Paco Hulpiau

MLKL Compromises Plasma Membrane Integrity by Binding to Phosphatidylinositol Phosphates

Although mixed lineage kinase domain-like (MLKL) protein has emerged as a specific and crucial protein for necroptosis induction, how MLKL transduces the death signal remains poorly understood. Here, we demonstrate that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death. Moreover, we found that a patch of positively charged amino acids on the surface of the 4HBD binds to phosphatidylinositol phosphates (PIPs) and allows recruitment of MLKL to the plasma membrane. Importantly, we found that recombinant MLKL, but not a mutant lacking these positive charges, induces leakage of PIP-containing liposomes as potently as BAX, supporting a model in which MLKL induces necroptosis by directly permeabilizing the plasma membrane. Accordingly, we found that inhibiting the formation of PI(5)P and PI(4,5)P2 specifically inhibits tumor necrosis factor (TNF)-mediated necroptosis but not apoptosis.

Molecular evolution of the cadherin superfamily

This review deals with the large and pleiotropic superfamily of cadherins and its molecular evolution. We compiled literature data and an in-depth phylogenetic analysis of more than 350 members of this superfamily from about 30 species, covering several but not all representative branches within metazoan evolution. We analyzed the sequence homology between either ectodomains or cytoplasmic domains, and we reviewed protein structural data and genomic architecture. Cadherins and cadherin-related molecules are defined by having an ectodomain in which at least two consecutive calcium-binding cadherin repeats are present. There are usually 5 or 6 domains, but in some cases as many as 34. Additional protein modules in the ectodomains point at adaptive evolution. Despite the occurrence of several conserved motifs in subsets of cytoplasmic domains, these domains are even more diverse than ectodomains and most likely have evolved separately from the ectodomains. By fine tuning molecular classifications, we reduced the number of solitary superfamily members. We propose a cadherin major branch, subdivided in two families and 8 subfamilies, and a cadherin-related major branch, subdivided in four families and 11 subfamilies. Accordingly, we propose a more appropriate nomenclature. Although still fragmentary, our insight into the molecular evolution of these remarkable proteins is steadily growing. Consequently, we can start to propose testable hypotheses for structure-function relationships with impact on our models of molecular evolution. An emerging concept is that the ever evolving diversity of cadherin structures is serving dual and important functions: specific cell adhesion and intricate cell signaling.

Mutations in the area composita protein αT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes. METHODS AND RESULTS We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and β-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells. CONCLUSION These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.

Passenger Mutations Confound Interpretation of All Genetically Modified Congenic Mice

Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.

Mx Proteins: Antiviral Gatekeepers That Restrain the Uninvited

SUMMARY Fifty years after the discovery of the mouse Mx1 gene, researchers are still trying to understand the molecular details of the antiviral mechanisms mediated by Mx proteins. Mx proteins are evolutionarily conserved dynamin-like large GTPases, and GTPase activity is required for their antiviral activity. The expression of Mx genes is controlled by type I and type III interferons. A phylogenetic analysis revealed that Mx genes are present in almost all vertebrates, usually in one to three copies. Mx proteins are best known for inhibiting negative-stranded RNA viruses, but they also inhibit other virus families. Recent structural analyses provide hints about the antiviral mechanisms of Mx proteins, but it is not known how they can suppress such a wide variety of viruses lacking an obvious common molecular pattern. Perhaps they interact with a (partially) symmetrical invading oligomeric structure, such as a viral ribonucleoprotein complex. Such an interaction may be of a fairly low affinity, in line with the broad target specificity of Mx proteins, yet it would be strong enough to instigate Mx oligomerization and ring assembly. Such a model is compatible with the broad “substrate” specificity of Mx proteins: depending on the size of the invading viral ribonucleoprotein complexes that need to be wrapped, the assembly process would consume the necessary amount of Mx precursor molecules. These Mx ring structures might then act as energy-consuming wrenches to disassemble the viral target structure.

Evolutionary functional analysis and molecular regulation of the ZEB transcription factors

ZEB1 and ZEB2, which are members of the ZEB family of transcription factors, play a pivotal role in the development of the vertebrate embryo. However, recent evidence shows that both proteins can also drive the process of epithelial-mesenchymal transition during malignant cancer progression. The understanding of how both ZEBs act as transcription factors opens up new possibilities for future treatment of advanced carcinomas. This review gives insight into the molecular mechanisms that form the basis of the multitude of cellular processes controlled by both ZEB factors. By using an evolutionary approach, we analyzed how the specific organization of the different domains and regulatory sites in ZEB1 and ZEB2 came into existence. On the basis of this analysis, a detailed overview is provided of the different cofactors and post-translational mechanisms that are associated with ZEB protein functionality.

New Insights into the Evolution of Metazoan Cadherins

Mining newly sequenced genomes of basal metazoan organisms reveals the evolutionary origin of modern protein families. Specific cell-cell adhesion and intracellular communication are key processes in multicellular animals, and members of the cadherin superfamily are essential players in these processes. Mammalian genomes contain over 100 genes belonging to this superfamily. By a combination of tBLASTn and profile hidden Markov model analyses, we made an exhaustive search for cadherins and compiled the cadherin repertoires in key organisms, including Branchiostoma floridae (amphioxus), the sea anemone Nematostella vectensis, and the placozoan Trichoplax adhaerens. Comparative analyses of multiple protein domains within known and novel cadherins enabled us to reconstruct the complex evolution in metazoa of this large superfamily. Five main cadherin branches are represented in the primitive metazoan Trichoplax: classical (CDH), flamingo (CELSR), dachsous (DCHS), FAT, and FAT-like. Classical cadherins, such as E-cadherin, arose from an Urmetazoan cadherin, which progressively lost N-terminal extracellular cadherin repeats, whereas its cytoplasmic domain, which binds the armadillo proteins p120ctn and β-catenin, remained quite conserved from placozoa to man. The origin of protocadherins predates the Bilateria and is likely rooted in an ancestral FAT cadherin. Several but not all protostomians lost protocadherins. The emergence of chordates coincided with a great expansion of the protocadherin repertoire. The evolution of ancient metazoan cadherins points to their unique and crucial roles in multicellular animal life.

ConTra v2: a tool to identify transcription factor binding sites across species, update 2011

Transcription factors are important gene regulators with distinctive roles in development, cell signaling and cell cycling, and they have been associated with many diseases. The ConTra v2 web server allows easy visualization and exploration of predicted transcription factor binding sites in any genomic region surrounding coding or non-coding genes. In this new version, users can choose from nine reference organisms ranging from human to yeast. ConTra v2 can analyze promoter regions, 5′-UTRs, 3′-UTRs and introns or any other genomic region of interest. Hundreds of position weight matrices are available to choose from, but the user can also upload any other matrices for detecting specific binding sites. A typical analysis is run in four simple steps of choosing the gene, the transcript, the region of interest and then selecting one or more transcription factor binding sites. The ConTra v2 web server is freely available at http://bioit.dmbr.ugent.be/contrav2/index.php.

ConTra: a promoter alignment analysis tool for identification of transcription factor binding sites across species

Transcription factors (TFs) are key components in signaling pathways, and the presence of their binding sites in the promoter regions of DNA is essential for their regulation of the expression of the corresponding genes. Orthologous promoter sequences are commonly used to increase the specificity with which potentially functional transcription factor binding sites (TFBSs) are recognized and to detect possibly important similarities or differences between the different species. The ConTra (conserved TFBSs) web server provides the biologist at the bench with a user-friendly tool to interactively visualize TFBSs predicted using either TransFac (1) or JASPAR (2) position weight matrix libraries, on a promoter alignment of choice. The visualization can be preceded by a simple scoring analysis to explore which TFs are the most likely to bind to the promoter of interest. The ConTra web server is available at http://bioit.dmbr.ugent.be/ConTra/index.php.

p53 promotes VEGF expression and angiogenesis in the absence of an intact p21-Rb pathway.

There is growing evidence that the p53 tumour suppressor downregulates vascular endothelial growth factor (VEGF) expression, although the underlying mechanisms remain unclear and controversial. Here we provide insights from in vitro experiments and in vivo xenotransplantation assays that highlight a dual role for p53 in regulating VEGF during hypoxia. Unexpectedly, and for the first time, we demonstrate that p53 rapidly induces VEGF transcription upon hypoxia exposure by binding, in an HIF-1α-dependent manner, to a highly conserved and functional p53-binding site within the VEGF promoter. However, during sustained hypoxia, p53 indirectly downregulates VEGF expression via the retinoblastoma (Rb) pathway in a p21-dependent manner, which is distinct from its role in cell-cycle regulation. Our findings have important implications for cancer therapy, especially for tumours that harbour wild-type TP53 and a dysfunctional Rb pathway.