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The Mechanisms of Degradation of Telomere Associated Centrosomal Protein TACP1 and Formation of ALT-associated PML Bodies

Author: YuJian
Tutor: HuangHe
School: Zhejiang University
Course: Internal Medicine
Keywords: Telomere Centrosome TACP1 yeast two-hybrid screen HECTD3 ubiquitin-proteasome system degradation ubiquitin-protein ligases (E3s) HECT E3 ubiquitin ligases TRF1 PML3 Alternative lengthening of telomeres (ALT) ALT-associated PML bodies (APBs) PML nuclear bodies (PNBs)
CLC: R730.2
Type: PhD thesis
Year: 2008
Downloads: 142
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Abstract

Telomeres are specialized protective structures at the extreme ends of linearly eukaryotic chromosomes.Telomere dysfunction is associated with chromosomal instability which is implicated in ageing and carcinogenesis.Centrosomes,the principal microtubule organizing center(MTOC)in nearly all higher eukaryotic cells, have a pivotal role in regulating cell division in mitotic cells.During mitosis, centrosomes govern assembly and orientation of the bipolar mitotic spindle that is essential for correct chromosome segregation and cytokinesis.Centrosome aberrations may compromise the fidelity of cell division and cause chromosomal instability.There is compelling evidence to suggest a strong link between telomere maintenance and cell cycle control,especially mitotic regulation.However,little is known about the identity and function of the signaling molecule(s)connecting telomere maintenance and mitotic regulation.Recent studies showed that TRF1 translocates to centrosome and/or spindle pole during mitosis and TRF1 overexpression induces mitotic entry in cells with short telomeres,suggesting the role of TRF1 in mitotic regulation by providing a functional link between telomere and centrosome.In our previous study,a novel TRF1 interacting protein was identified from mitotic HeLa cell lysates by employing immunoaffinity isolatin and mass spectrometry(MS).We refer to the protein as Telomere Associated Centrosomal Protein 1(TACP1)since it distinguishes from other TRF1 binding proteins and traffics toward centrosome during mitosis.Further studies revealed that depletion of TACP1 by small interfering RNA leads to multipolar spindle formation and abolishes the centrosomal localization of TRF1 and Tankyrase 1.To further elucidate the role of TACP1 in connecting telomere maintenance and mitotic regulation,we performed a yeast two-hybrid screen with TACP1 as a bait and identified 23 novel TACP1-binding proteins including KIAA0649 and HECTD3. Further yeast co-transformation assay and pull-dwon assay confirmed the interaction between TACP1 and HECTD3 1-75.The ubiquitin-proteasome system,the most important protein degradation pathway in eukaryotic cells,regulates a host of critical cellular functions such as cell cycle progression and apoptosis through mediating the selective and time-dependent degradation of short-lived regulatory proteins.Many centrosomal proteins are regulated in a cell cycle-dependent fashion,and most of them are degraded through ubiqultin-proteasome pathway.Our previous studies showed that TACP1,as a novel telomere associated centrosomal protein,is a cell cycle-regulated protein.To clarify the degradation mechanism of TACP1,we carded out in vivo ubiquitination assay to determine whether TACP1 is ubiquitinated in vivo.The results showed that TACP1 is ubiquitinated in vivo and treatment of cells with the proteasome inhibitor MG132 causes a robust increase of TACP1 ubuiquitination,suggesting that TACP1 is the substrate for proteasome.In the ubiquitin-proteasome system,the selectivity of substrate appears to be determined through interaction between specific ubiquitin-protein ligase(E3)and substrate.According to computational analysis,we hypothesized that HECTD3,a putative member of HECT E3 ubiquitin ligases,is a specific E3 ligase for TACP1.To confirm this hypothesis,we first validated that TACP1 interacts with HECTD3 in vitro and in vivo.Further studies showed that HECTD3 overexpression enhances the ubiquitination of TACP1 and promotes the turnover of TACP1,where depletion of HECTD3 decreases TACP1 degradation. More notably,depletion of TACP1 leads to multipolar spindle formation.All these findings suggest that HECTD3 is a specific E3 ligase for TACP1 and may facilitate cell cycle progression via regulation ubiquitination and degradation of TACP1.In normal somatic human cells,telomeres shorten by 50-150 base pairs(bp)per cell division owing to the end-replication problem of the lagging strand,which ultimately leads to replicative senescence when the telomere length reaches a critical point(4～5 kb).Therefore,telomeres have been proposed to act as a counting mechanism for cellular proliferation.To achieve unlimited replicative potential,most cancer cells activate telomerase to elongate telomeres.However,some cancer cells, including sarcomas,astrocytomas,and tumors of Li-Fraumeni syndrome,cannot activate telomerase and use telomere homologous recombination to elongate telomeres,a mechanism termed altemative lengthening of telomeres(ALT).A hallmark of ALT cells is the presence of ALT-associated PML bodies(APBs).It has been suggested that APBs may have an integral role in the ALT mechanism,but the exact mechanism of APBs formation remains unclear.Recent studies revealed that TRF1 and its sumoylation is essential for the formation of APBs,but the detailed mechanism of the recruitment of TRF1 to APBs is unknown.PML,as the most prominent component of PML nuclear bodies(PNBs),has a fundamental role in assembly of PML nuclear bodies.According to C-terminal sequence variation,seven PML isoforms have been identified.Up to date,most studies on these isoforms concentrate on PML3,which has been shown to recruit P53 to PML nuclear bodies and increase its transcriptional activity.Taking the function of PML3,we hypothesized that PML3 may assist the recruitment of TRF1 to APBs.To confirmed this,we first validated the interaction between PML3 and TRF1 by co-immunoprecipitation and GST pull-down assays. Immunofluorescence studies showed that TRF1 colocalizes with PML3 at APBs in a subset of ALT cells and the percentage of cells with co-localization of TRF1 with APBs greatly increased in cells enriched in G2/M.Further studies revealed that overexpression of PML3 strengthens the localization of TRF1 to PML nuclear bodies, while depletion of PML3 inhibits the recruitment of TRF1 and TRF2 to APBs.Taken together,our results suggest that PML3 interacts with TRF1 and is essential for APBs formation.

The Mechanisms of Degradation of Telomere Associated Centrosomal Protein TACP1 and Formation of ALT-associated PML Bodies

Abstract

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