IP3RPEP6, a novel peptide inhibitor of IP3 receptor channels that does not affect connexin-43 hemichannels.

AIM: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca2+ -release channels with crucial roles in cell function. Current IP3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP3 R subtypes. We developed a novel peptide inhibitor of IP3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP3 R stimulation. METHODS: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP3 R2 channels and carbachol-induced IP3 -mediated Ca2+ responses in IP3 R1, 2 or 3 expressing cells, triple IP3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3 Rs were verified by co-immunoprecipitation and affinity pull-down assays. RESULTS: IP3RPEP6 concentration-dependently reduced the open probability of IP3 R2 channels and competitively inhibited IP3 Rs in an IC50 order of IP3 R2 (~3.9 µM) < IP3 R3 (~4.3 µM) < IP3 R1 (~9.0 µM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP3 R2 but not with IP3 R1; interaction with IP3 R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol-induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3 -Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP3 R-inhibiting peptide. CONCLUSION: IP3RPEP6 inhibits IP3 R2/R3 at concentrations that have limited effects on IP3 R1. IP3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP3 -triggered Ca2+ responses.

Functions of the RIP kinase family members in the skin.

The receptor interacting protein kinases (RIPK) are a family of serine/threonine kinases that are involved in the integration of various stress signals. In response to several extracellular and/or intracellular stimuli, RIP kinases engage signaling cascades leading to the activation of NF-κB and mitogen-activated protein kinases, cell death, inflammation, differentiation and Wnt signaling and can have kinase-dependent and kinase-independent functions. Although it was previously suggested that seven RIPKs are part of the RIPK family, phylogenetic analysis indicates that there are only five genuine RIPKs. RIPK1 and RIPK3 are mainly involved in controlling and executing necroptosis in keratinocytes, while RIPK4 controls proliferation and differentiation of keratinocytes and thereby can act as a tumor suppressor in skin. Therefore, in this review we summarize and discuss the functions of RIPKs in skin homeostasis as well as the signaling pathways involved.

Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.

The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of ß-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.

Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Armc8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions.

Armadillo-repeat-containing protein 8 (Armc8) belongs to the family of armadillo-repeat containing proteins, which have been found to be involved in diverse cellular functions including cell-cell contacts and intracellular signaling. By comparative analyses of armadillo repeat protein structures and genomes from various premetazoan and metazoan species, we identified orthologs of human Armc8 and analyzed in detail the evolutionary relationship of Armc8 genes and their encoded proteins. Armc8 is a highly ancestral armadillo protein although not present in yeast. Consequently, Armc8 is not the human ortholog of yeast Gid5/Vid28.Further, we performed a candidate approach to characterize new protein interactors of Armc8. Interactions between Armc8 and specific δ-catenins (plakophilins-1, -2, -3 and p0071) were observed by the yeast two-hybrid approach and confirmed by co-immunoprecipitation and co-localization. We also showed that Armc8 interacts specifically with αE-catenin but neither with αN-catenin nor with αT-catenin. Degradation of αE-catenin has been reported to be important in cancer and to be regulated by Armc8. A similar process may occur with respect to plakophilins in desmosomes. Deregulation of desmosomal proteins has been considered to contribute to tumorigenesis.

Ancient Origin of the CARD-Coiled Coil/Bcl10/MALT1-Like Paracaspase Signaling Complex Indicates Unknown Critical Functions.

The CARD-coiled coil (CC)/Bcl10/MALT1-like paracaspase (CBM) signaling complexes composed of a CARD-CC family member (CARD-9, -10, -11, or -14), Bcl10, and the type 1 paracaspase MALT1 (PCASP1) play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians). Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects) lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD-CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD-CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD-CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein-protein interactions, NF-κB signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD-CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-κB-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1.

Nanos genes and their role in development and beyond.

The hallmark of Nanos proteins is their typical (CCHC)2 zinc finger motif (zf-nanos). Animals have one to four nanos genes. For example, the fruit fly and demosponge have only one nanos gene, zebrafish and humans have three, and Fugu rubripes has four. Nanos genes are mainly known for their evolutionarily preserved role in germ cell survival and pluripotency. Nanos proteins have been reported to bind the C-terminal RNA-binding domain of Pumilio to form a post-transcriptional repressor complex. Several observations point to a link between the miRNA-mediated repression complex and the Nanos/Pumilio complex. Repression of the E2F3 oncogene product is, indeed, mediated by cooperation between the Nanos/Pumilio complex and miRNAs. Another important interaction partner of Nanos is the CCR4-NOT deadenylase complex. Besides the tissue-specific contribution of Nanos proteins to normal development, their ectopic expression has been observed in several cancer cell lines and various human cancers. An inverse correlation between the expression levels of human Nanos1 and Nanos3 and E-cadherin was observed in several cancer cell lines. Loss of E-cadherin, an important cell-cell adhesion protein, contributes to tumor invasion and metastasis. Overexpression of Nanos3 induces epithelial-mesenchymal transition in lung cancer cell lines partly by repressing E-cadherin. Other than some most interesting data from Nanos knockout mice, little is known about mammalian Nanos proteins, and further research is needed. In this review, we summarize the main roles of Nanos proteins and discuss the emerging concept of Nanos proteins as oncofetal antigens.

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.

Efficient analysis of mouse genome sequences reveal many nonsense variants.

Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1.

MALT1 is not alone after all: identification of novel paracaspases.

Paracaspases and metacaspases are two families of caspase-like proteins identified in 2000. Up until now paracaspases were considered a single gene family with one known non-metazoan paracaspase in the slime mold Dictyostelium and a single animal paracaspase called MALT1. Human MALT1 is a critical signaling component in many innate and adaptive immunity pathways that drive inflammation, and when it is overly active, it can also cause certain forms of cancer. Here, we report the identification and functional analysis of two new vertebrate paracaspases, PCASP2 and PCASP3. Functional characterization indicates that both scaffold and protease functions are conserved across the three vertebrate paralogs. This redundancy might explain the loss of two of the paralogs in mammals and one in Xenopus. Several of the vertebrate paracaspases currently have incorrect or ambiguous annotations. We propose to annotate them accordingly as PCASP1, PCASP2, and PCASP3 similar to the caspase gene nomenclature. A comprehensive search in other metazoans and in non-metazoan species identified additional new paracaspases. We also discovered the first animal metacaspase in the sponge Amphimedon. Comparative analysis of the active site suggests that paracaspases constitute one of the several subclasses of metacaspases that have evolved several times independently.

Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

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.

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 ß-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.

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.