Research Overview

A small number of key signalling pathways collaborate to confer stem-cell properties to cells, and the Wnt/beta-catenin pathway is a prototypic example for such a pathway. Wnt/beta-catenin signalling plays important roles in embryonic development, regeneration and adult organ homeostasis. It is dysregulated in a number of different cancer types, most prominently in colorectal cancers, the vast majority of which bear mutations in components of the pathway.

At the same time, stem and most cancer cells rely on active telomerase to prevent erosion of their telomeres and maintain their unlimited replicative potential. Recent findings show that Wnt/beta-catenin signalling and telomere homeostasis are closely intertwined at multiple levels and form an integrated self-renewal programme, relevant to normal tissue regeneration, ageing and cancer.

The poly-ADP-ribosyltransferase tankyrase both promotes Wnt/beta-catenin signalling and is essential for normal telomere extension in humans, thereby providing an important link between both processes.

Our overarching goal is to understand the precise molecular mechanisms that underlie Wnt/beta-catenin signalling, telomere maintenance and their control by poly(ADP-ribosyl)ation. We have a long-standing interest in deciphering the structural basis and molecular mechanisms of tankyrase function.

We take a multidisciplinary approach to study Wnt/beta-catenin signalling, telomere maintenance and their regulation by poly(ADP-ribosyl)ation. Structural biology is at the centre of our work. (Images modified from Mariotti et al., 2016)

Research Projects

Regulation and molecular mechanisms of tankyrase

ADP-ribosylation is a post-translational modification carried out by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. ADP-ribosylation controls many aspects of cell function, including DNA repair, cell division, telomere maintenance, chromatin dynamics, apoptosis and various signal transduction processes. Given their roles in DNA repair, telomere homeostasis and cancer-relevant signalling pathways, several ARTs are being explored as potential cancer therapy targets.

In humans, the family of intracellular ARTs encompasses 17 members with similar catalytic domains but greatly diverse non-catalytic accessory domains. Different catalytically active ARTs can either transfer a single unit of ADP-ribose or attach ADP-ribose processively, thereby constructing poly(ADP-ribose) (PAR) chains, which can be of varying length and structure. Enzymes in the latter group are known as poly-ADP-ribosyltransferases. Compared to other types of post-translational modification, such as phosphorylation, PARylation remains understudied.

We take a particular interest in the PARP enzyme tankyrase, which fulfils a wide range of biological functions, many of which are relevant to cancer. The human genome encodes two highly similar tankyrase paralogues, TNKS and TNKS2. Both share a C-terminal catalytic PARP domain, a set of five N-terminal ankyrin repeat clusters (ARCs) responsible for substrate recruitment, and a polymerising sterile alpha motif (SAM) domain in between.

Our previous structure-function work has revealed the mechanisms of substrate recognition and polymerisation by tankyrase and shown that tankyrase can act as a scaffolding protein, independently of its catalytic function. We now aim to use both X-ray crystallography and cryo-electron microscopy to understand how tankyrase’s various domains act together. Moreover, we work with chemists to develop novel approaches to modulate tankyrase function.

(A) Functional modules in tankyrase (TNKS, TNKS2). Tankyrase uses its ankyrin repeat clusters (ARCs) to recruit binding partners, many of which are also PARylated by tankyrase’s PARP domain. ARCs recognise degenerate peptide motifs found in many proteins. Our earlier work (Guettler et al., 2011) has revealed the substrate recognition mechanism and explained how the rare human disease Cherubism is caused. The sterile alpha motif (SAM) domain enables filamentous polymerisation of tankyrase, critical to both catalytic and non-catalytic functions of tankyrase (Mariotti, Templeton and Ranes et al., 2016; Pillay and Mariotti et al., 2022). (B) Cryo-electron microscopy revealed functionally important inter-domain contacts established through self-assembly of tankyrase into filamentous polymers (see Pillay and Mariotti et al., 2022). (Images modified from Guettler et al., 2011; Mariotti et al., 2016; Pollock et al., 2017; Pillay and Mariotti et al., 2022)

Molecular mechanisms of Wnt/beta-catenin signalling, telomere maintenance and their regulation by poly(ADP-ribosyl)ation

We take a reductionist approach to study how large macromolecular complexes coordinate Wnt/beta-catenin signalling and telomere length homeostasis and how they are controlled by tankyrase-dependent poly(ADP-ribosyl)ation. We combine biochemical assays with cryo-electron microscopy and X-ray crystallography to uncover the detailed mechanisms governing the functions of these complexes and their regulation.

Besides uncovering fundamental mechanisms underlying stem and cancer cell function, we endeavour to understand the molecular basis of disease mutations and open up new opportunities for pharmacological intervention.

We use purified proteins to reconstitute Wnt/beta-catenin signalling in vitro, interrogating signalling mechanisms at the levels of beta-catenin destruction complex assembly and its biochemical activities. The latter encompass phosphorylation, ubiquitylation and proteasomal degradation.

Top: Wnt/beta-catenin signalling revolves around controlling the levels of the transcriptional co-activator beta-catenin. A multi-protein beta-catenin destruction complex captures cytoplasmic beta-catenin and initiates its phosphorylation- and ubiquitylation-dependent proteasomal degradation. Destruction complex function is impaired in the vast majority of colorectal cancers. Wnt signals remodel the destruction complex into a membrane-localised “Wnt signalosome” incapable of destabilising beta-catenin. Tankyrase controls the receptiveness of cells to incoming Wnt signals by PARylating AXIN, thereby destabilising the destruction complex or promoting Wnt signalosome formation. (Images modified from Mariotti et al., 2017)
Bottom: The image, which was created by our multi-talented PhD students Saira Sakalas and Yexin Xie, shows armadillos (the namesakes of beta-catenin in Drosophila and the structural fold of beta-catenin) being captured by a fishing net, whose ropes signify AXIN polymers and APC in the beta-catenin destruction complex. Capturing beta-catenin/armadillo in the cytoplasm prevents its function as a transcriptional co-activator and oncogenic driver in the nucleus. — Top: Wnt/beta-catenin signalling revolves around controlling the levels of the transcriptional co-activator beta-catenin. A multi-protein beta-catenin destruction complex captures cytoplasmic beta-catenin and initiates its phosphorylation- and ubiquitylation-dependent proteasomal degradation. Destruction complex function is impaired in the vast majority of colorectal cancers. Wnt signals remodel the destruction complex into a membrane-localised “Wnt signalosome” incapable of destabilising beta-catenin. Tankyrase controls the receptiveness of cells to incoming Wnt signals by PARylating AXIN, thereby destabilising the destruction complex or promoting Wnt signalosome formation. (Images modified from Mariotti et al., 2017) Bottom: The image, which was created by our multi-talented PhD students Saira Sakalas and Yexin Xie, shows armadillos (the namesakes of beta-catenin in Drosophila and the structural fold of beta-catenin) being captured by a fishing net, whose ropes signify AXIN polymers and APC in the beta-catenin destruction complex. Capturing beta-catenin/armadillo in the cytoplasm prevents its function as a transcriptional co-activator and oncogenic driver in the nucleus.

Novel approaches to inhibit tankyrase function

Protein-observed NMR identifies the binding site of a fragment molecule to a substrate-binding ankyrin repeat cluster (ARC) of tankyrase. The binding site of the fragment overlaps with that of tankyrase binders and substrates, which are recruited to tankyrase ARCs through ARC-binding peptide motifs. (Image modified from Pollock et al., 2019)

We have observed that tankyrase can drive Wnt/beta-catenin signalling independently of its catalytic PARP activity, at least when tankyrase levels are high, and non-catalytic functions of tankyrase as a scaffolding protein are emerging. To interrogate these scaffolding functions, we collaborate with Professor Ian Collins (ICR Division of Cancer Therapeutics) to develop inhibitors of the tankyrase:substrate protein:protein interaction. Using structure-function studies, we have previously shown that the substrate-binding ankyrin repeat clusters (ARCs) of tankyrase are critical for its scaffolding activity, suggesting that targeting the ARCs by small molecules will provide a means to interrogate the non-catalytic functions of tankyrase.

Collaborators

Professor Chris Lord	The Institute of Cancer Research, London, UK
Professor Ian Collins	The Institute of Cancer Research, London, UK
Dr Frank Sicheri	The Lunenfeld-Tanenbaum Research Institute, Toronto, Canada
Dr Edward Morris	The Institute of Cancer Research, London, UK
Dr Mark Pfuhl	King's College, London, UK

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