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Previously, we showed that an aberrant accumulation of activated Ras in mitochondria correlates with an increase in apoptosis. In this article, we show that lack of trehalose‐6P‐synthase, known to trigger apoptosis in Saccharomyces cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above‐mentioned correlation. Next, by characterizing the ras1Δ and ras2Δ mutants, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid (a pro‐apoptotic stimulus), are likely the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro‐survival molecular machinery. Finally, by characterizing the gpa2Δ and cyr1Δ mutants, in which the cAMP/PKA pathway is compromised, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway. [ABSTRACT FROM AUTHOR]

The Ras proteins, which regulate intracellular signaling by a cyclic process involving interconversion between active GTP-bound and inactive GDP-bound states, play an essential role in controlling the activity of several crucial signaling pathways regulating normal cellular proliferation. Mutational activation of RAS genes can induce cancer in humans and other mammals. About 30% of human tumors contain an altered oncogenic Ras; therefore, inhibitors of Ras activation are potentially antineoplastic drugs. In this work we describe original molecules acting as Ras inhibitors. Recently a new class of inhibitors of the Ras nucleotide exchange process was described by Taveras et al . These molecules are able to form a noncovalent complex with Ras-GDP, inhibiting the GDP-GTP nucleotide exchange. We synthesized molecule SCH- 53870 and we found that it inhibits p21-hRas nucleotide exchange in vitro , but it has very low solubility in water and undergoes rapid degradation at room temperature when dissolved in water-DMSO mixtures. This chemical instability could prejudice pharmacological activity in vivo. With the aim to improve solubility and chemical stability, we designed and synthesized other original bioactive molecules that have been characterized in vitro using purified human and yeast Ras proteins and in vivo using suitable Saccharomyces cerevisiae strains. In the long term we hope that the knowledge we derive from these compounds will help in the development of an alternative therapy targeting Ras for a specific inhibition of transformed cell proliferation. [ABSTRACT FROM AUTHOR]

In the yeast Saccharomyces cerevisiae , the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related ‘wild-type’ laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for l ack of c AMP r esponses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35 , encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo . Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+ -dependent adenylate cyclase activity in isolated plasma membranes of the cyr1 met1876 (lcr1 ) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+ -dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo . Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1 met1876 (lcr1 ) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by... [ABSTRACT FROM AUTHOR]

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids, An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40 °C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B. [ABSTRACT FROM AUTHOR]

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2val19 allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the iraΔ iraΔ strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Gα-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2val19 inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2val19 with adenylate cyclase apparently prevents its activation by both agonists. [ABSTRACT FROM AUTHOR]