SUMMARY
Rho GTPases impact a number of activities important for oncogenesis. Here we describe a small molecule inhibitor which blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NFκB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism, and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.
integration ͉ structure ͉ retrovirus ͉ transcription ͉ host factor L ike all retroviruses, HIV-1 must integrate a reversetranscribed copy of its viral RNA genome into a host cell chromosome to establish a productive infection. Integration is mediated by the viral integrase (IN) protein acting on the DNA attachment sites at the ends of the linear reverse transcript. IN acts within the context of a higher-order preintegration complex (PIC) that is derived from the core of the infecting virion. IN catalyzes two sequential reactions, initially removing 3Ј terminal GT nucleotides from both ends of HIV-1 cDNA. After nuclear entry, IN inserts the processed 3Ј termini into opposing strands of chromosomal DNA. Repair of single-strand gaps by host cell enzymes completes the integration process (for a review, see ref.
Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.glutamine metabolism | Warburg effect
Background: GAC supplies for increased metabolic needs of tumors because of exclusive localization and kinetic properties. Results: Higher than tetramer oligomers are the active form in in vitro and in cellular assays. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide disrupts oligomers. Conclusion: A novel molecular mechanism for GAC activation is proposed. Significance: The data affect the development of therapies targeting GAC in tumors, with emphasis on allosteric inhibitors.
SUMMARY
Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo, in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by alleles of a long ncRNA.
The binding of capped RNAs to the cap-binding complex (CBC) in the nucleus, and their dissociation from the CBC in the cytosol, represent essential steps in RNA-processing. Here we show how the nucleocytoplasmic transport proteins, importin-α and importin-β, play key roles in regulating these events. As a first step toward understanding the molecular basis for this regulation, we determined a 2.2 Å resolution x-ray structure for a CBC-importin-α complex that provides a detailed picture for how importin-α binds to the CBP80 subunit of the CBC. Through a combination of biochemical studies, x-ray crystallographic information, and small-angle scattering experiments, we then determined how importin-β binds to the CBC through its CBP20 subunit. Together, these studies enable us to propose a model describing how importin-β stimulates the dissociation of capped RNA from the CBC in the cytosol following its nuclear export.
The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.
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