Telomere-binding Protein TRF2 Research Articles

Telomeres cooperate with the nuclear envelope to maintain genome stability.

Mammalian telomeres have evolved safeguards to prevent their recognition as DNA double-stranded breaks by suppressing the activation of various DNA sensing and repair proteins. We have shown that the telomere-binding proteins TRF2 and RAP1 cooperate to prevent telomeres from undergoing aberrant homology-directed recombination by mediating t-loop protection. Our recent findings also suggest that mammalian telomere-binding proteins interact with the nuclear envelope to maintain chromosome stability. RAP1 interacts with nuclear lamins through KU70/KU80, and disruption of RAP1 and TRF2 function result in nuclear envelope rupture, promoting telomere-telomere recombination to form structures termed ultrabright telomeres. In this review, we discuss the importance of the interactions between shelterin components and the nuclear envelope to maintain telomere homeostasis and genome stability.

MCCC2 is a novel mediator between mitochondria and telomere and functions as an oncogene in colorectal cancer

BackgroundThe mitochondrial gene MCCC2, a subunit of the heterodimer of 3-methylcrotonyl-CoA carboxylase, plays a pivotal role in catabolism of leucine and isovaleric acid. The molecular mechanisms and prognostic value still need to be explored in the context of specific cancers, including colorectal cancer (CRC).MethodsIn vitro and in vivo cell-based assays were performed to explore the role of MCCC2 in CRC cell proliferation, invasion, and migration. Mitochondrial morphology, membrane potential, intracellular reactive oxygen species (ROS), telomerase activity, and telomere length were examined and analyzed accordingly. Protein complex formation was detected by co-immunoprecipitation (CO-IP). Mitochondrial morphology was observed by transmission electron microscopy (TEM). The Cancer Genome Atlas (TCGA) CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the MCCC2 expression level. The association between MCCC2 expression and various clinical characteristics was analyzed by chi-square tests. CRC patients’ overall survival (OS) was analyzed by Kaplan–Meier analysis.ResultsEctopic overexpression of MCCC2 promoted cell proliferation, invasion, and migration, while MCCC2 knockdown (KD) or knockout (KO) inhibited cell proliferation, invasion, and migration. MCCC2 KD or KO resulted in reduced mitochondria numbers, but did not affect the gross ATP production in the cells. Mitochondrial fusion markers MFN1, MFN2, and OPA1 were all upregulated in MCCC2 KD or KO cells, which is in line with a phenomenon of more prominent mitochondrial fusion. Interestingly, telomere lengths of MCCC2 KD or KO cells were reduced more than control cells. Furthermore, we found that MCCC2 could specifically form a complex with telomere binding protein TRF2, and MCCC2 KD or KO did not affect the expression or activity of telomerase reverse transcriptase (TERT). Finally, MCCC2 expression was heightened in CRC, and patients with higher MCCC2 expression had favorable prognosis.ConclusionsTogether, we identified MCCC2 as a novel mediator between mitochondria and telomeres, and provided an additional biomarker for CRC stratification.

Intact mitochondrial function in the setting of telomere-induced senescence.

Mitochondria play essential roles in metabolic support and signaling within all cells. Congenital and acquired defects in mitochondria are responsible for several pathologies, including premature entrance to cellar senescence. Conversely, we examined the consequences of dysfunctional telomere-driven cellular senescence on mitochondrial biogenesis and function. We drove senescence in vitro and in vivo by deleting the telomere-binding protein TRF2 in fibroblasts and hepatocytes, respectively. Deletion of TRF2 led to a robust DNA damage response, global changes in transcription, and induction of cellular senescence. In vitro, senescent cells had significant increases in mitochondrial respiratory capacity driven by increased cellular and mitochondrial volume. Hepatocytes with dysfunctional telomeres maintained their mitochondrial respiratory capacity in vivo, whether measured in intact cells or purified mitochondria. Induction of senescence led to the upregulation of overlapping and distinct genes in fibroblasts and hepatocytes, but transcripts related to mitochondria were preserved. Our results support that mitochondrial function and activity are preserved in telomere dysfunction-induced senescence, which may facilitate continued cellular functions.

A Novel Role of Prostate-Specific Membrane Antigen in Telomere Stability in Prostate Cancer Cells.

Role of PSMA in telomere stability suggests a strong correlation between PSMA expression and prostate cancer progression.

Localization of Telomere-Binding Protein TRF2 in Spermatogenic Cells of Hibernating Frogs Rana temporaria

Localization of Telomere-Binding Protein TRF2 in Spermatogenic Cells of Hibernating Frogs Rana temporaria

Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1–TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

From the wings to the center stage of chromosomes

Telomere-binding protein TRF2 protects the linear chromosome ends, telomeres, from being recognized as damaged DNA. TRF2 also regulates gene expression outside telomeres, but the detailed mechanism has not been fully understood. Mukherjee and colleagues have employed ChIP-Seq and biochemical analyses to identify G-quadruplexes at gene promoters across the genome as nontelomeric TRF2-binding sites. TRF2 occupancy on such target sites leads to epigenetic gene repression, implicating TRF2-G-quadruplex interaction as a sophisticated regulator of gene expression.

The karyosphere capsule in Rana temporaria oocytes contains structural and DNA-binding proteins

ABSTRACTDuring the last stages of oogenesis, oocyte chromosomes condense and come close together, forming the so-called karyosphere. Karyosphere formation is accompanied by an essential decrease in transcriptional activity. In the grass frog Rana temporaria, the karyosphere is surrounded by an extrachromosomal capsule that separates the chromosomes from the rest of the nucleoplasm. The karyosphere capsule (KC) of R. temporaria has been investigated in detail at the ultrastructural level, but its protein composition remained largely unknown. We demonstrate here that nuclear actin, especially F-actin, as well as lamins A/C and B are the most abundant proteins of the KC. Key proteins of nuclear pore complexes, such as Nup93 and Nup35, are also detectable in the KC. New antibodies recognizing the telomere-binding protein TRF2 allowed us to localize TRF2 in nuclear speckles. We also found that the R. temporaria KC contains some proteins involved in chromatin remodeling, including topoisomerase II and ATRX. Thus, we believe that KC isolates the chromosomes from the rest of the nucleoplasm during the final period of oocyte growth (late diplotene) and represents a specialized oocyte nuclear compartment to store a variety of factors involved in nuclear metabolism that can be used in future early development.Abbreviations: BrUTP: 5-bromouridine 5’-triphosphate; CytD: cytochalasin D; IGCs: interchromatin granule clasters; IgG: immunoglobulin G; KC: karyosphere capsule; Mw: molecular weight; NE: nuclear envelope; PBS: phosphate buffered saline; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; Topo II: topoisomerase II

Abstract 1469: T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of T-oligo in cancer cells remains elusive. Recent studies confirmed that T-oligo can form intermolecular G-quadruplexes (G4), which are stabilized by hydrogen bonding of guanine residues, and cause disruption of the protective shelterin complex of telomeres. We hypothesize that single-stranded (SS) T-oligo and G4 T-oligo may modulate the shelterin proteins TRF2 and POT2 and thus induce DDRs. In this study, we utilized a pull-down assay using T-oligo, showing that T-oligo is co-localized with TRF2 and POT1 and indicating that T-oligo may interact with these telomeric proteins. We further investigated the modulation of these proteins by western blotting, showing that T-oligo treatment upregulates TRF2 by 2.2 and 3.0-fold (p<0.01) at 48h and 72h, respectively, and POT1 by 3.0-fold (p<0.02) both at 48h and 72h in melanoma cells (MM-AN). Immunofluorescence studies confirmed upregulation of TRF2 (2.4-fold) and POT1 (2.0-fold). Additionally we found that T-oligo can co-localize with telomere binding proteins TRF2 (88.4±4.5%) and POT1 (84.5±8%) using immunofluorescence. Using qRT-PCR, we found that T-oligo inhibited mRNA expression of hTERT, a catalytic subunit of telomerase, by 50%. It has been suggested that JNK activation may lead to downregulation of hTERT, hence we investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. Recently, it has been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. Furthermore, this study aims to investigate the potential of 6-thio-dG in combination with T-oligo as an anticancer therapeutic. We first studied the concentrations of 6-thio-dG that are effective in inhibiting the growth of melanoma cells. We found that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6 and 3.8-fold, respectively (p<0.01). However, treatment of melanoma cells in combination with both 10μM T-oligo and 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone. T-oligo may downregulate hTERT, which is required for telomerase activity, and/or disrupt the shelterin complex, both of which are necessary for 6-thio-dG mediated inhibition of growth of melanoma cells. These results indicate that T-oligo and 6-thio-dG may induce their effects by a similar mechanism of action. Citation Format: Nabiha Haleema Khan, Zachary Schrank, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri, Gagan Chhabra. T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1469.

Inhibition of TRF1 Telomere Protein Impairs Tumor Initiation and Progression in Glioblastoma Mouse Models and Patient-Derived Xenografts

Glioblastoma multiforme (GBM) is a deadly and common brain tumor. Poor prognosis is linked to high proliferation and cell heterogeneity, including glioma stem cells (GSCs). Telomere genes are frequently mutated. The telomere binding protein TRF1 is essential for telomere protection, and for adult and pluripotent stem cells. Here, we find TRF1 upregulation in mouse and human GBM. Brain-specific Trf1 genetic deletion in GBM mouse models inhibited GBM initiation and progression, increasing survival. Trf1 deletion increased telomeric DNA damage and reduced proliferation and stemness. TRF1 chemical inhibitors mimicked these effects in human GBM cells and also blocked tumor sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Thus, targeting telomeres throughout TRF1 inhibition is an effective therapeutic strategy for GBM.

Distinct TERB1 Domains Regulate Different Protein Interactions in Meiotic Telomere Movement

Meiotic telomeres attach to the nuclear envelope (NE) and drive the chromosome movement required for the pairing of homologous chromosomes. The meiosis-specific telomere proteins TERB1, TERB2, and MAJIN are required to regulate these events, but their assembly processes are largely unknown. Here, we developed a germ-cell-specific knockout mouse of the canonical telomere-binding protein TRF1 and revealed an essential role for TRF1 in directing the assembly of TERB1-TERB2-MAJIN. Further, we identified a TERB2 binding (T2B) domain in TERB1 that is dispensable for the TRF1-TERB1 interaction but is essential for the subsequent TERB1-TERB2 interaction and therefore for telomere attachment to the NE. Meanwhile, cohesin recruitment at telomeres, which is required for efficient telomere movement, is mediated by the MYB-like domain of TERB1, but not by TERB2-MAJIN. Our results reveal distinct protein interactions through various domains of TERB1, which enable the sequential assembly of the meiotic telomere complex for their movements.

Abstract 4128: Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of action of T-oligo in cancer cells is largely unknown. Recent studies suggest that GROs can form G-quadruplexes (G4) which are stabilized by the hydrogen-bonding of guanine residues. This study aims to examine the G4-forming capabilities of T-oligo in vitro and investigates the molecular mechanism of single-stranded (SS) and G4-T-oligo induced DDRs in melanoma cells (MM-AN). G4-formation by T-oligo was confirmed using the SS-T-oligo and G4-T-oligo on a polyacrylamide gel under non-denaturing conditions. NMR studies for T-oligo in KCl confirmed that T-oligo forms G4 structures. Immunofluorescence studies conducted with an anti-G-quadruplex antibody (BG4), a G4 detecting antibody, showed 88.4% co-localization of T-oligo and BG4 in the nuclei of melanoma cells confirming the ability of T-oligo to form G-quadruplexes inside melanoma cells. While G4-T-oligo was found more stable in nuclease degradation assay by DNase I, it had a decreased anti-proliferative effects compared to SS-T-oligo. However, G4-T-oligo had similar cellular uptake as SS-T-oligo. Further, two shelterin complex proteins TRF2 and POT1 which are mainly found at the telomere ends were found to be upregulated (2.0 fold) by T-oligo suggesting TRF2 and POT1 mediated telomere overhang dissociation. We also found that T-oligo can co-localize with telomere binding proteins TRF2 and POT1 by 88.4±4.5% (n=12) and 84.5±8% (n=10) respectively. Western blot analysis results also showed upregulation of both p-JNK and total JNK by 4.0- and 2.0-fold respectively. To further confirm the involvement of p-JNK in T-oligo mediated apoptosis we used a specific JNK inhibitor SP600125. Western blot analysis showed that T-oligo mediated upregulation of p-JNK was reversed in presence of SP600125. Results from an MTT assay showed a 73.8% decrease in cell viability after T-oligo treatment alone; however, cell viability was decreased to 45.8%, and 25.3% when SP600125 was present at concentrations of 10 μM, and 12 μM respectively, in comparison to diluent. T-oligo also inhibited mRNA expression of hTERT; a catalytic subunit of telomerase by 50% .We further investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. In conclusion, these studies demonstrate that T-oligo can form G-quadruplexes and the anti-proliferative mechanism of T-oligo may be mediated through POT1 and TRF2 as well as via JNK-activation inducing hTERT-inhibition in melanoma cells. Citation Format: Gagan Chhabra, Luke Wojdyla, Ankita Sanjali, Mark Frakes, Marko Ivancich, Pooja Vinay, Zachary Schrank, Benjamin E. Ramirez, Neelu Puri. Mechanism of action of G-quadruplex forming oligonucleotide homologous to the telomere overhang in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2017-4128

The telomere-binding protein TRF2 is required for metronomic therapeutic effects of gemcitabine and capecitabine

Gemcitabine and capecitabine are two effective anticancer agents against solid tumors. The pharmacological mechanisms have been known as incorporation into DNA and thereby inhibition of DNA synthesis. When used as metronomic chemotherapy, they may inhibit angiogenesis and induce immunity. In our previous study, we showed that low-dose gemcitabine caused telomere shortening by stabilizing TRF2 that was required for XPF-dependent telomere loss. In this report, we established a SKOV3.ip1 ascites cell model. Tumor-bearing mice were treated with low-dose gemcitabine (GEM) or capecitabine (CAP). Both GEM and CAP caused telomere shortening and increased expression of TRF2 with improved ascites in nude mice and decreased in vitro clonogenic activity. TRF2 knockdown altered telomeres to a shortened but new status that may evade XPF-dependent telomere loss and conferred resistance of SKOV3.ip1 ascites cells to low-dose GEM and CAP. Our study provides a new mechanism of metronomic chemotherapy i.e. TRF2 is required for metronomic therapeutic effects of gemcitabine and capecitabine.

Expression of Telomere Repeat Binding Factor 1 and TRF2 in Prostate Cancer and Correlation with Clinical Parameters.

Objective The objective of this study was to investigate the expression of telomere repeat binding factor 1 (TRF1) and TRF2 in prostate cancer and their relationships with clinicopathological features. Methods In total 50 prostate cancer tissues and paired benign prostate hyperplasia tissues were analyzed. The telomere-binding proteins TRF1 and TRF2 were measured using immunohistochemical method. Correlation analyses were used to evaluate the association between immunohistochemical score and clinical parameters. Results The expression of TRF1 was significantly higher in prostate cancer tissue than in benign prostate hyperplasia tissue (χ2 = 62.69, P < 0.01). Elevated levels of TRF2 were observed in both prostate cancer and benign prostate hyperplasia tissue (χ2 = 1.13, P = 0.76). TRF1 expression was significantly positively correlated with surgical capsular invasion (Spearman's r = 0.43, P = 0.002), seminal vesicle invasion (Spearman's r = 0.35, P = 0.01), lymph nodes metastases (Spearman's r = 0.41, P = 0.003), total prostate specific antigen (r = 0.61, P < 0.05), and Gleason score (r = 0.47, P = 0.01). However, there were no significant statistical differences between prostate volume (r = 0.06, P = 0.75) and age (r = 0.14, P = 0.09). Conclusion Both TRF1 and TRF2 were overexpressed in prostate cancer. There was no specificity of TRF2 in prostate cancer, while TRF1 may be associated with prostate cancer progression.

Nucleolus-like body of mouse oocytes contains lamin A and B and TRF2 but not actin and topo II.

BackgroundDuring the final stages of oocyte development, all chromosomes join in a limited nuclear volume for the final formation of a single complex chromatin structure – the karyosphere. In the majority of mammalian species, the chromosomes surround a round protein/fibrillar body known as the central body, or nucleolus-like body (NLB). Nothing seems to unite the inner portion of the karyosphere with the nucleolus except position at its remnants. Nevertheless, in this study we will use term NLB as the conventional one for karyosphere with the central body. At the morphological level, NLBs consist of tightly-packed fibres of 6–10 nm. The biochemical structure of this dense, compact NLB fibre centre remains uncertain.ResultsThe aim of this study was to determine which proteins represent the NLB components at final stages of karyosphere formation in mouse oogenesis. To determine this, three antibodies (ABs) have been examined against different actin epitopes. Examination of both ABs against the actin N-end provided similar results: spots inside the nucleus. Double staining with AB against SC35 and actin revealed the colocalization of these proteins in IGCs (interchromatin granule clusters/nuclear speckles/SC35 domains). In contrast, examination of polyclonal AB against peptide at the C-end reveals a different result: actin is localized exclusively in connection with the chromatin. Surprisingly, no forms of actin or topoisomerase II are present as components of the NLB. It was discovered that: (1) lamin B is an NLB component from the beginning of NLB formation, and a major portion of it resides in the NLB at the end of oocyte development; (2) lamin A undergoes rapid movement into the NLB, and a majority of it remains in the NLB; (3) the telomere-binding protein TRF2 resides in the IGCs/nuclear speckles until the end of oocyte development, when significant part of it transfers to the NLB.ConclusionsNLBs do not contain actin or topo II. Lamin B is involved from the beginning of NLB formation. Both Lamin A and TRF2 exhibit rapid movement to the NLB at the end of oogenesis. This dynamic distribution of proteins may reflect the NLB’s role in future chromatin organization post-fertilisation.

Telomeres are partly shielded from ultraviolet-induced damage and proficient for nucleotide excision repair of photoproducts.

Ultraviolet light induces cyclobutane pyrimidine dimers (CPD) and pyrimidine(6–4)pyrimidone photoproducts, which interfere with DNA replication and transcription. Nucleotide excision repair (NER) removes these photoproducts, but whether NER functions at telomeres is unresolved. Here we use immunospot blotting to examine the efficiency of photoproduct formation and removal at telomeres purified from UVC irradiated cells at various recovery times. Telomeres exhibit approximately twofold fewer photoproducts compared with the bulk genome in cells, and telomere-binding protein TRF1 significantly reduces photoproduct formation in telomeric fragments in vitro. CPD removal from telomeres occurs 1.5-fold faster than the bulk genome, and is completed by 48 h. 6–4PP removal is rapidly completed by 6 h in both telomeres and the overall genome. A requirement for XPA protein indicates the mechanism of telomeric photoproduct removal is NER. These data provide new evidence that telomeres are partially protected from ultraviolet irradiation and that NER preserves telomere integrity.

Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination.

Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination status of the human REST4 protein in cultured cells, a neural alternative splicing isoform of REST (RE-1 silencing transcription factor), that antagonizes the repressive function of REST on neural differentiation and neuron formation. Using this protocol, we show that the telomere binding protein TRF2 stabilizes the expression of the human REST4 by inhibiting its ubiquitination. This indicates that TRF2 plays a positive role in neural differentiation (Ovando-Roche et al., 2014). This protocol is also useful for the detection of ubiquitination of other proteins of interest.

PRODUCING OF A RECOMBINANT DOMAIN OF TELOMERE-BINDING PROTEIN TRF2 AND ANTIBODIES TO IT

A nucleotide sequence encoding udTRF2 domain of the human telomere-binding protein TRF2 was cloned in the expression vector pET32a(+). The construct (pET32a-udTRF2) was introduced by transformation in a bacterial strain E. coli RosettaB- lue(DE3). The obtained E. coli RosettaBlue(DE3)-pET32a-udTRF2 strain was shown to produce the udTRF2 recombinant polypeptide. The latter was used to obtain polyclonal antibodies to the udTRF2 domain. The capacity of the antibodies to react with TRF2 in a nuclear lysate from mouse liver was shown which points out a possibility of using them for the investigation of the udTRF2 functions.

Modulation of a Specific Pattern of microRNAs, Including miR-29a, miR-30a and miR-34a, in Cultured Human Skin Fibroblasts, in Response to the Application of a Biofunctional Ingredient that Protects against Cellular Senescence &amp;lt;i&amp;gt;in Vitro&amp;lt;/i&amp;gt;

Skin aging is a process of structural and compositional remodeling that can be manifested by wrinkling and sagging. Remarkably, the dermis plays a dominant role in the aging process. Recent studies suggest that microRNAs are implicated in the regulation of gene expression during aging. However, studies about age-related microRNAs and how they modulate skin aging remain limited. In the present work, a complex of hydrolyzed natural yeast proteins (Saccharomyces cerevisiae) and hydrolyzed natural soya bean was developed and showed the ability to modulate the expression of telomere-binding protein TRF2, which is a key factor for telomere protection and to prevent cellular senescence in vitro and DNA damage. The aim of the study was to identify microRNAs specifically modulated after application of the ingredient complex to cultured fibroblasts, and their possible involvement in remodeling of the human extracellular matrix and fibroblast senescence. Consequently, human skin fibroblasts were cultured and treated with 1% of the ingredient complex for 48 h before analyzing microRNA modulation by RT-qPCR. The use of bioinformatics allowed us to predict the target genes for modulated microRNAs. Results show that the ingredient complex modulated a pattern of microRNAs including the down-regulation of miR-29a-3p, miR-30a-5p and miR-34a-5p, which are associated with fibroblast senescence and remodeling of the human dermal extracellular matrix. In conclusion, our results indicate that miR-29a-3p, miR-30a-5p and miR-34a-5p possibly represent key microRNAs that impact human fibroblast senescence and remodeling of the dermal extracellular matrix.