Application of proteomics in the study of tumor metastasis

Tumor metastasis is the dominant cause of death in cancer patients. However, the molecular and cellular mechanisms underlying tumor metastasis are still elusive.The identification of protein molecules with their expressions correlated to the metastatic process would help to understand the metastatic mechanisms and thus facilitate the development of strategies for the therapeutic interventions and clinical management of cancer. Proteomics is a systematic research approach aiming to provide the global characterization of protein expression and function under given conditions. Proteomic technology has been widely used in biomarker discovery and pathogenetic studies including tumor metastasis. This article provides a brief review of the application of proteomics in identifying molecular factors in tumor metastasis process. The combination of proteomics with other experimental approaches in biochemistry, cell biology, molecular genetics and chemistry, together with the development of new technologies and improvements in existing methodologies will continue to extend its application in studying cancer metastasis.     

Expression and activation of an esterase from Pseudomonas aeruginosa 1001 in Escherichia coli

An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage.     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

(-)-3,5-Dicaffeoyl-muco-quinic acid isolated from Aster scaber contributes to the differentiation of PC12 cells: through tyrosine kinase cascade signaling

Aster scaber T. (Asteraceae) has been used in traditional Korean and Chinese medicine to treat bruises, snakebites, headaches, and dizziness. (-)-3,5-Dicaffeoyl-muco-quinic acid (DQ) isolated from A. scaber induced neurite outgrowth in PC12 cells. It has been reported that the activation of the extracellular signal regulated kinase 1/2 (Erk 1/2) and phosphoinositide 3 (PI3) kinase plays a crucial role in the NGF-induced differentiation of PC12 cells. This study showed that the effect of DQ on neurite outgrowth is mediated via the Erk 1/2 and PI3 kinase-dependent pathways like NGF. Furthermore, DQ stimulated the phosphorylation of Trk A. Overall, DQ elicited the differentiation of PC12 cells through Trk A phosphorylation followed by Erk 1/2 and PI3 kinase activation.     

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid,the Lathyrus sativus Neurotoxin

-N-oxalyl-l-,-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 M concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca²⁺ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

低温胁迫下凤眼莲叶片的适应——脱落酸和可溶性蛋白质含量升高

本文报道低温胁迫下风眼莲叶片脱落酸(ABA)、可溶性蛋白质和水势的测定结果。低温胁迫时脱落酸和可溶性蛋白质含量远高于对照,(前者含量最高可达对照的4倍,后者可达到对照的2.75倍),而且脱落酸和蛋白质含量随温度降低而升高。蛋白质的生物合成抑制剂亚胺环己酮证明,可溶性蛋白质含量升高,原因是有部分是新合成的。在各种低温处理下获得了几乎相同于对照的叶片水势。我们推测:低温胁迫下,脱落酸水平的相应变化不是由于低温诱导水分胁迫所致,而是低温胁迫本身诱导。     

饲料蛋白水平对宝石鲈生长和体组成影响研究

选择体重一致、健康无病、初始体重104.13±0.46g宝石鲈105尾,随机分为5组,每组3重复,分别喂以蛋白水平为23.64%、28.45%、33.50%、3.04%和41.62%的等能颗粒饲料(以白鱼粉为蛋白源),在15口水泥池中控温养殖8周,研究饲料蛋白水平对宝石鲈生长和体组成的影响.饲养试验结果表明:饲料蛋白水平为33.50%时,宝石鲈的增重率最大,与饲料蛋白水平23.64%组相比差异极显著(P0.05).试验不同组间鱼体肥满度、肝体指数、蛋白质净利用率和饵料系数没有显著差异(P>0.05).通过增重率的折线法分析结果表明,宝石鲈获得最佳增重率的饲料蛋白需要量为33.94%.饲料蛋白水平对鱼体组成和肌肉成分研究表明,饲料蛋白水平为33.50%时鱼体蛋白含量最高,与蛋白水平23.64%组相比,差异极显著(P0.05).       

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.