Sexually dimorphic RNA helicases DDX3X and DDX3Y differentially regulate RNA metabolism through phase separation.

Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.

Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client.

A brain specific alternatively spliced isoform of nonmuscle myosin IIA lacks its mechanoenzymatic activities.

Recent genomic studies reported that 90 to 95% of human genes can undergo alternative splicing, by which multiple isoforms of proteins are synthesized. However, the functional consequences of most of the isoforms are largely unknown. Here, we report a novel alternatively spliced isoform of nonmuscle myosin IIA (NM IIA), called NM IIA2, which is generated by the inclusion of 21 amino acids near the actin-binding region (loop 2) of the head domain of heavy chains. Expression of NM IIA2 is found exclusively in the brain tissue, where it reaches a maximum level at 24 h during the circadian rhythm. The actin-dependent Mg2+-ATPase activity and in vitro motility assays reveal that NM IIA2 lacks its motor activities but localizes with actin filaments in cells. Interestingly, NM IIA2 can also make heterofilaments with NM IIA0 (noninserted isoform of NM IIA) and can retard the in vitro motility of NM IIA, when the two are mixed. Altogether, our findings provide the functional importance of a previously unknown alternatively spliced isoform, NM IIA2, and its potential physiological role in regulating NM IIA activity in the brain.

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

Flipping and other astonishing transporter dance moves in fungal drug resistance.

In all domains of life, transmembrane proteins from the ATP-binding cassette (ABC) transporter family drive the translocation of diverse substances across lipid bilayers. In pathogenic fungi, the ABC transporters of the pleiotropic drug resistance (PDR) subfamily confer antibiotic resistance and so are of interest as therapeutic targets. They also drive the quest for understanding how ABC transporters can generally accommodate such a wide range of substrates. The Pdr5 transporter from baker's yeast is representative of the PDR group and, ever since its discovery more than 30 years ago, has been the subject of extensive functional analyses. A new perspective of these studies has been recently provided in the framework of the first electron cryo-microscopy structures of Pdr5, as well as emergent applications of machine learning in the field. Taken together, the old and the new developments have been used to propose a mechanism for the transport process in PDR proteins. This mechanism involves a "flippase" step that moves the substrates from one leaflet of the bilayer to the other, as a central element of cellular efflux.

Endogenous IAA affected fluoranthene accumulation by regulating H+-ATPase and SOD activity in ryegrass.

This study explores the role of endogenous indole-3-acetic acid (IAA) in modulating plant responses to pollution stress and its effect on pollutant accumulation, with a focus on fluoranthene (Flu) in ryegrass. To elucidate the mechanism, we employed an IAA promoter (α-aminobutyric acid [α-AB]) and an IAA inhibitor (naphthylphthalamic acid [NPA]) to regulate IAA levels and analyze Flu uptake characteristics. The experimental setup included a Flu treatment group (ryegrass with Flu addition) and a control group (ryegrass without Flu). Our findings demonstrate that Flu treatment enhanced IAA content and plant growth in ryegrass compared to the control. The Flu+AB treatment further enhanced these effects, while the Flu+NPA treatment exhibited a contrasting trend. Moreover, Flu+AB treatment led to increased Flu accumulation, in contrast to the inhibitory effect observed with Flu+NPA treatment. Flu treatment also enhanced the activities of key antioxidant enzymes (SOD, POD, CAT) and increased soluble sugar and protein levels, indicative of enzymatic and nonenzymatic defense responses, respectively. The Flu+AB treatment amplified these responses, whereas the Flu+NPA treatment attenuated them. Significantly, Flu treatment raised H+-ATPase activity compared to the control, an effect further elevated by Flu+AB treatment and diminished by Flu+NPA treatment. A random forest analysis suggested that Flu accumulation dependency varied under different treatments: it relied more on H+-ATPase activity under Flu+AB treatment and more on SOD activity under Flu+NPA treatment. Additionally, Flu+AB treatment boosted the transpiration rate in ryegrass, thereby increasing the Flu translocation factor, a trend reversed by Flu+NPA treatment. This research highlights crucial factors influencing Flu accumulation in ryegrass, offering potential new avenues for controlling the gathering of contaminants within plant systems.

Binding of the Mycobacterium tuberculosis EccCb1 ATPase double hexameric ring to the EsxAB virulence factor is enhanced by ATP.

The EccC enzyme of Mycobacterium tuberculosis ESX-1 secretion system is involved in EsxAB virulence factor secretion and offers an attractive target for antivirulence inhibitors development against M. tuberculosis. The EccCb1 polypeptide of the EccC enzyme contains two Ftsk/SpoIIIE type ATPase domains (D2 and D3) and binds to the EsxAB factor at the C-terminal region of the D3 domain. In the current study, we have determined a low-resolution structure of EccCb1, and its mechanism involved in ATPase activity and EsxAB factor binding. Small-angle X-ray scattering data yielded a double hexameric ring structure of EccCb1 in solution and was further confirmed by SEC-MALS and dynamic light scattering. ATPase activity of wild-type, D2, and D3 mutants showed that D2-K90A and D3-K382A mutations led to a complete loss of enzyme activity. The full-length EccCb1 showed ∼3.7-fold lower catalytic efficiency than D2 domain and ∼1.7 fold lower than D3 domain. The EsxAB factor binds EccCb1 with Kd ∼ 11.3 ± 0.6 nM and its affinity is enhanced ∼2 fold in presence of ATP + Mg2+. These data indicate the involvement of ATPase activity in EsxAB factor translocation. Molecular dynamics simulation on wild-type, ATP + Mg2+, and EsxAB + ATP + Mg2+ bound EccCb1 double-ring structure showed enhanced stability of enzyme upon ATP + Mg2+ and EsxAB binding. Overall, our study showed a low-resolution structure of EccCb1, and the mechanism involved in ATPase activity and EsxAB factor recognition, which can be targeted for the development of antivirulence drugs against M. tuberculosis.

Synergistic Inhibitory Effect of Quercetin and Cyanidin-3O-Sophoroside on ABCB1.

The human ABCB1 (P-glycoprotein, Pgp) protein is an active exporter expressed in the plasma membrane of cells forming biological barriers. In accordance with its broad substrate spectrum and tissue expression pattern, it affects the pharmacokinetics of numerous chemotherapeutic drugs and it is involved in unwanted drug-drug interactions leading to side effects or toxicities. When expressed in tumor tissues, it contributes to the development of chemotherapy resistance in malignancies. Therefore, the understanding of the molecular details of the ligand-ABCB1 interactions is of crucial importance. In a previous study, we found that quercetin (QUR) hampers both the transport and ATPase activity of ABCB1, while cyandin-3O-sophroside (C3S) stimulates the ATPase activity and causes only a weak inhibition of substrate transport. In the current study, when QUR and C3S were applied together, both a stronger ATPase inhibition and a robust decrease in substrate transport were observed, supporting their synergistic ABCB1 inhibitory effect. Similar to cyclosporine A, a potent ABCB1 inhibitor, co-treatment with QUR and C3S shifted the conformational equilibrium to the "inward-facing" conformer of ABCB1, as it was detected by the conformation-selective UIC2 mAb. To gain deeper insight into the molecular details of ligand-ABCB1 interactions, molecular docking experiments and MD simulations were also carried out. Our in silico studies support that QUR and C3S can bind simultaneously to ABCB1. The most favourable ligand-ABCB1 interaction is obtained when C3S binds to the central substrate binding site and QUR occupies the "access tunnel". Our results also highlight that the strong ABCB1 inhibitory effect of the combined treatment with QUR and C3S may be exploited in chemotherapy protocols for the treatment of multidrug-resistant tumors or for improving drug delivery through pharmacological barriers.

Direct Effects of Toxic Divalent Cations on Contractile Proteins with Implications for the Heart: Unraveling Mechanisms of Dysfunction.

The binding of calcium and magnesium ions to proteins is crucial for regulating heart contraction. However, other divalent cations, including xenobiotics, can accumulate in the myocardium and enter cardiomyocytes, where they can bind to proteins. In this article, we summarized the impact of these cations on myosin ATPase activity and EF-hand proteins, with special attention given to toxic cations. Optimal binding to EF-hand proteins occurs at an ionic radius close to that of Mg2+ and Ca2+. In skeletal Troponin C, Cd2+, Sr2+, Pb2+, Mn2+, Co2+, Ni2+, Ba2+, Mg2+, Zn2+, and trivalent lanthanides can substitute for Ca2+. As myosin ATPase is not a specific MgATPase, Ca2+, Fe2+, Mn2+, Ni2+, and Sr2+ could support myosin ATPase activity. On the other hand, Zn2+ and Cu2 significantly inhibit ATPase activity. The affinity to various divalent cations depends on certain proteins or their isoforms and can alter with amino acid substitution and post-translational modification. Cardiac EF-hand proteins and the myosin ATP-binding pocket are potential molecular targets for toxic cations, which could significantly alter the mechanical characteristics of the heart muscle at the molecular level.

Effect of bacteriophage-encoded chaperonins on amyloid transformation of α-synuclein.

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity.

Protective Effects of Inosine on Memory Consolidation in a Rat Model of Scopolamine-Induced Cognitive Impairment: Involvement of Cholinergic Signaling, Redox Status, and Ion Pump Activities.

This study investigated the effects of inosine on memory acquisition and consolidation, cholinesterases activities, redox status and Na+, K+-ATPase activity in a rat model of scopolamine-induced cognitive impairment. Adult male rats were divided into four groups: control (saline), scopolamine (1 mg/kg), scopolamine plus inosine (50 mg/kg), and scopolamine plus inosine (100 mg/kg). Inosine was pre-administered for 7 days, intraperitoneally. On day 8, scopolamine was administered pre (memory acquisition protocol) or post training (memory consolidation protocol) on inhibitory avoidance tasks. The animals were subjected to the step-down inhibitory avoidance task 24 hours after the training. Scopolamine induced impairment in the acquisition and consolidation phases; however, inosine was able to prevent only the impairment in memory consolidation. Also, scopolamine increased the activity of acetylcholinesterase and reduced the activity of Na+, K+-ATPase and the treatment with inosine protected against these alterations in consolidation protocol. In the animals treated with scopolamine, inosine improved the redox status by reducing the levels of reactive oxygen species and thiobarbituric acid reactive substances and restoring the activity of the antioxidant enzymes, superoxide dismutase and catalase. Our findings suggest that inosine may offer protection against scopolamine-induced memory consolidation impairment by modulating brain redox status, cholinergic signaling and ion pump activity. This compound may provide an interesting approach in pharmacotherapy and as a prophylactic against neurodegenerative mechanisms involved in Alzheimer's disease.

Freshwater adaptation in prickly sculpin (Pisces: Cottidae): intraspecific comparisons reveal evidence for water pH and Na+ concentration driving diversity in gill H+-ATPase and ion regulation.

Phenotypic divergence is a hallmark of adaptive radiation. One example involves differentiation in physiological traits involved in ion regulation among species with contrasting lifestyles and living in distinct environments. Differentiation in ion regulation and its ecological implications among populations within species are, however, less well understood. To address this knowledge gap, we collected prickly sculpin (Cottus asper) from distinct habitat types including coastal rivers connected to estuaries, coastal lakes and interior lakes, all from British Columbia, Canada. We tested for differences in plasma Na+ and Cl-, gill Na+/K+-ATPase and H+-ATPase activity and protein abundance as well as changes in body mass and arterial blood pH in fish sampled from the field and acclimated to two different freshwater conditions in the laboratory: artificial lake water (ALW) and ion-poor water (IPW). We also tested for links between environmental water chemistry and the physiological characteristics associated with ion regulation. Transfer to IPW resulted in upregulation of gill Na+/K+-ATPase and H+-ATPase activity as well as increases in gill H+-ATPase protein expression level in each habitat compared with that in the common ALW treatment. Despite the presence of population-within-habitat-type differences, significant habitat-type effects were revealed in most of the ion regulation characteristics examined under different acclimation conditions. Significantly lower plasma Cl- was detected in fish from coastal rivers than in fish from the other two habitat types during the IPW treatment, which was also significantly lower compared with that in ALW. Similarly, gill Na+/K+-ATPase activity was lower in the coastal river populations in IPW than in fish from coastal and interior lakes, which was not in accordance with the protein expression in the gill. For gill H+-ATPase, fish from interior lake populations had the highest level of activity across all habitat types under all conditions, which was related to the protein levels in the gill. The activity of gill H+-ATPase was positively correlated with the combined effect of water Na+ and pH under the ALW treatment. Our results suggest that variation in habitat may be an important factor driving differences in gill Na+/K+-ATPase and H+-ATPase activity across populations of C. asper. Further, the combined effect of water Na+ and pH may have played a key role in physiological adaptation in C. asper during post-glacial freshwater colonization and dispersal.

Decreased Na+/K+ pump activity in the erythrocyte membrane due to malondialdehyde in rheumatoid arthritis: an in vivo and in silico study.

Apart from demonstrating the interaction behavior of malondialdehyde (MDA) with Na+/K+-ATPase using in silico, the current study aims to investigate the effect of rheumatoid arthritis-related oxidative stress on Na+/K+-ATPase activity that is present in the erythrocyte cell membrane, which is rich in proteins vulnerable to damage from MDA and other free radicals. The target population of this study consists of 28 rheumatoid arthritis patients and 20 healthy volunteers whose MDA levels and Na+/K+-ATPase activity were determined. It was shown that MDA levels of rheumatoid arthritis patients increased (p < 0.001) and their Na+/K+-ATPase activity noticeably decreased when compared to those of healthy individuals. Also, according to this in silico modeling, MDA decreased Na+/K+-ATPase activity in line with the correlation analyses. Consequently, while elevated levels of MDA in the rheumatoid arthritis group were suggestive of oxidative stress, a decreased Na+/K+-ATPase-activity led us to speculate that the cellular membrane had sustained injury. Therefore, our results could be useful in explaining how MDA affects Na+/K+-ATPase activity in the interior of a specific molecular pathway.

Analysis of reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin.

Cypermethrin contamination was a potential threat to soil organisms. In the present work, reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin was investigated. It was found that earthworms could absorb and accumulate residual cypermethrin in soil, and also earthworm activities helped accelerate the degradation of cypermethrin in soil. The accumulation of cypermethrin in earthworms induced sperm damage, and cypermethrin not only caused the imbalance of calcium homeostasis in earthworm sperm cells by inhibiting earthworm sperm Ca2+-ATP and Ca2+-Mg2+-ATP enzyme activities but also caused barriers in acrosome reaction. It also affected sperm energy supply of earthworms by inhibiting the activity of Na+-K+-ATPase and Mg2+-ATPase of earthworm sperm. Meanwhile, the inhibition of acrosome enzyme activity of earthworm sperm by cypermethrin led to hinder fertilization and reduced cocoon production of earthworms, and the damage of cypermethrin to sperm of earthworm was a significant cause of its reproductive toxicity. The results of the evaluation of IBR index showed that reproductive toxicity of cypermethrin to earthworms reduced with the increasing time. The decreased reproductive toxicity of cypermethrin to earthworms at the later stage of exposure (42-56 d) might be due to a combination of reduced absorption of cypermethrin in soil by earthworms, decreased accumulation of cypermethrin in the body, and improved sperm capacitation.

Rotenoids from Clitoria fairchildiana R. Howard (Fabaceae) seeds affect the cellular metabolism of larvae of Aedes aegypti L. (Culicidae).

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Activation of Piezo1 Increases Na,K-ATPase-Mediated Ion Transport in Mouse Lens.

Lens ion homeostasis depends on Na,K-ATPase and NKCC1. TRPV4 and TRPV1 channels, which are mechanosensitive, play important roles in mechanisms that regulate the activity of these transporters. Here, we examined another mechanosensitive channel, piezo1, which is also expressed in the lens. The purpose of the study was to examine piezo1 function. Recognizing that activation of TRPV4 and TRPV1 causes changes in lens ion transport mechanisms, we carried out studies to determine whether piezo1 activation changes either Na,K-ATPase-mediated or NKCC1-mediated ion transport. We also examined channel function of piezo1 by measuring calcium entry. Rb uptake was measured as an index of inwardly directed potassium transport by intact mouse lenses. Intracellular calcium concentration was measured in Fura-2 loaded cells by a ratiometric imaging technique. Piezo1 immunolocalization was most evident in the lens epithelium. Potassium (Rb) uptake was increased in intact lenses as well as in cultured lens epithelium exposed to Yoda1, a piezo1 agonist. The majority of Rb uptake is Na,K-ATPase-dependent, although there also is a significant NKCC-dependent component. In the presence of ouabain, an Na,K-ATPase inhibitor, Yoda1 did not increase Rb uptake. In contrast, Yoda1 increased Rb uptake to a similar degree in the presence or absence of 1 µM bumetanide, an NKCC inhibitor. The Rb uptake response to Yoda1 was inhibited by the selective piezo1 antagonist GsMTx4, and also by the nonselective antagonists ruthenium red and gadolinium. In parallel studies, Yoda1 was observed to increase cytoplasmic calcium concentration in cells loaded with Fura-2. The calcium response to Yoda1 was abolished by gadolinium or ruthenium red. The calcium and Rb uptake responses to Yoda1 were absent in calcium-free bathing solution, consistent with calcium entry when piezo1 is activated. Taken together, these findings point to stimulation of Na,K-ATPase, but not NKCC, when piezo1 is activated. Na,K-ATPase is the principal mechanism responsible for ion and water homeostasis in the lens. The functional role of lens piezo1 is a topic for further study.

A Preparative Method for the Isolation of Calponin from Molluscan Catch Muscle.

We describe the development of a preparative method to isolate molluscan catch muscle, calponin. This method is based on the ability of calponin to interact with actin in a temperature-dependent manner. After extracting thin filaments, as previously described, the extract was ultracentrifuged at 2 °C. While other surface proteins of thin filaments co-precipitated with actin, calponin, along with some minor contaminants, remained in the supernatant. Calponin was purified through cation-exchange chromatography. The yield of pure protein was four-fold higher than that achieved through high-temperature extraction. To evaluate functionally isolated proteins, we determined the effect of calponin on Mg2+-ATPase activity of hybrid and non-hybrid actomyosin. The degree of ATPase inhibition was consistent with previously published data but strongly dependent on the environmental conditions and source of actin and myosin used. Furthermore, at low concentrations, calponin could induce the ATPase activity of hybrid actomyosin. This result was consistent with data indicating that calponin can modulate actin conformation to increase the relative content of "switched on" actin monomers in thin filaments. We assume that calponin obtained by the isolation method proposed herein is a fully functional protein that can both inhibit and induce the ATPase activity.

Conservation and Diversification of tRNA t6A-Modifying Enzymes across the Three Domains of Life.

The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon-codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3-/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD-TsaB -TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway-Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure-function relationship from perspectives of conservation and diversity.

New Structures, Spectrometric Quantification, and Inhibitory Properties of Cardenolides from Asclepias curassavica Seeds.

Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias, cardenolides in milkweed plants have an established role in plant-herbivore and predator-prey interactions, based on their ability to inhibit the membrane-bound Na+/K+-ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica. We report the structures of new compounds: 3-O-ß-allopyranosyl coroglaucigenin (1), 3-[4'-O-ß-glucopyranosyl-ß-allopyranosyl] coroglaucigenin (2), 3'-O-ß-glucopyranosyl-15-ß-hydroxycalotropin (3), and 3-O-ß-glucopyranosyl-12-ß-hydroxyl coroglaucigenin (4), as well as six previously reported cardenolides (5-10). We test the in vitro inhibition of these compounds on the sensitive porcine Na+/K+-ATPase. The least inhibitory compound was also the most abundant in the seeds-4'-O-ß-glucopyranosyl frugoside (5). Gofruside (9) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant-herbivore interactions.