The citrus flavonoid naringenin stimulates DNA repair in prostate cancer cells

As part of a systematic study of the effects of phytochemicals beyond antioxidation on cancer prevention, we investigated whether naringenin (NR), a citrus flavonoid, stimulates DNA repair following oxidative damage in LNCaP human prostate cancer cells. The 8-hydroxydeoxyguanosine (8-OH-dG) to deoxyguanosine (dG) ratio was measured after cells were treated with 200 micromol/L of ferrous sulfate in serum-free medium followed by NR exposure for 24 h in growth medium. The results demonstrated that exposure to 10-80 micromol/L of NR led to a significant decrease in the ratio of 8-OH-dG to 10(6) dG. Because cells were treated with NR after ferrous sulfate was removed, we conclude that we demonstrated an effect on DNA repair beyond antioxidation. In support of this conclusion, we determined the induction of mRNA expression over time after oxidative stress followed by NR administration of three major enzymes in the DNA base excision repair (BER) pathway: 8-oxoguanine-DNA glycosylase 1 (hOGG1), apurinic/apyrimidinic endonuclease and DNA polymerase beta (DNA poly beta). hOGG1 and DNA poly beta mRNA expression in cells after 24-h exposure to NR was increased significantly compared with control cells without NR. The intracellular concentration of NR after exposure to 80 micromol/L was 3 pmol/mg protein, which is physiologically achievable in tissues. In conclusion, the cancer-preventive effects of citrus fruits demonstrated in epidemiological studies may be due in part to stimulation of DNA repair by NR, which by stimulating BER processes may prevent mutagenic changes in prostate cancer cells.     

Interactome analysis reveals versatile functions of Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 in RNA processing within the nucleus and cytoplasm

Arabidopsis COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3) shares an RNA chaperone function with E. coli cold shock proteins and regulates freezing tolerance during cold acclimation. Here, we screened for AtCSP3-interacting proteins using a yeast two-hybrid system and 38 candidate interactors were identified. Sixteen of these were further confirmed in planta interaction between AtCSP3 by a bi-molecular fluorescence complementation assay. We found that AtCSP3 interacts with CONSTANS-LIKE protein 15 and nuclear poly(A)-binding proteins in nuclear speckles. Three 60S ribosomal proteins (RPL26A, RPL40A/UBQ2, and RPL36aB) and the Gar1 RNA-binding protein interacted with AtCSP3 in the nucleolus and nucleoplasm, suggesting that AtCSP3 functions in ribosome biogenesis. Interactions with LOS2/enolase and glycine-rich RNA-binding protein 7 that are cold inducible, and an mRNA decapping protein 5 (DCP5) were observed in the cytoplasm. These data suggest that AtCSP3 participates in multiple complexes that reside in nuclear and cytoplasmic compartments and possibly regulates RNA processing and functioning.     

The glioma-associated protein SETA interacts with AIP1/Alix and ALG-2 and modulates apoptosis in astrocytes

Expression of the src homology 3 (SH3) domain-containing expressed in tumorigenic astrocytes (SETA) gene is associated with the tumorigenic state in astrocytes. SETA encodes a variety of adapter proteins containing either one or two SH3 domains, as suggested by the sequence heterogeneity of isolated cDNAs. Using both SH3 domains in a yeast two-hybrid screen of a glial progenitor cell cDNA library, we isolated the rat homolog of the ALG-2-interacting protein 1 or ALG-2-interacting protein X (AIP1/Alix). In vitro confrontation experiments showed that the SH3-N domain of SETA interacted with the proline-rich C terminus of AIP1. In co-immunoprecipitation experiments, SETA and AIP1 interacted and could form a complex with apoptosis-linked gene 2 protein. Endogenous SETA and AIP1 proteins showed similar patterns of staining in primary rat astrocytes. Misexpression of a variety of SETA protein isoforms in these astrocytes revealed that they localized to the actin cytoskeleton. Furthermore, SETA proteins containing the SH3-N domain were able to sensitize astrocytes to apoptosis induced by UV irradiation. Expression of the isolated SH3-N domain had the greatest effect in these experiments, indicating that interference in the interaction between endogenous SETA and AIP1 sensitizes astrocytes to apoptosis in response to DNA damage.     

Direct interaction between cohesin complex and DNA replication machinery

Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.     

The relationship between PthA expression and the pathogenicity of Xanthomonas axonopodis pv. citri

Citrus canker disease, caused by Xanthomonas axonopodis pv. citri, affects almost all citrus species and cultivars and hascaused severe damage to the citrus industry worldwide. PthA is considered the main pathogenesis effector of the pathogen. This research aimed to temporally and spatially analyze the expression of the PthA protein of the bactrium during its culture, and then try to understand the relationship between the PthA expression levels and the pathogenicity. The relationship between the expression of PthA and the pathogenicity of X. axonopodis pv. citri was fully investigated by using SDS-PAGE, Western blot, ELISA and field inoculation, It was found that bacteria cultured for 36 h had the highest expression of PthA and showed the most virulent pathogenicity. The conservation duration of the pathogen isolates influenced their PthA expression and the pathogenicity, and negative relationship between the duration and the expression of PthA and pathogenicity. When the stored pathogen bacteria were cultured in liquid LB medium, they were able to regain activated, showing higher PthA expression level and enhanced pathogenicity, even though the activity was inferior, in terms of both PthA expression and pathogenicity, than the freshly isolated ones. Seven isolates from different citrus orchards displayed almost identical protein expression profiles. It could conclude that the expressions of PthA was positively related to pathogenicity.     

Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4:dCsLOB1.3 infection

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker‐resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker‐resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBE_PthA4‐CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBE_PthA4‐CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBE_PthA4‐T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild‐type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker‐resistant plants. This work lays the groundwork to generate canker‐resistant citrus varieties via Cas9/sgRNA in the future.