Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced staining for β-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

/pdf/extension-of-life-span-by-introduction-of-telomerase-into-4ucjiz7vl1.pdf

Extension of life-span by introduction of telomerase into normal human cells

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

https://science.sciencemag.org/content/sci/277/5328/955.full.pdf

Telomerase catalytic subunit homologs from fission yeast and human

https://courses.washington.edu/gs466/readings/meyerson.pdf

hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

https://science.sciencemag.org/content/sci/276/5312/561.full.pdf

Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase

Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span

The primary determinant for telomere replication is the enzyme telomerase, responsible for elongating the G-rich strand of the telomere. The only component of this enzyme that has been identified in Saccharomyces cerevisiae is the TLC1 gene, encoding the telomerase RNA subunit. However, a yeast strain defective for the EST1 gene exhibits the same phenotypes (progressively shorter telomeres and a senescence phenotype) as a strain deleted for TLC1, suggesting that EST1 encodes either a component of telomerase or some other factor essential for telomerase function. We designed a multitiered screen that led to the isolation of 22 mutants that display the same phenotypes as est1 and tlc1 mutant strains. These mutations mapped to four complementation groups: the previously identified EST1 gene and three additional genes, called EST2, EST3 and EST4. Cloning of the EST2 gene demonstrated that it encodes a large, extremely basic novel protein with no motifs that provide clues as to function. Epistasis analysis indicated that the four EST genes function in the same pathway for telomere replication as defined by the TLC1 gene, suggesting that the EST genes encode either components of telomerase or factors that positively regulate telomerase activity.

https://www.genetics.org/content/genetics/144/4/1399.full.pdf

Senescence Mutants of Saccharomyces cerevisiae With a Defect in Telomere Replication Identify Three Additional EST Genes

In addition to the activation of classical progestin receptor-dependent genomic pathway, progesterone (P) can activate nonclassical, membrane-initiated signaling pathways in the brain. We recently demonstrated rapid P activation of second-messenger kinases, protein kinase A, and protein kinase C in the ventromedial nucleus (VMN) and preoptic area (POA) of rat brain. To determine whether P can activate yet another Ca+2 dependent kinase, we examined the rapid P modulation of calcium and calmodulin-dependent protein kinase II (CaMKII) in the VMN and POA in female rats. A rapid P-initiated activation of CaMKII basal activity was observed in the VMN but not the POA at 30 min. Estradiol benzoate (EB) priming enhanced this CaMKII basal activity in both the VMN and POA. CaMKII protein levels and phosphorylation of Thr-286 moiety on CaMKII, however, remained unchanged with EB and/or P treatments, suggesting that the changes in the CaMKII kinase activity are due to rapid P modulation of the kinase activity and not its synthesis or autoactivation. Furthermore, intracerebroventricular (icv) administration of a CaMKII-specific inhibitor, KN-93, 30 min prior to the P infusion, in EB-primed, ovariectomized female rats inhibited CaMKII activation but not protein kinase A and protein kinase C activities. Interestingly, icv administration of KN-93 30 min prior to P infusion (icv) resulted in a reduction but not total inhibition of P-facilitated lordosis response in EB-primed female rats. These observations suggest a redundancy or, alternately, a hierarchy in the P-regulated activation of kinase signaling cascades in female reproductive behavior.

Nonclassical mechanisms of progesterone action in the brain: II. Role of calmodulin-dependent protein kinase II in progesterone-mediated signaling in the hypothalamus of female rats.

The modulation of gene regulation by progesterone (P) and its classical intracellular regulation by progestin receptors in the brain, resulting in alterations in physiology and behavior has been well studied. The mechanisms mediating the short latency effects of P are less well understood. Recent studies have revealed rapid nonclassical signaling action of P involving the activation of intracellular signaling pathways. We explored the involvement of protein kinase C (PKC) in P-induced rapid signaling in the ventromedial nucleus of the hypothalamus (VMN) and preoptic area (POA) of the rat brain. Both the Ca2+-independent (basal) PKC activity representing the activation of PKC by the in vivo treatments and the Ca+2-dependent (total) PKC activity assayed in the presence of exogenous cofactors in vitro were determined. A comparison of the two activities demonstrated the strength and temporal status of PKC regulation by steroid hormones in vivo. P treatment resulted in a rapid increase in basal PKC activity in the VMN but not the POA. Estradiol benzoate priming augmented P-initiated increase in PKC basal activity in both the VMN and POA. These increases were inhibited by intracerebroventricular administration of a PKC inhibitor administered 30 min prior to P. The total PKC activity remained unchanged demonstrating maximal PKC activation within 30 min in the VMN. In contrast, P regulation in the POA significantly attenuated total PKC activity +/- estradiol benzoate priming. These rapid changes in P-initiated PKC activity were not due to changes in PKC protein levels or phosphorylation status.

/pdf/nonclassical-mechanisms-of-progesterone-action-in-the-brain-6v3crrrbsd.pdf

Bhuvana Balasubramanian

Papers

Senescence Mutants of Saccharomyces cerevisiae With a Defect in Telomere Replication Identify Three Additional EST Genes

Nonclassical mechanisms of progesterone action in the brain: II. Role of calmodulin-dependent protein kinase II in progesterone-mediated signaling in the hypothalamus of female rats.

Nonclassical mechanisms of progesterone action in the brain: I. Protein kinase C activation in the hypothalamus of female rats.