Subunit Of Holoenzyme Research Articles

The architecture of Tetrahymena telomerase (926.2)

Telomerase is an RNA‐protein complex that extends the ends of linear chromosomes, and is a highly regulated determinant of cellular aging, stem cell renewal, and tumorigenesis. We have obtained the 3D structure of endogenously assembled Tetrahymena thermophila telomerase holoenzyme at 25 Å resolution using electron microscopy (EM). The 7 protein subunits and the stem‐loop 2 region of TER have been localized in the 3D structure by affinity labeling. Fitting with the available high‐resolution structures, including a p65‐TER complex, reveals the organization of TERT, TER, and p65 in the RNP catalytic core. Among the other holoenzyme proteins, p50 has an unanticipated role as a hub between the RNP catalytic core, p75‐p19‐p45 subcomplex, and the DNA‐binding Teb1. A complete in vitro holoenzyme reconstitution correlates activity with structure. This first physical and functional network architecture of a telomerase holoenzyme provides unprecedented detail about the structure of the RNP catalytic core and reveals the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres.J. Jiang, E.J. Miracco, K. Hong, B. Eckert, H. Chan, D.D. Cash, B. Min, Z. H. Zhou, K. Collins, and J. Feigon, “The architecture of Tetrahymena telomerase holoenzyme”, Nature, 496, 187‐182 (2013).K. Hong, H. Upton, E.J. Miracco, J. Jiang, Z.H. Zhou, J. Feigon, and K. Collins, “Tetrahymena telomerase holoenzyme assembly, activation, and inhibition by domains of the p50 central hub”, Mol Cell Biol. 33, 3962‐3971 (2013).M. Singh, Z. Wang, B.‐K. Koo, A. Patel, D. Cascio, K. Collins, and J. Feigon: “Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65”, Mol Cell 47, 16‐26 (2012).Grant Funding Source: NSF MCB1022379, NIH GM48123

Abstract P5-07-06: Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis

Abstract Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Activation of the cyclin D1 oncogene, often by amplification or rearrangement, is a major driver of multiple types of human tumors including breast and squamous cell cancers, B-cell lymphoma, myeloma, and parathyroid adenoma. The cyclin D1 gene is amplified or overexpressed in up to half of human breast cancers and its mammary-targeted overexpression induces mammary tumorigenesis in mice. Cyclin D1 encodes the regulatory subunit of the cyclin-dependent kinase (CDK) holoenzyme that phosphorylates several substrates including the retinoblastoma protein (pRb) to advance the G1S cell cycle checkpoint, promote DNA synthesis and regulate NRF-1 to inhibit mitochondrial biogenesis thereby coordinating nuclear and mitochondrial functions. In addition to cyclin D1's function as a regulatory subunit of a CDK holoenzyme, several CDK-independent functions have been identified. Tumors overexpressing cyclin D1 tend to display normal levels of proliferation and expression of E2F target genes, which contrasts with tumors overexpressing cyclin E or an activator for pRb. Breast cancers overexpressing cyclin D1 that are wild type for pRb have relatively normal proliferation rates, in contrast to those caused by genetic inactivation of pRb, which show significantly increased proliferation rates. Furthermore, the alternate splice form of cyclin D1, (cyclin D1b), has potent transforming ability, which does not correlate with the ability to phosphorylate the pRb protein. Several other properties of cyclin D1 have been identified including the induction of cellular migration and enhanced angiogenesis, inhibition of mitochondrial biogenesis, and mediating DNA-damage repair signaling. Cyclin D1 binding proteins participating in these putatively CDK-independent functions include PACSIN2, NRF1, and p27KIP1; binding to p27KIP1 and PACSIN2 contribute to the pro-migratory function of cyclin D1. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1-/- mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1WT or cyclin D1KE in the mammary gland was sufficient to induce the CIN signature within 7 days. Sustained expression of cyclin D1KE induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1WT and cyclin D1KE to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-06.

The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator

Author SummaryThe CK2 kinase is an ancient enzyme known to be at the heart of self-sustaining circadian clocks in animals, plants, and fungi. Circadian clocks are responsible for daily circadian rhythms in molecular, physiological, and behavioral processes. Their mechanism relies on transcriptional activators and repressors that constitute a feedback loop. The CLOCK (CLK) activator is required to initiate the transcription of the period (per) and timeless (tim) genes in the late day. The PER and TIM repressors accumulate in a delayed manner and translocate to the nucleus to repress their own gene. Degradation of these repressors allows the activator to start a new cycle. In the fruit fly Drosophila melanogaster, CK2-mediated phosphorylation of the PER and TIM repressors targets them for degradation. Here we find that the CLK activator is also regulated by CK2. In contrast to PER and TIM, CLK is stabilized by CK2α phosphorylation; we also show that PER and TIM are dispensable for this stabilization of CLK. The identification of CK2–CLK protein complexes and the in vitro phosphorylation of CLK by CK2 hint at a direct action of the kinase on the activator. Although transcription factor stabilization is generally expected to be associated with increased activity, genetic analysis indicates that CK2 represses CLK. Hence CK2 phosphorylation seems to be a key signal for the transcriptional activator complex to adopt a proper activation state at a given point of the circadian cycle.

Impaired expression of protein phosphatase 2A subunits enhances metastatic potential of human prostate cancer cells through activation of AKT pathway

Background:Protein phosphatase 2A (PP2A) is a dephosphorylating enzyme, loss of which can contribute to prostate cancer (PCa) pathogenesis. The aim of this study was to analyse the transcriptional and translational expression patterns of individual subunits of the PP2A holoenzyme during PCa progression.Methods:Immunohistochemistry (IHC), western blot, and real-time PCR was performed on androgen-dependent (AD) and androgen-independent (AI) PCa cells, and benign and malignant prostate tissues for all the three PP2A (scaffold, regulatory, and catalytic) subunits. Mechanistic and functional studies were performed using various biochemical and cellular techniques.Results:Through immunohistochemical analysis we observed significantly reduced levels of PP2A-A and -B′γ subunits (P<0.001 and P=0.0002) in PCa specimens compared with benign prostate. Contemporarily, there was no significant difference in PP2A-C subunit expression between benign and malignant tissues. Similar to the expression pattern observed in tissues, the endogenous levels of PP2A-A and B′γ subunits were abrogated from the low metastatic to high metastatic and AD to AI cell line models, without any change in the catalytic subunit expression. Furthermore, using in vitro studies we demonstrated that PP2A-Aα scaffold subunit has a role in dampening AKT, β-catenin, and FAK (focal adhesion kinase) signalling.Conclusion:We conclude that loss of expression of scaffold and regulatory subunits of PP2A is responsible for its altered function during PCa pathogenesis.

A solution to the telomerase puzzle

Telomerase synthesizes DNA sequences that protect the integrity of chromosome ends. A model for how the components of this enzyme complex co-assemble offers insight into its structure and function. See Article p.187 The ends of linear chromosomes in eukaryotes are maintained by a ribonucleoprotein complex known as telomerase. Juli Feigon and colleagues have obtained a long-awaited structure of the ciliate (Tetrahymena thermophila) telomerase by electron microscopy. Modelling with crystal structures of various components allowed identification of the catalytic core and subunit interactions, and a complete in vitro reconstitution of the enzyme was performed. The structure provides unprecedented detail about the architecture of the ribonucleoprotein catalytic core and reveals the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres.

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

BackgroundInduction of the death pathway resulting from the specific interaction of the PP2A1 phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides. MethodsUsing biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences. ResultsWe identified overlapping sequences, located within the N-terminal domain E4orf423-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A1 holoenzyme. We characterized DPT-E4orf44 and TAT-E4orf44, two bi-partite cell penetrating peptides containing the 12 PP2A1 binding residues of the canine type 2 E4orf427-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf44, in contrast to inactive TAT-E4orf44, adopted a well defined α-helical structure and co-precipitated PP2A1 from HeLa cell extracts. DPT-E4orf44 also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf44 or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf44 also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts. ConclusionsWe characterized the transducing properties of a new α-helical DPT-E4orf44 peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein. General significanceOur results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.

Probes of the mitochondrial cAMP-dependent protein kinase

The development of a fluorescent assay to detect activity of the mitochondrial cAMP-dependent protein kinase (PKA) is described. A peptide-based sensor was utilized to quantify the relative amount of PKA activity present in each compartment of the mitochondria (the outer membrane, the intermembrane space, and the matrix). In the process of validating this assay, we discovered that PKA activity is regulated by the protease calpain. Upon exposure of bovine heart mitochondria to digitonin, Ca2+, and a variety of electron transport chain inhibitors, the regulatory subunits of the PKA holoenzyme (R2C2) are digested, releasing active catalytic subunits. This proteolysis is attenuated by calpain inhibitor I (ALLN). This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Fluorescence Polarization and Fluctuation Analysis Reveals CAMKIIα Catalytic Domain Pair Extension Triggered by Ca2+/Calmodulin

The autoinhibited calcium/calmodulin-dependent protein kinases-II (CaMKII) assembles to form a holoenzyme structure with multiple catalytic domain pairs radiating out from a central core composed of oligomerization domains. Activation of CaMKII upon binding calcium2+/calmodulin (CaM) is known to initiate a biochemical cascade resulting in CaMKII translocation to synaptic spines and the induction of long-term potentiation. Little is known about the structural changes in the intact CaMKII holoenzyme responsible for this activation. Fluorescence polarization and fluctuation analysis (FPFA) microscopy, a method that combines time-resolved anisotropy and fluorescence correlation spectroscopy (FCS), was used to monitor Venus-CaMKIIα (where a Venus fluorescent-protein is tagged onto the N-terminus of CaMKIIα) before and after activation with CaM. FPFA simultaneously monitors brightness (a measure of the number of subunits in the holoenzyme), homo-FRET (a catalytic domain proximity detector) and diffusion time (a parameter sensitive to the mass and hydrodynamic volume of the holoenzyme). Together these measurements can potentially detect an array of different covert structural changes in the CaMKII holoenzyme triggered by CaM. In homogenates prepared from HEK cells expressing Venus-CaMKIIα, the addition of CaM triggered T286 autophosphorylation, an indicator of kinase activation. FPFA revealed a large increase in the holoenzyme diffusion time. Because changes in holoenzyme subunit stoichiometry were not observed, the change in diffusion time most likely indicates a large change in hydrodynamic volume, such as catalytic domain pair extension. This change was not observed in Venus-CaMKIIα-TT305/6DD, a mutant that cannot bind calmodulin. Holoenzyme activation by CaM did trigger a small, dose-dependent biphasic change in anisotropy (homo-FRET between Venus-tagged catalytic domains), but its magnitude was too small to be interpreted as catalytic domain un-pairing. Interpretation of these experiments will be discussed.

Introduction: Current Themes on Cell Cycle and Cancer

Introduction: Current Themes on Cell Cycle and Cancer

Heterotypic Three-dimensional &lt;em&gt;In Vitro&lt;/em&gt; Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades. Whilst stage I tumors have 5-year survival rates >85%, survival rates for stage III/IV disease are <40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF) cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Neuronal excitability and calcium/calmodulin‐dependent protein kinase type II: Location, location, location

Calcium/calmodulin-dependent protein kinase type II (CaMKII) is a highly abundant serine/threonine kinase comprising a significant fraction of total protein in mammalian forebrain and forming a major component of the postsynaptic density. CaMKII is essential for certain forms of synaptic plasticity and memory consolidation and this is mediated through substrate binding and intramolecular phosphorylation of holoenzyme subunits. CaMKII is multifunctional; it targets a variety of cellular substrates, and this diversity depends on holoenzyme subunit composition. CaMKII comprises homooligomeric and heterooligomeric complexes generated from four subunits (α, β, δ, and γ) encoded by separate genes that are further expanded by extensive alternative splicing to more than 30 different isoforms. Much attention has been paid to understanding the regulation of CaMKII function through its structural diversity and/or substrate specificity. However, given the importance of subunit composition to holoenzyme activity, it is likely that specificity of cellular expression of CaMKII isoforms also plays a major role in regulation of enzyme function. Herein we review the cellular colocalization of CaMKII isoforms with special regard to the cell-type specificity of isoform expression in brain. In addition, we highlight the remarkable specificity of subcellular localization by the CaMKIIα isoform. In addition, we discuss the role that this cellular specificity of expression might play in propagating the type of recurrent neuronal activity associated with disorders such as temporal lobe epilepsy.

Roles of Telomerase Reverse Transcriptase N-terminal Domain in Assembly and Activity of Tetrahymena Telomerase Holoenzyme

Telomerase extends chromosome ends by the addition of single-stranded telomeric repeats. To support processive repeat synthesis, it has been proposed that coordination occurs between DNA interactions with the telomerase RNA template, the active site in the telomerase reverse transcriptase (TERT) core, a TERT N-terminal (TEN) domain, and additional subunits of the telomerase holoenzyme required for telomere elongation in vivo. The roles of TEN domain surface residues in primer binding and product elongation have been studied largely using assays of minimal recombinant telomerase enzymes, which lack holoenzyme subunits that properly fold and conformationally stabilize the ribonucleoprotein and/or control its association with telomere substrates in vivo. Here, we use Tetrahymena telomerase holoenzyme reconstitution in vitro to assess TEN domain sequence requirements in the physiological enzyme context. We find that TEN domain sequence substitutions in the Tetrahymena telomerase holoenzyme influence synthesis initiation and elongation rate but not processivity. Functional and direct physical interaction assays pinpoint a conserved TEN domain surface required for holoenzyme subunit association and for high repeat addition processivity. Our results add to the understanding of telomerase holoenzyme architecture and TERT domain functions with direct implications for the unique mechanism of single-stranded repeat synthesis.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Proteolytic Regulation of the Mitochondrial cAMP-Dependent Protein Kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.

Single-Molecule Analysis of the Functional Structure of Telomerase RNP

Telomerase is a cellular ribonucleoprotein (RNP) complex comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The activity of the telomerase enzyme is essential for the maintenance of genome stability and normal cell development. Despite the biomedical importance of telomerase activity, detailed structural models for the enzyme remain to be established. Here we report the development and application of a single-molecule assay for direct structural analysis of catalytically active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity, allowing precise discrimination between inactive, active and processive enzyme binding events. FRET signals from individual enzyme molecules during the active and processive binding events were then used to determine the global organization of telomerase RNA within catalytically active holoenzymes. Using this assay, we have identified an active conformation of telomerase among a heterogeneous population of enzymes with distinct structures. In particular, we have established that the phylogenetically conserved pseudoknot motif within telomerase RNA is properly folded in catalytically active enzymes. Interestingly, the pseudoknot motif is misfolded in the absence of proteins, and the protein subunits of the telomerase holoenzyme counteract RNA misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of various telomerase RNA regions such as the pseudoknot but also establish a direct method for elucidating how the structure of telomerase enables its function.

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes (1-3). The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression(4,5). These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues (6). The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis (7-10). CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance (11). We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

TIN2 Protein Dyskeratosis Congenita Missense Mutants Are Defective in Association with Telomerase

Dyskeratosis congenita (DC) is a progressive and heterogeneous congenital disorder that affects multiple systems and is characterized by bone marrow failure and a triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. One common feature for all DC patients is abnormally short telomeres and defects in telomere biology. Most of the known DC mutations have been found to affect core components of the telomerase holoenzyme. Recently, multiple mutations in the gene encoding the telomeric protein TIN2 have been identified in DC patients with intact telomerase genes, but the molecular mechanisms underlying TIN2 mutation-mediated DC remain unknown. Here, we demonstrate that ectopic expression of TIN2 with DC missense mutations in human cells led to accelerated telomere shortening, similar to the telomere phenotypes found in DC patients. However, this telomere shortening was not accompanied by changes in total telomerase activity, localization of TIN2, or telomere end protection status. Interestingly, we found TIN2 to participate in the TPP1-dependent recruitment of telomerase activity. Furthermore, DC mutations in TIN2 led to its decreased ability to associate with TERC and telomerase activity. Taken together, our data suggest that TIN2 mutations in DC may compromise the telomere recruitment of telomerase, leading to telomere shortening and the associated pathogenesis.

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.

HTERT Overexpression Alleviates Intracellular ROS Production, Improves Mitochondrial Function, and Inhibits ROS-Mediated Apoptosis in Cancer Cells

The human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase holoenzyme. Evidence is accumulating to link hTERT to activities other than telomere maintenance and immortalization. Here, we show that hTERT overexpression not only reduces the basal cellular reactive oxygen species (ROS) levels but also inhibits endogenous ROS production in response to stimuli that induce intracellular ROS generation. Conversely, siRNA-mediated gene silencing of hTERT potentiated the increase in cellular ROS levels following exposure to oxidative stress. This antioxidant effect of hTERT is mediated via a significant increase in the ratio of reduced to oxidized glutathione (GSH:GSSG) as well as efficient recovery of the oxidized peroxiredoxin to its nonoxidized form. Our data also provide evidence for mitochondrial localization of hTERT, and a significantly higher activity of cytochrome C oxidase, the rate-limiting enzyme in the mitochondrial electron transport chain, in hTERT overexpressing cells. To ascertain whether the improved mitochondrial function and antioxidant effect of hTERT could provide cancer cells with a survival advantage, the effect of oxidative stress on mitochondrial apoptosis was evaluated. Indeed, hTERT overexpressing cells inhibited cytosolic acidification, translocation of Bax, the drop in mitochondrial transmembrane potential, the release of cytochrome C to the cytosol, and cell death. Taken together, these data demonstrate a hitherto undefined role of hTERT in alleviating cellular ROS levels by way of potentiating the cellular antioxidant defense systems, and in doing so endowing cancer cells with the ability to evade death stimuli.