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Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 10 10 –10 11 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Summary Glutaredoxins (GRXs) are essential for reactive oxygen species (ROS) homeostasis in responses of plants to environment changes. We previously identified several drought‐responsive CC‐type GRXs in cassava, an important tropical crop. However, how CC‐type GRX regulates ROS homeostasis of cassava under drought stress remained largely unknown. Here, we report that a drought‐responsive CC‐type GRX, namely MeGRXC3, was associated with activity of catalase in the leaves of 100 cultivars (or unique unnamed genotypes) of cassava under drought stress. MeGRXC3 negatively regulated drought tolerance by modulating drought‐ and abscisic acid‐induced stomatal closure in transgenic cassava. It antagonistically regulated hydrogen peroxide (H2O2) accumulation in epidermal cells and guard cells. Moreover, MeGRXC3 interacted with two catalases of cassava, MeCAT1 and MeCAT2, and regulated their activity in vivo. Additionally, MeGRXC3 interacts with a cassava TGA transcription factor, MeTGA2, in the nucleus, and regulates the expression of MeCAT7 through a MeTGA2‐MeMYB63 pathway. Overall, we demonstrated the roles of MeGRXC3 in regulating activity of catalase at both transcriptional and post‐translational levels, therefore involving in ROS homeostasis and stomatal movement in responses of cassava to drought stress. Our study provides the first insights into how MeGRXC3 may be used in molecular breeding of cassava crops.

Loss of iron-sulfur cluster function predisposes cancer cells to ferroptosis by upregulating iron-starvation response, but the role of glutaredoxin 5 (GLRX5) silencing in ferroptosis remains unknown. We examined the role of GLRX5 functional loss in promoting ferroptosis in cisplatin-resistant head and neck cancer (HNC) cells. The effects of sulfasalazine treatment and GLRX5 gene silencing were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species (ROS) and mitochondrial iron production, labile iron pool, mRNA/protein expression, and malondialdehyde assays. Cyst(e)ine deprivation, erastin, or sulfasalazine induced ferroptosis in HNC cells, which was relatively less sensitive in cisplatin-resistant HNC cells. Sulfasalazine or cyst(e)ine deprivation-induced ferroptosis resulted from increased lipid peroxidation and intracellular free iron, which were significantly promoted by short-interfering RNA or short hairpin RNA (shRNA) targeting GLRX5 ( <0.05). GLRX5 silencing activated iron-starvation response and boosted up intracellular free iron through the iron-responsive element-binding activity of increased iron regulatory protein (increased transferrin receptor and decreased ferritin). These effects were rescued by resistant GLRX5 cDNA but not by catalytically inactive mutant GLRX5 K101Q. The same results were noted in an mouse model transplanted with vector or shGLRX5-transduced HNC cells and treated with sulfasalazine. Our data suggest that inhibition of GLRX5 predisposes therapy-resistant HNC cells to ferroptosis.

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins. Glutaredoxins play a central role in numerous biological processes including cellular redox homeostasis and Fe-S cluster biogenesis. Here the authors establish the molecular basis for glutaredoxin redox catalysis through comprehensive biochemical and structural analyses.

Class I glutaredoxins (GRXs) are nearly ubiquitous proteins that catalyse the glutathione (GSH)-dependent reduction of mainly glutathionylated substrates. In land plants, a third class of GRXs has evolved (class III). Class III GRXs regulate the activity of TGA transcription factors through yet unexplored mechanisms. Here we show that Arabidopsis thaliana class III GRX ROXY9 is inactive as an oxidoreductase on widely used model substrates. Glutathionylation of the active site cysteine, a prerequisite for enzymatic activity, occurs only under highly oxidizing conditions established by the GSH/glutathione disulfide (GSSG) redox couple, while class I GRXs are readily glutathionylated even at very negative GSH/GSSG redox potentials. Thus, structural alterations in the GSH binding site leading to an altered GSH binding mode likely explain the enzymatic inactivity of ROXY9. This might have evolved to avoid overlapping functions with class I GRXs and raises questions of whether ROXY9 regulates TGA substrates through redox regulation. Plant-specific class III glutaredoxins regulate the activity of TGA transcription factors. Here, the authors show that that ROXY9, a member of the class III of glutaredoxins, lacks oxidoreductase activity due to unfavourable positioning of glutathione. Consequently, class III glutaredoxins may not regulate gene expression through redox modifications of target proteins.

The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1–ISCA2–IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1–ISCA2–IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1–ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factorassisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion.

Plants modulate the efficiency of root nitrogen (N) acquisition in response to shoot N demand. However, molecular components directly involved in this shoot-to-root communication remain to be identified. Here, we show that phloem-mobile CEPD-like 2 (CEPDL2) polypeptide is upregulated in the leaf vasculature in response to decreased shoot N status and, after translocation to the roots, promotes high-affinity uptake and root-to-shoot transport of nitrate. Loss of CEPDL2 leads to a reduction in shoot nitrate content and plant biomass. CEPDL2 contributes to N acquisition cooperatively with CEPD1 and CEPD2 which mediate root N status, and the complete loss of all three proteins severely impairs N homeostasis in plants. Reciprocal grafting analysis provides conclusive evidence that the shoot CEPDL2 / CEPD1/2 genotype defines the high-affinity nitrate uptake activity in root. Our results indicate that plants integrate shoot N status and root N status in leaves and systemically regulate the efficiency of root N acquisition. Plants regulate nitrate uptake in roots to meet nitrogen demand in shoots. Here Ota et al. identify CEPDL2, a polypeptide that is induced during nitrogen deficiency in leaves, and show that it moves via the phloem to promote high-affinity nitrate uptake and root-to-shoot nitrate transport.

C - TERMINALLY ENCODED PEPTIDE (CEP) and cytokinin hormones act over short and long distances to control plant responses to environmental cues. CEP and cytokinin pathway mutants share phenotypes, however, it is not known if these pathways intersect. We show that CEP and cytokinin signalling converge on CEP DOWNSTREAM (CEPD) glutaredoxins to inhibit primary root growth. CEP inhibition of root growth was impaired in mutants defective in trans -zeatin ( t Z)-type cytokinin biosynthesis, transport, perception, and output. Concordantly, mutants affected in CEP RECEPTOR 1 showed reduced root growth inhibition in response to t Z, and altered levels of t Z-type cytokinins. Grafting and organ-specific hormone treatments showed that t Z-mediated root growth inhibition involved CEPD activity in roots. By contrast, root growth inhibition by CEP depended on shoot CEPD function. The results demonstrate that CEP and cytokinin pathways intersect, and utilise signalling circuits in separate organs involving common glutaredoxin genes to coordinate root growth. C-terminally encoded peptide (CEP) and cytokinin (CK) hormones modulate plant root architecture in response to environmental cues. The results show that CEP and CK pathways utilise CEPD glutaredoxins in separate organs to curb primary root growth.

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body’s reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

• TGACG-BINDING FACTORs (TGAs) control the developmental or defense-related processes. In Arabidopsis thaliana, the functions of at least TGA2 and PERIANTHIA (PAN) can be repressed by interacting with CC-type glutaredoxins, which have the potential to control the redox state of target proteins. As TGA1 can be redox modulated in planta, we analyzed whether some of the 21 CC-type glutaredoxins (ROXYs) encoded in the Arabidopsis genome can influence TGA1 activity in planta and whether the redox active cysteines of TGA1 are functionally important. • We show that the tga1 tga4 mutant and plants ectopically expressing ROXY8 or ROXY9 are impaired in hyponastic growth. As expression of ROXY8 and ROXY9 is activated upon transfer of plants from hyponasty-inducing low light to normal light, they might interfere with the growth-promoting function of TGA1/TGA4 to facilitate reversal of hyponastic growth. • The redox-sensitive cysteines of TGA1 are not required for induction or reversal of hyponastic growth. • TGA1 and TGA4 interact with ROXYs 8, 9, 18, and 19/GRX480, but ectopically expressed ROXY18 and ROXY19/GRX480 do not interfere with hyponastic growth. Our results therefore demonstrate functional specificities of individual ROXYs for distinct TGAs despite promiscuous protein–protein interactions and point to different repression mechanisms, depending on the TGA/ROXY combination.