Structural basis of tankyrase activation by polymerization.

The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT-ß-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3-5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT-ß-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.

Transmembrane and coiled-coil 2 associates with Alzheimer's disease pathology in the human brain.

Transmembrane and coiled-coil 2 (TMCC2) is a human orthologue of the Drosophila gene dementin, mutant alleles of which cause neurodegeneration with features of Alzheimer's disease (AD). TMCC2 and Dementin further have an evolutionarily conserved interaction with the amyloid protein precursor (APP), a protein central to AD pathogenesis. To investigate if human TMCC2 might also participate in mechanisms of neurodegeneration, we examined TMCC2 expression in late onset AD human brain and age-matched controls, familial AD cases bearing a mutation in APP Val717, and Down syndrome AD. Consistent with previous observations of complex formation between TMCC2 and APP in the rat brain, the dual immunocytochemistry of control human temporal cortex showed highly similar distributions of TMCC2 and APP. In late onset AD cases stratified by APOE genotype, TMCC2 immunoreactivity was associated with dense core senile plaques and adjacent neuronal dystrophies, but not with Aß surrounding the core, diffuse Aß plaques or tauopathy. In Down syndrome AD, we observed in addition TMCC2-immunoreactive and methoxy-X04-positive pathological features that were morphologically distinct from those seen in the late onset and familial AD cases, suggesting enhanced pathological alteration of TMCC2 in Down syndrome AD. At the protein level, western blots of human brain extracts revealed that human brain-derived TMCC2 exists as at least three isoforms, the relative abundance of which varied between the temporal gyrus and cerebellum and was influenced by APOE and/or dementia status. Our findings thus implicate human TMCC2 in AD via its interactions with APP, its association with dense core plaques, as well as its alteration in Down syndrome AD.

Neurodegeneration in a Drosophila model for the function of TMCC2, an amyloid protein precursor-interacting and apolipoprotein E-binding protein.

We previously identified TMCC2 as a protein that interacted differentially with normal versus Alzheimer's disease-risk forms of both apolipoprotein E (apoE) and the amyloid protein precursor (APP). We hypothesized that disrupted function of TMCC2 would affect neurodegeneration. To test this hypothesis, we investigated the Drosophila orthologue of TMCC2, that we have named Dementin. We showed that Dementin interacts genetically both with human APP and its Drosophila orthologue, the APP-like protein (APPL). Ectopic expression of Dementin in Drosophila rescued developmental and behavioral defects caused by expression of human APP. Both a hypomorphic lethal mutation in the dementin gene (dmtn(1)) and RNAi for Dementin caused the accumulation of fragments derived from APPL. We found that Dementin was required for normal development of the brain, and that glial Dementin was required for development of the Drosophila medulla neuropil. Expression of wild-type Dementin in either the neurons or glia of dmtn(1) flies rescued developmental lethality. Adult dmtn(1) flies rescued by expression of wild-type Dementin in glia, i.e. whose neurons expressed only dmtn(1), showed pathological features resembling early onset Alzheimer's disease, accumulation of abnormal APPL metabolites, synaptic pathology, mis-localized microtubule-binding proteins, neurodegeneration, and early death.

The impact of a novel apolipoprotein E and amyloid-ß protein precursor-interacting protein on the production of amyloid-ß.

Alzheimer's disease (AD) is characterized by disrupted metabolism of the amyloid-ß protein precursor (AßPP) and deposition of a byproduct, the amyloid-ß (Aß) peptide, into plaques. AD is also genetically linked to the gene for apolipoprotein E (apoE). We have identified a novel apoE-binding protein (TMCC2) that also forms a complex with AßPP. TMCC2 is a neuronal, predominantly ER-localized, protein that co-migrated with AßPP during native gel electrophoresis of rat brain extracts, and co-immunoprecipitated with AßPP from transfected human cell lysates. TMCC2 bound apoE in an isoform-specific manner in vitro and co-immunoprecipitated with apoE from cell lysates. Co-expression of apoE and TMCC2 stimulated Aß production from the "Swedish" variant of AßPP (K595 M/N596L) by up to 1.5-fold (p < 0.05), and also from the 99-amino acid C-terminal fragment of AßPP (AßPP-C99) that is the direct precursor to Aß by 1.5- to 2-fold (p < 0.0005), this effect was greater with apoE4 than apoE3 (p = 0.02); both apoE3 and apoE4 stimulated a greater increase in Aß1-42 than Aß1-40 production from AßPP-C99 in the presence of TMCC2. The interaction between TMCC2 and apoE may therefore contribute to disrupted AßPP metabolism and altered Aß production, as observed in AD.

Evidence for differential effects of apoE3 and apoE4 on HDL metabolism.

We present a murine model that examines the effects of macrophage-produced apolipoprotein E3 (apoE3) and apoE4 on VLDL and high density lipoprotein (HDL) metabolism. Mice expressing apoE3 on the Apoe(-/-) background had substantially lower VLDL levels than mice expressing apoE4. In addition, there were differences between the HDL of apoE3- and apoE4-expressing mice. Apoe(-/-) mice have low levels of HDL. Low level expression of either apoE3 or apoE4 was able to restore near-normal HDL levels, which increased dramatically when the mice were challenged with a high-fat diet. ApoE4-expressing mice had smaller HDL than apoE3-expressing mice on both chow and high-fat diets. In addition, plasma from apoE4-expressing mice was less efficient at transferring apoA-I from VLDL to HDL and at generating HDL in vitro than that from apoE3-expressing mice. Thus, we present experimental evidence for differential effects of apoE3 and apoE4 on HDL metabolism that supports epidemiological observations made in humans, which suggested that individual homozygous for the epsilon 4 allele had lower HDL than others.