the P-Ser262, 285, and 289 complex plays an important role in the formation of paired helical filaments (PHFs) by forming a stable dimer. Generally, it is important to identify how structural features of segments in tau protein change when the phosphorylated sites increase from one to three sites and their effects on the formation of PHFs for drug design and diagnostic biomarkers.

Stabilization of Zn group dimers: A theoretical study

AbstractQuantum chemical calculations have been carried out to investigate the possibility of Group 12 (Zn, Cd, Hg) dimer formation supported by the B3ring. All these complexes have significantly shorter M‐M (M = Zn, Cd, Hg) distances compared to their bare dimers, M2. The staggered and eclipsed forms of these complexes were found to be nearly degenerate. They have significant bond strengths as revealed from the significantly higher values of Wiberg bond indices as well as bond dissociation energies. The topological feature of electron density reveals significant covalency in the M‐M interaction which has also been confirmed from natural orbital for chemical valence analysis coupled with extended transition state (ETS‐NOCV) calculations.

Influence of Ionic Liquids on the Aggregation and Pre-aggregation Phenomena of Asphaltenes in Model Solvent Mixtures by Molecular Dynamics Simulations and Quantum Mechanical Calculations

This work presents a systematic study of asphaltene pre-aggregation phenomena in the absence and in the presence of ionic liquids (ILs) from the family of 1-alkyl-3-methylimidazolium by molecular dynamics simulations. The effects of the alkyl chain length of the cation (studying ILs with alkyl chains between C4 and C10) and of the dimension of the anion (testing chloride and bromide) on the aggregation behavior of asphaltenes have been studied. To correlate the results obtained with the direct interaction between each additive and asphaltene, the latter was investigated both by the analysis of the radial distribution functions obtained by molecular dynamics simulations and quantum mechanical calculations. The DFT method was used to calculate the relative stability of the asphaltene-ionic liquid dimers and also the energy, shape, and spatial distribution of frontier orbitals. It was found that all the ionic liquids studied present a dispersing effect on asphaltene in model solvents, except for mixtures rich in toluene where, in most cases, the opposite effect is observed. This is accompanied by the interaction intensity as measured by radial distribution functions. The effects of the alkyl side chain length of the cation and of the anion radius are subtler, but it seems that the asphaltene dispersion effect increases with the length of the cation’s alkyl side chain and decreases with the radius of the anion; these effects are more clearly observed in the mixtures richer in n-heptane. These trends were corroborated by DFT calculations, which showed that the energetic stability of the asphaltene-additive dimer is as higher as the alkyl chain is longer and the anion is smaller. Pre-aggregation phenomena were also studied in mixtures containing CO2, which proved to be a precipitating agent as observed experimentally. The relative performances of the IL studied were not altered by the presence of CO2.

Analysis of amyloidogenic transthyretin mutations using continuum solvent free energy calculations.

Many proteins can undergo pathological conformational changes that result in the formation of amyloidogenic fibril structures. Various neurodegenerative diseases are associated with such pathological fibril formation of specific proteins. Transthyretin (TTR) is a tetrameric globular transport protein in the blood plasma that can dissociate, unfold, and form long and stable fibrils. Many TTR mutations are known that promote (TTR) amyloidosis and cause severe diseases. TTR amyloidosis has been studied extensively using biochemical methods and structures of various mutations in the globular form have been characterized. Recently, also the structure of a TTR fibril has been determined. In an effort to better understand why some mutations increase or decrease the tendency of amyloid formation, we have applied a combined molecular dynamics and continuum solvent approach to calculate the energetic influence of residue changes in the globular versus fibril form. For 29 out of 36 tested TTR single residue mutations, the approach correctly predicts the increased or decreased tendency for amyloidosis allowing us also to elucidate the origins of the tendency. We find that indeed the destabilization of the globular monomer or changes in dimer and tetramer stability due to mutation has a dominant influence on the amyloidogenic tendency. The continuum solvent model predicts a significantly more favorable mean energy per residue of the fibril form compared to the globular form. This effect is only slightly modulated by single-point mutations preserving the energetic preference for fibril formation upon protein unfolding. It explains why no correlation between experimental amyloidosis and calculated change in fibril stability was observed.

A New Way to Predict the Efficiency of Optical Limiters without Providing an Experiment: Processing of TDDFT Calculations for the Case of Pure Two-Photon Absorption.

Laser-power-limiting devices play a predominant role in photonics because of their potential for protecting human eyes and optical devices that are sensitive to intense laser beams. This paper describes a new methodology for predicting the efficiency of optical limiting based on electric-field-induced changes in absorption spectra calculated with the TDDFT quantum-chemical method. Analytical equations are derived to evaluate the optical thresholds and speed of switching on, the dynamic range, and the degree of nonlinear attenuation of the radiation fluxes for the case of two-photon absorption. Thus, the researcher does not need to conduct costly experiments to evaluate the suitability of nonlinear absorbers for the creation of optical limiters. The possibility of developing a forecasting model is demonstrated by an example of a series of stable slipped-cofacial phthalocyanine J-type dimers, which were synthesized and investigated previously.

HUG Domain Is Responsible for Active Dimer Stabilization in an NrdJd Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.

A computational and theoretical study of some heavy metal heteronuclear dimers

Heteronuclear metal dimers have several applications in scientific domains which entail the need for understanding their properties. Accordingly, a computational and theoretical analysis based on the Conceptual Density Functional Theory (CDFT) descriptors is reported to explain electronic properties and stability of CuM, RhM, YM, LaM and AuM-type heteronuclear dimers, where M = metal. Moreover, CuM-type dimers are revealed to be the most stable while LaM-type dimers the least. A reasonable correlation exists between the selected descriptors and HOMO-LUMO gap. Computational and empirically calculated data run parallel supporting the consistency of our computations. It is believed that the present examination would help in exploring the most competent and stable hetero-metal dimer for potent applications.

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule.

The β2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β1 isoform have been shown to trans-interact in a species-specific mode with the β1 subunit on the epithelial neighboring cell, the β2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β2 or described its adhesion mechanism. Until now, the interactions pronounced for β2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β2 is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β2 subunit show that the β2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein–protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer’s RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

The interaction between STING and NCOA4 exacerbates lethal sepsis by orchestrating ferroptosis and inflammatory responses in macrophages

The discovery of STING-related innate immunity has recently provided a deep mechanistic understanding of immunopathy. While the detrimental effects of STING during sepsis had been well documented, the exact mechanism by which STING causes lethal sepsis remains obscure. Through single-cell RNA sequence, genetic approaches, and mass spectrometry, we demonstrate that STING promotes sepsis-induced multiple organ injury by inducing macrophage ferroptosis in a cGAS- and interferon-independent manner. Mechanistically, Q237, E316, and S322 in the CBD domain of STING are critical binding sites for the interaction with the coiled-coil domain of NCOA4. Their interaction not only triggers ferritinophagy-mediated ferroptosis, but also maintains the stability of STING dimers leading to enhanced inflammatory response, and reduces the nuclear localization of NCOA4, which impairs the transcription factor coregulator function of NCOA4. Meanwhile, we identified HET0016 by high throughput screening, a selective 20-HETE synthase inhibitor, decreased STING-induced ferroptosis in peripheral blood mononuclear cells from patients with sepsis and mortality in septic mice model. Our findings uncover a novel mechanism by which the interaction between STING and NCOA4 regulates innate immune response and ferroptosis, which can be reversed by HET0016, providing mechanistic and promising targets insights into sepsis.

Abstract 676: ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis

Abstract Lung cancer is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into invasive mesenchymal cells. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in a panel of lung adenocarcinoma cell lines. We hypothesize that ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis. We determined that the acetyl-deficient mutant (125kDa) exhibits a decreased half-life compared to WT and acetylated ZEB1, suggesting that disruption of acetylation hinders dimerization and protein stability. However, the acetyl-mimetic mutant (250kDa) protects ZEB1 from proteasomal degradation brought by the action of the E3 ubiquitin ligase SIAH1. siRNA-mediated silencing of SIAH1 increased the mRNA and protein levels of ZEB1 WT and both acetylation mutants. Importantly, silencing of SIAH1 markedly increased the half-life of 250kDa ZEB1 WT from 18.0hrs to 25.4hrs and acetyl-mimetic from 12.3hrs to 24.0hrs, as well as 125kDa ZEB1 acetyl-deficient from 9.7hrs to 18.3hrs. Although we determined that 250kDa ZEB1 is significantly more stable than 125kDa ZEB1, the increase in the stability by silencing SIAH1 contributes to ZEB1 acetyl-deficient binding at the promoter of its target genes mir200c-141 and SEMA3F. 250kDa ZEB1 WT and acetyl-mimetic recruits the histone deacetylases (HDACs) containing the nucleosome remodeling and deacetylase (NuRD) complex, to promote lung adenocarcinoma invasion and metastasis compared to ZEB1 acetyl-deficient. We further established that the stability of ZEB1 is integral to promoting these mesenchymal features. Our results suggest that ZEB1_K811ac regulates its dimerization and stability to promote lung adenocarcinoma via EMT. Future studies are focused on understanding the role of ZEB1 protein stability to recruit NuRD co-repressor and subsequently mediate tumorigenesis and metastasis. Citation Format: Mabel G. Perez-Oquendo, Roxsan Manshouri, Don Gibbons. ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 676.

Effect of Differential Backbone Di‐substitution of Gamma Amino Acid Residues on the Conformation and Assembly of their Fmoc Derivatives in Solid and Solution States

We studied the effect of variable backbone dimethyl-substitution of γ amino acid residues (γ2,2 , γ3,3 and γ4,4 ) on the conformation and assembly, in crystals and solution of their Fmoc derivatives. The crystal structure of γ2,2 and γ4,4 derivatives showed distinct conformations (open/close for γ2,2 /γ4,4 ) that differed in torsion angles, hydrogen-bonding and most importantly the π-π Fmoc-stacking interactions (relatively favourable for γ4,4 -close). Fmoc derivatives existed in an equilibrium between major-monomeric (low energy, non-hydrogen bonded) and minor-dimeric (high energy, hydrogen bonded) populations in solution. The rate of major/minor population exchange was dependent on the position of substitution, highest being for γ4,4 derivative. In solution, assembly of Fmoc derivatives was solvent dependent, but it was independent of the position of geminal substitution. Crystallization was primarily governed by the stabilization of high-energy dimer by favourable π-π stacking involving Fmoc moieties. High free-energy of the dimers (γ2,2 -close, γ3,3 -open/close) offset favourable stacking interactions and hindered crystallization.

Photolysis by UVA-visible light and thermal degradation of TNT in aqueous solutions according to aerodynamic thermal breakup droplet ionization mass spectrometry

The mechanism of photolytic degradation of 2-4-6-trinitrotoluene (TNT) in an aqueous solution (initial concentration 102 mg L−1) by UVA-visible light (>320 nm) was investigated by aerodynamic thermal breakup droplet ionization mass spectrometry. For photolysis, a DRK-120 mercury-quartz lamp was used. The final and intermediate products of the photolysis reaction were compared with known products of TNT transformation in the environment. The first step in the photolysis process under our conditions was found to be the association of TNT molecules into a fairly stable dimer. The TNT dimer formation proceed through the formation of a Meisenheimer complex. Thermal degradation of the TNT dimer in microdroplets within a heated capillary used as a flow reactor was studied next. It was shown that the degradation of the TNT dimer in microdroplets occurs very quickly (2.1 ms) at heating (up to 200–250 °C). Of course, heating an aqueous aerosol requires energy, but the transformation of TNT can be controlled using a combination of photo- and thermal treatments. We hope that these results will help to reduce the toxicity of TNT contaminants and remove nitroaromatic compounds from water at ambient conditions. In this work, the TNT dimer formed at the first stage is transformed with the loss of NO2 fragments or water. In addition, further polymerization of TNT is observed.

Distinct dissociation rates of murine and human norovirus P-domain dimers suggest a role of dimer stability in virus-host interactions

Norovirus capsids are icosahedral particles composed of 90 dimers of the major capsid protein VP1. The C-terminus of the VP1 proteins forms a protruding (P)-domain, mediating receptor attachment, and providing a target for neutralizing antibodies. NMR and native mass spectrometry directly detect P-domain monomers in solution for murine (MNV) but not for human norovirus (HuNoV). We report that the binding of glycochenodeoxycholic acid (GCDCA) stabilizes MNV-1 P-domain dimers (P-dimers) and induces long-range NMR chemical shift perturbations (CSPs) within loops involved in antibody and receptor binding, likely reflecting corresponding conformational changes. Global line shape analysis of monomer and dimer cross-peaks in concentration-dependent methyl TROSY NMR spectra yields a dissociation rate constant koff of about 1 s−1 for MNV-1 P-dimers. For structurally closely related HuNoV GII.4 Saga P-dimers a value of about 10−6 s−1 is obtained from ion-exchange chromatography, suggesting essential differences in the role of GCDCA as a cofactor for MNV and HuNoV infection.

Beyond single-atom catalysts: Exploration of Cu dimer and trimer for CO2 reduction to methane

We extend the single-atom catalysts (SACs) concept to metal-dimer and trimer catalysts anchored on graphene and explore their characteristic performance in catalyzing CO2RR into methane using first-principles calculations. In designing the catalyst, we first identify highly energetically stable Cu dimer and trimer atomic configurations in graphene host and then compute the Gibbs free energy changes in the reaction pathway from carbon dioxide to methane. Remarkably, compared to Cu SAC, the limiting potential is 0.60 V for the Cu-trimer and 1.22 V for Cu-dimer, which is lower than 1.82 V for SAC. The Cu dimer is suggested to be the optimal choice because it demonstrates reasonably high activity to CO2RR and high selectivity against HER than SAC. The methodology study suggested that the adsorption term plays the key role in the whole CO2RR process. The present work points out a new strategy for designing high-performance catalysts beyond single atoms for CO2RR.

Theoretical studies on 2,3,5,6-Tetra(1H-tetrazol-5-yl)pyrazine and 1,1-diamino-2,2-Dinitroethylene blending system

To study the properties of 2,3,5,6-tetra(1H-tetrazol-5-yl)pyrazine (H4TTP) and 1,1′-diamino-2,2′-dinitroethylene (FOX-7) blending system, the structures of H4TTP, FOX-7, and H4TTP/FOX-7 dimers were optimized using density functional theory (DFT), and the mechanical properties and cohesive energy densities (CED) of H4TTP/FOX-7 blends with different mass ratios were calculated by molecular dynamics (MD) simulation. The results show that the HOMO of H4TTP is distributed on the pyrazine and tetrazole rings, while the LUMO is mainly distributed on the pyrazine ring, with a small contribution from the tetrazole ring. The HOMO of FOX-7 molecules is mainly located on the CC bonds, while the LUMO is mainly located on the nitro groups. The most stable dimer, (I), was formed when the interaction between frontier MOs is possible and hydrogen bond is formed between two monomers, which was confirmed by the Reduced Density Gradient (RDG) isosurface graph. MD studies were carried out to examine the mechanical properties and cohesive energy density of the blending systems. In monomer systems, FOX-7 has the strongest rigidity and best ductility, while H4TTP has the largest elasticity and best toughness. In the blending systems, we found that various mechanical properties and CED values were different from those of monomers, which improves the sensitivity of H4TTP and the safety of explosives.

Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders.

Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.