Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

Antibody reactivity to heat shock protein 70 and inner ear-specific proteins in patients with idiopathic sensorineural hearing loss.

Deafness is attributable to autoimmunity in a subset of adult patients with sensorineural hearing loss (SNHL) of unknown aetiology. To determine the roles of self-antigens in the pathogenesis of idiopathic SNHL, we analysed antibody responses to the inner ear-specific proteins, cochlin and beta-tectorin as well as the non-specific heat shock protein 70 (HSP70). Recombinant cochlin and beta-tectorin proteins were used in a qualitative Western blot assay for the detection of antigen-specific IgG antibodies in 58 patients with idiopathic SNHL and 28 healthy blood donors. In the same study cohort, we also used a Western blot assay to assess IgG antibody responses to the recombinant human HSP70. Of the 58 patient samples analysed, 19 tested positive to the HSP70, eight to cochlin and one to beta-tectorin, giving a prevalence of 33, 14 and 2%, respectively. Only one patient sample was reactive for HSP70, cochlin and beta-tectorin, seven of the remaining eight cochlin IgG antibody-positive samples were monospecific. Thus, cochlin-specific antibodies were observed predominantly in HSP70 IgG-negative patients demonstrating an additive value for testing this antibody response in patients with idiopathic SNHL.

Characterization of the Trypanosoma cruzi Cdc2p-related protein kinase 1 and identification of three novel associating cyclins.

Several Cdc2p-related protein kinases (CRKs) have been described in trypanosomatids but their role in the control of the cell cycle nor their biological functions have been addressed. In Trypanosoma cruzi two CRKs have been identified, TzCRK1 and TzCRK3. In this work we further characterize T. cruzi CRK1 and report the identification of three novel associating cyclins. We demonstrate that CRK1 levels and localization do not vary during the cell cycle, and show that it is localized in the cytoplasm, discrete regions of the nucleus, and is highly concentrated in the mitochondrion DNA (kinetoplast), suggesting a putative control function in this organelle. Using purified anti-CRK1 IgGs, we immunoprecipitated from the soluble fraction of T. cruzi epimastigote forms a protein kinase activity which is not inhibited by CDK inhibitors. In addition, we co-precipitated with p13Suc1p beads a kinase activity that was inhibited by the CDK inhibitor flavopiridol and olomoucine. Lastly, using the yeast two-hybrid system we identified three novel cyclin-like proteins able to associate with TzCRK1, and demonstrate that two of these cyclins also bind the T. cruzi CRK3 protein, indicating that these two CRKs are cyclin-dependent kinases.

Differential toxicities of anticancer agents among DNA repair and checkpoint mutants of Saccharomyces cerevisiae.

Most cytotoxic anticancer agents damage DNA directly, interfere with DNA metabolism or chromosome segregation, and are particularly toxic in dividing cells. Although a considerable amount of information on the mechanisms of action of these agents is available, the molecular bases for selective tumor cell killing by chemotherapy are largely unknown. Many genetic alterations found in sporadic and hereditary cancers affect functions in DNA repair and cell cycle control and result in sensitivity to DNA damaging agents. We have therefore set out to determine the effects of these cancer mutations on sensitivity or resistance to various chemotherapeutic agents. Because most of the affected genes are well conserved among eukaryotes, we have carried out a comprehensive analysis of a panel of isogenic yeast strains, each defective in a particular DNA repair or cell cycle checkpoint function, for sensitivity to the Food and Drug Administration-approved cytotoxic anticancer agents. Widely different toxicity profiles were observed for 23 agents and X-rays, indicating that the type of DNA repair and cell cycle checkpoint mutations in individual tumors could strongly influence the outcome of a particular chemotherapeutic regimen.

Integrating genetic approaches into the discovery of anticancer drugs.

The discovery of anticancer drugs is now driven by the numerous molecular alterations identified in tumor cells over the past decade. To exploit these alterations, it is necessary to understand how they define a molecular context that allows increased sensitivity to particular compounds. Traditional genetic approaches together with the new wealth of genomic information for both human and model organisms open up strategies by which drugs can be profiled for their ability to selectively kill cells in a molecular context that matches those found in tumors. Similarly, it may be possible to identify and validate new targets for drugs that would selectively kill tumor cells with a particular molecular context. This article outlines some of the ways that yeast genetics can be used to streamline anticancer drug discovery.

Requirement of S. pombe exonuclease II, a homologue of S. cerevisiae Sep1, for normal mitotic growth and viability.

Exonuclease II (ExoII) from Schizosaccharomyces pombe is a 5'-->3' single-stranded DNA exonuclease. We have cloned its gene, exo2, whose nucleotide sequence revealed that ExoII is a homologue of the multifunctional Saccharomyces cerevisiae Sep1 protein (also called Kem1, Xrn1, Rar5, Dst2). S. pombe exo2 null mutants were cold-sensitive for growth, had increased cell size at the restrictive temperature, were hypersensitive to the mitotic inhibitor thiabendazol and to caffeine, and died rapidly in stationary phase. Many of these phenotypes are similar to those of sep1 (kem1 or xrn1) mutants of S. cerevisiae. In contrast, the exo2 mutation had only a moderate effect on progression through meiosis and no significant effect on meiotic recombination. We discuss possible functions of the multifunctional ExoII protein.

A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction.

Exonuclease I (Exo I) from Schizosaccharomyces pombe, a 5'-->3' double-stranded DNA exonuclease, is induced during meiotic prophase I. The exo1 gene is a member of a family of related DNA repair genes, including RAD2/rad13/xpgc and YKL510/rad2, conserved from yeast to humans. An exo1 mutant displays a mutator phenotype and alters activity of the ade6-M387 marker effect. These results suggest that Exo I acts in a pathway that corrects mismatched base pairs.

Regulation of p105wee1 and p34cdc2 during meiosis in Schizosaccharomyces pombe.

Temperature-sensitive pat1 mutants of the fission yeast Schizosaccharomyces pombe can be induced to undergo meiosis at the restrictive temperature, irrespective of the mat1 configuration and the nutritional conditions. Using a combination of exit from stationary phase and thermal inactivation of the 52-kilodalton protein kinase that is encoded by the pat1 (also called ran1) gene, highly synchronous meiotic cultures were obtained. Synthesis and tyrosyl phosphorylation of p34cdc2 was evident during meiotic G1 and S phases. During this period there was increased expression of p105wee1, a protein kinase implicated in the tyrosyl phosphorylation of p34cdc2. Following a relatively brief G2 period, during which a reduction in the steady-state level of p105wee1 occurred, there was an approximately 19-fold increase in the histone H1 phosphotransferase activity of p34cdc2. Only a single peak of histone H1 kinase activation was observed, which implies that unlike meiosis in amphibians and echinoderms, p34cdc2 is functional only during one of the meiotic divisions in S. pombe, presumably meiosis II. Stimulation of the kinase activity of p34cdc2 was associated with its tyrosyl dephosphorylation. This is analogous to mitotic M phase and suggests parallels in the mechanism of activation of p34cdc2 during mitosis and one of the meiotic divisions in S. pombe.

A single-stranded DNA exonuclease from Schizosaccharomyces pombe.

We have purified to near homogeneity a DNA exonuclease from meiotic cells of Schizosaccharomyces pombe. The enzyme, designated exonuclease II (ExoII), had an apparent molecular weight of 134,000 and was abundant in the cell. It specifically degraded single-stranded DNA in the 5'----3' direction with an apparent Km for 5' DNA ends of 3.6 x 10(-11) M and produced 5' deoxynucleoside monophosphates. Its mode of degradation is similar to that of the RecJ protein from Escherichia coli; ExoII may, therefore, be involved in genetic recombination and DNA damage repair.

A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe.

In meiotic cells of the fission yeast Schizosaccharomyces pombe, a DNA exonuclease activity increased approximately 5-fold after premeiotic S-phase and decreased to the initial level before the meiotic divisions. We have purified this activity, designated exonuclease I, to near homogeneity. The activity co-purified with a polypeptide with an apparent molecular weight of 36,000. With a linear double-stranded DNA substrate, exonuclease I degraded only the 5'-ended strand from each end to produce 3'-single-stranded tails. The enzyme also acted on nicked circular DNA with comparable affinity. The meiotic induction of exonuclease I and its mode of action, similar to that of recombination-promoting exonucleases from bacteria, suggest that exonuclease I is involved in meiotic homologous recombination in S. pombe.

DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26.

The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.

Functional complementation between mutations in a yeast suppressor tRNA gene reveals potential for evolution of tRNA sequences.

Successive rounds of mutagenesis of a Schizosaccharomyces pombe strain bearing the UGA-reading sup3 tRNASer suppressor have been carried out for two cycles of inactivation and reactivation of the suppressor. The suppressor phenotype at each stage was found to involve different combinations of three mutations, A30, A53, and A67, in the sup3-UGA gene. Single mutations A30 and A53 inactivate the suppressor as does the presence of all three mutations. A67 by itself is phenotypically neutral, but in combination with either A30 or A53 suppressor function is restored. The frequency with which these and other complementation events occur in S. pombe demonstrates a significant potential for nucleotide sequence evolution in tRNA. Differential expression of the S. pombe genes in Saccharomyces cerevisiae suggests that the two yeasts have diverged at the transcriptional and RNA processing level. Processing of the mutant tRNA precursors in S. cerevisiae reveals a hierarchy of structural domains within the tRNA that vary in their importance for RNase P cleavage.

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation.

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.