Gene Duplication and Loss of AANAT in Mammals Driven by Rhythmic Adaptations.

Arylalkylamine N-acetyltransferase (AANAT) plays a crucial role in synchronizing internal biological functions to circadian and circannual changes. Generally speaking, only one copy of AANAT gene has been found in mammals, however, three independent duplications of this gene were detected in several cetartiodactyl lineages (i.e., Suidae, Hippopotamidae, and Pecora), which originated in the middle Eocene, a geological period characterized with the increased climate seasonality. Lineage-specific expansions of AANAT and the associated functional enhancement in these lineages strongly suggest an improvement in regulating photoperiodic response to adapt to seasonal climate changes. In contrast, independent inactivating mutations or deletions of the AANAT locus were identified in the four pineal-deficient clades (cetaceans, sirenians, xenarthrans, and pangolins). Loss of AANAT function in cetaceans and sirenians could disrupt the sleep-promoting effects of pineal melatonin, which might contribute to increasing wakefulness, adapting these clades to underwater sleep. The absence of AANAT and pineal glands in xenarthrans and pangolins may be associated with their body temperature maintenance. The present work demonstrates a far more complex and intriguing evolutionary pattern and functional diversity of mammalian AANAT genes than previously thought and provides further evidence for understanding AANAT evolution as driven by rhythmic adaptations in mammals.

Prevention of medulla neuron dedifferentiation by Nerfin-1 requires inhibition of Notch activity.

The Drosophila larval central nervous system comprises the central brain, ventral nerve cord and optic lobe. In these regions, neuroblasts (NBs) divide asymmetrically to self-renew and generate differentiated neurons or glia. To date, mechanisms of preventing neuron dedifferentiation are still unclear, especially in the optic lobe. Here, we show that the zinc-finger transcription factor Nerfin-1 is expressed in early-stage medulla neurons and is essential for maintaining their differentiation. Loss of Nerfin-1 activates Notch signaling, which promotes neuron-to-NB reversion. Repressing Notch signaling largely rescues dedifferentiation in nerfin-1 mutant clones. Thus, we conclude that Nerfin-1 represses Notch activity in medulla neurons and prevents them from dedifferentiation.

The TEA domain family transcription factor TEAD4 represses murine adipogenesis by recruiting the cofactors VGLL4 and CtBP2 into a transcriptional complex.

The Hippo signaling pathway is known to play an important role in multiple physiological processes, including adipogenesis. However, whether the downstream components of the Hippo pathway are involved in adipogenesis remains unknown. Here we demonstrate that the TEA domain family (TEAD) transcription factors are essential for adipogenesis in murine 3T3-L1 preadipocytes. Knockdown of TEAD1-4 stimulated adipogenesis and increased the expression of adipocyte markers in these cells. Interestingly, we found that the TEAD4 knockdown-mediated adipogenesis proceeded in a Yes-associated protein (YAP)/TAZ (Wwtr1)-independent manner and that adipogenesis suppression in WT cells involved formation of a ternary complex comprising TEAD4 and the transcriptional cofactors C-terminal binding protein 2 (CtBP2) and vestigial-like family member 4 (VGLL4). VGLL4 acted as an adaptor protein that enhanced the interaction between TEAD4 and CtBP2, and this TEAD4-VGLL4-CtBP2 ternary complex dynamically existed at the early stage of adipogenesis. Finally, we verified that TEAD4 directly targets the promoters of major adipogenesis transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) and adiponectin, C1Q, and collagen domain-containing (Adipoq) during adipogenesis. These findings reveal critical insights into the role of the TEAD4-VGLL4-CtBP2 transcriptional repressor complex in suppression of adipogenesis in murine preadipocytes.

Importin α1 Mediates Yorkie Nuclear Import via an N-terminal Non-canonical Nuclear Localization Signal.

The Hippo signaling pathway controls organ size by orchestrating cell proliferation and apoptosis. When the Hippo pathway was inactivated, the transcriptional co-activator Yorkie translocates into the nucleus and forms a complex with transcription factor Scalloped to promote the expression of Hippo pathway target genes. Therefore, the nuclear translocation of Yorkie is a critical step in Hippo signaling. Here, we provide evidence that the N-terminal 1-55 amino acids of Yorkie, especially Arg-15, were essential for its nuclear localization. By mass spectrometry and biochemical analyses, we found that Importin α1 can directly interact with the Yorkie N terminus and drive Yorkie into the nucleus. Further experiments show that the upstream component Hippo can inhibit Importin α1-mediated Yorkie nuclear import. Taken together, we identified a potential nuclear localization signal at the N-terminal end of Yorkie as well as a critical role for Importin α1 in Yorkie nuclear import.

Involvement of the Hippo pathway in regeneration and fibrogenesis after ischaemic acute kidney injury: YAP is the key effector.

Renal tubule cells can recover after they undergo AKI (acute kidney injury). An incomplete repair of renal tubules can result in progressive fibrotic CKD (chronic kidney disease). Studies have revealed the relationship between tubular epithelial cells and kidney fibrogenesis. However, the underlying mechanism remains unclear. Hippo pathway components were evaluated in complete/incomplete repair of I/R (ischaemia/reperfusion) AKI rat models, HK-2 cells and AKI human renal biopsy samples. We found that the expression levels of the Hippo pathway components changed dynamically during kidney regeneration and fibrogenesis in rat models of I/R-induced AKI and human renal biopsy samples. The transcription cofactor YAP (Yes-associated protein) might be a key effector of renal regeneration and fibrogenesis. Our results showed further that YAP might elicit both beneficial and detrimental effects on I/R AKI. After I/R injury occurred, YAP could promote the repair of the injured epithelia. The constant YAP increase and activation might be related to interstitial fibrosis and abnormal renal tubule differentiation. These results indicate that the proper modulation of the Hippo pathway, specifically the transcription cofactor YAP, during repair might be a potent therapeutic target in AKI-CKD transition after I/R injury.

Par-1 regulates tissue growth by influencing hippo phosphorylation status and hippo-salvador association.

The evolutionarily conserved Hippo (Hpo) signaling pathway plays a pivotal role in organ size control by balancing cell proliferation and cell death. Here, we reported the identification of Par-1 as a regulator of the Hpo signaling pathway using a gain-of-function EP screen in Drosophila melanogaster. Overexpression of Par-1 elevated Yorkie activity, resulting in increased Hpo target gene expression and tissue overgrowth, while loss of Par-1 diminished Hpo target gene expression and reduced organ size. We demonstrated that par-1 functioned downstream of fat and expanded and upstream of hpo and salvador (sav). In addition, we also found that Par-1 physically interacted with Hpo and Sav and regulated the phosphorylation of Hpo at Ser30 to restrict its activity. Par-1 also inhibited the association of Hpo and Sav, resulting in Sav dephosphorylation and destabilization. Furthermore, we provided evidence that Par-1-induced Hpo regulation is conserved in mammalian cells. Taken together, our findings identified Par-1 as a novel component of the Hpo signaling network.

Evaluation of antiphospholipid antibody assays using latent class analysis to address the lack of a reference standard.

BACKGROUND: Method evaluation of new assays for the detection of antiphospholipid antibodies (aPL) such as anti-cardiolipin (aCL) or anti-ß2-glycoprotein I (aß2-GPI) is challenging, as no internationally accepted reference material is available yet. Besides a lack of standardization, unacceptable inter-laboratory comparability of established tests is regularly observed. Owing to the absence of a commonly accepted reference standard, the evaluation of two research surface plasmon resonance (SPR) biosensor assays was performed using statistical methods from latent class analysis (LCA). METHODS: aCL and aß2-GPI IgG and IgM were measured in sera from 63 antiphospholipid syndrome patients, fulfilling the Sydney criteria, and in 34 healthy controls with four commercial assays. LCA was performed on the results and sera were assigned to the antibody-positive or antibody-negative group. Sera were subsequently evaluated in the SPR assays for aCL and aß2-GPI. Optimal cutoffs and diagnostic performances of the research systems were established employing the LCA-derived gold standard. RESULTS: With area under the curve results of 0.96 and 0.89 for the detection of aCL and aß2-GPI, the research SPR assays discriminated well between antibody-positive and antibody-negative sera. Their sensitivities and specificities were comparable to the investigated commercial immunoassays. CONCLUSIONS: SPR assays are a suitable tool for the detection of aCL and aß2-GPI with diagnostic performances not different from currently available commercial tests. LCA enabled the calculation of sensitivities and specificities for aPL assays in absence of a reference standard.

Drosophila casein kinase 2 (CK2) promotes warts protein to suppress Yorkie protein activity for growth control.

Drosophila Hippo signaling regulates Wts activity to phosphorylate and inhibit Yki in order to control tissue growth. CK2 is widely expressed and involved in a variety of signaling pathways. In this study we report that Drosophila CK2 promotes Wts activity to phosphorylate and inhibit Yki activity, which is independent of Hpo-induced Wts promotion. In vivo, CK2 overexpression suppresses hpo mutant-induced expanded (Ex) up-regulation and overgrowth phenotype, whereas it cannot affect wts mutant. Consistent with this, knockdown of CK2 up-regulates Hpo pathway target expression. We also found that Drosophila CK2 is essential for tissue growth as a cell death inhibitor as knockdown of CK2 in the developing disc induces severe growth defects as well as caspase3 signals. Taken together, our results uncover a dual role of CK2; although its major role is promoting cell survive, it may potentially be a growth inhibitor as well.

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites.

BACKGROUND: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. METHODS: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. RESULTS: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. CONCLUSIONS: These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites.

Hippo signaling in epithelial stem cells.

Over the past decade, discoveries on Hippo signaling have revealed a complex signaling network integrating various signaling pathways to modulate tissue homeostasis, organ size control, tissue repair, and regeneration. Malfunction of the Hippo pathway is associated with tumor and cancer development. Moreover, Hippo signaling has been proposed to act in numerous stem cells in a variety of organisms. Recently, more attention has been paid to define the functions of the Hippo pathway in tissue-specific stem cells, which have great potential to be used in cell-based therapies. Here we provide an overview of its roles in regulating stem cells in epithelial tissues and its potential implications in related cancers.

Development of a new parallelized, optical biosensor platform for label-free detection of autoimmunity-related antibodies.

Autoimmune diseases are characterized by the presence of autoantibodies in serum of affected patients. The heterogeneity of autoimmune relevant antigens creates a variety of different antibodies, which requires a simultaneous detection mode. For this reason, we developed a tool for parallelized, label-free, optical detection that accomplishes the characterization of multiple antigen-antibody interactions within a single measurement on a timescale of minutes. Using 11-aminoundecyltrimethoxysilane, we were able to immobilize proteinogenic antigens as well as an amino-functionalized cardiolipin on a glass surface. Assay conditions were optimized for serum measurements with a single spot antigen chip on a single spot 1-λ detection system. Minimized background signal allows a differentiation between patients and healthy controls with a good sensitivity and specificity. Applying polarized imaging reflectometric interference spectroscopy, we evaluated samples from three APS patients and three control subjects for this proof-of-principle and already obtained good results for ß2-glycoprotein I and cardiolipin.

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum.

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of ß2-glycoprotein I (ß2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of ß2-GPI to the cardiolipin surface. Using the switchSENSE sensor, ß2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between ß2-GPI-dependent autoimmune- and ß2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Comparative analyses of aging-related genes in long-lived mammals provide insights into natural longevity.

Extreme longevity has evolved multiple times during the evolution of mammals, yet its underlying molecular mechanisms remain largely underexplored. Here, we compared the evolution of 115 aging-related genes in 11 long-lived species and 25 mammals with non-increased lifespan (control group) in the hopes of better understanding the common molecular mechanisms behind longevity. We identified 16 unique positively selected genes and 23 rapidly evolving genes in long-lived species, which included nine genes involved in regulating lifespan through the insulin/IGF-1 signaling (IIS) pathway and 11 genes highly enriched in immune-response-related pathways, suggesting that the IIS pathway and immune response play a particularly important role in exceptional mammalian longevity. Interestingly, 11 genes related to cancer progression, including four positively selected genes and seven genes with convergent amino acid changes, were shared by two or more long-lived lineages, indicating that long-lived mammals might have evolved convergent or similar mechanisms of cancer resistance that extended their lifespan. This suggestion was further corroborated by our identification of 12 robust candidates for longevity-related genes closely related to cancer.

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

Synthesis and biological evaluation of a novel cardiolipin affinity matrix.

Cardiolipin (1) is a dimeric phospholipid found in the mitochondrial membranes of both plants and animals. In order to understand better its role, we report the preparation of an immobilised analogue (2) using phosphoramidite chemistry; the probe has been used successfully to bind a recombinant protein containing a cardiolipin-binding domain.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded.

Non-receptor tyrosine kinase activated cdc42 kinase was reported to participate in several types of cancers in mammals. It is also believed to have an anti-apoptotic function in Drosophila. Here, we report the identification of Drosophila activated cdc42 kinase as a growth promoter and a novel Hippo signaling pathway regulator. We find that activated cdc42 kinase promotes tissue growth through modulating Yorkie activity. Furthermore, we demonstrate that activated cdc42 kinase interacts with Expanded and induces tyrosine phosphorylation of Expanded on multiple sites. We propose a model that activated cdc42 kinase negatively regulates Expanded by changing its phosphorylation status to promote tissue growth. Moreover, we show that ack genetically interacts with merlin and expanded. Thus, we identify Drosophila activated cdc42 kinase as a Hippo pathway regulator.

Taiman acts as a coactivator of Yorkie in the Hippo pathway to promote tissue growth and intestinal regeneration.

The Hippo signaling pathway regulates tissue growth and organ size through controlling cell growth, proliferation and apoptosis. During these processes, the coactivator Yorkie partners with the transcription factor Scalloped to mediate Hippo pathway-regulated cellular functions. Here, we demonstrate that Taiman facilitates the activity of Yorkie. First, Taiman overexpression upregulates Hippo pathway-responsive genes and induces tissue overgrowth. Second, the loss of tai downregulates the expression of Hippo pathway target genes and reduces organ size as well as tissue overgrowth caused by Yorkie overexpression. Furthermore, we provide evidence that Taiman binds to Yorkie and facilitates the activity of Yorkie-Scalloped to activate the transcription of several Hippo pathway target genes. Moreover, we found that the C-terminus of Taiman is indispensable for the function of Taiman in Hippo signaling. Finally, we demonstrate that Taiman is also required in intestinal stem cell proliferation. Our findings suggest Taiman is an essential coactivator of Yorkie.

Growth suppressor lingerer regulates bantam microRNA to restrict organ size.

The evolutionarily conserved Hippo signaling pathway plays an important role in organ size control by regulating cell proliferation and apoptosis. Here, we identify Lingerer (Lig) as a growth suppressor using RNAi modifying screen in Drosophila melanogaster. Loss of lig increases organ size and upregulates bantam (ban) and the expression of the Hippo pathway target genes, while overexpression of lig results in diminished ban expression and organ size reduction. We demonstrate that Lig C-terminal exhibits dominant-negative function on growth and ban expression, and thus plays an important role in organ size control and ban regulation. In addition, we provide evidence that both Yki and Mad are essential for Lig-induced ban expression. We also show that Lig regulates the expression of the Hippo pathway target genes partially via Yorkie. Moreover, we find that Lig physically interacts with and requires Salvador to restrict cell growth. Taken together, we demonstrate that Lig functions as a critical growth suppressor to control organ size via ban and Hippo signaling.

Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis.

Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. Here we report that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. We show that Su(dx) targets Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induces proliferation in the fly midgut epithelium, which can be rescued by overexpressed Pez. We also demonstrate that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, we find that Kibra, a binding partner of Pez, stabilizes Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut.