Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Effects of land preparation and maize cultivar on efficiency of N-fertilizer applied at different times and by different methods in maize—mungbean association using15N

Summary A field experiment was conducted using¹⁵N-labelled urea on a Reddish Brown Lateritic (Peleustult) soil. Growing two crops on flat land and on soil ridges of 15 cm height produced similar comparative effects from fertilizer on maize. However, fertilizer applied by broadcasting on maize with a 50 cm effective band followed by incorporating was more useful to mungbean than that applied by banding below the cereal seed rows when crops were grown on flat land. The reverse was observed when crops were grown on ridges. It was deduced that the maize cultivar was not likely to affect comparative efficiencies of fertilizer. For fertilizer application at sowing, broadcasting in 50 cm maize effective band followed by incorporating was slightly superior to banding below maize seed rows. Side-dressing of fertilizer to maize at 4 weeks after sowing was superior to application at sowing. Evenly-split application, at sowing and at 4 weeks after sowing, was either only slightly superior or comparable to non-split application by banding below maize seed rows at sowing, depending on placement method of the first application. Soil moisture status as a possible factor rendering discrepancy in the comparative efficiencies obtained by different authors is discussed.     

环氧树脂固定化的Bacillus sp.DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用

环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp. DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:p H 8. 0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e. e. p=97. 5%的(R)-1-苯乙醇(产率为45. 0%)和e. e. s=99. 2%的(S)-乙酸苏合香酯(产率为83. 9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e. e. p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。     

Taste receptor activity and feeding behaviour reveal mechanisms of white spruce natural resistance to Eastern spruce budworm Choristoneura fumiferana

The pattern of feeding of Eastern spruce budworm Choristoneura fumiferana (Clem.) (Lepidoptera, Tortricidae) is compared on foliage from white spruce Picea glauca (Moench) Voss. (Pinaceae) trees previously determined to be susceptible and resistant to defoliation by budworm. No differences are observed in electrophysiological responses from taste sensilla to aqueous extracts of the two foliage types, nor is there a preference for either extract type in a choice test. Acetone extracts from the two foliage types are both preferred to a control sucrose solution, although neither elicits a preference relative to the other. These results suggest that there is no difference in phagostimulatory power of internal leaf contents of the two foliage types. Longer‐term observation of feeding behaviour in a no‐choice situation shows no difference in meal duration, confirming the lack of difference in phagostimulatory power. However, on average, intermeal intervals are twice as long on the resistant foliage, leading to an overall lower food consumption during the assay. This result suggests an anti‐digestive or toxic effect of the resistant foliage that slows behaviour and limits food intake. Previous research has shown that waxes of the resistant foliage deter initiation of feeding by the spruce budworm and that this foliage contains higher levels of tannins and monoterpenes. The data suggest that the resistant foliage contains a post‐ingestive second line of defence against the spruce budworm.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M₄₁₂) and light-induced proton release under steady-state conditions in aqueous suspensions of ‘purple membrane’ derived from Halobacterium halobium. The decay of M₄₁₂ was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M₄₁₂ which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M₄₁₂ was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M₄₁₂ without affecting the level of M₄₁₂ which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca²⁺ and their antagonists La³⁺ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.