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Mitochondrial dysfunction induces a signaling pathway, which culminates in changes in the expression of many nuclear genes. This retrograde response, as it is called, extends yeast replicative life span. It also results in a marked increase in the cellular content of extrachromsomal ribosomal DNA circles (ERCs), which can cause the demise of the cell. We have resolved the conundrum of how these two molecular mechanisms of yeast longevity operate in tandem. About 50% of the life-span extension elicited by the retrograde response involves processes other than those that counteract the deleterious effects of ERCs. Deletion of RTG2, a gene that plays a central role in relating the retrograde response signal to the nucleus, enhances the generation of ERCs in cells with (grande) or in cells without (petite) fully functional mitochondria, and it curtails the life span of each. In contrast, overexpression of RTG2 diminishes ERC formation in both grandes and petites. The excess Rtg2p did not augment the retrograde response, indicating that it was not engaged in retrograde signaling. FOB1, which is known to be required for ERC formation, and RTG2 were found to be in converging pathways for ERC production. RTG2 did not affect silencing of ribosomal DNA in either grandes or petites, which were similar to each other in the extent of silencing at this locus. Silencing of ribosomal DNA increased with replicative age in either the presence or the absence of Rtg2p, distinguishing silencing and ERC accumulation. Our results indicate that the suppression of ERC production by Rtg2p requires that it not be in the process of transducing the retrograde signal from the mitochondrion. Thus, RTG2 lies at the nexus of cellular metabolism and genome stability, coordinating two pathways that have opposite effects on yeast longevity.

The yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, or budding. In each cell division, the daughter cell is usually smaller and younger than the mother cell, as defined by the number of divisions it can potentially complete before it dies. Although individual yeast cells have a limited life span, this age asymmetry between mother and daughter ensures that the yeast strain remains immortal. To understand the mechanisms underlying age asymmetry, we have isolated temperature-sensitive mutants that have limited growth capacity. One of these clonal-senescence mutants was in ATP2, the gene encoding the [beta]-subunit of mitochondrial [F.sub.1], [F.sub.0]-ATPase. A point mutation in this gene caused a valine-to-isoleucine substitution at the ninetieth amino acid of the mature polypeptide. This mutation did not affect the growth rate on a nonfermentable carbon source. Life-span determinations following temperature shift-down showed that the clonal-senescence phenotype results from a loss of age asymmetry at 36[degrees], such that daughters are born old. It was characterized by a loss of mitochondrial membrane potential followed by the lack of proper segregation of active mitochondria to daughter cells. This was associated with a change in mitochondrial morphology and distribution in the mother cell and ultimately resulted in the generation of cells totally lacking mitochondria. The results indicate that segregation of active mitochondria to daughter cells is important for maintenance of age asymmetry and raise the possibility that mitochondrial dysfunction may be a normal cause of aging. The finding that dysfunctional mitochondria accumulated in yeasts as they aged and the propensity for old mother cells to produce daughters depleted of active mitochondria lend support to this notion. We propose, more generally, that age asymmetry depends on partition of active and undamaged cellular components to the progeny and that this 'filter' breaks down with age.

Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl+ K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5 is predominantly present in the cytoplasm and the survival of the Jak2(V617F)+ HEL cells is impeded through the inhibition of the cytoplasmic functions of Stat5. [ABSTRACT FROM AUTHOR]

Background: Signal transducer and activator of transcription 3 and 5 (Stat3 and Stat5) play important roles in cell differentiation, proliferation, apoptosis and inflammation. They are transiently activated by ligand-receptor interactions in normal cells but are often found to be constitutively active in cancer cells. Analysis of their activation pattern is therefore important for the description of developmental processes and the understanding of cellular transformation. Materials and methods: To visualize Stat3 and Stat5 transactivation activity in different cell types, we designed novel reporter constructs. These constructs comprise Stat3 or Stat5 specific promoter elements and reporter genes encoding bgalactosidase or fluorescent proteins. These constructs were integrated into lentiviral gene transfer vectors facilitating efficient transduction of most cell types. Results: The lentiviral reporter constructs were used to infect different cell types and their inducibility by activated Stat3 or Stat5 was measured. The Stat3-mCherry reporter was active in transduced tumor cells, which exhibit high levels of phosphorylated Stat3 and it was inducible in HepG2 liver cells by interleukin-6 treatment. The Stat5-LacZ reporter was active in cultured cells upon hormone induction of Stat5 and in primary mammary epithelial cells transplanted into cleared fat pads of mice during late pregnancy. Conclusion: These novel reporter constructs are valuable tools to investigate and to distinguish between Stat3 and Stat5 activity in primary cells and cancer cells. They will also be useful in the discovery of drugs targeting Stat3 or Stat5. They can also be employed to generate transgenic mice and track Stat activity during development. [ABSTRACT FROM AUTHOR]

Investigates the mechanisms of age asymmetry between mother and daughter cells in Saccharomyces cerevisiae. Association of mitochondrial dysfunction with aging; Reproduction of yeast S. cerevisiae by asymmetric cell division; Importance of mitochondrial segregation in the maintenance of age asymmetry.

Peptide aptamers are molecules that bind to protein targets and are able to interfere with their functions. In the past, important achievements have been made using such peptide aptamers in different approaches and for various purposes. Peptide aptamers are comprised of a variable peptide region of 8 to 20 amino acids in length, which is displayed by a scaffold protein. An overview of the numerous scaffold proteins that have been investigated for their suitability to present peptide aptamers will be given. To identify peptide aptamers efficiently and specifically binding to a predetermined target, two eukaryotic systems have been used in multiple studies: a modified version of the Gal4 yeast-two-hybrid system and the optimized LexA interaction trap system. The two yeast systems are compared and the design of high-complexity peptide aptamer libraries for these systems is described. Although the yeast-two-hybrid system is based on intracellular interactions mammalian screens, performed in cell culture experiments, are sometimes preferred or required. We will give an overview of the mammalian selection systems available, which are based on the expression of peptide aptamers in retroviral or lentiviral vectors. We will show that the isolation and use of peptide aptamers as inhibitors of individual signaling components represents a new challenge for drug development.