Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach

Pollock, Katie; Ranes, Michael; Collins, Ian; Guettler, Sebastian

doi:10.1007/978-1-4939-6993-7_28

Cited by 14 publications

(25 citation statements)

References 39 publications

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“…Disruption of this interaction by mutation of a glycine in the TBM in axin abolishes proteasomal-mediated degradation of axin and ADP-ribosylation by PARP5A 73 . Sequence alignments demonstrate that TBMs are found in many proteins, of which several have been validated as targets of PARP5A/B 74 .…”

Section: Target Recognition and Amino Acid Selectionmentioning

confidence: 99%

Insights into the biogenesis, function, and regulation of ADP-ribosylation

2018

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ADP-ribosylation—the transfer of ADP-ribose (ADPr) from NAD+ onto target molecules—is catalyzed by members of the ADP-ribosyltransferase (ART) superfamily of proteins, found in all kingdoms of life. Modification of amino acids in protein targets by ADPr regulates critical cellular pathways in eukaryotes and underlies the pathogenicity of certain bacteria. Several members of the ART superfamily are highly relevant for disease; these include the poly(ADP-ribose) polymerases (PARPs), recently shown to be important cancer targets, and the bacterial toxins diphtheria toxin and cholera toxin, long known to be responsible for the symptoms of diphtheria and cholera that result in morbidity. In this Review, we discuss the functions of amino acid ADPr modifications and the ART proteins that make them, the nature of the chemical linkage between ADPr and its targets and how this impacts function and stability, and the way that ARTs select specific amino acids in targets to modify.

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Section: Target Recognition and Amino Acid Selectionmentioning

confidence: 99%

Insights into the biogenesis, function, and regulation of ADP-ribosylation

2018

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“…Our pilot studies showed that among all TNKS2 single ARCs that we could produce recombinantly (ARCs 1, 4 and 5) 49 , ARC5 displayed the lowest melting temperature (Tm) and the largest shift in melting temperature upon addition of the 3BP2 TBM peptide (DTm) ( Figure 2A). Therefore, we chose TNKS2 ARC5 for screening by DSF, anticipating the largest signal window for measuring changes in Tm upon fragment binding.…”

Section: Primary Fragment Screening By Dsfmentioning

confidence: 99%

“…Tankyrase ARC constructs were produced as previously described (see Pollock et al, 2017 for construct details) 49 antibiotics as before. Cultures were grown at 37 °C with shaking (180 rpm) to an optical density of 0.6, measured at 600 nm.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Cells were harvested by centrifugation (4000 x g, 30 min). The pellet was stored at -80 °C until purification following the previously described method 49 .…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Tankyrase contains five N-terminal ankyrin repeat clusters (ARCs), four of which, namely ARCs 1, 2, 4 and 5, can recruit binders and substrates 4,47,48 (Figure 1A). ARCs bind conserved but degenerate six-to eight-amino-acid peptide motifs, termed the tankyrasebinding motif (TBM, consensus R-X-X-[small hydrophobic or G]-[D/E/I/P]-G-[no P]-[D/E]) 4,49 .…”

Section: Introductionmentioning

confidence: 99%

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Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

Pollock

Liu

Zaleska

et al. 2019

Preprint

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The PARP enzyme and scaffolding protein tankyrase (TNKS, TNKS2) uses its ankyrin repeat clusters (ARCs) to bind a wide range of proteins and thereby controls diverse cellular functions. A number of these are implicated in cancer-relevant processes, including Wnt/bcatenin signaling and telomere maintenance. The ARCs recognise a conserved tankyrasebinding peptide motif (TBM). All currently available tankyrase inhibitors target the catalytic domain and inhibit tankyrase's poly(ADP-ribosyl)ation function. However, there is emerging evidence that catalysis-independent "scaffolding" mechanisms contribute to tankyrase function. Here we report a fragment-based screening program against tankyrase ARC domains, using a combination of biophysical assays, including differential scanning fluorimetry (DSF) and nuclear magnetic resonance (NMR). We identify fragment molecules that will serve as starting points for the development of tankyrase substrate binding antagonists. Such compounds will enable probing the scaffolding functions of tankyrase, and may, in the future, provide potential alternative therapeutic approaches to inhibiting tankyrase activity in cancer and other conditions.

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Solution NMR assignment of the ARC4 domain of human tankyrase 2

et al. 2019

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Tankyrases are poly(ADP-ribose)polymerases (PARPs) which recognize their substrates via their ankyrin repeat cluster (ARC) domains. The human tankyrases (TNKS/TNKS2) contain five ARCs in their extensive N-terminal region; of these, four bind peptides present within tankyrase interactors and substrates. These short, linear segments, known as tankyrase-binding motifs (TBMs), contain some highly conserved features: an arginine at position 1, which occupies a predominantly acidic binding site, and a glycine at position 6 that is sandwiched between two aromatic side chains on the surface of the ARC domain. Tankyrases are involved in a multitude of biological functions, amongst them Wnt/β-catenin signaling, the maintenance of telomeres, glucose metabolism, spindle formation, the DNA damage response and Hippo signaling. As many of these are relevant to human disease, tankyrase is an important target candidate for drug development. With the emergence of non-catalytic (scaffolding) functions of tankyrase, it seems attractive to interfere with ARC function rather than the enzymatic activity of tankyrase. To study the mechanism of ARC-dependent recruitment of tankyrase binders and enable protein-observed NMR screening methods, we have as the first step obtained a full backbone and partial side chain assignment of TNKS2 ARC4. The assignment highlights some of the unusual structural features of the ARC domain.

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Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach

Cited by 14 publications

References 39 publications

Insights into the biogenesis, function, and regulation of ADP-ribosylation

Insights into the biogenesis, function, and regulation of ADP-ribosylation

Fragment-based screening identifies molecules targeting the substrate-binding ankyrin repeat domains of tankyrase

Solution NMR assignment of the ARC4 domain of human tankyrase 2

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