Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3′ ssDNA extension of human telomeres. POT1–TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1–TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1–TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1–TPP1, in contrast to other single amino acid mutations. Chimeric human–fish telomerases that contained the human TEN domain were active but not stimulated by POT1–TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A–mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1–POT1–telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.     

CTLA4—lg融合蛋白基因的表达及其生物学功能的初步研究

获得ＣＴＬＡ４全长基因并在真核表达系统中表达。方法通过ＲＴ－ＰＣＲ方法，从ＭＴ２细胞系克隆ＣＴ－ＬＡ４全长基因；经直物重叠和半巢式ＰＣＲ改造，获得含抑瘤素Ｍ信号肽的ＣＴＬＡ４胞外段ＤＮＡ，然后克隆至真核表达载体ｐＩｇ，转染ＣＯＳ和ＣＨＯ－Ｋ细胞表达。结论真核表达体系表达了有生物学活性的ＣＴＬＡ４－Ｉｇ。     

Pdx1与NeuroD1联合诱导L02细胞Nkx6.1和GLUT2的表达

目的：应用分子生物学技术,构建胰腺十二指肠同源框蛋白1(Pdx1)和神经分化因子1(NeuroD1)转录因子真核表达质粒,检测其能否在真核细胞中有效表达并诱导人肝细胞转分化的能力。方法:以人胚胎胰腺组织mRNA为模板经RT-PCR扩增获得Pdx1和NeuroD1基因,分别克隆到真核双表达载体pIRES质粒的多克隆位点A(MCSA)和B(MCSB)中,构建pI/Pdx1/NeuroD1真核表达质粒,并转染L02细胞。用RT-PCR、免疫组化、间接荧光法和Western blotting检测目的基因及NK转录因子相关的Nkx6.1(Nkx6.1)和葡萄糖运载体2(GLUT2)的表达情况。结果:目的基因克隆正确且在真核细胞中有效表达;并可诱导肝细胞表达胰腺β细胞功能相关的Nkx6.1和GLUT2因子。结论:pI/Pdx1/NeuroD1质粒成功构建和在人真核细胞表达,并诱导人肝细胞表达β细胞功能相关的Nkx6.1转录因子和GLUT2,提示Pdx1与NeuroD1可诱导肝细胞向胰腺内分泌细胞分化。     

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.     

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.     

A POT1 mutation implicates defective telomere end fill-in and telomere truncations in Coats plus

Coats plus (CP) can be caused by mutations in the CTC1 component of CST, which promotes polymerase α (polα)/primase-dependent fill-in throughout the genome and at telomeres. The cellular pathology relating to CP has not been established. We identified a homozygous POT1 S322L substitution (POT1^CP) in two siblings with CP. POT1^CP induced a proliferative arrest that could be bypassed by telomerase. POT1^CP was expressed at normal levels, bound TPP1 and telomeres, and blocked ATR signaling. POT1^CP was defective in regulating telomerase, leading to telomere elongation rather than the telomere shortening observed in other telomeropathies. POT1^CP was also defective in the maintenance of the telomeric C strand, causing extended 3′ overhangs and stochastic telomere truncations that could be healed by telomerase. Consistent with shortening of the telomeric C strand, metaphase chromosomes showed loss of telomeres synthesized by leading strand DNA synthesis. We propose that CP is caused by a defect in POT1/CST-dependent telomere fill-in. We further propose that deficiency in the fill-in step generates truncated telomeres that halt proliferation in cells lacking telomerase, whereas, in tissues expressing telomerase (e.g., bone marrow), the truncations are healed. The proposed etiology can explain why CP presents with features distinct from those associated with telomerase defects (e.g., dyskeratosis congenita).     

NEK6‐mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment

Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB‐fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6‐mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB‐fold domains. Ser255 is located in the intervening region between the telomerase‐recruiting OB‐fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB‐fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment.     

Human POT1 is required for efficient telomere C-rich strand replication in the absence of WRN

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.     

Characterization of POT1 tumor predisposition syndrome: Tumor prevalence in a clinically diverse hereditary cancer cohort

PurposeGermline variants in POT1 have been implicated in predisposition to melanoma, sarcoma, and glioma in limited studies. Here, we determine the prevalence of cancer types in individuals with POT1 pathogenic variants (PVs) undergoing multigene panel testing (MGPT) for a broad variety of cancer indications.MethodsWe performed a retrospective review of data provided on clinical documents from individuals with POT1 PVs identified via MGPT over a 5-year period. Tumor prevalence in POT1 PV heterozygotes was compared with MGPT-negative wild-type (WT) controls using χ² test.ResultsPOT1 PVs were identified in 227 individuals. POT1 PV and WT (n = 13,315) cohorts had a similar proportion of reported tumors (69.6% and 69.2%, respectively); however, POT1 PV heterozygotes were more likely to be diagnosed with multiple tumors (18.9% vs 8.7%; P < .001). Compared with POT1 WT, we identified a significant increase in melanoma (odds ratio 7.03; 95% CI 4.7-10.5; P < .001) and sarcoma (odds ratio 6.6; 95% CI 3.1-13.9; P < .001).ConclusionThis analysis of the largest POT1 PV cohort to date validates the inclusion of POT1 in hereditary cancer MGPT and has the potential to impact clinical management recommendations, particularly for patients and families at risk for melanoma and sarcoma.     

Teaching problem-oriented therapy

The therapeutic concept is problem- and patient-oriented. In analogy to it the didactic concept is problem- and therapist-oriented. The essential point is learning in groups from practical cases. Problem-oriented in this context means that the problems-solving process in which the patient and the therapist are engaged is supported by the observer group. Therapist-oriented means that the learning process should take account of the different preferences and experiences of the therapists. That can only be accomplished by problem-oriented learning. Each psychotherapy training group consists of 4 assistant doctors and a supervisor and stays together about 1 year. The strongly structured concept of problem-oriented therapy (POT) [Blaser et al., 1988] offers the beginner a framework by which he can guide the dialogue with the patient. The eclecticism of POT allows the more experienced therapist to try out new methods without losing track of the problem. The constantly changing role of being observer or therapist supports the group coherence, and furthermore it promotes an important element of therapeutic competence, the ability to get into a close relation with the patient and at the same time being able to observe oneself, the patient and the therapeutic process from a more distant view. In addition to the POT training group tutorials in special psychotherapy methods and single supervision sessions are offered.     

介绍一种改进的、简便的凝胶迁移滞留试验法

目的 :改进凝胶迁移滞留试验 (EMSA)方法 ,运用该法检测新蛋白。方法 :抽提与定量核蛋白、DNA探针标记与纯化 ,探针与蛋白质的结合、电泳及放射自显影。结果 :采用本法简便、节约、灵敏度高、图像清晰、有可比性及重复性 ,成功运用于NF-κB及POT蛋白的检测。结论 :此法在本实验组运用过 ,可靠 ,值得应用与推广。     

Relations between psychometric profiles and cardiovascular autonomic regulation in physical education students

This study was designed to investigate physical education (PE) students the link between mood disturbances, caused by psychological or physical stressors associated with studying, and the autonomic nervous system modifications. PE students completed the profile of mood state (POMS) questionnaire at the end of the university year. Heart rate variability (HRV) was then measured during a head-up tilt test (HUT) in those with the highest and lowest total mood disturbance (TMD) scores on three successive POMS. Among the 218 students who completed the POMS (85 female and 137 male), 65 had high TMD scores, suggesting mood disturbances and fatigue. The final sample included 12 subjects in the potentially overtrained (POT) group and 16 subjects in the control (CTL) group. A greater decrease of two indices of the autonomic system (SD1 and RMSSD) was observed during the HUT in the POT than in the CTL group (P < 0.05). The depression (Dep) and vigor (Vig) subscales of POMS were correlated with several HRV indices. More specifically, in the POT group, the Vig score was correlated with autonomous activity in the supine position, and the Dep score with percentages of change of sympatho-vagal activity during the HUT. This suggests that (1) POT students could present a weaker autonomic response to HUT, (2) Dep and Vig subscales of the POMS questionnaire may indicate autonomic dysregulations.     

Mutations in POT1 predispose to familial cutaneous malignant melanoma

POT1 loss‐of‐function variants predispose to familial melanoma Robles‐Espinoza et al. (2014) Nat Genet 46(5):478–481 Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma Shi et al. (2014) Nat Genet 46(5):482–486     

Theory and technique of problem-oriented therapy

Problem-oriented therapy (POT) features the following essentials: problem orientation, eclecticism, patient orientation and brevity. These ingredients, above all the eclecticism, are detailed: diverse psychotherapeutic interventions are administered to foster awareness, modification of thought patterns, modification of behavior, emotionality, awareness of the body, ability to relax, and support. The required abilities of the therapist are mainly flexibility and transparency of his therapeutic actions. The definition of the problem which is negotiated together with the patient is decisive for the selection of the intervention strategy. An example illustrates the use of POT especially as in contrast to school-consistent traditional therapies.     

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.     

Entrainment of the rat's activity rhythm by cyclic light following lateral geniculate nucleus lesions.

The rhythmic running-wheel activity of rats is entrained by sensory input via the visual system. The retinal projection transmitting visual information essential for entrainment of the activity rhythm is unknown. Lesions of the lateral geniculate nucleus (LGN) disrupt two projections, the primary optic tract (POT) and the superior accessory optic tract (AOT-SF). It was found that activity remained entrained to a light cycle following LGN lesions. This indicates that the POT and the AOT-SF are not necessary for entrainment. The two remaining projections, the retinohypothalamic pathway and the inferior accessory optic tract (AOT-IF), are sufficient to maintain entrainment. In light of other investigations, it is suggested that the retinohypothalamic pathway is the key projection for synchrony of the activity rhythm with environmental illumination. It was also found that LGN lesions severely impaired the subjects' ability to learn a brightness discrimination.     

Primary orthostatic tremor: analysis of three cases

Primary orthostatic tremor (POT) is a rare form of tremor characterized by unsteadiness and quivering of lower extremities while standing. These symptoms relieve when sitting or reclining It is much less apparent when leaning against an object or during walking. The rhythmic tremor activity with a frequency of 13-18 Hz can be obtained electromyography recordings. Here we report three cases that have typical clinical and electrophysiological findings of POT.     

酵母三杂交系统:一种在真核细胞水平研究RNA-蛋白质相互作用的有效方法

RNA-蛋白质之间的相互作用是真核细胞RNA在细胞内执行其功能的基础,这种相互作用在基因表达的多个水平上均发挥着重要的调控作用。要研究RNA对基因表达的调控就必须对RNA-蛋白质之间相互作用的方式进行研究。本文介绍的酵母三杂交系统(yeast three-hybrid system)是一种近年来在酵母双杂交系统的基础上发展而来的,可用于鉴定先前未知的,在自然界中存在且具有生物学意义的RNA-蛋白质相互作用的实验方法。该方法提供了一个在真核细胞内研究RNA-蛋白质相互作用的技术平台。它可以根据酵母细胞所表现出的简单表型及易于辨别的酶反应,对体内复杂环境中发生的RNA-蛋白质的相互作用进行遗传学分析。     

Shelterin: the protein complex that shapes and safeguards human telomeres

Added by telomerase, arrays of TTAGGG repeats specify the ends of human chromosomes. A complex formed by six telomere-specific proteins associates with this sequence and protects chromosome ends. By analogy to other chromosomal protein complexes such as condensin and cohesin, I will refer to this complex as shelterin. Three shelterin subunits, TRF1, TRF2, and POT1 directly recognize TTAGGG repeats. They are interconnected by three additional shelterin proteins, TIN2, TPP1, and Rap1, forming a complex that allows cells to distinguish telomeres from sites of DNA damage. Without the protective activity of shelterin, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways. How does shelterin avert these events? The current data argue that shelterin is not a static structural component of the telomere. Instead, shelterin is emerging as a protein complex with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. Six shelterin subunits: TRF1, TRF2, TIN2, Rap1, TPP1, and POT1.     

Oral administration of proteinase inhibitor II from potatoes reduces energy intake in man

The effect of a proteinase inhibitor extracted from potatoes (POT II) which increases CCK release, on food intake was examined in 11 lean subjects. They received 1.5 g POT II in a high-protein soup vehicle (70 kcal), the soup vehicle alone, or a no-soup control five minutes before being presented with a lunchtime test meal, according to a double-blind, within-subjects design. Consuming the soup alone led to a nonsignificant 3% reduction in energy intake. The addition of 1.5 g POT II to the soup significantly reduced energy intake by a further 17.5%. Premeal ratings of motivation to eat and food preferences did not predict the reduction in energy intake by the proteinase inhibitor. These findings suggest that endogenous CCK may be important in the control of food intake and that proteinase inhibition may have therapeutic potential for reducing food intake.