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.

Molecular simulation approach integrated with deep learning model for the discovery of Tankyrase inhibitors

Tankyrases belong to the poly (ADP-ribose) polymerase family epitomized as a novel group of medicinal targets with various prospective diseased conditions and it is appraised to be a challenging drug target for the intervention of multiple cancers. Thus, the principal objective of our study is to explore the dual-site selective tankyrase 1 inhibitor by employing the pharmacophore strategy. Initially, the ligand-based pharmacophore study generated five featured pharmacophore hypothesis, which was then employed for the database screening. The screened molecules were scrutinized through docking, MM/GBSA calculations and molecular simulation alongside pharmacokinetics properties. The analysis yielded potent dual-site tankyrase 1 inhibitors such as nebivolol and ondansetron from the DrugBank repository. Notably, the recognized lead molecules were perceived to have higher XP GScore and binding energy scores. Subsequently, simulation studies were executed to validate the structural stability of the lead molecules. It is worth mentioning that the existence of benzopyran and carbazole scaffolds in the lead molecules displayed anti-neoplastic activity and also facilitate the effective binding with tankyrase 1 protein. Ultimately, the IC50 values of the lead molecules were examined against the NCI-H596 cell line using a deep learning model. Indeed, these results are of immense importance and provide a clue to the experimental biologist in developing a potent tankyrase 1 dual-site inhibitor.

Tankyrases as modulators of pro-tumoral functions: molecular insights and therapeutic opportunities

Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) are two homologous proteins that are gaining increasing importance due to their implication in multiple pathways and diseases such as cancer. TNKS1/2 interact with a large variety of substrates through the ankyrin (ANK) domain, which recognizes a sequence present in all the substrates of tankyrase, called Tankyrase Binding Motif (TBM). One of the main functions of tankyrases is the regulation of protein stability through the process of PARylation-dependent ubiquitination (PARdU). Nonetheless, there are other functions less studied that are also essential in order to understand the role of tankyrases in many pathways. In this review, we concentrate in different tankyrase substrates and we analyze in depth the biological consequences derived of their interaction with TNKS1/2. We also examine the concept of both canonical and non-canonical TBMs and finally, we focus on the information about the role of TNKS1/2 in different tumor context, along with the benefits and limitations of the current TNKS inhibitors targeting the catalytic PARP domain and the novel strategies to develop inhibitors against the ankyrin domain. Available data indicates the need for further deepening in the knowledge of tankyrases to elucidate and improve the current view of the role of these PARP family members and get inhibitors with a better therapeutic and safety profile.

Arpin Regulates Migration Persistence by Interacting with Both Tankyrases and the Arp2/3 Complex

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for its interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two-hybrid screening and coimmunoprecipitation with full-length Arpin as bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal-binding site on its acidic tail, which overlaps with the Arp2/3-binding site. Arpin was found to dissolve the liquid–liquid phase separation of TNKS upon overexpression. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased migration persistence of the Arpin knockout cells using random plasmid integration or compensating knock-ins at the ARPIN locus. Arpin mutations impairing interactions with either Arp2/3 or TNKS were insufficient to fully abolish Arpin activity. Only the mutation that affected both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, whereby Arpin controls the migration persistence.

Arpin regulates migration persistence by interacting with both tankyrases and the Arp2/3 complex

During cell migration, protrusion of the leading edge is driven by the polymerization of Arp2/3-dependent branched actin networks. Migration persistence is negatively regulated by the Arp2/3 inhibitory protein Arpin. To better understand Arpin regulation in the cell, we looked for interacting partners and identified both Tankyrase 1 and 2 (TNKS) using a yeast two hybrid screen and co-immunoprecipitation with full-length Arpin as a bait. Arpin interacts with ankyrin repeats of TNKS through a C-terminal binding site on its acidic tail overlapping with the Arp2/3 binding site. To uncouple the interactions of Arpin with TNKS and Arp2/3, we introduced point mutations in the Arpin tail and attempted to rescue the increased persistence of the Arpin knock-out using random plasmid integration or compensating knock-in at the ARPIN locus. Arpin mutations impairing either Arp2/3- or TNKS-interaction were insufficient to fully abolish Arpin activity. Only the mutation that affects both interactions rendered Arpin completely inactive, suggesting the existence of two independent pathways, by which Arpin controls migration persistence. Arpin was found to dissolve liquid-liquid phase separation of TNKS upon overexpression. Together these data suggest that TNKS might be mediating the function of Arpin rather than regulating Arpin.

Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death

ABSTRACTTumor necrosis factor (TNF) is an inflammatory cytokine that, upon binding to its receptor TNFR1, can drive cytokine production, cell survival, or cell death and is a major component of an organism’s anti-pathogen repetoire1,2. TNF stimulation leads to the formation of two distinct signalling complexes, a well-defined membrane bound complex (complex 1), and a less well characterised cytosolic death inducing complex (complex 2). Using mass spectrometry, we identified the ADP-ribosyltransferase, tankyrase-1 (TNKS1/TNKS/ARTD5/PARP5a) as a novel native complex 2 component. Following a TNF-induced death stimulus TNKS1 is recruited to complex 2, resulting in complex 2 poly(ADP-ribosyl)ation (PARylation). Tankyrase inhibitors sensitise cells to TNF-induced death, which is correlated with increased complex 2 assembly. Tankyrase-mediated PARylation promotes recruitment of the E3 ligase RNF146 and RNF146 deficiency or proteasome inhibition results in increased levels of complex 2, suggesting that RNF146 causes proteasomal degradation of complex 2. Several viruses express ADP-ribose binding macrodomain proteins, and expression of the SARS-CoV-2 or VEEV macrodomain markedly sensitises cells to TNF-induced death. This suggests that ADP-ribosylation serves as yet another mechanism to detect pathogenic interference of TNF signalling and retaliate with an inflammatory cell death.

TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

ABSTRACTThe shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3’ overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2’s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

Discovery of Novel Inhibitor for WNT/β-Catenin Pathway by Tankyrase 1/2 Structure-Based Virtual Screening

Aberrant activation of the WNT/β-catenin signaling pathway is implicated in various types of cancers. Inhibitors targeting the Wnt signaling pathway are intensively studied in the current cancer research field, the outcomes of which remain to be determined. In this study, we have attempted to discover novel potent WNT/β-catenin pathway inhibitors through tankyrase 1/2 structure-based virtual screening. After screening more than 13.4 million compounds through molecular docking, we experimentally verified one compound, LZZ-02, as the most potent inhibitor out of 11 structurally representative top hits. LiCl-induced HEK293 cells containing TOPFlash reporter showed that LZZ-02 inhibited the transcriptional activity of β-catenin with an IC50 of 10 ± 1.2 μM. Mechanistically, LZZ-02 degrades the expression of β-catenin by stabilizing axin 2, thereby diminishing downstream proteins levels, including c-Myc and cyclin D1. LZZ-02 also inhibits the growth of colonic carcinoma cell harboring constitutively active β-catenin. More importantly, LZZ-02 effectively shrinks tumor xenograft derived from colonic cell lines. Our study successfully identified a novel tankyrase 1/2 inhibitor and shed light on a novel strategy for developing inhibitors targeting the WNT/β-catenin signaling axis.