Use of PARP inhibitors in prostate cancer: from specific to broader application.

Prostate cancer (PC) is one of the major health issues of elderly men in the word. It is showed that there were approximately 1.414 million patients with PC in 2020 worldwide, with a high mortality rate in metastatic cases. In the present choices of treatment in PC, androgen deprivation therapy has long been as a backbone of them. But the clinical outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) were not ideal because of their poor prognosis, more effective therapeutic approaches are still necessary to further improve this problem. Poly (ADP-ribose) polymerase (PARP) inhibitors lead to the single-strand DNA breaks and/or double-strand DNA breaks, and result in synthetic lethality in cancer cells with impaired homologous recombination genes. It is estimated that approximately 20~25% of patients with mCRPC have a somatic or germinal DNA damage repair gene mutation. Furthermore, in "BRCAness" cases, which has been used to describe as tumors that have not arisen from a germline BRCA1 or BRCA2 mutation, there were also a number of studies sought to extend these promising results of PARP inhibitors. It is worth noting that an interaction between androgen receptor signaling and synthetic lethality with PARP inhibitors has been proposed. In this review, we discussed the mechanism of action and clinical research of PARP inhibitors, which may benefit population from "specific" to the "all-comer" in patients with PC when combined with novel hormonal therapies.

Not just protons: Chloride also activates lysosomal acidic hydrolases.

Lysosomal hydrolases require an acidic lumen for their optimal activities. In this issue, two independent groups (Wu et al. 2023. J. Cell Biol.https://doi.org/10.1083/jcb.202208155; Zhang et al. 2023. J. Cell. Biol.https://doi.org/10.1083/jcb.202210063) report that hydrolase activation also requires high intralysosomal Cl-, which is established by the lysosomal Cl-/H+ exchanger ClC-7.

Toxoplasma ERK7 protects the apical complex from premature degradation.

Accurate cellular replication balances the biogenesis and turnover of complex structures. In the apicomplexan parasite Toxoplasma gondii, daughter cells form within an intact mother cell, creating additional challenges to ensuring fidelity of division. The apical complex is critical to parasite infectivity and consists of apical secretory organelles and specialized cytoskeletal structures. We previously identified the kinase ERK7 as required for maturation of the apical complex in Toxoplasma. Here, we define the Toxoplasma ERK7 interactome, including a putative E3 ligase, CSAR1. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by mislocalization from the parasite residual body to the apical complex. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage.

The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

Targeting Enterococcus faecalis HMG-CoA reductase with a non-statin inhibitor.

HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development of novel antibiotics. In this study, we report the crystal structures of HMGR from Enterococcus faecalis (efHMGR) in the apo and liganded forms, highlighting several unique features of this enzyme. Statins, which inhibit the human enzyme with nanomolar affinity, perform poorly against the bacterial HMGR homologs. We also report a potent competitive inhibitor (Chembridge2 ID 7828315 or compound 315) of the efHMGR enzyme identified by a high-throughput, in-vitro screening. The X-ray crystal structure of efHMGR in complex with 315 was determined to 1.27 Å resolution revealing that the inhibitor occupies the mevalonate-binding site and interacts with several key active site residues conserved among bacterial homologs. Importantly, 315 does not inhibit the human HMGR. Our identification of a selective, non-statin inhibitor of bacterial HMG-CoA reductases will be instrumental in lead optimization and development of novel antibacterial drug candidates.

Checkpoint Kinase 1 Is a Key Signal Transducer of DNA Damage in the Early Mammalian Cleavage Embryo.

After fertilization, remodeling of the oocyte and sperm genome is essential for the successful initiation of mitotic activity in the fertilized oocyte and subsequent proliferative activity of the early embryo. Despite the fact that the molecular mechanisms of cell cycle control in early mammalian embryos are in principle comparable to those in somatic cells, there are differences resulting from the specific nature of the gene totipotency of the blastomeres of early cleavage embryos. In this review, we focus on the Chk1 kinase as a key transduction factor in monitoring the integrity of DNA molecules during early embryogenesis.

PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Cellulose synthase-like D movement in the plasma membrane requires enzymatic activity.

Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate ß-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis. Actin is required to target CSLD to cell tips concomitant with cell expansion, but not to cell plates, which depend on actin and CSLD for structural support. Like Cellulose Synthase (CESA), CSLD requires catalytic activity to move in the plasma membrane. We discovered that CSLD moves significantly faster, with shorter duration and less linear trajectories than CESA. In contrast to CESA, CSLD movement was insensitive to the cellulose synthesis inhibitor isoxaben, suggesting that CSLD and CESA function within different complexes possibly producing structurally distinct cellulose microfibrils.

TnpB structure reveals minimal functional core of Cas12 nuclease family.

The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3-5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB-reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.

Evolution and expression analysis of the caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene family in jute (Corchorus L.).

BACKGROUND: Jute is considered one of the most important crops for fiber production and multipurpose usages. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a crucial enzyme involved in lignin biosynthesis in plants. The potential functions of CCoAOMT in lignin biosynthesis of jute have been reported in several studies. However, little is known about the evolution of the CCoAOMT gene family, and either their expression level at different developing stages in different jute cultivars, as well as under abiotic stresses including salt and drought stress. RESULTS: In the present study, 66 CCoAOMT genes from 12 species including 12 and eight CCoAOMTs in Corchorus olitorius and C. capsularis were identified. Phylogenetic analysis revealed that CCoAOMTs could be divided into six groups, and gene expansion was observed in C. olitorius. Furthermore, gene expression analysis of developing jute fibers was conducted at different developmental stages (15, 30, 45, 60, and 90 days after sowing [DAS]) in six varieties (Jute-179 [J179], Lubinyuanguo [LB], and Qiongyueqing [QY] for C. capsularis; Funong No.5 [F5], Kuanyechangguo [KY], and Cvlv [CL] for C. olitorius). The results showed that CCoAOMT1 and CCoAOMT2 were the dominant genes in the CCoAOMT family. Of these two dominant CCoAOMTs, CCoAOMT2 showed a constitutive expression level during the entire growth stages, while CCoAOMT1 exhibited differential expression patterns. These two genes showed higher expression levels in C. olitorius than in C. capsularis. The correlation between lignin content and CCoAOMT gene expression levels indicated that this gene family influences the lignin content of jute. Using real-time quantitative reverse transcription PCR (qRT-PCR), a substantial up-regulation of CCoAOMTs was detected in stem tissues of jute 24 h after drought treatment, with an up to 17-fold increase in expression compared to that of untreated plants. CONCLUSIONS: This study provides a basis for comprehensive genomic studies of the entire CCoAOMT gene family in C. capsularis and C. olitorius. Comparative genomics analysis among the CCoAOMT gene families of 12 species revealed the close evolutionary relationship among Corchorus, Theobroma cacao and Gossypium raimondii. This study also shows that CCoAOMTs are not only involved in lignin biosynthesis, but also are associated with the abiotic stress response in jute, and suggests the potential use of these lignin-related genes to genetically improve the fiber quality of jute.

PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Roles of cuproptosis-related gene DLAT in various cancers: a bioinformatic analysis and preliminary verification on pro-survival autophagy.

Background: Studies have shown that the expressions and working mechanisms of Dihydrolipoamide S-acetyltransferase (DLAT) in different cancers vary. It is necessary to analyze the expressions and regulatory roles of DLAT in tumors systematically. Methods: Online public-platform literature on the relationships between DLAT expression levels and tumor prognosis, methylation status, genetic alteration, drug sensitivity, and immune infiltration has been reviewed. The literature includes such documents as The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), Tumor Immune Estimation Resource 2.0 (TIMER2.0), Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Receiver Operating Characteristic plotter (ROC plotter). The molecular mechanisms of DLAT were explored with the Gene Set Enrichment Analysis (GSEA). The relationship between down-regulated DLAT and autophagy in two liver hepatocellular carcinoma (LIHC) cell lines was confirmed with the western blot method, colony formation assay, and transmission electron microscopy. Tissue microarrays were validated through the immunohistochemical staining of DLAT. Results: DLAT is upregulated in the LIHC, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), and stomach adenocarcinoma (STAD) tumors but is down-regulated in the head and neck squamous cell carcinoma (HNSC) and kidney renal clear cell carcinoma (KIRC) tumors in comparison with normal tissues. For LIHC patients treated with 5-Fluorouracil and Lenvatinib, the DLAT levels of those in the drug-resistant group are significantly high. In LIHC cells, autophagy will be inhibited, and cell death will be induced when DLAT breaks down. Moreover, there exist positive correlations between DLAT expression levels and infiltration of B cells, DC cells, Tregs, and CD8+ T cells in kidney chromophobe (KICH), breast invasive carcinoma (BRCA), prostate adenocarcinoma (PRAD), LIHC and HPV+ HNSC. In LIHC, markers of Tregs are positively correlated with DLAT. Compared with those of normal tissues, the staining intensity of DLAT and the amount of Tregs marker CD49d in LIHC increase. Conclusions: Through this study, the expressions of DLAT in various cancer types can be understood comprehensively. It suggests that DLAT may be a prognostic marker for LIHC, LUAD, LUSC, STAD and KIRC. A high DLAT expression in LIHC may promote tumorigenesis by stimulating autophagy and inhibiting anti-tumor immunity.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

In situ structure of the red algal phycobilisome-PSII-PSI-LHC megacomplex.

In oxygenic photosynthetic organisms, light energy is captured by antenna systems and transferred to photosystem II (PSII) and photosystem I (PSI) to drive photosynthesis1,2. The antenna systems of red algae consist of soluble phycobilisomes (PBSs) and transmembrane light-harvesting complexes (LHCs)3. Excitation energy transfer pathways from PBS to photosystems remain unclear owing to the lack of structural information. Here we present in situ structures of PBS-PSII-PSI-LHC megacomplexes from the red alga Porphyridium purpureum at near-atomic resolution using cryogenic electron tomography and in situ single-particle analysis4, providing interaction details between PBS, PSII and PSI. The structures reveal several unidentified and incomplete proteins and their roles in the assembly of the megacomplex, as well as a huge and sophisticated pigment network. This work provides a solid structural basis for unravelling the mechanisms of PBS-PSII-PSI-LHC megacomplex assembly, efficient energy transfer from PBS to the two photosystems, and regulation of energy distribution between PSII and PSI.

Functional characterization of a Flavonol 3-O-rhamnosyltransferase and two UDP-rhamnose synthases from Hypericum monogynum.

Rhamnosyltransferase (RT) and rhamnose synthase (Rhs) are the key enzymes that are responsible for the biosynthesis of rhamnosides and UDP-l-rhamnose (UDP-Rha) in plants, respectively. How to discover such enzymes efficiently for use is still a problem to be solved. Here, we identified HmF3RT, HmRhs1, and HmRhs2 from Hypericum monogynum, which is abundant in flavonol rhamnosides, with the help of a full-length and high throughput transcriptome sequencing platform. HmF3RT could regiospecifically transfer the rhamnose moiety of UDP-Rha onto the 3-OH position of flavonols and has weakly catalytic for UDP-xylose (UDP-Xyl) and UDP-glucose (UDP-Glc). HmF3RT showed well quercetin substrate affinity and high catalytic efficiency with Km of 5.14 µM and kcat/Km of 2.21 × 105 S-1 M-1, respectively. Docking, dynamic simulation, and mutagenesis studies revealed that V129, D372, and N373 are critical residues for the activity and sugar donor recognition of HmF3RT, mutant V129A, and V129T greatly enhance the conversion rate of catalytic flavonol glucosides. HmRhs1 and HmRhs2 convert UDP-Glc to UDP-Rha, which could be further used by HmF3RT. The HmF3RT and HmRhs1 co-expressed strain RTS1 could produce quercetin 3-O-rhamnoside (quercitrin), kaempferol 3-O-rhamnoside (afzelin), and myricetin 3-O-rhamnoside (myricitrin) at yields of 85.1, 110.7, and 77.6 mg L-1, respectively. It would provide a valuable reference for establishing a better and more efficient biocatalyst for preparing bioactive flavonol rhamnosides by identifying HmF3RT and HmRhs.

Unfolding Individual Domains of BmrA, a Bacterial ABC Transporter Involved in Multidrug Resistance.

The folding and stability of proteins are often studied via unfolding (and refolding) a protein with urea. Yet, in the case of membrane integral protein domains, which are shielded by a membrane or a membrane mimetic, urea generally does not induce unfolding. However, the unfolding of α-helical membrane proteins may be induced by the addition of sodium dodecyl sulfate (SDS). When protein unfolding is followed via monitoring changes in Trp fluorescence characteristics, the contributions of individual Trp residues often cannot be disentangled, and, consequently, the folding and stability of the individual domains of a multi-domain membrane protein cannot be studied. In this study, the unfolding of the homodimeric bacterial ATP-binding cassette (ABC) transporter Bacillus multidrug resistance ATP (BmrA), which comprises a transmembrane domain and a cytosolic nucleotide-binding domain, was investigated. To study the stability of individual BmrA domains in the context of the full-length protein, the individual domains were silenced by mutating the existent Trps. The SDS-induced unfolding of the corresponding constructs was compared to the (un)folding characteristics of the wild-type (wt) protein and isolated domains. The full-length variants BmrAW413Y and BmrAW104YW164A were able to mirror the changes observed with the isolated domains; thus, these variants allowed for the study of the unfolding and thermodynamic stability of mutated domains in the context of full-length BmrA.

The role of cyanoalanine synthase and alternative oxidase in promoting salt stress tolerance in Arabidopsis thaliana.

BACKGROUND: Cyanide is a toxic chemical that inhibits cellular respiration. In plants, cyanide can be produced by themselves, especially under stressful conditions. Cyanoalanine synthase (CAS) is a key enzyme involved in plant cyanide detoxification. There are three genes encoding CAS in Arabidopsis thaliana, but the roles of these genes in the plant's response to stress are less studied. In addition, it is known that alternative oxidase (AOX) mediates cyanide-resistant respiration, but the relationship between CAS and AOX in regulating the plant stress response remains largely unknown. RESULTS: Here, the effects of the overexpression or mutation of these three CAS genes on salt stress tolerance were investigated. The results showed that under normal conditions, the overexpression or mutation of the CAS genes had no significant effect on the seed germination and growth of Arabidopsis thaliana compared with wild type (WT). However, under 50, 100, and 200 mM NaCl conditions, the seeds overexpressing CAS genes showed stronger salt stress resistance, i.e., higher germination speed than WT seeds, especially those that overexpressed the CYS-C1 and CYS-D1 genes. In contrast, the seeds with CAS gene mutations exhibited salt sensitivity, and their germination ability and growth were significantly damaged by 100 and 200 mM NaCl. Importantly, this difference in salt stress resistance became more pronounced in CAS-OE, WT, and mutant seeds with increasing salt concentration. The CAS-OE seeds maintained higher respiration rates than the WT and CAS mutant seeds under salt stress conditions. The cyanide contents in CAS mutant seeds were approximately 3 times higher than those in WT seeds and more than 5 times higher than those in CAS-OE seeds. In comparison, plants overexpressing CYS-C1 had the fastest detoxification of cyanide and the best salt tolerance, followed by those overexpressing CYS-D1 and CYS-D2. Furthermore, less hydrogen sulfide (H2S) was observed in CAS-OE seedlings than in WT seedlings under long-term salt stress conditions. Nonetheless, the lack of AOX impaired CAS-OE-mediated plant salt stress resistance, suggesting that CAS and AOX interact to improve salt tolerance is essential. The results also showed that CAS and AOX contributed to the reduction in oxidative damage by helping maintain relatively high levels of antioxidant enzyme activity. CONCLUSION: In summary, the findings of the present study suggest that overexpression of Arabidopsis CAS family genes plays a positive role in salt stress tolerance and highlights the contribution of AOX to CAS-mediated plant salt resistance, mainly by reducing cyanide and H2S toxicity.

Differential Structural Features of Two Mutant ADAR1p150 Zα Domains Associated with Aicardi-Goutières Syndrome.

The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins.