Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1.

In cancer cells, the mammalian target of rapamycin complex 1 (mTORC1) that requires hormonal and nutrient signals for its activation, is constitutively activated. We found that overexpression of pyruvate kinase M2 (PKM2) activates mTORC1 signaling through phosphorylating mTORC1 inhibitor AKT1 substrate 1 (AKT1S1). An unbiased quantitative phosphoproteomic survey identified 974 PKM2 substrates, including serine202 and serine203 (S202/203) of AKT1S1, in the proteome of renal cell carcinoma (RCC). Phosphorylation of S202/203 of AKT1S1 by PKM2 released AKT1S1 from raptor and facilitated its binding to 14-3-3, resulted in hormonal- and nutrient-signals independent activation of mTORC1 signaling and led accelerated oncogenic growth and autophagy inhibition in cancer cells. Decreasing S202/203 phosphorylation by TEPP-46 treatment reversed these effects. In RCCs and breast cancers, PKM2 overexpression was correlated with elevated S202/203 phosphorylation, activated mTORC1 and inhibited autophagy. Our results provided the first phosphorylome of PKM2 and revealed a constitutive mTORC1 activating mechanism in cancer cells.

Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel.

Cellulosome is a kind of multienzyme complex that displays high activity, selectivity, and stability. Here, we report a novel, non-cellulolytic, cellulosome-like multienzyme complex that produced by the Cellulosimicrobium cellulans wild-type strain F16 isolated from soil microflora. This multienzyme complex, with excellent catalytic efficiency of kcat 13.2 s(-1) to remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (10-DAXP), has an outstanding tolerance against organic solvents and an excellent general stability, with the long half-life of 214 hours. This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones. The key catalytic subunit responsible for the ß-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family. The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

[The application of comparative proteomic analysis to screen proteins associated with mechanical properties of engineered cartilage: a preliminary study].

OBJECTIVE: To study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis. METHODS: Human chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient. RESULTS: All these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05). CONCLUSIONS: Comparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.

Trilinear decomposition method applied to removal of three-dimensional background drift in comprehensive two-dimensional separation data.

A novel technique for removal of three-dimensional background drift in comprehensive two-dimensional (2D) liquid chromatography coupled with diode array detection (LCxLC-DAD) data is proposed. The basic idea is to perform trilinear decomposition on the instrumental response data, which is based on the alternating trilinear decomposition (ATLD) algorithm. In model construction, the background drift is modeled as one component or factor as well as the analytes of interest, hence, the drift is explicitly included into the calibration. The method involves performing trilinear decomposition on the raw data, then extracting the background component and subtracting this background data from the raw data, leaving the analytes' signal on a flat baseline. Simultaneous evaluation of three-dimensional background drift and true signals may improve the quality of the data. This method is applied to the determination and removal of three-dimensional background drifts in simulated multidimensional data as well as experimental comprehensive two-dimensional liquid chromatographic data. It is shown that this technique yield a good removal of background drift, without the need to perform a blank chromatographic run, and required no prior knowledge about the sample composition.

Separation of compounds interacting with liposome membrane in combined prescription of traditional Chinese medicines with immobilized liposome chromatography.

Immobilized liposome chromatography (ILC), the stationary phase of which has been regarded as a mimic biomembranes system was used to separate and analyze compounds interacting with liposome membrane in Danggui Buxue decoction, a combined prescription of traditional Chinese medicines (CPTCMs), and its compositions Radix Astragli and Radix Angelica Sinensis. More than 10 main peaks in the extract of Danggui Buxue decoction were resolved on the ILC column, suggesting that more than 10 components in the prescription have significant retention on ILC column. Ligustilide, astragaloside IV and formononetin, three main bioactive ingredients in Danggui Buxue decoction, were found to have relatively significant, while ferulic acid, another bioactive ingredient in the prescription, relatively weak retention on ILC column. Effects of the eluent pH and amount of immobilized phosphatidylcholine (PC) on separation of interactional compounds in the extract of Danggui Buxue decoction were also investigated. It was found that these two factors strongly affected the retention of some interactional compounds. In addition, the fractions partitioned with different solvents from water extract of this combined prescription were evaluated with this ILC column system.

Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification.

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in <10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry.

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.

[Preparation of molecularly imprinted chiral monolithic column and its applications for separation of diastereomers].

A cinchonine imprinted chiral monolithic column was prepared for the separation of the diastereomers of cinchonine and cinchonidine by in-situ molecular imprinting technique. This type of molecularly imprinted column can be prepared only by a single-step procedure. In order to improve the selectivity and efficiency, a new in-situ molecular imprinting polymerization system was introduced by adopting porogenic solvents of toluene and 1-dodecanol with relatively low polarity and appropriate ratios of polymerization mixture. Diastereomers of cinchonine and cinchonidine were fully separated both under isocratic and gradient elutions on the chiral monolithic column. The broad peak shown in isocratic elution could be improved in gradient elution. Effects of mobile phase composition, flow rate and temperature on retention and separation factor were investigated. Due to the large throughput pores in the chiral monolithic column, low backpressure was observed during the separation process and a separation factor of 3.18 was obtained at 1.0 mL/min. The increase of temperature could improve the separation factor and a maximum separation factor of 5.40 was obtained at 60 degrees C.

[Application of genetic algorithm in optimization of mobile phase composition in high performance liquid chromatography].

Referring to traditional optimal methods, a method for the optimization of isocratic elution mobile phase composition in high performance liquid chromatography has been developed. In this method, the genetic algorithm based on line-crossover and plane-mutation is used. The principle of genetic algorithm and the process of optimization of mobile phase composition in reversed-phase ion-pair high performance liquid chromatography using genetic algorithm are introduced in details. With the concentrations of acetonitrile and ion-pair reagent sodium octane sulfonate chosen as the optimal parameters, the optimum was obtained by three times of optimization procedures. The mean relative error between the predicted and experimental values was 0.75% at the optimum and the optimization results were satisfactory.

[Simultaneous determination of trace Ni2+, Mn2+, Zn2+ and Cu2+ with microwave derivatization/ion-pair high performance liquid chromatography/meso-tetra(4-sulfophenyl)-porphyrin spectroscopy].

The best conditions for the reaction of meso-tetra(4-sulfophenyl)-porphyrin (TPPS4) with Mn2+, Ni2+, Zn2+ and Cu2+ have been investigated. A method for the fast determination of Ni2+, Mn2+, Zn2+ and Cu2+ has been developed: microwave derivatization at 750 W for 3 min, C18 column, acetonitrile-water(22.5:77.5, volume ratio) as mobile phase with tetraethylammonium bromide (TEABr) as ion-pair reagent and detection wavelength of 415 nm. The detection limits for Zn2+, Cu2+, Ni2+ and Mn2+ were 0.05 microgram/L, 0.01 microgram/L, 0.10 microgram/L and 0.40 microgram/L, respectively. This method has been applied to analyze the concentrations of these four metal ions in marketed tea.