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.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

Nucleotide sequence of Suncus murinus immunoglobulin mu gene and comparison with mouse and human mu genes

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [-³³P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly⁸⁴³ and Met⁸⁹⁵ of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.     

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures

The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Stable steady state conditions with tightly coupled growth were reached at various dilution rates between 0.02 and 0.14 h^-1. Both ethanol and H₂ steady state concentrations increased with growth rate and were lower in cocultures with the sulfate reducer < methanogen < homoacetogen. Due to the higher affinity for H₂, D. desulfuricans outcompeted M. bryantii, and this one A. woodii when inoculated in cocultures with P. acetylenicus. Cocultures with A. woodii had lower H₂ steady state concentrations when bicarbonate reduction was replaced by the energetically more favourable caffeate reduction. Similarly, cocultures with D. desulfuricans had lower H₂ concentrations with nitrate than with sulfate as electron acceptor. The Gibbs free energy (G) available to the H₂-producing P. acetylenicus was independent of growth rate and the H₂-utilizing partner, whereas the G available to the latter increased with growth rate and the energy yielding potential of the H₂ oxidation reaction. The critical Gibbs free energy (G_c), i.e. the minimum energy required for H₂ production and H₂ oxidation, was-5.5 to-8.0 kJ mol^-1 H₂ for P. acetylenicus,-5.1 to-6.3 kJ mol^-1 H₂ for A. woodii,-7.5 to-9.1 kJ mol^-1 H₂ for M. bryantii, and-10.3 to-12.3 kJ mol^-1 H₂ for D. desulfuricans. Obviously, the potentially available energy was used more efficiently by homoacetogens > methanogens > sulfate reducers.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

Irreversible Activation of the Opiate Receptor of Neuroblastoma × Glioma Hybrid Cells by an Alkylating Benzomorphan Derivative

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.     

天冬酰胺酶及PEG_2-天冬酰胺酶对L-天冬酰胺、谷氨酰胺亲和性的研究

本文报导了天冬酰胺酶及PEG_2-天冬酰胺酶对废物L-天冬酰胺、谷氨酰胺亲和性的研究,结果表明:PEG_2-天冬酰胺酶对谷氨酰胺的亲和性明显强于天冬酰胺酶(Km值分别为7.35×10~(-3)mol/L和7.14×10~(-2)mol/L),对天冬酰胺的亲和性略强于天冬酰胺酶(Km值分别为2.9×10~(-5)mol/L和4.0×10~(-5)mol/L)。天冬酰胺酶和PEG_2-天冬酰胺酶的CD光谱表明:天冬酰胺和谷氨酰胺对天冬酰胺酶和PEG_2-天冬酰胺酶的构象影响较大,但天冬酰胺酶和PEG_2-天冬酰胺酶的构象变化趋势有明显的不同。     

白百利烟草愈伤组织细胞细胞分裂素结合蛋白的研究

利用BA-Sepharose 4B亲和层析技术丛白百利烟草(Nicotiana tabacum Baibaili)愈伤组织细胞分离提纯了分子量为4400±100道尔顿的细胞分裂素结合蛋白(CB-蛋白)。在细胞表面、核糖体、线粒体、叶绿体和细胞核上以及在细胞液中都有CB-蛋白存在,而核糖体上的CB-蛋白含量量高。探讨了CB-蛋白的功能。