YUCCA2 (YUC2)-Mediated 3-Indoleacetic Acid (IAA) Biosynthesis Regulates Chloroplast RNA Editing by Relieving the Auxin Response Factor 1 (ARF1)-Dependent Inhibition of Editing Factors in Arabidopsis thaliana.

Although recent research progress on the abundant C-to-U RNA editing events in plant chloroplasts and mitochondria has uncovered many recognition factors and their molecular mechanisms, the intrinsic regulation of RNA editing within plants remains largely unknown. This study aimed to establish a regulatory relationship in Arabidopsis between the plant hormone auxin and chloroplast RNA editing. We first analyzed auxin response elements (AuxREs) present within promoters of chloroplast editing factors reported to date. We found that each has more than one AuxRE, suggesting a potential regulatory role of auxin in their expression. Further investigation unveiled that the depletion of auxin synthesis gene YUC2 reduces the expression of several editing factors. However, in yuc2 mutants, only the expression of CRR4, DYW1, ISE2, and ECD1 editing factors and the editing efficiency of their corresponding editing sites, ndhD-2 and rps14-149, were simultaneously suppressed. In addition, exogenous IAA and the overexpression of YUC2 enhanced the expression of these editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These results suggested a direct effect of auxin upon the editing of the ndhD-2 and rps14-149 sites through the modulation of the expression of the editing factors. We further demonstrated that ARF1, a downstream transcription factor in the auxin-signaling pathway, could directly bind to and inactivate the promoters of CRR4, DYW1, and ISE2 in a dual-luciferase reporter system, thereby inhibiting their expression. Moreover, the overexpression of ARF1 in Arabidopsis significantly reduced the expression of the three editing factors and the editing efficiency at the ndhD-2 and rps14-149 sites. These data suggest that YUC2-mediated auxin biosynthesis governs the RNA-editing process through the ARF1-dependent signal transduction pathway.

Computed tomography imaging features of malignant 'triton' tumor to facilitate its clinical diagnosis: report of two cases.

BACKGROUND: Malignant 'triton' tumor is an extremely rare subtype of malignant periphery nerve sheath tumors. Clinical diagnosis of malignant triton tumor is difficult before surgery due to its low incidence and the lack of knowledge. Therefore, to describe and summarize the CT imaging characteristics of malignant triton tumor is of great assistance for early and preoperative diagnosis. CASE PRESENTATION: Two cases suspected of MTT by CT scan before operation were closely observed. The diagnosis of malignant triton tumor was eventually confirmed by immunochemical assay, which verified speculation of CT scans. Huge, irregular, well-circumscribed lobulated mass-like shadows can be observed from these patients by CT scans. Besides, heterogeneity of density within the body of tumor was well-established by CT scans, together with linear septum. Meanwhile, CT scans demonstrated that calcifications were remarkable at the margin of tumor body. CONCLUSIONS: Some CT image features from two cases were presented as a reference for the preoperative consideration of MTT: (i) enormity of mass-like shadow; (ii) presence of well-circumscribed lobulated shape; (iii) septum within the well-defined mass accompanied with hemorrhage, necrosis and cystic changes as well as calcification, especially within neurofibromatosis type 1 patients.

Structural Determinants within the Adenovirus Early Region 1A Protein Spacer Region Necessary for Tumorigenesis.

It has long been established that group A human adenoviruses (HAdV-A12, -A18, and -A31) can cause tumors in newborn rodents, with tumorigenicity related to the presence of a unique spacer region located between conserved regions 2 and 3 within the HAdV-A12 early region 1A (E1A) protein. Group B adenoviruses are weakly oncogenic, whereas most of the remaining human adenoviruses are nononcogenic. In an attempt to understand better the relationship between the structure of the AdE1A spacer region and oncogenicity of HAdVs, the structures of synthetic peptides identical or very similar to the adenovirus 12 E1A spacer region were determined and found to be α-helical using nuclear magnetic resonance (NMR) spectroscopy. This contrasts significantly with some previous suggestions that this region is unstructured. Using available predictive algorithms, the structures of spacer regions from other E1As were also examined, and the extent of the predicted α-helix was found to correlate reasonably well with the tumorigenicity of the respective virus. We suggest that this may represent an as-yet-unknown binding site for a partner protein required for tumorigenesis.IMPORTANCE This research analyzed small peptides equivalent to a region within the human adenovirus early region 1A protein that confers, in part, tumor-inducing properties to various degrees on several viral strains in rats and mice. The oncogenic spacer region is α-helical, which contrasts with previous suggestions that this region is unstructured. The helix is characterized by a stretch of amino acids rich in alanine residues that are organized into a hydrophobic, or "water-hating," surface that is considered to form a major site of interaction with cellular protein targets that mediate tumor formation. The extent of α-helix in E1A from other adenovirus species can be correlated to a limited degree to the tumorigenicity of that virus. Some serotypes show significant differences in predicted structural propensity, suggesting that the amino acid type and physicochemical properties are also of importance.

TRAIL and Celastrol Combinational Treatment Suppresses Proliferation, Migration, and Invasion of Human Glioblastoma Cells via Targeting Wnt/ß-catenin Signaling Pathway.

OBJECTIVE: To investigate the mechanistic basis for the anti-proliferation and anti-invasion effect of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) and celastrol combination treatment (TCCT) in glioblastoma cells. METHODS: Cell counting kit-8 was used to detect the effects of different concentrations of celastrol (0-16 µmol/L) and TRAIL (0-500 ng/mL) on the cell viability of glioblastoma cells. U87 cells were randomly divided into 4 groups, namely control, TRAIL (TRAIL 100 ng/mL), Cel (celastrol 0.5 µmol/L) and TCCT (TRAIL 100 ng/mL+ celastrol 0.5 µmol/L). Cell proliferation, migration, and invasion were detected by colony formation, wound healing, and Transwell assays, respectively. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to assess the levels of epithelial-mesenchymal transition (EMT) markers (zona occludens, N-cadherin, vimentin, zinc finger E-box-binding homeobox, Slug, and ß-catenin). Wnt pathway was activated by lithium chloride (LiCl, 20 mol/L) and the mechanism for action of TCCT was explored. RESULTS: Celastrol and TRAIL synergistically inhibited the proliferation, migration, invasion, and EMT of U87 cells (P<0.01). TCCT up-regulated the expression of GSK-3ß and down-regulated the expression of ß-catenin and its associated proteins (P<0.05 or P<0.01), including c-Myc, Cyclin-D1, and matrix metalloproteinase (MMP)-2. In addition, LiCl, an activator of the Wnt signaling pathway, restored the inhibitory effects of TCCT on the expression of ß-catenin and its downstream genes, as well as the migration and invasion of glioblastoma cells (P<0.05 or P<0.01). CONCLUSIONS: Celastrol and TRAIL can synergistically suppress glioblastoma cell migration, invasion, and EMT, potentially through inhibition of Wnt/ß-catenin pathway. This underlies a novel mechanism of action for TCCT as an effective therapy for glioblastoma.

The DYW domain of RARE1 plays an indispensable role in regulating accD-C794 RNA editing in Arabidopsis thaliana.

The Arabidopsis pentatricopeptide repeat (PPR) proteins, required for accD RNA editing 1 (RARE1) and early chloroplast biogenesis 2 (AtECB2), each contain a DYW domain deemed essential for cytosine deamination at the accD-C794 RNA editing site in chloroplasts. Complementation assays using the rare1 mutant investigate the correlation between these PPRs and their respective DYW domain functions in RNA editing of accD-C794. The results demonstrate that the coding sequence of AtECB2 cannot replace that of RARE1. Moreover, rare1 mutants complemented with DYW-deleted RARE1 failed to recover the RNA editing of accD-C794 even in the presence of the highly similar DYW domain of the AtECB2 protein. These findings indicate that RARE1 and AtECB2 possess divergent roles in RNA editing, with specificity for accD-C794 directly attributable to DYW domain within RARE1. Structural modeling data suggest this functioning pertains to a local α-helical motif that residues slightly N-terminal to the consensus glutamate and CXXCH motif in the DYW domain for cytidine deamination during C-to-U editing by RARE1 that is absent within AtECB2.

Identification of Adenovirus E1B-55K Interaction Partners through a Common Binding Motif.

The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.

The PPR protein RARE1-mediated editing of chloroplast accD transcripts is required for fatty acid biosynthesis and heat tolerance in Arabidopsis.

It has been reported that Arabidopsis chloroplast accD transcripts undergo RNA editing and that loss of accD-C794 RNA editing does not affect plant growth under normal conditions. To date, the exact biological role of accD-C794 editing has remained elusive. Here, we reveal an unexpected role for accD-C794 editing in response to heat stress. Loss of accD-C794 editing results in a yellow and dwarf phenotype with decreased chloroplast gene expression under heat stress, and artificial improvement of C794-edited accD gene expression enhances heat tolerance in Arabidopsis. These data suggest that accD-C794 editing confers heat tolerance in planta. We also found that treatment with the product of acetyl coenzyme A carboxylase (ACCase) could allay mutant phenotypic characteristics and showed that a mutation in the CAC3 gene for the α-subunit of ACCase was associated with dwarfism under heat stress. These observations indicate that defective accD-C794 editing may be intrinsic to reduced ACCase activity, thereby contributing to heat sensitivity. ACCase catalyzes the committed step of de novo fatty acid (FA) biosynthesis. FA content analysis revealed that unsaturated oleic (C18:1) and linoleic acids (C18:2) were low in the accD-C794 editing-defective mutant but high in the C794-edited accD-overexpressing plants compared with the wild type. Supplying exogenous C18:1 and C18:2 could rescue the mutant phenotype, suggesting that these FAs play an essential role in tolerance to heat stress. Transmission electron microscopy observations showed that heat stress seriously affected the membrane architecture in accD editing-defective mutants but not in accD-overexpressing plants. These results provide the first evidence that accD-C794 editing regulates FA biosynthesis for maintenance of membrane structural homeostasis under heat stress.

MORF2 tightly associates with MORF9 to regulate chloroplast RNA editing in Arabidopsis.

RNA editing in chloroplasts and mitochondria is performed by hypothetical editosomes. The MORF family proteins are essential components of these editosomes. In Arabidopsis, MORF2 and MORF9 are involved in the editing of most sites in chloroplasts. In this work, we performed immunoprecipitation and mass spectrometry assays of transgenic lines expressing MORF2-4xMYC and MORF9-4xMYC to identify interacting proteins. We found that MORF2 and MORF9 are present in the same complex. Blue-Native PAGE analysis of chloroplast protein complexes also revealed that both MORF2 and MORF9 are part of a complex of approximately 140 kDa, suggesting the existence of tight MORF2-MORF9 interaction in chloroplasts. The editing of ndhD-1 (ndhD-C2) site was reported to be blocked in both morf2 and morf9. RNA immunoprecipitation assays showed that MORF2 and MORF9 are tightly associated with the editing site of ndhD-1. However, in an RNA-EMSA assay MORF2 and MORF9 could not directly bind to transcripts harboring the editing site of ndhD-1. Taken together, these results indicate that the MORF2-MORF9 heterodimer is the core members of editosomes in chloroplasts, while they are not responsible for RNA editing site recognition.

The effect of CtBP1 binding on the structure of the C-terminal region of adenovirus 12 early region 1A.

Adenovirus early region 1A (AdE1A) binds to the C-terminal binding protein 1 (CtBP1) primarily through a highly conserved PXDLS motif located close to its C-terminus. Purified synthetic peptides equivalent to this region of AdE1A have been shown to form a series of beta-turns. In this present study the effect of CtBP1 binding on the conformation of C-terminal region of Ad12E1A has been investigated. Using one- and two-dimensional (1)H NMR spectroscopy, the conformation of 20-residue peptides equivalent to amino acids I(241)-V(260) and E(247)-N(266) of Ad12E1A were examined in the absence of CtBP1. Whilst the latter peptide forms a series of beta-turns in its C-terminal half as reported previously, the former peptide is alpha-helical over the region D(243)-Q(253). Upon interaction with CtBP1 the conformation of the backbone in the region (255)PVDLCVK(261) of the Ad12E1A E(247)-N(266) peptide reorganises from a predominately beta-turn to an alpha-helical conformation. This structural isomerisation is characterised by a shift upfield of 0.318 ppm for the delta-CH(3) proton resonance of V(256). 2-D NOESY experiments showed new signals in the amide-alpha region which correlate to transferred NOEs from the protein to the peptide residues E(251), V(256) and K(261). In further analyses the contribution of individual amino acids within the sequence (254)VPVDLS(259) was assessed for their importance in determining structure and consequently affinity of the peptide for CtBP. It has been concluded that Ad12E1A residues (255)P-V(260) serve initially as a recognition site for CtBP and then as an anchor through a beta-turns-->alpha-helix conformational rearrangement. In addition it has been predicted that regions N-terminal to the PXDLS motif in AdE1As from different virus serotypes and from mammalian proteins form alpha-helices.

Predicted consequences of site-directed mutagenesis and the impact of species variation on prion protein misfolding through the N-terminal domain.

Variant Creutzfeldt-Jacob disease (vCJD) is considered to afflict humans through the acquisition of variant isomers and misfolding of the normal cellular prion polypeptide, PrP(C). Although the exact mechanism of the misfolding is not been yet clearly understood, this paper provides four additional pieces of evidence in support of the hypothesis that misfolding within PrP(C) involves N-terminal residues, up to and including Asn178. Structural predictions for N-terminal residues between Leu4 and Gly124 revealed that Leu4-Leu19 might adopt a helical conformation. Furthermore, measurement of C(alpha) distance variations, as determined from available NMR solution structures of wild type, as well as the biologically significant Val166, Asn170 and Lys220 variants of PrP(C), revealed previously unreported global and local conformational differences may occur in PrP(C) as a result of these amino-acid substitutions. Notably, three regions, His140-Tyr150 and Met166-Phe175 showed deviations greater than 3 A in their C(alpha)-coordinates (cf wild type) indicating that the majority of the N-terminal domain is likely to contribute to the misfolding of PrP(C). Minor variations in the orientation of amino acids Thr193-Glu200, located towards the C terminus of the protein, were also noted. This most likely indicates the presence of a hinge mechanism, inherent to a Helix-Loop-helix (HLH) motif formed by amino acids within alpha2, LIII and alpha3, in order to accommodate reorientation of the motif in response to misalignment of the N-terminal domain. An unexpected 3 angstroms deviation from the coordinates of the wild type polypeptide, absent from either Val166, Asn170 variants was observed over the region Arg154-Tyr155 within the Val166 form of PrP(C). This may contribute to the explanation as to why patients carrying the Val166 isoform of PrP(C) may be more susceptible to vCJD.