Molecular architecture of the human tRNA ligase complex

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B, and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair, and mRNA transport. Here, we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex and provide a structural framework for understanding its functions in cellular RNA metabolism

Kinetic behavior of MgH2-transition metal composites: towards hydrogen storage

Hydrogen as an energy vector represents great potential, due to its high gravimetric density and low mass, as well as the fact that combustion does not emit harmful chemical byproducts. Hydrogen has the highest energy density per unit mass compared to any other fuel but a rather low energy density per unit volume. Further, hydrogen storage is a key technology for developing a hydrogen and fuel cell-based economy [1]. Metal hydrides as alternative hydrogen carriers have a wide range of performance parameters such as operating temperature, sorption kinetics, activation conditions, cyclic options, and equilibrium hydrogen pressure. These parameters can be improved or adjusted to meet the technical requirements of different applications. The most commonly used method for hydride destabilization is nanostructuring by mechanical milling which leads to a reduction in the particle and crystallite size of the MgH2 powder. Nanostructuring is often combined with catalyst addition and composite formation [2,3]. The most of research is focused on the morphological, structural, and thermodynamic effects typical for long milling times, while in this work we have followed the changes taking place under short milling times. The thermal stability of magnesium hydride is related to - changes in the crystallites and powder particle size. The analysis also considered the changes in activation energy. MgH2-M composites were prepared by mechanical milling of the as-received MgH2 powder (Alfa Aesar, 98% purity) with the addition of 2 and 5 wt.% of M (M= V, W, Mo). Mechanical milling was performed in s SPEX 5100 Mixer Mill using 8mm diameter milling ball. Samples were milled for 15-45 minutes under the inert atmosphere of argon and a ball-to-powder ratio 10:1 Figure 1. shows the kinetic curves obtained for composites with 5wt% of vanadium. To investigate the desorption process in detail, different models of solid-state kinetics were used as implemented in the code developed in our group. The ratelimiting step of the desorption reaction was determined using the iso-conversional kinetic method due to better accuracy of obtained apparent activation energies. As shown in Table 1 a decrease in apparent activation energies has been observed. It is obvious that the sorption kinetics is affected by material preparation because the reactivity of magnesium with hydrogen is strongly modified by changes in several surface parameters that govern the chemisorption, the dissociation of molecular hydrogen, and hydride nucleation7th MESC-IS 2023 : International Symposium on Materials for Energy Storage and Conversion : 11th INESS : International Conference on Nanomaterials & Adv. Energy Storage Systems : October 7-10, Baku, 2023

The Catalytic Effect of Vanadium on Sorption Properties of MgH2-Based Nanocomposites Obtained Using Low Milling Time

The effects of catalysis using vanadium as an additive (2 and 5 wt.%) in a high-energy ball mill on composite desorption properties were examined. The influence of microstructure on the dehydration temperature and hydrogen desorption kinetics was monitored. Morphological and microstructural studies of the synthesized sample were performed by X-ray diffraction (XRD), laser particle size distribution (PSD), and scanning electron microscopy (SEM) methods, while differential scanning calorimetry (DSC) determined thermal properties. To further access amorph species in the milling blend, the absorption spectra were obtained by FTIR-ATR analysis (Fourier transform infrared spectroscopy attenuated total reflection). The results show lower apparent activation energy (Eapp) and H2 desorption temperature are obtained for milling bland with 5 wt.% added vanadium. The best explanation of hydrogen desorption reaction shows the Avrami-Erofeev model for parameter n = 4. Since the obtained value of apparent activation energy is close to the Mg-H bond-breaking energy, one can conclude that breaking this bond would be the rate-limiting step of the process

The oxidoreductase PYROXD1 uses NAD(P)+ as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism

The oxidoreductase PYROXD1 uses NAD(P) + as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism. Keywords: NADH; NADPH; PYROXD1; RtcB; UPR; copper; metalloenzyme; myopathy; oxidative stress; oxidoreductase; pre-tRNA splicing; tRNA ligase complex

Molecular architecture of the human tRNA ligase complex

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B, and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair, and mRNA transport. Here, we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex and provide a structural framework for understanding its functions in cellular RNA metabolism.ISSN:2050-084