METTL16 exerts an m6A-independent function to facilitate translation and tumorigenesis.

METTL16 has recently been identified as an RNA methyltransferase responsible for the deposition of N6-methyladenosine (m6A) in a few transcripts. Whether METTL16 methylates a large set of transcripts, similar to METTL3 and METTL14, remains unclear. Here we show that METTL16 exerts both methyltransferase activity-dependent and -independent functions in gene regulation. In the cell nucleus, METTL16 functions as an m6A writer to deposit m6A into hundreds of its specific messenger RNA targets. In the cytosol, METTL16 promotes translation in an m6A-independent manner. More specifically, METTL16 directly interacts with the eukaryotic initiation factors 3a and -b as well as ribosomal RNA through its Mtase domain, thereby facilitating the assembly of the translation-initiation complex and promoting the translation of over 4,000 mRNA transcripts. Moreover, we demonstrate that METTL16 is critical for the tumorigenesis of hepatocellular carcinoma. Collectively, our studies reveal previously unappreciated dual functions of METTL16 as an m6A writer and a translation-initiation facilitator, which together contribute to its essential function in tumorigenesis.

Acyl-Coa Thioesterases: A Rheostat That Controls Activated Fatty Acids Modulates Dengue Virus Serotype 2 Replication.

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. The loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that functional differences and substrate specificities due to the location (cytosol and mitochondria, respectively) of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell, highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

The Iron Maiden. Cytosolic Aconitase/IRP1 Conformational Transition in the Regulation of Ferritin Translation and Iron Hemostasis.

Maintaining iron homeostasis is fundamental for almost all living beings, and its deregulation correlates with severe and debilitating pathologies. The process is made more complicated by the omnipresence of iron and by its role as a fundamental component of a number of crucial metallo proteins. The response to modifications in the amount of the free-iron pool is performed via the inhibition of ferritin translation by sequestering consensus messenger RNA (mRNA) sequences. In turn, this is regulated by the iron-sensitive conformational equilibrium between cytosolic aconitase and IRP1, mediated by the presence of an iron-sulfur cluster. In this contribution, we analyze by full-atom molecular dynamics simulation, the factors leading to both the interaction with mRNA and the conformational transition. Furthermore, the role of the iron-sulfur cluster in driving the conformational transition is assessed by obtaining the related free energy profile via enhanced sampling molecular dynamics simulations.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

The ubiquitin ligase Ozz decreases the replacement rate of embryonic myosin in myofibrils.

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669-C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle-specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP-Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP-Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.

Cloning, purification and characterisation of cytosolic fructose-1,6-bisphosphatase from mung bean (Vigna radiata).

To improve the crop yield and quality, the cytosolic fructose-1,6-bisphosphatase (cFBPase) from mung bean (Vigna radiata), a rate-limiting enzyme in gluconeogenesis, was cloned, purified, and structurally characterised. To function it required Mg2+ and Mn2+ at 0.01-10 mM. The Michaelis-Menton constant and adenosine monophosphate (AMP) inhibitory constant (Ki) were 7.96 and 111.09 µM, respectively. The functional site residues of AMP binding (Arg30, Asp32, and Phe33) and the active site residues (Asn218 and Met251) were tested via site-directed mutagenesis and molecular docking. Asn218 and Met251 were replaced by Tyr and Leu, respectively. The M251L mutant showed enhanced substrate affinity and activity, resulting from decreased binding energy (-2.58 kcal·mol-1) and molecular distance (4.2 Å). AMP binding site mutations changed the enzyme activities, indicating a connection between the binding and active sites. Furthermore, Ki and docking analysis revealed that Asp32 plays a key role in maintaining the AMP binding conformation.

Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases.

Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

Phagocytic activity of splenic macrophages is enhanced and accompanied by cytosolic alkalinization in TRPM7 kinase-dead mice.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.

Purification of a Src family tyrosine protein kinase from bovine retinas.

Since tyrosine phosphorylation appears to play important functions in photoreceptor cells, we searched here for retinal nonreceptor tyrosine kinases of the Src family. We demonstrated that Src family tyrosine kinases were present in the cytosolic fraction of extracted bovine retinas. A Src family tyrosine kinase with an apparent molecular mass of about 62 kDa was purified to homogeneity from the soluble fraction of dark-adapted bovine retinas after three consecutive purification steps: ω-aminooctyl-agarose hydrophobic chromatography, Cibacron blue 3GA-agarose pseudo-affinity chromatography, and α-casein-agarose affinity chromatography. The purified protein was subjected to N-terminal amino acid sequencing and the sequence Gly-Ile-Ile-Lys-Ser-Glu-Glu was obtained, which displayed homology with the first seven residues of the Src family tyrosine kinase c-Yes from Bos taurus (Gly-Cys-Ile-Lys-Ser-Lys-Glu). Although the cytosolic fraction from dark-adapted retinas contained tyrosine kinases of the Src family capable of phosphorylating the α-subunit of transducin, which is the heterotrimeric G protein involved in phototransduction, the purified tyrosine kinase was not capable of using transducin as a substrate. The cellular role of this retinal Src family member remains to be found.

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)-two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 ± 3.6 µM and a Ki of 23.2 ± 3.2 µM. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum.

Benzoic acid-derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate-coenzyme A (benzoate-CoA) ligase (BZL), which activates benzoic acid to benzoyl-CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate-degrading microorganisms. Here, a transcript for a plant acyl-activating enzyme (AAE) was cloned from xanthone-producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway-related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl-CoA in Hypericum by the CoA-dependent non-ß-oxidative route. A luciferase-based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4-coumarate-CoA ligase and cinnamate-CoA ligase enzymes, HcAAE1 did not accept 4-coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

Structural Basis for the Selective Inhibition of HDAC10, the Cytosolic Polyamine Deacetylase.

The cytosolic class IIb histone deacetylase HDAC10 is an emerging target for drug design. As an inducer of autophagy, its selective inhibition suppresses the autophagic response that otherwise attenuates the efficacy of cytotoxic cancer chemotherapy drugs. HDAC10 is a zinc-dependent polyamine deacetylase exhibiting maximal catalytic activity against N8-acetylspermidine. As revealed in the structure of Danio rerio (zebrafish) HDAC10, two conserved structural motifs direct this narrow substrate specificity: a 310 helix containing the P(E,A)CE motif that sterically constricts the active site and an electrostatic "gatekeeper," E274, that confers selectivity for cationic polyamine substrates. To accelerate drug design efforts targeting human HDAC10, we now report the preparation of "humanized" zebrafish HDAC10 in which two amino acid substitutions, A24E and D94A, yield an active site contour more similar to that of human HDAC10. X-ray crystal structures of this HDAC10 variant complexed with Tubastatin A and indole analogues bearing pendant tertiary amines reveal that inhibitors capable of hydrogen bonding with gatekeeper E274 exhibit high affinity and selectivity for HDAC10 over HDAC6 (the other class IIb isozyme). Moreover, these structures reveal that the P(E,A)CE motif helix can shift by up to 2 Å to accommodate the binding of bulky inhibitors. Thus, slender polyamine-like inhibitor structures are not exclusively required for selective, high affinity binding to HDAC10. Indeed, the flexibility of the P(E,A)CE motif helix could conceivably enable the binding of certain protein substrates.

pH gradient reversal fuels cancer progression.

pH gradient reversal refers to intracellular alkalization and extracellular acidification commonly seen in malignant tumors. To meet their high anabolic demand, cancer cells rewire their glucose metabolism from oxidative phosphorylation to lactate fermentation, which results in the excessive generation of protons. To avoid lethal cytosolic acidification, lactate-fermenting cancer cells activate multiple acid removal pathways leading to the acidification of the extracellular space. This acidification is often further intensified by the defective capacity of the disorganized tumor vasculature to dilute protons away from the cancer tissue. The cancer-specific proton equilibrium with highly alkaline cytosol and acidic extracellular space is emerging as a fundamental driving force for cancer growth. Here, we discuss how cancer cells establish and maintain reversed pH gradient, how pH gradient reversal fuels cancer progression, and how these mechanisms can be targeted in cancer therapy.

Protein kinase CK2 impact on intracellular calcium homeostasis in prostate cancer.

Protein kinase CK2 plays multiple roles in cell function in normal and disease states. CK2 is elevated in numerous types of cancer cells, and CK2 suppression of apoptosis represents a key link to the cancer cell phenotype. CK2 regulation of cell survival and death involves diverse processes, and our previous work suggested that mitochondrial machinery is a key locus of this function. One of the earliest responses of prostate cells to inhibition of CK2 is a change in mitochondrial membrane potential, possibly associated with Ca2+ signaling. Thus, in the present work, we investigated early impact of CK2 on intracellular Ca2+ dynamics. Three prostate cancer (PCa) cell lines, PC3-LN4, C4-2B, and 22Rv1, were studied. PCa cells were treated with the CK2 small molecule inhibitors 4,5,6,7-tetrabrombenzotriazole and CX-4945 followed by analysis of Ca2+ levels in various cellular compartments over time. The results showed dose-dependent loss in cytosolic Ca2+ levels starting within 2 min and reaching maximal loss within 5-10 min. There was a concomitant increase in Ca2+ in the endoplasmic reticulum (ER) and mitochondrial compartments. The results suggest that inhibition of CK2 activity results in a rapid movement of Ca2+ out of the cytosol and into the ER and mitochondria, which may be among the earliest contributory factors for induction of apoptosis in cells subjected to inhibition of CK2. In cells with death-inducing levels of CK2 inhibition, total cellular Ca2+ levels dropped at 2 h post-treatment. These novel observations represent a potential mechanism underlying regulation of cell survival and death by CK2 activity.

MESH1 is a cytosolic NADPH phosphatase that regulates ferroptosis.

Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate. Ferroptosis-inducing erastin or cystine deprivation elevates MESH1, whose overexpression depletes NADPH and sensitizes cells to ferroptosis, whereas MESH1 depletion promotes ferroptosis survival by sustaining the levels of NADPH and GSH and by reducing lipid peroxidation. The ferroptotic protection by MESH1 depletion is ablated by suppression of the cytosolic NAD(H) kinase, NADK, but not its mitochondrial counterpart NADK2. Collectively, these data shed light on the importance of cytosolic NADPH levels and their regulation under ferroptosis-inducing conditions in mammalian cells.

Towards identification of molecular mechanism in which the overexpression of wheat cytosolic and plastid glutamine synthetases in tobacco enhanced drought tolerance.

Glutamine synthetases (GS) play an essential role in Nitrogen assimilation. Nonetheless, information respecting the molecular functions of GS in drought tolerance (DT) is limited. Here we show that overexpressing cytosolic GS1 or plastidic GS2 gene in tobacco enhanced DT of both root and leaf tissues of the two transgenic seedlings (named as GS1-TR and GS2-TR). RNA-seq analysis on root tissues showed that 83 aquaporin (AQP) genes were identified. Among them, 37 differential expression genes (DEGs) were found in the GS1-TR roots under normal condition, and all were down-regulated; no any DEGs in the GS2-TR roots were found. Contrastingly, under drought, 28 and 32 DEGs of AQP were up-regulated in GS1-TR and GS2-TR roots, respectively. GC-MS analysis on leaf tissues showed that glutamine (Gln) concentrations were negatively correlated AQP expressions in the all four conditions, which suggests that Gln, as a signal molecule, can negatively regulate many AQP expressions. Prestress accumulation of sucrose and proline (Pro) and chlorophyll, and had higher activities of ROS scavengers also contribute the plant DT in both of the two transgenic plants under drought. In addition, 5-aminolevulinic acid (ALA) was up-accumulated in GS2-TR leaves solely under normal condition, which leads to its net photosynthetic rate higher than that in GS1-TR leaves. Last but not the less, the PYL-PP2C-SnRK2 core ABA-signaling pathway was uniquely activated in GS1-TR independent of drought stress (DS). Therefore, our results suggest a possible model reflecting how overexpression of wheat TaGS1 and TaGS2 regulate plant responses to drought.

Defective arginine metabolism impairs mitochondrial homeostasis in Caenorhabditiselegans.

Arginine catabolism involves enzyme-dependent reactions in both mitochondria and the cytosol, defects in which may lead to hyperargininemia, a devastating developmental disorder. It is largely unknown if defective arginine catabolism has any effects on mitochondria. Here we report that normal arginine catabolism is essential for mitochondrial homeostasis in Caenorhabditiselegans. Mutations of the arginase gene argn-1 lead to abnormal mitochondrial enlargement and reduced adenosine triphosphate (ATP) production in C. elegans hypodermal cells. ARGN-1 localizes to mitochondria and its loss causes arginine accumulation, which disrupts mitochondrial dynamics. Heterologous expression of human ARG1 or ARG2 rescued the mitochondrial defects of argn-1 mutants. Importantly, genetic inactivation of the mitochondrial basic amino acid transporter SLC-25A29 or the mitochondrial glutamate transporter SLC-25A18.1 fully suppressed the mitochondrial defects caused by argn-1 mutations. These findings suggest that mitochondrial damage probably contributes to the pathogenesis of hyperargininemia and provide clues for developing therapeutic treatments for hyperargininemia.

Cathepsin B in neurodegeneration of Alzheimer's disease, traumatic brain injury, and related brain disorders.

Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.