Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Hmo1:A versatile member of the high mobility group box family of chromosomal architecture proteins

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.

Characterization and analysis of ribosomal proteins in two marine calanoid copepods

Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the c DNAs of cytoplasmic ribosomal proteins(c RPs) of two calanoid copepods, P seudodiaptomus poplesia and A cartia pacifi ca. We obtained 79 c RP c DNAs from P. poplesia and 67 from A. pacifi ca by c DNA library construction/sequencing and rapid amplifi cation of c DNA ends. Analysis of the nucleic acid composition showed that the copepod c RP-encoding genes had higher GC content in the protein-coding regions(CDSs) than in the untranslated regions(UTRs), and single nucleotide repeats(>3 repeats) were common, with "A" repeats being the most frequent, especially in the CDSs. The 3′-UTRs of the c RP genes were signifi cantly longer than the 5′-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the c RPs contained high proportions of positively charged residues and had high p I values. This is the fi rst report of a complete set of c RP-encoding genes from copepods. Our results shed light on the characteristics of c RPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod c RP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

Isolation of a genomic DNA for Jatropha curcas ribosome inactivating protein and its tobacco transformation

Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.

Cloning of Novel Tumor Metastasis-Related Genes from the Highly Metastatic Human Lung Adenocarcinoma Cell Line Anip973

A cDNA library was successfully constructed from Anip973, a human lung adenocarcinoma cell line with high metastatic potential. NIH3T3 cells were stably transfected using this cDNA library and screened for morphological changes in a soft agar assay. Genomic DNA was isolated from putative clones and the integrated sequence was retrieved by PCR and sequencing. Three known genes, ribosomal protein L23, hypothetical protein FLJ22104, and serine protease inhibitor, kazal type 6 and a number of 5＇-terminally truncated sequences were identified. Furthermore, cells transfected with ribosomal protein L23 was highly invasive compared with the empty vector as control （P 〈 0.02）. These results indicate that the expression cloning of cDNA libraries in NIH3T3 cells and subsequent screening for loss of contact inhibition in soft agar is a viable tool for identifying tumor-related genes and ribosomal protein L23 gene plays a role in cell movement and metastasis.

Molecular Cloning and Bioinformatical Analysis of a cDNA Encoding Mitochondrial 50S Ribosomal Protein L21 from Hevea brasiliensis Muell. Arg.

[Objective] ＂Tapping panel dryness （TPD）＂, a syndrome known as tapping incision blocked partly or entirely during latex exploiting, has become the most important factor causing great losses for rubber production. Aiming to elucidate the molecular mechanism of tapping panel dryness occurrence, this study carried out molecular cloning and bioinformatical analysis of a mRPL21 cDNA sequence, a gene associated with TPD. [Method] In a preliminary study, an expressed sequence tag （EST） encoding a deduced protein homologous to mitochondrial 50S ribosomal protein L21 （mRPL21）, which showed to be down-regulated in the latex of TPD-affected rubber trees, was isolated by suppression subtractive hybridization （SSH）. After ESTs assembling and RT-PCR validation, an 853 bp cDNA sequence with an open reading frame （ORF） was cloned, which was named as HbmRPL21 under GenBank accession number of HM230670. [Result] Bioinformatical analysis suggests that HbmRPL21 encodes a deduced polypeptide of 271 amino acids with a theoretical molecular weight （Mw） of 30.52 kDa and isolectric point （pI） of 8.40, and HbmRPL21 is a mitochondrion-targeted protein with a conserved domain of Ribosomal_L21p involving translation. Homology analysis reveals high amino acid sequence identity of mRPL21 from plants, while diversity of that between plant and animal kingdom. [Conclusion] This study laid the basis for further revealing the biological functions of mRPL21 in TPD-affected rubber trees.

Effect of moxibustion on mTOR-mediated autophagy in rotenone-induced Parkinson's disease model rats

Defects in autophagy-mediated clearance of α-synuclein may be one of the key factors leading to progressive loss of dopaminergic neurons in the substantia nigra. Moxibustion therapy for Parkinson’s disease has been shown to have a positive effect, but the underlying mechanism remains unknown. Based on this, we explored whether moxibustion could protect dopaminergic neurons by promoting autophagy mediated by mammalian target of rapamycin （mTOR）, with subsequent elimination of α-syn. A Parkinson’s disease model was induced in rats by subcutaneous injection of rotenone at the back of their necks, and they received moxibustion at Zusanli （ST36）, Guanyuan （CV4）and Fengfu （GV16）, for 10 minutes at every point, once per day, for 14 consecutive days. Model rats without any treatment were used as a sham control. Compared with the Parkinson’s disease group, the moxibustion group showed significantly greater tyrosine hydroxylase immunoreactivity and expression of light chain 3-II protein in the substantia nigra, and their behavioral score, α-synuclein immunoreactivity,the expression of phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase （p-p70S6K） in the substantia nigra were significantly lower. These results suggest that moxibustion can promote the autophagic clearance of α-syn and improve behavioral performance in Parkinson’s disease model rats. The protective mechanism may be associated with suppression of the mTOR/p70S6K pathway.

Ribosomal proteins:functions beyond the ribosome

Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation,their ribosomeindependent functions have also been greatly appreciated.Over the past decade,more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress.In addition,these ribosomal proteins are involved in various physiological and pathological processes.This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins,as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis,immune signaling,and development.Wealso propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics.

Discovery and validation of prognostic markers in gastric cancer by genome-wide expression profiling

AIM: To develop a prognostic gene set that can predict patient overall survival status based on the whole genome expression analysis. METHODS: Using Illumina HumanWG-6 BeadChip followed by semi-supervised analysis, we analyzed the expression of 47 296 transcripts in two batches of gastric cancer patients who underwent surgical resection. Thirty-nine samples in the first batch were used as the training set to discover candidate markers correlated to overall survival, and thirty-three samples in the second batch were used for validation. RESULTS: A panel of ten genes were identified as prognostic marker in the first batch samples and classified patients into a lowand a high-risk group with significantly different survival times (P = 0.000047). This prognostic marker was then verified in an independent validation sample batch (P = 0.0009). By comparing with the traditional Tumor-node-metastasis (TNM) staging system, this ten-gene prognostic marker showed consistent prognosis results. It was the only independent prognostic value by multivariate Cox regression analysis (P = 0.007). Interestingly, six of these ten genes are ribosomal proteins, suggesting a possible association between the deregulation of ribosome related gene expression and the poor prognosis. CONCLUSION: A ten-gene marker correlated with overall prognosis, including 6 ribosomal proteins, was identified and verified, which may complement the predictive value of TNM staging system.

Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

Inhibitory effects of rapamycin on the different stages of hepatic fibrosis

AIM: To investigate and compare the inhibitory effects of rapamycin in the different stages of liver fibrosis.

Identification of differentially expressed genes in human uterine leiomyomas using differential display

In searching of differentially expressed genes in human uterine leiomyomas, differential display was used with twelve pairs of primers to compare human uterine leiomyomas with matched myometrium. False positives were eliminated by reverse Northern analysis. Positives were confirmed by Northern blot analysis. RESULTS: Four of 69 cDNA fragments (3 up-regulated named L1, L2 and L3 and 1 down-regulated named M1 in leiomyoma) were confirmed by Northern analysis. Sequence comparison and Northern analysis proved that L1 is exactly the human ribosomal protein S19. It was present ubiquitously in 13 tissues tested but in various levels and even in different size. L1 was highly expressed in parotidean cystadenocarcinoma, pancreatic cancer and breast cancer examined. No mutations have been found in human uterine leiomyomas (n=6). CONCLUSIONS: hRPS19 overexpression might be a universal signal in rapid cell growth tissues.

Molecular Replacement Studies of Cucurmosin from Cucurbita Moschata:Structure Homology with Trichosanthin

High diffraction quality crystals of cucurmosin, a type I ribosome inactivating protein isolated from the sarcocarp of Cucurbita moschata (pumpkin), have been grown under newly optimised conditions. With in-house rotating anode X-ray source, these crystals diffract to 1.65 ?resolution which is much higher than that of the previously reported crystals that diffracted only to 3 ?resolution. The crystals belong to space group P212121 with cell parameters a = 41.5, b = 58.4 and c = 99.3 . Molecular replacement studies indicate that the cucurmosin structure is homologous to trichosanthin. The initial structural model has been obtained and the model fitting/ refinement is in progress.

18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells

BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the mechanism by which 18β-glycyrrhetinic acid(18β-GRA)regulates mitochondrial ribosomal protein L35(MRPL35)related signal proteins to inhibit the proliferation of GC cells.METHODS Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823.The apoptosis and cell cycle were assessed by flow cytometry.Cell invasion and migration were evaluated by Transwell assay,and cell scratch test was used to detect cell migration.Furthermore,a tumor model was established by hypodermic injection of 2.5×106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect MRPL35 expression in the engrafted tumors in mice.We used the term tandem mass tag(TMT)labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins(DEPs)extracted from GC cells and control cells after 18β-GRA intervention.A detailed bioinformatics analysis of these DEPs was performed,including Gene Ontology annotation and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis,and so on.Moreover,STRING database(https://string-db.org/)was used to predict proteinprotein interaction(PPI)relationships and Western blot was used to detect the expression of proteins of interest in GC cells.RESULTS The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose-and timedependent manner.It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase.The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose,and the migration and invasiveness of GC cells were inhibited.The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice,and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice.Using TMT technology,609 DEPs,among which 335 were up-regulated and 274 were down-regulated,were identified in 18β-GRA intervention compared with control.We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways,such as cellular processes,biological regulation,and TP53 signaling pathway.Notably,after the drug intervention,MRPL35 expression was significantly down-regulated(P=0.000247),TP53 expression was up-regulated(P=0.02676),and BCL2L1 was down-regulated(P=0.01699).Combined with the Retrieval of Interacting Genes/Proteins database,we analyzed the relationship between MRPL35,TP53,and BCL2L1 signaling proteins,and we found that COPS5,BAX,and BAD proteins can form a PPI network with MRPL35,TP53,and BCL2L1.Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells,MRPL35,TP53,and BCL2L1 showed dose-dependent up/down-regulation,and the expression of COPS5,BAX,and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35,COPS5,TP53,BCL2L1,BAX,and BAD.

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant,large RNA-protein complexes that are the sites of all protein synthesis in cells.Defects in ribosomal proteins(RPs),including proteoforms arising from genetic variations,alternative splicing of RNA transcripts,post-translational modifications and alterations of protein expression level,have been linked to a diverse range of diseases,including cancer and aging.Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets.In the present work,using E.coli 70S RPs as an example,we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry(MS)system,and then applied it to the HeLa 80S ribosome.The results were complemented by a bottom-up approach.In total,50 out of 55 RPs were identified using the top-down approach.Among these,more than 30 RPs were found to have their N-terminal methionine removed.Additional modifications such as methylation,acetylation,and hydroxylation were also observed,and the modification sites were identified by bottomup MS.In a HeLa 80S ribosomal sample,we identified 98 ribosomal proteoforms,among which multiple truncated 80S ribosomal proteoforms were observed,the type of information which is often overlooked by bottom-up experiments.Although their relevance to diseases is not yet known,the integration of topdown and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Validation of Housekeeping Genes for Gene Expression Analysis in Iwagaki Oyster(Crassostrea nippona)Under Salinity Stress by Quantitative Real-Time PCR

Hypo-salinity can reduce the immunological reaction in Crassostrea nippona,even lead to massive mortality.It is important to understand the molecular mechanism of oyster defense system,while quantitative real-time PCR can be employed in the study.However,the accuracy of quantitative real-time PCR relies on the use of suitable reference genes.In this study,the expression stability of 14 candidate reference genes including traditional housekeeping genes EF1A,TUB,TUA,GAPDH,RO21,as well as new candidate reference genes RPL5,RPL8,RPS27,RPL14,RPL4,CO3,RPS8,RPS4,CYTB in different tissues of C.nippona under salinity stress has been validated by quantitative real-time PCR.Ribosomal protein genes selected through expression analysis of transcriptome data from C.nippona generally were more stable than traditional reference genes.According to the geNorm analysis,RPL4 and RPS4 could be used as internal controls for studying gene expression in C.nippona with real-time PCR under salinity stress.

The Study of Anti-tumor Activity of Trichosanthin by Cyclic Voltammogram

The anti-tumor activity of Trichosanthin (TCS) has been frequently reported in recent years. In our experiments, electrochemical methods were applied to detect the effects of TCS on human leukemia cells U937. 50 mu g/ml TCS treatment for 40 hours can cause irreversible negative effects on the viability of U937 cells. This effect largely depends on the concentration of TCS and the time period of treatment.

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world. However, it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. In this study, the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique. The lines were the T2 of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene, and were positive by Southern Blot analysis. The resistance difference was studied through comparing the death percentage of transgenic soybean with the control. The results showed that four lines were more resistant to P sojae, whereas other one had no significant difference in comparison with the control. These transgenic soybean lines with enhanced resistance to P sojae will be useful in soybean resistance breeding.

Antibodies against ribosomal protein S29(RPS29)fused with glutathione's transferase specially react with native RPS29 in mouse and human cells

The ribosomal protein S29 also known as RPS29, is not only a component of the 40S subunit of ribosome, but also involved in embryonic development, oncogenesis and other pathologic conditions. However, rare commercial antibody against RPS29 restricts the discovery of precise physiological and pathological function of this protein. In this study, the whole RPS29 gene was inserted into plasmid pGEX-6p-1 to express glutathione＇s transferase （GST） fusion proteins in Escherichia eoli （E. coli） strain BL21. High yields of soluble recombinant proteins were obtained. Mice were immunized with the recombinant RPS29 protein. The serum from the immunized mice could specially react with purified recombinant RPS29 proteins and native RPS29 proteins in CCE cells by western blotting, immunofluorescence staining and flow cytometric analysis. Further more the polyclonal antibodies also reacted specifically with human cell strain ECV304, which showed typical cytoplasmatic fluorescence. The polyclonal antibodies we prepared would be an available tool for studying the roles of RPS29 in embryonic development and human diseases.

Knockdown of 60S ribosomal protein L14‑2 reveals their potential regulatory roles to enhance drought and salt tolerance in cotton

Background:Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally.The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions.However,plants have evolved a number of survival strategies,among them is the induction of various stress-responsive genes such as the ribosomal protein large(RPL)gene.The RPL gene families encode critical proteins,which alleviate the effects of drought and salt stress in plants.In this study,comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses.Results:Based on the genome-wide evaluation,26,8,and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum,G.raimondii,and G.arboreum,respectively.Furthermore,through bioinformatics analysis,key cis-regulatory elements related to RPL14B genes were discovered.The Myb binding sites(MBS),abscisic acid-responsive element(ABRE),CAAT-box,TATA box,TGACG-motif,and CGTCA-motif responsive to methyl jasmonate,as well as the TCA-motif responsive to salicylic acid,were identified.Expression analysis revealed a key gene,Gh_D01G0234(RPL14B),with significantly higher induction levels was further evaluated through a reverse genetic approach.The knockdown of Gh_D01G0234(RPL14B)significantly affected the performance of cotton seedlings under drought/salt stress conditions,as evidenced by a substantial reduction in various morphological and physiological traits.Moreover,the level of the antioxidant enzyme was significantly reduced in VIGS-plants,while oxidant enzyme levels increased significantly,as demonstrated by the higher malondialdehyde concentration level.Conclusion:The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes,which are key in oxidizing the various oxidants.The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.