SummaryTAp63α, a homolog of the p53 tumor suppressor, is a quality control factor in the female germline. Remarkably, already undamaged oocytes express high levels of the protein, suggesting that TAp63α's activity is under tight control of an inhibitory mechanism. Biochemical studies have proposed that inhibition requires the C-terminal transactivation inhibitory domain. However, the structural mechanism of TAp63α inhibition remains unknown. Here, we show that TAp63α is kept in an inactive dimeric state. We reveal that relief of inhibition leads to tetramer formation with ∼20-fold higher DNA affinity. In vivo, phosphorylation-triggered tetramerization of TAp63α is not reversible by dephosphorylation. Furthermore, we show that a helix in the oligomerization domain of p63 is crucial for tetramer stabilization and competes with the transactivation domain for the same binding site. Our results demonstrate how TAp63α is inhibited by complex domain-domain interactions that provide the basis for regulating quality control in oocytes.

Mitochondrial cristae morphology is highly variable and altered under numerous pathological conditions. The protein complexes involved are largely unknown or only insufficiently characterized. Using complexome profiling we identified apolipoprotein O (APOO) and apolipoprotein O-like protein (APOOL) as putative components of the Mitofilin/MINOS protein complex which was recently implicated in determining cristae morphology. We show that APOOL is a mitochondrial membrane protein facing the intermembrane space. It specifically binds to cardiolipin in vitro but not to the precursor lipid phosphatidylglycerol. Overexpression of APOOL led to fragmentation of mitochondria, a reduced basal oxygen consumption rate, and altered cristae morphology. Downregulation of APOOL impaired mitochondrial respiration and caused major alterations in cristae morphology. We further show that APOOL physically interacts with several subunits of the MINOS complex, namely Mitofilin, MINOS1, and SAMM50. We conclude that APOOL is a cardiolipin-binding component of the Mitofilin/MINOS protein complex determining cristae morphology in mammalian mitochondria. Our findings further assign an intracellular role to a member of the apolipoprotein family in mammals.

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ∼13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63.

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional a-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family. p53, a well-known tumor suppressor that is mutated in more than 50% of all human tumors, induces genes leading either to cell-cycle arrest or to apoptosis. The discovery of two proteins with a high sequence identity to p53, called p63 and p73, has sparked speculations that tumor suppression is carried out by the combined action of several members of this protein family, for example, by direct interaction through heterooligomerization. Knockout mouse studies with p63 and p73, the two ancestral members, 1 have shown functional roles distinct from p53: p63 is essential for maintaining epithelial stem cells 2,3 and for protecting the genomic stability of oocytes, 4 whereas p73 is involved in neurogenesis, sensory pathways and homeostatic control. 5 p73 is further known as an important inducer of apoptosis in response to DNA damage. 6 Although only few mutations of p63 and p73 have been found in human tumors so far, overexpression of p63 is often observed in squamous cell carcinoma, 7-10 which has been shown to suppress p73-dependent apoptosis. 11 Among all p53 family members, including those from invertebrate species, the DNA binding domain is the most conserved domain, 12-15 followed by the oligomerization domain (OD), which is indispensable for the biological function of all p53 protein family members. [16][17][18] It is a structural domain that forms a tetramer, and mutations within the OD that inhibit tetramerization of p53 result in greatly reduced transcriptional activity. 19 In addition, several protein-protein interactions and posttranslational modifications require the tetrameric state as well, 16 and mutations in the OD of p53 that prevent oligomerization have been identified in human cancers. 20,21 Owing to its functional importance, the OD of p53 has been the target of several structure determination projects. 22,23 The p53 tetramer consists of a dimer of dimers, with each monomer contributing one b-strand and o...

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Chromatin remodelling precedes transcriptional and structural changes in heart failure. A body of work suggests roles for the developmental Wnt signalling pathway in cardiac remodelling. Hitherto, there is no evidence supporting a direct role of Wnt nuclear components in regulating chromatin landscapes in this process. We show that transcriptionally active, nuclear, phosphorylated(p)Ser675-β-catenin and TCF7L2 are upregulated in diseased murine and human cardiac ventricles. We report that inducible cardiomyocytes (CM)-specific pSer675-β-catenin accumulation mimics the disease situation by triggering TCF7L2 expression. This enhances active chromatin, characterized by increased H3K27ac and TCF7L2 occupancies to cardiac developmental and remodelling genes in vivo. Accordingly, transcriptomic analysis of β-catenin stabilized hearts shows a strong recapitulation of cardiac developmental processes like cell cycling and cytoskeletal remodelling. Mechanistically, TCF7L2 co-occupies distal genomic regions with cardiac transcription factors NKX2–5 and GATA4 in stabilized-β-catenin hearts. Validation assays revealed a previously unrecognized function of GATA4 as a cardiac repressor of the TCF7L2/β-catenin complex in vivo, thereby defining a transcriptional switch controlling disease progression. Conversely, preventing β-catenin activation post-pressure-overload results in a downregulation of these novel TCF7L2-targets and rescues cardiac function. Thus, we present a novel role for TCF7L2/β-catenin in CMs-specific chromatin modulation, which could be exploited for manipulating the ubiquitous Wnt pathway.

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Thermal optimization of autoclave molds is essential to increase part quality, reduce manufacturing costs and increase autoclave capacity. Previous experience-based tooling designs allowed an optimization only after the mold was manufactured and tested. Manufacturing process simulation provides the capability for virtual tooling optimization within the design phase. Thereby, the range of possible optimizations increases and the tooling cost decreases. In order to use manufacturing process simulation efficiently, fast but accurate simulation methods must be available. The so-called shift factor approach was previously presented by the authors. This paper takes up the given approach and explains different influences on mold heat-up and how they can be covered in a thermal tooling simulation on an industrial scale. Proof of the simulation accuracy under realistic manufacturing conditions is provided together with an example of its application.