Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism

Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.     

Alterations in phosphoproteome under salt stress in Thellungiella roots

High salinity stress is a major environmental factor that limits plant’s distribution and productivity. An Arabidopsis-related halophyte, Thellungiella halophila, is an emerging model system used for plant abiotic stress tolerance research. Previous studies have suggested that protein phosphorylation has a crucial role in the high salinity response in plants. However, the phosphoproteome differential expression under high salinity stress in halophytes has not been well studied. In this report, phosphoproteome differential expression was analyzed under high salinity stress in Thellungiella roots. Twenty-six putative phosphoproteins were found to have changed expression pattern at the post-translational level. Twenty of these were identified by mass spectrometric analysis, including 18 upregulated and two downregulated phosphoproteins. These proteins were involved in a variety of cellular processes, such as signal transduction, ROS detoxification, energy pathway, protein synthesis and protein folding. While most of these salt-responsive putative phosphoproteins are known salt-stress-related proteins, some of them have not been previously reported. Our results provide not only new insights into salt stress responses in Thellungiella but also a good foundation for further investigation of these high salinity-regulated phosphoproteins.     

蛋白质磷酸化修饰研究进展

 蛋白质磷酸化是由蛋白质激酶催化的磷酸基转移反应,是最常见、最重要的蛋白质翻译后修饰方式之一,是一种普遍的生命活动调节方式,在细胞信号转导过程中起重要作用。本文介绍了蛋白质磷酸化修饰的主要类型与功能、磷酸化蛋白的鉴定及磷酸化位点的预测等方面研究进展,并着重介绍了一些灵敏度高、特异性强的以同位素标记、免疫印迹-化学发光法等作为核心的磷酸化蛋白质分析方案。Western blot方法被证明是鉴别磷蛋白的灵敏、特异方法,而NanoPro100/1000超微量蛋白分析系统等又在此基础上加以改善。蛋白磷酸化分析工具和软件的发展也很迅猛。     

DDR相关蛋白缺陷引起的原发性免疫缺陷表型研究进展

在细胞内可变区基因(多样化基因)连接区基因片段重组(variable(diversity)joining recombination,V(D)J)与免疫球蛋白的类别转换重组(class switch recombination,CSR)过程中会产生程序性DNA双链断裂(DNA double strand break,DSB).当检测到DSB发生时DNA损伤反应(DNA damage response,DDR)被启动.DDR缺陷的病人具有原发性免疫缺陷表型(primary immunodeficiency,PID).总结了V(D)J重组与CSR产生DDR的分子机制,综述了V(D)J重组与CSR过程中DDR相关蛋白缺陷引起的原发性免疫缺陷表型.     

Identification of protein superfamily from structurebased sequence motif

The structure-based sequence motif of the distant proteins in evolution, protein tyrosine phosphatases (PTP) Ⅰ and Ⅱ superfamilies, as an example, has been defined by the structural comparison, structure-based sequence alignment and analyses on substitution patterns of residues in common sequence conserved regions. And the phosphatases Ⅰ and Ⅱ can be correctly identified together by the structure-based PTP sequence motif from SWISS-PROT and TrEBML databases. The results show that the correct rates of identification are over 98%. This is the first time to identify PTP Ⅰ and Ⅱ together by this motif.     

Chromosomal localization of a novel retinoic acid induced geneRA28 and protein distribution of its encoded protein

GeneRA28 is a retinoic acid induced novel gene isolated in our laboratory previously. All-trans retinoic acid (ATRA) was used to induce lung adenocarcinoma cell line GLC-82, andRA28 was obtained by subtractive hybridization. Green fluorescent protein (GFP) has emerged as a unique tool for examining introcellular phenomena in living cells. GFP possesses an intrinsic fluorescence at 488 nm that does not require other co-factors. In this report, an eukaryotic expression plasmid pEGFP-C1-RA28 was constructed and transfected with parental cell line GLC-82 to analyze protein expression and its distribution in living cells. Moreover, radiation hybrid (RH) technique was used to localizeRA28 to the chromosome. The results show that geneRA28 is mapped to the chromosome 19q13.1 region, its encoded protein is distributed on cell membrane. All the results further demonstrate that GFP and RH techniques are accurate, fast, repetitive, and will be powerful methods for investigating the gene and protein localization.     

肝大部切除前后热休克处理对热休克蛋白、酸性磷酸酶和碱性磷酸酶活性影响的分析

综合了磷酸酶的原位复性电泳、体视学分析、比色分析等方法分析①切除大白鼠大部分肝后的肝再生期间,②热休克处理大白鼠（４６℃、３０ｍｉｎ）恢复８ｈ,再切除大部分肝后的肝再生期间,③切除大部分肝恢复４ｈ,再热休克处理（４６℃、３０ｍｉｎ）后的肝再生期间热休克蛋白（ＨＳＣ７０／ＨＳＰ６８）、酸性磷酸酶（ＡＣＰ）和碱性磷酸酶（ＡＫＰ）动态变化的资料,从６个方面比较分析了ＨＳＣ７０／ＨＳＰ６８,ＡＣＰ和ＡＫＰ在肝再生中的相互关系及对肝再生的可能作用     

A dopamine- and cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions

Several mammalian neurotransmitter candidates, for example, serotonin, dopamine and noradrenaline, may exert some of their synaptic effects by regulating protein phosphorylation systems. Comparison of the regional distribution of brain phosphoproteins with neurotransmitter systems may help to identify the specific phosphoproteins involved in the functions of particular neurotransmitters. Here we report the association of one such phosphoprotein with the dopamine pathways in brain. This protein, of apparent molecular weight (MW) 32,000 (32K), seems to be present only in nervous tissue. Its regional distribution within the brain is very similar to the pattern of dopamine-containing nerve terminals; more specifically, the protein appears to be enriched in those dopaminoceptive neurones which possess D-1 receptors (dopamine receptors coupled to adenylate cyclase). The state of phosphorylation of the protein in these dopaminoceptive neurones can be regulated by both dopamine and cyclic AMP. These results suggest that the phosphoprotein may mediate certain of the trans-synaptic effects of dopamine acting on dopaminoceptive neurones.     

Site-specific DICER and DROSHA RNA products control the DNA-damage response

Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication

Early tumorigenesis is associated with the engagement of the DNA-damage checkpoint response (DDR). Cell proliferation and transformation induced by oncogene activation are restrained by cellular senescence. It is unclear whether DDR activation and oncogene-induced senescence (OIS) are causally linked. Here we show that senescence, triggered by the expression of an activated oncogene (H-RasV12) in normal human cells, is a consequence of the activation of a robust DDR. Experimental inactivation of DDR abrogates OIS and promotes cell transformation. DDR and OIS are established after a hyper-replicative phase occurring immediately after oncogene expression. Senescent cells arrest with partly replicated DNA and with DNA replication origins having fired multiple times. In vivo DNA labelling and molecular DNA combing reveal that oncogene activation leads to augmented numbers of active replicons and to alterations in DNA replication fork progression. We also show that oncogene expression does not trigger a DDR in the absence of DNA replication. Last, we show that oncogene activation is associated with DDR activation in a mouse model in vivo. We propose that OIS results from the enforcement of a DDR triggered by oncogene-induced DNA hyper-replication.     

Fragile X mental retardation protein interacts with TDG

Fragile X syndrome is the most common form of inherited mental retardation disease, resulting from absent of expression of its disease geneFMR1. To study the function of the fragile X mental retardation protein (FMRP) through protein/protein interaction, a mouse embryo cDNA library was screened by the yeast two-hybrid system. A clone was found to interact specifically with FMRP. The cDNA of this clone (Genbank accession number af 102875) encoded a protein highly homologous to human G/T mismatch-specific DNA thymine glycosylase (hTDG). Interactions between various alternatively spliced FMRP isoforms and a series of mTDG deletion proteins were further studied in the yeast two-hybrid system and their interaction amino acid regions were determined. Interaction between FMRP and TDG existed inside exon 13 of FMRP (amino acid residue 397–425) and around amino acid residue 122–346 of TDG. These results will be helpful to the study of the biological role of FMRP.     

Phytochrome controlled, long-day photoperiod-inducible protein in rice leaves

A new protein in the leaves of NK58S and NK58 (Oryza sativa L. subsp.japonica), which can be induced by 10 d-long-day photoperiod (14 h lightid) and cannot be induced by 10 d-short-day photoperiod (10 h light/d), has been found by two-dimensional gel electrophoresis. The protein, whose molecular weight and isoelectric point are 36 ku and pH 5.2 respectively, is found to be controlled by phytochrome as shown by the experiment of red light induction-far red light reversion.The existence of this protein in both NK58S and NK58 reflects that some of the responses of NK58S and NK58 might be similar in response to long-day photoperiod, a mild stress.     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

金黄色葡萄球菌sPP2C的克隆、表达及性质研究

从金黄色葡萄球菌基因文库中克隆了一条新的双特异性磷酸酶，命名为sPP2C (protein phosphatase 2C, Staphylococcus aureus). sPP2C基因具有741个碱基，编码的蛋白有247个氨基酸，具有一个蛋白磷酸酶2C的催化结构域.sPP2C的分子量为26.1 kD，等电点为4.95.在E.Coli. Rossetta中表达蛋白sPP2C.高纯度的sPP2C用亲和层析的方法纯化得到.酶学研究结果表明：sPP2C对磷酸酶的通用底物硝基苯磷酸 (p-nitrophenyl　phosphate, pNPP)不起作用，而与pSer/Thr和pTyr的寡肽均有去磷酸化作用.这些实验结果说明sPP2C是一个新的双特异性磷酸酶.     

人WDR70蛋白结构与功能的生物信息学预测

人WDR70蛋白作为一个衔接蛋白,可介导多蛋白相互作用,参与DNA损伤修复、胚胎发育等生物过程。采用生物信息学软件对人WDR70蛋白的结构和功能进行分析,发现WDR70蛋白的相对分子质量约为73 kD,pI为5.94,半衰期为30h,不含有信号肽和跨膜区结构;同源蛋白的多重序列比对和进化分析显示,人WDR70蛋白与黑猩猩、白枕白眉猴、食蟹猕猴的WDR70蛋白具有高度的同源性,并且在系统发育树上聚为一类;无规则卷曲是WDR70蛋白二级结构主要的结构形式,三级结构的预测结果与之吻合;功能分类预测其为结合蛋白,参与蛋白的泛素化和转运过程。开展WDR70蛋白的生物信息学分析,为了解该蛋白的结构和功能提供信息依据,为后续WDR70功能研究提供新思路。     

Isolation and cloning of a novel cDNALDB1 encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldbl. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldbl,Xenopus Xldbl andDrosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been namedLDB1 ( LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts ofLDBI in heart, brain and lung were considerably higher than those in other tissues.     

Structural basis of protein phosphatase 1 regulation

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 A resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits.