Tankyrase-1 regulates RBP-mediated mRNA turnover to promote muscle fiber formation.

Poly(ADP-ribosylation) (PARylation) is a post-translational modification mediated by a subset of ADP-ribosyl transferases (ARTs). Although PARylation-inhibition based therapies are considered as an avenue to combat debilitating diseases such as cancer and myopathies, the role of this modification in physiological processes such as cell differentiation remains unclear. Here, we show that Tankyrase1 (TNKS1), a PARylating ART, plays a major role in myogenesis, a vital process known to drive muscle fiber formation and regeneration. Although all bona fide PARPs are expressed in muscle cells, experiments using siRNA-mediated knockdown or pharmacological inhibition show that TNKS1 is the enzyme responsible of catalyzing PARylation during myogenesis. Via this activity, TNKS1 controls the turnover of mRNAs encoding myogenic regulatory factors such as nucleophosmin (NPM) and myogenin. TNKS1 mediates these effects by targeting RNA-binding proteins such as Human Antigen R (HuR). HuR harbors a conserved TNKS-binding motif (TBM), the mutation of which not only prevents the association of HuR with TNKS1 and its PARylation, but also precludes HuR from regulating the turnover of NPM and myogenin mRNAs as well as from promoting myogenesis. Therefore, our data uncover a new role for TNKS1 as a key modulator of RBP-mediated post-transcriptional events required for vital processes such as myogenesis.

APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

TERRA R-loops connect and protect sister telomeres in mitosis.

Resolution of cohesion between sister telomeres in human cells depends on TRF1-mediated recruitment of the polyADP-ribosyltransferase tankyrase to telomeres. In human aged cells, due to insufficient recruitment of TRF1/tankyrase to shortened telomeres, sisters remain cohered in mitosis. This persistent cohesion plays a protective role, but the mechanism by which sisters remain cohered is not well understood. Here we show that telomere repeat-containing RNA (TERRA) holds sister telomeres together through RNA-DNA hybrid (R-loop) structures. We show that a tankyrase-interacting partner, the RNA-binding protein C19orf43, is required for repression of TERRA R-loops. Persistent telomere cohesion in C19orf43-depleted cells is counteracted by RNaseH1, confirming that RNA-DNA hybrids hold sisters together. Consistent with a protective role for persistent telomere cohesion, depletion of C19orf43 in aged cells reduces DNA damage and delays replicative senescence. We propose that the inherent inability of shortened telomeres to recruit R-loop-repressing machinery permits a controlled onset of senescence.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

MicroRNA-9-1 Attenuates Influenza A Virus Replication via Targeting Tankyrase 1.

An unstable influenza genome leads to the virus resistance to antiviral drugs that target viral proteins. Thus, identification of host factors essential for virus replication may pave the way to develop novel antiviral therapies. In this study, we investigated the roles of the poly(ADP-ribose) polymerase enzyme, tankyrase 1 (TNKS1), and the endogenous small noncoding RNA, miR-9-1, in influenza A virus (IAV) infection. Increased expression of TNKS1 was observed in IAV-infected human lung epithelial cells and mouse lungs. TNKS1 knockdown by RNA interference repressed influenza viral replication. A screen using TNKS1 3'-untranslation region (3'-UTR) reporter assays and predicted microRNAs identified that miR-9-1 targeted TNKS1. Overexpression of miR-9-1 reduced influenza viral replication in lung epithelial cells as measured by viral mRNA and protein levels as well as virus production. miR-9-1 induced type I interferon production and enhanced the phosphorylation of STAT1 in cell culture. The ectopic expression of miR-9-1 in the lungs of mice by using an adenoviral viral vector enhanced type I interferon response, inhibited viral replication, and reduced susceptibility to IAV infection. Our results indicate that miR-9-1 is an anti-influenza microRNA that targets TNKS1 and enhances cellular antiviral state.

WWOX binds MERIT40 and modulates its function in homologous recombination, implications in breast cancer.

The tumor suppressor gene WWOX is localized in an unstable chromosomal region and its expression is decreased or absent in several types of cancer. A low expression of WWOX is associated with a poor prognosis in breast cancer (BC). It has recently been shown that WWOX contributes to genome stability through its role in the DNA damage response (DDR). In breast cancer cells, WWOX inhibits homologous recombination (HR), and thus promotes the repair of DNA double-stranded breaks (DSBs) by non-homologous end joining (NHEJ). The fine-tuning modulation of HR activity is crucial. Its under or overstimulation inducing genome alterations that can induce cancer. MERIT40 is a positive regulator of the DDR. This protein is indispensable for the function of the multi-protein complex BRCA1-A, which suppresses excessive HR activity. MERIT40 also recruits Tankyrase, a positive regulator of HR, to the DSBs to stimulate DNA repair. Here, we identified MERIT40 as a new molecular partner of WWOX. We demonstrated that WWOX inhibited excessive HR activity induced by overexpression of MERIT40. We showed that WWOX impaired the MERIT40-Tankyrase interaction preventing the role of the complex on DSBs. Furthermore, we found that MERIT40 is overexpressed in BC and that this overexpression is associated to a poor prognosis. These results strongly suggest that WWOX, through its interaction with MERIT40, prevents the deleterious impact of excessive HR on BC development by inhibiting MERIT40-Tankyrase association. This inhibitory effect of WWOX would oppose MERIT40-dependent BC development.

The interplay between telomeric complex members and BCR::ABL1 oncogenic tyrosine kinase in the maintenance of telomere length in chronic myeloid leukemia.

PURPOSE: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by recurrent genetic aberration in leukemic stem cells, namely Philadelphia chromosome caused by reciprocal translocation t(9;22)(q34;q11). In our study, we analyzed the telomeric complex expression and function in the molecular pathogenesis of CML. METHODS: We employed CD34+ primary leukemic cells, comprising both leukemic stem and progenitor cell populations, isolated from peripheral blood or bone marrow of CML patients in chronic and blastic phase to analyze the telomere length and telomeric-associated proteins. RESULTS: The reduction in telomere length during disease progression was correlated with increased expression of BCR::ABL1 transcript and the dynamic changes were neither associated with the enzymatic activity of telomerase nor with gene copy number and expression of telomerase subunits. Increased expression of BCR::ABL1 was positively correlated with expression of TRF2, RAP1, TPP1, DKC1, TNKS1, and TNKS2 genes. CONCLUSIONS: The dynamics of telomere length changes in CD34+ CML cells is dependent on the expression level of BCR::ABL, which promotes the expression of certain shelterins including RAP1 and TRF2, as well as TNKS, and TNKS2, and results in telomere shortening regardless of telomerase activity. Our results may allow better understanding of the mechanisms responsible for the genomic instability of leukemic cells and CML progression.

PARsylation-mediated ubiquitylation: lessons from rare hereditary disease Cherubism.

Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway.

Effect and interaction of TNKS genetic polymorphisms and environmental factors on telomere damage in COEs-exposure workers.

Coke oven emissions (COEs) contain many carcinogenic polycyclic aromatic hydrocarbons (PAHs). Telomere damage is an early biological marker reflecting long-term COEs-exposure. Whereas, whether the genetic variations of telomere-regulated gene TNKS have an effect on the COEs-induced telomere damage is unknown. So we detected the environmental exposure levels, relative telomere length (RTL), and TNKS genetic polymorphisms among 544 COEs-exposure workers and 238 healthy participants. We found that the RTL of the wild homozygous GG genotype in rs1055328 locus was statistically shorter compared with the CG+CC genotype for the healthy participants using covariance analysis(P = 0.008). In the Generalized linear model (GLM) analysis, TNKS rs1055328 GG could accelerate telomere shortening (P = 0.011); and the interaction between TNKS rs1055328 GG and COEs-exposure had an effect on RTL (P = 0.002). In conclusion, this study was the first to discover the role of TNKS rs1055328 locus in COEs-induced telomere damage, and proved that chromosomal damage was a combined consequence of environmental and genetic factors.

An Evolutionary Perspective on the Origin, Conservation and Binding Partner Acquisition of Tankyrases.

Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species may exist. In this work, we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identify the single-celled choanoflagellates as earliest origin of tankyrases. We further show that the sequences and structural aspects of TNKSs are well-conserved even between distantly related species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners varies greatly between species, indicating that tankyrases may have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, and that their regulatory functions in cells have likely changed considerably throughout evolution.

TAB182 aggravates progression of esophageal squamous cell carcinoma by enhancing ß-catenin nuclear translocation through FHL2 dependent manner.

TAB182 (also named TNKS1BP1), a binding protein of tankyrase 1, has been found to participate in DNA repair. Our previous study has revealed the involvement of TAB182 in the radioresistance of esophageal squamous cell carcinoma (ESCC) cells. However, whether TAB182 contributes to the ESCC tumorigenesis and progression remains unclear. In this study, we found that highly expressed TAB182 is closely associated with a poor prognosis of patients with ESCC. TAB182 silencing reduced ESCC cell proliferation and invasion in vitro, tumorigenicity and metastasis in vivo. RNA-seq and IP-MS analysis revealed that TAB182 could affect the ß-catenin signaling pathway via interacting with ß-catenin. Furthermore, TAB182 prevented ß-catenin to be phosphorylated by GSK3ß and recruited four and a half of LIM-only protein 2 (FHL2), which thereby promoted ß-catenin nucleus translocation to result in activation of the downstream targets transcription in ESCC cells. Our findings demonstrate that TAB182 enhances tumorigenesis of esophageal cancer by promoting the activation of the ß-catenin signaling pathway, which provides new insights into the molecular mechanisms by which TAB182 accelerates progression of ESCC.

Stabilizing and upregulating Axin with tankyrase inhibitor reverses 5-fluorouracil chemoresistance and proliferation by targeting the WNT/caveolin-1 axis in colorectal cancer cells.

Chemoresistance is a main obstacle for colorectal cancer treatment. In this study, we evaluated the effects and mechanisms of the WNT/ß-catenin signaling pathway on the chemoresistance of SW480 and SW620 colorectal cancer cells. The activity of ß-catenin was activated/inhibited by the small molecule compound GSK-3 inhibitor 6-bromo-indirubin-3'-oxime and the tankyrase inhibitor XAV939. The downstream target genes of the WNT/ß-catenin signaling pathway were screened using a cDNA microarray and bioinformatics analysis. Apoptosis induced by 5-Fu, cell cycle distribution and expression levels of WNT/ß-catenin/TCF12/caveolin-1 and multidrug resistance proteins were examed by flow cytometry and western blot after ß-catenin activation/inhibition and caveolin-1 overexpression/interference. The effect and mechanism of XAV939 on proliferation and apoptosis induced by 5-Fu in xenograft tumors of nude mice were evaluated by immunohistochemistry and TUNEL staining. 6-Bromo-indirubin-3'-oxime treatment increased ß-catenin expression by regulating GSK-3ß phosphorylation, accompanied by upregulation of TCF12, caveolin-1, P-gp, and MRP2 and downregulation of apoptosis induced by 5-Fu. Conversely, XAV939 treatment decreased ß-catenin expression by upregulating Axin, accompanied by downregulation of TCF12, Caveolin-1, P-gp, and MRP2 and upregulation of apoptosis induced by 5-Fu. The caveolin-1 gene was identified as an important downstream gene of the WNT/ß-catenin signaling pathway. Caveolin-1 overexpression upregulated ß-catenin expression, increased P-gp and MRP2 expression and decreased apoptosis induced by 5-Fu; conversely, caveolin-1 interference caused the opposite effects. In addition, in vivo experiments showed that XAV939 treatment reduced ß-catenin expression, increased apoptosis induced by 5-Fu and repressed xenograft tumor growth. Our findings suggested that inhibition of WNT/ß-catenin/TCF12/caveolin-1 provides a new promising therapeutic strategy for colorectal cancer treatment.

Tankyrases inhibit innate antiviral response by PARylating VISA/MAVS and priming it for RNF146-mediated ubiquitination and degradation.

During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked ß-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation (PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.

Tankyrase represses autoinflammation through the attenuation of TLR2 signaling.

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response.

miR-582-5p inhibits migration and chemo-resistant capabilities of colorectal cancer cells by targeting TNKS2.

BACKGROUND: Metastasis and chemo-resistance are still important factors that limit the overall efficacy of colorectal cancer treatment. Understanding the detailed molecular mechanism and identifying potential biomarkers are of great value in prognosis prediction and risk stratification. OBJECTIVE: We investigated the role of miR-582-5p in colorectal cancer pathogenesis, progression and chemo-resistance. Furthermore, we explored the underlying molecular mechanism of miR-582-5p in modulation of malignant behaviors of colorectal cancer cells. METHODS: Clinical samples and colorectal cancer cell lines were applied to explore miR-582-5p expression level and its significance on tumor cell metastasis and chemo-resistance. Transwell study and cellular survivability study were performed to explore the influences of miR-582-5p expression modulation on tumor cell chemo-resistance and invasion/migration. Dual-luciferase reporter gene assay was conducted to explore the influences of miR-582-5p on its target gene TNKS2. RESULTS: Colorectal cancer patients with lymph node or distal organ metastatic diseases exhibited significantly lower level of miR-582-5p. In vitro studies have indicated that miR-582-5p inhibition significantly increased migration and chemo-resistant capabilities of tumor cells. And dual-luciferase reporter gene assay demonstrated that miR-582-5p exhibited its influences on the biological behavior of tumor cells by targeting TNKS2. CONCLUSIONS: Our study demonstrated for the first time that miR-582-5p played an important role for colorectal tumor cell metastasis and chemo-resistance. Our research also indicated that miR-582-5p and its target gene TNKS2 could be novel biomarkers for metastatic disease prediction, overall prognosis evaluation, as well as potential therapeutic target for colorectal cancer patients.

PARP5B is required for nonhomologous end joining during tumorigenesis in vivo.

Poly(ADP-ribose) polymerases (PARP) act as DNA damage sensors that produce poly(ADP-ribose) (PAR) chains at double-strand breaks, facilitating the recruitment of repair factors. Cancers with homologous recombination defects are sensitive to small molecule PARP inhibitors. Despite PARP5B gene copy number changes in many cancers, the effects of this genetic alteration on tumor phenotype are largely unknown. To better understand this clinical finding, we characterized a PARP5B null mutation in a carcinogen-induced in vivo head and neck squamous cell carcinoma (SCC) model. Reduced PARP5B expression inhibited tumor growth, induced primary tumor differentiation and apoptosis, and inhibited cell proliferation and metastasis. Loss of PARP5B expression-induced ataxia telangiectasia and Rad3 related (ATR) activation and depleted the cancer stem cell fraction. PARP5B null tumor cells lacked 53BP1+ double-strand break foci, ATM activation, and p53 induction compared to PARP5B+/+ cancers. PARP5B null SCC expresses a multiprotein complex containing PML, pRPA, Rad50, Rad51, XRCC1, proliferating cell nuclear antigen (PCNA), and Mcm2, suggesting an HR-mediated repair mechanism at DNA replication foci. Low doses of etoposide combined with the PARP5B inhibitor XAV939 induced senescence and apoptosis in human SCC lines. NBS1 overexpression in these cells inhibited the effects of low-dose etoposide/XAV939 treatment. Our results indicate that PARP5B inhibition is new targeted cancer therapy.

Down-regulation of hsa_circ_0045474 induces macrophage autophagy in tuberculosis via miR-582-5p/TNKS2 axis.

Macrophage autophagy plays a major role in the control and elimination of invading Mycobacterium tuberculosis. However, the function and mechanism of circRNA on macrophage autophagy in tuberculosis remain unclear. Therefore, this study aimed to explore the role of circRNA underlying macrophage autophagy in tuberculosis. Quantitative real-time polymerase chain reaction was used to detect the expression of hsa_circ_0045474, miR-582-5p and TNKS2. Autophagy was detected by LC3B immunofluorescence and transmission electron microscopy. Dual-luciferase reporter assays were used to detect the relationship of miR-582-5p and hsa_circ_0045474 or TNKS2. Western blot was used to detect the expression of LC3-І and LC3-ІІ. The results showed that hsa_circ_0045474 was down-regulated in monocytes from patients with tuberculosis and induced autophagy in macrophages. hsa_circ_0045474 sponged miR-582-5p and negatively regulated miR-582-5p expression. Overexpression of miR-582-5p affected by hsa_circ_0045474 induced autophagy in macrophages. TNKS2 served as a target of miR-582-5p and down-regulation of TNKS2 induced autophagy in macrophages regulated by miR-582-5p. In conclusion, our results demonstrated that hsa_circ_0045474 down-regulation induced macrophage autophagy in tuberculosis via miR-582-5p/ TNKS2 axis, implying a novel strategy to treat the occurrence of active pulmonary tuberculosis caused by immune escape of M. tuberculosis.

Tankyrase-1-mediated degradation of Golgin45 regulates glycosyltransferase trafficking and protein glycosylation in Rab2-GTP-dependent manner.

Altered glycosylation plays an important role during development and is also a hallmark of increased tumorigenicity and metastatic potentials of several cancers. We report here that Tankyrase-1 (TNKS1) controls protein glycosylation by Poly-ADP-ribosylation (PARylation) of a Golgi structural protein, Golgin45, at the Golgi. TNKS1 is a Golgi-localized peripheral membrane protein that plays various roles throughout the cell, ranging from telomere maintenance to Glut4 trafficking. Our study indicates that TNKS1 localization to the Golgi apparatus is mediated by Golgin45. TNKS1-dependent control of Golgin45 protein stability influences protein glycosylation, as shown by Glycomic analysis. Further, FRAP experiments indicated that Golgin45 protein level modulates Golgi glycosyltransferease trafficking in Rab2-GTP-dependent manner. Taken together, these results suggest that TNKS1-dependent regulation of Golgin45 may provide a molecular underpinning for altered glycosylation at the Golgi during development or oncogenic transformation.

Tankyrase regulates epithelial lumen formation via suppression of Rab11 GEFs.

Rab11 GTPase proteins are required for cytokinesis, ciliogenesis, and lumenogenesis. Rab11a is critical for apical delivery of podocalyxin (PODXL) during lumen formation in epithelial cells. SH3BP5 and SH3BP5L are guanine nucleotide exchange factors (GEFs) for Rab11. We show that SH3BP5 and SH3BP5L are required for activation of Rab11a and cyst lumen formation. Using proximity-dependent biotin identification (BioID) interaction proteomics, we have identified SH3BP5 and its paralogue SH3BP5L as new substrates of the poly-ADP-ribose polymerase Tankyrase and the E3 ligase RNF146. We provide data demonstrating that epithelial polarity via cyst lumen formation is governed by Tankyrase, which inhibits Rab11a activation through the suppression of SH3BP5 and SH3BP5L. RNF146 reduces Tankyrase protein abundance and restores Rab11a activation and lumen formation. Thus, Rab11a activation is controlled by a signaling pathway composed of the sequential inhibition of SH3BP5 paralogues by Tankyrase, which is itself suppressed by RNF146.

Association of relative leukocyte telomere length and genetic variants in telomere-related genes (TERT, TERT-CLPTM1, TRF1, TNKS2, TRF2) with atrophic age-related macular degeneration.

Background: In an experimental model, telomere shortening inhibits neovascularization. It is thus possible that telomere shortening might have a role in the pathogenesis of geographic atrophy in case of age-related macular degeneration (AMD). This is why we aimed to find any associated differences of telomere length and genetic variants in telomere-related genes (TERT, TERT-CLPTM1, TRF1, TNKS2, and TRF2) in patients with atrophic AMD compared to healthy controls.Methods: The study enrolled patients with atrophic AMD (n = 56) and healthy (n = 73) controls. Samples of DNA from peripheral blood leukocytes were extracted by DNA salting-out method. The genotyping of TERT rs2736098, rs401681 in TERT-CLPTM1 locus, TRF1 rs1545827, rs10107605, TNKS2 rs10509637, rs10509639, and TRF2 rs251796 and relative leukocyte telomere length (T/S) measurement were carried out using a real-time polymerase chain reaction method. The results were assessed using the statistical analysis method of "IBM SPSS Statistics 20.0".Results: We found statistically significantly higher T/S in atrophic AMD patients than in healthy controls (T/S, median (IQR): 1.638 (1.110) vs. 0.764 (0.801), p < .001). Also, statistically significant differences were found in TRF1 rs10107605 allele (A and C) distributions between the atrophic AMD and control groups (88.36% and 11.64% vs. 95.54% and 4.46%, respectively, p = .041), as well as between the short telomere and long telomere groups (86.92% and 13.08% vs. 96.09% and 3.91%, respectively, p = .008).Conclusions: Our research revealed the leukocyte telomere length having a role in atrophic AMD development, also the association between TRF1 rs10107605 and the telomere length.